A research group led by Prof. Katsuhiko Mikoshiba from Shanghai Institute for Advanced Immunochemical Studies (SIAIS) at ShanghaiTech University proposes a novel and simple method for creating a stable cytoplasmic antibody, termed STAND, which can be correctly folded and stably expressed in the cytoplasm of the cells. Without the need for complicated amino acid substitutions, it successfully converts aggregation-prone antibodies to various STANDs that are fully functional in vivo.
Antibodies are powerful tools in basic research and proven candidates for therapeutic development because they specifically bind to antigens and interfere with targeted molecular pathways by inhibiting protein-protein interactions. While many key drug targets are intracellular molecules, most antibody-based therapies are limited to extracellular targets. Cytoplasmic expression of single-chain Fv (scFv), a most popular format of antibody genes, has been a long-standing challenge due to the tendency to misfold and aggregate; the cytoplasm is a reducing environment, where disulfide bridge formation within the Fvs of the light and heavy chains of scFv molecules can be prevented, thereby causing intracellular aggregation (Fig. 1). Currently, there is no reliable method to engineer stable cytoplasmic intrabodies in mammalian cells.
Here, the research group found a statistically significant negative correlation between aggregation and the net negative charge at pH 6.6, but not at pH 7.4 (Fig. 2a); we also found a statistically significant positive correlation between their pI value and aggregation rate (Fig. 2b). These results indicate that the low pI and strong net negative charge of the scFv antibodies at pH 6.6, but not at pH 7.4, are critical parameters for the stability of cytoplasmic antibodies. Fusion of the peptide tags with the strong net negative charge at pH 6.6 converts aggregation-prone scFvs to STANDs. The STAND for synaptotagmin, a Ca2+ sensor for neurotransmitter release, can be stably expressed in the cytoplasm of dopaminergic neurons (Fig. 3) and inhibit striatal dopamine release, thereby causing motor skill learning (Fig. 4). They also successfully convert aggregation-prone anti-Kras scFv to a STAND, which inhibits in vivo cancer proliferation by mutated https://doi.org/10.1038/s41467-019-13654-9
