CLINICAL TRIAL: EBV-SPECIFIC CYTOTOXIC T-LYMPHOCYTES FOR EBV-POSITIVE NASOPHARYN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在这项研究中，我们希望产生多克隆的EBV特异性细胞毒性T细胞，并过继地将它们转移给活动性鼻咽癌患者。用于产生多克隆CTL的淋巴母细胞系表达包括LMP-1和LMP-2在内的所有EBV衍生基因产物。尽管LMP特异性CTL为数不多，但即使在少数情况下，它们也有可能识别和杀伤表达LMP-1的细胞，包括鼻咽癌肿瘤细胞。 本项目的具体目标是：1)确定从活动性鼻咽癌患者中产生EBV特异性细胞毒T细胞株的可行性。2)探讨鼻咽癌患者静脉注射两种自体EBV特异性细胞毒性T淋巴细胞(CTL)的安全性。3)检测EBV特异性细胞毒T淋巴细胞株的存活率、免疫效力和抗肿瘤作用。 鼻咽癌是一种恶性疾病，其发病率随年龄、地理位置、种族和EB病毒(EBV)的暴露而变化。在美国，它的年发病率接近每100,000名儿童中就有1例。对于大多数患者(75-91%)来说，综合治疗的远期预后良好。在最初治疗后复发的10%-15%的患者中，只有40%的患者会进入第二次缓解。对于其余未能挽救化疗或第二次复发的患者，预后很差。在成人中，放射治疗后的总5年存活率与分期有关，I期为70%至80%，IV期为20%至40%。对于更晚期的疾病，成人综合治疗的总存活率为50%。 尽管儿童的总体存活率很好，但目前的治疗方案仍然很不理想。鼻咽癌治疗后的晚期医学并发症包括生长激素缺乏、甲状腺功能减退和肺纤维化。在台湾对1,549名患者进行了一项大规模的回顾研究，以评估鼻咽癌患者放疗+化疗后继发恶性肿瘤的风险。患者年龄10-80岁，中位年龄46.3岁，最长随访16年。致命的肿瘤并发症包括与烷化剂化疗相关的继发性白血病和头颈癌(最有可能是辐射引起的)，以及胃癌。因此，开发新的治疗方法是可取的，它可以提高复发/难治性患者的无病存活率，并最终可能减少所有患者长期治疗相关并发症的发生率。 地方性鼻咽癌的病因包括EB病毒、环境危险因素和遗传易感性。鼻咽癌和EB病毒之间的病因学联系首先是基于血清学证据。免疫球蛋白和免疫球蛋白A抗体滴度升高是常见的。随后证实了EBV与鼻咽癌之间的联系，证实了鼻咽癌肿瘤细胞中存在EBV DNA，鼻咽癌活检标本中的EBV DNA是克隆性的，来自单个EBV感染的细胞。EBV在几乎所有未分化的非角化性鼻咽癌中都被检测到。另一方面，鳞状细胞鼻咽癌在其EBV与在亚洲等高发地区出现的较高EBV阳性肿瘤的相关性方面似乎显示出地理上的差异性。此外，鳞状细胞鼻咽癌似乎代表了一组更加异质性的肿瘤，其他辅助因素，如吸烟和人乳头瘤病毒(HPV)，促进了致病过程。然而，鼻咽癌与EB病毒之间的密切联系仍然存在。因此，鼻咽癌可能是对包括病毒感染、职业和环境刺激在内的几个病理过程的最终共同反应，可能与基因有关。 在原发感染中，EBV的主要进入途径是通过口咽上皮。病毒在这些细胞中的复制随后允许B淋巴细胞感染，从而导致转化的B细胞的多克隆扩增。体液免疫和细胞免疫在EB病毒的控制中都发挥着重要作用。EBV特异性CD8+CTL被认为是抵抗EBV感染B细胞生长的最重要的防御机制。这些细胞识别来自病毒抗原的多肽片段，这些片段表达在与MHC分子相关的抗原提呈细胞表面。 EBV阳性的鼻咽癌细胞有规律地表达核抗原ENBA1，部分肿瘤似乎也呈潜伏膜蛋白LMP1阳性。转录分析表明，LMP2在大多数肿瘤活检组织中都有表达。此外，在鼻咽癌细胞中还检测到BARF0基因的转录。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In this study we wish to generate polyclonal EBV specific cytotoxic T cells and adoptively transfer them to patients with active Nasopharyngeal Carcinoma. The lymphoblastoid cell lines, which are used to generate polyclonal CTLs, express the entire range of EBV derived gene products including LMP-1 and LMP-2. Although LMP-specific CTLs are in a minority, it is possible that even in small numbers they will have the ability to recognize and kill LMP-1 expressing cells including the NPC tumor cells. The Specific Aims of this project are: 1) To determine the feasibility of generating EBV specific cytotoxic T cell lines from patients with active Nasopharyngeal Carcinoma. 2) To determine the safety of two intravenous injections of autologous EBV specific cytotoxic T-lymphocytes (CTL) in patients with Nasopharyngeal Carcinoma. 3) To determine the survival, immunological efficacy and anti-tumor effects of EBV specific cytotoxic T-lymphocyte lines. Nasopharyngeal carcinoma, is a malignant disease with a variable range of incidence depending on age, geographical place, race and Epstein-Barr virus (EBV) exposure. It has an annual incidence of nearly 1 case per 100,000 children < 21yrs in the USA. For the majority (75-91%) of patients, the long-term prognosis is favorable with combined modality therapy. Of the 10-15% of patients who relapse after initial therapy, only 40% will enter a second remission. For the remainder who fail salvage chemotherapy or relapse for a second time, the prognosis is poor. In adults, the overall 5-year survival rates following radiotherapy are stage dependant and range