WO2020177071A1 - 体外诱导和/或扩增tscm的组合物、培养基和方法 - Google Patents

体外诱导和/或扩增tscm的组合物、培养基和方法 Download PDF

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WO2020177071A1
WO2020177071A1 PCT/CN2019/076957 CN2019076957W WO2020177071A1 WO 2020177071 A1 WO2020177071 A1 WO 2020177071A1 CN 2019076957 W CN2019076957 W CN 2019076957W WO 2020177071 A1 WO2020177071 A1 WO 2020177071A1
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antibody
ligand
cells
medium
scm
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French (fr)
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徐建青
张晓燕
廖启彬
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Shanghai Sinobay Biotechnology Co Ltd
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Shanghai Sinobay Biotechnology Co Ltd
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Priority to PCT/CN2019/076957 priority Critical patent/WO2020177071A1/zh
Priority to US17/436,306 priority patent/US20220177839A1/en
Priority to EP19917821.1A priority patent/EP3936611A4/en
Publication of WO2020177071A1 publication Critical patent/WO2020177071A1/zh
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Definitions

  • the invention belongs to the field of cell culture. Specifically, the present invention relates to compositions, media, and methods for inducing and/or expanding cells. More specifically, the present invention relates to a composition, medium and method for inducing and/or expanding stem cell-like memory T cells in vitro.
  • T cells have strong anti-infection and anti-tumor abilities. They are the backbone of the body's adaptive immune system to defend against pathogens, tumor cells and other foreign bodies. It can not only quickly respond to foreign bodies or neoplasms, but also provide longer-term immune protection and memory. . Studies have confirmed that by inducing and expanding memory T cells in vitro and transfused back into patients, many diseases can be treated, especially cancers that seriously threaten human health.
  • T cells At present, in the in vitro induction and expansion methods of memory T cells, the more mature is the use of anti-CD3 antibody and anti-CD28 antibody combined with IL-2 to stimulate differentiation, which stimulates the proliferation of differentiated T cells and significantly reduces naive T cells.
  • the number of T cells in the terminal state has increased significantly.
  • Such T cells have a short survival time in the body, cannot provide long-term immune protection, and cannot meet the needs of clinical treatment.
  • T SCM stem cell-like memory T cell
  • T SCM is located upstream of memory T cells, has the characteristics of stem cells, has stronger multi-directional differentiation potential, and can differentiate into central memory T cells (T CM ), effector memory T cells (T EM ) and effector T cells (T EF ), which produces a large number of effectors such as IFN- ⁇ , and has a more powerful killing ability.
  • T SCM has the potential for multi-directional differentiation as well as the ability to self-renew, so as to maintain its own homeostasis in the absence of antigens. Studies have shown that T SCM has a more powerful anti-tumor ability, and its self-renewal ability, secondary immune response strength and survival time in vivo are better than traditional memory T cells.
  • the adoptive reinfusion of immune cell therapy is facing a bottleneck, especially the rapid development of chimeric antigen receptor T cell (CAR-T) immunotherapy, which requires sufficient immunization within a short period of time (14 days)
  • CAR-T chimeric antigen receptor T cell
  • the current induced and expanded T cells are mostly cells in the terminal stage, especially the more commonly used anti-CD3 antibody and anti-CD28 antibody combined with IL-2 culture method, the proportion of T SCM obtained is low, accounting for only 9%.
  • T SCM multi-directional differentiation potential and self-renewal characteristics of T SCM have just solved the clinical problems of adoptive reinfusion of immune cell therapy in in vitro induction and expansion, and poor efficacy in vivo.
  • T SCM has a more powerful anti-tumor ability in vivo.
  • the reason may be that the strong viability of T SCM greatly prolongs the survival time of CAR-T cells in vivo. Therefore, the powerful anti-tumor ability makes T SCM the most effective cell subset in immunotherapy. Therefore, the use of antigen-specific T SCM , TCR-T SCM or CAR-T SCM induced and amplified in vitro for immunotherapy will become a new approach and new means of cell therapy.
  • the purpose of the present invention is to provide a composition and culture medium for inducing and/or expanding stem cell-like memory T cells in vitro to overcome the deficiencies of the prior art.
  • the invention also provides a method for in vitro induction and/or expansion of stem cell-like memory T cells using the combination of the invention.
  • the ratio of CAR-T SCM to CAR-T cells is significantly increased, and can be directly used for the reinfusion therapy of CAR-T therapy of patients.
  • the present invention provides a composition for inducing and/or expanding stem cell-like memory T cells (TSCM ) in vitro.
  • the composition is composed of an inducer, and the inducer comprises an interleukin- 7 (IL-7) and Interleukin-21 (IL-21).
  • the inducer further comprises one or more of IL-3, IL-12, IL-15 and IL-18; preferably, the inducer further comprises IL- 15.
  • composition according to the present invention wherein the inducer further comprises a GSK-3 ⁇ inhibitor; preferably, the GSK-3 ⁇ inhibitor is TWS119;
  • composition according to the present invention wherein the inducer comprises IL-7, IL-21, IL-15 and GSK-3 ⁇ inhibitor;
  • composition according to the present invention wherein the inducer is added to the culture medium in the form of free protein, coupled to magnetic beads, coated on a cell culture plate, or expressed on a cell.
  • the working concentration of each inducer is as follows:
  • the working concentration of each inducer except the GSK-3 ⁇ inhibitor is in the range of 1-100ng/mL, and the working concentration of the GSK-3 ⁇ inhibitor is in the range of 1-50 ⁇ M.
  • composition according to the present invention wherein the working concentration of each inducer in the composition is as follows:
  • the composition further comprises 1-100ng/mL IL-15;
  • the composition further comprises 1-50 ⁇ M GSK-3 ⁇ inhibitor.
  • the present invention provides a medium for inducing and/or expanding stem cell-like memory T cells (TSCM ) in vitro, wherein the medium comprises a T cell growth basal medium and the composition .
  • TSCM stem cell-like memory T cells
  • the preparation method of the culture medium or culture medium for in vitro induction and/or expansion of T SCM according to the present invention includes the step of adding the composition to the T cell growth basal medium;
  • feeder cells infected or transfected with genetic material encoding one or more components of the composition in the form of a vector to a T cell growth basal medium, and co-cultured with the cultured target cells;
  • exogenous cells to express one or more components of the composition so that it can function in the cell supernatant cultured using T cell growth basal medium.
  • one or a combination of methods can be used to prepare the medium.
  • the present invention also provides the use of the composition and/or medium for inducing and/or amplifying TSCM in vitro.
  • T SCM is T SCM is the peripheral blood mononuclear cells (PBMC), CD4 + T cells, CD8 + T cells or CD4 - CD8 - T cells.
  • PBMC peripheral blood mononuclear cells
  • T SCM is CAR-T cell, TCR-T cell or tumor infiltrating lymphocyte.
  • the present invention provides a method for inducing and/or expanding T SCM in vitro, said method comprising the step of inducing and/or expanding cells using the composition and/or culture medium of the present invention;
  • the method includes the following steps:
  • PBMC peripheral blood mononuclear cells
  • step 2 Put the cells obtained in step 1 in the medium of the present invention or in the T cell growth basal medium while adding the composition and adding the stimulant, and culture for 6-7 days;
  • the stimulant is anti-CD3 antibody, anti-CD28 antibody/CD28 ligand, anti-CD137 antibody/CD137 ligand, anti-OX40 antibody/OX40 ligand, anti-CD160 antibody/CD160 ligand, Toll-like receptor 1 ( TLR1) ligand, Toll-like receptor 2 (TLR2) ligand, Toll-like receptor 5 (TLR5) ligand, Toll-like receptor 6 (TLR6) ligand and retinoic acid inducible gene protein (RIG-I)
  • TLR1 Toll-like receptor 1
  • TLR2 Toll-like receptor 2
  • TLR5 Toll-like receptor 5
  • TLR6 Toll-like receptor 6
  • ligand retinoic acid inducible gene protein
  • the stimulating agent is a magnetic bead that couples an anti-CD3 antibody and an anti-CD28 antibody;
  • step 3 Put the cells obtained in step 2 in the medium of the present invention or in the T cell growth basal medium while adding the composition, supplement the medium of the present invention or supplement the T cell growth basal medium every 2-4 days The composition is added at the same time until the end of the cell culture cycle.
