Background Clinical outcomes in CAR T therapy correlate with engraftment, expansion, and persistence of CAR T cells. In order to facilitate engraftment and expansion, a lymphodepleting regimen consisting of cyclophosphamide and fludarabine precedes CAR T infusion. This creates niches for infused CAR T cells and stimulates beneficial homeostatic cytokine production. As these compounds are also toxic to CAR T cells, administering the proper doses of both the conditioning drugs and the cell therapies with appropriate timing can be a challenge.
Methods To protect CAR T cells from fludarabine toxicity, we have knocked down deoxycytidine kinase (dCK), which converts fludarabine from the prodrug form to an active compound. This was accomplished using an RNAi sequence featuring a dCK-specific shRNA sequence embedded into a micro-RNA backbone. The resulting RNAi sequence demonstrated the potency of shRNA and the stability of a microRNA. Using Precision BioSciences’ ARCUS® gene editing technology and AAV-mediated targeted transgene insertion strategy, we disrupted the endogenous T cell receptor and inserted a transgene encoding a CD19-specific CAR and a dCK-specific RNAi sequence. Cells produced in this manner were exposed to CD19+ target cells in vitro and in immune-deficient mice and CAR T proliferation and target killing were monitored in the presence and absence of fludarabine.
Results We observed that the inclusion of the RNAi feature resulted in 70% reduction in dCK mRNA abundance, and conferred resistance to fludarabine in vitro. Moreover, treatment of tumor-bearing mice with fludarabine and dCK knockdown CAR T cells resulted in enhanced tumor clearance and survival compared to mice receiving CAR T cells alone or fludarabine plus dCK replete CAR T cells.
Conclusions CAR T cells expressing a dCK-specific RNAi gene exhibited resistance to fludarabine in vitro and in vivo. This drug resistance feature may enable allogeneic CAR-T cells to be simultaneously administered with fludarabine, suppressing rejection of CAR T and improving CAR T engraftment and expansion. This synergy between conditioning and CAR T therapy may improve clinical outcomes by enhancing effector persistence and tumor clearing.
Acknowledgements I would like to thank Aaron J. Martin, PhD and Daniel T. Macleod, PhD for their excellent mentorship and the Precision Biosciences Vivarium team for their support during this study.
Macleod DT, et al. Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells. Mol Ther 2017; 25(4):949–961.
Fellmann C, et al. An Optimized microRNA Backbone for Effective Single-Copy RNAi. Cell Reports 2013;5:1704–1713.
Ethics Approval The animal study conducted was approved by the Institutional Animal Care and Use Committee (IACUC) of Mispro Biotech.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.