394. Generation of a Novel Allogeneic CAR T Cell Platform Utilizing an Engineered Meganuclease and AAV Donor Template to Achieve Efficient Disruption of T Cell Receptor Expression and Simultaneous Homology-Directed Insertion of a CD19 CAR

Chimeric antigen receptor (CAR) T cell therapies have achieved dramatic results treating hematological malignancies by redirecting the specificity of T cells to target CD19 expressing cancer cells. Clinical trials reporting these impressive results have used autologous therapies, which pose significant manufacturing, logistical, and cost issues, complicating the implementation of such therapies on a larger scale. Furthermore, CAR T cells are typically generated by using Lenti- or Retro- viral vectors, resulting in random integration of the CAR gene, heterogeneous expression of the CAR on the cell surface, and the potential for insertional mutagenesis. We have developed a platform to address these issues by using a streamlined process to insert a CAR gene into the T cell receptor (TCR) alpha chain locus of T cells obtained from healthy donors using a highly optimized engineered meganuclease. The resulting gene-edited allogeneic CAR T cells do not express an endogenous TCR and therefore should not be capable of eliciting graft versus host disease upon adoptive transfer. We first produced and validated an engineered meganuclease targeting TCRα, and developed a scalable process for electroporating T cells with meganuclease mRNA that routinely results in greater than 60% knockout of the TCR, as measured by flow cytometry. To test our gene knock-in approach, we treated cells with our meganuclease to induce a double-strand break (DSB) in TCRα. We then transduced these cells using an AAV6 vector containing a donor DNA template consisting of a second generation CD19 CAR driven by an exogenous promoter and flanked by homology arms to the target site in the TCRα sequence. Homology directed repair of the DSB utilizing this donor template results in insertion of the CD19 CAR at this site in the genome. Incorporating this process into a 14 day procedure for generation and expansion of TCR knockout CAR T cells, we successfully generated a large population of TCR knockout cells with >70% of those cells stably expressing the CD19 CAR. Importantly, these CAR T cells proliferated when co-cultured with CD19+ target cells, released cytokines including IFN-γ and IL-2, and exhibited potent cytotoxic activity against CD19+ target cells. Studies to confirm in vivo antitumor activity are currently being conducted. In summary, we describe a novel, highly efficient, and scalable method to produce allogeneic CAR T cells in a streamlined process using an engineered meganuclease in combination with an AAV6 donor template.