Engineering second-generation TCR-T cells by site-specific integration of TRAF-binding motifs into the CD247 locus

BACKGROUND: The incorporation of co-stimulatory signaling domains into second-generation chimeric antigen receptors (CARs) significantly enhances the proliferation and persistence of CAR-T cells in vivo, leading to successful clinical outcomes. METHODS: To achieve such functional enhancement in transgenic T-cell receptor-engineered T-cell (TCR-T) therapy, we designed a second-generation TCR-T cell in which CD3ζ genes modified to contain the intracellular domain (ICD) of the 4-1BB receptor were selectively inserted into the CD247 locus. RESULTS: This modification enabled the simultaneous recruitment of key adaptor molecules for signals 1 and 2 on TCR engagement. However, the addition of full-length 4-1BB ICD unexpectedly impaired the expression and signaling of TCRs, leading to suboptimal antitumor activity of the resulting TCR-T cells in vivo. We found that the basic-rich motif (BRM) in the 4-1BB ICD was responsible for the undesirable outcomes, and that fusion of minimal tumor necrosis factor receptor-associated factor (TRAF)-binding motifs at the C-terminus of CD3ζ (zBB(ΔBRM)) was sufficient to recruit TRAF2, the key adaptor molecule in 4-1BB signaling, while retaining the expression and proximal signaling of the transgenic TCR. Consequently, TCR-T cells expressing zBB(ΔBRM) exhibited improved persistence and expansion in vitro and in vivo, resulting in superior antitumor activity in a mouse xenograft model. CONCLUSIONS: Our findings offer a promising strategy for improving the intracellular signaling of TCR-T cells and their application in treating solid tumors.