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 (zBBΔBRM) 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 zBBΔBRM 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.

CAR T Cell Immunotherapy Beyond Haematological Malignancy.

Chimeric antigen receptor (CAR) T cells, which express a synthetic receptor engineered to target specific antigens, have demonstrated remarkable potential to treat haematological malignancies. However, their transition beyond haematological malignancy has so far been unsatisfactory. Here, we discuss recent challenges and improvements for CAR T cell therapy against solid tumors: Antigen heterogeneity which provides an effective escape mechanism against conventional mono-antigen-specific CAR T cells; and the immunosuppressive tumor microenvironment which provides physical and molecular barriers that respectively prevent T cell infiltration and drive T cell dysfunction and hypoproliferation. Further, we discuss the application of CAR T cells in infectious disease and autoimmunity.