Identification and engineering of human variable regions that allow expression of stable single-chain T cell receptors

Single-chain antibody fragments (scFv), consisting of two linked variable regions (V-H and V-L), are a versatile format for engineering and as potential antigen-specific therapeutics. Although the analogous format for T cell receptors (TCRs), consisting of two linked V regions (V alpha and V beta; referred to here as scTv), could provide similar opportunities, all wild-type scTv proteins examined to date are unstable. This obstacle has prevented scTv fragments from being widely used for engineering or therapeutics. To further explore whether some stable human scTv fragments could be expressed, we used a yeast system in which display of properly folded domains correlates with ability to express the folded scTv in soluble form. We discovered that, unexpectedly, scTv fragments that contained the human V alpha 2 region (IMGT: TRAV12 family) were displayed and properly associated with different V beta regions. Furthermore, a single polymorphic residue (Ser(alpha 49)) in the framework region conferred additional thermal stability. These stabilized V alpha 2-containing scTv fragments could be expressed at high levels in Escherichia coli, and used to stain target cells that expressed the specific pep-HLA-A2 complexes. Thus, the scTv fragments can serve as a platform for engineering TCRs with diverse specificities, and possibly for therapeutic or diagnostic applications.