Effective generation of tumor-targeted t cells derived from pluripotent stem cells

Abstract

The present invention relates to the field of adoptive immunotherapy. The invention provides methods for generating phenotypically defined, functional, and/or expandable T cells from pluripotent stem cells engineered through safe genetic modifications. The engineered cells may provide one or more of: 1) targeting a specific predetermined antigen expressed on the cell surface of a target cell in an HLA independent manner, 2) enhanced survival and functional potential 3) “off-the-shelf” T cells for administration to multiple recipients, eventually across immunogenic barriers, and/or 4) cytotoxic potential and anti-tumor activity.

1 .- 49 . (canceled) 
     
     
         50 . A pluripotent stem cell that comprises a rearranged T-cell receptor (TCR) locus and expresses a chimeric antigen receptor (CAR), wherein the pluripotent stem cell is derived from a T cell. 
     
     
         51 . The pluripotent stem cell of  claim 50 , wherein (i) the pluripotent stem cell is transduced with the CAR; (ii) pluripotent stem cell does not express TCR on the cell surface; and/or (iii) said CAR binds to a tumor antigen. 
     
     
         52 . The pluripotent stem cell of  claim 51 , wherein said tumor antigen is selected from the group consisting of carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1). 
     
     
         53 . A method of producing a cell of  claim 50 , comprising,
 a) providing,
 i) a pluripotent stem cell bearing a rearranged TCR locus (T-PSC), and 
 ii) a CAR expression vector encoding an antigen binding domain and at least a portion of CD3ξ polypeptide, and 
   b) transducing said T-PSC with said CAR expression vector under conditions thereby obtaining a CAR-expressing and a rearranged T-cell receptor (TCR)-bearing iPSC (CAR-T-PSC).   
     
     
         54 . The method of  claim 53 , wherein said CAR expression vector comprises a heterologous gene encoding at least one costimulatory signaling region or a costimulatory ligand. 
     
     
         55 . The method of  claim 54 , wherein (i) the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, or an ICOS polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide; and/or (ii) the costimulatory ligand is selected from the group consisting of CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD-L1. 
     
     
         56 . The method of  claim 53 , wherein the pluripotent stem cell bearing a rearranged TCR locus is (i) an embryonic stem cell; (ii) an induced pluripotent stem cell; or (iii) an induced pluripotent stem cell obtained from reprogramming a T cell. 
     
     
         57 . The method of  claim 53 , further comprising:
 c) culturing said CAR-T-PSC under conditions such that a CAR-expressing T-PSC-derived T cell (CAR-T-PSC-derived T cell) is produced.   
     
     
         58 . The method of  claim 57 , wherein said c) culturing said CAR-T-PSC under conditions to obtain a CAR-T-PSCs-derived T cell comprises:
 (a) providing,
 i) said CAR-T-PSC, 
 ii) a first cell culture medium for mesoderm induction, 
 iii) a second cell culture medium for hematopoietic specification and expansion, 
 iv) a third cell culture medium for T-lymphoid differentiation, and 
 v) a feeder cell line that induces T lymphoid commitment in hematopoietic cells, 
   (b) incubating said CAR-T-PSC with said first cell culture medium for up to about 4 days under conditions such that a mesoderm cell is produced,   (c) incubating said mesoderm cell with said second cell culture medium for up to about 6 days under conditions such that a hematopoietic cell is produced and expanded, and   (d) incubating said expanded hematopoietic cell and said feeder cell line with said third cell culture medium for at least about 5 days for inducing T lymphoid commitment in said expanded hematopoietic cell to produce a CAR-expressing T-PSC-derived T cell.   
     
     
         59 . The method of  claim 57 , wherein
 (i) said T cell has at least one of the following characteristics:
 (a) targeting specifically to one specific antigen and antigen specificity of said T cells is HLA-independent; 
 (b) expresses the CAR of the CAR-T-PSC; 
   (ii) said CAR-T-PSC is at least one of the followings:
 (a) an embryonic stem cell; 
 (b) an induced pluripotent stem cell (iPSC); or 
 (c) an induced pluripotent stem cell obtained from reprogramming a T cell (T-PSC); 
 (d) being obtained from transducing a T-PSC with a CAR expression vector; and/or 
   (iii) said CAR expression vector comprises a heterologous polynucleotide encoding a CAR comprising an antigen binding domain, and at least one costimulatory signaling region or a costimulatory ligand.   
     
