Selection and characterization of Her2 binding-designed ankyrin repeat proteins.
Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (Kd=7.3 nm) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.
Zahnd
Christian
C
Department of Biochemistry, University of Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
Pecorari
Frédéric
F
Straumann
Nadine
N
Wyler
Emanuel
E
Plückthun
Andreas
A
D000595:Amino Acid Sequence; D017487:Ankyrins / Q000737:chemistry* / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D001940:Breast / Q000378:metabolism; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D005260:Female; D005786:Gene Expression Regulation; D018734:Genes, erbB-2 / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012270:Ribosomes; D000068878:Trastuzumab
D017487:Ankyrins; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C485206:pertuzumab; D000068878:Trastuzumab