Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
Por:
Rueda A., Mendoza J.I., Alba-Castellon L., Parladé E., Voltà-Durán E., Paez D., Aviño A., Eritja R., Vázquez E., Villaverde A., Mangues R., Unzueta U.
Publicada:
1 ene 2023
Ahead of Print:
1 sep 2023
Resumen:
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.
Filiaciones:
Rueda A.:
Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), Sant Quintí 77-79, Barcelona, 08041, Spain
Josep Carreras Leukaemia Research Institute, IJC Campus Sant Pau, Barcelona, 08041, Spain
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Mendoza J.I.:
Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), Sant Quintí 77-79, Barcelona, 08041, Spain
Josep Carreras Leukaemia Research Institute, IJC Campus Sant Pau, Barcelona, 08041, Spain
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Alba-Castellon L.:
Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), Sant Quintí 77-79, Barcelona, 08041, Spain
Josep Carreras Leukaemia Research Institute, IJC Campus Sant Pau, Barcelona, 08041, Spain
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Parladé E.:
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra08193, Spain
Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain
Voltà-Durán E.:
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra08193, Spain
Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain
Paez D.:
Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau. Barcelona, Barcelona, 08025, Spain
CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, 28029, Spain
Aviño A.:
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, 08034, Spain
Eritja R.:
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, 08034, Spain
Vázquez E.:
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra08193, Spain
Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain
Villaverde A.:
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra08193, Spain
Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain
Mangues R.:
Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), Sant Quintí 77-79, Barcelona, 08041, Spain
Josep Carreras Leukaemia Research Institute, IJC Campus Sant Pau, Barcelona, 08041, Spain
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Unzueta U.:
Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), Sant Quintí 77-79, Barcelona, 08041, Spain
Josep Carreras Leukaemia Research Institute, IJC Campus Sant Pau, Barcelona, 08041, Spain
CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, 28029, Spain
Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain
Bronze, All Open Access; Bronze
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