Screening for scFv-fragments that are stable and active in the cytosol

Intrabodies are antibodies that are not secreted but bind to their antigens inside the cell producing them. Intrabodies targeting antigens in the endoplasmatic reticulum were successfully used in vitro and in vivo. However, many target antigens interesting for research or therapy are located in the reducing environment of the cytosol, where correct folding and formation of disulfide bonds cannot be ensured. The majority of different scFv fragments, when expressed in the cytosol of the cell, do not fold correctly, are not stable or cannot bind their antigen. Such scFv antibodies are therefore not suited as intrabodies.In this study, we evaluated fast and simple screening methods to identify scFv fragments that are stable and functional in the cytosol. We analyzed various phage display derived human scFv antibodies recognizing extracellular signal-regulated kinase 2 (Erk2) for stability and antigen binding under reducing and non-reducing conditions. Further, we developed an assay allowing to measure the interaction of the scFv intrabodies with their antigen in the cytosol of in living cells, by using a Split-Luciferase (Split-Luc) assay. ScFv fragments showing antigen binding in the cytosol could successfully be identified.