T cell expansion

Abstract

A method of treating a cancer in a subject is disclosed, comprising: (1) isolating T cells from a subject; generating or expanding a population of T cells specific for a virus by a method comprising: stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of the virus, wherein 10 to 25% of the media in which the cells are cultured is conditioned media obtained from a stimulation culture comprising T cells and APCs presenting a peptide of the virus; and (3) administering the generated or expanded population of T cells to a subject. Also disclosed are methods for generating or expanding a population of T cells specific for a virus.

Claims

1. A method of treating a cancer in a subject, the method comprising: (1) isolating T cells from a subject; (2) generating or expanding a population of T cells specific for Epstein-Barr Virus (EBV) by a method comprising: stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of EBV, wherein 12.5% to 17.5% of the media in which the cells are cultured is cell-free conditioned media obtained from a separate stimulation culture comprising T cells and APCs presenting a peptide of EBV; and (3) administering the generated or expanded population of T cells to a subject, wherein the cancer is an EBV-positive cancer.

2. The method according to claim 1, wherein the cell-free conditioned media is obtained from a separate stimulation culture comprising T cells (responders) and APCs presenting a peptide of EBV (stimulators) at a responder:stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days.

3. The method according to claim 1, wherein stimulating T cells (responders) by culture in the presence of APCs presenting a peptide of EBV (stimulators) comprises culture in the presence of EBV-transformed lymphoblastoid cell lines (LCLs) at a responder to stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days, in media comprising: (a) cell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% fetal bovine serum (FBS), and 1-5 mM L-glutamine, (b) 12.5% to 17.5% cell free conditioned media obtained by a method comprising: stimulating T cells by culture in the presence of EBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1 in cell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2 at a final concentration of 10-200 IU/ml, for a period of 1 to 8 days, and (c) added IL-2 at a final concentration of 10-200 IU/ml.

4. The method according to claim 1, wherein the APCs presenting a peptide of EBV are EBV-transformed lymphoblastoid cell line (LCL) cells.

5. The method according to claim 1, wherein the cancer is EBV-positive nasopharyngeal carcinoma (NPC).

6. The method according to claim 1, wherein about 15% of the media in which the cells are cultured is cell-free conditioned media.

7. The method according to claim 1, wherein step (2) additionally comprises: collecting the generated or expanded population of T cells.

8. A method of treating a cancer in a subject, the method comprising: (1) isolating T cells from a subject; (2) generating or expanding a population of T cells specific for Epstein-Barr Virus (EBV) by a method comprising: stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of EBV, wherein 12.5% to 17.5% of the media in which the cells are cultured is cell-free conditioned media, wherein the cell-free conditioned media is obtained from a separate stimulation culture comprising T cells (responders) and APCs presenting a peptide of EBV (stimulators) at a responder:stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days; and (3) administering the generated or expanded population of T cells to a subject, wherein the cancer is an EBV-positive cancer.

9. The method according to claim 8, wherein stimulating T cells (responders) by culture in the presence of APCs presenting a peptide of EBV (stimulators) comprises culture in the presence of EBV-transformed lymphoblastoid cell lines (LCLs) at a responder to stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days, in media comprising: (a) cell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% fetal bovine serum (FBS), and 1-5 mM L-glutamine, (b) 12.5% to 17.5% cell-free conditioned media obtained by a method comprising: stimulating T cells by culture in the presence of EBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1 in cell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2 at a final concentration of 10-200 IU/ml, for a period of 1 to 8 days, and (c) added IL-2 at a final concentration of 10-200 IU/ml.

10. The method according to claim 8, wherein the cancer is EBV-positive nasopharyngeal carcinoma (NPC).

11. The method according to claim 8, wherein the 12.5% to 17.5% cell-free conditioned media is about 15% cell-free conditioned media.

12. The method according to claim 8, wherein step (2) additionally comprises: collecting the generated or expanded population of T cells.

