Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells

Targeted gene transfer by nonviral vectors can be achieved through incorpor ation 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 ScF v, 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 extracellu lar domain of the tumor surface protein, ErbB2. Two approaches were taken: (a) development of a vector that is composed of a bifunctional fusion prote in capable of binding DNA with the ErbB2-specific ML39 ScFv at its N-termin us and a truncated form of human protamine at its C-terminus, and (b) formu lation and evaluation of delivery vectors consisting of three independent c omponents including ML39 ScFv, protamine, and cationic lipids. We demonstra te 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, protamin e, and lipids in vitro could even more efficiently deliver the reporter gen e into ErbB2( +) cells with approximately 5-fold increase in gene expressio n in ErbB2( +) cell as compared to ErbB2( -) cells. Expression and refoldin g of the ScFv fusion proteins, in addition to determination of optimal cond itions for vector development using these approaches, are discussed.