@article {Moritzeaav0860, author = {Moritz, Andreas and Anjanappa, Raghavendra and Wagner, Claudia and Bunk, Sebastian and Hofmann, Martin and Pszolla, Gabriele and Saikia, Ankur and Garcia-Alai, Maria and Meijers, Rob and Rammensee, Hans-Georg and Springer, Sebastian and Maurer, Dominik}, title = {High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules}, volume = {4}, number = {37}, elocation-id = {eaav0860}, year = {2019}, doi = {10.1126/sciimmunol.aav0860}, publisher = {Science Immunology}, abstract = {Recombinant MHC molecules displaying single peptides in their peptide binding cleft are valuable reagents for identifying T cells that bind specific peptide-MHC complexes. Two studies in this week{\textquoteright}s issue show that disulfide-stabilized (DS) MHC class I molecules offer a more efficient path to preparing large libraries of MHC-peptide reagents. Saini et al. developed DS versions of three MHC class I molecules and used a library of DS HLA-A2{\textendash}peptide multimers to rapidly screen T cells infiltrating human melanoma tumors for neoantigen reactivity. Moritz et al. prepared libraries of DS HLA-A2{\textendash}peptide complexes to screen an affinity-matured TCR for cross-reactivity with self-peptide{\textendash}MHC complexes. Empty MHC class I molecules that are stable and easily loaded with peptide will facilitate the wider use of MHC-peptide reagents for T cell detection. See related Research Article by Saini et al. in this issue.Major histocompatibility complex (MHC) class I molecules present short peptide ligands on the cell surface for interrogation by cytotoxic CD8+ T cells. MHC class I complexes presenting tumor-associated peptides such as neoantigens represent key targets of cancer immunotherapy approaches currently in development, making them important for efficacy and safety screenings. Without peptide ligand, MHC class I complexes are unstable and decay quickly, making the production of soluble monomers for analytical purposes labor intensive. We have developed a disulfide-stabilized HLA-A*02:01 molecule that is stable without peptide but can form peptide-MHC complexes (pMHCs) with ligands of choice in a one-step loading procedure. We illustrate the similarity between the engineered mutant and the wild-type molecule with respect to affinity of wild-type or affinity-matured T cell receptors (TCRs) and present a crystal structure corroborating the binding kinetics measurements. In addition, we demonstrate a high-throughput binding kinetics measurement platform to analyze the binding characteristics of bispecific TCR (bsTCR) molecules against diverse pMHC libraries produced with the disulfide-stabilized HLA-A*02:01 molecule. We show that bsTCR affinities for pMHCs are indicative of in vitro function and generate a bsTCR binding motif to identify potential off-target interactions in the human proteome. These findings showcase the potential of the platform and the engineered HLA-A*02:01 molecule in the emerging field of pMHC-targeting biologics.}, URL = {https://immunology.sciencemag.org/content/4/37/eaav0860}, eprint = {https://immunology.sciencemag.org/content/4/37/eaav0860.full.pdf}, journal = {Science Immunology} }