3D-organoid culture supports differentiation of human CAR + iPSCs into highly functional CAR T cells.

Unlimited generation of chimeric antigen receptor (CAR) T cells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR T cell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ T cell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L ⁺ naive and memory T cells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR T cell characteristics. Expanded iPSC CD19-CAR T cells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR T cells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR T cells also mediated potent antitumor activity in vivo, prolonging survival of mice with CD19 ⁺ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR T cells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.
Competing Interests: Declaration of interests C.E.B., S.J.F., and Z.W. are listed on patent(s) relating to this work: Forman, S.J., Brown, C.E., and Wang, Z. Generation of chimeric antigen receptor modified T cells from stem cells and therapeutic uses thereof. U.S. application n. 62/931125. PCT application no. PCT/US2020/059216. A.M.H., C.S.S., and G.M.C. are co-founders of Pluto Immunotherapeutics, which holds certain rights over intellectual property relating to the ATO system.
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