EBV-SPECIFIC CYTOTOXIC T-LYMPHOCYTES FOR EBV-POSITIVE NASOPHARYNGEAL CANCER PTS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在这项研究中，我们希望产生多克隆EBV特异性细胞毒性T细胞，并将其过继转移到活动性鼻咽癌患者体内。用于产生多克隆CTL的淋巴母细胞样细胞系表达包括LMP-1和LMP-2在内的全部EBV衍生基因产物。 尽管LMP特异性CTL是少数，但即使是少量，它们也可能具有识别和杀死表达LMP-1的细胞（包括NPC肿瘤细胞）的能力。 本课题的具体目的是：1）确定从活动期鼻咽癌患者中产生EBV特异性细胞毒性T细胞系的可行性。2)目的探讨鼻咽癌患者静脉注射两种EB病毒特异性细胞毒性T淋巴细胞（CTL）的安全性。3)探讨EB病毒特异性细胞毒性T淋巴细胞系的存活率、免疫效力和抗肿瘤作用。 鼻咽癌是一种恶性疾病，其发病率范围取决于年龄、地理位置、种族和EB病毒（EBV）暴露。在美国，它的年发病率为每10万名<21岁的儿童中近1例。 对于大多数（75-91%）患者，联合治疗的长期预后良好。在初始治疗后复发的10-15%患者中，只有40%会进入第二次缓解。对于挽救化疗失败或第二次复发的其余患者，预后较差。在成人中，放射治疗后的总体5年生存率取决于分期，I期为70%至80%，IV期为20-40%。对于更晚期的疾病，在成人中使用综合疗法可使总生存率达到50%。 虽然儿童的总体生存率很高，但目前的治疗方案仍远未达到理想水平。鼻咽癌治疗后的晚期并发症包括生长激素缺乏症、甲状腺功能减退症和肺纤维化。在台湾进行了一项大型回顾性研究，纳入1，549例患者，以评估鼻咽癌患者放疗+/-化疗后继发性恶性肿瘤的风险。患者年龄范围为10- 80岁（中位数46.3岁），最长随访时间为16年。致死性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（最可能是放射诱导的）以及胃癌。因此，需要开发新的疗法，其可以改善复发/难治性患者的无病生存期，并且其可以最终降低所有患者中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。 NPC和EBV之间的病因学联系首先基于血清学证据。IgG和伊加抗体滴度升高是常见的。随后通过显示EBV DNA存在于NPC肿瘤细胞中并且NPC活检样品中的EBV DNA是克隆的，由单个EBV感染的细胞产生，证实了EBV和NPC之间的关联。几乎在所有未分化的非角化性NPC病例中均检测到EBV。另一方面，鳞状细胞NPC似乎显示出其EBV与高EBV阳性肿瘤相关的地理变异性，这些肿瘤见于高发地区，如亚洲。此外，鳞状细胞鼻咽癌似乎代表了一个更异质性的肿瘤组，其他辅助因素如吸烟和人乳头瘤病毒（HPV）有助于致病过程。然而，NPC与EBV的强关联仍然存在。因此，NPC可能代表了对几种病理过程的最终共同反应，这些病理过程包括病毒感染、职业和环境刺激，可能具有遗传贡献。 在原发性感染中，EBV进入的主要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淋巴细胞的感染，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EBV的控制中起重要作用。EBV特异性CD 8 + CTL被认为是对抗EBV感染的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肽片段，所述肽片段在抗原呈递细胞的表面上与MHC分子结合表达。 EBV阳性的NPC细胞有规律地表达核抗原ENBA 1，并且肿瘤的一个子集似乎也是潜伏膜蛋白LMP 1阳性的。转录分析表明，LMP 2 mRNA的表达在大多数肿瘤活检。此外，已在NPC细胞中检测到BARF 0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.