Glioblastoma multiforme (GBM) is the most common neuroepithelial brain tumor and among the most clinically aggressive of all human tumor types. Due to its invasive nature, complete resection or radiation of this tumor is practically impossible, leading to recurrence in almost all patients. Since current therapeutics are not life-saving, there is a high unmet need for targeted therapies that eliminate residual cancer cells, while minimizing damage to healthy tissue. The epidermal growth factor receptor (EGFR) is often amplified and/or mutated in GBM of which the most common form is the EGFR mutant variant III (EGFRvIII). This unique tumor-restrictive biomarker for GBM is present in up to 60% of reported cases. Importantly, as opposed to wild-type EGFR, this mutated form of EGFR doesn’t occur in healthy tissues, which makes it an ideal marker for targeted therapies. Under guidance of the CMIM and ICMI research groups at VUB, nanobodies have been developed as novel vehicle for innovative diagnostic and therapeutic applications. Nanobodies are the smallest natural antigen-binding fragments, occurring from heavy-chain-only Camelidae antibodies. Their small size leads to better tissue penetration, favorable pharmacological properties and ability to recognize small, buried epitopes, such as present in EGFRvIII. Labeled with appropriate radionuclides, nanobodies can be used as a targeted radionuclide therapy (TRNT) option for EGFRvIII-expressing tumors. This project’s major aim is to generate nanobodies binding an EGFRvIII-specific epitope (i.e. nanobodies that do not recognize wild-type EGFR) as a tumor-restrictive vehicle for the development of a novel GBM molecular therapy. The specificity of the acquired nanobodies will be validated in vitro and in mouse models of EGFRvIII-positive GBM. As a first therapy option, nanobodies will be coupled to appropriate radionuclides and will be optimized to specifically deliver a radiotoxic load in, or in the vicinity of expanding EGFRvIII-positive cancer cells, including those developing in the brain. In conclusion, we will exploit the triple specificity of an exclusive tumor marker targeted with a highly specific nanobody and labeled with a short-range therapeutic radionuclide. We believe this will offer a more efficient and less toxic alternative than existing therapies and could provide a solution for cancer patients with yet untreatable cancers.