title: Receptor-targeted lentiviral vectors are exceptionally sensitive toward the biophysical properties of the displayed single-chain Fv.
abstract: An increasing number of applications require the expression of single-chain variable fragments (scFv) fusion proteins in mammalian cells at the cell surface membrane. Here we assessed the CD30-specific scFv HRS3, which is used in immunotherapy, for its ability to retarget lentiviral vectors (LVs) to CD30 and to mediate selective gene transfer into CD30-positive cells. Fused to the C-terminus of the type-II transmembrane protein hemagglutinin (H) of measles virus and expressed in LV packaging cells, gene transfer mediated by the released LV particles was inefficient. A series of point mutations in the scFv framework regions addressing its biophysical properties, which substantially improved production and increased the melting temperature without impairing its kinetic binding behavior to CD30, also improved the performance of LV particles. Gene transfer into CD30-positive cells increased ∼100-fold due to improved transport of the H-scFv protein to the plasma membrane. Concomitantly, LV particle aggregation and syncytia formation in packaging cells were substantially reduced. The data suggest that syncytia formation can be triggered by trans-cellular dimerization of H-scFv proteins displayed on adjacent cells. Taken together, we show that the biophysical properties of the targeting ligand have a decisive role for the gene transfer efficiency of receptor-targeted LVs.
authors.affiliation: Section of Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Paul-Ehrlich-Str. 51-59, Langen 63225, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Friedel
authors.affiliation: Roche Pharmaceutical Research and Early Development, Protein Engineering Group, Roche Innovation Center Zürich, Schlieren 8952, Switzerland.
authors.forename: Lydia J
authors.initials: LJ
authors.lastname: Hanisch
authors.affiliation: Section of Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Paul-Ehrlich-Str. 51-59, Langen 63225, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Muth
authors.affiliation: Department of Biochemistry, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Honegger
authors.affiliation: Center for Molecular Medicine Cologne, University of Cologne, Cologne 50931, Germany Department I of Internal Medicine, University Hospital Cologne, Cologne 50931, Germany.
authors.forename: Hinrich
authors.initials: H
authors.lastname: Abken
authors.affiliation: Department of Biochemistry, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
authors.affiliation: Section of Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Paul-Ehrlich-Str. 51-59, Langen 63225, Germany christian.buchholz@pei.de.
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Buchholz
authors.affiliation: Section of Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Paul-Ehrlich-Str. 51-59, Langen 63225, Germany.
authors.forename: Irene C
authors.initials: IC
authors.lastname: Schneider
mesh_terms: D000818:Animals; D002460:Cell Line; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007167:Immunotherapy; D017730:Ki-1 Antigen / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D016086:Lentivirus / Q000235:genetics; D008024:Ligands; D051379:Mice; D017510:Protein Folding; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D007128:Immunoglobulin Fragments; D017730:Ki-1 Antigen; D008024:Ligands; D057127:Single-Chain Antibodies
keywords: CD30; HRS3; lentiviral vector; particle aggregation; scFv interface