Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
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-mediate...
| Autores: | , , , , , , , , , , , |
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| Formato: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | España |
| Recursos: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/360912 |
| Acesso em linha: | http://hdl.handle.net/10261/360912 https://api.elsevier.com/content/abstract/scopus_id/85172098368 |
| Access Level: | acceso abierto |
| Palavra-chave: | THIOCAP Cysteine coupling http://metadata.un.org/sdg/3 Ensure healthy lives and promote well-being for all at all ages |
| Resumo: | 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.[Figure not available: see fulltext.] |
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