An efficient site-selective, dual bioconjugation approach exploiting N-terminal cysteines as minimalistic handles to engineer tailored anti-HER2 affibody conjugates

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 esters 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 exhibited an exacerbated cytoxicity against HER2-expressing cell lines as compared to a combination of two monoconjugates, demonstrating a potent synergistic effect. Consistently, affibody-drug conjugates did not decrease the viability of HER2-negative cells, confirming their specificity for the target. [Display omitted] •Straightforward strategy to exploit N-terminal cysteine (NCys) residues for dual functionalization approaches.•Native Chemical Ligation -based mechanism to harness non-selective NHS ester reagents as NCys-directed bioconjugation tools.•Chemical approach to generate dual affibody conjugates 7a and 7b, displaying at once different probes and cytotoxic payloads.•Anti-HER2 affibody conjugate 7b, displaying two drugs at once reveals a synergystic effect in killing HER2-positive cells.