N-terminal cysteine as minimalistic handle for dual, site-selective bioconjugation: application to an anti-HER2 affibody reveals synergy of two conjugated drugs

Site-selective, dual-conjugation approaches for the incorporation of distinct payloads are key for the development of molecularly targeted biomolecules, such as antibody conjugates, endowed with better properties. Combinations of cytotoxic drugs, imaging probes, or pharmacokinetics modulators enabled for improved outcomes in both molecular imaging, and therapeutic settings. We have developed an efficacious dual-bioconjugation strategy to target the N-terminal cysteine of a chemically-synthesized third-generation anti-HER2 affibody. Such two-step, one-purification approach can be carried out under mild conditions (without chaotropic agents, neutral pH) by means of a slight excess of commercially available N-hydroxysuccinimidyl- and maleimido-functionalized payloads, to generate dual conjugates displaying drugs (DM1/MMAE) or probes (sulfo-Cy5/biotin) in high yields and purity. Remarkably, the double drug conjugate exhibits an exacerbated cytoxicity against HER2-expressing cell lines as compared to a combination of two monoconjugates, demonstrating a potent synergistic effect.