Methods of making chimeric antigen receptor-expressing cells

Abstract

The invention provides methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to express a chimeric antigen receptor (CAR), and compositions and reaction mixtures comprising the same.

1 . A method of making a population of immune effector cells that is depleted of T regulatory cells and can be engineered to express a CAR, the method comprising:
 providing a population of immune effector cells, and   removing T regulatory cells from the population, to thereby provide a population of T regulatory-depleted cells, that are suitable for expression of a CAR.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the population of immune effector cells are cells of a subject having a hematologic cancer. 
     
     
         4 . The method of  claim 3 , wherein the population of T regulatory-depleted cells contains less than 50% of CD25+ cells and less than 50% of tumor cells. 
     
     
         5 . The method of  claim 1 , wherein the T regulatory cells are removed from the population using an anti-CD25 antibody, or fragment thereof. 
     
     
         6 .- 8 . (canceled) 
     
     
         9 . The method of  claim 1 , the method further comprising:
 (i) removing cells from the population which express a tumor antigen, to thereby provide a population of T regulatory-depleted and tumor antigen depleted cells that are suitable for expression of a CAR; or   (ii) removing cells from the population which express a check point inhibitor to thereby provide a population of T regulatory-depleted and check point inhibitor depleted cells.   
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the population of immune effector cells provided has been selected based upon the expression of one or more markers, chosen from CD3, CD28, CD4, CD8, CD45RA, and CD45RO. 
     
     
         12 . The method of  claim 1 , further comprising: one or both of activating the population of T regulatory-depleted cells or transducing a cell from the population of T regulatory-depleted cells with a vector comprising a nucleic acid encoding a CAR. 
     
     
         13 . The method of  claim 12 , further comprising expanding the population of T regulatory-depleted cells. 
     
     
         14 . The method of  claim 13 , wherein the population of cells is expanded:
 (i) for a period of 8 days or less, or wherein the population of cells is expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions;   (ii) by culturing the cells in the presence of an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the cell; or   (ii) in an appropriate media that includes one or more interleukin that result in at least a 200-fold increase in cells over a 14 day expansion period.   
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 13 , wherein the population of the cells is cryopreserved after the appropriate expansion period. 
     
     
         18 . The method of  claim 1  further comprising contacting the population of immune effector cells with a nucleic acid encoding a telomerase subunit. 
     
     
         19 . A reaction mixture of immune effector cells, comprising a population of T regulatory-depleted cells containing less than 50% of CD25+ cells, wherein a plurality of the cells of the population in the reaction mixture comprise a nucleic acid molecule. 
     
     
         20 .- 26 . (canceled) 
     
     
         27 . A method of making a population of immune effector cells engineered to express a CAR, the method comprising:
 providing a population of immune effector cells, wherein a plurality of the immune effector cells comprise a nucleic acid encoding a CAR, and   expanding the cells of the population in the presence of one or more interleukin that result in at least a 200-fold increase in cells over a 14 day expansion period.   
     
     
         28 . The method of  claim 27 , wherein the population of cells is expanded in the presence of IL-15 IL-7, or both of IL-15 and IL-7. 
     
     
         29 . The method of  claim 27 , wherein the population of cells is expanded for a period of less than 8 days or 3 days. 
     
     
         30 . The method of  claim 27 , wherein the cells are expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. 
     
     
         31 . The method of  claim 30 , wherein the cells expanded for 5 days show at least a one fold increase in cells doublings upon antigen stimulation as compared to the same cells expanded in culture for 9 days under the same culture conditions, or wherein the cells are expanded in culture for 5 days, and the resulting cells exhibit higher proinflammatory cytokine production as compared to the same cells expanded in culture for 9 days under the same culture conditions. 
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 27 , wherein the provided population of immune effector cells is a population of T regulatory-depleted cells containing less than 50% of CD25+ cells, or wherein the provided population of immune effector cells is a population of T regulatory-depleted cells containing less than 50% of CD25+ cells and less than 50% of CD25 expressing tumor cells. 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 33 , wherein the provided population of immune effector cells also contains less than 50% of a checkpoint inhibitor expressing cells. 
     
     
         36 . The method of  claim 33 , further comprising contacting the population of immune effector cells with a nucleic acid encoding a telomerase subunit. 
     
     
         37 . A reaction mixture comprising a population of immune effector cells, wherein a plurality of the cells of the population in the reaction mixture comprise a nucleic acid molecule that comprises a CAR encoding sequence, and IL-7 or IL-15 or both of IL-7 and IL-15. 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . A method of making a population of immune effector cells the method comprising:
 providing a population of immune effector cells, and   contacting the population of immune effector cells with a nucleic acid encoding a CAR and a RNA encoding a telomerase subunit under conditions suitable for expression of the CAR and the telomerase subunit.   
     
     
         41 .- 46 . (canceled)