基于Affibody结构的新型靶向肝细胞癌标志物GPC3的正电子分子影像探针的构建

目前对于肝细胞癌（HCC）的诊断临床上尚无理想的特异性影像学方法，亦未见特异性分子影像探针应用。已发现磷脂酰肌醇蛋白聚糖3（GPC3）为HCC新的标志物。前期工作构建的64Cu-anti-GPC3 MAb在高表达GPC3的HCC细胞和肿瘤组织中高度摄取，但是肿瘤/肝比值不高。小分子亲和蛋白Affibody具有血液清除快、靶/本底（肝）比值高、高亲和力、高稳定性及位点特异性标记等优点。本项目拟利用噬菌体展示技术筛选、鉴定特异性高度亲和GPC3的Affibody的氨基酸序列，合成并制备18F-Affibody分子影像探针，通过体外研究、体内生物学分布及肿瘤模型活体显像鉴定其与GPC3的亲和力及特异性，评价其应用于HCC显像的可行性和效能。为HCC的早期诊断、鉴别、探查转移和复发、监测治疗及评估预后提供有效的新途径，并为以Affibody为载体携带基因、毒素、药物、核素靶向治疗HCC打下基础。

Early detection of hepatocellular carcinoma (HCC) is crucial and may significantly change its outcome. Considering the poor performance of current imaging modalities for HCC diagnosis, there is an urgent need to develop new molecular imaging techniques which can dramatically improve the HCC detection. GPC3 is a novel and promising biomarker for HCC earlier detection. A positron emission tomography (PET) probe which can recognize GPC3 in vivo will likely provide a powerful non-invasive imaging technique for HCC diagnosis..Our previous study showed 64Cu-anti-GPC3 monoclonal antibody (MAb) PET probe can specifically differentiate the GPC3 positive and negative HCC tumors in vivo. More importantly, 64Cu-anti-GPC3-MAb exhibited much better HCC imaging quality than that of a conventional tumor PET tracer, 18F-FDG. But the tumor-to-liver contrast is still not high because of the hepatobiliary clearance of large size of antibodies. Therefore we propose to explore new molecular platforms to develop an ideal PET probe targeting GPC3 for HCC early detection..Affibody molecules, a class of engineered affinity proteins with favorable properties such as small size, high stability, simple and no-cysteine containing structure, and high specific binding affinities to targets (KD generally ranges in nM and pM), have been proven to be ideal for the diagnostic applications, as well as many other biotechnological applications. .This study aims to screen a novel and specific Affibody molecule against GPC3 and further develop the Affibody-based PET probe targeting GPC3. Such a molecule is also expected to hold potential for HCC targeted therapy when it is used to carry therapeutic genes, toxins, chemotherapeutics and radionuclides..In this study, an Affibody molecule library will be constructed and displayed on phages, followed by biopanning against GPC3 protein. The phage clones binding to GPC3 protein will be isolated, and the Affibody proteins expressed on the surface of selected phage clones will be purified by affinity chromatography and subsequently characterized by SDS electrophoresis, CD spectroscopy and biosensor analysis. According to the DNA sequence and amino acid sequence, the anti-GPC3 Affibody will be chemically synthesized and purified by high-performance liquid chromatography (HPLC) and then characterized by mass spectroscopy (MS). The anti-GPC3 Affibody will be labeled with a fluorescent dye, Cy5.5 and used for in vivo evaluation by optical imaging technique. The PET probe 18F-anti-GPC3-Affibody will then be prepared and studied in vitro for specific cell uptake in GPC3-high and GPC3-low/negative HCC cell lines. Pharmacokinetic study and tumor PET imaging of 18F-anti-GPC3-Affibody will be further performed in vivo using quantitative PET imaging and biodistribution analysis, in nude mice bearing HCC xenografts with high or low GPC3 expression. Blocking studies will be performed to demonstrate the in vivo targeting specificity of the probes as well.