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.
Author Info: (1) Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany. (2) Department of Biology II, Ludwig Maximilian University o
Author Info: (1) Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany. (2) Department of Biology II, Ludwig Maximilian University of Munich, 82152 Planegg-Martinsried, Germany. (3) Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany. (4) Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany. (5) Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany. (6) Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany. National Center for Infection Research, 85748 Munich, Germany; and. (7) Department of Biology II, Ludwig Maximilian University of Munich, 82152 Planegg-Martinsried, Germany. (8) Department of Biology II, Ludwig Maximilian University of Munich, 82152 Planegg-Martinsried, Germany. (9) Department of Biology II, Ludwig Maximilian University of Munich, 82152 Planegg-Martinsried, Germany. (10) Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany; dirk.busch@tum.de. National Center for Infection Research, 85748 Munich, Germany; and. Clinical Cell Processing and Purification Focus Group, Institute for Advanced Study, Technical University of Munich, 81675 Munich, Germany.
