COMPOSITION, CULTURE MEDIUM AND METHOD FOR INDUCING AND/OR AMPLIFYING TSCM IN VITRO

Abstract

A composition for inducing and/or amplifying T.sub.SCM in vitro, a culture medium including the composition, and a method for inducing and/or amplifying T.sub.SCM in vitro are provided, wherein the composition comprises inducing agents including IL-7 and IL-21. The chimeric antigen receptor T-memory stem cells induced differentiated and amplified by adding the composition can be used directly for reinfusion therapy of patients.

Claims

1. A composition for inducing and/or amplifying T.sub.SCM in vitro, the composition comprising inducing agents, the inducing agents comprising IL-7 and IL-21.

2. The composition of claim 1, wherein the inducing agents further comprise one or more of IL-3, IL-12, IL-15 and IL-18.

3. The composition of claim 1, wherein the inducing agents further comprise a GSK-3β inhibitor.

4. The composition of claim 1, wherein working concentrations of various inducing agents in the composition are as follows: a working concentration of each of inducing agents, excluding a GSK-3β inhibitor, ranges from 1 to 100 ng/mL, and a working concentration of a GSK-3β inhibitor ranges from 1 to 50 μM.

5. A culture medium for inducing and/or amplifying T.sub.SCM in vitro, wherein the culture medium comprises a T cell growth basal culture medium and a composition of claim 1.

6. (canceled)

7. A method for inducing and/or amplifying T.sub.SCM in vitro, wherein the method comprises the step of inducing and/or amplifying cells by using a composition of claim 1 and/or a culture medium comprising a T cell growth basal culture medium and a composition of claim 1.

8. The method of claim 7, wherein the method comprises the steps of: 1) separating PBMC, CD4.sup.+T cells, CD8.sup.+T cells or CD4.sup.−CD8.sup.−T cells; 2) placing the cells obtained in step 1) in a culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in a T cell growth basal culture medium while adding a composition of claim 1, adding a stimulant, and culturing for 6 to 7 days; and 3) placing the cells obtained in step 2) in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, and supplementing the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or the T cell growth basal culture medium while adding the composition of claim 1 every 2 to 4 days until the end of a cell culture cycle.

9. The method of claim 7, wherein the method is a method for inducing and/or amplifying CAR-T.sub.SCM in vitro and comprises the steps of: 1) separating PBMC, CD4.sup.+T cells, CD8.sup.+T cells or CD4.sup.−CD8.sup.−T cells; 2) placing the cells obtained in step 1) in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, adding a stimulant, and culturing for 1 to 2 days; 3) transfecting or infecting the cells obtained in step 2) with a vector carrying a chimeric antigen receptor; and 4) placing the cells obtained in step 3) in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, adding a stimulant, continuing culturing for 5 to 6 days, and then removing the stimulant; and supplementing the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or the T cell growth basal culture medium while adding the composition of claim 1 every 2 to 4 days from Day 4 until the end of a culture cycle of CAR-T cells.

10. The method of claim 7, wherein the method is a method for inducing and/or amplifying TCR-T.sub.SCM in vitro and comprises the steps of: 1) separating PBMC, CD4.sup.+T cells, or CD8.sup.+T cells; 2) placing the cells obtained in step 1) in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, adding a stimulant, and culturing for 1 to 2 days; and 3) transfecting or infecting the cells obtained in step 2) with a vector carrying a T cell receptor; and 4) placing the cells obtained in step 3) in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, adding a stimulant, continuing culturing for 5 to 6 days, and then removing the stimulant; and supplementing the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or the T cell growth basal culture medium while adding the composition of claim 1 every 2 to 4 days from Day 4 until the end of a culture cycle of TCR-T cells.

11. T.sub.SCM prepared by a method of claim 7.

12. A pharmaceutical composition for treating tumors comprising T.sub.SCM prepared by a method of claim 7 and a pharmaceutically acceptable carrier.

13. A method for cellular immunotherapeutically treating tumors in a subject, comprising administering a therapeutically effective amount of T.sub.SCM prepared by a method of claim 7 to a subject in need thereof.

