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.

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 .