T Cell Expansion

Abstract

A method for 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 at least 10% 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. Also disclosed are methods for accelerating the rate of expansion of a virus-specific T cell population, and methods for treating or preventing diseases or disorders using the generated or expanded T cell population.

Claims

1. A method for generating or expanding a population of T cells specific for a virus, comprising stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of the virus, wherein at least 10% 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.

2. A method for accelerating the rate of expansion of a virus-specific T cell population, the method comprising stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of the virus, wherein at least 10% 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.

3. A method for generating or expanding a population of T cells specific for a virus, comprising: (i) stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of the virus; and (ii) re-stimulating the T cells by culture in the presence of APCs presenting a peptide of the virus, wherein at least 10% of the media in which the cells are cultured is conditioned media obtained from a stimulation culture of T cells and APCs presenting a peptide of the virus.

4. A method for generating or expanding a population of T cells specific for a virus, comprising: (i) stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of the virus; (ii) collecting the cells obtained by step (i), and; (iii) re-stimulating the T cells by culture in the presence of APCs presenting a peptide of the virus, wherein at least 10% of the media in which the cells are cultured is conditioned media obtained from a stimulation culture of T cells and APCs presenting a peptide of the virus.

5. A method for generating or expanding a population of T cells specific for a virus, wherein the method comprises: (i) stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of the virus; (ii) collecting the cells obtained by step (i); (iii) re-stimulating the T cells by culture in the presence of APCs presenting a peptide of the virus; (iv) collecting the cells obtained by step (iii); and (v) re-stimulating the T cells by culture in the presence of APCs presenting a peptide of the virus, wherein at least 10% of the media in which the cells are cultured is conditioned media obtained from a stimulation culture of T cells and APCs presenting a peptide of the virus.

6. The method according to paragraph 5, wherein the conditioned media is obtained from the stimulation culture of step (iii).

7. The method according to any one of paragraphs 1 to 6, wherein the conditioned media is obtained from a stimulation culture of T cells and APCs presenting a peptide of the virus after a culture period of 1 to 8 days.

8. The method according to any one of paragraphs 1 to 7, wherein the conditioned media is obtained from a stimulation culture of T cells and APCs at a responder:stimulator ratio of 1:1 to 10:1.

9. The method according to any one of paragraphs 1 to 8, wherein the APCs presenting a peptide of the virus are EBV-transformed lymphoblastoid cell line (LCL) cells.

10. The method according to any one of paragraphs 1 to 9, wherein the at least 10% of conditioned media is 20 to 40% of conditioned media.

11. A method for generating or expanding a population of Epstein-Barr Virus (EBV)-specific T cells, 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 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% FBS, and 1-5 mM L-glutamine, (b) at least 10% 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.

12. A method for accelerating the rate of expansion of a population of Epstein-Barr Virus (EBV)-specific T cells, 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 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% FBS, and 1-5 mM L-glutamine, (b) at least 10% 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.

13. A method for generating or expanding a population of Epstein-Barr Virus (EBV)-specific T cells, comprising: (i) stimulating T cells by culturing PBMCs in the presence of EBV-transformed LCLs at a responder to stimulator ratio of 10:1 to 80:1 in cell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine for a period of 7 to 14 days; (ii) collecting the cells obtained by step (i); (iii) re-stimulating the T cells by culturing cells collected at step (ii) 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; (iv) collecting the cells obtained by step (iii), and; (v) re-stimulating the T cells by culturing cells collected at step (iv) in the presence of EBV-transformed 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% FBS, and 1-5 mM L-glutamine, (b) at least 10% conditioned media obtained at the end point of step (iii), and (c) added IL-2 at a final concentration of 10-200 (e.g. 40-100) IU/ml.

14. The method according to paragraph 13, wherein the method additionally comprises: (vi) collecting the cells obtained by step (v), and; (vii) re-stimulating the T cells by culturing cells collected at step (vi) in the presence of EBV-transformed 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% FBS, and 1-5 mM L-glutamine, (b) at least 10% conditioned media obtained at the end point of step (v), and (c) added IL-2 at a final concentration of 10-200 (e.g. 40-100) IU/ml.

