Quantitative ^99mTc Labeling Kit for HYNIC-Conjugated Single Chain Antibody Fragments Targeting Malignant Mesothelioma

Abstract

Single chain antibody fragment (scFv) is a promising agent for imaging and targeted therapy. The objective of the study is to evaluate a kit formulation for 99m Tc labeling of scFv for tumor imaging. The scFv was engineered to contain a cysteine tag to accommodate the specific conjugation of HYNIC and subsequent 99m Tc labeling. The labeling conditions were formulated to allow instantaneous one-pot quantitative labeling. The reproducibility of labeling was evaluated at various time points during kit storage at −20 °C. In vitro cell binding experiments and HPLC analysis were performed to assess binding affinity and radiolabel stability, respectively. In vivo tumor targeting study was performed in xenograft models with biodistribution studied at 1, 3, and 24 h post-injection. The optimized kit with 5 μg SnF 2 , pH 5.5, and 50 μg GH along with as low as 15 μg of HYNIC-cys-scFv provided high labeling yield (>95%), high specific activity (1.8 × 10 7 Ci/Mol), and robust reproducibility with shelf life up to 90 days when stored at −20 °C. The in vitro cell binding study showed the labeled scFv maintained the binding capability with an apparent K D of ∼27 nM. The animal study using tumor-bearing mice showed high tumor uptake at 16.9%ID/g 24 h post-injection along with rapid blood clearance (0.18%ID/g) and kidney excretion (44%ID/g), resulting in very high contrast (tumor/muscle >200:1). A kit formulation for 99m Tc labeling of scFvs targeting mesothelioma was developed based on specific HYNIC conjugation and GH (Glucoheptonate) as a coligand, producing not only high specific activity, but also improved tumor uptake. This convenient one-pot labeling method has the potential for translation into clinical use and is applicable to other scFvs as well.

Development and Characterization of 99mTc-scFvD2B as a Potential Radiopharmaceutical for SPECT Imaging of Prostate Cancer

“…99m Tc-HscFv1 ( 99m Tc-HYNIC-GGC-scFvD2B-Tag) was obtained after a 60 min incubation of HscFv1 with [ 99m Tc]-pertechnetate at 37 °C and neutral pH in the presence of two coligands (EDDA and tricine) to complete the metal coordination sphere. The radiolabeling yield (RLY) was assessed using SE-HPLC chromatography (using method A), as reported in the literature for other single-chain antibody fragments conjugated to HYNIC [ 36 ]. HPLC-chromatograms showed a main peak (RLY > 99%) with an Rt overlapping that of unlabeled HscFv1, and no presence of pertechnetate was observed ( Figure 5 A).…”

Synthesis and Bioevaluation of Novel Technetium-99m-Labeled Complexes with Norfloxacin HYNIC Derivatives for Bacterial Infection Imaging

“…The radiolabeling routes of HYNICNF are shown in Scheme . The speculative structures of 99m Tc-labeled HYNICNF compounds were referred to the previous literature. − The radiochemical purities of the complexes were determined by HPLC. The HPLC results of the complexes are shown in Figure .…”