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

Authors

David H. Aggen, University of Illinois
Adam S. Chervin, University of Illinois
Francis K. Insaidoo, University of Notre Dame
Kurt H. Piepenbrink, University of Notre DameFollow
Brian M. Baker, University of Notre Dame
David M. Kranz, University of Illinois

Date of this Version

4-24-2011

Document Type

Article

Citation

Protein Engineering Design and Selection 2011 Apr; 24(4): 361–372. Published online 2010 Dec 14. doi: 10.1093/protein/gzq113 PMCID: PMC3049343 PMID: 21159619

Abstract

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 (Vα and Vβ; 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 Vα2 region (IMGT: TRAV12 family) were displayed and properly associated with different Vβ regions. Furthermore, a single polymorphic residue (Ser_α49) in the framework region conferred additional thermal stability. These stabilized Vα2-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.