  • the method includes the following steps:
  • step 3 Put the cells obtained in step 2 in the medium of the present invention or in the T cell growth basal medium while adding the composition. From the 4th day onwards, supplement with fresh culture medium or supplement with fresh T cell every 3 days. The cell growth basal medium is simultaneously added to the composition until the end of the cell culture cycle.
  • the method is an in vitro method for inducing and/or expanding stem cell-like memory chimeric antigen receptor T cells (CAR-T SCM ), which includes the following steps:
  • PBMC peripheral blood mononuclear cells
  • step 2) Put the cells obtained in step 1) in the culture medium of the present invention or in the T cell growth basal medium while adding the composition and adding the stimulant, and culture for 1-2 days;
  • the stimulant is anti-CD3 antibody, anti-CD28 antibody/CD28 ligand, anti-CD137 antibody/CD137 ligand, anti-OX40 antibody/OX40 ligand, anti-CD160 antibody/CD160 ligand, Toll-like receptor 1 ( TLR1) ligand, Toll-like receptor 2 (TLR2) ligand, Toll-like receptor 5 (TLR5) ligand, Toll-like receptor 6 (TLR6) ligand and retinoic acid inducible gene protein (RIG-I)
  • TLR1 Toll-like receptor 1
  • TLR2 Toll-like receptor 2
  • TLR5 Toll-like receptor 5
  • TLR6 Toll-like receptor 6
  • ligand retinoic acid inducible gene protein
  • the stimulating agent is a magnetic bead that couples an anti-CD3 antibody and an anti-CD28 antibody;
  • step 2) Transfect or infect the cells obtained in step 2) with vectors carrying chimeric antigen receptors;
  • step 4) The cells obtained in step 3) are placed in the medium of the present invention or placed in the T cell growth basal medium while adding the composition, and adding the stimulant to continue culturing for 5-6 days before removing the stimulant. From the 4th day, supplement the medium or supplement the T cell growth basal medium every 2-4 days and add the composition at the same time until the CAR-T cell culture cycle ends;
  • the stimulant is anti-CD3 antibody, anti-CD28 antibody/CD28 ligand, anti-CD137 antibody/CD137 ligand, anti-OX40 antibody/OX40 ligand, anti-CD160 antibody/CD160 ligand, Toll-like receptor 1 ( TLR1) ligand, Toll-like receptor 2 (TLR2) ligand, Toll-like receptor 5 (TLR5) ligand, Toll-like receptor 6 (TLR6) ligand and retinoic acid inducible gene protein (RIG-I)
  • TLR1 Toll-like receptor 1
  • TLR2 Toll-like receptor 2
  • TLR5 Toll-like receptor 5
  • TLR6 Toll-like receptor 6
  • ligand retinoic acid inducible gene protein
  • the stimulating agent is a magnetic bead coupled with an anti-CD3 antibody and an anti-CD28 antibody.
  • the method includes the following steps:
  • PBMC peripheral blood mononuclear cells
  • CD8 + T cells CD8 + T cells
  • step 2) Place the cells obtained in step 1) in the culture medium of the present invention or in the T cell growth basal medium while adding the composition, and adding magnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody, and culture for 1 day ;
  • step 4) After centrifugation, the cells obtained in step 3) are cultured overnight, placed in the culture medium of the present invention or in the T cell growth basal medium while adding the composition, and adding the coupled anti-CD3 antibody and anti-CD28 antibody After the magnetic beads were cultured for 5-6 days, the magnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody were removed. From the 4th day, supplement the medium or supplement the T cell growth basal medium every 3 days while adding the composition, and continue the culture until the CAR-T cell culture cycle ends;
  • the stimulant is anti-CD3 antibody, anti-CD28 antibody/CD28 ligand, anti-CD137 antibody/CD137 ligand, anti-OX40 antibody/OX40 ligand, anti-CD160 antibody/CD160 ligand, Toll-like receptor 1 ( TLR1) ligand, Toll-like receptor 2 (TLR2) ligand, Toll-like receptor 5 (TLR5) ligand, Toll-like receptor 6 (TLR6) ligand and retinoic acid inducible gene protein (RIG-I)
  • TLR1 Toll-like receptor 1
  • TLR2 Toll-like receptor 2
  • TLR5 Toll-like receptor 5
  • TLR6 Toll-like receptor 6
  • ligand retinoic acid inducible gene protein
  • the stimulating agent is a magnetic bead coupled with an anti-CD3 antibody and an anti-CD28 antibody.
  • the method is an in vitro method for inducing and/or expanding stem cell-like memory T cell receptor T cells (TCR- TSCM ), which includes the following steps:
  • PBMC peripheral blood mononuclear cells
  • CD4 + T cells CD8 + T cells
  • step 2) Put the cells obtained in step 1) in the culture medium of the present invention or in the T cell growth basal medium while adding the composition and adding the stimulant, and culture for 1-2 days;
  • the stimulant is anti-CD3 antibody, anti-CD28 antibody/CD28 ligand, anti-CD137 antibody/CD137 ligand, anti-OX40 antibody/OX40 ligand, anti-CD160 antibody/CD160 ligand, Toll-like receptor 1 ( TLR1) ligand, Toll-like receptor 2 (TLR2) ligand, Toll-like receptor 5 (TLR5) ligand, Toll-like receptor 6 (TLR6) ligand and retinoic acid inducible gene protein (RIG-I)
  • TLR1 Toll-like receptor 1
  • TLR2 Toll-like receptor 2
  • TLR5 Toll-like receptor 5
  • TLR6 Toll-like receptor 6
  • ligand chimeric antigen receptor target antigen
  • PD-1 blocking antibody CTLA-4 blocking antibody
  • LAG-3 blocking antibody Tm-3 blocking antibody
  • Tm-3 blocking antibody and BTLA blocking antibody Tm-3 blocking antibody
  • the stimulating agent is a magnetic bead that couples an anti-CD3 antibody and an anti-CD28 antibody;
  • step 2) Transfect or infect the cells obtained in step 2) with vectors carrying T cell receptors;
  • step 4) The cells obtained in step 3) are placed in the medium of the present invention or placed in the T cell growth basal medium while adding the composition, and adding the stimulant to continue culturing for 5-6 days before removing the stimulant. Starting from the 4th day, supplement the medium or supplement the T cell growth basal medium every 2-4 days while adding the composition until the end of the TCR-T cell culture cycle.
  • the method includes the following steps:
  • PBMC peripheral blood mononuclear cells
  • CD4 + T cells CD8 + T cells
  • step 2) Place the cells obtained in step 1) in the culture medium of the present invention or in the T cell growth basal medium while adding the composition, and adding magnetic beads coupling anti-CD3 antibody and anti-CD28 antibody, and culture for 1 day;
  • step 4) After centrifugation, the cells obtained in step 3) are cultured overnight, placed in the medium of the present invention or placed in the T cell growth basal medium while adding the composition, and adding the magnetic coupling anti-CD3 antibody and anti-CD28 antibody After the beads were cultured for 5-6 days, the magnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody were removed. Starting from the 4th day, supplement the medium or supplement the T cell growth basal medium every 3 days while adding the composition, and continue the culture until the end of the TCR-T cell culture cycle.
  • the present invention provides T SCM prepared according to the method of the present invention.
  • the T SCM is CAR-T SCM , TCR-T SCM or tumor infiltrating lymphocytes.
  • the invention also provides the use of the T SCM of the invention in preparing a medicine for treating tumors.
  • the present invention provides a cellular immunotherapy method for tumors, the method comprising using the T SCM of the present invention.
  • the inventors of the present invention found that the addition of cytokines IL-7, IL-15 and IL-21 during T cell culture can efficiently induce and/or expand T cells into T SCM in vitro, and T SCM accounts for The proportion of T cells has increased significantly, and the number of cells has increased significantly.
  • the method disclosed in the present invention can be used for in vitro induction and/or expansion of stem cell-like memory chimeric antigen receptor T cells (CAR-T SCM ), and can also be used for stem cell-like memory T cell receptor T cells (TCR-T SCM ) in vitro induction and/or amplification.
  • the composition used in the present invention is safe and easy to obtain.
  • the TCR-T SCM or CAR-T SCM induced and/or amplified by the composition can be used for cell therapy of tumor patients, providing a new strategy for cancer immunotherapy. Has important development and promotion value.