14. The composition of claim 1, wherein the GSK-3β inhibitor is TWS119.

15. The composition of claim 1, wherein the inducing agents comprise IL-7, IL-21, IL-15 and a GSK-3β inhibitor.

16. The method of claim 8, wherein the stimulant is one or more of an anti-CD3 antibody, an anti-CD28 antibody/CD28 ligand, an anti-CD137 antibody/CD137 ligand, an anti-OX40 antibody/OX40 ligand, an anti-CD160 antibody/CD160 ligand, a TLR1 ligand, a TLR2 ligand, a TLR5 ligand, a TLR6 ligand, an RIG-I ligand, a chimeric antigen receptor target antigen, a PD-1 blocking antibody, a CTLA-4 blocking antibody, an LAG-3 blocking antibody, a Tm-3 blocking antibody, and a BTLA blocking antibody.

17. The method of claim 8, comprising the steps of: 1) selecting CD8.sup.+T cells by a magnetic bead negative selection method; 2) placing the cells obtained in step 1) in the T cell growth basal culture medium, adding the composition of claim 1, adding magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody, and culturing for 7 days; and 3) placing the cells obtained in step 2) in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, and supplementing the fresh culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or the fresh T cell growth basal culture medium while adding the composition of claim 1 every 3 days from Day 4 until the end of a cell culture cycle.

18. The method of claim 9, wherein the stimulant is one or more of an anti-CD3 antibody, an anti-CD28 antibody/CD28 ligand, an anti-CD137 antibody/CD137 ligand, an anti-OX40 antibody/OX40 ligand, an anti-CD160 antibody/CD160 ligand, a TLR1 ligand, a TLR2 ligand, a TLR5 ligand, a TLR6 ligand, an RIG-I ligand, a chimeric antigen receptor target antigen, a PD-1 blocking antibody, a CTLA-4 blocking antibody, an LAG-3 blocking antibody, a Tm-3 blocking antibody, and a BTLA blocking antibody.

19. The method of claim 9, comprising the steps of: 1) separating PBMC or CD8.sup.+T cells; 2) placing the cells obtained in step 1) in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, adding magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody, and culturing for 1 day; 3) adding a lentiviral vector LV-CAR carrying the chimeric antigen receptor into the cells obtained in step 2) in a proportion of multiplicity of infection (MOI) of 2-15, and performing centrifugal infection at 30 to 32° C. for 2 h; and 4) after centrifugation is finished, culturing the cells obtained in step 3) overnight, placing the cells in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, adding magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody, continuing culturing for 5 to 6 days, and then removing the magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody; and supplementing the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or the T cell growth basal culture medium while adding the composition of claim 1 every 3 days from Day 4, and continuing culturing until the end of a culture cycle of CAR-T cells.

20. The method of claim 10, wherein the stimulant is one or more of an anti-CD3 antibody, an anti-CD28 antibody/CD28 ligand, an anti-CD137 antibody/CD137 ligand, an anti-OX40 antibody/OX40 ligand, an anti-CD160 antibody/CD160 ligand, a TLR1 ligand, a TLR2 ligand, a TLR5 ligand, a TLR6 ligand, an RIG-I ligand, a chimeric antigen receptor target antigen, a PD-1 blocking antibody, a CTLA-4 blocking antibody, an LAG-3 blocking antibody, a Tm-3 blocking antibody, and a BTLA blocking antibody.

21. The method of claim 10, comprising the steps of: 1) separating PBMC, CD4.sup.+T cells, or CD8.sup.+T cells; 2) placing the cells obtained in step 1) in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, adding magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody, and culturing for 1 day; 3) adding a lentiviral vector LV-TCR carrying a chimeric antigen receptor into the cells obtained in step 2) in a proportion of multiplicity of infection (MOI) of 2-15, and performing centrifugal infection at 30 to 32° C. for 2 h; and 4) after centrifugation is finished, culturing the cells obtained in step 3) overnight, placing the cells in the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or in the T cell growth basal culture medium while adding the composition of claim 1, adding magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody, continuing culturing for 5 to 6 days, and then removing the magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody; and supplementing the culture medium comprising a T cell growth basal culture medium and a composition of claim 1 or the T cell growth basal culture medium while adding the composition of claim 1 every 3 days from Day 4, and continuing culturing until the end of a culture cycle of TCR-T cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The embodiments of the present invention will be described below in combination with drawings in detail.

