The present invention belongs to the field of cell culture. In particular, the present invention relates to a composition, culture medium and method for inducing and/or amplifying cells. More particularly, the present invention relates to a composition, culture medium and method for inducing and/or amplifying T-memory stem cells in vitro.
T cells, with strong anti-infection and anti-tumor abilities, are the backbone force of the body's adaptive immune system to defend against pathogens, tumor cells and other foreign bodies. They can not only quickly respond to foreign bodies or body neoplasms, but also provide relatively long-term immune protection memory. Studies have shown that by transfusing memory T cells that are induced and amplified in vitro back into patients' bodies, a variety of diseases, especially cancer that are serious threats to human health, can be treated.
At present, among in-vitro induction and amplification methods of memory T cells, the more mature method is to combine an anti-CD3 antibody, an anti-CD28 antibody, and an IL-2 for stimulating induction and differentiation, and by this method, the proliferation of T cells subjected to stimulated differentiation is significantly accelerated, the number of naive T cells is greatly reduced, while the number of terminally differentiated T cells is significantly increased. Such T cells have a short survival time in vivo, cannot provide a long-term immune protection, and cannot meet the needs of clinical treatment.
In recent years, a new T cell subset has been identified and named T-memory stem cell (TSCM). TSCM, which is in the upstream of memory T cells, has the characteristics of stem cells, has a stronger multi-directional differentiation potential, and can differentiate into central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEF), thus producing a large number of effector molecules such as IFN-γ which have a stronger killing ability. Moreover, TSCM also has a self-renewal ability while having the multi-directional differentiation potential, thereby maintaining its homeostasis in the absence of antigens. Studies have shown that TSCM has a stronger anti-tumor ability, and its in-vivo self-renewal ability, secondary immune response intensity and in-vivo survival time are superior to those of traditional memory T cells.
The adoptive reinfusion of an immune cell therapy has encountered a bottleneck problem in clinical practice, and especially a recently rapidly developed chimeric antigen receptor T cell (CAR-T) immunotherapy is facing the problems of how to complete induction and amplification culture of sufficient immune cells within a short time (14 days), and maintain the activity of immune cells as much as possible and maintain “young” immune cells. At present, however, most of the T cells cultured by induction and amplification are terminally differentiated cells, and especially TSCM, which is obtained by the commonly used culture method of combining an anti-CD3 antibody, an anti-CD28 antibody, and an IL-2, has low percentage (only 9 to 20%). First, such cells are difficult to survive for a long time under the conditions of in-vitro culture, and thus it is difficult to obtain a sufficient amount of immune cells; and second, long-term immune protection is difficult to be obtained even if such immune cells are transfused back into patients' bodies. However, the multi-directional differentiation potential and self-renewal ability of TSCM have solved the problems of difficulties in in-vitro induction and amplification and poor in-vivo efficacy of the adoptive reinfusion of the immune cell therapy in clinical practice. Studies have shown that the percentage of TSCM-like CD8+CD45RA+CCR7+CAR-T cells that are induced, amplified and cultured in vitro is positively correlated with the clinical efficacy of tumor immunotherapy, which indicates that TSCM has a stronger in-vivo anti-tumor ability. The reason may be that the strong viability of TSCM greatly prolongs the in-vivo survival time of transfused CAR-T cells. Thus, the strong anti-tumor ability makes TSCM the most effective cell subset in immunotherapy. Therefore, immunotherapy using antigen-specific TSCM, TCR-TSCM or CAR-TSCM that are induced and amplified in vitro will become a new approach and new means of cell therapy.
Based on the above objectives, it is of great practical significance to develop and improve the technical methods for inducting and amplifying TSCM in vitro on purpose and target and to find economical, convenient cytokines or drugs with slight side effects. However, there is still a lack of a convenient and efficient method for massively inducting and amplifying TSCM in vitro in the prior art.
The aim of the present invention is to provide a composition and culture medium for inducing and/or amplifying T-memory stem cells in vitro to overcome the shortcomings of the prior art. The present invention further provides a method for inducing and/or amplifying T-memory stem cells in vitro by using the composition provided by the present invention. In accordance with the method of the present invention, the proportion of CAR-TSCM in CAR-T cells is significantly increased and the CAR-TSCM can be used directly for a reinfusion therapy of a CAR-T therapy for a patient.
