Open AccessA bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences
Author(s) -
Korbinian N. Kropp,
Tim J. Schäufele,
Martina Fatho,
Michael Volkmar,
Roland Conradi,
Matthias Theobald,
Thomas Wölfel,
Catherine Wölfel
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0238875
Subject(s) - t cell receptor , cloning (programming) , expression cloning , computational biology , biology , t cell , microbiology and biotechnology , genetics , complementary dna , gene , computer science , immune system , programming language
To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αβTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCRα and TCRβ V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of the human αβTCR constant regions with their codon-optimized murine counterparts for chimerization, addition of a second interchain disulfide bond and arrangement of the TCR chains in the order β-P2A-α. We exemplified the utility of our vector backbone by cloning and functional testing of three melanoma-reactive TCRs in primary human T cells.