Abstract
Chimeric antigen receptor (CAR) T cell mediated trogocytosis (CMT) involves the transfer of the CAR binding antigen to the surface of T cells, leading to CAR T cell fratricide and the emergence of low antigen expressing tumor cells. There have been different strategies employed to mitigate trogocytosis, but none have identified the mechanism underlying the phenomenon. We have previously shown that phosphoinositide 3-kinase (PI3K) is an important pathway that regulates CMT. Knockdown or inhibition of PI3K p110δ in CAR T cells reduces the loss of cognate antigen from the tumor cells. PI3K inhibition suppressed Rac1 activation and disrupted F-actin polymerization, indicating a role of cytoskeletal dynamics in regulation of CART trogocytosis. Building on this, we now study the effects of transient PI3K inhibition on the efficacy of CAR T cells in vitro and in vivo.
We used a selective PI3K p110δ inhibitor, ME401, to study the effects of blocking PI3K activity in CAR T cells. We treated CAR T cells with 1mM of ME401 or DMSO (control) for 20 hours and then co-cultured them with NALM6 cells for 15 minutes and determined the expression level of CD19 by flow cytometry. We found that there was a significant decrease in CD19 expression on NALM6 cells in control (DMSO) treated CAR T cells compared to ME401 treated cells. Reciprocally we saw an increase in CD19 expression on CAR T cells treated with DMSO compared to ME401, suggesting that PI3K inhibition reduces trogocytosis mediated transfer of cognate antigens. Next, we co-cultured NALM6 cells labelled with Cell trace far red (CTFR) with ME401 or DMSO treated CD19 CAR T cells for one hour and determined cytotoxicity using flow cytometry. We observed improved killing of NALM6 cells by ME401 treated CD19 CAR T cells at 5:1 effector: target ratio. Moreover, there was a consistent increase in CAR T cells in the ME401 treated group compared to DMSO treated groups across all E:T ratios tested, with 2.5:1 ratio showing the most pronounced difference. Furthermore, there was significantly reduced levels of IFN-g in cell culture supernatant of ME401 treated CD19 CAR T cells compared to DMSO treated CD19 CAR T cells co-cultured with NALM6 cells. We also found that CD19 CAR T cells treated with ME401 showed evidence of decreased apoptosis as measured by Annexin V staining after co-culturing with NALM6 cells for 4 hours. ME401 treated CD19 CAR T cells also demonstrated reduced expression of exhaustion markers like LAG3, TIM3, TIGIT and PD1 when co-cultured with NALM6 cells. We observed similar improvement in CAR T cell function using BCMA CAR T cells in a myeloma model with OPM2 cell lines.
For in vivo experiments, NRG mice were engrafted with 1x10^6 NALM6 cells expressing firefly luciferase after sublethal radiation with 5 cGy. Two weeks after NALM6 injection, we confirmed that the tumor growth reached 10^10 total flux by IVIS imaging. Then, we proceeded to treat the mice with 6x10^6 of either ME401 or DMSO treated CD19 CAR T cells. Tumor volume was determined by IVIS imaging the day prior and one day after CAR T cell administration. we found that the rate of the tumor growth was significantly reduced in the ME401 treated mice compared to the DMSO treated group. Finally, one day after CD19 CAR T cell injection we harvested and processed the spleens to analyze CD19 CAR T cells numbers and exhaustion markers by flow cytometry. We found a 3-fold relative increase in CD19 CAR T cells numbers in the ME401 treated group compared to the DMSO group. Moreover, the ME401 treated CD19 CAR T cells exhibited a lower PD1 and LAG3 expression compared to the DMSO treated cells.
In summary, we have demonstrated that PI3K is an important mediator of CAR T cell trogocytosis and that treatment of CAR T cells with PI3K p110 δ inhibitor improves cytotoxicity and reduces CAR T cell fratricide. This improvement in fratricide translated into improved CAR T cell persistence in vitro and in vivo and led to a less exhausted phenotype and better tumor control in xenograft model of ALL.