15. The method according to paragraph 13 or 14, wherein the method comprises additional steps of collecting cells, and re-stimulating the T cells by culturing the collected cells in the presence of EBV-transformed LCLs (e.g. irradiated, EBV-transformed 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% FBS, and 1-5 mM L-glutamine, (b) at least 10% conditioned media obtained at the end point of the preceding stimulation step, and (c) added IL-2 at a final concentration of 10-200 IU/ml.

16. 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 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.

17. The method according to claim 16, wherein the conditioned media is obtained from a stimulation culture comprising T cells and APCs presenting a peptide of the virus at a responder:stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days.

18. The method according to claim 16 or claim 17, wherein stimulating T cells by culture in the presence of APCs presenting a peptide of the virus comprises culture in the presence of EBV-transformed 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% FBS, and 1-5 mM L-glutamine, (b) 10% to 25% 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.

19. The method according to claim 16 or claim 17, wherein the APCs presenting a peptide of the virus are EBV-transformed lymphoblastoid cell line (LCL) cells.

20. The method according to any one of claims 16 to 19, wherein the cancer is an EBV-positive cancer.

21. The method according to any one of claims 16 to 20, wherein the cancer is EBV-positive nasopharyngeal carcinoma (NPC).

22. The method according to any one of claims 16 to 21, wherein about 15% of the media in which the cells are cultured is conditioned media.

23. The method according to any one of claims 16 to 22, wherein step (2) additionally comprises: collecting the generated or expanded population of T cells.

24. 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 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, wherein the conditioned media is obtained from a stimulation culture comprising T cells and APCs presenting a peptide of the virus 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.

25. The method according to claim 24, wherein stimulating T cells by culture in the presence of APCs presenting a peptide of the virus comprises culture in the presence of EBV-transformed 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% FBS, and 1-5 mM L-glutamine, (b) 10% to 25% 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.

26. The method according to claim 24 or claim 25, wherein the cancer is an EBV-positive cancer.

27. The method according to any one of claims 24 to 26, wherein the cancer is EBV-positive nasopharyngeal carcinoma (NPC).

28. The method according to any one of claims 24 to 27, wherein the 10% to 25% conditioned media is about 15% conditioned media.

29. The method according to any one of claims 24 to 28, wherein step (2) additionally comprises: collecting the generated or expanded population of T cells.

30. A method for generating or expanding a population of T cells specific for a virus, 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.

31. The method according to claim 30, wherein the conditioned media is obtained from a stimulation culture comprising T cells and APCs presenting a peptide of the virus at a responder:stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days.

32. The method according to claim 30 or claim 31, wherein stimulating T cells by culture in the presence of APCs presenting a peptide of the virus comprises culture in the presence of EBV-transformed 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% FBS, and 1-5 mM L-glutamine, (b) 10% to 25% 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.

33. The method according to claim 30 or claim 31, wherein the APCs presenting a peptide of the virus are EBV-transformed lymphoblastoid cell line (LCL) cells.

34. The method according to any one of claims 30 to 33, wherein the 10% to 25% of conditioned media is about 15% of conditioned media.

35. The method according to any one of claims 30 to 34, wherein the method additionally comprises: collecting the generated or expanded population of T cells.

36. The method according to any one of claims 30 to 34, wherein the method additionally comprises: mixing the generated or expanded population of T cells with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

37. A population of T cells specific for a virus, wherein the population of T cells is obtained by, obtainable by, or is the product of, a method according to any one of claims 1 to 15 or 30 to 36.

38. A pharmaceutical composition comprising a population of T cells according to claim 37 and a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

39. A population of T cells according to claim 37, or a pharmaceutical composition according to claim 38, for use in the treatment or prevention of a disease or disorder.

40. Use of a population of T cells according to claim 37, or a pharmaceutical composition according to claim 38, in the manufacture of a medicament or vaccine for use in the treatment or prevention of a disease or disorder.

41. A method of treating or preventing a disease or disorder in a subject, comprising administering to a subject a therapeutically or prophylactically effective amount of a population of T cells according to claim 37, or a pharmaceutical composition according to claim 38.

42. The population of T cells or pharmaceutical composition for use according to claim 39, the use according to claim 40, or the method according to claim 41, wherein the disease or disorder is caused or exacerbated by infection with the virus for which the T cells are specific, or is a disease or disorder for which infection with the virus for which the T cells are specific is a risk factor.

43. The population of T cells or pharmaceutical composition for use, the use, or the method according to any one of claims 39 to 42, wherein the disease or disorder is a cancer.

