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

• Main Authors: Zettlitz, Kirstin A, Waldmann, Christopher M, Tsai, Wen-Ting K, Tavaré, Richard, Collins, Jeffrey, Murphy, Jennifer M, Wu, Anna M
• Format: Article in Journal/Newspaper
• Language: unknown
• Published: eScholarship, University of California 2019
• Subjects: Biomedical and Clinical Sciences; Clinical Sciences; Bioengineering; Biomedical Imaging; Cancer; Prostate Cancer; Biotechnology; Urologic Diseases; Animals; Antibodies; Antigens; Neoplasm; Carbocyanines; Cyclooctanes; Cysteine; Fluorescence; Fluorescent Dyes; Fluorine Radioisotopes; GPI-Linked Proteins; Humans; Immunoglobulin Fragments; Male; Mice; Microscopy; Neoplasm Proteins; Neoplasm Transplantation; Optical Imaging; Positron-Emission Tomography; Prostatic Neoplasms; Radiopharmaceuticals; Tissue Distribution; immuno-PET; fluorescence image; guided surgery; cys-diabody; F-18; click chemistry; 18F; fluorescence image-guided surgery; Nuclear Medicine & Medical Imaging; DML
• Online Access: https://escholarship.org/uc/item/7512h39h

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 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 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 18F-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 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-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 ...