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

Adam S. Chervin, David H. Aggen, John M. Raseman, David M Kranz

Research output: Contribution to journalArticle

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 CDR3α 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/K b (SIY/K b ). The selected TCR contained a sequence motif in the CDR3α 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.

Original languageEnglish (US)
Pages (from-to)175-184
Number of pages10
JournalJournal of Immunological Methods
Volume339
Issue number2
DOIs
StatePublished - Dec 31 2008

Fingerprint

T-Cell Antigen Receptor
T-Lymphocytes
Hybridomas
CD3 Antigens
Protein Engineering
Neoplasm Antigens
Cell- and Tissue-Based Therapy
Major Histocompatibility Complex
Libraries
Organism Cloning
Clone Cells
Yeasts
Antigens
Peptides

Keywords

  • Affinity
  • Peptide-MHC
  • Protein engineering
  • Retroviral vectors
  • T cell receptors

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Engineering higher affinity T cell receptors using a T cell display system. / Chervin, Adam S.; Aggen, David H.; Raseman, John M.; Kranz, David M.

In: Journal of Immunological Methods, Vol. 339, No. 2, 31.12.2008, p. 175-184.

Research output: Contribution to journalArticle

Chervin, Adam S. ; Aggen, David H. ; Raseman, John M. ; Kranz, David M. / Engineering higher affinity T cell receptors using a T cell display system. In: Journal of Immunological Methods. 2008 ; Vol. 339, No. 2. pp. 175-184.
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