title: Downregulation of EGFR by a novel multivalent nanobody-liposome platform.
abstract: The epidermal growth factor receptor (EGFR) is a recognized target for tumor therapy and monoclonal antibodies (mAbs, e.g. cetuximab) have been developed to inhibit receptor activation. Besides blocking ligand (e.g. EGF) binding to the receptor, reports have shown that mAbs promote slow receptor internalization and degradation in lysosomes, i.e. downregulation. The efficacy of receptor downregulation was recently shown to depend on the size of receptor clusters formed at the cell surface. In this study, a multivalent platform is presented, consisting of nanobodies recognizing the ectodomain of EGFR (EGa1) coupled to PEG-liposomes, and the in vitro and in vivo effects of this system on EGFR internalization and downregulation were investigated. Nanobodies are the smallest functional antigen-binding immunoglobulin fragments and the EGa1 nanobody has been described as an EGFR-antagonist. EGa1-liposomes (EGa1-L) induced a more than 90% removal of EGFR from the cell surface, as a result of receptor internalization. Furthermore, this massive sequestration of EGFR mediated by EGa1-L lead to receptor degradation, while no degradation was detected with the monovalent nanobody. The downregulatory capacity here reported was found to be independent of the epitope on EGFR recognized by the grafted nanobody, and exclusive to the nanobody-liposomes, as anti-EGFR single chain variable fragments (scFv) coupled to liposomes were unable to induce this effect. Importantly, EGa1-L induced a significant inhibition of tumor cell proliferation, in vitro, an effect likely mediated by the combination of receptor downregulation and receptor antagonism. Also in vivo, EGFR downregulation was observed in tumors of mice intravenously injected with EGa1-L, indicating that this multivalent platform blocks ligand binding to the receptor and simultaneously induces the downregulation of EGFR.
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Oliveira
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Schiffelers
authors.forename: Joris
authors.initials: J
authors.lastname: van der Veeken
authors.forename: Roy
authors.initials: R
authors.lastname: van der Meel
authors.forename: Ranitha
authors.initials: R
authors.lastname: Vongpromek
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen En Henegouwen
authors.forename: Gert
authors.initials: G
authors.lastname: Storm
authors.forename: Rob C
authors.initials: RC
authors.lastname: Roovers
mesh_terms: D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects*; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005260:Female; D019085:Fluorescent Antibody Technique, Direct; D006258:Head and Neck Neoplasms / Q000473:pathology / Q000628:therapy; D006801:Humans; D008024:Ligands; D008081:Liposomes / Q000276:immunology / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: D000939:Epitopes; D008024:Ligands; D008081:Liposomes; D066246:ErbB Receptors