Novel EBV LMP-2-affibody and affitoxin in molecular imaging and targeted therapy of nasopharyngeal carcinoma

Epstein-Barr virus (EBV) infection is closely linked to several human malignancies including endemic Burkitt’s lymphoma, Hodgkin’s lymphoma and nasopharyngeal carcinomas (NPC). Latent membrane protein 2 (LMP-2) of EBV plays a pivotal role in pathogenesis of EBV-related tumors and thus, is a potential target for diagnosis and targeted therapy of EBV LMP-2+ malignant cancers. Affibody molecules are developing as imaging probes and tumor-targeted delivery of small molecules. In this study, four EBV LMP-2-binding affibodies (ZEBV LMP-212, ZEBV LMP-2132, ZEBV LMP-2137, and ZEBV LMP-2142) were identified by screening a phage-displayed LMP-2 peptide library for molecular imaging and targeted therapy in EBV xenograft mice model. ZEBV LMP-2 affibody has high binding affinity for EBV LMP-2 and accumulates in mouse tumor derived from EBV LMP-2+ xenografts for 24 h after intravenous (IV) injection. Subsequent fusion of Pseudomonas exotoxin PE38KDEL to the ZEBV LMP-2 142 affibody led to production of Z142X affitoxin. This fused Z142X affitoxin exhibits high cytotoxicity specific for EBV+ cells in vitro and significant antitumor effect in mice bearing EBV+ tumor xenografts by IV injection. The data provide the proof of principle that EBV LMP-2-speicifc affibody molecules are useful for molecular imaging diagnosis and have potentials for targeted therapy of LMP-2-expressing EBV malignancies.EB病毒（Epstein-Barr virus，EBV）感染与多种人类恶性肿瘤密切相关，包括地方性伯基特淋巴瘤、霍奇金淋巴瘤及鼻咽癌（nasopharyngeal carcinomas，NPC）。EB病毒潜伏膜蛋白2（Latent membrane protein 2，LMP-2）在EBV相关肿瘤的发病机制中发挥关键作用，因此是EBV LMP-2阳性恶性肿瘤的诊断及靶向治疗的潜在靶点。亲和体分子（Affibody molecules）正被开发为成像探针及小分子肿瘤靶向递送载体。本研究通过筛选噬菌体展示的LMP-2肽库，在EBV异种移植小鼠模型中鉴定出4种可结合EBV LMP-2的亲和体（ZEBV LMP-212、ZEBV LMP-2132、ZEBV LMP-2137及ZEBV LMP-2142），用于分子成像与靶向治疗。ZEBV LMP-2亲和体对EBV LMP-2具有高结合亲和力，经静脉（intravenous，IV）注射后，可在源自EBV LMP-2阳性异种移植瘤的小鼠肿瘤组织中蓄积长达24小时。后续将铜绿假单胞菌外毒素PE38KDEL与ZEBV LMP-2 142亲和体融合，制备得到Z142X亲和毒素。该融合型Z142X亲和毒素在体外对EBV阳性细胞表现出高特异性细胞毒性，且经静脉注射后，对携带EBV阳性肿瘤异种移植物的小鼠具有显著的抗肿瘤效果。本研究数据从原理上验证了EBV LMP-2特异性亲和体分子可用于分子成像诊断，并在表达LMP-2的EBV相关恶性肿瘤的靶向治疗中具有应用潜力。