Targeting Human T Cells by Retroviral Vectors Displaying Antibody Domains Selected from a Phage Display Library
| Autor: | Te-Hua Tearina Chu, Klaus Cichutek, Reinhard Kurth, Martin Engelstädter, Maria Bobkova, Ralph Dornburg, Michael Baier, Christian J. Buchholz, Nicola Holtkamp, Jörn Stitz |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Radioimmunoprecipitation Assay
Phage display T-Lymphocytes viruses T cell Blotting Western Genetic Vectors Molecular Sequence Data Jurkat cells Cell Line Mice Transduction (genetics) Viral envelope Peptide Library Genetics medicine Animals Humans Amino Acid Sequence Immunoglobulin Fragments Molecular Biology B-Lymphocytes Mice Inbred BALB C biology HEK 293 cells Gene Transfer Techniques Antibodies Monoclonal Flow Cytometry Virology Molecular biology Retroviridae medicine.anatomical_structure Cell culture Leukocytes Mononuclear biology.protein Molecular Medicine Antibody HeLa Cells |
| Zdroj: | Human Gene Therapy. 11:293-303 |
| ISSN: | 1557-7422 1043-0342 |
| Popis: | To generate T cell-specific retroviral vectors an scFv phage display library derived from immunized mice was selected for binding to the human T cell line Molt-4/8. The scFv cDNAs recovered from the selected phages were transiently expressed as an N-terminal fusion of the spleen necrosis virus (SNV) transmembrane protein (TM) subunit of the viral envelope protein (Env) in the cell line DSH-cxl, which packages the beta-galactosidase gene into SNV particles. Screening of supernatants from about 150 transfections resulted in the identification of 5 scFvs that mediated efficient transduction of Molt-4/8 cells. Using stable packaging cell lines vector preparations with titers greater than 10(4) EFU/ml on human T cells were obtained. The scFv 7A5 in particular was able to mediate selective transduction of human T cells with high efficiency. Titers of up to 106 EFU/ml were reached on Molt-4/8, Jurkat, and A301 cells, while titers on HeLa cells, TE671 cells, 293T cells, and HT1080 cells were below 102 EFU/ml. Transduction of stimulated primary human peripheral blood cells, which consisted mainly of T cells, was about fivefold more efficient than transduction of B cells. Western blot analysis of supernatant from the 7A5 packaging cells demonstrated incorporation of 7A5-TM into vector particles and indicated proteolytic processing of the coexpressed unmodified TM during particle formation. Binding of bacterially expressed 7A5-scFv to a panel of cell lines correlated well with the transduction results. These data provide the first proof of concept that a general approach can be taken to obtain scFvs able to mediate selective gene transfer into target cells. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|