TCR Transduction for EBV Specific Immunotherapy

描述（由申请人提供）：EBV特异性TCR转导 免疫治疗。用多克隆细胞毒性T细胞系进行免疫治疗 (CTL)在移植后的治疗中取得了临床成功， 淋巴瘤，一种EB病毒（EBV）相关的恶性肿瘤，表达 大多数免疫显性EBV潜伏抗原。此策略不适用于 另外两种EBV相关的恶性肿瘤，霍奇金病（Hodgkin's disease，appx.的百分之50 例与EB病毒相关）和鼻咽癌（100 EBV相关）。这些恶性肿瘤仅表达EBV潜伏抗原 LMP-1、LMP-2和EBNA-1;它们都不诱导强烈的免疫应答。这些 代表亚显性肿瘤相关抗原。这个项目的目标是 为患有这些疾病的患者提供免疫选择， 通过克隆单个T细胞受体分子（TCR）， LMP-1和LMP-2，并将这些重组体导入 TCR进入HLA-A2淋巴细胞。为了实现这一目标，LMP-2特异性CTL克隆 将产生TCR α和β链的分子克隆， 转移到逆转录病毒表达载体中。这些向量将用于 对已知特异性的CTL克隆以及活化的初级CTL克隆， 在大量培养中的淋巴细胞。该项目的具体目标是 确定哪些TCR是遗传转导的最佳候选者， 将原始细胞的CTL活性与新的裂解活性进行比较， 赋予被转导的细胞。目前还有待确定， TCR的一级序列或转导细胞的生理学， 决定了新受体赋予的细胞溶解活性。我们还将 确定转导细胞中TCR-CD3复合物的结构， 检查大量转导的淋巴细胞群体， 能够表达转导的受体。如果逆转录病毒载体 在这些研究中使用的未提供转导的TCR的长期表达，我们 我还提出了新一代的逆转录病毒载体， 而不是.一旦转导，新表达的TCR-α和β链将 必须与内源性TCR元件竞争与CD3 受体复合物，随后转运到细胞表面。获得的数据 该项目将使逆转录病毒基因水平之间的相关性 转导、mRNA表达、细胞内蛋白表达（组装 ICR亚基在内质网中的表达）， 转导的TCR和待产生的裂解功能。更好地了解 这些第一原则的功能ICR组装在初级淋巴细胞，其他 具有已知肿瘤相关抗原的恶性肿瘤可以被这种方法靶向。 方法也。

DESCRIPTION (provided by applicant): TCR TRANSDUCTION FOR EBV-SPECIFIC IMMUNOTHERAPY. Adoptive immunotherapy with polyclonal cytotoxic T cell lines (CTL) has met with clinical success in the treatment of post-transplant lymphoma, an Epstein-Barr virus (EBV)-associated malignancy that expresses the most immunodominant EBV latency antigens. This strategy is not applicable to two other EBV-associated malignancies, Hodgkin's disease (appx. 50 percent of cases are EBV-associated) and nasopharyngeal carcinoma (100 percent EBV-associated). These malignancies only express the EBV latency antigens LMP-1, LMP-2, and EBNA-1; none of which induce a strong immune response. These represent sub-dominant tumor-associated antigens. The goal of this project is to provide an immunotherapeutic option to patients suffering from these diseases by cloning individual T cell receptor molecules (TCR) that recognize LMP-1 and LMP-2 in an HLA-restricted manner, and introducing these recombinant TCR into HLA-A2 lymphocytes. Toward that goal, CTL clones specific for LMP-2 will be generated, the TCR alpha and beta chains molecularly cloned, and then transferred to retroviral expression vectors. These vectors will then be used to transduce CTL clones of known specificity as well as activated primary lymphocytes in bulk culture. The specific aims of this project seek to determine which TCRs are the best candidates for genetic transduction by comparing the CTL activity of the original cell to the lytic activity newly conferred upon the transduced cell. It remains to be determined whether it is the primary sequence of the TCR or the physiology of the transduced cell that determines the cytolytic activity conferred by the new receptor. We will also determine the structure of the TCR-CD3 complex in transduced cells, and in examining the bulk transduced lymphocyte population determine which cells are capable of expressing the transduced receptor. Should the retroviral vector used in these studies not give long-term expression of the transduced TCR, we also propose a newer generation of retroviral vectors that would be used instead. Once transduced, the newly expressed TCR-alpha and beta chains will have to compete with the endogenous TCR elements for association with the CD3 receptor complex and subsequent transit to the cell surface. Data obtained from this project will allow correlation between levels of retroviral gene transduction, mRNA expression, intracellular protein expression (the assembly of ICR subunits in the endoplasmic reticulum), cell surface expression of transduced TCR, and lytic function to be made. With a better understanding of these first principles of functional ICR assembly in primary lymphocytes, other malignancies with known tumor-associated antigens could be targeted by this approach as well.