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 (, ) 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.

中文翻译：

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使用基于 99mTc-MAG3-Cet-F(ab)2 的 SPECT/CT 成像对消化道肿瘤的 EGFR 表达进行无创评估

目的。本研究旨在调查基于西妥昔单抗 (Cet) F(ab ) ₂片段 (Cet-F(ab ) ₂ -) 的单光子发射断层扫描/计算机断层扫描 (SPECT/CT) 用于评估表皮生长因子受体的可行性(EGFR) 在消化道肿瘤小鼠模型中的表达。方法。Cet-F(ab ) _2是使用化脓性链球菌的免疫球蛋白 G 降解酶合成的(IdeS) 蛋白酶并用蛋白 A 珠纯化。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析产物及其在生理盐水和 1% 牛血清白蛋白中的体外稳定性。使用蛋白质印迹和免疫细胞化学验证人结肠肿瘤细胞系HT29和人胃肿瘤细胞系MGC803中的EGFR表达。Cet-F(ab ) ₂与 5(6)-羧基四甲基罗丹明琥珀酰亚胺酯缀合以证明其与 MGC803 和 HT29 细胞的结合能力。Cet-F(ab ) ₂与 NHS-MAG ₃结合用于^99m Tc 放射性标记。最佳成像时间使用生物分布测定在注射后 1、4、16 和 24 小时确定^99m Tc-MAG ₃ -Cet-F(ab ) ₂示踪剂。此外，对MGC803和HT29荷瘤裸鼠进行了^99m Tc-MAG ₃ -Cet-F(ab ) _{2 SPECT/CT。}结果。HT29细胞具有低EGFR表达，而MGC803细胞表现出高EGFR表达。Cet-F(ab ) ₂和完整的西妥昔单抗对 MGC803 细胞显示出相似的高结合能力，但对 HT29 细胞没有。Cet-F(ab ) ₂和^99m Tc-MAG ₃ -Cet-F(ab ) ₂表现出优异的体外稳定性。生物分布测定表明，目标与非目标的比率在 16 h 时最高（, )示踪剂注射后。基于^99m Tc-MAG ₃ -Cet-F(ab ) ₂的 SPECT/CT 成像显示在 MGC803 肿瘤中而不是在具有高图像对比度的 HT29 肿瘤中快速和持续地吸收示踪剂，这与体外结果一致。结论。^使用99m Tc-MAG ₃ -Cet-F(ab ) ₂的SPECT/CT 成像能够评估小鼠 EGFR 阳性肿瘤中的 EGFR 表达，表明在无创评估肿瘤中 EGFR 表达的潜在效用。