title: Inhibiting HER3-mediated tumor cell growth with affibody molecules engineered to low picomolar affinity by position-directed error-prone PCR-like diversification.
abstract: The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Malm
authors.forename: Nina
authors.initials: N
authors.lastname: Kronqvist
authors.forename: Hanna
authors.initials: H
authors.lastname: Lindberg
authors.forename: Lindvi
authors.initials: L
authors.lastname: Gudmundsdotter
authors.forename: Tarek
authors.initials: T
authors.lastname: Bass
authors.forename: Fredrik Y
authors.initials: FY
authors.lastname: Frejd
authors.forename: Ingmarie
authors.initials: I
authors.lastname: Höidén-Guthenberg
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Varasteh
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
mesh_terms: D000409:Alanine; D000818:Animals; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D061505:Cell Surface Display Techniques; D002942:Circular Dichroism; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D051379:Mice; D009154:Mutation; D009369:Neoplasms / Q000378:metabolism*; D019151:Peptide Library; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D020893:Receptor, ErbB-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D008024:Ligands; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D020893:Receptor, ErbB-3; D000409:Alanine