Abstract 1158: Inosine endows CAR T cells with features of increased stemness and anti-tumor potency
Authors: Klysz Dorota , Freitas Katie , Malipatlolla Meena , Maier Stefanie , Fowler Carley , Labanieh Louai , Stuani Lucille , Daniel Bence , Sandor Katalin , Xu Peng , Huang Jing , Leruste Amaury , Keerthi Vimal , Gkitsas Nikolaos , Patel Shabnum , Arredondo-Guerrero Justin , Fisher Chris , Asano Kyle , Patel Sunny , Satpathy Ansuman , Davis Kara , Sotillo Elena , Feldman Steven , Mackall Crystal 
Published: April 2023

Abstract
Chimeric antigen receptors (CARs) are synthetic receptors that link an extracellular tumor specific domain to intracellular signaling domains. Despite of remarkable results against refractory B cell malignancies, intense effort is underway to augment the potency of CAR T cells in order to overcome the suppressive tumor microenvironment, which is associated with T cell exhaustion. Adenosine is a major mediator of immune suppression. Ectoenzyme CD39 plays a central role in the generation of adenosine by catalyzing the metabolism of ATP into ADP/AMP. Then CD73 subsequently metabolizes ADP/AMP into adenosine which mediates immune suppression through adenosine associated receptor signaling. CD39 is also expressed by exhausted CD8+ and tumor reactive T cells within the tumor microenvironment, where it is associated with tumor progression, but it remains unclear whether exhausted and/or tumor reactive CD39+CD8+ T cells mediate immune suppression via the purigenic pathway. We developed a high affinity version of the disialoganglioside (GD2)-targeting chimeric antigen receptor (HA-GD2) that spontaneously clusters on the surface of human T cells in the absence of antigen and mimics chronic antigen exposure leading to T cell exhaustion. Using this model, we demonstrate that exhausted CD39+CD8+ CAR T cells actively produce adenosine and mediate immune suppression. In an attempt to generate adenosine resistance and enhance the function of exhausted CAR T cells, we knocked out CD39, CD73, or A2aR (adenosine A2a receptor) but observed only modest changes in phenotype and transcriptomics. In contrast, overexpression of transmembrane-bound adenosine deaminase, which metabolizes adenosine to inosine, induced higher frequency of stem- and central- like memory T cells, and a simultaneous decrease of exhausted T cell subpopulations. Direct exposure of HA-GD2 CAR T cells to high inosine concentration during cell manufacturing process, lead to a higher frequency of central-like memory cells and significant fitness enhancement associated with broad changes at the metabolic level. RNAseq and cyTOF analysis indicated decreased glycolytic flux, increased mitochondrial activity driven by glutamine and polyamine metabolism. Further, inosine altered the epigenetic state of HA-GD2 CAR T cells. We observed significant enrichment of IRF and NF-κB transcription factor motifs and motifs associated with memory differentiation in T cells grown in the presence of inosine. Finally, we showed that production of exhausted HA-GD2 and clinical GD2 CAR T cells in inosine-containing culture media enhances their in vivo efficacy, leading to improved survival in GD2+ tumor-bearing mice. In conclusion, we propose introducing inosine during GMP cell manufacturing as a novel strategy to improve CAR T function and subsequent clinical outcomes of CAR T cell therapy.
Citation Format: Dorota Klysz, Katie Freitas, Meena Malipatlolla, Stefanie Maier, Carley Fowler, Louai Labanieh, Lucille Stuani, Bence Daniel, Katalin Sandor, Peng Xu, Jing Huang, Amaury Leruste, Vimal Keerthi, Nikolaos Gkitsas, Shabnum Patel, Justin Arredondo-Guerrero, Chris Fisher, Kyle Asano, Sunny Patel, Ansuman Satpathy, Kara Davis, Elena Sotillo, Steven Feldman, Crystal Mackall. Inosine endows CAR T cells with features of increased stemness and anti-tumor potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1158.