With the goal of being able to perform antigen-specific T cell identification, T cell isolation, and TCR avidity characterization using only one type of pMHC precursor for all uses, Effenberger, Stengl, and Schober et al. designed “FLEXamers” – novel, double-tagged pMHC constructs that allow for reversible multimerization via a Strep-tag and versatile functionalization with any probe (biotin, fluorophore, DNA-oligonucleotides, etc.) via a Tub-tag. FLEXamers performed equivalently with currently used methods, but were generated via a much simpler process, suggesting potential utility in personalized immunotherapy approaches.
Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor ("FLEXamer"). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general.