[0071] FIG. 1 illustrates an in-vitro induction effect of a culture medium according to the present invention on a CD8.sup.+T.sub.SCM, wherein FIG. 1A illustrates the percentage of T.sub.SCM in CD8.sup.+T cells in healthy human peripheral blood; FIG. 1B is a comparison diagram illustrating effects of inducing CD8.sup.+T cells to differentiate into T.sub.SCM by using the culture medium according to the present invention at Day 7 and Day 14 of culture, and the higher the T.sub.SCM proportion, the better the induction effect.

[0072] FIG. 2 illustrates an in-vitro induction and amplification effect of the culture medium according to the present invention on the CD8.sup.+T.sub.SCM, wherein FIG. 2A is a cell count diagram illustrating the CD8.sup.+T.sub.SCM induced and amplified by the culture medium according to the present invention. For the same culture time, the more CD8.sup.+T.sub.SCM cells, the better the induction and amplification effects of the composition; and FIG. 2B illustrates induction and amplification multiples of CD8.sup.+T.sub.SCM at Day 7 and Day 14 of culture by using the culture medium according to the present invention.

[0073] FIG. 3 illustrates in-vitro induced differentiation and amplification effects of two culture media according to the present invention on CD19 CAR-T.sub.SCM, wherein FIG. 3A illustrates a result of flow cytometry analysis of T.sub.SCM in CD19 chimeric antigen receptor T cells (CD19 CAR-T) on Day 11 of culture; FIG. 3B is a statistical diagram of the percentages of CD19 CAR-T.sub.SCM in T cells at Day 7 and Day 11 of culture; and FIG. 3C illustrates in-vitro amplification effects of CD19 CAR-T.sub.SCM at Day 7 and Day 11 of culture.

[0074] FIG. 4 illustrates in-vitro induced differentiation and amplification effects of two culture media according to the present invention on CD19 CAR-CD8.sup.+T.sub.SCM, wherein FIG. 4A illustrates a result of flow cytometry analysis of T.sub.SCM in CD19 CAR-CD8.sup.+T cells at Day 11 of culture; FIG. 4B is a statistical diagram of the percentages of the CD19 CAR-CD8.sup.+T.sub.SCM in CD8.sup.+T cells at Day 7 and Day 11 of culture; and FIG. 4C illustrates in-vitro amplification effects of CD19 CAR-CD8.sup.+T.sub.SCM at Day 7 and Day 11 of culture.

[0075] FIG. 5 illustrates an in-vitro activation effect of CD19 CAR-CD8.sup.+T cells cultured by using two culture media according to the present invention.

[0076] FIG. 6 illustrates an in-vitro killing activity of CD19 CAR-CD8.sup.+T cells cultured by using two culture media according to the present invention.

[0077] FIG. 7 illustrates an anti-tumor effect of CD19.sup.+CAR-T cells cultured by using two culture media according to the present invention on severe combined immunodeficiency (NSG) mice.

BEST MODE FOR CARRYING OUT THE INVENTION

[0078] The following description of the present application is merely an illustration of various embodiments of the present application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. Multiple equivalents, changes, and modifications can be readily made by those skilled in the art without departing from the scope of the present application, and it should be understood that such equivalent embodiments are to be included within the scope of the present invention. All documents, including publications, patents, and patent applications, cited in the present application, are hereby incorporated by reference in their entirety.

[0079] The present invention will now be further described in detail with reference to the accompanying drawings and specific experiments. Unless otherwise indicated, reagents, instruments, devices, and methods used in the present invention are those conventional and commercially available reagents, instruments, devices, and methods used in the art.

[0080] Culture media B to E of the present invention were prepared, and a culture medium A was prepared for control, wherein the media contained the following common components:

[0081] a T cell growth basal culture medium: a serum-free culture medium for lymphocytes (KBM581, Corning Inc.);

[0082] The different components of the media were as follows:

[0083] culture medium A contained 50 ng/mL IL-2;

[0084] culture medium B contained 5 ng/mL IL-7 and 30 ng/mL IL-21;

[0085] culture medium C contained 5 ng/mL IL-7, 30 ng/mL IL-21, and 5 μM TWS119;

[0086] culture medium D contained 5 ng/mL IL-7, 30 ng/mL IL-21 and 10 ng/mL IL-15; and

[0087] culture medium E contained 5 ng/mL IL-7, 30 ng/mL IL-21, 10 ng/mL IL-15, and 5 μM TWS119.

