Zhou and Cao et al. fused the cytoplasmic tail of CTLA-4 (CCT) with CAR T cells to fine-tune CAR dynamics and improve efficacy. CAR-CCT cells showed reduced surface CAR expression through ongoing endocytosis, recycling, and degradation, leading to reduced trogocytosis of target ligands and tonic signaling at baseline and preserved responsiveness to repeated stimulations. CCT fusion improved the survival and proliferation of CAR T cells and impaired apoptosis associated with trogocytosis. CARs with monomeric or duplex CCTs showed increased persistence and TCM differentiation and superior antitumor efficacy in a relapsed leukemia model.
Contributed by Shishir Pant
ABSTRACT: Chimeric antigen receptor (CAR)-T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail, we reprogram CAR function and substantially enhance CAR-T efficacy in vivo. CAR-T cells with monomeric, duplex or triplex CTLA-4 cytoplasmic tails (CCTs) fused to the C terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of proinflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior antitumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.
Author Info: (1) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biolog
Author Info: (1) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (2) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (3) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (4) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. MD-PhD Program, Yale University, New Haven, CT, USA. Department of Immunobiology, Yale University, New Haven, CT, USA. (5) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. (6) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Yale College, New Haven, CT, USA. (7) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (8) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. MD-PhD Program, Yale University, New Haven, CT, USA. Department of Immunobiology, Yale University, New Haven, CT, USA. Immunobiology Program, Yale University, New Haven, CT, USA. (9) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. (10) System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Department of Biomedical Engineering, Yale University, New Haven, CT, USA. (11) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (12) System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Department of Biomedical Engineering, Yale University, New Haven, CT, USA. (13) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu. System Biology Institute, Yale University, West Haven, CT, USA. sidi.chen@yale.edu. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. sidi.chen@yale.edu. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. sidi.chen@yale.edu. MD-PhD Program, Yale University, New Haven, CT, USA. sidi.chen@yale.edu. Immunobiology Program, Yale University, New Haven, CT, USA. sidi.chen@yale.edu. Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu. Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu. Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
