TY - JOUR T1 - Engineering second-generation TCR-T cells by site-specific integration of TRAF-binding motifs into the <em>CD247</em> locus JF - Journal for ImmunoTherapy of Cancer JO - J Immunother Cancer DO - 10.1136/jitc-2022-005519 VL - 11 IS - 4 SP - e005519 AU - Sangjoon Lah AU - Segi Kim AU - In Kang AU - Hyojin Kim AU - Cedric Hupperetz AU - Hyuncheol Jung AU - Hyeong Ryeol Choi AU - Young-Ho Lee AU - Hyeon-Ki Jang AU - Sangsu Bae AU - Chan Hyuk Kim Y1 - 2023/04/01 UR - http://jitc.bmj.com/content/11/4/e005519.abstract N2 - 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.Data are available upon reasonable request. ER -