Computational design of optimized and preferentially paired human TCR constant regions for improved T-cell function

TCR gene transfer is promising for cancer immunotherapy, but its efficacy is limited due to mispairing of TCR chains, which reduces surface expression and could lead to off-target effects. We applied the PROSS and FuncLib algorithms to engineer human TCR constant regions for improved stability and orthogonal pairing. After several screening rounds, we identified a variant, termed Structurally Enhanced TCR (SET), which exhibits markedly enhanced surface expression, functional avidity, and reduced mispairing.

SET-expressing T-cells secreted higher cytokine levels (ranging from threefold to sixteen-fold), displayed increased activation as measured using 41BB, CD25, and CD69, and mediated superior cytotoxicity. Notably, SET preserved function in CD4+ T-cells without CD8 co-receptor support and demonstrated potent tumour control in xenograft models, significantly delaying growth and improving survival.

SET’s benefits were reproducible across five unrelated TCRs, both cancer-associated and of viral origin, underscoring its broad applicability. These findings highlight the potential of rational design to improve TCR-based therapies by enhancing expression, specificity, and in vivo efficacy without the need for additional genetic engineering.