TY  - JOUR
AU  - Kropp, Korbinian N
AU  - Schäufele, Tim J
AU  - Fatho, Martina
AU  - Volkmar, Michael
AU  - Conradi, Roland
AU  - Theobald, Matthias
AU  - Wölfel, Thomas
AU  - Wölfel, Catherine
TI  - A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.
JO  - PLOS ONE
VL  - 15
IS  - 9
SN  - 1932-6203
CY  - San Francisco, California, US
PB  - PLOS
M1  - DKFZ-2020-01828
SP  - e0238875 -
PY  - 2020
N1  - HI-TRON
AB  - 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.
LB  - PUB:(DE-HGF)16
C6  - pmid:32903281
DO  - DOI:10.1371/journal.pone.0238875
UR  - https://inrepo02.dkfz.de/record/163037
ER  -