Synthesis and In Vitro Evaluation of a HER2-Specific ImmunoSCIFI Probe

Secondary Cerenkov-induced fluorescence imaging (SCIFI) is an emerging biomedical optical imaging modality that leverages Cerenkov luminescence, primarily generated by β-emitting radioisotopes, to excite fluorophores that offer near-infrared emissions with optimal tissue penetrance. Dual-functionalized immunoconjugates composed of an antibody, a near-infrared fluorophore, and a β-emitting radioisotope have potential utility as novel SCIFI constructs with high specificity for molecular biomarkers of disease. Here, we report the synthesis and characterization of [⁸⁹Zr]Zr-DFO-trastuzumab-BOD665, a self-excitatory HER2-specific "immunoSCIFI" probe capable of yielding near-infrared fluorescence in situ without external excitation. The penetration depth of the SCIFI signal was measured in hemoglobin-infused optical tissue phantoms that indicated a 2.05-fold increase compared to ⁸⁹Zr-generated Cerenkov luminescence. Additionally, the binding specificity of the immunoSCIFI probe for HER2 was evaluated in a cellular assay that showed significantly higher binding to SKBR3 (high HER2 expression) relative to MDA-MB-468 (low HER2) breast cancer cells based on measurements of total flux in the near-infrared region with external excitation blocked. Taken together, the results of this study indicate the potential utility of immunoSCIFI constructs for interrogation of molecular biomarkers of disease.