Example 1 In-Vitro Induction and Amplification of CD8.SUP.+.T.SUB.SCM .in the Culture Media of the Present Invention

[0088] 1) Pre-prepared human primary T cells (Shanghai Sinobay Bio-Tech Co., Ltd., hereinafter referred to as “Sinobay”) were selected by a magnetic bead negative selection method to obtain human primary CD8.sup.+T cells (CD3.sup.+CD8.sup.+), and the human primary CD8.sup.+T cells (CD3.sup.+CD8.sup.+) were placed in a 96-well U-bottom culture plate, with 5×10.sup.4 CD8.sup.+T cells in each well;

[0089] 2) subsequently, culture media A to E and magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody were added into the culture plates containing the CD8.sup.+T cells, wherein a ratio of the number of the magnetic beads to the number of the cells was 1:1; and fresh culture media A to E were supplemented at Day 4, Day 7, Day 10 and Day 13 of cell culture; and

[0090] 3) the cells were counted at Day 0, Day 7 and Day 14 of cell culture respectively, flow cytometry antibodies (CD45RA, CCR7 and CD95) for detecting T.sub.SCM were incubated, and the T.sub.SCM was detected by using a flow cytometry.

[0091] Experimental results are shown in FIG. 1 and FIG. 2.

[0092] FIG. 1 is a comparison diagram illustrating in-vitro induction effects of the culture media A to E on CD8.sup.+T.sub.SCM. FIG. 1A illustrates the percentage of T.sub.SCM in CD8.sup.+T cells (namely, primary cells) in healthy human peripheral blood. As shown in FIG. 1A, the percentage of T.sub.SCM in primary CD8.sup.+T cells was very low, which is less than 2%, and FIG. 1B is a comparison diagram illustrating effects of inducing CD8.sup.+T cells to differentiate into T.sub.SCM by using the culture media A to E at Day 7 and Day 14 of culture. Compared with commonly used IL-2, other media B to E could effectively induce CD8.sup.+T cells to differentiate into T.sub.SCM, and had much better induction effects than IL-2. The medium A had an average induction efficiency of less than 10% at Day 14 of culture. However, at Day 14 of culture, the percentage of T.sub.SCM of the culture medium B group in the whole primary CD8.sup.+T cells, namely the induction efficiency, was up to 78.7%, and the induction efficiency of T.sub.SCM of the culture medium D group was slightly lower (72.0%). The culture medium C group and E group, having TWS119 combined in the medium, achieved an induction efficiency of T.sub.SCM of 87.0%. Furthermore, the inventors found that the inducing agents of the present invention could effectively induce the formation of T.sub.SCM in the following concentration ranges: IL-7 (1 to 100 ng/mL), IL-15 (1 to 100 ng/mL), IL-21 (1 to 100 ng/mL), and TWS119 (1 to 50 μM).

[0093] FIG. 2 illustrates in-vitro induction and amplification effects of the culture media A to E according to the present invention on CD8.sup.+T.sub.SCM.

[0094] FIG. 2A is a cell counting diagram illustrating CD8.sup.+T.sub.SCM induced and amplified by using the culture media A to E according to the present invention. At Day 14 of culture, the induction and amplification effects of the culture medium A on CD8.sup.+T.sub.SCM were the lowest, and the induction and amplification effects of the culture medium B group according to the present invention on CD8.sup.+T.sub.SCM were significantly superior to those of the culture medium C group according to the present invention, while the induction and amplification effects of the culture medium D group according to the present invention on CD8.sup.+T.sub.SCM were even better.

