Restoration of LAT signaling through the expression of a novel Adjunctive LAT-Activating CAR (ALA-CART) enhances the antigen-sensitivity and persistence of CAR T cells
Chimeric antigen receptor (CAR) T cell therapy have demonstrated remarkable success in treating B-cell malignancies that are refractory to standard therapies; however, preclinical and clinical studies have demonstrated that CAR T cell efficacy is greatly reduced against antigen-low tumors. This was exemplified in a clinical trial of CD22-directed CAR T cells where post-CAR relapses were driven by a decrease in the surface expression of the CD22 antigen. To address the poor response of CAR T cells to antigen-low tumor cells, we performed global phosphoproteomics using a SILAC/mass spectrometry approach to examine signal transduction events within CAR T cells in response to high- and low-levels of CD22 antigen. Stimulation with low levels of CD22 antigen resulted in decreased activation of several canonical T cell signaling pathways in CAR T cells, suggesting poor utilization of LAT. To overcome this inefficiency of LAT activation, 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 demonstrated enhanced phosphorylation of LAT and restored downstream signaling in response to low levels of CD22. In xenograft models, ALA-CART cells completely eradicated CD22-low leukemia, significantly extending survival of mice in comparison to the clinically-active, standard CD22-BBz CAR T cells. Compared to the standard CD22-BBz CAR T cells, ALA-CART cells exhibited transcriptional differences 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. Thus, through the identification of CAR signaling deficits, we rationally designed the ALA-CART platform which improves the sensitivity and persistence of CAR T cells to target antigen-low cells, overcoming a mechanism of resistance to standard CAR therapies. Overall design: To investigate transcriptional differences between the clinically active CD22-BBz CAR and the ALA-CART, we isolated CD4+ (by negative magnetic bead separation) and CD8+ CAR-T cells (by positive magnetic bead separation) of purified CD22-BBz and 22ALA-CART cells. CAR T cells were added to wells of 6-well tissue culture plates that were either uncoated (unstimulated CAR T cells) or wells coated with 3 ug/mL 22Fc-antigen (stimulated CAR T cells). CAR T cells were incubated at 37C for 30 mins and lysed using buffer RLT followed by extraction using RNeasy kit (Qiagen). Comparative gene expression analysis was performed on CD4 and CD8 CAR T cells with and without stimulation.