  • Figure 1 shows the effect of the medium according to the present invention on the induction of CD8 + T SCM in vitro; among which, Figure 1A shows the proportion of T SCM in CD8 + T cells in the peripheral blood of healthy people; Figure 1B shows the effect of the medium according to the present invention on Comparison of the effect of inducing CD8 + T cells to differentiate into T SCM on the 7th and 14th day of culture. The higher the ratio of T SCM , the better the induction effect.
  • Fig. 2 shows the effect of the medium according to the present invention on the induction and expansion of CD8 + T SCM in vitro; among them, Fig. 2A is a cell count chart of the medium according to the present invention inducing and expanding CD8 + T SCM . For the same culture time, the more CD8 + T SCM cells, the better the induction and expansion effect of the composition; Figure 2B shows the CD8 + T of the medium according to the present invention on the 7th and 14th days of culture SCM induction and amplification factor.
  • Figure 3 shows the in vitro differentiation and expansion effects of the two media according to the present invention on CD19 CAR-T SCM ; among them, Figure 3A shows the CD19 chimeric antigen receptor T cells (CD19 CAR-T) on the 11th day of culture. The flow cytometry results of SCM ; Figure 3B is a statistical diagram of the proportion of CD19 CAR-T SCM in T cells on the 7th and 11th day of culture; Figure 3C is the in vitro expansion of CD19 CAR-T SCM on the 7th and 11th day of culture Increase effect.
  • Figure 4 shows the effects of two media according to the present invention on the in vitro differentiation and expansion of CD19 CAR-CD8 + T SCM .
  • Figure 4A shows the flow cytometry results of T SCM in CD19 CAR-CD8 + T cells on the 11th day of culture
  • Figure 4B shows the ratio of CD19 CAR-CD8 + T SCM to CD8 + T cells on the 7th and 11th day of culture Statistics
  • Figure 4C shows the in vitro expansion effect of CD19 CAR-CD8 + T SCM on the 7th and 11th day of culture.
  • Figure 5 shows the in vitro activation effect of CD19 CAR-CD8 + T cells cultured in two media according to the present invention.
  • Figure 6 shows the in vitro killing activity of CD19 CAR-CD8 + T cells cultured in two media according to the present invention.
  • Fig. 7 shows the anti-tumor effects of CD19 + CAR-T cells cultured in two media according to the present invention in severe combined immunodeficiency (NSG) mice.
  • the medium B-E of the present invention is prepared, and the medium A for the control is prepared at the same time, wherein the common components of the medium are as follows:
  • T cell growth basal medium lymphocyte serum-free medium (KBM581, Corning);
  • the different components of the medium are as follows:
  • Medium A contains 50ng/mL IL-2;
  • Medium B contains 5ng/mL IL-7 and 30ng/mL IL-21;
  • Medium C contains 5ng/mL IL-7, 30ng/mL IL-21 and 5 ⁇ M TWS119;
  • Medium D contains 5ng/mL IL-7, 30ng/mL IL-21 and 10ng/mL IL-15;
  • Medium E contains 5ng/mL IL-7, 30ng/mL IL-21, 10ng/mL IL-15, and 5 ⁇ M TWS119
  • Example 1 The culture medium of the present invention induces and amplifies CD8 + T SCM in vitro
  • Xinwan Company Take pre-prepared human primary T cells (Shanghai Xinwan Biotechnology Co., Ltd., hereinafter referred to as "Xinwan Company"), and use magnetic bead negative selection to obtain human primary CD8 + T cells (CD3 + CD8 + ), And spread it in a 96-well U-bottom culture plate, 5 ⁇ 10 4 CD8 + T cells per well;
  • Figure 1 is a comparison diagram of the in vitro induction effect of medium AE on CD8 + T SCM .
  • Figure 1A shows the proportion of T SCM in CD8 + T cells (primary) in the peripheral blood of healthy people.
  • the proportion of T SCM in primary CD8 + T cells is extremely low, accounting for less than 2%.
  • Figure 1B shows that the medium AE induces CD8 + T cell differentiation on the 7th and 14th day of culture Compare the effects of T SCM .
  • the other medium BE can effectively induce the differentiation of CD8 + T cells into T SCM , and the induction effect of IL-2 is much better.
  • the average induction efficiency of medium A was lower than 10% on the 14th day of culture.
  • the ratio of T SCM in the medium B group to the entire primary CD8+ T cells was as high as 78.7%, and the T SCM induction efficiency in the medium D group was slightly lower (72.0%).
  • the medium C group and E group that is, the combined use of TWS119, can reach 87.0% T SCM induction efficiency.
  • inducing agent of the present invention may be effective to induce T SCM concentration range formed as follows: IL-7 (1-100ng / mL ), IL-15 (1-100ng / mL), IL-21 (1 -100ng/mL), and TWS119 (1-50 ⁇ M).
  • Figure 2 shows the effect of the medium AE according to the present invention on the induction and expansion of CD8 + T SCM in vitro.
  • Fig. 2A is a cell count chart of CD8 + T SCM induced and expanded by medium AE according to the present invention.
  • the medium A On the 14th day of culture, the medium A has the lowest effect on inducing and amplifying CD8 + T SCM .
  • the medium B group according to the present invention has a significantly better effect on inducing and amplifying CD8 + T SCM than the medium C group according to the present invention.
  • the induction and expansion effect of the medium group D according to the present invention is better.
  • Figure 2B shows the CD8 + T SCM induction and expansion folds of the medium AE according to the present invention on the 7th and 14th days of culture.
  • the effects of inducing and expanding CD8 + T SCM are as follows: the medium A group according to the present invention is 800 times, the medium B group according to the present invention reaches 1162 times, and the medium D according to the present invention Group up to 5269 times.
  • IL-7, IL-15, and IL-21 effectively amplified CD8 + T SCM with a concentration range of 1-100ng/mL.
  • Medium B and D of Example 2 of the present invention is S C M vitro CD19 CAR-T inducing differentiation and Amplifications
  • PBMC peripheral blood mononuclear cells
  • Figure 3 shows the effect of two T SCM induction and expansion medium B or D on the in vitro differentiation and expansion of CD19 CAR-T SCM .
  • Fig. 3A shows the flow cytometry detection process of CD19 CAR-T SCM on the 11th day of culture and the corresponding cell subpopulation ratio.
  • Figure 3B shows the statistical results of the proportion of CD19 CAR-T SCM to CD19 CAR-T cells on the 7th and 11th day of culture.
  • the proportion of T SCM CD45RA + CCR7 + CD95 + induced to differentiate in Medium B and Medium D reached 81.3% and 64.4%, respectively, indicating that both mediums can Effectively induce the differentiation of CD19 CAR-T SCM .
  • Figure 3C shows the effects of two T SCM induction and expansion media on the in vitro differentiation and expansion of CD19 CAR-T SCM .
  • Experimental results show that medium D can more effectively induce and amplify CD19CAR-T SCM .
  • Example 3 Medium B and D of the present invention can induce differentiation and differentiation of CD19 CAR-CD8 + T SCM in vitro Amplification effect
  • CD19 chimeric antigen receptor CD8 + T cells CD19 CAR-CD8 + T cells
  • PBMC peripheral blood mononuclear cells
  • the infection was centrifuged at 1200g for 2 hours at 32°C. After centrifugation, the culture was continued overnight, the supernatant was discarded and supplemented with medium B or D according to the present invention.
  • the magnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody were removed and supplemented with medium B or D according to the present invention.
  • Figure 4 shows the effect of two media B or D on the in vitro differentiation and expansion of CD19 CAR-CD8 + T SCM .
  • Fig. 4A shows the flow cytometry detection process of CD19 CAR-CD8 + T SCM cultured for 11 days and the corresponding cell subpopulation ratio.
  • Figure 4B shows the statistical results of the proportion of CD19 CAR-CD8 + T SCM in CD19 CAR-CD8 + T cells.
  • the proportion of T SCM CD45RA + CCR7 + CD95 + induced by medium B and D reached 93.2% and 82.5%, respectively, indicating that both cytokine combinations can effectively induce CD19 CAR -CD8 + T SCM differentiation.
  • Figure 4C shows the effects of two media B or D on the induction of differentiation and expansion of CD19 CAR-CD8 + T SCM in vitro. Experimental results show that medium D can more effectively induce and amplify CD19 CAR-CD8 + T SCM .
