An Engineered Peptide for Non-Invasive Optical Imaging of Brain Tumors

The majority of central nervous system (CNS) tumors carry grave clinical prognoses due to the limited effectiveness of surgical resection, radiation, and chemotherapy.  Thus, improved strategies are critically needed for brain tumor visualization and targeted treatment.  We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine-knot (knottin) peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrin receptors.  This integrin-binding knottin, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB.  Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared to the normal surrounding brain tissue, as measured by optical imaging. The intensity of the imaging signal correlated with tumor volume.  Due to its unique ability to bind to α₅β₁ integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared to other engineered integrin-binding knottin peptides and to c(RGDfK), a well-studied integrin-binding peptidomimetic.  Next, EETI 2.5F was fused to an antibody Fc domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also access intracranial brain tumors.  EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following intravenous injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity.  These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging.