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  • Title: Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells.
    Author: Li X, Stuckert P, Bosch I, Marks JD, Marasco WA.
    Journal: Cancer Gene Ther; 2001 Aug; 8(8):555-65. PubMed ID: 11571533.
    Abstract:
    Targeted gene transfer by nonviral vectors can be achieved through incorporation of specific ligand(s) into the vectors. In this study, the effects of incorporation of an anti-ErbB2 single-chain antibody fragment (ScFv) into nonviral vectors for targeted gene delivery were investigated. The ML39 ScFv, selected from a human ScFv phage display library and affinity matured in vitro (K(d)=1 x 10(-9) M), was used as ligand specific for the extracellular domain of the tumor surface protein, ErbB2. Two approaches were taken: (a) development of a vector that is composed of a bifunctional fusion protein capable of binding DNA with the ErbB2-specific ML39 ScFv at its N-terminus and a truncated form of human protamine at its C-terminus, and (b) formulation and evaluation of delivery vectors consisting of three independent components including ML39 ScFv, protamine, and cationic lipids. We demonstrate that fusion proteins comprised of the ML39 ScFv and a truncated form of protamine, denoted as ScFv-P-S, can selectively deliver exogenous DNA into ErbB2(+) cells, with an 8- to 10-fold increase in expression levels of the luciferase reporter gene in ErbB2(+) cells as compared to ErbB2(-) cells. In addition, vectors formulated by appropriately mixing DNA, ScFv, protamine, and lipids in vitro could even more efficiently deliver the reporter gene into ErbB2(+) cells with approximately 5-fold increase in gene expression in ErbB2(+) cell as compared to ErbB2(-) cells. Expression and refolding of the ScFv fusion proteins, in addition to determination of optimal conditions for vector development using these approaches, are discussed.
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