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Directed evolution of human T-cell receptors with picomolar affinities by phage display


Peptides derived from almost all proteins, including disease-associated proteins, can be presented on the cell surface as peptide–human leukocyte antigen (pHLA) complexes. T cells specifically recognize pHLA with their clonally rearranged T-cell receptors (TCRs), whose natural affinities are limited to 1–100 μM1. Here we describe the display of ten different human TCRs on the surface of bacteriophage, stabilized by a nonnative interchain disulfide bond2. We report the directed evolution of high-affinity TCRs specific for two different pHLAs: the human T-cell lymphotropic virus type 1 (HTLV-1) tax11–19 peptide–HLA-A*0201 complex3 and the NY-ESO-1157–165 tumor-associated peptide antigen–HLA-A*0201 complex4, with affinities of up to 2.5 nM and 26 pM, respectively, and we demonstrate their high specificity and sensitivity for targeting of cell-surface pHLAs.

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Figure 1: Biacore SPR binding of high-affinity TCRs compared to their parent wild-type TCRs.
Figure 2: High-affinity TCR targeting of cell-surface pHLAs.


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We would like to thank the following for supplying plasmids containing wild-type TCR genes: W.E. Biddison for A6, V. Cerundolo for 1G4 and MM15, E. Gostick for ILAK, S. Burrows for LC13, G.F. Gao for JM22, H. Gaston for AH1.23, S. Gadola for CD1d and Paul Bowness for GRb. We would like to thank M. Sami, P. Todorov and A. Johnson for assistance with protein purification, Martin Green, R. Ashfield and N. Lissin for helpful discussions and critical reading of the manuscript, B. Laugel and A.K. Sewell for additionally sharing data before publication and D. Sutton for assistance in preparing figures.

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Correspondence to Jonathan M Boulter.

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Competing interests

Authors with affiliation to Avidex Ltd. are or have been employees of Avidex. J.M.B. and B.K.J. are also minor shareholders. Avidex Ltd. is developing high affinity TCRs as therapeutic targeting agents. We do not believe that this has in any way influenced our presentation or interpretation of the data presented in this manuscript.

Supplementary information

Supplementary Fig. 1

Global fit analysis of 1G4c113 binding (PDF 86 kb)

Supplementary Fig. 2

Competition analysis of 1G4c113 binding (PDF 199 kb)

Supplementary Table 1

Peptide-HLA complexes used for testing A6c134 specificity. Peptide-HLA complexes used for testing 1G4c113 specificity. (PDF 50 kb)

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Li, Y., Moysey, R., Molloy, P. et al. Directed evolution of human T-cell receptors with picomolar affinities by phage display. Nat Biotechnol 23, 349–354 (2005).

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