Abstract
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Objectives Human epidermal growth factor receptor type 2 (HER2) is overexpressed in various carcinomas. It has become a definite target of diagnosis and treatment for HER2 overexpression tumors. It is very important to monitor HER2 target treatment by molecular imaging methods. The aim of this study was to develop a simple method for preparation 99mTc labeled ZHER2:V2 Affibody molecule with C-terminal engineered cysteine and to evaluate its properties for combining with HER2 receptors in vitro or in vivo, with an eye towards clinical translation for imaging HER2-overexpression tumors.
Methods The HER2-binding ZHER2:342 Affibody molecule with a C-terminal chelating sequence -G (Gly) GGC (Cys), also named as ZHER2:V2, was synthesized by Fmoc/tBu solid phase synthesis, the Affibody molecule was labeled with 99mTc. The labeling efficiency of 99mTc-labeled ZHER2:V2 was determined by reversed-phase high-performance liquid chromatography (HPLC). Cellular uptake, retention, binding specificity, and stability of the probe were studied in vitro. Biodistribution and SPECT imaging were performed in BALB/c nude mice bearing SKOV-3 (high HER2 expression) or MCF-7 (low HER2 expression) carcinoma xenografts.
Results The average labeling efficiency of 99mTc-ZHER2:V2 was 98.99% ± 0.99% (mean±SD, n=6), even after 6h at room temperature, the labeling efficiency exceed 97%. 99mTc-ZHER2:V2 was highly stable in normal saline and fresh human serum at 37°C in vitro. The peak of cellular uptake of SKOV3 cells appeared at 8h about 6.3% ± 0.25%, the retention ratio of the probe was 48.58 ± 4.52% at 6h after interrupted incubation, even after 24h, the retention ratio was still 35.16 ± 11.23%. Meanwhile, 99mTc-ZHER2:V2 cellular uptake could be blocked by excess unlabeled ZHER2:V2 in SKOV3 HER2 overexpression cell lines. Biodistribution demonstrating the radioactive accumulation in SKOV-3 xenografts was higher than that of organs except for the kidneys or bladder. The tumor uptake was 4.14±1.61 % ID/g, 6.47±2.09 % ID/g, 5.04±2.58 % ID/g, 10.07±0.33 % ID/g at 1, 2, 4 and 6 h, respectively, however, tumor uptake in MCF-7 xenografts was very lower than that of SKOV-3 xenografts at 1, 2, 4 and 6 h (p<0.05, n=5). The SPECT imaging showed clear localization of 99mTc-ZHER2:V2 in the SKOV3 xenografts shortly after injection but not in liver and other organs except for the kidneys or bladder. Ratios of 99mTc-ZHER2:V2 radioactive counts in tumors to those in the contralateral equivalent non-tumor regions (T/NT ratios) were 3.78±0.71, 7.57±1.69, 9.98±3.22, 12.07±3.47, and 13.23±4.20, at 1, 2, 4, 6 and 8 h, respectively. However, there was no obvious tumor uptake in low HER2-expressing MCF-7 xenografts on SPECT images at any time after the injection of 99mTc-ZHER2:2891. The T/NT ratios in MCF-7 xenografts were 1.29±0.12, 2.11±0.31, 3.79±0.67, 5.29±1.10, 4.94±1.04, at 1, 2, 4, 6 and 8 h, respectively. The molecular imaging showed high uptake in HER2-expressing SKOV3 xenografts, whereas the MCF-7 xenografts with low HER2 expression were moderately imaged at any time after the injection of 99mTc-ZHER2:V2. Meanwhile, the SKOV3 xenografts can be blocked by pre-saturation of receptors with unlabelled ZHER2:V2 before injection.
Conclusions 99mTc-ZHER2:V2 could be labeled easily and quickly with good labeling yields and radiochemical purity. 99mTc-ZHER2:V2 specifically and efficiently target HER2 overexpressing tumors suggesting that it may be a promising SPECT imaging agent for diagnosing HER2 overexpression tumors and evaluating the efficacy of HER2 target therapy. Fund:This work was supported by the National Natural Science Foundation of China (NSFC) project (81571702) and Hebei Province Government Funding for Clinical Excellence Talent Project.