Covalent Peptide Engagers (CPEs) Reprogram Antibodies for Dual-Antigen Tumor Targeting and Enhanced Phagocytosis

Site-specific antibody modification is crucial for enhancing therapeutic function, particularly in cancer immunotherapy. Current methods, such as lysine- or cysteine-based conjugations, often yield heterogeneous products, while protein engineering is resource-intensive. This creates a gap for efficient, selective antibody functionalization. Affinity-guided covalent strategies using antibody-binding peptides offer a promising chemical alternative, but face challenges in integrating reactive electrophiles into complex peptide ligands, limiting their broader application.

Researchers developed covalent peptide engagers (CPEs) designed to equip antibodies with new tumour-targeting capabilities. These CPEs integrate an Fc-binding peptide, a SuFEx electrophile, and a target-engager moiety. CPEs were synthesized and evaluated for covalent labelling kinetics and selectivity using SDS-PAGE gels and biolayer interferometry (BLI). Functional activity was assessed in tumour cell-immune cell co-culture assays, specifically measuring antibody-dependent cellular phagocytosis of urokinase-type plasminogen activator receptor (uPAR)-expressing tumour cells. The study also explored functionalizing Herceptin with uPAR-CPE.

A first-in-class CPE was successfully synthesized, incorporating a latent protein-reactive electrophile within a complex Fc-binding peptide. These CPEs demonstrated sufficient selectivity and kinetics for site-selective antibody modifications. Compatibility of the SuFEx peptide ligand was found to be dependent on the presence of internal tyrosine or histidine residues, which could inactivate the covalent peptide or enhance its hydrolysis rate. The anti-tumour function of IgG-CPE was validated through antibody-dependent cellular phagocytosis assays. > This demonstrated robust phagocytosis of uPAR-expressing tumour cells, confirming the new targeting function. Furthermore, Herceptin functionalized with uPAR-CPE enabled dual-antigen targeting, facilitating phagocytosis of cells co-expressing both HER2 and uPAR, consistent with avidity effects from engaging two tumour antigens simultaneously.