(PA-034) One-time ex-vivo Conditioning of CAR-T Cells with a Novel Metabolic Modulator

Metabolism is altered during tumor progression in both tumor cells and immune cells, including T cells, contributing to immunosuppression in the tumor microenvironment. These metabolic changes highlight the importance of targeting metabolic enzymes to enhance the efficacies of tumor immunotherapy. We previously identified that carnitine palmitoyltransferase 1A (CPT1A), which is known as a rate limiting enzyme in fatty acid oxidation (FAO), has a moonlight enzymatic activity as lysing succinyltransferase (KSTase). Our studies in cancer metabolism reveal that CPT1A can directly succinylate fatty acid synthase (FASN) to maintain its stability in cells, thereby promoting de novo lipogenesis (DNL). Based on these findings, we first developed a novel small molecule inhibitor against the KSTase activity of CPT1A (KSTi) that inhibits FASN succinylation in-vitro. Subsequently, we developed a prodrug of KSTi to allow for improved targeted drug delivery that also decreases FASN succinylation and expression leading to reduced intracellular levels of palmitate (PA) without affecting FAO.


Methods:

Since DNL and saturated fatty acid accumulation have been reported to impair T cell function while FAO supports the longevity of T cells, we hypothesized if KSTi prodrug that decreases FASN while sparing FAO would affect any CART cell function. To test this hypothesis, we conditioned anti-BCMA CAR-T cells with KSTi prodrug on the last day of manufacturing and performed in vitro cytotoxicity analysis of these ex-vivo conditioned anti-BCMA CAR-T cells against myeloma cells. We also measured their cytokine production and metabolite levels using flow cytometry and mass spectrometry, respectively. Finally, we evaluated their in vivo efficacy in myeloma cell line xenograft models in which 2.5 x 10^5 myeloma OPM2 cells were intravenously injected into NSG mice followed by another intravenous injection of 1.5 x 10^5 ex-vivo conditioned anti-BCMA CAR-T cells 21 days after OPM2 cell injection. Myeloma disease progression was assessed by monitoring for signs of paralysis.    


Results:

KSTi prodrug treatment decreases total PA levels in anti-BCMA CAR-T cells while increasing the intracellular levels of TNF-α and IFN-γ and more importantly, one-time ex-vivo conditioning of CART cells with KSTi prodrug significantly increases their efficacy and durability against myeloma cells in vitro and in mice. The median survival rate for the group of mice with anti-BCMA CAR-T cells alone was 50 days after CAR-T cell injection while that with ex-vivo conditioned anti-BCMA CAR-T cells had not yet been reached at 80 days.


Conclusions:

These results support our hypothesis that KSTase activity of CPT1A is a novel druggable driver of lipogenesis that can be exploited to improve the efficacy and durability of anti-BCMA CAR-T cells against myeloma.