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Directed evolution of a stable scaffold for T-cell receptor engineering

Nature Biotechnology volume 18, pages 754–759 (2000)Cite this article

Abstract

Here we have constructed a single-chain T-cell receptor (scTCR) scaffold with high stability and soluble expression efficiency by directed evolution and yeast surface display. We evolved scTCRs in parallel for either enhanced resistance to thermal denaturation at 46°C, or improved intracellular processing at 37°C, with essentially equivalent results. This indicates that the efficiency of the consecutive kinetic processes of membrane translocation, protein folding, quality control, and vesicular transport can be well predicted by the single thermodynamic parameter of thermal stability. Selected mutations were recombined to create an scTCR scaffold that was stable for over an hour at 65°C, had solubility of over 4 mg ml−1, and shake-flask expression levels of 7.5 mg l−1, while retaining specific ligand binding to peptide–major histocompatibility complexes (pMHCs) and bacterial superantigen. These properties are comparable to those for stable single-chain antibodies, but are markedly improved over existing scTCR constructs. Availability of this scaffold allows engineering of high-affinity soluble scTCRs as antigen-specific antagonists of cell-mediated immunity. Moreover, yeast displaying the scTCR formed specific conjugates with antigen-presenting cells (APCs), which could allow development of novel cell-to-cell selection strategies for evolving scTCRs with improved binding to various pMHC ligands in situ.

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Figure 1: Screening and analysis of yeast-displayed scTCR libraries for enhanced stability or expression at elevated temperatures.
Figure 2: Stability and expression properties of selected scTCR mutants.
Figure 3: Peptide–MHC binding by soluble mutant scTCR.
Figure 4: Formation of specific scTCR-yeast–APC complexes.
Figure 5: Locations of mutations in the LWHI scTCR, shown on the wild-type 2C TCR structure41.

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Acknowledgements

Thanks to Roy Mariuzza and Peter Andersen for the superantigen mutant SEC3/1A4, Sean O'Herrin and Jeff Bluestone for QL9/Ld dimers, and Steve Jameson and Mark Daniels for SIYR/Kb tetramers. This work was funded by NIH GM55757 and NSF BES 95-31407. We would like to acknowledge Dr. Michael Glaser and Jonah Chan for their assistance in performing the fluorescence microscopy, and Gary Durack of the University of Illinois Flow Cytometry Facility for his assistance. Also, special thanks to Dr. Marti Ware for her rosette assay expertise.

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Authors and Affiliations

  1. Department of Chemical Engineering, University of Illinois, Urbana, 61801, IL

    Eric V. Shusta & K.Dane Wittrup

  2. Department of Biochemistry, University of Illinois, Urbana, 61801, IL

    Phillp D. Holler, Michele C. Kieke & David M. Kranz

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  1. Eric V. Shusta
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Correspondence to David M. Kranz or K.Dane Wittrup.

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Shusta, E., Holler, P., Kieke, M. et al. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat Biotechnol 18, 754–759 (2000). https://doi.org/10.1038/77325

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