Method for preparing genetically-modified t cells which express chimeric antigen receptor

Abstract

Provided is a method for preparing genetically-modified T cells expressing chimeric antigen receptor, comprising: (i) a step of preparing non-proliferative cells holding a viral peptide antigen, which are obtained by stimulating a group of cells comprising T cells using an anti-CD3 antibody and an anti-CD28 antibody followed by culturing in the presence of the viral peptide antigen and a treatment for causing the cells to lose their proliferation capability; (ii) a step of obtaining genetically-modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced using a transposon method; (iii) a step of mixing the non-proliferative cells prepared by step (i) with the genetically-modified T cells obtained by step (ii), and co-culturing the mixed cells; and (iv) a step of collecting the cells after culture.

1 - 6 . (canceled) 
     
     
         7 . A method for preparing genetically-modified T cells expressing chimeric antigen receptor, comprising the following steps (i-a) or (i-b), and (ii) to (iv):
 (i-a) a step of preparing non-proliferative cells holding a viral peptide antigen, which are obtained by stimulating a group of cells comprising T cells using an anti-CD3 antibody and an anti-CD28 antibody followed by culturing in the presence of the viral peptide antigen and a treatment for causing the cells to lose their proliferation capability;   
       or
 (i-b) a step of preparing viral peptide-holding non-proliferative peripheral blood mononuclear cells, which are prepared by culturing peripheral blood mononuclear cells in the presence of the viral peptide antigen, and a treatment for causing the cells to lose their proliferation capability; and 
 (ii) a step of introducing a target antigen-specific chimeric antigen receptor gene into T cells using a transposon method and thereby obtaining the genetically-modified T cells; 
 (iii) a step of mixing the non-proliferative cells prepared by step (i-a) or the viral peptide-holding non-proliferative peripheral blood mononuclear cells prepared by step (i-b), with the genetically-modified T cells obtained by step (ii), and co-culturing the mixed cells; and 
 (iv) a step of collecting the cells after culture; 
 wherein the step (i-a) or (i-b) is conducted in advance of step (ii). 
 
     
     
         8 . The preparation method according to  claim 7 , wherein, after the cells are co-cultured, a step of culturing the co-cultured cells in the presence of a T-cell growth factor is carried out between step (iii) and step (iv). 
     
     
         9 . The preparation method according to  claim 7 , wherein the period of the co-culturing in step (iii) is one day to 14 days. 
     
     
         10 . The preparation method according to  claim 7 , wherein step (iii) is carried out in the presence of a T-cell growth factor. 
     
     
         11 . The preparation method according to  claim 10 , wherein the T-cell growth factor is IL-15. 
     
     
         12 . The preparation method according to  claim 10 , wherein the T-cell growth factor is a combination of IL-15 and IL-7. 
     
     
         13 . The preparation method according to claim  1 , wherein the group of cells comprising T cells is peripheral blood mononuclear cells (PBMCs). 
     
     
         14 . The preparation method according to claim  1 , wherein the treatment for losing the proliferation capability is irradiation. 
     
     
         15 . The preparation method according to claim  1 , wherein the transposon method comprises preparing a vector including the gene coding transposase and a vector having a structure wherein the gene coding a target protein is sandwiched between inverted repeat sequences, and introducing these vectors to the target cell. 
     
     
         16 . The preparation method according to claim  1 , wherein the target antigen is the CD19, GD2, GMCSF receptor or the IGF receptor. 
     
     
         17 . The preparation method according to claim  1 , wherein the non-proliferative cells or the viral peptide-holding non-proliferative peripheral blood mononuclear cells, and the genetically-modified T cells are derived from an identical individual. 
     
     
         18 - 20 . (canceled) 
     
     
         21 . The preparation method according to claim  1 , which comprises step (i-a). 
     
     
         22 . The preparation method according to claim  1 , which comprises step (i-b).