769 CAR T cells undergoing epigenetic reprogramming by low-dose decitabine enhances persistent anti-tumor efficacy in vivo

Background Anti-CD19-directed chimeric antigen receptor (CAR) T-cell therapy has had a resounding effect on the treatment of B-ALL. However, CAR T cells have been less effective against B-cell non-Hodgkin lymphoma (B-NHL), in part because they become a exhausted state triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function.1-4 It has recently been demonstrated that de novo DNA methylation promoted T-cell exhaustion, whereas methylation inhibition enhanced ICB-mediated T-cell rejuvenation in vivo.5 6 FDA-approved DNA demethylating agents, such as decitabine (DAC), may provide a means to modify exhaustion-associated DNA methylation programs that restrict ICB-responsiveness. Methods We treated CAR (CAR-CD19-expressing) T cells with low-dose DAC (dCAR T cells), to determine its effects on antitumor activities, exhaustion- and memory-associate cell phenotype change, cell cytokine production, and cell proliferation. Its impact on antitumor activities was evaluated in vitro functional assays and mouse in vivo studies. We also conducted western blot, flow cytometry, methylation analysis, RNA in situ hybridization and high throughput RNA sequencing to determine the underlying mechanisms of dCAR T cell function. Results The low-dose, short-term DAC treatment in vitro enhanced the central memory (Tcm) population and the ration of CD4/CD8, and induced degradation of DNMT3a.CAR T cell treated by DAC developing into less-differention status by enhancing memory. dCAR T cells exhibit enhanced antitumour reactivity and the maintenance of a memory-like phenotype at low effector:target ratios. Especially shown by the ‘stress test’, the dCAR T cells at very low doses could efficiently control tumours with a very large burden, and have effective recall responses upon tumour re-challenge in vivo. Importantly, the dCAR T cells maintained a higher proportion of cells with a memory phenotype than did the CAR T cells under long-term tumour stimulation. Transcription of gene sets involved in memory maintenance, proliferation, cytokine production and anti-inhibitor processes was triggered by antigen-expressing target cells upon DAC exposure before antigen stimulation. dCAR T cells avoided the exhaustion programme induced during tumour cell stimulation; they did not upregulate the expression of genes encoding inhibitory receptors and retained relatively high expression of memory related transcription factors and genes. Conclusions CAR T cells underwent DNA reprogramming after DAC treatment, which induced significant sustained cell expansion, cytotoxicity, and cytokine production and reduced exhaustion after antigen exposure. Acknowledgements We thank Professor Lin Xin of Tsinghua University and Professor Mingzhou Guo of Chinese PLA General Hospital for support of data analysis. References Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 2015;15:486–499. doi:10.1038/nri3862 Wherry EJ, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 2007;27:670–684. doi:10.1016/j.immuni.2007.09.006 Schietinger A, et al. Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis. Immunity 2016;45:389–401. doi:10.1016/j.immuni.2016.07.011 Schietinger A, Greenberg PD. Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Trends Immunol 2014;35:51–60. doi:10.1016/j.it.2013.10.001 Ghoneim HE, et al. De novo epigenetic programs inhibit PD-1 blockade-mediated T cell rejuvenation. Cell 2017;170:142–157.e119. doi:10.1016/j.cell.2017.06.007 Pauken KE, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354:1160–1165. doi:10.1126/science.aaf2807