Dual targeting of cancer metabolome and stress antigens affects transcriptomic heterogeneity and efficacy of engineered T cells.

Few cancers can be targeted efficiently by engineered T cell strategies. Here, we show that γδ T cell antigen receptor (γδ TCR)-mediated cancer metabolome targeting can be combined with targeting of cancer-associated stress antigens (such as NKG2D ligands or CD277) through the addition of chimeric co-receptors. This strategy overcomes suboptimal γ9δ2 TCR engagement of αβ T cells engineered to express a defined γδ TCR (TEGs) and improves serial killing, proliferation and persistence of TEGs. In vivo, the NKG2D-CD28 chimera enabled control only of liquid tumors, whereas the NKG2D-4-1BB chimera prolonged persistence of TEGs and improved control of liquid and solid tumors. The CD277-targeting chimera (103-4-1BB) was the most optimal co-stimulation format, eradicating both liquid and solid tumors. Single-cell transcriptomic analysis revealed that NKG2D-4-1BB and 103-4-1BB chimeras reprogram TEGs through NF-κB. Owing to competition with naturally expressed NKG2D in CD8 TEGs, the NKG2D-4-1BB chimera mainly skewed CD4 TEGs toward adhesion, proliferation, cytotoxicity and less exhausted signatures, whereas the 103-4-1BB chimera additionally shaped the CD8 subset toward a proliferative state.