  • the results of the experiment are shown in Figure 5.
  • the CD19CAR-CD8 + T cells induced and expanded in the two media B and D can effectively activate and secrete a large number of functional effector molecules IFN- ⁇ after contacting tumor target cells. , But the activation effect of medium D is better.
  • T cells in the untransduced control group (UTD) do not produce the effector molecule IFN- ⁇ .
  • Medium B and D of Example 5 of the present invention is cultured in vitro CD19 CAR-CD8 + T cell killing effect.
  • eFluor 450 is used to label K562-CD19 cells. Wash the cells twice with PBS or serum-free RPMI1640 medium, 500g, and centrifuge for 5 minutes to remove residual serum from the cells. The cells were resuspended in PBS at a density of 2 ⁇ 10 7 /mL (PBS at least 500 ⁇ L). Dilute the eFluor450 stock solution with PBS to 10 ⁇ M, and mix it with the cell suspension 1:1 (total volume 1 mL). 37°C water bath, avoid light and react for 10 minutes. Add 200 ⁇ L of pre-chilled FBS to stop the reaction, and incubate on ice for 5 minutes. Then it was washed 3 times with PBS and labeled with the cell membrane dye PKH26.
  • PKH26 to label the above target cells: Refer to the manufacturer's instructions in the PKH26 kit for the labeling method. Use Diluent C equipped in the kit to prepare 2 ⁇ single cell suspension, and control the cell density at 2 ⁇ 10 7 /mL. Then use Diluent C to prepare 2 ⁇ PKH26 staining solution, add 2 ⁇ single cell suspension to 2 ⁇ PKH26 staining solution at a ratio of 1:1, and react at room temperature for 5 minutes. Finally, add an equal volume of FBS to react for 1 minute to stop staining. Wash 3 times with complete medium RPMI1640 (10% FBS), and the cells are ready for use.
  • the experimental results are shown in Figure 6.
  • the CD19 CAR-CD8 + T cells induced and expanded by medium B and D can both kill tumor target cells in a short period of time, and medium D has a better killing effect on effector cells.
  • the T cells that were not transduced with UTD in the control group hardly killed tumor cells.
  • Medium B and E of Example 6 of the present invention is cultured CD19 CAR-T SCM vivo antitumor effect.
  • mice 6 days after inoculation with tumor cells, tumors appeared under the skin on the right side of NSG mice.
  • the tumor-forming mice were randomly divided into three groups, 5 in each group.
  • Three groups of mice were intravenously infused with different cells (all from Examples 2 and 3), including untransduced control (UTD) T cells, and the treatment group of CD19CAR-T SCM prepared by two types of methods (medium B Group and medium group D).
  • tumor volume (long diameter of tumor ⁇ width diameter of tumor 2 )/2.
  • Figure 7A shows the time schedule of the in vivo experiment in mice.
  • Fig. 7B and Fig. 7C are the anti-tumor effects of CD19 CAR-T SCM cultured in medium B and D in vivo.
  • the CD19 CAR-T SCM induced and amplified by the two media can effectively inhibit the growth of tumors.
  • the tumor clearance rate of the medium B group is 80%
  • the medium D group combined culture group The prepared CD19 CAR-T SCM has a better anti-tumor effect in vivo, realizing rapid and efficient removal of tumors in the body, and the tumor removal rate is as high as 100%.
  • the UTD T cells in the control group were unable to inhibit tumor growth.
  • Figure 7D is a graph of survival curves of mice in each group after CD19 CAR-T SCM treatment. As shown in the figure, all mice treated with CD19 CAR-T SCM induced and expanded by the two media survived, while the mortality rate of mice in the control group UTD T cell group was as high as 80%.

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Abstract

提供了一种用于体外诱导和/或扩增T SCM的组合物、包含所述组合物的培养基，以及体外诱导和/或扩增T SCM的方法。其中，所述组合物包扩含有IL-7和IL-21在内的诱导剂。加入组合物诱导分化和扩增的干细胞样记忆性嵌合抗原受体T细胞可直接用于患者回输治疗。

Description

体外诱导和/或扩增T SCM的组合物、培养基和方法 技术领域
本发明属于细胞培养领域。具体地，本发明涉及用于诱导和/或扩增细胞的组合物、培养基和方法。更具体地，本发明涉及用于体外诱导和/或扩增干细胞样记忆性T细胞的组合物、培养基和方法。
背景技术
T细胞具有强大的抗感染、抗肿瘤能力，是机体适应性免疫系统防御病原体、肿瘤细胞等异物的中坚力量，不仅能够快速应答外来异物或机体赘生物，而且还能够提供较为长期的免疫保护记忆。研究已经证实通过体外诱导和扩增记忆性T细胞并回输至患者体内，可以治疗多种疾病，特别是严重威胁人类健康的癌症。
