Abstract
Rationale: Antibody-based dual-modality (PET/fluorescence) imaging enables both pre-surgery antigen-specific immunoPET for non-invasive whole-body evaluation and intra-operative fluorescence for visualization of superficial tissue layers for image guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates 1) site-specific conjugation to a cysteine residue bearing antibody fragment; 2) introduction of a commercially available fluorescent dye (via amine-reactive prosthetic group); 3) rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment based imaging agent, the DML was labeled with the far-red dye sulfo-Cy5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2 (A2cDb), and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immunoPET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a dual-modality linker (DML) for conjugation of a fluorescent dye and an 18F-radioisotope. The anti-PSCA A2cDb was site-specifically conjugated with either DML or DML-sCy5 and radiolabeled via click chemistry with 18F-TCO. ImmunoPET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h post injection. Rapid renal clearance of the 50 kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The dual-modality linker (DML) enables site-specific conjugation away from the antigen-binding site of antibody fragments, with controlled linker-to-protein ratio and combines signaling moieties for two imaging systems into one molecule. Dual-modality imaging could provide both non-invasive whole body imaging with organ level biodistribution and fluorescent image guided identification of tumor margins during surgery.
- Molecular Imaging
- Optical
- Radioimmunoimaging
- ImmunoPET
- click chemistry
- cys-diabody
- fluorescence image-guided surgery
- fluorine-18
- Copyright © 2019 by the Society of Nuclear Medicine and Molecular Imaging, Inc.