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 - 104 . (canceled) 
     
     
         105 . A method of manufacturing engineered cells comprising
 obtaining a sample of cells; and   electroporating a sample of cells with a DNA comprising a chimeric antigen receptor and a fusion protein comprising IL-15 and IL-15Ra and a DNA encoding a transposase, to obtain a population of engineered cells.   
     
     
         106 . The method of  claim 105 , further comprising administering engineered cells to a patient once one or more release criteria is met. 
     
     
         107 . The method of  claim 106 , wherein release criteria includes sterility, phenotype, viability, or cell number. 
     
     
         108 . The method of  claim 105 , wherein the population of engineered cells is immediately infused into a subject. 
     
     
         109 . The method of  claim 105 , further comprising culturing the population of engineered T-cells ex vivo for no more than 2 days. 
     
     
         110 . The method of  claim 105 , wherein the fusion protein comprises an amino acid sequence which is at least 90% identical to SEQ ID NO: 6. 
     
     
         111 . The method of  claim 105 , wherein the fusion protein is encoded by a DNA comprising a sequence which is at least 90% identical to SEQ ID NO: 7. 
     
     
         112 . The method of  claim 105 , wherein the transposase is a Sleeping Beauty transposase or piggyBac. 
     
     
         113 . The method of  claim 112 , wherein the Sleeping Beauty transposase is SB10, SB11 or SB100x transposase. 
     
     
         114 . The method of  claim 105 , wherein the transposase is provided as a DNA expression vector. 
     
     
         115 . The method of  claim 105 , wherein the DNA further comprises a Sleeping Beauty transposon. 
     
     
         116 . The method of  claim 105 , wherein the CAR targets a cancer cell antigen. 
     
     
         117 . The method of  claim 116 , wherein the cancer cell antigen is CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, folate binding protein, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination or HER 1-HER2 in combination. 
     
     
         118 . The method of  claim 105 , wherein the sample of cells is obtained by apheresis. 
     
     
         119 . The method of  claim 105 , wherein the sample of cells is a peripheral blood sample from a subject. 
     
     
         120 . The method of  claim 105 , wherein the sample of cells is a cryopreserved sample. 
     
     
         121 . The method of  claim 105 , wherein the sample of cells is from umbilical cord blood. 
     
     
         122 . The method of  claim 105 , wherein the sample of cells are T-cells, T-cell precursors or NK cells. 
     
     
         123 . The method of  claim 105 , wherein the T-cells or T-cell progenitor cells are allogeneic cells. 
     
     
         124 . The method of  claim 123 , wherein the T-cells or T-cell progenitor cells are inactivated for expression of an endogenous T-cell receptor and/or endogenous HLA. 
     
     
         125 . A method of manufacturing engineered cells comprising obtaining a sample of cells; and
 transducing a sample of cells with a viral vector comprising a chimeric antigen receptor and a membrane-bound Cγ cytokine, to obtain a population of engineered cells.   
     
     
         126 . The method of  claim 125 , wherein the viral vector is a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector. 
     
     
         127 . The method of  claim 125 , wherein the membrane-bound Cγ cytokine is a membrane bound IL-7, IL-15 or IL-21 
     
     
         128 . A method of treating cancer comprising:
 obtaining a sample of cells from a patient;   electroporating the sample of cells with a DNA encoding a transposon comprising a chimeric antigen receptor and a fusion protein comprising IL-15 and IL-15Ra and a DNA encoding a transposase, to obtain a population of engineered cells; and   administering an effective amount of said population of engineered cells to the patient.