PT  - JOURNAL ARTICLE
AU  - Zettlitz, Kirstin A.
AU  - Waldmann, Christopher M.
AU  - Tsai, Wen-Ting K.
AU  - Tavaré, Richard
AU  - Collins, Jeffrey
AU  - Murphy, Jennifer M.
AU  - Wu, Anna M.
TI  - A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for <sup>18</sup>F-Immuno-PET and Fluorescence Imaging
AID  - 10.2967/jnumed.118.223560
DP  - 2019 Oct 01
TA  - Journal of Nuclear Medicine
PG  - 1467--1473
VI  - 60
IP  - 10
4099  - http://jnm.snmjournals.org/content/60/10/1467.short
4100  - http://jnm.snmjournals.org/content/60/10/1467.full
SO  - J Nucl Med2019 Oct 01; 60
AB  - 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 biodistribution and fluorescence image–guided identification of tumor margins during surgery.