PS1210 DEVELOPMENT OF A NEXT GENERATION ALLOGENEIC CAR‐T CELL PLATFORM WITHOUT GENE EDITING

Background: Autologous CAR‐T cell therapy is now a proven breakthrough technology in the treatment of B cell malignancies and holds promise in the therapy of all types of cancer. However, autologous CAR‐T cell therapy does have significant challenges relating to logistics and product consistency. Allogeneic CAR‐T therapeutic products could allow treatment of multiple patients with cells from the same healthy donor, increasing product consistency, likely reducing manufacturing costs and avoiding the time delay required to generate an autologous T cell product. Aims: Allogeneic CAR‐T cell therapy is dependent upon eliminating the activity of the endogenous T Cell Receptor (TCR) within the engineered T cell, thereby preventing the induction of a graft versus host response and resultant potentially life‐threatening toxicity. We have explored RNA interference to modulate the TCR and to assess the potential of short hairpin RNA (shRNA) as a platform technology for Allogeneic CAR T cell therapy. Methods: shRNAs targeting the CD3ε and CD3ζ chains of the TCR complex were expressed in primary T cells where the efficacy of the individual shRNAs to downregulate the CD3 component and subsequent impact upon TCR expression assessed. The optimal shRNAs were tested in a head to head fashion against the state‐of‐the‐art gene editing technology, CRISPR‐Cas9, in vitro for response against mitogenic stimulation and in animal models for control of xenoGvHD. Results: shRNAs specific for CD3ε and CD3ζ reduced the transcriptional expression of their relevant targets, as well as the cell surface expression of the TCR. Downregulation of TCR expression and inhibition of the in vitro mitogenic response when challenged with anti‐CD3 antibodies was similar between shRNAs and CRISP‐Cas9 targeting CD3ζ. Importantly, in vivo experiments with T cells adoptively transferred to NSG mice showed that shRNAs targeting CD3ζ protected animals from GvHD, while T cell persistence was significantly higher compared to T cells genetically engineered with CRISP‐Cas9 targeting CD3ζ. Summary/Conclusion: This work demonstrates that the expression of shRNA in a standard retroviral vector provides a one‐step solution to produce cells suitable for Allogeneic CAR T cell therapy. Our testing continues, involving the examination of the anti‐tumor potency of such shRNA‐Allo CAR‐T cells. Importantly, this approach provides a one vector solution that offers an alternative to strategies such as gene editing to eliminate the TCR, while also requiring no major alteration to current CAR‐T cell manufacturing, that should enable the rapid adoption of the approach into clinical testing.