Expanding the repertoire of chemically induced covalent neoantigens

The ubiquitin-proteasome system generates peptide fragments that are displayed on class I major histocompatibility complex molecules. Hapten-modified peptides, or covalent neoantigens, arise from proteins covalently modified by small molecules and have long been proposed as components of a mechanism for immune recognition. Here we report a platform to investigate this mechanism and harness covalent neoantigens for immune cell recruitment and activation. Using cysteine-reactive probes, we demonstrated that covalently modified intracellular proteins could be processed and presented as covalent neoantigens in multiple cell lines. Immunopeptidomics confirmed the presence of probe-modified peptides within the class I major histocompatibility complex immunopeptidome. We further developed a bioorthogonal strategy for immune cell engagement by combining covalent neoantigens bearing a bioorthogonal handle with complementary immune cell recruiters, leading to activation of CD32- and CD3-expressing reporter immune cells. These findings expand the scope of covalent neoantigen formation and provide a generalizable strategy for immune targeting of covalent neoantigens.