Engineering higher affinity T cell receptors using a T cell display system.
The T cell receptor (TCR) determines the cellular response to antigens, which are presented on the surface of target cells in the form of a peptide bound to a product of the major histocompatibility complex (pepMHC). The response of the T cell depends on the affinity of the TCR for the pepMHC, yet many TCRs have been shown to be of low affinity, and some naturally occurring T cell responses are poor due to low affinities. Accordingly, engineering the TCR for increased affinity for pepMHC, particularly tumor-associated antigens, has become an increasingly desirable goal, especially with the advent of adoptive T cell therapies. For largely technical reasons, to date there have been only a handful of TCRs engineered in vitro for higher affinity using well established methods of protein engineering. Here we report the use of a T cell display system, using a retroviral vector, for generating a high-affinity TCR from the mouse T cell clone 2C. The method relies on the display of the TCR, in its normal, signaling competent state, as a CD3 complex on the T cell surface. A library in the CDR3alpha of the 2C TCR was generated in the MSCV retroviral vector and transduced into a TCR-negative hybridoma. Selection of a high-affinity, CD8-independent TCR was accomplished after only two rounds of flow cytometric sorting using the pepMHC SIYRYYGL/Kb (SIY/Kb). The selected TCR contained a sequence motif in the CDR3alpha with characteristics of several other TCRs previously selected by yeast display. In addition, it was possible to directly use the selected T cell hybridoma in functional assays without the need for sub-cloning, revealing that the selected TCR was capable of mediating CD8-independent activity. The method may be useful in the direct isolation and characterization of TCRs that could be used in therapies with adoptive transferred T cells.
Department of Biochemistry, University of Illinois, 600 S. Matthews Ave., Urbana, IL 61801, USA.
Adam S
AS
Chervin
David H
DH
Aggen
John M
JM
Raseman
David M
DM
Kranz
D000818:Animals; D016827:CD8 Antigens / Q000235:genetics / Q000276:immunology; D022801:Complementarity Determining Regions / Q000235:genetics* / Q000276:immunology*; D005822:Genetic Vectors; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D009842:Oligopeptides / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D012190:Retroviridae; D013601:T-Lymphocytes / Q000276:immunology* / Q000637:transplantation; D014161:Transduction, Genetic / Q000379:methods
D016827:CD8 Antigens; D022801:Complementarity Determining Regions; D009842:Oligopeptides; C406862:superagonist SIYR