Engineering higher affinity T cell receptors using a T cell display system

J Immunol Methods. 2008 Dec 31;339(2):175-84. doi: 10.1016/j.jim.2008.09.016. Epub 2008 Oct 12.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD8 Antigens / genetics
  • CD8 Antigens / immunology
  • Complementarity Determining Regions / genetics*
  • Complementarity Determining Regions / immunology*
  • Genetic Vectors
  • Hybridomas / immunology
  • Mice
  • Oligopeptides / immunology*
  • Protein Engineering / methods*
  • Retroviridae
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / transplantation
  • Transduction, Genetic / methods

Substances

  • CD8 Antigens
  • Complementarity Determining Regions
  • Oligopeptides
  • superagonist SIYR