[0095] FIG. 2B illustrates induction and amplification multiples of CD8.sup.+T.sub.SCM at Day 7 and Day 14 of culture by using the culture media A to E according to the present invention. After Day 14 of culture, the induction and amplification effects on CD8.sup.+T.sub.SCM were as follows: the induction and amplification multiple of the culture medium A group according to the present invention was 800 folds, the induction and amplification multiple of the culture medium B group according to the present invention was 1162 folds, and the induction and amplification multiple of the culture medium D group according to the present invention was 5269 folds. The concentrations of IL-7, IL-15 and IL-21 for effectively amplifying CD8.sup.+T.sub.SCM all ranged from 1 to 100 ng/mL.

Example 2 In-Vitro Induced Differentiation and Amplification Effects of the Culture Media B and D of the Present Invention on CD19 CAR-T.SUB.SCM

[0096] 1) Pre-prepared and frozen healthy peripheral blood mononuclear cells (PBMCs) (Sinobay) were thawed and added into a 48-well cell culture plate (1×10.sup.6 PBMCs/well).

[0097] 2) Subsequently, magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody (the number of cells: the number of magnetic beads=1:1) and the culture medium B or D according to the present invention were added into the above cell culture plate. Three parallel wells were provided for each treatment.

[0098] 3) After the cells were cultured for 1 day, a lentiviral vector LV-CD19CAR (Sinobay) was added into the above culture system, wherein multiplicity of infection (MOI) was equal to 5. After the cells were centrifugally infected at 1200 g and 32° C. for 2 hours, a supernatant was discarded, the culture medium B or D was supplemented, and culturing was continued.

[0099] At Day 4 of cell culture, the whole of the above cell culture system was transferred to a 12-well plate, and the culture medium B or D was supplemented.

[0100] At Day 7 of cell culture, the magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody were removed, and the culture medium B or D was supplemented.

[0101] At Day 10 of cell culture, a fresh culture medium B or D was supplemented repeatedly as that at Day 7.

[0102] 4) The cells were counted at Day 7 and Day 11 of cell culture respectively, and T.sub.SCM (CD45RA, CCR7 and CD95) was detected by using a flow cytometry.

[0103] Experimental results are shown in FIG. 3.

[0104] FIG. 3 illustrates in-vitro induced differentiation and amplification effects of two T.sub.SCM induction and amplification culture media B or D on CD19 CAR-T.sub.SCM.

[0105] FIG. 3A illustrates a detection flow of the flow cytometry for CD19 CAR-T.sub.SCM at Day 11 of culture and percentages of the corresponding cell subsets.

[0106] FIG. 3B illustrates a statistical result of the percentages of CD19 CAR-T.sub.SCM in CD19 CAR-T cells at Day 7 and Day 11 of culture. The percentages of T.sub.SCM(CD45RA.sup.+CCR7.sup.+CD95.sup.+) induced and differentiated by the culture medium B and the culture medium D reached 81.3% and 64.4% respectively in CD19 CAR-T cells cultured at Day 11, indicating that both media can effectively induce the differentiation of CD19 CAR-T.sub.SCM.

[0107] FIG. 3C illustrates in-vitro induced differentiation and amplification effects of two T.sub.SCM induction and amplification culture media on CD19 CAR-T.sub.SCM. The experimental results showed that culture medium D could induce and amplify CD19 CAR-T.sub.SCM more effectively.

Example 3 In-Vitro Induced Differentiation and Amplification Effects of the Culture Media B and D of the Present Invention on CD19 CAR-CD8.SUP.+.T.SUB.SCM

[0108] CD19 chimeric antigen receptor CD8.sup.+T cells (CD19 CAR-CD8.sup.+T cells) were prepared:

[0109] 1) Pre-prepared and frozen healthy peripheral blood mononuclear cells (PBMCs) (Sinobay) were thawed, and then CD8.sup.+T cells were selected by a magnetic bead negative selection method, and were added into a 48-well cell culture plate (1×10.sup.6 CD8.sup.+T cells/well).

[0110] 2) Subsequently, magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody (the number of cells: the number of magnetic beads=1:1) and the culture medium B or D according to the present invention were added into the above cell culture plate. Three parallel wells were provided for each treatment.