目前，记忆性T细胞的体外诱导和扩增方法中，较为成熟的是使用抗CD3抗体和抗CD28抗体联合IL-2刺激诱导分化，刺激分化的T细胞增殖明显加快，幼稚性T细胞大大减少，而终末状态的T细胞明显增多，这种T细胞在体内存活时间较短，不能提供长期的免疫保护，难以满足临床治疗的需求。
近年来，研究发现并鉴定了一个新的T细胞亚群，这群细胞被命名为干细胞样记忆性T细胞(T-memory stem cell，T SCM)。T SCM处于记忆性T细胞的上游，具备干细胞的特征，具有更强的多向分化潜能，可以分化为中枢性记忆T细胞(T CM)、效应性记忆T细胞(T EM)和效应T细胞(T EF)，从而产生大量的IFN-γ等效应分子，具备更为强大的杀伤能力。此外，T SCM具备多向分化潜能的同时亦有自我更新的能力，从而在抗原不存在的情况下维持自身稳态。研究表明T SCM具有更为强大的抗肿瘤能力，其在体内的自我更新能力、二次免疫应答强度及体内存活时间均优于传统的记忆性T细胞。
临床上免疫细胞治疗的过继回输面临着一个瓶颈，特别是目前快速发展的嵌合抗原受体T细胞(CAR-T)免疫治疗，既要在较短时间内(14天内)完成足量免疫细胞的诱导和扩增培养，又要尽量保持免疫细胞的活性、维持“年轻态”的免疫细胞。然而，目前诱导和扩增培养的T细胞多为处于终末阶段的细胞，特别是较为常用的抗CD3抗体和抗CD28抗体联合IL-2的培养方法获得的T SCM比例较低，仅占9-20％，该类细胞一是难以在体外培养的条件下长期存活，从而难以获得足够量的免疫细胞；二是这种免疫细胞即使回 输回患者体内，也很难获得较为长期的免疫保护作用。然而，T SCM所具备的多向分化潜能和自我更新特性则恰好解决了临床上免疫细胞治疗的过继回输体外诱导和扩增难、体内疗效差的问题。研究表明，体外诱导和扩增培养的T SCM样的CD8 +CD45RA +CCR7 +CAR-T细胞比例与肿瘤免疫治疗临床疗效呈现正相关，表明T SCM具有更为强大的体内抗肿瘤能力。原因可能是T SCM具备的强大生存能力使得回输的CAR-T细胞的体内生存时间大大延长。因此，强大的抗肿瘤能力使得T SCM成为免疫治疗中最为有效的细胞亚群。因此，利用体外诱导和扩增的抗原特异性T SCM、TCR-T SCM或CAR-T SCM进行免疫治疗将成为细胞治疗的新途径、新手段。
基于上述目的，有目的、有针对性的开发并改良体外诱导和扩增T SCM的技术方法，并找到经济、方便、且副作用小的细胞因子或者药物，具有极为重要的现实意义。然而，目前的技术方法中仍然缺乏简便且高效的体外大量诱导和扩增T SCM的方法。
发明内容
本发明的目的是针对现有技术的不足，提供一种用于体外诱导和/或扩增干细胞样记忆性T细胞的组合物和培养基。本发明还提供了使用本发明的组合物体外诱导和/或扩增干细胞样记忆性T细胞的方法。本发明提供的方法中，CAR-T SCM占CAR-T细胞的比例有明显提高，可直接用于患者CAR-T治疗的回输治疗。
一方面，本发明提供了一种用于体外诱导和/或扩增干细胞样记忆性T细胞(T SCM)的组合物，所述组合物由诱导剂组成，所述诱导剂包含白细胞介素-7(IL-7)和白细胞介素-21(IL-21)。
根据本发明所述的组合物，其中，所述诱导剂还包含IL-3、IL-12、IL-15和IL-18的一种或多种；优选地，所述诱导剂还包含IL-15。
根据本发明所述的组合物，其中，所述诱导剂还包含GSK-3β抑制剂；优选地，所述GSK-3β抑制剂为TWS119；
根据本发明所述的组合物，其中，所述诱导剂包含IL-7、IL-21、IL-15和GSK-3β抑制剂；
根据本发明所述的组合物，其中，所述诱导剂以游离蛋白形式加至培养基、耦联于磁珠上，包被于细胞培养板上，或表达于细胞上。
根据本发明所述的组合物，其中，各诱导剂的工作浓度如下：
除了GSK-3β抑制剂之外的每种诱导剂的工作浓度在1-100ng/mL的范围内，GSK-3β抑制剂的工作浓度在1-50μM的范围内。
根据本发明所述的组合物，其中，所述组合物中各诱导剂的工作浓度如 下：
1-100ng/mL的IL-7、1-100ng/m的IL-21；
优选地，所述组合物还包含1-100ng/mL的IL-15；
优选地，所述组合物还包含1-50μM的GSK-3β抑制剂。
另一方面，本发明提供了一种用于体外诱导和/或扩增干细胞样记忆性T细胞(T SCM)的培养基，其中所述培养基包含T细胞生长基础培养基和所述组合物。
根据本发明所述的用于体外诱导和/或扩增T SCM的培养基或培养基的制备方法，其中包括在T细胞生长基础培养基中加入所述组合物的步骤；
优选地，通过包括以下步骤的方式实现：
将所述组合物的一种或多种组分以制备的游离蛋白分子添加到T细胞生长基础培养基；
将所述组合物的一种或多种组分包被在磁珠上并添加至T细胞生长基础培养基；
将编码所述组合物的一种或多种组分的遗传物质以载体形式感染或转染的饲养细胞加至T细胞生长基础培养基，与培养的目的细胞共培养；
将编码所述组合物的一种或多种组分的遗传物质以载体形式感染或转染目的细胞，并用T细胞生长基础培养基培养；
或利用外源细胞表达所述组合物的一种或多种组分，使其在使用T细胞生长基础培养基培养的细胞上清中起作用。
优选地，可以使用方法的一种或多种的联合来制备所述培养基。
本发明还提供了所述组合物和/或培养基用于体外诱导和/或扩增T SCM的用途。
根据本发明所述的用途，其中所述T SCM为所述T SCM为外周血单个核细胞(PBMC)、CD4 +T细胞、CD8 +T细胞或CD4 -CD8 -T细胞。
根据本发明所述的用途，其中所述T SCM为CAR-T细胞、TCR-T细胞或肿瘤浸润性淋巴细胞。
再一方面，本发明提供了一种体外诱导和/或扩增T SCM的方法，所述方法包括使用本发明的组合物和/或培养基诱导和/或扩增细胞的步骤；
根据本发明所述的体外诱导和/或扩增T SCM的方法，其中所述方法包括以下步骤：
①分离外周血单个核细胞(PBMC)、CD4 +T细胞、CD8 +T细胞或CD4 -CD8 -T细胞；
②将步骤①得到的细胞置于本发明的培养基或置于T细胞生长基础培养基同时加入所述组合物，并加入刺激剂，培养6-7天；
优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、Toll样受体1(TLR1)配体、Toll样受体2(TLR2)配体、Toll样受体5(TLR5)配体、Toll样受体6(TLR6)配体和视黄酸诱导基因蛋白(RIG-I)的配体、嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体中的一种或多种；
更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠；
③将步骤②得到的细胞置于本发明的培养基或置于T细胞生长基础培养基同时加入所述组合物，每隔2-4天补充本发明的培养基或补充T细胞生长基础培养基同时加入所述组合物，直至细胞培养周期结束。
在一个优选的实施方案中，所述方法包括以下步骤：
①磁珠阴选出CD8 +T细胞；
②将步骤①得到的细胞置于T细胞生长基础培养基，并加入工作浓度的组合物，加入耦联抗CD3抗体和抗CD28抗体的磁珠，培养7天；
③将步骤②得到的细胞置于本发明的培养基或置于T细胞生长基础培养基同时加入所述组合物，从第4天起，每隔3天补充新鲜的培养基或补充新鲜的T细胞生长基础培养基同时加入所述组合物，直至细胞培养周期结束。
在一个优选的实施方案中，所述方法为体外诱导和/或扩增干细胞样记忆性嵌合抗原受体T细胞(CAR-T SCM)的方法，包括以下步骤：
1)分离外周血单个核细胞(PBMC)、CD4 +T细胞、CD8 +T细胞或CD4 -CD8 -T细胞；
2)将步骤1)得到的细胞置于本发明的培养基或置于T细胞生长基础培养基同时加入组合物，并加入刺激剂，培养1-2天；
优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、Toll样受体1(TLR1)配体、Toll样受体2(TLR2)配体、Toll样受体5(TLR5)配体、Toll样受体6(TLR6)配体和视黄酸诱导基因蛋白(RIG-I)的配体、嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体中的一种或多种；
更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠；
3)使步骤2)得到的细胞转染或感染携带嵌合抗原受体的载体；
4)将步骤3)得到的细胞置于本发明的培养基或置于T细胞生长基础培养基同时加入组合物，并加入刺激剂继续培养5-6天后去除刺激剂。从第4天起，每隔2-4天补充培养基或补充T细胞生长基础培养基同时加入组合物，直至CAR-T细胞培养周期结束；
优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、Toll样受体1(TLR1)配体、Toll样受体2(TLR2)配体、Toll样受体5(TLR5)配体、Toll样受体6(TLR6)配体和视黄酸诱导基因蛋白(RIG-I)的配体、嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体中的一种或多种；
更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠。
在一个优选的实施方案中，所述方法包括以下步骤：
1)分离外周血单个核细胞(PBMC)或CD8 +T细胞；
2)将步骤1)得到的细胞置于本发明的培养基或置于T细胞生长基础培养基同时加入所述组合物，并加入耦联抗CD3抗体和抗CD28抗体的磁珠，培养1天；
