T Cell Expansion
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.
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 .