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 dualbioconjugation strategy to target the N-terminal cysteine of a chemically-synthesized, third-generation antiHER2 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.

Automated summary

Site-selective, dual-conjugation approaches are important for the development of targeted biomolecules with improved properties. Combinations of drugs and probes can enhance molecular imaging and therapeutic outcomes. We have developed an efficient dual-bioconjugation strategy that can generate high-yield, high-purity dual conjugates under mild conditions. These dual conjugates exhibit potent cytotoxicity against HER2-expressing cells and show specificity for the target.