Previously, we used directed evolution to engineer mutants of the Ecballium elaterium
trypsin inhibitor (EETI-II)
knottin that bind to αvβ3 and αvβ5
integrin receptors with low nanomolar affinity, and showed that Cy5.5- or (64)Cu-DOTA-labeled
knottin peptides could be used to image
integrin expression in mouse
tumor models using near-infrared fluorescence (NIRF) imaging or positron emission tomography (PET). Here, we report the development of a dual-labeled
knottin peptide conjugated to both NIRF and PET imaging agents for multimodality imaging in living subjects. We created an orthogonally protected
peptide-based linker for stoichiometric coupling of (64)Cu-DOTA and
Cy5.5 onto the
knottin N-terminus and confirmed that conjugation did not affect binding to αvβ3 and αvβ5
integrins. NIRF and PET imaging studies in
tumor xenograft models showed that
Cy5.5 conjugation significantly increased kidney uptake and retention compared to the
knottin peptide labeled with (64)Cu-DOTA alone. In the
tumor, the dual-labeled (64)Cu-
DOTA/
Cy5.5 knottin peptide showed decreased wash-out leading to significantly better retention (p < 0.05) compared to the (64)Cu-DOTA-labeled
knottin peptide.
Tumor uptake was significantly reduced (p < 0.05) when the dual-labeled
knottin peptide was coinjected with an excess of unlabeled competitor and when tested in a
tumor model with lower levels of
integrin expression. Finally, plots of
tumor-to-background tissue ratios for
Cy5.5 versus (64)Cu uptake were well-correlated over several time points post injection, demonstrating pharmacokinetic cross validation of imaging labels. This dual-modality NIRF/PET imaging agent is promising for further development in clinical applications where high sensitivity and high resolution are desired, such as detection of
tumors located deep within the body and image-guided surgical resection.