title: A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth.
abstract: The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies have been successfully used, such as cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In our study, we aimed to improve the efficacy of these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single biparatopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or cetuximab to EGFR and that did not compete for each others' binding. A combination of nanobodies from both epitope groups into the biparatopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this biparatopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, monospecific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of biparatopic nanobody-based anticancer therapeutics may yield potent lead molecules for further development.
authors.affiliation: Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands. r.c.roovers@uu.nl
authors.forename: Rob C
authors.initials: RC
authors.lastname: Roovers
authors.forename: Maria J W D
authors.initials: MJ
authors.lastname: Vosjan
authors.forename: Toon
authors.initials: T
authors.lastname: Laeremans
authors.forename: Rachid
authors.initials: R
authors.lastname: el Khoulati
authors.forename: Renée C G
authors.initials: RC
authors.lastname: de Bruin
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Ferguson
authors.forename: Arie J
authors.initials: AJ
authors.lastname: Verkleij
authors.forename: Guus A M S
authors.initials: GA
authors.lastname: van Dongen
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen en Henegouwen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000915:Antibody Affinity; D000918:Antibody Specificity; D002294:Carcinoma, Squamous Cell / Q000628:therapy*; D045744:Cell Line, Tumor; D000068818:Cetuximab; D000939:Epitopes; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000276:immunology*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D057127:Single-Chain Antibodies / Q000627:therapeutic use*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C574875:CONAN-1 nanobody; D000939:Epitopes; D057127:Single-Chain Antibodies; D066246:ErbB Receptors; D000068818:Cetuximab