Genetically engineered T cells are at the vanguard of an emerging wave of scientific breakthrough focused on harnessing the power of the immune system to treat cancer and other immune disorders. Perhaps the most dramatic clinical outcome to date has been demonstrated in clinical trials evaluating autologous chimeric antigen receptor (CAR) therapy for the treatment of refractory B cell ALL where complete responses in the majority of the patients have been reported. While adoptive CAR-T cell treatments hold great promise, pressing challenges remain to ensure multi-parameter genetically engineered T-cell immunotherapies can be successfully derived, cost-effectively and consistently manufactured, and safely and reliably delivered at the scale necessary to support wide patient base commercialization.Human induced pluripotent stem cell (hiPSC)-derived T cells uniquely represent a practical and renewable supply of well-defined engineered CAR-T cells for various therapeutic applications. We have previously described a novel platform to facilitate multi-gene locus-specific engineering of hiPSCs at the single cell level to establish highly characterized master cell banks which can then be repeatedly applied to our stage-specific lymphocyte directed differentiation process to reproducibly and reliably generate engineered cytotoxic T cells.Here we present the first set of pre-clinical data for FT819, a first-of-kind off-the-shelf hiPSC-derived CAR-T cell product. To generate FT819, we successfully combined reprogramming of peripheral blood derived T cells with targeted insertion of a CD19 CAR into the T cell receptor α (TRAC) locus under the transcriptional control of its endogenous regulatory elements to generate a single cell-derived clonal TRAC-targeted CAR expressing master hiPSC line. The clone was characterized to be pluripotent (>95% SSEA4 / TRA181) and consisted of bi-allelic disruption of TRAC locus. During the stage-specific differentiation, the hiPSC line faithfully converted into CD34 positive cells which were then differentiated towards CD8 positive cells with uniform CAR expression (95 +/- 5%) in the absence of TCR expression, eliminating the likelihood of GvHD. In vitro functional studies demonstrated that FT819 elicits an efficient cytotoxic T lymphocyte response to CD19 antigen challenge with production of effector cytokines (IFNg, TNFa, IL2), degranulation (CD107a/b, Perforin, Granzyme B), proliferation (>85% entry into cell cycle) and upregulation of activation markers CD69 and CD25. Importantly, FT819 targets tumor in an antigen specific manner as demonstrated by lysis of CD19+, but not CD19-, Raji and Nalm6 tumor cell lines. In summary, FT819 holds the promise of a safe and efficacious off-the-shelf cytotoxic CAR-T cell product derived in a renewable and highly reproducible process analogous to biopharmaceutical drug products.

Citation Format: Raedun L. Clarke, Sjoukje van der Stegen, Tom Lee, Jorge Mansilla-Soto, Chia-wei Chang, Jeffrey Sasaki, Mushtaq Husain, Eigen Peralta, Ian Hardy, Edwin Ortiz, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. Generation of off-the-shelf TCR-less CAR-targeted cytotoxic T cells from renewable pluripotent cells for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-108.