Metabolic conditioning for enhanced CAR T cell function

Abstract

Abstract Introduction Durable benefits in adoptive immunotherapies are often linked to metabolic reprogramming, differentiation states, and epigenetic remodeling. Previously, we showed that CAR design has a profound impact on T cell metabolism. Implicit in these earlier discoveries is that T cell metabolism is not fixed and can be dynamically modified to suit the target environment. Despite these advances, genetic targets and/or conditioning strategies to overcome metabolic conditions encountered in hostile environments are underdefined. Here, we developed an arginine conditioning strategy to enhance human CAR T cell metabolic fitness and sustain tumor control in several xenograft models of cancer. Methods To provide mechanistic insights into the value of arginine for CAR T cells, we expanded primary human CAR T cells in medium spiked with 5X arginine for 7 days. We tested anti-tumor function and persistence in our clinically validated Nalm6-xenograft model of leukemia. We examined differentiation status by flow cytometry as well as ATAC seq analyses. To understand how arginine conferred long-lasting efficacy, we screened histone modifications by ultra-high resolution mass spectrometry. We combined this with genomic metabolic modeling and a multimodal analyses including metabolomics, RNA seq, ATAC seq and machine-learning. We then validated the unique impact of arginine conditioning on methionine, glutamine, and branched chain amino acid metabolism via 13C tracer technology. Finally, we performed targeted LC-MS to examine the abundance of arginine and its related metabolites in responder vs nonresponder CAR T cell patients. Results We report that arginine promotes central memory differentiated T cells with enhanced mitochondrial volume. Arginine induces a strong epigenetic signature distinguished by methylation as well as acetylation of several histone proteins. S-adenosyl methionine (SAM) sits at the intersection of histone modification and polyamine biosynthesis. In novel insights, we propose that the fate of SAM is nutritionally-regulated via an arginine-mediated upregulation of ASS1. After its metabolism to ornithine, arginine can support polyamine synthesis (SAM dependent) or be metabolized by ASS1. As ASS1 is an ornithine-consuming enzyme, it deploys ornithine in the arginine-citrulline cycle. We propose that this intrinsic buffering role of ASS1 limits the commitment of ornithine to polyamine biosynthesis; sparing SAM for transmethylation. Importantly, this novel concept has never been described previously. Disproportionate increases in ornithine relative to SAM activate SAM production in the folate and methionine cycle. Modeling insights, RNA seq and ATAC-seq analyses highlight an important role for one carbon metabolism including induction of the synergistic gene pair MTHFD1L and SHMT2, as well as methionine synthase and the twin axis of methyl transferase enzymes MAT2A as well as MAT2B in the methionine cycle. Using a 13C metabolic tracer, we verify that arginine conditioning significantly enhances the replenishment of SAM via methionine. We show that the induction of cystathionine beta synthase (CBS) is a hallmark of arginine-treatment. We propose that CBS provides a relief valve for homocysteine, which facilitates high methionine cycle activity and SAM production. PRMTs are a family of proteins that catalyze the transfer of methyl groups to proteins including histones. We provide pharmacologic evidence that the benefits of arginine conditioning are regulated in part by PRMT3. We also identified that several arginine-related metabolites were elevated in the serum metabolome of “responder” patients CAR T cell treatment against lymphoma. Conclusions This study provides mechanistic insights into how arginine supports T cell anti-tumor function. Anchored to their epigenetic footprint, arginine-treated CAR T cells form progeny with enhanced mitochondrial fitness, lasting anti-tumor function in several models, and central memory differentiation in vivo. Our findings have immediate translational relevance as clinical outcomes depend on the cytolytic potency and metabolic fitness of the therapeutic product.