TCR Transduction for EBV Specific Immunotherapy

描述(申请人提供)：EBV特异性TCR转导 免疫疗法。多克隆细胞毒性T细胞的过继免疫治疗 (CTL)在移植后的治疗中取得了临床成功 淋巴瘤是一种与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。这一策略不适用于 另外两种与EBV相关的恶性肿瘤，霍奇金氏病(appx.50%的 与EB病毒相关的病例)和鼻咽癌(100% 与EBV相关)。这些恶性肿瘤只表达EBV潜伏抗原。 LMP-1、LMP-2和EBNA-1；它们都不能诱导强烈的免疫反应。这些 代表次要的肿瘤相关抗原。这个项目的目标是 为患有这些疾病的患者提供免疫治疗选择 通过克隆识别疾病的单个T细胞受体分子(TCR) LMP-1和LMP-2，并引入这些重组 TCR转化为人类白细胞抗原A2淋巴细胞。为了实现这一目标，CTL克隆了LMP-2的特异性 将被产生，TCRα和β链被分子克隆，然后 转移到逆转录病毒表达载体上。然后将使用这些向量 转导已知特异性的CTL克隆以及激活的原代 批量培养中的淋巴细胞。这个项目的具体目标是寻求 确定哪些TCR是最适合进行基因转导的候选基因 比较原始细胞的CTL活性和新的裂解细胞活性 授予换能器细胞。它是否是还有待确定。 TCR的初级序列或转导细胞的生理学 确定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复合体的结构，并在 检查大量转导的淋巴细胞群体确定哪些细胞是 能够表达被转导的受体。逆转录病毒载体是否应该 在这些研究中使用不能给出转导的TCR的长期表达，我们 还提出了新一代逆转录病毒载体，将用于 取而代之的是。一旦被转导，新表达的TCR-α和β链将 必须与内源性TCR元件竞争与CD3的关联 受体复合体和随后的转运到细胞表面。数据获取自 该项目将允许逆转录病毒基因水平之间的关联 转导、mRNA表达、细胞内蛋白表达(组装 内质网中ICR亚基的表达)，细胞表面表达 换能式TCR，并可制作溶解功能。更好地理解 这些初级淋巴细胞中功能性ICR组装的基本原则，其他 具有已知肿瘤相关抗原的恶性肿瘤可以通过这种方法进行靶向 也可以进场。

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.