Remodeling DNA Methylation Landscapes to Prolong CAR T-cell Activity to Overcome Cancer Relapse
Introduction
Although chimeric antigen receptor (CAR) T-cell based immunotherapy has achieved promising outcomes in patients with lymphoma and leukemia over the past five years, antigen escape and gradual reduction of T-cell activity led to cancer relapse in nearly 50% of patients after initial treatment. Recent studies uncovered the critical role of DNA methylation in boosting CAR T activity, hence pointing to the posibility of overcoming cancer relapse through precise control over the DNA methylation regulatory pathways in therapeutic T-cells.
Methods
Novel classes of DNMT enzyme inhibitors and TET protein engineering technologies are used to remodel the DNA methylation landscapes in CAR T-cells. In vitro co-culture of CD19 expressed Raji lymphoma cells and CAR T-cells and in vivo injection of CAR T-cells into tumor bearing mice systems are applied to test the CAR T-cell activities after remodeling their DNA methylation landscapes.
Results
We identified two classes of small molecules which might have inhibitory effects on DNMT1 enzyme. In vitro enzymatic activity assay indicated that several candidate compounds had strong DNMT1 inhibition. The inhibitory effect of these candidates was further tested in a co-culture system composed of CD19-positive Raji lymphoma cells and engineered CAR T-cell activity. To modulate TET activity, we developed more than 15 nanobodies anginst TET2. Immunofluorescence staining and immunoprecipitation experiments showed that two nanobodies showed strong colocalization with TET2, suggesting that they can recognize TET2 efficiently. In parallel, the nanobodies angainst TET2 were fused with various proteasomal degradation systems to modulate intracellular TET2 protein levels and will be used in the following experiments to modulate TET2 activity in CAR T-cells.
Conclusion
We identified candidates that can specifically inhibit DNMT1 activity and boost CAR T-cell activity in vitro and produced TET2-specific nanobodies, the latter of which can be used to modulate TET2 activity. The CAR T-cell modulating activities of both DNMT1 inhibitors and TET2 nanobodies will be tested in mouse models of CD19-positive tumor, thereby the preclinical rationale of manipulating the DNA methylation landscapes to benefit CAR T-cell therapy.