The fate of an anti-HER2 nanobody: mode of action studies in cancer cells

Breast cancer is the leading cause of female cancer death worldwide. Human epidermal growth factor 2 (HER2) is a transmembrane receptor, overexpressed in about 20-30% of breast cancer and when activated, is related to tumor cell multiplication and invasion, resulting in distant metastases. HER2 is thus a potent target for effective therapeutic strategy. In a previous project in the lab, we developed a highly potent camelid-derived nanobody, designated NB46, targeting HER2 in its active conformation (i.e., as a HER2/3 heterodimer). In the current project, we focused on its mode of action in breast cancer cells. Cell-based assays, using HEK293 cells overexpressing HER2, HER3, or both, showed HER3-independent, specific binding of NB46 to HER2. Importantly, we show that there is no competition in binding to cells between NB46 and Trastuzumab and Pertuzumab, monoclonal antibodies that are currently in clinical use. In addition, NB46 has no effect on HER2 recycling, in contrast to treatment with Trastuzumab and Pertuzumab, which cause degradation of HER2, its depletion and consequently drug resistance mechanisms. HER2-dependent binding and internalization of NB46 were confirmed using HER2 silencing. Live imaging showed that NB46 internalizes into the cancer cell cytoplasm in HER2 dependent process. NB46 had no effect on activation (i.e., phosphorylation) of HER2 and cell viability in breast cancer cells. Our results therefore suggest that NB46 has a potential as a drug delivery agent for the treatment of breast cancer.