Inosine induces stemness features in CAR-T cells and enhances potency

Dorota D Klysz; Carley Fowler; Meena Malipatlolla; Lucille Stuani; Katherine A Freitas; Yiyun Chen; Stefanie Meier; Bence Daniel; Katalin Sandor; Peng Xu; Jing Huang; Louai Labanieh; Vimal Keerthi; Amaury Leruste; Malek Bashti; Janette Mata-Alcazar; Nikolaos Gkitsas; Justin A Guerrero; Chris Fisher; Sunny Patel; Kyle Asano; Shabnum Patel; Kara L Davis; Ansuman T Satpathy; Steven A Feldman; Elena Sotillo; Crystal L Mackall

doi:10.1016/j.ccell.2024.01.002

Inosine induces stemness features in CAR-T cells and enhances potency

Cancer Cell. 2024 Feb 12;42(2):266-282.e8. doi: 10.1016/j.ccell.2024.01.002. Epub 2024 Jan 25.

Authors

Dorota D Klysz¹, Carley Fowler¹, Meena Malipatlolla¹, Lucille Stuani², Katherine A Freitas¹, Yiyun Chen¹, Stefanie Meier³, Bence Daniel⁴, Katalin Sandor⁵, Peng Xu¹, Jing Huang¹, Louai Labanieh¹, Vimal Keerthi¹, Amaury Leruste¹, Malek Bashti¹, Janette Mata-Alcazar¹, Nikolaos Gkitsas¹, Justin A Guerrero¹, Chris Fisher¹, Sunny Patel¹, Kyle Asano¹, Shabnum Patel¹, Kara L Davis⁶, Ansuman T Satpathy⁷, Steven A Feldman¹, Elena Sotillo¹, Crystal L Mackall⁸

Affiliations

¹ Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA.
² Department of Pediatrics, Division of Pediatric Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
³ Parker 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.
⁴ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA.
⁵ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
⁶ Center 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.
⁷ Center 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.
⁸ Center 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.

PMID: 38278150
PMCID: PMC10923096 (available on 2025-02-12)
DOI: 10.1016/j.ccell.2024.01.002

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.

Inosine induces stemness features in CAR-T cells and enhances potency

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