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Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable results in the treatment of hematological malignancies. However, several challenges remain, including achieving predictable efficacy and safety profiles and extending this approach to solid tumors. As such, the development of approaches that generate engineered T cells with superior persistence and efficacy are needed. Currently, the generation of CAR-T requires that they be activated and expanded ex vivo, generally via beads coated with anti-CD3/anti-CD28 mAbs. T cell activation provides three fundamental signals; (i) primary triggering of TCR signals; (ii) secondary co-stimulatory signals and, (iii) synthesis of cytokines to direct T cell expansion and differentiation. We hypothesize that a CAR-T response can be modulated by tuning the: (i) pattern, (ii) magnitude and, (iii) duration of the input T cell activation signals. To this end, we used a novel, soluble, tunable activation platform based on DNA hybridization - utilizing concatenated single stranded DNA (ssDNA) polymers to cluster complementary oligonucleotide-modified antibodies targeted at T-cell surface receptors. This DNA-Based-T-cell-Activation (DBTA) platform was compared with conventional bead-bound CD3/CD28 antibodies (Dynabead) in the generation and function of T cells engineered to express a CD19-targeted CAR harboring a 4-1BB co-stimulatory domain. The two methods did not result in any significant differences in CAR-T expansion nor in memory/activation phenotypes. Notably, though, these cells exhibited higher cytokine secretion in response to CAR stimulation, and in vitro killing of DBTA-generated CART was significantly higher than that of bead-activated CART. Indeed, only the former showed cytotoxicity against Nalm6 leukemic cells expressing low levels of the CD19 target antigen CART. Furthermore, in an in vivo NSG model with Nalm6 leukemia, the adoptive transfer of low numbers of DBTA-generated CART (2e6) resulted in leukemia eradication and delayed leukemia relapse. Mechanistically, the enhanced function of DBTA-generated CAR was associated with increased metabolic activity in the pre-infusion product. These results suggest that calibrating the activation potential of CAR-T cells can result in the generation of a product with enhanced therapeutic potential. Citation Format: Mehdi Benzaoui, Sooraj R. Achar, Vandana Keskar, Anup Sood, Brian M. Davis, Steven L. Highfill, Grégoire Altan-Bonnet, Christopher D. Chien, Naomi Taylor. Novel DNA-based-T-cell-activation for the generation of chimeric antigen receptor T cells with enhanced anti-leukemia cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB103.