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.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to methods for generating and / or expanding populations of virus-specific T cells.BACKGROUND TO THE INVENTION

[0002] Adoptive transfer of virus-specific T cells is a promising strategy for the prevention and treatment of viral disease.

[0003] Adoptive transfer requires the use of a large number of T cells. For example, Chia et al., Mol Ther (2014) 22(1): 132-139 describe treatment of EBV-positive nasopharyngeal carcinoma (NPC) by adoptive transfer of a median total number of 9.6×108 EBV-CTLs (ranging from 6.3 to 10.3×108 CTLs).

[0004] A major problem for therapy by adoptive T cell transfer is the period of time taken to generate sufficiently large numbers of virus-specific T cells for administration. Chia et al. (supra) reported that the median time taken to produce and the first dose of CTLs was 13 weeks (ranging from 8 to 22 weeks). Moreover, it is reported that in this study that for 3 patients there was deviation from the schedu...

Examples

example 1

ercentage 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 b...

example 2

n 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, 15×106 peripheral blood mononuclear cells (PBMCs) are incubated with concentrated supernatant of B95-8 cultures, in the presence of 1 μg / 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]60×106 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 ...

example 3

fficacy

[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.

FIELD OF THE INVENTION

[0001] The present invention relates to methods for generating and / or expanding populations of virus-specific T cells.BACKGROUND TO THE INVENTION

[0002] Adoptive transfer of virus-specific T cells is a promising strategy for the prevention and treatment of viral disease.

[0003] Adoptive transfer requires the use of a large number of T cells. For example, Chia et al., Mol Ther (2014) 22(1): 132-139 describe treatment of EBV-positive nasopharyngeal carcinoma (NPC) by adoptive transfer of a median total number of 9.6×108 EBV-CTLs (ranging from 6.3 to 10.3×108 CTLs).

[0004] A major problem for therapy by adoptive T cell transfer is the period of time taken to generate sufficiently large numbers of virus-specific T cells for administration. Chia et al. (supra) reported that the median time taken to produce and the first dose of CTLs was 13 weeks (ranging from 8 to 22 weeks). Moreover, it is reported that in this study that for 3 patients there was deviation from the schedu...

Examples

example 1

ercentage 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 b...

example 2

n 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, 15×106 peripheral blood mononuclear cells (PBMCs) are incubated with concentrated supernatant of B95-8 cultures, in the presence of 1 μg / 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]60×106 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 ...

example 3

fficacy

[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.

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