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Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment, demonstrating clinical success in B-cell malignancies that are refractory to standard therapies. Relapses after CAR therapy has demonstrated the limitations of current CAR T cell therapies to target antigen-low tumors and induce consistent persistence. Furthermore, the success of CAR T cell therapy in B-lineage malignancies has not yet been translated to acute myeloid leukemia (AML). The targeting of myeloid-restricted antigens, such as CD33, with standard 2nd generation CAR T cells has not yielded similar clinical responses as ALL. Therefore, we hypothesize that novel CAR T cell strategies will be required to improve responses in non-B cell malignancies. We identified that CAR T cells inefficiently activate the scaffolding protein LAT in response to low levels of antigen, leading to diminished efficacy. We designed a bicistronic construct consisting of a clinically active 2nd generation CD22-BBz CAR along with a novel Adjunctive LAT Activating CAR incorporating the intracellular signaling domain of LAT (ALA-CART) to overcome this deficiency of signaling in CAR T cells. In xenograft models, 22ALA-CART cells completely eradicated CD22-low leukemia, which is not responsive to standard CD22-BBz CAR T cells. We have extended these findings to CD19-directed ALA-CART cells, which eradicate CD19-low leukemia that is otherwise resistant to standard CD19-BBz CAR T cells. To understand how ALA-CART improves responses to antigen-low leukemia, we evaluated the in vivo potency of ALA-CART relative to 2nd generation CARs and found ALA-CART cells could eradicate ALL in xenograft models at lower doses than standard CAR T cells. RNA-Seq analysis demonstrated global differences in ALA-CART cells in the resting state reflecting a less differentiated state, which corresponded to an enrichment of T stem cell memory cells and enhanced in vivo persistence. Stimulation through the ALA-CART receptor led to widespread changes in transcription factor activity in T cells which were distinct from standard CAR activation, particularly inducing significantly lower interferon response factor activity which has been associated with CAR T cell failure in patients. As strategies targeting myeloid lineage-restricted antigens with 2nd generation CAR T cells have not yet produced the clinical success seen with ALL-directed CAR therapy, we tested whether the ALA-CART platform could improve the activity of CD33-directed CAR T cells. CD33 ALA-CART cells demonstrated improved in vivo potency and exhibited enhanced memory potential like our results with ALL-directed ALA-CART. Thus, through the reversal of signaling deficits intrinsic to 2nd generation CARs, the ALA-CART platform improves the potency and persistence of CAR T cell therapy against both lymphoid and myeloid acute leukemias. Citation Format: Catherine Danis, Lillie Leach, Christopher Ebmeier, Amanda Novak, Etienne Danis, M. Eric Kohler. ALA-CART induces global transcriptional changes leading to enhanced potency and persistence of CAR T cells against both lymphoid and myeloid acute leukemias [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3190. |
