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Journal of Biomedicine and Biotechnology
Volume 2006, Article ID 68091, 9 pages
http://dx.doi.org/10.1155/JBB/2006/68091
Methods Report

Cloning and Expression of Human Membrane-Bound and Soluble Engineered T Cell Receptors for Immunotherapy

Department of Immunology, School of Medicine, University of Pittsburgh, PA, Pittsburgh 15261, USA

Received 28 June 2005; Revised 15 November 2005; Accepted 21 November 2005

Copyright © 2006 Nehad M. Alajez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

We report here the design and construction of several gene vectors for expression in mammalian cells of membrane-bound and soluble human T cell receptors (TR). We designed a vector (TR-ALPHA-IRES-TR-BETA pEF4) that encodes high-level expression of the full-length TR on the surface of T cells. Furthermore, we engineered TR that does not require the presence of endogenous CD3 molecules for surface expression and thus expression is not limited to T cells. We also constructed a vector encoding a single-chain TR (scTR) as a fusion protein of V-ALPHA-V-BETA-C-BETA with CD3Z. Since it is encoded and expressed as a single molecule, this scTR is well suited for gene therapy. Lastly, we successfully used a mammalian expression vector for generation of soluble human TR. The approaches we used here for manipulation of a human tumor-specific TR can be useful for other investigators interested in TR-based immunotherapy.