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

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

机构地区：[1]Institut d'Investigació Biomèdica Sant Pau(IIB Sant Pau),Sant Quintí 77-79,08041 Barcelona,Spain [2]Josep Carreras Leukaemia Research Institute(IJC Campus Sant Pau),08041 Barcelona,Spain [3]CIBER de Bioingeniería,Biomateriales y Nanomedicina,Instituto de Salud Carlos Ⅲ,28029 Madrid,Spain [4]Institut de Biotecnologia i de Biomedicina,Universitat Autònoma de Barcelona,08193 Bellaterra,Spain [5]Departament de Genètica i de Microbiologia,Universitat Autònoma de Barcelona,08193 Bellaterra,Spain [6]Department of Medical Oncology,Hospital de la Santa Creu i Sant Pau.Barcelona,08025 Barcelona,Spain [7]CIBER de Enfermedades Raras(CIBERER),Instituto de Salud Carlos Ⅲ,28029 Madrid,Spain [8]Institute for Advanced Chemistry of Catalonia(IQAC),CSIC,08034 Barcelona,Spain

出　　处：《Science China Materials》2023年第10期4109-4120,共12页中国科学（材料科学（英文版）

基　　金：to ISCIII(PI20/00400)cofunded by European Regional Development Fund(ERDF,a way to make Europe);to CIBER-BBN(project NANOSCAPE and NANOLINK)granted to Unzueta U;to AEI(PID2019-105416RB-I00/AEI/10.13039/501100011033);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);to AGAUR(2021 SGR-01140)granted to Mangues R;to CIBER-BBN(VENOM4CANCER);AGAUR(2021 SGR-00092)granted to Villaverde A;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);by CERCA programme(Generalitat de Catalunya);supported by Miguel Servet contract(CP19/00028)from ISCIII co-funded by European Social Fund(ESF investing in your future);supported by the Spanish Association Against Cancer-AECC(POSTD20070ALBA);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);support from the National Institutes of Health R01-GM129325;the Office of Cyber Infrastructure and Computational Biology,National Institute of Allergy and Infectious Diseases。

摘　　要：The development of a new generation of nonantibody 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 onestep 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 nanocarrier 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.

关 键 词：DISULFIDE directed BRIDGES 

分 类 号：TQ460.1[化学工程—制药化工]