基于全人源抗LMP1抗体IgG的ENKTL分子靶向治疗及分子机制研究

Extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTL) is an aggressive subtype of non-Hodgkin's lymphoma (NHL) associated with Epstein-Barr virus (EBV). It has a unique geographic distribution and it is diagnosed more commonly in Asian, especially in southern China and is characterized by highly aggressive clinical course and poor prognosis. In recent years, the incidence rate of this destructive malignancy is increasing and the people's health is severely threatened. Lately, it is reported that latent membrane protein 1 (LMP1) is a membrane-spanning polypeptide which encoded by the Epstein-Barr virus (EBV) and has been implicated in the maintenance of viral latency. It has also been suggested that this protein might be a driving factor in the tumorigenesis of EBV-associated malignancies, such as Hodgkin's lymphoma, NK/T cell lymphoma, Burkitt's lymphoma and nasopharyngeal carcinoma. In our previous research, we observed high expression of LMP1 in cell membrane of ENKTL and LMP1 may serve as a novel prognostic factor in ENKTL. Consequently, we believed that LMP1 could be an ideal target for the treatment of this malignant lymphoma. We have selected and screened a human anti-LMP1 antibody Fab from a human na?ve Fab phage library successfully, tested its character and detected the antitumor effect preliminary. Inhibitory effect of lymphoma cell growth and apoptosis of lymphoma cell were witnessed in our vitro study. Based on the above results, we try to construct the eukaryotic expression system, express and purify a novel anti-LMP1 full length IgG antibody. Furthermore, we will investigate the inhibitory effects of this novel IgG antibody on ENKTL and analyze the potential molecular mechanism, in vitro and in vivo.

结外鼻型NK/T细胞淋巴瘤(ENKTL)是一种起源于NK细胞的非霍奇金淋巴瘤，与EB病毒感染高度相关, 多发于东南亚及中国南方地区。该疾病发展迅速、生存期短、预后差，近年来发病率明显上升。研究发现，LMP1是一种EB病毒编码的潜伏跨膜蛋白，与多种EBV相关恶性肿瘤的发生、发展关系密切，在ENKTL中呈明显高表达并与疾病预后不佳高度相关，因此LMP1可能成为ENKTL靶向治疗的新靶点。针对该靶标，本课题组从全人源噬菌体抗体库中筛选出抗LMP1抗体片段Fab，该Fab可特异性识别LMP1，抑制淋巴瘤细胞增殖并诱导其凋亡。本研究拟在前期制备抗LMP1抗体片段Fab的基础上，构建包含全分子IgG抗体的真核表达系统，表达抗LMP1全分子抗体IgG，并在细胞和个体水平上，分析该全分子抗体IgG对ENKTL的细胞增殖及远处转移的抑制作用，探讨其分子机制。

项目背景：.结外鼻型NK/T 细胞淋巴瘤(ENKTL)是一种起源于NK 细胞的非霍奇金淋巴瘤，与EB 病毒感染高度相关, 多发于东南亚及中国南方地区。该疾病发展迅速、生存期短、预后差，近年来发病率明显上升。研究发现，LMP1 是一种EB 病毒编码的潜伏跨膜蛋白，与多种EBV 相关恶性肿瘤的发生、发展关系密切，在ENKTL 中呈明显高表达并与疾病预后不佳高度相关，因此LMP1 可能成为ENKTL 靶向治疗的新靶点。.研究内容：.针对该靶标，本课题组从全人源噬菌体抗体库中筛选出抗LMP1 抗体片段Fab，该Fab 可特异性识别LMP1，抑制淋巴瘤细胞增殖并诱导其凋亡。本研究拟在前期制备抗LMP1 抗体片段Fab 的基础上，构建包含全分子IgG抗体的真核表达系统，表达抗LMP1 全分子抗体IgG，并在细胞和个体水平上，分析该全分子抗体IgG 对ENKTL 的细胞增殖及远处转移的抑制作用，探讨其分子机制。.重要结果：.完成全人源抗LMP1全分子抗体IgG的构建、表达、纯化、鉴定；检测LMP1-IgG对ENKTL一系列生物学行为的影响；LMP1-IgG抑制ENKTL的作用机制之一可能为抑制JAK/STAT信号通路；.关键数据：.1. 成功构建全人源LMP1-IgG抗体，并通过ELISA、WB、亲和力实验及IHC检测证明LMP1-IgG可成功识别ENKTL细胞及组织表达的肿瘤抗原LMP1；.2. CCK-8、MTT、凋亡实验、ADCC/CDC证明LMP1-IgG抗体可明显抑制ENKTL生长，可诱导ENKTL发生细胞凋亡，LMP1-IgG可发挥明显ADCC/CDC效应；.3. JAK3及STAT3磷酸化作用可被LMP1-IgG抗体抑制，将LMP1表达Si后，则对LMP1-IgG对JAK3及STAT3磷酸化的抑制作用明显减弱。.科学意义：.1. 合成具有中和作用的人源全分子LMP1-IgG抗体；.2. LMP1-IgG抗体可发挥明显抑瘤作用；.3. JAK/STAT信号通路的抑制可能是LMP1-IgG抗体发挥抑瘤作用的机制之一；.4. 为ENKTL的靶向治疗及免疫治疗提供新思路。