Expanding the repertoire of chemically induced covalent neoantigens

The ubiquitin-proteasome system generates peptide fragments that are displayed on MHC-I molecules. Recent studies revealed that covalent small molecules can hijack this process, leading to the presentation of hapten-modified peptide fragments, or covalent neoantigens. Here, we report a chemical proteomics platform to systematically investigate this process and harness covalent neoantigens for immune cell recruitment and activation. Using a bioorthogonal cysteine-reactive probe (IA-mTz), we demonstrate that diverse intracellular proteins can be modified, processed, and presented as covalent neoantigens across multiple cell lines. Mass spectrometry confirmed the presence of IA-mTz-modified peptides within the MHC-I immunopeptidome, and structural modeling revealed stable peptide-MHC-I interactions. We further integrated a bioorthogonal strategy that enables immune engagement: IA-mTz-modified cells conjugated with trans-cyclooctene (TCO)-IgG elicited Fc receptor activation in a reporter assay, supporting immune recruitment via antibody-dependent cellular phagocytosis. This approach was extended to additional cysteine-reactive probes and to 2,4-dinitrochlorobenzene (DNCB), a common skin sensitizer, which we show induces covalent neoantigen formation in a cell- and HLA-dependent manner. These findings reveal a previously underappreciated breadth of covalent neoantigen formation and provide a generalizable strategy for immune targeting of covalent neoantigens.