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bioRxiv · 10.1101/2025.07.08.663715

Expanding the repertoire of chemically induced covalent neoantigens

Zhou, C.·Huang, W.·Jin, X.·Wu, H.·McGregor, L. M.·Schirle, M.·Zhang, C.·Zhang, X.
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Abstract

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.

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Zhou, C., Huang, W., Jin, X., Wu, H., McGregor, L. M., Schirle, M., Zhang, C., Zhang, X.. 2025-07-12. Expanding the repertoire of chemically induced covalent neoantigens. https://doi.org/10.1101/2025.07.08.663715

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