13. A method for generating or expanding a population of T cells specific for Epstein-Barr Virus (EBV), comprising stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of EBV, wherein 12.5% to 17.5% of the media in which the cells are cultured is cell-free conditioned media obtained from a separate stimulation culture comprising T cells and APCs presenting a peptide of EBV.

14. The method according to claim 13, wherein the cell-free conditioned media is obtained from a separate stimulation culture comprising T cells (responders) and APCs presenting a peptide of EBV (stimulators) at a responder:stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days.

15. The method according to claim 13, wherein stimulating T cells (responders) by culture in the presence of APCs presenting a peptide of EBV (stimulators) comprises culture in the presence of EBV-transformed lymphoblastoid cell lines (LCLs) at a responder to stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days, in media comprising: (a) cell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% fetal bovine serum (FBS), and 1-5 mM L-glutamine, (b) 12.5% to 17.5% cell-free conditioned media obtained by a method comprising: stimulating T cells by culture in the presence of EBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1 in cell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2 at a final concentration of 10-200 IU/ml, for a period of 1 to 8 days, and (c) added IL-2 at a final concentration of 10-200 IU/ml.

16. The method according to claim 13, wherein the APCs presenting a peptide of EBV are EBV-transformed lymphoblastoid cell line (LCL) cells.

17. The method according to claim 13, wherein the 12.5% to 17.5% of cell-free conditioned media is about 15% of cell-free conditioned media.

18. The method according to claim 13, wherein step (2) additionally comprises: collecting the generated or expanded population of T cells.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

(2) FIGS. 1A and FIG. 1B. Graph showing the effect of conditioned media on EBV-specific T cell expansion from cells obtained from (1A) Donor 1 and (1B) Donors 2 and 3. Increasing percentages of conditioned media are added to culture media. After one week of co-culture with LCL, the number of expanded T cells is compared to the original number of seeded T cells.

(3) FIG. 2. Graph showing the effect of conditioned media on EBV-specific T cell expansion from cells obtained from three donors, in a Grex-100 bioreactor. Increasing percentages of conditioned media are added to culture media. After one week of co-culture with LCL, the number of expanded T cells is compared to the original number of seeded T cells.

(4) FIG. 3. Graph showing specific killing of EBV-transformed LCL cells by EBV-Specific T cells. T cells expanded by culture in 0% and 30% conditioned medium are co-cultured with LCLs at different effector/target cell ratios, and specific lysis of the LCL cells is measured after 4 hours.

(5) FIG. 4. Graph showing the effect of the presence of different percentages of conditioned media on fold expansion of EBV-specific CTLs, cultured in Grex-100 bioreactor culture vessels.

EXAMPLES

(6) In the following Examples the inventors describe expansion of EBV-specific T cells including culture in the presence of conditioned media. The inventors describe experiments for determining the optimum percentage of conditioned media to include in cultures, and for confirming cytotoxic activity of the expanded CTLs.

Example 1: Optimum Percentage of Conditioned Media

(7) To determine the optimum percentage of conditioned medium for T cell expansion, in T cell expansions performed in 24 well plates, cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine is mixed with 10%, 20%, 30%, 40%, 50%, and 60% of conditioned medium obtained from a co-culture of T cells with LCL cells.

(8) After one week of co-culture with LCL cells, the number of T cells is counted and compared to the number of seeded cells, to determine fold expansion.

(9) The expected expansion results are shown on FIG. 1A. T cells are expanded at a faster rate by the methods including culture in the presence of conditioned media. The optimum percentage of conditioned media in a method step according to B for greatest fold expansion is expected to be 20-30% conditioned media.

(10) The same experiment is performed on cells obtained from two further, different donors (Donors 2 and 3). It is expected that the results will be similar as for the Donor 1, with maximum T cell expansion observed at 20-30% conditioned media (see FIG. 1B).

(11) Because large-scale expansion of T cells will be performed in bioreactors such as e.g. Grex-100 culture vessels, the optimised percentage of conditioned media is further investigated in bioreactor culture.

