Novel Single-Chain Fv′ Formats for the Generation of Immunoliposomes by Site-Directed Coupling
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- 作者：Sylvia K. E. Messerschmidt ; Anke Kolbe ; Dafne Mü ; ller ; Michael Knoll ; Jü ; rgen Pleiss ; Roland E. Kontermann
- 刊名：Bioconjugate Chemistry
- 出版年：2008
- 出版时间：January 2008
- 年：2008
- 卷：19
- 期：1
- 页码：362 - 369
- 全文大小：1176K
- 年卷期：v.19,no.1(January 2008)
- ISSN：1520-4812
文摘
Immunoliposomes generated by coupling of antibodies to the liposomal surface allow for an active targeting of entrapped compounds to diseased areas. Single-chain Fv fragments (scFv) represent the smallest part of an antibody containing the entire antigen-binding site. They can be coupled in a defined and site-directed manner through genetically engineered cysteine residues, for example, those added at the C-terminus. Here, we have performed a comparative analysis of various scFv′ variants with cysteine residues present at the end of a C-terminal extension of varying length and composition (HC variants) or introduced in the linker sequence connecting the variable heavy and light chain domain (LC variants). Using a scFv fragment directed against fibroblast activation protein (FAP) as a model antibody, we could show that all variants can be employed for the generation of active immunoliposomes, although the presence of three additional cysteine residues in one scFv′ molecule resulted in decreased binding of immunoliposomes compared to that of immunoliposomes generated with scFv′ molecules containing only one additional cysteine residue. In order to further improve the scFv′ format by reducing the number of additional amino acid residues, we also generated molecules with the hexahistidyl-tag incorporated into the linker sequence together with a cysteine residue either at position 1 or 3 of the linker sequence (LCH variants). These newly designed scFv′ molecules may be particularly suitable for the generation of immunoliposomes and other antibody conjugates, limiting the number of additional residues in these antibody molecules to a minimum.