Human application of engineered chimeric antigen receptor (car) t-cells

Abstract

The present invention concerns methods and compositions for immunotherapy employing a modified T cell comprising a chimeric antigen receptor (CAR). In particular aspects, CAR-expressing T-cells are producing using electroporation in conjunction with a transposon-based integration system to produce a population of CAR-expressing cells that require minimal ex vivo expansion or that can be directly administered to patients for disease (e.g., cancer) treatment.

1 . A method of culturing engineered T-cells comprising:
 (a) obtaining a sample of cells from a subject, the sample comprising T-cells or T-cell progenitors;   (b) transfecting the cells with a DNA encoding a transposon-flanked chimeric antigen receptor (CAR) and a transposase effective to integrate the DNA encoding the CAR into the genome of the cells, to provide a population of engineered CAR-expressing T-cells; and   (c) culturing the population of engineered CAR cells ex vivo in a medium that selectively enhances proliferation of CAR-expressing T-cells, wherein the engineered CAR T-cells are cultured for no more than 21 days.   
     
     
         2 . A method of stimulating the proliferation and/or survival of engineered T-cells comprising:
 (a) obtaining a sample of cells from a the subject, the sample comprising T-cells or T-cell progenitors;   (b) transfecting the cells with a DNA encoding a transposon-flanked chimeric antigen receptor (CAR) and a transposase effective to integrate the DNA encoding the CAR into the genome of the cells, to provide a population of engineered CAR-expressing T-cells;   (c) transfecting the cells with a DNA encoding a fusion protein comprising IL-15 and IL-15Rα, and   (d) optionally, culturing the population of engineered CAR cells ex vivo for 2 days or less.   
     
     
         3 . The method of  claim 1 , further comprising purifying or enriching T-cells in the sample prior to step (b). 
     
     
         4 . The method of  claim 3 , wherein enriching T-cells in the sample comprises collecting a mononuclear cell fraction. 
     
     
         5 . The method of  claim 1  , wherein the engineered CAR T-cells are cultured no more than 14 days. 
     
     
         6 - 14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the engineered CAR T-cells are inactivated for expression of an endogenous T-cell receptor and/or endogenous HLA. 
     
     
         16 . The method of  claim 1 , wherein obtaining the sample of cells comprises obtaining the cells from a 3rd party. 
     
     
         17 . The method of  claim 1 , wherein transfecting the cells with DNA comprises electroporating the cells. 
     
     
         18 . The method of  claim 2 , wherein transfecting the cells with DNA comprises electroporating the cells. 
     
     
         19 . The method of  claim 1 , wherein transfecting the cells furthers comprises transfecting the cells with a nucleic acid encoding a membrane-bound Cy cytokine. 
     
     
         20 . The method of  claim 19 , wherein the membrane-bound Cy cytokine is a membrane bound IL-7, IL-15 or IL-21. 
     
     
         21 . The method of  claim 19 , wherein the membrane-bound Cy cytokine is a fusion protein comprising IL-15 and IL-15Rα. 
     
     
         22 . The method of  claim 1 , wherein the DNA encoding a CAR is a plasmid. 
     
     
         23 . The method of  claim 1 , wherein the transposase is provided as a DNA expression vector. 
     
     
         24 . The method of  claim 1 , wherein the transposase is provided as an mRNA. 
     
     
         25 . The method of  claim 24 , wherein the mRNA comprises a capped and/or PolyA tail. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein the transposase is salmonid-type Tc1-like transposase (SB). 
     
     
         28 . The method of  claim 27 , wherein the transposase is the SB11 or SB100× transposase. 
     
     
         29 . The method of  claim 1 , wherein culturing the engineered CAR cells (c) may comprise culturing the engineered CAR cells in the presence of dendritic cells or artificial antigen presenting cells (aAPCs) that stimulate expansion of the CAR-expressing T-cells. 
     
     
         30 . The method of  claim 29 , wherein the aAPCs are engineered K562 cells. 
     
     
         31 . The method of  claim 29 , wherein the aAPCs comprises (i) an antigen targeted by the CAR expressed on the engineered CAR cells; (ii) CD64; CD86; (iii) CD 137L; and/or (v) membrane-bound IL-15, expressed on the surface of the aAPCs. 
     
     
         32 - 37 . (canceled) 
     
     
         38 . The method of  claim 29 , wherein culturing the engineered CAR cells in the presence of aAPCs comprises culturing the transgenic CAR cells in a medium comprising IL-21 and/or IL-2. 
     
     
         39 . The method of  claim 29 , wherein culturing the engineered CAR cells in the presence of aAPCs comprises culturing the cells at a ratio of about 10:1 to about 1:10 (CAR cells to aAPCs). 
     
     
         40 - 42 . (canceled) 
     
     
         43 . The method of  claim 1 , wherein the engineered cells are not cultured ex vivo in the presence of aAPCs. 
     
     
         44 . The method of  claim 2 , wherein the engineered cells are not cultured ex vivo in the presence of aAPCs. 
     
     
         45 - 50 . (canceled) 
     
     
         51 . The method of  claim 1 , wherein the CAR is targeted to a cancer-cell antigen. 
     
     
         52 . The method of  claim 51 , wherein the cancer cell antigen is CD 19, CD20, ROR1, CD22 carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30 , CD56, c-Met, meothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination or HER 1-HER2 in combination. 
     
     
         53 . The method of  claim 52 , wherein the cancer cell antigen is CD 19 and the CAR is a CD19-targeted CAR. 
     
     
         54 - 59 . (canceled) 
     
     
         60 . A T-cell composition made by a method of  claim 1 . 
     
     
         61 - 104 . (canceled)