Messerschmidt Sylvia K E, Kolbe Anke, Müller Dafne, Knoll Michael, Pleiss Jürgen, Kontermann Roland E
Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Bioconjug Chem. 2008 Jan;19(1):362-9. doi: 10.1021/bc700349k. Epub 2007 Nov 8.
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.
通过将抗体偶联到脂质体表面而产生的免疫脂质体能够将包封的化合物主动靶向到病变区域。单链Fv片段（scFv）代表抗体中包含整个抗原结合位点的最小部分。它们可以通过基因工程改造的半胱氨酸残基，例如在C末端添加的那些残基，以确定的、位点定向的方式进行偶联。在此，我们对各种scFv'变体进行了比较分析，这些变体在不同长度和组成的C末端延伸（HC变体）末端或连接可变重链和轻链结构域的接头序列（LC变体）中存在半胱氨酸残基。使用针对成纤维细胞活化蛋白（FAP）的scFv片段作为模型抗体，我们可以表明所有变体都可用于生成活性免疫脂质体，尽管与仅含有一个额外半胱氨酸残基的scFv'分子生成的免疫脂质体相比，一个scFv'分子中存在三个额外半胱氨酸残基会导致免疫脂质体的结合减少。为了通过减少额外氨基酸残基的数量进一步改进scFv'形式，我们还生成了接头序列中同时含有六组氨酸标签以及接头序列第1或3位的半胱氨酸残基的分子（LCH变体）。这些新设计的scFv'分子可能特别适合用于生成免疫脂质体和其他抗体偶联物，将这些抗体分子中的额外残基数量限制到最少。
