Development of brain-permeable masked nanobody-drug conjugates to eliminate glioma stem cells

Glioblastoma multiforme (GBM) is one of the most prevalent brain tumours and is currently incurable. Patients die from tumour recurrence after resection and chemotherapy. New treatments need to overcome two great challenges: reaching the tumour margins, where the blood-brain barrier (BBB) is intact, and eradicating glioma stem cells (GSCs), which cause tumour relapse. Targeting receptors overexpressed on cancer cells with antibody-drug conjugates (ADC) has shown great promise in cancer treatment. However, most receptors on GSCs are also present in normal stem cells. Thus, targeting these receptors with antibody-drug conjugates may lead to severe side effects. The main objective of this proposal is to develop a masked nanobody-drug conjugate with the capacity to overcome the BBB and to be activated only in the tumour to selectively target GSCs. Nanobodies have increased capacity to penetrate tissues and biological barriers but none is yet available to target the GSC marker CD133. Therefore, I will generate new nanobodies against this receptor. Subsequently, I will set up a phage display platform to develop masked nanobodies that can be selectively unmasked by tumour proteases. The masked nanobody will be conjugated to a drug and then to a BBB-shuttle peptide. A panel of conjugates will be screened in vitro for target selectivity, efficient activation and BBB transport. Finally, the efficacy of the most promising candidates will be tested in a GBM mouse model. Our unique combination will generate the first bispecific nanobody-drug conjugates targeting GSCs to treat GBM. The concept of GSC-targeting with activatable ADC may change the current paradigm for the treatment of brain tumours. Furthermore, our workflow may be applied to dramatically enhance the efficacy of other biotherapeutics with on-target off-site dose-limiting effects.