Development and characterization of protein kinase B/AKT isoform-specific nanobodies

The serine/threonine protein kinase AKT is frequently over-activated in cancer and is associated with poor prognosis. As a central node in the PI3K/AKT/mTOR pathway, which regulates various processes considered to be hallmarks of cancer, this kinase has become a prime target for cancer therapy. However, AKT has proven to be a highly complex target as it comes in three isoforms (AKT1, AKT2 and AKT3) which are highly homologous, yet non-redundant. The isoform-specific functions of the AKT kinases can be dependent on context (i.e. different types of cancer) and even opposed to one another. To date, there is no isoform-specific inhibitor available and no alternative to genetic approaches to study the function of a single AKT isoform. We have developed and characterized nanobodies that specifically interact with the AKT1 or AKT2 isoforms. These new tools should enable future studies of AKT1 and AKT2 isoform-specific functions. Furthermore, for both isoforms we obtained a nanobody that interferes with the AKT-PIP3-interaction, an essential step in the activation of the kinase. The nanobodies characterized in this study are a new stepping stone towards unravelling AKT isoform-specific signalling.