258 Highly multiplexed allogeneic CAR T-cell engineering using in process DNA-PK inhibition

<h3>Background</h3> Donor-derived allogeneic CAR T-cell therapeutics require several layers of engineering to 1) ensure safety and 2) to improve drug product efficacy. When using gene editing-based engineering, it is critical to maximize the intended product fraction containing all desired edits to boost potency and process yield. DNA-PK plays an instrumental role in repairing DNA double-stranded breaks induced by CRISPR/Cas nucleases as part of the non-homologous end joining (NHEJ) repair pathway. By inhibiting DNA-PK (DNA-PKi), DNA repair is skewed toward homology-directed repair, improving gene knock-in frequency, product homogeneity and yield. <h3>Methods</h3> T-cells were electroporated with up to 4 RNPs and synthetic DNA repair templates for non-viral cells or transduced with up to 2 AAV post RNP electroporation (4 knockout, 2 knock-in). DNA-PKi was added immediately following electroporation for 24 hours followed by dilution of the inhibitor with T-cell media. CAR T-cells were cryopreserved at the end of production, and later thawed and tested in functional assays. <h3>Results</h3> In a multiplex cell product engineering context, DNA-PKi treatment significantly increased the frequency (1.7-fold) and yield (1.3-fold) of AAV engineered CAR T-cells containing all four knockouts and both knock-ins. Treated cells were phenotypically similar to untreated cells, however higher cell-surface CAR expression was observed, as measured by mean fluorescence intensity. Importantly, we found DNA-PKi treatment had no impact on CAR cytoxicity, proliferation, or cytokine production over acute and chronic stimulation with target antigen in vitro. The increase in fully edited T-cell yield was scalable, as a 1.6-fold increase was observed in our at-scale manufacturing process. DNA-PKi also improved non-viral CAR T-cell production, with a 2.5-fold increase in fully edited CAR T-cell frequency and increased CAR T-cell yield. The concentration of DNA-PKi used in CAR T-cell production was below the genotoxic threshold as determined by acute cytotoxicity and micronuclei quantification. <h3>Conclusions</h3> DNA-PKi treatment enables the complex cellular engineering required for donor derived allogeneic CAR T-cell products. In the context of multiplexed gene editing, DNA-PKi treatment significantly increased fully edited CAR T-cell yields and product homogeneity without compromising genomic integrity or in vitro CAR T-cell performance.