TY - JOUR
T1 - Directed Evolution to Low Nanomolar Affinity of a Tumor-Targeting Epidermal Growth Factor Receptor-Binding Affibody Molecule
AU - Friedman, Mikaela
AU - Orlova, Anna
AU - Johansson, Eva
AU - Eriksson, Tove L.J.
AU - Höidén-Guthenberg, Ingmarie
AU - Tolmachev, Vladimir
AU - Nilsson, Fredrik Y.
AU - Ståhl, Stefan
N1 - Funding Information:
The authors would like to thank Prof. Per-Åke Nygren, John Löfblom (Department of Molecular Biotechnology, Royal Institute of Technology, Stockholm, Sweden), Andreas Jonsson, Magnus Hjertman, Gunilla Fant, Olof Widmark, Sara Nystedt, Barbro Baastrup, Lovisa Göstring, Anna Sjöberg, and Per Jonasson (Affibody AB, Bromma, Sweden) for valuable advice and experimental assistance. This study was funded by grant P25882-1 from the Swedish Governmental Agency for Innovation Systems (VINNOVA).
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2008/3/7
Y1 - 2008/3/7
N2 - The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (Kd = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, Kd, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (Kd = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.
AB - The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (Kd = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, Kd, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (Kd = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.
KW - Affibody
KW - affinity maturation
KW - EGFR
KW - phage display selection
KW - tumor targeting
UR - http://www.scopus.com/inward/record.url?scp=39149087035&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=39149087035&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2007.12.060
DO - 10.1016/j.jmb.2007.12.060
M3 - Article
C2 - 18207161
AN - SCOPUS:39149087035
VL - 376
SP - 1388
EP - 1402
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 5
ER -