A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.

Korbinian N Kropp,Michael Volkmar,Roland Conradi,Matthias Theobald,Catherine Wölfel,Tim J Schäufele,Thomas Wölfel,Martina Fatho,Chen Ling

doi:10.1371/journal.pone.0238875

Abstract

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.

Highlights

Adoptive transfer of in vitro expanded tumor infiltrating lymphocytes (TIL) naturally expressing cancer-reactive T-cell receptors (TCR) has yielded promising results in metastatic melanoma [1] and in epithelial cancers [2]
We report on a vector backbone for the generation of αβTCR constructs
Hu et al generated full-length TCR constructs by Golden Gate cloning, but ligated only the hypervariable CDR3 region into components of an extensive vector backbone library, Seamless αβTCR cloning members of which each encode one of the different TCR α and β variable sequences [22]

Summary

Introduction

Adoptive transfer of in vitro expanded tumor infiltrating lymphocytes (TIL) naturally expressing cancer-reactive T-cell receptors (TCR) has yielded promising results in metastatic melanoma [1] and in epithelial cancers [2]. This treatment option is confined only to patients, in whom the expansion of functional TIL is successful [3]. ΑβTCR expression constructs often share optimizations that favor expression of transgenic TCRs and reduce putative mispairing with endogenous α and β TCR chains. These, amongst others, comprise expression of the α and β TCR chains from the same promoter by linking the individual genes with a 2A

Methods

Results

Conclusion