Broad reactivity of HLA-E restricted TCRs highlights challenges in universal T cell therapy development 2335164

Introduction T cell receptor—engineered therapies (TCR-Ts) redirect T cells to tumor antigens presented by human leukocyte antigen (HLA) molecules but are limited by the extreme polymorphism of classical HLAs, restricting broad applicability. HLA-E is a non-classical, functionally monomorphic HLA molecule that presents host- and virus-derived peptides and elicits CD8 T cell responses. Virus-associated malignancies disproportionately affect diverse populations, highlighting the need for broadly applicable immunotherapies. We investigated HLA-E—restricted TCRs as a platform for universal cellular therapy using a cytomegalovirus-derived epitope. Methods Peripheral blood mononuclear cells from CMV-seropositive donors were stimulated with antigen-presenting cells expressing HLA-E—VL9 (VMAPRTLLL). VL9-specific T cells were isolated by tetramer sorting and single-cell TCR sequencing. An HLA-E—null NFAT-luciferase reporter line expressing the VL9-TCR was used to assess activation against HLA-E—peptide complexes and cancer cell lines. Primary CD8 T cells underwent endogenous TCR and HLA-E knockout before VL9-TCR transduction. Results The VL9-TCR exhibited unexpected cross-reactivity to multiple HLA-E—bound peptides, corroborated by structural modeling. Reporter cells showed strong activation against HTLV-1+ and EBV+ leukemia and lymphoma lines, with moderate activation toward HPV+ cancers and healthy primary cells, indicating off-tumor activity. VL9-TCR—Ts demonstrated preferential HLA-E—dependent cytotoxicity against HTLV-1+ targets compared with healthy PBMCs. Conclusion HLA-E—VL9 TCR-Ts exhibit strong anti-tumor activity but also substantial peptide cross-reactivity, highlighting key safety challenges. Although the VL9-TCR itself is unlikely to be clinically viable, these results support HLA-E’s potential as a universal immunotherapy platform. Ongoing work focuses on identifying tumor-specific HLA-E—binding peptides and integrating synNotch logic-gated circuits to determine whether specificity can be enhanced.