Enhancing the anti-tumor activity of TCR-engineered T cells by modified-CD3zeta fused with TRAF-binding motifs

Abstract

The major therapeutic modality for treating cancer is the adoptive transfer of chimeric antigen receptor (CAR)- or transgenic T cell receptor (TCR)-T cells. The main advantages of the synthetic receptors are that it enables not only the redirection of T cell antigen specificity, but also the rewiring of signaling components to achieve optimal T cell activation. Indeed, the major breakthrough in the clinical success of CAR-T therapy has been the incorporation of a costimulatory domain in the cytoplasmic domain of a second-generation CAR, which led to a dramatic increase in their proliferation and persistence. Unlike CAR-T therapy, however, research on TCR-T therapy has so far focused primarily on identifying antigen-specific TCR repertoires.In this study, I developed second-generation TCR-T cells that simultaneously deliver TCR and co-stimulatory signals upon TCR stimulation. To achieved this, I generated a modified-CD3ζ that is fused with the signaling domain of the 4-1BB. The modified-CD3ζ recruited tumor necrosis factor receptor-associated factor 2 (TRAF2), a key adaptor of 4-1BB signaling, but exhibited diminished TCR signaling, resulting in a suboptimal anti-tumor effect in vivo. Based on the results of mutational studies, I further identified that a basic-rich motif (BRM) located in the 4-1BB signaling domain is responsible for the undesirable outcomes, likely due to the reinforced association between the modified-CD3ζ and the plasma membrane. Thus, I generated a modified-CD3ζ with BRM-truncated 4-1BB, termed zBBΔBRM. This modification was sufficient to recruit TRAF2 while preserving TCR signaling. Consequently, zBBΔBRM-based TCR-T cells demonstrated enhanced persistence with a higher expression of anti-apoptotic molecules upon repeated encounters with target cells. Subsequently, I confirmed significantly enhanced anti-tumor activity of my second-generation TCR-T cells, as compared to that of their first-generation counterparts, in a xenograft mouse model. Collectively, my findings offer a strategy for engineering signaling components of TCR-T cells, and in addition, provide a potential therapeutic approach for treatment of cancer.