3)在步骤2)得到的细胞中以感染复数MOI＝2-15的比例加入携带嵌合抗原受体慢病毒载体LV-CAR，在30-32℃下，离心感染2h；
4)离心结束后，将步骤3)得到的细胞培养过夜，置于本发明的培养基或置于T细胞生长基础培养基同时加入所述组合物，并加入耦联抗CD3抗体和抗CD28抗体的磁珠继续培养5-6天后去除耦联抗CD3抗体和抗CD28抗体的磁珠。从第4天起，每隔3天补充培养基或补充T细胞生长基础培养基同时加入所述组合物，继续培养直至CAR-T细胞培养周期结束；
优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、Toll样受体1(TLR1)配体、Toll样受体2(TLR2)配体、Toll样受体5(TLR5)配体、Toll样受体6(TLR6)配体和视黄酸诱导基因蛋白(RIG-I)的配体、嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体中的一种或多种；
更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠。
在一个优选的实施方案中，所述方法为体外诱导和/或扩增干细胞样记忆性T细胞受体T细胞(TCR-T SCM)的方法，包括以下步骤：
1)分离外周血单个核细胞(PBMC)、CD4 +T细胞或CD8 +T细胞；
2)将步骤1)得到的细胞置于本发明的培养基或置于T细胞生长基础培养基同时加入所述组合物，并加入刺激剂，培养1-2天；
优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、Toll样受体1(TLR1)配体、Toll样受体2(TLR2)配体、Toll样受体5(TLR5)配体、Toll样受体6(TLR6)配体和视黄酸诱导基因蛋白(RIG-I)的配体、 嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体的一种或多种；
更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠；
3)使步骤2)得到的细胞转染或感染携带T细胞受体的载体；
4)将步骤3)得到的细胞置于本发明的培养基或置于T细胞生长基础培养基同时加入组合物，并加入刺激剂继续培养5-6天后去除刺激剂。从第4天起，每隔2-4天补充培养基或补充T细胞生长基础培养基同时加入组合物，直至TCR-T细胞培养周期结束。
在一个优选的实施方案中，所述方法包括以下步骤：
1)分离外周血单个核细胞(PBMC)、CD4 +T细胞或CD8 +T细胞；
2)将步骤1)得到的细胞置于本发明的培养基或置于T细胞生长基础培养基同时加入组合物，并加入耦联抗CD3抗体和抗CD28抗体的磁珠，培养1天；
3)在步骤2)得到的细胞中以感染复数MOI＝2-15的比例加入携带嵌合抗原受体慢病毒载体LV-TCR，在30-32℃下，离心感染2h；
4)离心结束后，将步骤3)得到的细胞培养过夜，置于本发明的培养基或置于T细胞生长基础培养基同时加入组合物，并加入耦联抗CD3抗体和抗CD28抗体的磁珠继续培养5-6天后去除耦联抗CD3抗体和抗CD28抗体的磁珠。从第4天起，每隔3天补充培养基或补充T细胞生长基础培养基同时加入组合物，继续培养直至TCR-T细胞培养周期结束。
再一方面，本发明提供了根据本发明的方法制备的T SCM；
优选地，所述T SCM为CAR-T SCM、TCR-T SCM或肿瘤浸润性淋巴细胞。
本发明还提供了本发明的T SCM制备用于治疗肿瘤的药物中的用途。
再另一方面，本发明提供了一种肿瘤的细胞免疫治疗方法，所述方法包括使用本发明的T SCM。
本发明的发明人发现，在T细胞培养过程中添加细胞因子IL-7、IL-15和IL-21，可以在体外高效地将T细胞诱导诱导和/或扩增为T SCM，T SCM占T细胞的比例显著提升，且细胞数目显著增加。本发明公开的方法可用于干细胞样记忆性嵌合抗原受体T细胞(CAR-T SCM)的体外诱导和/或扩增，亦可用于干细胞样记忆性T细胞受体T细胞(TCR-T SCM)的体外诱导和/或扩增。本发明使用的组合物安全性好、易获取，组合物诱导和/或扩增的TCR-T SCM或CAR-T SCM可用于肿瘤患者的细胞治疗，为癌症免疫治疗提供了一种新策略，具有重要的开发和推广价值。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为根据本发明的培养基对CD8 +T SCM体外诱导的效果；其中，图1A为健康人外周血CD8 +T细胞中T SCM所占的比例；图1B为根据本发明的培养基在培养第7天和第14天时诱导CD8 +T细胞分化为T SCM的效果比较图，其中T SCM比例越高，说明诱导效果越好。
图2为根据本发明的培养基对CD8 +T SCM体外诱导和扩增的效果；其中，图2A为根据本发明的培养基诱导和扩增CD8 +T SCM的细胞计数图。相同的培养时间，CD8 +T SCM的细胞数目越多，说明该组合物的诱导和扩增效果越佳；图2B为根据本发明的培养基在培养第7天和第14天时的CD8 +T SCM的诱导和扩增倍数。
图3为根据本发明的两种培养基对CD19 CAR-T SCM体外诱导分化和扩增效果；其中，图3A为培养第11天CD19嵌合抗原受体T细胞(CD19 CAR-T)中T SCM的流式检测结果；图3B为培养第7天和第11天CD19 CAR-T SCM占T细胞比例的统计图；图3C为培养第7天和第11天的CD19 CAR-T SCM体外扩增效果。
图4为根据本发明的两种培养基对CD19 CAR-CD8 +T SCM体外诱导分化和扩增效果。其中，图4A为培养第11天CD19 CAR-CD8 +T细胞中T SCM的流式检测结果；图4B为培养第7天和第11天CD19 CAR-CD8 +T SCM占CD8 +T细胞比例的统计图；图4C为培养第7天和第11天的CD19 CAR-CD8 +T SCM体外扩增效果。
图5为根据本发明的两种培养基培养的CD19 CAR-CD8 +T细胞体外活化效果。
图6为根据本发明的两种培养基培养的CD19 CAR-CD8 +T细胞体外杀伤活性。
图7为根据本发明的两种培养基培养的CD19 +CAR-T细胞在重症联合免疫缺陷(NSG)小鼠体内的抗肿瘤作用。
实施发明的最佳方式
本申请的以下描述只为说明本申请的多种实施方式。因此，此处讨论的具体修改方式不应理解为对申请范围的限制。本领域的技术人员在不偏离本申请范围的情况下即可很容易地得出多种等同方式、变化和修改，应理解这样的等同实施方式包括在本发明范围内。在本申请中引用的所有文献，包括公开出版物、专利和专利申请都通过引用的方式全文并入。
下面结合附图和具体实验进一步详细说明本发明。除非特别说明，本发明所用试剂、仪器、设备和方法均为本技术领域的常规市购试剂、仪器、设 备和方法。
制备本发明的培养基B-E，同时制备用于对照的培养基A，其中，所述培养基共有组分如下：
T细胞生长基础培养基：淋巴细胞无血清培养基(KBM581，康宁公司)；
所述培养基的差异组分如下：
培养基A含有50ng/mL IL-2；
培养基B含有5ng/mL IL-7和30ng/mL IL-21；
培养基C含有5ng/mL IL-7、30ng/mL IL-21和5μM TWS119；
培养基D含有5ng/mL IL-7、30ng/mL IL-21和10ng/mL IL-15；
培养基E含有5ng/mL IL-7、30ng/mL IL-21、10ng/mL IL-15和5μM TWS119
实施例1本发明的培养基体外诱导和扩增CD8 +T SCM
1)取预先制备的人原代T细胞(上海鑫湾生物科技有限公司，以下简称“鑫湾公司”)，采用磁珠阴选方法获得人原代CD8 +T细胞(CD3 +CD8 +)，并铺在96孔U底培养板中，每孔5×10 4个CD8 +T细胞；
2)随后，向含有CD8 +T细胞的培养板中加入培养基A-E，并加入含有抗CD3抗体和抗CD28抗体耦联的磁珠，其中磁珠：细胞数＝1：1；并在细胞培养的第4天、第7天、第10天、第13天补充新鲜培养基A-E。
3)分别在细胞培养第0天，第7天和第14天进行细胞计数，并孵育检测T SCM的流式抗体(CD45RA、CCR7和CD95)，采用流式细胞仪进行T SCM的检测。
实验结果如图1和图2所示。
图1为培养基A-E对CD8 +T SCM体外诱导效果比较图。其中，图1A为健康人外周血CD8 +T细胞(即原代)中T SCM所占的比例。如图1A所示，原代CD8 +T细胞中的T SCM的比例极低，所占比例低于2％，图1B为培养基A-E在培养第7天和第14天时诱导CD8 +T细胞分化为T SCM的效果比较。相对于常用的IL-2，其余培养基B-E均能有效地诱导CD8 +T细胞分化为T SCM，且大大优于IL-2的诱导效果。培养至第14天，培养基A的平均诱导效率低于10％。然而，培养第14天，培养基B组的T SCM占整个原代CD8+T细胞的比例即诱导效率高达78.7％，培养基D组的T SCM诱导效率稍低(72.0％)。培养基C组和E组，也就是联合使用TWS119后，均能达到87.0％的T SCM诱导效率。此外，发明人发现，本发明的诱导剂可有效诱导T SCM的形成的浓度范围如下：IL-7(1-100ng/mL)、IL-15(1-100ng/mL)、IL-21(1-100ng/mL)、以及和TWS119(1-50μM)。
图2为根据本发明的培养基A-E对CD8 +T SCM体外诱导和扩增的效果。
