Antigen-independent selection of intracellular stable antibody frameworks.
The intracellular expression of highly specific antibody fragments ("intrabodies") in eukaryotes has a great potential in functional genomics and therapeutics. However, since the intracellular reducing environment prevents formation of the conserved intrachain disulfide bonds, most antibodies do not fold properly and are therefore inactive inside cells. The few antibodies that have been found to function in an intracellular environment and that have been characterized for their biophysical properties have generally shown a high degree of stability and solubility. Thus, for intracellular expression and application, very stable antibody frameworks are needed that can correctly fold even in the absence of disulfide bonds and that do not aggregate. Here, we present and discuss a novel method, named "Quality Control," which allows selection of stable and soluble antibody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single-chain antibody fragments (scFvs) fused to a selectable marker that can control gene expression and cell growth. The activity of such a selectable marker fused to various scFvs that have been biophysically characterized correlated with the solubility and stability of the scFv moieties. This antigen-independent intrabody selection system was applied to screen scFv libraries for identifying stable and soluble frameworks, which subsequently served as acceptor backbones to construct intrabody libraries by randomization of hypervariable loops.
ESBATech AG, Wagistrasse 21, CH-8952 Zurich-Schlieren, Switzerland.
Adrian
A
Auf der Maur
Kathrin
K
Tissot
Alcide
A
Barberis
D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D000941:Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology
D000906:Antibodies; D000941:Antigens