A covalent peptide engager approach to equip immunotherapeutic monoclonal and pan IgG serum antibodies with new tumour‐targeting function

BACKGROUND AND PURPOSE: Site-specific antibody modification is a useful chemical strategy to elaborate antibody therapeutic function. Conventional lysine- or cysteine-based conjugation methods generate heterogeneous products, while protein engineering approaches are resource-intensive. Affinity-guided covalent strategies using antibody binding peptides offer a chemical alternative for selective antibody functionalisation but are limited by challenges with incorporating reactive electrophiles into complex peptide ligands. EXPERIMENTAL APPROACH: We developed covalent peptide engagers (CPEs) that equip antibodies with new tumour-targeting function. CPEs integrate an Fc-binding peptide, SuFEx electrophile and a target-engager moiety. CPEs were synthesised and evaluated in covalent labelling kinetics and selectivity assays using SDS-PAGE gels and biolayer interferometry (BLI). Functional activity was evaluated using tumour cell-immune cell co-culture assays. KEY RESULTS: A first-in-class CPE was synthesised incorporating a latent protein-reactive electrophile within a complex Fc-binding peptide. CPEs demonstrate sufficient selectivity/kinetics for site-selective antibody modifications. We observed SuFEx peptide ligand compatibility is dependent on the presence of internal tyrosine or histidine residues, which can inactivate the covalent peptide and/or enhance hydrolysis rate. Anti-tumour function of IgG-CPE was validated by antibody-dependent cellular phagocytosis, demonstrating robust phagocytosis of urokinase-type plasminogen activator receptor (uPAR)-expressing tumour cells. Herceptin functionalised with uPAR-CPE enabled dual-antigen-targeting via phagocytosis of cells co-expressing HER2 and uPAR, consistent with avidity effects associated with engagement of two tumour antigens. CONCLUSIONS AND IMPLICATIONS: CPEs enable covalent reprogramming of endogenous IgG and monoclonal antibodies, offering an alternative to protein engineering for the rapid generation of multi-specific antibodies and therapeutic engagement of humoral immunity across cancer and infectious disease.