Structure-Based Rational and General Strategy for Stabilizing Single-Chain T-Cell Receptors to Enhance Affinity

The T-cell receptor (TCR) is a crucial molecule in cellular immunity. The single-chain T-cell receptor (scTCR) is a potential format in TCR therapeutics because it eliminates the possibility of αβ-TCR mispairing. However, its poor stability and solubility impede the in vitro study and manufacturing of therapeutic applications. In this study, some conserved structural motifs are identified in variable domains regardless of germlines and species. Theoretical analysis helps to identify those unfavored factors and leads to a general strategy for stabilizing scTCRs by substituting residues at exact IMGT positions with beneficial propensities on the consensus sequence of germlines. Several representative scTCRs are displayed to achieve stability optimization and retain comparable binding affinities with the corresponding αβ-TCRs in the range of μM to pM. These results demonstrate that our strategies for scTCR engineering are capable of providing the affinity-enhanced and specificity-retained format, which are of great value in facilitating the development of TCR-related therapeutics.