In one aspect, the present invention provides a composition for inducing and/or amplifying T-memory stem cells (TSCM) in vitro. The composition consists of inducing agents comprising interleukin-7 (IL-7) and interleukin-21 (IL-21).
In accordance with the composition of the present invention, the inducing agents further comprise one or more of IL-3, IL-12, IL-15 and IL-18; and preferably, the inducing agents further comprise IL-15.
In accordance with the composition of the present invention, the inducing agents further comprise a GSK-3β inhibitor, preferably, the GSK-3β inhibitor is TWS119;
In accordance with the composition of the present invention, the inducing agents comprise IL-7, IL-21, IL-15 and a GSK-3β inhibitor, and
in accordance with the composition of the present invention, the inducing agents are added to a culture medium, coupled to magnetic beads, coated on a cell culture plate, or expressed on cells in the form of free proteins.
In accordance with the composition of the present invention, working concentrations of various inducing agents 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.
In accordance with the composition of the present invention, the working concentrations of various inducing agents in the composition are as follows:
1 to 100 ng/mL of IL-7, and 1 to 100 ng/mL of IL-21;
preferably, the composition further comprises 1 to 100 ng/mL of IL-15; and preferably, the composition further comprises 1 to 50 μM of a GSK-3β inhibitor.
In another aspect, the present invention provides a culture medium for inducing and/or amplifying T-memory stem cells (TSCM) in vitro, wherein the culture medium comprises a T cell growth basal culture medium and the composition.
In accordance with the culture medium for inducing and/or amplifying TSCM in vitro or a preparation method thereof of the present invention, the method comprises a step of adding the composition into the T cell growth basal culture medium;
preferably, the above step is completed in a manner comprising the steps of:
adding one or more components of the composition in the form of prepared free protein molecules into the T cell growth basal culture medium;
coating one or more components of the composition on magnetic beads and adding the magnetic beads to the T cell growth basal culture medium;
adding feeder cells which are infected or transfected with genetic materials encoding one or more components of the composition in the form of a vector into the T cell growth basal culture medium, and co-culturing the feeder cells and cultured objective cells;
infecting or transfecting the objective cells with genetic materials encoding one or more components of the composition in the form of a vector, and culturing the objective cells with the T cell growth basal culture medium; or
expressing one or more components of the composition by using exogenous cells to allow the exogenous cells to act in a cell supernatant cultured by using the T cell growth basal culture medium.
Preferably, the culture medium may be prepared by using one or a combination of the method.
The present invention further provides use of the composition and/or culture medium for inducing and/or amplifying TSCM in vitro.
In accordance with the use of the present invention, the TSCM is a peripheral blood mononuclear cell (PBMC), a CD4+T cell, a CD8+T cell or a CD4+CD8+T cell.
In accordance with the use of the present invention, the TSCM is a CAR-T cell, a TCR-T cell or a tumor infiltrating lymphocyte.
In still another aspect, the present invention provides a method for inducing and/or amplifying TSCM in vitro, which comprises the step of inducing and/or amplifying cells by using the composition and/or culture medium of the present invention;
in accordance with the method for inducing and/or amplifying TSCM in vitro of the present invention comprises the steps of:
{circle around (1)} separating peripheral blood mononuclear cells (PBMCs), CD4+T cells, CD8+T cells or CD4+CD8+T cells;
{circle around (2)} placing the cells obtained in step {circle around (1)} in the culture medium of the present invention or in a T cell growth basal culture medium while adding the composition, adding a stimulant, and culturing for 6 to 7 days, preferably, 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 Toll-like receptor 1 (TLR1) ligand, a Toll-like receptor 2 (TLR2) ligand, a Toll-like receptor 5 (TLR5) ligand, a Toll-like receptor 6 (TLR6) ligand, a retinoic acid-induced gene protein (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, and
more preferably, the stimulant is magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody, and
{circle around (3)} placing the cells obtained in step {circle around (2)} in the culture medium of the present invention or in the T cell growth basal culture medium while adding the composition, supplementing the culture medium of the present disclosure or the T cell growth basal culture medium while adding the composition every 2 to 4 days until the end of a cell culture cycle.
