Engineering high-affinity human single-chain T cell receptors against MART1 cancer antigens

Single-chain T cell receptors consisting of two linked variable regions (Valpha and Vbeta; referred to as scTv) provide a useful alternative engineering format to single-chain antibody fragments (scFv). Previously, we described the engineering of two high affinity human T-cell receptors (TCRs) in which the usage of the highly stable Valpha2 region when properly paired with different Vbeta regions allowed the TCR to be expressed on the surface of yeast as a scTv, and correlated with the ability to express soluble scTv fragments in E. coli. In this study, a wild-type TCR, called INR1-T1, which naturally uses the highly stable Valpha2 region, was engineered for high-affinity against variants of the Melan-A/MART-1 cancer antigen. We report an affinity improvement from 32 μM to 45 nM for the ELAGIGILTV peptide/HLA-A2 complex, as measured by surface plasmon resonance. Both the stabilized, wild-type scTv and the high affinity scTv could be expressed at high levels in E. coli, and the soluble protein could detect HLA-A2-positive antigen presenting cells pulsed with the specific MART-1 peptide. Alanine mutations of various CDR residues demonstrated the key role of particular residues in contacting the alpha helices of the HLA-A2. Furthermore, the full-length INR1-T1 construct with affinity mutations was introduced into primary human T cells to examine the specificity in either CD4 or CD8 T cells. The scTv fragments can serve as a platform for engineering TCRs with various affinities and specificities against human cancer antigens, toward potential therapeutic applications.