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 chimeric antigen receptor (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, suitable for expression of a CAR.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the population of immune effector cells are obtained from a subject having cancer. 
     
     
         4 . The method of  claim 3 , wherein the population of T regulatory-depleted cells contains less than 50%, of T regulatory 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 . (canceled) 
     
     
         7 . The method of  claim 3 , wherein the subject has a hematological cancer chosen from a leukemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), a lymphoma, or mantle cell lymphoma (MCL). 
     
     
         8 - 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: activating the population of T regulatory-depleted cells; and/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 . (canceled) 
     
     
         15 . The method of  claim 13 , wherein the population of cells is expanded 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 cells. 
     
     
         16 - 26 . (canceled) 
     
     
         27 . A method of making a population of chimeric antigen receptor (CAR)-expressing immune effector cells, the method comprising:
 (a) providing a population of immune effector cells, wherein a plurality of the immune effector cells in the population comprises a nucleic acid encoding a CAR; and   (b) contacting the population of immune effector cells with an interleukin-15 (IL-15); an interleukin-7 (IL-7); or a combination of IL-15 and IL-7.   
     
     
         28 . The method of  claim 27 , wherein the population of immune effector cells in step (b) is contacted with the IL-15 or a combination of the IL-15 and an IL-15 receptor alpha (Ra). 
     
     
         29 . The method of  claim 27 , wherein the population of immune effector cells is expanded for a period of less than 8 days. 
     
     
         30 . The method of  claim 27 , wherein the population of immune effector 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. 
     
     
         31 . The method of  claim 30 , wherein:
 (i) the population of immune effector cells expanded for 5 days shows at least a two fold increase in cell doublings upon antigen stimulation as compared to the same cells expanded in culture for 9 days under the same culture conditions; or   (ii) the population of immune effector cells is 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 . The method of  claim 27 , wherein the population of immune effector cells is expanded 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 cells. 
     
     
         33 . The method of  claim 27 , wherein the provided population of immune effector cells is a population of T regulatory-depleted cells containing:
 (i) less than 50% of CD25+ cells; or   (ii) less than 50% of CD25+ cells, and less than 50% of CD25 expressing tumor cells.   
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 27 , wherein the provided population of immune effector cells contains less than 50%. 
     
     
         36 - 46 . (canceled) 
     
     
         47 . A method of making a population of chimeric antigen receptor (CAR)-expressing immune effector cells, the method comprising:
 (a) providing a population of cells comprising immune effector cells from an apheresis sample from a patient with chronic lymphocytic leukemia;   (b) depleting CD25-expressing cells from the population of cells of step (a) to provide a population of CD25-depleted cells, wherein the population of CD25-depleted cells is a population that contains less than 50% CD25+ cells in the population of cells of step (a) when assessed by flow cytometry;   (c) contacting the CD25-depleted cells with anti-CD3 and anti-CD28 coated beads; and   (d) transducing the population of CD25-depleted cells with a lentivirus vector encoding a chimeric antigen receptor;   to thereby provide a population of CAR-expressing immune effector cells.   
     
     
         48 . The method of  claim 28 , wherein the combination of the IL-15 and the IL-15Ra is hetIL-15. 
     
     
         49 . The method of  claim 27 , wherein the population of immune effector cells is expanded for a period of less than 3 days. 
     
     
         50 . The method of  claim 27 , wherein the plurality of immune effector cells in the population comprises a nucleic acid encoding a CD19 CAR.