Proliferating tissues, including several breast tumors, harbor increased folate requirement and thereby often show high folate receptor 1 (FOLR1) expression while FOLR1 expression is restricted in most healthy tissues. Breast cancer is the second most common solid malignancy that metastasizes to the brain. Reaching the brain with therapeutics appears to be extremely challenging due to the presence of brain barriers. Interestingly, the host lab recently identified an anti-FOLR1 VHH that is able to enter the brain via blood-cerebrospinal fluid (CSF) barrier crossing. Based on these findings, we hypothesize that FOLR1-targeting VHHs have promising clinical potential to guide therapeutics and imaging agents to FOLR1 positive (brain) tumors. In this research project, I aim to identify anti-FOLR1 VHHs for (brain) tumor-targeted imaging and therapy. Therefore, I will screen an anti-FOLR1 VHH phage library, available in the host lab, through in vitro biopanning on FOLR1 positive choroid plexus epithelial and breast carcinoma cells. Additionally, I will screen this library through in vivo phage display using two orthotopic FOLR1 positive breast cancer mouse models. Relevant anti-FOLR1 VHHs will be characterized in detail in vitro and in vivo. Finally, the tumor and brain targeting activity of the selected anti-FOLR1 VHHs will be explored. In conclusion, this project will yield novel insights in the use of FOLR1-targeting VHHs as (brain) tumor-targeting anti-cancer therapeutics.