(12) For Illustrative Purposes:

(13) Cells are cultured at a cell density of 1?10.sup.6 cells/ml in 200 ml of media, of which varying percentages are conditioned media:

(14) TABLE-US-00001 Cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, Conditioned % Conditioned 2 mM L-glutamine media media 160 ml 40 ml 20% 140 ml 60 ml 30% 120 ml 80 ml 40% 100 ml 100 ml 50% 80 ml 120 ml 60%

(15) The expected results for T cell expansion are shown in FIG. 2. It is expected that the optimum percentage of conditioned media will be 20-30%, consistent across different donors.

Example 2: Generation of EBV-Transformed LCLs and Expansion of EBV-Specific CTLs

(16) Peripheral blood (40-60 mL) obtained from patients with EBV-positive NPC are used to generate both EBV-transformed lymphoblastoid B-cell lines (LCLs) and EBV-specific T cells.

(17) Generation of EBV-Transformed LCLs

(18) Briefly, for LCL generation, 15?10.sup.6 peripheral blood mononuclear cells (PBMCs) are incubated with concentrated supernatant of B95-8 cultures, in the presence of 1 ?g/mL cyclosporin A (Sandoz, Vienna, Austria) to establish an LCL.

(19) LCLs are irradiated at 60 Gy prior to being used for stimulations (on the day of the stimulation).

(20) Expansion of EBV-Specific T Cells

(21) Expansion of EBV-specific T cells by two different methods are compared.

(22) In both methods, a first stimulation is performed as follows: 60?10.sup.6 PBMCs are re-suspended in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine, and a viable cell count is performed. PBMCs are seeded at 2?10.sup.6 cells/well into wells of a 24 well plate. PBMCs are stimulated with irradiated, Acyclovir-treated autologous LCLs at a responder to stimulator ratio of 40:1. The cells are cultured for 9-12 days at 37? C. in a 5% CO.sub.2 atmosphere.

(23) In both methods, a second stimulation is then performed, as follows: Cells are collected at the end of the first stimulation, a viable cell count is performed, and the cells are re-suspended at 1?10.sup.6 cells/ml in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine. 1 ml of cells are added into wells of 24 well plates, or the cell suspension is added into bioreactors at a concentration of 15?10.sup.6 cells/GRex10, in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine, and the cells are re-stimulated with autologous irradiated LCLs at a responder to stimulator ratio of 4:1. The cells are cultured for 3-4 days, and then the cells are re-suspended in fresh cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine. Recombinant human IL-2 (rhIL-2, Proleukin; Chiron Emeryville, Calif.)) is added to the cell culture at a final concentration of 40-100 IU/ml.

(24) Subsequent Stimulations

(25) Following the second stimulation, cells are collected, a viable cell count is performed. Culture media (i.e. conditioned media) is retained for use in method B below. The cells are then re-suspended at 1?10.sup.6 cells/ml (in 24 well plate) or 0.5?10.sup.6 cells/ml (in GRex10) in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine and re-stimulated with autologous irradiated LCLs at a responder to stimulator ratio of 4:1.

(26) Once 200?10.sup.6 cells are achieved, cells are then stimulated according to method step A or method step B:

(27) TABLE-US-00002 Method Step A Method Step B 100 ? 10.sup.6 cells are re-suspended in 100 ? 10.sup.6 cells are re-suspended in: 200 ml cell culture media (1) 180 ml cell culture media comprising 45% RPMI 1640, 45% comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM Click's media, 10% FBS, 2 mM L-glutamine, transferred to GRex L-glutamine, and; 100, and re-stimulated with and the (2) 20 ml conditioned media obtained cells are re-stimulated with from the culture at the end of the autologous irradiated LCLs at a second stimulation (i.e. 10% responder to stimulator ratio of 4:1, conditioned media), transferred with added rhIL-2 at a final to GRex 100, and re-stimulated concentration of 40-100 IU/ml. with and the cells are re-stimulated The cells are cultured for 3-4 days with autologous irradiated LCLs at a at 37? C. in a 5% CO.sub.2 atmosphere. responder to stimulator ratio of 4:1, with added rhIL-2 at a final concentration of 40-100 IU/ml. The cells are cultured for 3-4 days at 37? C. in a 5% CO.sub.2 atmosphere.

