No abstract

T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC , components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor–engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12 -deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers—most notably for STAT and AP-1 transcription factors—and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.

are coinventors on a patent application for a CD93-CAR. C.L.M. is a cofounder and consultant for Lyell Immunopharma, and Syncopation Life Sciences, which are developing CAR-based therapies, and serves as an advisor and consultant for Roche, NeoImmune Tech, Immatics, Apricity and Nektar. R.G.M. is a cofounder of Syncopation. E.S. is a consultant for Lyell Immunopharma. R.G.M. is a consultant for Lyell Immunopharma, Gamma Delta Therapeutics, Zai Lab, and Aptorum Group. R.M. is on the Board of Directors of BeyondSpring Inc, on the Scientific Advisory Boards of Kodikaz Therapeutic Solutions Inc., Coherus Biosciences, and Zenshine Pharmaceuticals, and is an inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc. K.R.P., A.T.S., and HYC are cofounders of Cartography Biosciences. A.T.S is a scientific founder of Immunai and receives research funding from Arsenal Biosciences. H.Y.C is a cofounder of Accent Therapeutics and Boundless Bio, and an advisor to 10X Genomics, Arsenal Biosciences, and Spring Discovery. The remaining authors declare no competing financial interests.

Adoptively transferred T cells and agents designed to block the CD47/SIRPα axis are promising antitumor therapeutics, which activate distinct arms of the immune system. We administered anti-CD47 (αCD47) with adoptively transferred T cells with the goal of enhancing antitumor efficacy but observed rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors, which blunted therapeutic benefit. αCD47 mediated CAR T clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered a CD47 variant (47E) that engaged SIRPα and provided a "don't-eat-me" signal that was not blocked by αCD47 antibodies. TCR or CAR T cells expressing 47Ewere resistant to clearance by macrophages following αCD47, and mediated significant, sustained macrophage recruitment into the TME. Although many of the recruited macrophages manifested an M2-like profile, the combined therapy resulted in synergistic enhancement in antitumor efficacy. This work identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T cell directed therapeutics with those designed to activate macrophages. It further delivers a therapeutic approach capable of simultaneously harnessing the antitumor effects of T cells and macrophages that manifests markedly enhanced potency against solid tumors.

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing.