Abstract
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.
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Acknowledgements
The authors are indebted to ISCIII (PI20/00400) co-funded by European Regional Development Fund (ERDF, a way to make Europe), and to CIBER-BBN (project NANOSCAPE and NANOLINK) granted to Unzueta U; to AEI (PID2019-105416RB-I00/AEI/10.13039/501100011033) and to CIBER-BBN (NANOREMOTE) granted to Vázquez E; to ISCIII (PI21/00150) co-funded by European Regional Development Fund (ERDF, a way to make Europe), to CIBER-BBN (4NanoMets) and to AGAUR (2021 SGR-01140) granted to Mangues R; and to CIBER-BBN (VENOM4CANCER) and AGAUR (2021 SGR-00092) granted to Villaverde A. This work was also supported by CIBER-Consorcio Centro de Investigación Biomédica en Red- (CB06/01/1031 and CB06/01/0014), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and European Regional Development Fund (ERDF) and by CERCA programme (Generalitat de Catalunya). Unzueta U was supported by Miguel Servet contract (CP19/00028) from ISCIII co-funded by European Social Fund (ESF investing in your future). Alba-Castellon L was supported by the Spanish Association Against Cancer-AECC (POSTD20070ALBA). Rueda A was supported by a PFIS predoctoral fellowship (FI21/00012) from ISCIII co-funded by European Social Fund (ESF, investing in your future) and EVD by a predoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades (FPU18/04615). Villaverde A received an Icrea Academia award. Protein production was partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/ IBB, at the UAB http://www.nanbiosis.es/portfolio/u1-protein-production-platform-ppp/. Molecular graphics were performed with UCSF Chimera and ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from the National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.
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Author contributions Rueda A performed most of the experiments and prepared the figures; Mendoza JI prepared the humanized nanoconjugate and prepared the figures; Alba-Castellon L performed in-vitro cytotoxicity assays and prepared the figures; Voltá-Durán E and Parladé E performed insilico analysis and prepared the figures. Paez D, Aviño A and Eritja R analyzed the data and supervised the chemical part of the study. Vázquez E, Mangues R, Villaverde A and Unzueta U conceived and supervised the whole study; Unzueta U prepared the first draft of the manuscript. All authors contributed to the general discussion and approved the manuscript.
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Conflict of interest The authors declare that they have no conflict of interest. Mangues R, Vazquez E and Villaverde A are co-founders of Nanoligent SL, a company devoted to developing nanostructured drugs for cancer treatment. Mangues R, Vázquez E, Villaverde A, Parladé E and Unzueta U are inventors of a patent covering the use of HSNBT for biomedical applications.
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Ariana Rueda graduated in biotechnology in 2020 and obtained a Master’s degree in biochemistry, molecular biology and biomedicine. She is currently pursuing a PhD degree in biotechnology directed by Dr. Ugutz Unzueta and Dr. Ramón Mangues at the Sant Pau Biomedical Research Institute of Barcelona, Spain. Her research focuses on developing new protein-based drug nanoconjugates (nanomedicines) for targeted therapies in the field of oncology.
Esther Vázquez obtained an M.D. PhD degree at the University of Valladolid and completed her training at the University of Oviedo, the State University of New York and the Institute of Cancer Research in London. She is currently a Researcher at the Autonomous University of Barcelona. She leads a team focused on the development of new protein-based materials that serve as drug delivery systems, formed by multidomain proteins that self-assemble into nanoparticles for targeted therapy in cancer.
Ugutz Unzueta graduated in biotechnology in 2008 and got his PhD degree in 2013. His research career has been developed in Spain, United Kingdom and Italy. From 2020, he has been leading an independent research line dedicated to the design of self-assembling protein materials and drug nanoconjugates for precision nanomedicines at Sant Pau Biomedical Research Institute (Barcelona, Spain). He is also a member of the Spanish nanomedicine network CIBER-BBN and associated professor at the Autonomous University of Barcelona.
Ramón Mangues became a pharmacist (1981), specialist in hospital pharmacy (1987) and PharmD PhD (1988) at Navarra University in Spain. He was a postdoctoral (1988-1992) and associated (1992-1998) researcher at New York University Medical Center. He became a National Health System Researcher at Sant Pau Biomedical Research Institute (Spain), where he leads the Group of Oncogenesis and Antitumor Drugs focusing on targeted drug delivery to selectively eliminate cancer stem cells to obtain anti-metastic effect.
Supplementary information Experimental details and supporting data are available in the online version of the paper.
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Rueda, A., Mendoza, J.I., Alba-Castellon, L. et al. Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs). Sci. China Mater. 66, 4109–4120 (2023). https://doi.org/10.1007/s40843-023-2571-6
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DOI: https://doi.org/10.1007/s40843-023-2571-6