图2A为根据本发明的培养基A-E诱导和扩增CD8 +T SCM的细胞计数图。培养第14天，培养基A诱导和扩增CD8 +T SCM的效果最低，根据本发明的培养基B组诱导和扩增CD8 +T SCM的效果明显优于根据本发明的培养基C组，而根据本发明的培养基D组的诱导和扩增效果更佳。
图2B为根据本发明的培养基A-E在培养第7天和第14天时的CD8 +T SCM的诱导和扩增倍数。在培养第14天后，诱导和扩增CD8 +T SCM的效果如下：根据本发明的培养基A组为800倍、根据本发明的培养基B组达到1162倍，而根据本发明的培养基D组高达5269倍。IL-7、IL-15、IL-21有效扩增CD8 +T SCM浓度范围均为1-100ng/mL。
实施例2本发明的培养基B和D对CD19  CAR-T S C M体外诱导分化和扩增 效果
1)复苏预先制备并冻存的健康外周血单个核细胞(PBMC)(鑫湾公司)，加在48孔细胞培养板中(1×10 6个PBMC/孔)。
2)随后，向上述细胞培养板中加入含有抗CD3抗体和抗CD28抗体耦联的磁珠(细胞数：磁珠数＝1：1)和根据本发明的培养基B或D。每种处理设置三个平行孔。
3)培养1天后，向上述培养体系中加入的慢病毒载体LV-CD19 CAR(鑫湾公司)，感染复数MOI＝5。在32℃下，以1200g离心感染2小时后，弃去上清并补充培养基B或D，继续培养。
在细胞培养的第4天，将上述细胞培养体系全部转移至12孔板，并补充含培养基B或D。
在细胞培养的第7天，移除抗CD3抗体和抗CD28抗体耦联的磁珠并补充培养基B或D。
在细胞培养的第10天，重复补充新鲜培养基B或D，同第7天。
4)分别在细胞培养第7天和第11天进行细胞计数，采用流式细胞仪检测T SCM(CD45RA、CCR7和CD95)。
实验结果如图3所示。
图3为两种T SCM诱导和扩增培养基B或D对CD19 CAR-T SCM体外诱导分化和扩增效果。
图3A为培养第11天的CD19 CAR-T SCM的流式细胞仪检测画门流程及相应的细胞亚群比例。
图3B为培养第7天和第11天的CD19 CAR-T SCM占CD19 CAR-T细胞的比例统计结果。在培养第11天的CD19 CAR-T细胞中，培养基B和培养 基D诱导分化的T SCM(CD45RA +CCR7 +CD95 +)的比例分别达到81.3％和64.4％，说明两种培养基均能有效诱导CD19 CAR-T SCM的分化。
图3C为两种T SCM诱导和扩增培养基对CD19 CAR-T SCM体外诱导分化并扩增的效果。实验结果显示，培养基D可更为有效地诱导并扩增CD19CAR-T SCM。
实施例3本发明的培养基B和D对CD19 CAR-CD8 +T SCM体外诱导分化和 扩增效果
制备CD19嵌合抗原受体CD8 +T细胞(CD19 CAR-CD8 +T细胞)：
1)复苏预先制备并冻存的健康外周血单个核细胞(PBMC)(鑫湾公司)，接着采用磁珠进一步阴选出CD8 +T细胞，加在48孔细胞培养板中(1×10 6个CD8 +T细胞/孔)。
2)随后，向上述细胞培养板中加入含有抗CD3抗体和抗CD28抗体耦联的磁珠(细胞数：磁珠数＝1：1)和和根据本发明的培养基B或D。每种处理设置三个平行孔。
3)培养1天后，向上述培养体系中加入的慢病毒载体LV-CD19 CAR(鑫湾公司)，感染复数MOI＝5。在32℃下，以1200g离心感染2小时。离心后继续培养过夜，弃去上清并补充加入根据本发明的培养基B或D。
在细胞培养的第4天，将上述细胞培养体系全部转移至12孔板，并补充根据本发明的培养基B或D。
在细胞培养的第7天，移除抗CD3抗体和抗CD28抗体耦联的磁珠并补充根据本发明的培养基B或D。
在细胞培养的第10天，重复补充新鲜的根据本发明的培养基B或D，同第7天。
4)分别在细胞培养第7天和第11天进行细胞计数，采用流式细胞仪检测CD8 +T SCM(CD45RA +CCR7 +CD95 +CD8 +T细胞)。
实验结果如图4所示。
图4为两种培养基B或D对CD19 CAR-CD8 +T SCM体外诱导分化和扩增效果。
图4A为培养11天的CD19 CAR-CD8 +T SCM的流式细胞仪检测画门流程及相应的细胞亚群比例。
图4B为CD19 CAR-CD8 +T SCM占CD19 CAR-CD8 +T细胞的比例统计结果。在CD19 CAR-CD8 +T细胞中，培养基B和D诱导分化的T SCM(CD45RA +CCR7 +CD95 +)的比例分别达到93.2％和82.5％，说明两种细胞因子组合均能有效诱导CD19 CAR-CD8 +T SCM的分化。
图4C为两种培养基B或D对CD19 CAR-CD8 +T SCM体外诱导分化并扩增的效果。实验结果显示，培养基D可更为有效地诱导并扩增CD19 CAR-CD8 +T SCM。
实施例4本发明的培养基B和D所培养的CD19 CAR-CD8 +T细胞体外活 化效果
1)自实施例3制备的CAR-T细胞中，取1×10 5个静息的效应细胞CD19CAR-CD8 +T细胞，并取1×10 5个K562-CD19靶细胞(鑫湾公司)，以效/靶细胞1：1的比例加至96孔U底细胞培养板中，400g，离心1分钟以促进效/靶细胞的接触；
2)效/靶细胞共培养24小时后，500g，离心5分钟，取培养细胞上清，采用ELISA测定上清中细胞因子IFN-γ的含量。
实验结果如图5所示，两种培养基B和D培养所诱导并扩增的CD19CAR-CD8 +T细胞，在接触肿瘤靶细胞后均可有效地活化并分泌大量的功能效应分子IFN-γ，但培养基D的活化效果更佳。未转导对照组(UTD)的T细胞则不产生效应分子IFN-γ。
实施例5本发明的培养基B和D所培养的CD19  CAR-CD8 +T细胞的体外 杀伤效果。
1)取制备的肿瘤细胞K562-CD19(鑫湾公司)作为靶细胞，标记染料分别采用细胞增殖染料eFluor 450(赛默飞世尔科技有限公司)和细胞膜染料PKH26(西格玛奥德里奇贸易有限公司)。
首先，采用eFluor 450标记K562-CD19细胞。用PBS或无血清RPMI1640培养基洗涤细胞两次，500g，离心5分钟以去除细胞中残余的血清。将细胞按2×10 7/mL的密度重悬在PBS中(PBS至少500μL)。用PBS稀释eFluor450储存液至10μM，然后和细胞悬液1：1混合(总体积1mL)。37℃水浴，避光反应10分钟。加200μL预冷的FBS终止反应，冰上孵育5分钟。随后用PBS洗涤3遍，进行细胞膜染料PKH26的标记。
2)采用PKH26标记上述靶细胞：标记方法参考PKH26试剂盒内的厂家说明书。采用试剂盒中配备的Diluent C制备2×单细胞悬液，细胞密度控制在2×10 7/mL。接着使用Diluent C制备2×的PKH26染色液，以1:1的比例将2×单细胞悬液加入到2×的PKH26染色液中，室温反应5分钟即可。最后，加入等体积的FBS反应1分钟，终止染色。用完全培养基RPMI1640(10％FBS)洗涤3遍，细胞待用。
3)将肿瘤靶细胞的数目固定为3×10 4，以效/靶比2：1、4：1、8：1 和16：1的比例分别加入实施例3中制备的效应细胞CD19 CAR-CD8 +T细胞，37℃，共孵育4小时，收集细胞悬液，采用流式细胞仪检测效应细胞的杀伤效率。
实验结果如图6所示，培养基B和D诱导并扩增的CD19 CAR-CD8 +T细胞在短时间内均能杀伤肿瘤靶细胞，培养基D的效应细胞杀伤效果更佳。未转导对照组UTD的T细胞则几乎不杀伤肿瘤细胞。
实施例6本发明的培养基B和E所培养的CD19  CAR-T SCM的体内抗肿瘤 作用。
1)采用6-8周龄的雌性重症联合免疫缺陷(NSG)小鼠(百奥赛图基因生物技术有限公司)为模型，在小鼠右侧皮下接种5×10 6个肿瘤细胞(CD19 +A549，鑫湾公司)，每只小鼠接种体积为125μL。
2)接种肿瘤细胞6天后，NSG小鼠右侧皮下均出现可见瘤体。将成瘤小鼠随机分为三组，每组5只。分别给三组小鼠静脉回输不同的细胞(均来自实施例2和3)，包括未转导对照组(UTD)T细胞、两类方法制备的CD19CAR-T SCM的治疗组(培养基B组和培养基D组)。细胞回输剂量为125μL(10 7细胞/只小鼠)。
3)细胞回输后每3天采用游标卡尺测量肿瘤的大小并记录每组的小鼠生存状况。
肿瘤体积的计算公式为：肿瘤体积＝(肿瘤的长径×肿瘤的宽径 2)/2。
实验结果如图7所示。
图7A为此次小鼠体内试验的时间进度安排。
图7B和图7C为培养基B和D所培养的CD19 CAR-T SCM体内抗肿瘤效果图。如图所示，两种培养基所诱导和扩增的CD19 CAR-T SCM均能有效地抑制肿瘤的生长，其中，培养基B组肿瘤清除率为80％，而培养基D组联合培养组制备的CD19 CAR-T SCM体内抗肿瘤效果更佳，实现了体内肿瘤的快速高效清除，肿瘤清除率高达100％。而对照组的UTD T细胞却不能抑制肿瘤的生长。
图7D为CD19 CAR-T SCM治疗后各组小鼠的生存曲线图。如图所示，接受两种培养基所诱导和扩增的CD19 CAR-T SCM治疗的小鼠全部存活，而回输了对照组UTD T细胞组的小鼠死亡率高达80％。

Claims (13)

  1. 一种用于体外诱导和/或扩增T SCM的组合物，所述组合物包含诱导剂，所述诱导剂包含IL-7和IL-21。
  2. 根据权利要求1所述的组合物，其中，所述诱导剂还包含IL-3、IL-12、IL-15和IL-18的一种或多种；优选地，所述诱导剂还包含IL-15。
  3. 根据权利要求1或2所述的组合物，其中，所述诱导剂还包含GSK-3β抑制剂；优选地，所述GSK-3β抑制剂为TWS119；
    优选地，所述诱导剂包含IL-7、IL-21、IL-15和GSK-3β抑制剂。