[0111] 3) After the cells were cultured for 1 day, a lentiviral vector LV-CD19CAR (Sinobay) was added into the above culture system, wherein multiplicity of infection (MOI) was equal to 5. The cells were centrifugally infected at 1200 g and 32° C. for 2 hour. After centrifugation, the culturing was continued overnight, a supernatant was discarded, and the culture medium B or D according to the present invention was supplemented.

[0112] At Day 4 of cell culture, the whole of the above cell culture system was transferred to a 12-well plate, and the culture medium B or D according to the present invention was supplemented.

[0113] At Day 7 of cell culture, the magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody were removed, and the culture medium B or D according to the present invention was supplemented.

[0114] At Day 10 of cell culture, a fresh culture medium B or D according to the present invention was supplemented repeatedly as that at Day 7.

[0115] 4) The cells were counted at Day 7 and Day 11 of cell culture respectively, and CD8.sup.+T.sub.SCM (CD45RA.sup.+CCR7.sup.+CD95.sup.+CD8.sup.+T cells) was detected by using a flow cytometry.

[0116] Experimental results are shown in FIG. 4.

[0117] FIG. 4 illustrates in-vitro induced differentiation and amplification effects of the two culture media B or D on CD19 CAR-CD8.sup.+T.sub.SCM.

[0118] FIG. 4A illustrates a detection flow of the flow cytometry for CD19 CAR-CD8.sup.+T.sub.SCM at Day 11 of culture and percentages of the corresponding cell subsets.

[0119] FIG. 4B illustrates a statistical result of the percentage of CD19 CAR-CD8.sup.+T.sub.SCM in CD19 CAR-CD8.sup.+T cells. The percentages of T.sub.SCM (CD45RA.sup.+CCR7.sup.+CD95.sup.+) induced and differentiated by the culture media B and D reached 93.2% and 82.5% respectively in CD19 CAR-CD8.sup.+T cells, indicating that the combination of two cytokines can effectively induce the differentiation of CD19 CAR-CD8.sup.+T.sub.SCM.

[0120] FIG. 4C illustrates in-vitro induced differentiation and amplification effects of the two culture media B or D on CD19 CAR-CD8.sup.+T.sub.SCM. The experimental results showed that the culture medium D could induce and amplify CD19 CAR-CD8.sup.+T.sub.SCM more effectively.

Example 4 In-Vitro Activation Effects of CD19 CAR-CD8.SUP.+.T Cells Cultured by the Culture Media B and D of the Present Invention

[0121] 1) 1×10.sup.5 resting effector cell CD19 CAR-CD8.sup.+T cells taken from the CAR-T cells prepared in Example 3 and 1×10.sup.5 K562-CD19 target cells (Sinobay) were added into a 96-well U-bottom cell culture plate in a ratio of effector cells to target cells of 1:1, the cells were centrifuged at 400 g for 1 min to promote the contact of the effector cells and the target cells;

[0122] 2) After the effector cells and the target cells were co-cultured for 24 hours, the cells were centrifuged at 500 g for 5 minutes, a supernatant of the cultured cells was taken, and the content of cytokines IFN-γ in the supernatant was detected by ELISA.

[0123] Experimental results are shown in FIG. 5, CD19 CAR-CD8.sup.+T cells induced and amplified by using the two culture media B and D could effectively activate and secrete a large amount of functional effector molecules IFN-γ after contacting tumor target cells, and the culture medium D had a better activation effect. T cells in the untransduced control group (UTD) did not produce the effector molecules IFN-γ.

Example 5 In-Vitro Killing Effects of CD19 CAR-CD8.SUP.+.T Cells Cultured by the Culture Media B and D According to the Present Invention

[0124] 1) Prepared tumor cells K562-CD19 (Sinobay) were used as target cells, and a cell proliferation dye eFluor 450 (Thermo Fisher Scientific Co., Ltd.) and a cell membrane dye PKH26 (Sigma-Aldrich Trading Co., Ltd.) were used as labeling dyes respectively.

