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

Abstract

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 residuebearing 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 farred 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 PSCApositive 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.

Tri-functional platform for construction of modular antibody fragments for in vivo18F-PET or NIRF molecular imaging

“…18a,19b Previously, our group reported a dual-modality linker for conjugation to a cys-diabody for imaging PSCA expression. 21 With a molecular weight of $53 kDa, below the threshold for rst-pass renal clearance, the initial clearance route for the bioconjugate was via the kidneys. Radiometabolites were reabsorbed and ultimately routed through the hepatobiliary system, resulting in extended blood pool activity and retention in the liver, kidneys and gastrointestinal tract (GI), presumably due to high lipophilicity of tracer radiometabolites.…”

“…Radiometabolites were reabsorbed and ultimately routed through the hepatobiliary system, resulting in extended blood pool activity and retention in the liver, kidneys and gastrointestinal tract (GI), presumably due to high lipophilicity of tracer radiometabolites. 21 Size, structure and charge distribution are key properties that inuence renal retention of radiolabeled metabolites. 22 As a result of this study, the tri-functional platform was designed to preserve kidney retention of radiometabolites, reduce liver and GI activity and provide a clean background in the lower abdomen by incorporating an anionic moiety to decrease lipophilicity.…”

“…3B). 21 Many PET probes are metabolized via the hepatobiliary route, resulting in high liver signal which hinders the ability to visualize liver metastasis. In order to maintain low background signal in the liver and generate high contrast images, renal clearance is highly desirable for PET molecular imaging probes.…”

A promising cancer diagnosis and treatment strategy: targeted cancer therapy and imaging based on antibody fragment

“…Dual-modality imaging, which provides non-invasive assessment of preoperative disease and provides real-time fluorescence imaging of tumour margins during surgery, is an important research direction in tumour imaging [64]. Dualmodality imaging results of multiple imaging can be mutually verified, so the imaging results of multimodal imaging are more accurate and reliable than single-modal imaging [65,66].…”

“…Dualmodality imaging results of multiple imaging can be mutually verified, so the imaging results of multimodal imaging are more accurate and reliable than single-modal imaging [65,66]. For example, a dual-labelled bispecific antibody 64 Cu-NOTA-heterodimer-ZW800 was synthesized though biorthogonal click chemistry [67]. The heterodimer antibody which can target both tissue factor and CD105 has stronger specific binding ability than homodimers antibody.…”