A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for (18)F-Immuno-PET and Fluorescence Imaging

• Published in: Journal of Nuclear Medicine
• Main Authors: Zettlitz, Kirstin A., Waldmann, Christopher M., Tsai, Wen-Ting K., Tavaré, Richard, Collins, Jeffrey, Murphy, Jennifer M., Wu, Anna M.
• Format: Text
• Language: English
• Published: Society of Nuclear Medicine 2019
• Subjects: Molecular Imaging; DML
• Online Access: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785789/; http://www.ncbi.nlm.nih.gov/pubmed/30877181; https://doi.org/10.2967/jnumed.118.223560

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue–bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with (18)F-labeled trans-cyclooctene ((18)F-TCO). To generate a dual-modality antibody fragment–based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with (18)F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and (18)F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with (18)F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after 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 DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level ...