Noninvasive Evaluation of EGFR Expression of Digestive Tumors Using ^99mTc-MAG₃-Cet-F(ab′)₂-Based SPECT/CT Imaging

Abstract

Purpose. This study is aimed at investigating the feasibility of cetuximab (Cet) F(ab ′ )2 fragment- (Cet-F(ab ′ )2-) based single photon emission tomography/computed tomography (SPECT/CT) for assessing the epidermal growth factor receptor (EGFR) expression in digestive tumor mouse models. Methods. Cet-F(ab ′ )2 was synthesized using immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) protease and purified with protein A beads. The product and its in vitro stability in normal saline and 1% bovine serum albumin were analyzed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The EGFR expression in the human colon tumor cell line HT29 and the human stomach tumor cell line MGC803 were verified using western blotting and immunocytochemistry. Cet-F(ab ′ )2 was conjugated with 5(6)-carboxytetramethylrhodamine succinimidyl ester to demonstrate its binding ability to the MGC803 and HT29 cells. Cet-F(ab ′ )2 was conjugated with NHS-MAG3 for 99mTc radiolabeling. The best imaging time was determined using a biodistribution assay at 1, 4, 16, and 24 h after injection of the 99mTc-MAG3-Cet-F(ab ′ )2 tracer. Furthermore, 99mTc-MAG3-Cet-F(ab ′ )2 SPECT/CT was performed on MGC803 and HT29 tumor-bearing nude mice. Results. HT29 cells had low EGFR expression while MGC803 cell exhibited the high EGFR expression. Cet-F(ab ′ )2 and intact cetuximab showed similar high binding ability to MGC803 cells but not to HT29 cells. Cet-F(ab ′ )2 and 99mTc-MAG3-Cet-F(ab ′ )2 showed excellent in vitro stability. The biodistribution assay showed that the target to nontarget ratio was the highest at 16 h ( 17.29 ± 5.72 , n = 4 ) after tracer injection. The 99mTc-MAG3-Cet-F(ab ′ )2-based SPECT/CT imaging revealed rapid and sustained tracer uptake in MGC803 tumors rather than in HT29 tumors with high image contrast, which was consistent with the results in vitro. Conclusion. SPECT/CT imaging using 99mTc-MAG3-Cet-F(ab ′ )2 enables the evaluation of the EGFR expression in murine EGFR-positive tumors, indicating the potential utility for noninvasive evaluation of the EGFR expression in tumors.

Preclinical Evaluation of 99mTc-MAG3-5-Fab Targeting TREM2 in Lung Cancer Mouse Models: A Comparison with 99mTc-MAG3-5-F(ab′)2

“…This may be related to the smaller molecular mass, better tissue penetration, and lower background uptake of 99m Tc-MAG 3 -5-Fab . Similarly, Cetuximab-Fab can obtain images with a high T/NT ratio within 0.5 h after injection; Cetuximab-F­(ab′) 2 required 16 h after the tracer injection. , The hTREM2 – A549 tumors could not be visualized, demonstrating the favorable specificity of 99m Tc-MAG 3 -5-Fab. The biodistribution analysis results supported the micro-SPECT/CT imaging results, and both probes were metabolized through the kidneys.…”

“…The 99m Tc-MAG 3 -5-F­(ab′) 2 and 99m Tc-MAG 3 -5-Fab preparation process is shown in Figure . The detailed experimental procedure for 99m Tc labeling was similar to that in our previous study . Briefly, an MAG 3 -5-Fab (45 μg)/ MAG 3 -5-F­(ab′) 2 (90 μg) conjugate was added to a combined solution of 45 μL of ammonium acetate (0.25 M) and 15 μL of tartrate buffer, and 25 μL of 99m TcO 4 – was added.…”

“…The detailed experimental procedure for 99m Tc labeling was similar to that in our previous study. 17 Briefly, an MAG 3 -5-Fab (45 μg)/ MAG 3 -5-F(ab′) 2 (90 μg) conjugate was added to a combined solution of 45 μL of ammonium acetate (0.25 M) and 15 μL of tartrate buffer, and 25 μL of 99m TcO 4 − was added. Immediately after vortexing, 3 μL of a freshly prepared 4 mg/mL SnCl 2 •2H 2 O solution was added.…”

Synthesis, Screening, and Evaluation of Theranostic Molecular CPCR4-Based Probe Targeting CXCR4

“…Radiochemical syntheses were performed according to the reported literature − 99m TcO 4 – and 131 I – were purchased from Shanghai Xinke Pharmaceutical (Shanghai, China) and Shanghai Atom Kexing Pharmaceuticals (Shanghai, China). 124 I – and 18 F – were produced by the Department of Nuclear Medicine, Zhongshan Hospital, Fudan University.…”