Engineering high-fidelity tapasin variants to enhance MHC-I antigen presentation.

Human leukocyte antigen (HLA) proteins are extremely polymorphic, with different allotypes exhibiting a wide range of dependencies on the chaperone tapasin for peptide loading, expression, and stability at the cell surface. Given its central role in antigen processing, tapasin is frequently downregulated across viral infections and cancers, impairing antigen presentation and hindering the identification of therapeutically relevant peptide antigens. We hypothesized that elucidating the mutational tolerance of tapasin surfaces, which mediate interactions with polymorphic HLA residues, can provide a means for fine-tuning its chaperoning function and reveal mechanistic epitopes that underlie its function. Using two complementary deep mutational scanning screens, we systematically mapped the tapasin/HLA-A∗02:01 interaction landscape, highlighting key regions involved in HLA-I assembly and repertoire optimization. We engineered two high-fidelity tapasin variants, tapasin-YTY and tapasin-I2, and show that these variants can increase cell surface HLA expression on a wild-type (WT) tapasin-deficient background by up to 50% and improve peptide-loading function across multiple HLA-A∗ allotypes. Our findings establish scanning mutagenesis as a general approach for fine-tuning chaperone interactions with HLA molecules to enhance antigen presentation in immunologically impaired settings.