In a preferred embodiment, the method comprises the steps of:
{circle around (1)} selecting CD8+T cells by a magnetic bead negative selection method;
{circle around (2)} placing the cells obtained in step {circle around (1)} in the T cell growth basal culture medium, adding the composition at the working concentration, adding magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody, and culturing for 7 days; and
{circle around (3)} placing the cells obtained in step {circle around (2)} in the culture medium of the present invention or in T cell growth basal culture medium while adding the composition, supplementing a fresh culture medium or a fresh T cell growth basal culture medium while adding the composition every 3 days from Day 4 until the end of a cell culture cycle.
In a preferred embodiment, the method is a method for inducing and/or amplifying chimeric antigen receptor T-memory stem cells (CAR-TSCM) in vitro, which comprises the steps of:
1) separating peripheral blood mononuclear cells (PBMCs), CD4+T cells, CD8+T cells or CD4−CD8−T cells;
2) placing the cells obtained in step 1) in the culture medium of the present invention or in the T cell growth basal culture medium while adding the composition, adding a stimulant, and culturing for 1 to 2 days, preferably, 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 Toll-like receptor 1 (TLR1) ligand, a Toll-like receptor 2 (TLR2) ligand, a Toll-like receptor 5 (TLR5) ligand, a Toll-like receptor 6 (TLR6) ligand, a retinoic acid-induced gene protein (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, and
more preferably, the stimulant is magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody;
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 of the present invention or in the T cell growth basal culture medium while adding the composition, adding a stimulant, continuing culturing for 5 to 6 days, and then removing the stimulant; supplementing the culture medium or the T cell growth basal culture medium while adding the composition every 2 to 4 days from Day 4 until the end of a culture cycle of CAR-T cell,
preferably, 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 Toll-like receptor 1 (TLR1) ligand, a Toll-like receptor 2 (TLR2) ligand, a Toll-like receptor 5 (TLR5) ligand, a Toll-like receptor 6 (TLR6) ligand, a retinoic acid-induced gene protein (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, and more preferably, the stimulant is magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody.
In a preferred embodiment, the method comprises the steps of:
1) separating peripheral blood mononuclear cells (PBMCs) or CD8+T cells;
2) placing the cells obtained in step 1) in the culture medium of the present invention or in the T cell growth basal culture medium while adding the composition, 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 of the present invention or in the T cell growth basal culture medium while adding the composition, 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. Supplementing the culture medium or the T cell growth basal culture medium while adding the composition every 3 days from Day 4, and continuing culturing until the end of a culture cycle of CAR-T cells,
preferably, 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 Toll-like receptor 1 (TLR1) ligand, a Toll-like receptor 2 (TLR2) ligand, a Toll-like receptor 5 (TLR5) ligand, a Toll-like receptor 6 (TLR6) ligand, a retinoic acid-induced gene protein (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, and
more preferably, the stimulant is magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody.
In a preferred embodiment, the method is a method for inducing and/or amplifying T cell receptor T-memory stem cells (TCR-TSCM) in vitro, which comprises the steps of:
1) separating peripheral blood mononuclear cells (PBMCs), CD4+T cells, or CD8+T cells;
2) placing the cells obtained in step 1) in the culture medium of the present invention or in the T cell growth basal culture medium while adding the composition, adding a stimulant, and culturing for 1 to 2 days, preferably, 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 Toll-like receptor 1 (TLR1) ligand, a Toll-like receptor 2 (TLR2) ligand, a Toll-like receptor 5 (TLR5) ligand, a Toll-like receptor 6 (TLR6) ligand, a retinoic acid-induced gene protein (RIG-1) 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, and
more preferably, the stimulant is magnetic beads coupled to an anti-CD3 antibody and an anti-CD28 antibody;
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 of the present invention or in the T cell growth basal culture medium while adding the composition, adding a stimulant, continuing culturing for 5 to 6 days, and then removing the stimulant. Supplementing the culture medium or the T cell growth basal culture medium while adding the composition every 2 to 4 days from Day 4 until the end of a culture cycle of TCR-T cells.
In a preferred embodiment, the method comprises the steps of:
1) separating peripheral blood mononuclear cells (PBMCs), CD4+T cells, or CD8+T cells;
2) placing the cells obtained in step 1) in the culture medium of the present invention or in the T cell growth basal culture medium while adding the composition, 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 of the present invention or in the T cell growth basal culture medium while adding the composition, 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. Supplementing the culture medium or the T cell growth basal culture medium while adding the composition every 3 days from Day 4, and continuing culturing until the end of a culture cycle of TCR-T cells.
