Abstract 3607: A novel adjunctive LAT-activating CAR T (ALA-CART) cell platform demonstrates enhanced antigen sensitivity and eradication of antigen-low leukemia

Abstract

Abstract CAR T cells have demonstrated clinical success in malignancies that are refractory to standard therapies; however, preclinical and clinical studies have demonstrated that CAR T cell efficacy is greatly reduced in settings of low levels of target antigen. This was exemplified in a clinical trial of CD22-directed CAR T cells where despite inducing complete remissions in >70% of patients, post-CAR relapses occurred in the majority of patients, driven by decreased expression of CD22 below the threshold required for adequate CAR T cell activation. To address this limitation of CAR T cells, we investigated the mechanism underlying suboptimal CAR responses to low levels of antigen. Using a SILAC/mass spectrometry approach, we performed global phosphoproteomics to examine CAR T cell signal transduction in response to high- and low-levels of CD22 antigen. Low levels of CD22 antigen resulted in decreased utilization of several canonical T cell signaling pathways in CAR T cells. We hypothesized that LAT, a scaffolding protein integral to the T cell signalosome, was inefficiently utilized in response to low levels of antigen. Indeed, while signaling events proximal to LAT, such as Zap70, were unaffected by CD22 antigen density, phosphorylation of LAT at multiple activating residues decreased in response to stimulation with CD22-low leukemia cells. To overcome the inefficient LAT activation by low levels of antigen, 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). ALA-CART cells expressed both CARs on the surface and demonstrated enhanced phosphorylation of LAT and PLCγ in response to CD22-low leukemia cells. In xenograft models, ALA-CART cells completely eradicated CD22-low leukemia, significantly extending survival of mice in comparison to the standard CD22-BBz CAR which only modestly slowed leukemia progression. Additionally, in mice engrafted with leukemia expressing WT-levels of CD22, we found the persistence of ALA-CART cell was significantly enhanced relative to standard CD22-BBz CAR T cells. Thus, by identifying deficits in CAR signaling, we rationally designed the ALA-CART platform to target antigen-low cells, overcoming a mechanism of resistance to standard CAR therapies with the potential to improve outcomes of patients receiving CAR therapies. Citation Format: Catherine Pham-Danis, Lillie Leach, Christopher Ebmeier, M. Eric Kohler. A novel adjunctive LAT-activating CAR T (ALA-CART) cell platform demonstrates enhanced antigen sensitivity and eradication of antigen-low leukemia [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 3607.