Abstract 3141: Restricting aerobic glycolysis provides functional improvement of chimeric antigen receptor (CAR)-modified T cells

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has proven effective at treating hematologic malignancies, however similar success has not been observed using CARs targeting other antigens. We have observed that several CARs, including a GD2-targeted CAR, are limited in efficacy due to T cell exhaustion induced by tonic, antigen-independent signaling. This phenomenon is triggered by oligomerization of scFvs on the surface of transduced T cells, leading to chronic CAR zeta signaling and early exhaustion characterized by a diminished proliferative potential, poor cytokine production, and overexpression of inhibitory receptors including PD1, TIM3 and LAG3. Early CAR T cell exhaustion is also associated with an altered cellular metabolism that is more highly dependent on glycolysis compared to CD19 CAR T cells or untransduced, activated control cells. Using a Seahorse Extracellular Flux Analyzer, we found that the rate of glycolysis (ECAR) in GD2 CAR T cells was twice that of CD19 CAR T cells or untransduced controls, and no deficiencies in oxidative phosphorylation in GD2 CAR T cells were observed (normal OCR and spare respiratory capacity). To evaluate whether increased glycolytic metabolism contributes to the development of CAR T cell exhaustion, we sought to restrict glycolysis by culturing GD2 CAR T cells with the hexokinase inhibitor 2-deoxyglucose (2DG) or in media supplemented with galactose instead of glucose. Restricting CAR T cell glycolysis was associated with decreased TIM3 and LAG3 expression and enhanced cytokine production of GD2 CAR T cells but a modest decrease in cytokine production of CD19 CAR T cells. Given the previously reported role of mTOR on promoting glycolytic metabolism, we used rapamycin (sirolimus) in the cell culture during transduction and expansion. Culturing GD2 CAR T cells with rapamycin similarly resulted in slower acidification of cell culture medium and diminished expression of TIM3 and LAG3. Rapamycin treated GD2 CAR T cells retain specific lytic ability of GD2-positive tumor cells compared to non-treated GD2 CARs as measured by chromium release, and these cells persist longer than non-treated cells in a murine xenograft model of GD2-positive 143b osteosarcoma. These results suggest that modulation of T cell metabolism may provide functional benefit and enhance antitumor activity of adoptively transferred tumor antigen specific T cells by diminishing the susceptibility of highly activated T cells to exhaustion. Citation Format: Jillian P. Smith, Adrienne H. Long, Crystal L. Mackall. Restricting aerobic glycolysis provides functional improvement of chimeric antigen receptor (CAR)-modified T cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3141. doi:10.1158/1538-7445.AM2015-3141