Targeting Of HER2 Overexpressing Gastric Cancer Xenograft With Site-specifically ¹⁸F Labeled Z_HER2:342 Modified With Hydrophilic Group

Objective:To investigate the effect of 18F labeled anti-HER2 affibody probe modified with hydrophilic Gly-Gly-Gly-Arg-Asp-Asn (GGGRDN) group on the targeting tracing of HER2 overexpressing human gastric cancer.Methods:The N-terminal cysteine (Cys) and GGGRDN modified HER2 specific affibody, Cys-GGGRDN-ZHER2:342, was obtained from chemical synthesis routes. The bifunctional maleimide derivative of 1,4,7-Triazacyclononane-1,4,7-triacetic acid (NOTA) was coupled to thiol-group of cysteine of ZHER2:342 to form the chelator-peptide conjugate. Then the newly produced 18F was labeled to the conjugate through 18FAl one step labeling method. The resulting radiolabeled product was analysed by high performance liquid chromatography (HPLC). The binding characteristics to different cells were assessed using the in vitro cell uptaking and blocking assays. Competition binding study was also conducted to detect the binding affinity with NCI N87 cell. MicroPET imaging and biodistribution experiments were performed to evaluate the in vivo targeting ability with gastric cancer xenograft models. Mice models bearing human gastric cancer were established and the immunohistochemical staining, western blot, were conducted to confirm the HER2 expression level of the studied cell lines and tumor tissues.Results:The radiochemical purity result of 18F labeled ZHER2:342 was more than 95%. The NCI N87 cell associated radioactivity was close to the platform (approximately (19.31±1.01)%ID/106 cells) as early as 15 minute after coincubation. Blocking analysis demonstrated that the binding of the probe to living HER2-overexpressing cells was receptor mediated because a significant decrease of binding activity happened after preblocking HER2 protein (p<0.01). Competition binding assay between FA1-NOTA-Mal-Cys-GGGRDN-ZHER2:342 and non-radioactive affibody molecules with NCI N87 indicated that the labeling product can be displaced by increasing concentration of unlabeled molecules. The calculated Half Maximal Inhibitory Concentration (IC50) was about 8.10 nmol/L. The micro PET imaging and biodistribution of human gastric cancer xenograft demonstrated that the radioactive probe could specifically accumulate in tumors at early time points and maintained at a high level with time extending. The protein level detection further confirmed the strong HER2 expression of NCI N87 cell, while weak of SGC7901 cell.Conclusions:The novel 18F labeled GGGRDN modified ZHER2:342 probe can be acquired through simple steps and can effectively targeting trace gastric cancer with HER2 overexpression.