Targeting human T cells by retroviral vectors displaying antibody domains selected from a phage display library

被引:36
作者
Engelstädter, M
Bobkova, M
Baier, M
Stitz, J
Holtkamp, N
Chu, THT
Kurth, R
Dornburg, R
Buchholz, CJ
Cichutek, K
机构
[1] Paul Ehrlich Inst, Dept Med Biotechnol, D-63225 Langen, Germany
[2] Thomas Jefferson Univ, Div Infect Dis, Philadelphia, PA 19107 USA
关键词
D O I
10.1089/10430340050016030
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 10(6) 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 10(2) 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.
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页码:293 / 303
页数:11
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