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

Abstract

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.

LINKED ARTICLES

This article is part of a themed issue Re-emergence of Covalent Modulation in Drug Discovery. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v183.22/issuetoc