Engineering bZIP family transcription factors for therapeutic T-cell persistence and effector function

PROJECT SUMMARY Cancer immunotherapy using T cells engineered with a chimeric antigen receptor or T-cell receptor directed against cancer antigens is a promising new therapeutic modality for treating many types of cancer. However, lack of efficacy due to T-cell dysfunction, particularly in the solid tumor setting, has stymied progress. Multiple stressors exist within tumors that drive T cells to a dysfunctional state, reducing their ability to clear tumors. Genes have been identified that improve T-cell resilience to different tumor stressors. However, these genetic modifications that improve T-cell exhaustion resistance often act via different mechanisms, creating uncertainty regarding the relative benefit of different approaches or how to combine them for maximum efficacy. Altering transcription factor activity is a particularly robust way to favor a functional therapeutic cell state. The field has identified transcription factors that can be overexpressed to promote retention of T-cell effector activity or a more persistent, stem-like state. Additional transcription factors have been identified that promote dysfunction downstream of chronic antigen signaling or in response to metabolic conditions in the tumor microenvironment and can be knocked out to favor retention of T-cell activity. Both approaches have yielded improvements in tumor control in preclinical models, but the use of different models and conflicting results leaves uncertainty about which modifications are most beneficial and their mechanism. We intend to use protein engineering to create protein-based inhibitors that bind and prevent the function of transcription factors when overexpressed in T cells. This will allow the creation of overexpression libraries with members that can both decrease and increase transcription factor activity to facilitate the comparison of genetic modifications in T cells. The goals of this Steven I. Katz proposal are to: 1) Understand the molecular mechanism of the transcription factors complexes regulating T-cell exhaustion, 2) Increase resistance to T-cell dysfunction through systematic multiplexing of transcription factor perturbations, and 3) Determine the relative benefit of transcription factor perturbations that counter different sources of T-cell dysfunction in the in vivo tumor microenvironment. As specified by the funding mechanism, this proposal sets forth an important new research direction for our lab in the areas of T-cell dysfunction and engineering of endogenous transcription factors and is well-based in published literature and supported by subject matter experts and collaboration. Through screening of pooled libraries and parallel characterization of a matrix of multiplexed engineered transcription factors using in vitro and in vivo models of tumor-induced T-cell dysfunction, this proposal will address the important question of which transcription factor perturbations are most effective in promoting resistance to T-cell dysfunction. Using cutting- edge single-cell RNA-seq mapped to transcription factor perturbations, we will determine the effect of these modifications on T cells in murine tumors. This grant will yield new transcription factor engineering solutions to improve therapeutic cells, generating biological insight and new research paths in cell therapy engineering.