In yet another aspect, the present invention provides a TSCM prepared according to the method of the present invention; and
preferably, the TSCM is a CAR-TSCM, a TCR-TSCM or a tumor infiltrating lymphocyte.
The present invention further provides use of the TSCM of the present invention in the manufacture of a medicament for treating tumors.
In still another aspect, the present invention provides a method for cellular immunotherapeutically treating tumors, which comprises using the TSCM of the present invention.
The inventors of the present invention have found that by adding cytokines IL-7, IL-15 and IL-21 during the culture of T cells, the T cells can be efficiently induced and/or amplified to TSCM in vitro, and the proportion of TSCM in the T cells and the number of TSCM are significantly increased. The method disclosed by the present invention can be used for inducing and/or amplifying chimeric antigen receptor T-memory stem cells (CAR-TSCM) in vitro and also for inducing and amplifying T cell receptor T-memory stem cells (TCR-TSCM) in vitro. The composition used in the present invention is safe and readily available; and TCR-TSCM or CAR-TSCM induced and/or amplified by the composition can be used in cellular therapy for tumor patients, which provides a new strategy for cancer immunotherapy and has an important value for development and popularization.
The embodiments of the present invention will be described below in combination with drawings in detail.
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.
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.
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:
a T cell growth basal culture medium: a serum-free culture medium for lymphocytes (KBM581, Corning Inc.);
The different components of the media were as follows:
culture medium A contained 50 ng/mL IL-2;
culture medium B contained 5 ng/mL IL-7 and 30 ng/mL IL-21;
culture medium C contained 5 ng/mL IL-7, 30 ng/mL IL-21, and 5 μM TWS119;
culture medium D contained 5 ng/mL IL-7, 30 ng/mL IL-21 and 10 ng/mL IL-15; and
culture medium E contained 5 ng/mL IL-7, 30 ng/mL IL-21, 10 ng/mL IL-15, and 5 μM TWS119.
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+T cells (CD3+CD8+), and the human primary CD8+T cells (CD3+CD8+) were placed in a 96-well U-bottom culture plate, with 5×104 CD8+T cells in each well;
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+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
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 TSCM were incubated, and the TSCM was detected by using a flow cytometry.
Experimental results are shown in
1) Pre-prepared and frozen healthy peripheral blood mononuclear cells (PBMCs) (Sinobay) were thawed and added into a 48-well cell culture plate (1×106 PBMCs/well).
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.
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.
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.
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.
At Day 10 of cell culture, a fresh culture medium B or D was supplemented repeatedly as that at Day 7.
4) The cells were counted at Day 7 and Day 11 of cell culture respectively, and TSCM (CD45RA, CCR7 and CD95) was detected by using a flow cytometry.
Experimental results are shown in
CD19 chimeric antigen receptor CD8+T cells (CD19 CAR-CD8+T cells) were prepared:
1) Pre-prepared and frozen healthy peripheral blood mononuclear cells (PBMCs) (Sinobay) were thawed, and then CD8+T cells were selected by a magnetic bead negative selection method, and were added into a 48-well cell culture plate (1×106 CD8+T cells/well).
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.
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.
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.
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.
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.
4) The cells were counted at Day 7 and Day 11 of cell culture respectively, and CD8+TSCM (CD45RA+CCR7+CD95+CD8+T cells) was detected by using a flow cytometry.
Experimental results are shown in
1) 1×105 resting effector cell CD19 CAR-CD8+T cells taken from the CAR-T cells prepared in Example 3 and 1×105 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;
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.
Experimental results are shown in
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.
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 2×107/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.
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 2×107/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.
3) The number of the tumor target cells was fixed at 3×104, the effector cells CD19 CAR-CD8+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.
Experimental results are shown in
1) 6-8-week-old female severe combined immunodeficiency (NSG) mice (Beijing Biocytogen Co., Ltd.) were used as models, and 5×106 tumor cells (CD19+A549, Sinobay) were inoculated subcutaneously in the right flank of each mouse in an inoculation volume of 125 μL per mouse.
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-TSCM 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 (107 cells/mouse).
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.
A calculation formula for tumor volume was: tumor volume=(long diameter of tumor×wide diameter2 of tumor)/2.
Experimental results are shown in
Filing Document | Filing Date | Country | Kind |
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PCT/CN2019/076957 | 3/5/2019 | WO | 00 |