Klysz et al. showed that CD39+ exhausted CAR T cells coexpressed CD73, which generates adenosine (Ado) and mediates immunosuppression through A2aR. However, CD39, CD73, or A2aR knockout induced modest changes in exhausted CAR T cells, whereas overexpression of adenosine deaminase, which metabolizes Ado to inosine (INO), significantly altered the transcriptome toward stemness and enhanced the CAR T cells’ fitness. INO also reprogrammed the metabolome and the epigenome toward greater stemness. Clinical scale manufacturing using INO-supplemented media generated CAR T cells with enhanced potency, meeting the criteria for clinical dosing.
Contributed by Shishir Pant
ABSTRACT: Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.
Author Info: 1Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA.
2Department of Pediatrics, Division of Pediatric Hematology,
Author Info: 1Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA.
2Department of Pediatrics, Division of Pediatric Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
3Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
4Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA.
5Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
6Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA; Department of Pediatrics, Division of Pediatric Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
7Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
8Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA; Department of Pediatrics, Division of Pediatric Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA; Department of Medicine, Division of Bone Marrow Transplantation and Cell Therapy, Stanford University School of Medicine, Stanford, CA, USA. Electronic address: cmackall@stanford.edu.
