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.