from 70% to 80% for stage I, and 20-40% for stage IV. For more advanced disease, the use of combined modality therapy in adults results in an overall survival of 50%. Although overall survival rates are good in children, current treatment regimens are still far from ideal. Late medical complications after treatment for NPC include growth hormone deficiency, hypothyroidism and pulmonary fibrosis. A large restrospective study in Taiwan of a cohort of 1,549 patients was performed to assess the risk of secondary malignancies in NPC patients post radiotherapy +/- chemotherapy. The patients ranged in age from 10-80years (median 46.3years) with a maximum follow-up of 16 years. Fatal neoplastic complications included secondary leukemia related to alkylating agent chemotherapy and head and neck cancers (most likely radiation induced), and gastric cancer. It is therefore desirable to develop novel therapies that could improve disease free survival in relapsed/refractory patients and which might ultimately reduce the incidence of long term treatment related complications in all patients. The etiological factors of endemic NPC include EBV, environmental risk factors and genetic susceptibility. The etiological link between NPC and EBV was first based on serological evidence. Elevated IgG and IgA antibody titers are frequently seen. The association between EBV and NPC was subsequently confirmed by showing that EBV DNA was present in the NPC tumor cells and that EBV DNA in NPC biopsy samples is clonal, arising from a single EBV infected cell. EBV has been detected in virtually all cases of undifferentiated non-keratinizing NPC. On the other hand, squamous cell NPC appear to show a geographical variability with regard to their EBV association with higher EBV positive tumors seen in high incidence areas such as Asia. In addition, squamous cell NPC appears to represent a more heterogeneous group of tumors with other co-factors such as smoking and human papilloma virus (HPV) contributing to the pathogenic process. Nevertheless, the strong association of NPC with EBV remains. Thus NPC may represent a final common response to several pathological processes that include viral infections, occupational and environmental stimuli with, perhaps, a genetic contribution. In primary infection, the main route of entry for EBV is via the oropharyngeal epithelium. Viral replication in these cells subsequently allows infection of B lymphocytes thus resulting in a polyclonal expansion of transformed B cells. Both humoral and cell mediated immunity have important roles to play in the control of EBV. EBV-specific CD8+ CTLs are thought to be the most important defense mechanism against outgrowth of EBV-infected B cells. These cells recognize peptide fragments, derived from viral antigens, expressed on the surface of antigen presenting cells in association with MHC molecules. EBV positive NPC cells regularly express the nuclear antigen ENBA1 and a subset of tumors also appear to be latent membrane protein LMP1 positive. Transcriptional analysis has shown expression of LMP2 mRNA in the majority of tumor biopsies. In addition, transcription of the BARF0 gene has been detected in NPC cells.