[0125] First, K562-CD19 cells were labeled with eFluor 450. The cells were washed twice with PBS or a serum-free RPMI1640 culture medium, and centrifuged at 500 g for 5 minutes to remove residual serum from the cells. The cells were resuspended in PBS (at least 500 μL of PBS) at a density of 2×10.sup.7/mL. An eFluor 450 stock solution was diluted to 10 μM by using PBS and then the diluted solution was mixed with the cell suspension in a ratio of 1:1 (total volume 1 mL). The mixture was put into a water bath at 37° C., and reacted in dark for 10 minutes. The reaction was stopped by adding 200 μL of precooled FBS and the mixture was incubated on ice for 5 minutes. And then, the cells were washed with PBS for 3 times, and labeled with the cell membrane dye PKH26.

[0126] 2) The above target cells were labeled with PKH26: refer to the manufacturer's instructions in a PKH26 kit for a labeling method. 2× single-cell suspension was prepared by using Diluent C provided in the kit, and the cell density was controlled at 2×10.sup.7/mL. Then, 2×PKH26 staining solution was prepared by using Diluent C, the 2× single-cell suspension was added into the 2×PKH26 staining solution in a ratio of 1:1, and the mixture was reacted at room temperature for 5 min. Finally, FBS in an equal volume was added for reacting for 1 min. and staining was stopped. The cells were washed with a complete medium RPMI1640 (10% FBS) for 3 times for later use.

[0127] 3) The number of the tumor target cells was fixed at 3×10.sup.4, the effector cells CD19 CAR-CD8.sup.+T cells prepared in Example 3 were added in a ratio of the effector cells to target cells of 2:1, 4:1, 8:1 and 16:1, respectively, the cells were co-incubated at 37° C. for 4 hours, a cell suspension was collected, and the killing efficiency of the effector cells was detected by using a flow cytometry.

[0128] Experimental results are shown in FIG. 6, CD19 CAR-CD8.sup.+T cells induced and amplified by both the culture media B and D could kill the tumor target cells in a short time, and the effector cells cultured by the culture medium D had a better killing effect T cells in the untransduced control group (UTD) hardly killed the tumor cells.

Example 6 In-Vivo Anti-Tumor Effects of CD19 CAR-T.SUB.SCM .Cultured by the Culture Media B and E According to the Present Invention

[0129] 1) 6-8-week-old female severe combined immunodeficiency (NSG) mice (Beijing Biocytogen Co., Ltd.) were used as models, and 5×10.sup.6 tumor cells (CD19.sup.+A549, Sinobay) were inoculated subcutaneously in the right flank of each mouse in an inoculation volume of 125 μL per mouse.

[0130] 2) Six days after inoculation of the tumor cells, a visible tumor appeared under the skin on the right flank of each NSG mouse. Tumor mice were randomly divided into three groups, with 5 mice in each group. Three groups of mice were reinfused intravenously with different cells (all from Examples 2 and 3), which included the untransduced control group (UTD) T cells and CD19 CAR-T.sub.SCM for treatment groups prepared by two methods (the culture medium B group and the culture medium D group). The cell reinfusion dose was 125 μL (10.sup.7 cells/mouse).

[0131] 3) The tumor size was measured every 3 days after cell reinfusion by using a vernier caliper, and survival conditions of mice of each group were recorded.

[0132] A calculation formula for tumor volume was: tumor volume=(long diameter of tumor×wide diameter.sup.2 of tumor)/2.

[0133] Experimental results are shown in FIG. 7.

[0134] FIG. 7A illustrates a schedule of in-vivo experiment of mice.

[0135] FIG. 7B and FIG. 7C are diagrams illustrating in-vivo anti-tumor effects of CD19 CAR-T.sub.SCM cultured by the culture media B and E. As shown in the figures, the CD19 CAR-T.sub.SCM induced and amplified by both the culture media could effectively inhibit the growth of tumor, wherein the tumor clearance rate of the culture medium B group was 80%, while the CD19 CAR-T.sub.SCM prepared by the combined culture group of the culture medium D group had a better in-vivo anti-tumor effect, achieving rapid and efficient tumor clearance in vivo, with the tumor clearance rate as high as 100%. However, UTD T cells of the control group could not inhibit the growth of tumor.

[0136] FIG. 7D is a diagram of survival curves of mice in each group after CD19 CAR-T.sub.SCM treatment. As shown in the figure, all the mice treated with CD19 CAR-T.sub.SCM induced and amplified by the two culture media survived, while the mortality rate of the mice reinfused with UTD T cells of the control group was as high as 80%.