Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)

Ariana Rueda, Julian I. Mendoza, Lorena Alba-Castellon, Eloi Parladé, Eric Voltà-Durán, David Paez, Anna Aviño, Ramon Eritja, Esther Vázquez*, Antonio Villaverde, Ramón Mangues*, Ugutz Unzueta*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

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The development of a new generation of non-antibody protein drug delivery systems requires site-directed conjugation strategies to produce homogeneous, reproducible and scalable nanomedicines. For that, the genetic addition of cysteine residues into solvent-exposed positions allows the thiol-mediated cysteine coupling of therapeutic drugs into protein-based nanocarriers. However, the high reactivity of unpaired cysteine residues usually reduces protein stability, consequently imposing the use of more methodologically demanding purification procedures. This is especially relevant for disulfide-containing nanocarriers, as previously observed in THIOMABs. Moreover, although many protein scaffolds and targeting ligands are also rich in disulfide bridges, the use of these methodologies over emerging non-antibody carrier proteins has been completely neglected. Here, we report the development of a simple and straightforward procedure for a one-step production and site-directed cysteine conjugation of disulfide-containing non-antibody thiolated carrier proteins (THIOCAPs). This method is validated in a fluorescent C-X-C chemokine receptor 4 (CXCR4)-targeted multivalent nano-carrier containing two intramolecular disulfide bridges and one reactive cysteine residue strategically placed into a solvent-exposed position (THIO-T22-GFP-H6) for drug conjugation and in a humanized alternative intended for clinical applications (T22-HSNBT-H6). Thus, we produce very stable, homogeneous and fully functional antitumoral nanoconjugates (THIO-T22-GFP-H6-MMAE and T22-HSNBT-H6-MMAE) that selectively eliminate target cancer cells via CXCR4-receptor. Altogether, the developed methodology appears as a powerful tool for the rational engineering of emerging non-antibody, cell-targeted protein nanocarriers that contain disulfide bridges together with a solvent-exposed reactive cysteine (THIOCAP). This should pave the way for the development of a new generation of stable, homogeneous and efficient nanomedicines.
Original languageEnglish
Pages (from-to)4109-4120
Number of pages12
JournalScience China Materials
Issue number10
Early online date22 Sept 2023
Publication statusPublished - 22 Sept 2023


  • Cysteine coupling
  • Disulfide-bonds
  • Nanoconjugates
  • Nanomedicine
  • Protein carriers
  • Thiocap


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