The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-Cβ antibody

Biochemical and Biophysical Research Communications
Tatsuhiko OzawaAtsushi Muraguchi

Abstract

The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-Cβ antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 × 10(-5)M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-Cβ antibody, its binding affinity...Continue Reading

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Citations

Sep 26, 2019·FEBS Letters·Flávio SádioGordana Wozniak-Knopp

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