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Targeting Human T Cells by Retroviral Vectors Displaying Antibody Domains Selected from a Phage Display Library
- Source :
- Human Gene Therapy. 11:293-303
- Publication Year :
- 2000
- Publisher :
- Mary Ann Liebert Inc, 2000.
-
Abstract
- 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.
- Subjects :
- 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
Subjects
Details
- ISSN :
- 15577422 and 10430342
- Volume :
- 11
- Database :
- OpenAIRE
- Journal :
- Human Gene Therapy
- Accession number :
- edsair.doi.dedup.....e36eefc8d0adfca5be08202d98845df7