(28) At the end of a stimulation according to method step A or method step B, cells are collected and re-stimulated according to the same method step A or B.

(29) For re-simulations according to method step B, conditioned media used is from the preceding stimulation according to B.

Example 3: Test of Efficacy

(30) The cytotoxic activity of CTLs expanded by methods including culture in the presence of conditioned media is compared to the cytotoxic activity of CTLs expanded without culture in the presence of conditioned media.

(31) T cells expanded from 0% and 30% conditioned media are added to LCL cells at different Effector/Target cell ratios (E/T ratio), and after 4 hours, specific lysis of the LCL cells is measured. It is expected that there will be very little or no difference between the specific lysis for cells expanded by culture in 0% and 30% conditioned media (FIG. 3).

(32) CTLs expanded at an increased rate are expected to retain the ability to specifically kill EBV-transformed LCL cells.

Example 4: Optimization of Epstein-Barr Virus-Specific T Cells (EB-VSTs) Growth Using Conditioned Medium

(33) The following example describes an investigation of the optimal percentage of conditioned media to be included in restimulations for maximum CTL expansion.

(34) Week 1: Lymphoblastoid cell line (LCL) samples were thawed and cultured for 1 week Requires minimally 10?10.sup.6 LCLs to initiate experiment

(35) Week 2: Frozen EB-VST03 cells from different patients were thawed, re-suspended in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine, and a viable cell count was performed EB-VST03 cells were obtained by culture of PMBCs according to Example 2, wherein the PBMCs underwent first and second stimulations, and then a third stimulation according to Method Step A (i.e. in the absence of conditioned media), after which point cells were harvested and frozen EB-VST03 cells were seeded at 1?10.sup.6 cells/well of a 24-well plate Cell suspensions were added to bioreactors (15?10.sup.6 cells/G-Rex 10) in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine, and re-stimulated with autologous irradiated LCLs at a Responder:Stimulator ratio of 4:1. The cells were cultured for 3-4 days, and then resuspended in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS and 2 mM L-glutamine. IL-2 was then added to the cell culture at a final concentration of 40-100 IU/ml.

(36) Week 3 Onwards: Cells were harvested, pooled, counted and stimulated as described under week 2 above until there were 120?10.sup.6 cells. 100?10.sup.6 cells were then seeded into G-Rex 100 flasks in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine, and different percentages of conditioned media (obtained from the culture at the end of the preceding stimulation culture), as follows:

(37) TABLE-US-00003 Cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, Conditioned % Conditioned 2 mM L-glutamine media media 200 ml 0 ml 0% 170 ml 30 ml 15% 140 ml 60 ml 30% 110 ml 90 ml 45% The cells were then cultured for 3-4 days at 37? C. in a 5% CO.sub.2 atmosphere. IL-2 was then added to the cell culture at a final concentration of 40-100 IU/ml. After one week, the cells were harvested and a viable cell count was performed.

(38) All viabilities were observed to be greater than 70%. The results are shown in FIG. 4.

(39) It was found that culture in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine and up to 15% conditioned media gave the highest yield of EB-VSTs, which was ?20% higher than the rate of EB-VST as compared to using 100% of cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine.

(40) Fold expansion of EB-VSTs decreased when higher percentages (i.e. 30%, 45%) of conditioned media were used.

(41) Fold expansion observed for Sample 3EB-VST was not as pronounced as observed for Sample 1 & Sample 2. This might be because Sample 3 EBV-VST underwent an additional cycle of stimulation, with the result that cell growth was overstressed.

(42) It is concluded that a combination of 15% conditioned media with 85% fresh culture media (complete Click's medium) produced highest EB-VST yield i.e. higher EB-VST expansion rate as compared to using 100% fresh culture media.

(43) Whilst the effect of conditioned medium on EB-VST growth varies among different individuals, it was observed that in every case, using 15% conditioned medium gave the best expansion rate.