Universal, open MHC-I for rapid ligand loading and enhanced complex stability across classical and non-classical allotypes.

Abstract

Abstract The instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents an important biochemical challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs) for autologous therapy development. Here, we leverage the coupled binding between ligand and the light chain (beta-2 microglobulin, β 2m) to the MHC-I heavy chain with the aim of developing conformationally stable molecules by engineering a disulfide bond to bridge the interface between the heavy and light chain. The resulting MHC-I molecules are stabilized in a ligand-receptive/open state, exhibit favorable ligand exchange properties, and are readily loaded with moderate affinity ligands to afford properly conformed protein complexes of enhanced stability compared to the wild type. We demonstrate the generality of the structural design using polymorphic HLA representatives covering multiple HLA-A and B supertypes, the nonclassical and oligomorphic HLA-Ib molecules, as well as MHC-like molecules, MR1 and CD1. Our platform allows spontaneous and rapid ligand exchange, which enables a range of approaches for ligand screening and the functional screening and characterization of polyclonal TCR repertoires. Supported by grants from NIH (R01AI143997, R35GM125034, U01DK112217).