title: DARPins: an efficient targeting domain for lentiviral vectors.
abstract: We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.
authors.affiliation: Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Münch
authors.forename: Michael D
authors.initials: MD
authors.lastname: Mühlebach
authors.forename: Thomas
authors.initials: T
authors.lastname: Schaser
authors.forename: Sabrina
authors.initials: S
authors.lastname: Kneissl
authors.forename: Christian
authors.initials: C
authors.lastname: Jost
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
authors.forename: Klaus
authors.initials: K
authors.lastname: Cichutek
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Buchholz
mesh_terms: D002460:Cell Line; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D016086:Lentivirus / Q000235:genetics*; D008459:Measles virus / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases; D057127:Single-Chain Antibodies; D014760:Viral Fusion Proteins