     
         60 . The method of  claim 58 , wherein (i) said first cell culture medium comprises bone morphogenetic protein 4 (BMP-4) and basic fibroblast growth factor (bFGF); and/or (ii) said second cell culture medium comprises Vascular endothelial growth factor (VEGF), bFGF, stem cell factor (SCF), FMS Like Tyrosine Kinase 3 Ligand (Flt3L), and at least one Th1 cytokine; and/or (iii) said third cell culture medium comprises SCF, at least one Th1 cytokine, and Flt3L. 
     
     
         61 . The method of  claim 60 , wherein said at least one Th1 cytokine is selected from the group consisting of Interleukin-3 (IL-3), IL-15, IL-7, IL-12 and IL-21. 
     
     
         62 . The method of  claim 59 , wherein
 (i) said at least one costimulatory signalling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide;   (ii) said costimulatory ligand is selected from the group consisting of CD80, CD86, CD70, OX40L 4-1BBL, CD48, TNFRSF14, and PD-L1; and/or   (iii) said antigen binding domain of the CAR specifically binds to a tumor antigen or a pathogen antigen;   (iv) said CAR specifically binds to a tumor antigen selected from the group consisting of carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF R2), and Wilms tumor protein (WT-1);   (v) said CAR expression vector comprises
 (a) a nucleic acid sequence that is integrated into said CAR-T-PSC's genome at a genomic safe harbor site; and/or 
 (b) a nucleic acid sequence encoding a fluorescent protein for expressing in said CAR-T-PSC. 
   
     
     
         63 . The method of  claim 57 , further comprising: d) exposing said CAR-T-PSC-derived T cell to an antigen-presenting cell under conditions for stimulating an activity of said CAR-T-PSC-derived T cell; e) inducing fluorescence in said CAR-T-PSC; and/or (f) tracking said CAR-T-PSCs. 
     
     
         64 . The method of  claim 63 , wherein said activity is selected from the group consisting of secretion of cytokine secretion, cell division, cytotoxicity and cytostatic inhibition of cell growth. 
     
     
         65 . The method of  claim 64 , wherein
 (i) said cytokine is a Th1 cytokine selected from the group consisting of IFN-γ, IL-2 and TNF-α;   (ii) said cytotoxicity is determined by killing a target cell expressing an antigen that binds to said CAR and measuring target cell death; and/or   (iii) cytostatic inhibition of cell growth comprises (a) inhibition of growth of a tumor cell; and/or (b) reduction in tumor size.   
     
     
         66 . The method of  claim 53 , wherein the pluripotent stem cell is obtained by a process comprising,
 a) providing,
 i) a cell selected from the group consisting of an isolated peripheral blood lymphocyte (PBL) and an isolated peripheral blood T-cell, and a combination thereof, and 
 ii) at least one reprogramming factor selected from the group consisting of octamer-binding transcription factor 4 (OCT4), Kruppel-like factor 4 (KLF4), myelocytomatosis viral oncogene homolog (c-MYC), and transcription factor SOX-2, and 
   b) transducing said cell with said at least one reprogramming factor under conditions for producing a pluripotent stem cell.   
     
     
         67 . A pluripotent stem cell that expresses a chimeric antigen receptor (CAR-T-PSC), wherein the cell is obtained using the method of  claim 53 . 
     
     
         68 . A CAR-expressing T-PSC-derived T cell (CAR-T-PSC-derived T cell), wherein the cell is obtained using the method of  claim 58 . 
     
     
         69 . A method of manufacturing therapeutic cells comprising a CAR-expressing T-PSC-derived T cell (CAR-T-PSC-derived T cell) according to  claim 58 . 
     
     
         70 . A process of using therapeutic cells of  claim 69  for adoptive cell therapy, comprising administering to a subject in need of the therapy one or multiple doses of said cells at a therapeutically sufficient amount. 
     
     
         71 . The process of  claim 70 , wherein (i) the therapeutic cells are comprised in a pharmaceutically acceptable carrier; and/or (ii) the subject has a neoplasia, a pathogen infection, an immune disorder, or an allogeneic transplant.