  4. 根据权利要求1-3中任一项所述的组合物，其中，所述组合物中各诱导剂的工作浓度如下：
    除了GSK-3β抑制剂之外的每种诱导剂的工作浓度在1-100ng/mL的范围内，GSK-3β抑制剂的工作浓度在1-50μM的范围内；
    优选地，所述组合物中各诱导剂的工作浓度如下：
    1-100ng/mL的IL-7、1-100ng/m的IL-21；
    优选地，所述组合物还包含1-100ng/mL的IL-15；
    优选地，所述组合物还包含1-50μM的GSK-3β抑制剂。
  5. 一种用于体外诱导和/或扩增T SCM的培养基，其中所述培养基包含T细胞生长基础培养基和权利要求1-4中任一项所述的组合物。
  6. 权利要求1-4中任一项所述的组合物或权利要求5所述的培养基用于体外诱导和/或扩增T SCM的用途；
    优选地，所述T SCM为PBMC、CD4 +T细胞、CD8 +T细胞或CD4 -CD8 -T细胞；
    优选地，所述T SCM为CAR-T细胞、TCR-T细胞或肿瘤浸润性淋巴细胞。
  7. 一种体外诱导和/或扩增T SCM的方法，所述方法包括使用权利要求1-4中任一项所述的组合物和/或权利要求5所述的培养基诱导和/或扩增细胞的步骤。
  8. 根据权利要求7所述的方法，其中，所述方法包括以下步骤：
    ①分离PBMC、CD4 +T细胞、CD8 +T细胞或CD4 -CD8 -T细胞；
    ②将步骤①得到的细胞置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，并加入刺激剂，培养6-7天；
    优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、TLR1配体、TLR2配体、TLR5配体、TLR6配体和RIG-I配体、嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体中的一种或多种；
    更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠；
    ③将步骤②得到的细胞置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，每隔2-4天补充权利要求5所述的培养基或补充T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，直至细胞培养周期结束；
    优选地，所述方法包括以下步骤：
    ①磁珠阴选出CD8 +T细胞；
    ②将步骤①得到的细胞置于T细胞生长基础培养基，并加入权利要求1-4中任一项所述的组合物，加入耦联抗CD3抗体和抗CD28抗体的磁珠，培养7天；
    ③将步骤②得到的细胞置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，从第4天起，每隔3天补充新鲜的权利要求5所述的培养基或补充新鲜的T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，直至细胞培养周期结束。
  9. 根据权利要求7所述的方法，其中，所述方法为体外诱导和/或扩增CAR-T SCM的方法，包括以下步骤：
    1)分离PBMC、CD4 +T细胞、CD8 +T细胞或CD4 -CD8 -T细胞；
    2)将步骤1)得到的细胞置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，并加入刺激剂，培养1-2天；
    优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、TLR1配体、TLR2配体、TLR5配体、TLR6配体和RIG-I配体、嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体中的一种或多种；
    更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠；
    3)使步骤2)得到的细胞转染或感染携带嵌合抗原受体的载体；
    4)将步骤3)得到的细胞置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，并加入刺激剂继续培养5-6天后去除刺激剂；从第4天起，每隔2-4天补充权利要求5所述的培养基或补充T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，直至CAR-T细胞培养周期结束；
    优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、TLR1配体、TLR2配体、TLR5配体、TLR6配体和RIG-I配体、嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体中的一种或多种；
    更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠；
    优选地，所述方法包括以下步骤：
    1)分离PBMC或CD8 +T细胞；
    2)将步骤1)得到的细胞置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，并加入耦联抗CD3抗体和抗CD28抗体的磁珠，培养1天；
    3)在步骤2)得到的细胞中以感染复数MOI＝2-15的比例加入携带嵌合抗原受体慢病毒载体LV-CAR，在30-32℃下，离心感染2h；
    4)离心结束后，将步骤3)得到的细胞培养过夜，置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，并加入耦联抗CD3抗体和抗CD28抗体的磁珠继续培养5-6天后去除耦联抗CD3抗体和抗CD28抗体的磁珠；从第4天起，每隔3天补充权利要求5所述的培养基或补充T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，继续培养直至CAR-T细胞培养周期结束。
  10. 根据权利要求7所述的方法，其中，所述方法为体外诱导和/或扩增TCR-T SCM的方法，包括以下步骤：
    1)分离PBMC、CD4 +T细胞或CD8 +T细胞；
    2)将步骤1)得到的细胞置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，并加入刺激剂，培养1-2天；
    优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、 TLR1配体、TLR2配体、TLR5配体、TLR6配体和RIG-I配体、嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体中的一种或多种；
    更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠；
    3)使步骤2)得到的细胞转染或感染携带T细胞受体的载体；
    4)将步骤3)得到的细胞置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，并加入刺激剂继续培养5-6天后去除刺激剂；从第4天起，每隔2-4天补充权利要求5所述的培养基或补充T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，直至TCR-T细胞培养周期结束；
    优选地，所述刺激剂为抗CD3抗体、抗CD28抗体/CD28配体、抗CD137抗体/CD137配体、抗OX40抗体/OX40配体、抗CD160抗体/CD160配体、TLR1配体、TLR2配体、TLR5配体、TLR6配体和RIG-I配体、嵌合抗原受体靶抗原、PD-1阻断抗体、CTLA-4阻断抗体、LAG-3阻断抗体、Tm-3阻断抗体和BTLA阻断抗体中的一种或多种；
    更优选地，所述刺激剂为耦联抗CD3抗体和抗CD28抗体的磁珠；
    优选地，所述方法包括以下步骤：
    1)分离PBMC、CD4 +T细胞或CD8 +T细胞；
    2)将步骤1)得到的细胞置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，并加入耦联抗CD3抗体和抗CD28抗体的磁珠，培养1天；
    3)在步骤2)得到的细胞中以感染复数MOI＝2-15的比例加入携带嵌合抗原受体慢病毒载体LV-TCR，在30-32℃下，离心感染2h；
    4)离心结束后，将步骤3)得到的细胞培养过夜，置于权利要求5所述的培养基或置于T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，并加入耦联抗CD3抗体和抗CD28抗体的磁珠继续培养5-6天后去除耦联抗CD3抗体和抗CD28抗体的磁珠；从第4天起，每隔3天补充权利要求5所述的培养基或补充T细胞生长基础培养基同时加入权利要求1-4中任一项所述的组合物，继续培养直至TCR-T细胞培养周期结束。
  11. 根据权利要求7-10中任一项所述的方法制备的T SCM。
  12. 如权利要求11的T SCM制备用于治疗肿瘤的药物中的用途。
  13. 一种肿瘤的细胞免疫治疗方法，所述方法包括使用如权利要求11 所述的T SCM。
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