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...

Full description

Bibliographic Details
Authors: Rueda, Ariana, Mendoza, Julian I., Alba-Castellon, Lorena, Parladé, Eloi, Voltà-Durán, Eric, Paez, David, Aviñó, Anna, Eritja Casadellà, Ramón, Vázquez, Esther, Villaverde, Antonio, Mangues, Ramón, Unzueta, Ugutz
Format: article
Status:Published version
Publication Date:2023
Country:España
Institution:Consejo Superior de Investigaciones Científicas (CSIC)
Repository:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/360912
Online Access:http://hdl.handle.net/10261/360912
https://api.elsevier.com/content/abstract/scopus_id/85172098368
Access Level:Open access
Keyword:THIOCAP
Cysteine coupling
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
id ES_3e7883899d20b32b9e8b4c8b2f20465d
oai_identifier_str oai:digital.csic.es:10261/360912
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
title Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
spellingShingle Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
Rueda, Ariana
THIOCAP
Cysteine coupling
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
title_short Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
title_full Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
title_fullStr Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
title_full_unstemmed Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
title_sort Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)
dc.creator.none.fl_str_mv Rueda, Ariana
Mendoza, Julian I.
Alba-Castellon, Lorena
Parladé, Eloi
Voltà-Durán, Eric
Paez, David
Aviñó, Anna
Eritja Casadellà, Ramón
Vázquez, Esther
Villaverde, Antonio
Mangues, Ramón
Unzueta, Ugutz
author Rueda, Ariana
author_facet Rueda, Ariana
Mendoza, Julian I.
Alba-Castellon, Lorena
Parladé, Eloi
Voltà-Durán, Eric
Paez, David
Aviñó, Anna
Eritja Casadellà, Ramón
Vázquez, Esther
Villaverde, Antonio
Mangues, Ramón
Unzueta, Ugutz
author_role author
author2 Mendoza, Julian I.
Alba-Castellon, Lorena
Parladé, Eloi
Voltà-Durán, Eric
Paez, David
Aviñó, Anna
Eritja Casadellà, Ramón
Vázquez, Esther
Villaverde, Antonio
Mangues, Ramón
Unzueta, Ugutz
author2_role author
author
author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv THIOCAP
Cysteine coupling
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
topic THIOCAP
Cysteine coupling
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
description 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.]
publishDate 2023
dc.date.none.fl_str_mv 2023
2024
2024
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/360912
https://api.elsevier.com/content/abstract/scopus_id/85172098368
url http://hdl.handle.net/10261/360912
https://api.elsevier.com/content/abstract/scopus_id/85172098368
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Science China Materials
https://doi.org/10.1007/s40843-023-2571-6
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Springer Nature
publisher.none.fl_str_mv Springer Nature
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869406541971456000
spelling Site-directed cysteine coupling of disulfide-containing non-antibody carrier proteins (THIOCAPs)Rueda, ArianaMendoza, Julian I.Alba-Castellon, LorenaParladé, EloiVoltà-Durán, EricPaez, DavidAviñó, AnnaEritja Casadellà, RamónVázquez, EstherVillaverde, AntonioMangues, RamónUnzueta, UgutzTHIOCAPCysteine couplinghttp://metadata.un.org/sdg/3Ensure healthy lives and promote well-being for all at all agesThe 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.]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.Peer reviewedSpringer NatureConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202420242023info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/360912https://api.elsevier.com/content/abstract/scopus_id/85172098368reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésScience China Materialshttps://doi.org/10.1007/s40843-023-2571-6Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3609122026-05-22T06:33:51Z
score 15,228081

Similar Items