44. The population of T cells or pharmaceutical composition for use, the use, or the method according to claim 43, wherein the cancer is an EBV-positive cancer.

45. The population of T cells or pharmaceutical composition for use, the use, or the method according to claim 43 or claim 44, wherein the cancer is an EBV-positive nasopharyngeal carcinoma (NPC).

46. A kit of parts comprising a predetermined quantity of a population of T cells according to claim 37, or the pharmaceutical composition of claim 38.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0248] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0249] FIG. 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.

[0250] 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.

[0251] 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.

[0252] 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

[0253] 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

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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).

[0258] 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.

[0259] For Illustrative Purposes:

[0260] Cells are cultured at a cell density of 110.sup.6 cells/ml in 200 ml of media, of which varying percentages are conditioned media:

TABLE-US-00001 Cell culture media comprising 45% RPMI 1640, 45% Click's Conditioned % Conditioned media, 10% FBS, 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%

[0261] 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

[0262] 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.

[0263] Generation of EBV-Transformed LCLs

[0264] Briefly, for LCL generation, 1510.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.

[0265] LCLs are irradiated at 60 Gy prior to being used for stimulations (on the day of the stimulation).

[0266] Expansion of EBV-Specific T Cells

[0267] Expansion of EBV-specific T cells by two different methods are compared.

[0268] In both methods, a first stimulation is performed as follows: [0269] 6010.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. [0270] PBMCs are seeded at 210.sup.6 cells/well into wells of a 24 well plate. [0271] PBMCs are stimulated with irradiated, Acyclovir-treated autologous LCLs at a responder to stimulator ratio of 40:1. [0272] The cells are cultured for 9-12 days at 37 C. in a 5% CO.sub.2 atmosphere.

[0273] In both methods, a second stimulation is then performed, as follows: [0274] Cells are collected at the end of the first stimulation, a viable cell count is performed, and the cells are re-suspended at 110.sup.6 cells/ml in cell culture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine. [0275] 1 ml of cells are added into wells of 24 well plates, or the cell suspension is added into bioreactors at a concentration of 1510.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. [0276] 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 (rhlL-2, Proleukin; Chiron Emeryville, Calif.)) is added to the cell culture at a final concentration of 40-100 IU/ml.

[0277] Subsequent Stimulations

[0278] Following the second stimulation, cells are collected, a viable cell count is performed.

[0279] Culture media (i.e. conditioned media) is retained for use in method B below. The cells are then re-suspended at 110.sup.6 cells/ml (in 24 well plate) or 0.510.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.

[0280] Once 20010.sup.6 cells are achieved, cells are then stimulated according to method step A or method step B:

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

[0281] 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.

[0282] For re-simulations according to method step B, conditioned media used is from the preceding stimulation according to B.

Example 3: Test of Efficacy

[0283] 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.

[0284] 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).

[0285] 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

[0286] The following example describes an investigation of the optimal percentage of conditioned media to be included in restimulations for maximum CTL expansion.

[0287] Week 1: [0288] Lymphoblastoid cell line (LCL) samples were thawed and cultured for 1 week [0289] Requires minimally 1010.sup.6 LCLs to initiate experiment

[0290] Week 2: [0291] 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 [0292] 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 [0293] EB-VST03 cells were seeded at 110.sup.6 cells/well of a 24-well plate [0294] Cell suspensions were added to bioreactors (1510.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. [0295] 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. [0296] IL-2 was then added to the cell culture at a final concentration of 40-100 IU/ml.

[0297] Week 3 Onwards: [0298] Cells were harvested, pooled, counted and stimulated as described under week 2 above until there were 12010.sup.6 cells. [0299] 10010.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:

TABLE-US-00003 Cell culture media comprising 45% RPMI 1640, 45% Click's Conditioned % Conditioned media, 10% FBS, 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% [0300] The cells were then cultured for 3-4 days at 37 C. in a 5% CO.sub.2 atmosphere. [0301] IL-2 was then added to the cell culture at a final concentration of 40-100 IU/ml. [0302] After one week, the cells were harvested and a viable cell count was performed.

[0303] All viabilities were observed to be greater than 70%. The results are shown in in FIG. 4.

[0304] 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.

[0305] Fold expansion of EB-VSTs decreased when higher percentages (i.e. 30%, 45%) of conditioned media were used.

[0306] 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.

[0307] 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.

[0308] 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.