Targeting human T cells by retroviral vectors displaying antibody domains selected from a phage display library.
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.
Engelstädter
M
M
Department of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany.
Bobkova
M
M
Baier
M
M
Stitz
J
J
Holtkamp
N
N
Chu
T H
TH
Kurth
R
R
Dornburg
R
R
Buchholz
C J
CJ
Cichutek
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D001402:B-Lymphocytes / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D015531:Radioimmunoprecipitation Assay; D012190:Retroviridae / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D019151:Peptide Library