Method for generating human dendritic cells for immunotherapy

Abstract

In various embodiments methods of producing a cell population enriched for CLEC9A+ dendritic cells are provided where the methods involve culturing stem cells and/or progenitor cells in a cell culture comprising culture medium, a notch ligand, stem cell factor (SCF), FLT3 ligand (FLT3L); thrombopoietin (TPO); and IL-3 and/or GMCSF.

1 - 92 . (canceled) 
     
     
         93 . A method of preparing a dendritic cell vaccine for a subject, said method comprising:
 preparing a cell population enriched for CLEC9A+ dendritic cells; and   pulsing or loading said dendritic cells with a tumor cell antigen, a tumor cell lysate or tumor cell preparation,   wherein said cell population enriched for CLEC9A+ dendritic cells is produced by a method comprising co-culturing stem cells or progenitor cells with stromal cells transduced or transfected with a nucleic acid that encodes and expresses a notch ligand,   wherein the notch ligand is DLL4, DLL1, Jagged 1 (JAG1), or Jagged 2 (JAG2), or a Notch-binding fragment of DLL4, DLL1, Jagged 1 (JAG1), or Jagged 2 (JAG2),   wherein said co-culturing comprises culture medium supplemented with L-alanyl-L-glutamine dipeptide.   
     
     
         94 . The method of  claim 93 , wherein said method further comprises providing a maturation signal to said dendritic cells. 
     
     
         95 . The method of  claim 94 , wherein said maturation signal comprises a TLR3 agonist. 
     
     
         96 . The method of  claim 94 , wherein said maturation signal comprises a TLR8 agonist. 
     
     
         97 . The method of  claim 93 , wherein said tumor cell antigen is WT1, MUC1, MP2, HPV E6 E7, EGFRvIII, HER-2/neu, diotype, MAGE A3, p53 nonmutant, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant, Proteinase3 (PR1), bcr-abl, Tyrosinase, Survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion), NA17, PAX3, ALK, Androgen receptor, Cyclin B1. Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, or Fos-related antigen 1. 
     
     
         98 . The method of  claim 93 , wherein said tumor cell antigen, tumor cell lysate or tumor cell preparation is derived from ovarian cancer, lung cancer, breast cancer, bladder cancer, colon and rectal cancer, endometrial cancer, kidney cancer, leukemia, lung cancer, melanoma, non-hodgkin lymphoma, pancreatic cancer, prostate cancer, or thyroid cancer. 
     
     
         99 . The method of  claim 93 , wherein method comprises pulsing or loading said dendritic cells with a tumor cell neoantigen. 
     
     
         100 . The method of  claim 99 , wherein said tumor cell neoantigen is derived from CDK4, MUM1, CTNNB1, CDC27, TRAPPC1, TPI, ASCC3, HHAT, FN1, OS-9, PTPRK, CDKN2A, HLA-A11, GAS7, GAPDH, SIRT2, GPNMB, SNRP116, RBAF600, SNRPD1, Prdx5, CLPP, PPP1R3B, EF2, ACTN4, ME1, NF-YC, HLA-A2, HSP70-2, KIAA1440, or CASP8. 
     
     
         101 . The method of  claim 93 , wherein said CLEC9A+ cells are autologous to a subject to whom said vaccine is to be administered. 
     
     
         102 . The method of  claim 93 , wherein said CLEC9A+ cells are heterologous to a subject to whom said vaccine is to be administered. 
     
     
         103 . The method of  claim 93 , wherein said CLEC9A+ cells are competent at cross-presenting antigen without adjuvant or without maturation. 
     
     
         104 . The method of  claim 93 , wherein said CLEC9A+ cells are competent at cross-presenting without TLR ligand. 
     
     
         105 . The method of  claim 93 , wherein said stem cells or progenitor cells comprise hematopoietic stem cells or hematopoietic progenitor cells. 
     
     
         106 . The method of  claim 93 , wherein said stem cells or progenitor cells are derived from bone marrow, umbilical cord, peripheral blood, or mobilized peripheral blood. 
     
     
         107 . The method of  claim 93 , wherein said stem cells comprise embryonic stem cells, adult stem cells, or induced pluripotent stem cells. 
     
     
         108 . The method of  claim 93 , wherein said stromal cells comprise stem cells. 
     
     
         109 . The method of  claim 108 , wherein said stem cells are mesenchymal stem cells. 
     
     
         110 . The method of  claim 108 , wherein said stromal cells comprise induced pluripotent stem cells (IPSCs), derivatives of IPSCs, or human embryonic stem cells.