ADMINISTRATION OF TGF-B RESISTANT LMP2A-SPECIFIC CYTOTOXIC T-LYMPHOCYTES TO P

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 摘要 放射化学疗法的改进相应地改善了霍奇金病（HD）和非霍奇金淋巴瘤（NHL）患者的预后。 然而，对标准治疗方法有抵抗力的患者具有极差的结果。 此外，治疗相关的死亡率和发病率显著降低了淋巴瘤治愈患者的预期寿命和生活质量。 目前治疗方案的这些局限性说明需要更有效的治疗方法复发的患者。 在HD和NHL中，高达49%的标本已被证明携带EBV-DNA并表达EBV基因。 我们的小组已经成功地在EBV阳性霍奇金淋巴瘤患者中产生了EBV特异性CTL。 输注后，这些回到肿瘤部位的CTL在循环中持续长达12个月，并产生短暂的临床益处。 在该方案中，淋巴母细胞样细胞系（LCL）用作EBV抗原呈递细胞（APC）。 LCL激活多克隆CTL群，其优先针对免疫显性EBNA 3A、3B和3C EBV蛋白。 这些免疫原性蛋白在HD/NHL肿瘤细胞中不表达。 相反，HD/NHL肿瘤细胞上的EBV抗原仅限于表达潜伏蛋白的一个子集，如LMP 2。 LCL在刺激针对这些亚显性蛋白的CTL方面具有有限的功效。 LMP 2表位显示在淋巴瘤活检样品中是保守的，在病毒株之间显示出很小的异质性。 因此，我们认为LMP 2可以被淋巴瘤住院患者的CTL靶向，并且在正在进行的研究中，我们的小组成功地从EBV +ev HD和NHL患者的外周血中产生靶向LMP 2的CTL。 在该现有方案中，LMP 2A特异性CTL能够消除一些EBV阳性淋巴瘤，并且LMP 2特异性CTL在肿瘤部位积累，在外周血中增加并产生短暂的临床益处。 然而，CTL在巨大疾病患者中效果最差。 因此，LMP 2A特异性CTL可能在体外具有良好的细胞毒性，但是当它们经受由肿瘤环境分泌的转化生长因子-β（TGFB）的高度免疫抑制活性时，这妨碍了有效的肿瘤消除。 因此，我们希望使CTL对TGFB具有抗性，以克服有效杀死肿瘤的问题。 在临床前研究中，我们已经将截短的显性阴性TGFB受体（DNR）克隆到逆转录病毒载体SFG和转导的CTL中。 在正常情况下，TGFB对已建立的CTL的增殖和细胞溶解活性具有深远的影响，并且TGFB在体外的持续存在导致培养物在两至三周内死亡。 相反，当用DNR转导的CTL在TGFB存在下培养时，它们保留了明显正常的细胞内信号传导，正常生长，继续分泌GM-CSF和γ IFN，并保留了它们的细胞溶解功能。 因此，反式显性阴性TGFB受体突变体能够在体外保护CTL免受TGFB的抑制作用。 重要的是，为了临床上使用这种方法的可行性，在不存在抗原刺激和生长因子的情况下培养CTL，导致细胞死亡与未转导的T细胞一样快。 我们还在小鼠模型中开发并完成了一项体内安全性研究，以确认该方法适用于临床使用。 在这项研究中，我们将使用与我们目前研究相同的方法产生LMP 2A特异性细胞毒性T细胞，并使用表达显性阴性TGFB受体II（DNRII）的逆转录病毒载体对其进行遗传修饰，使其对TGFB的免疫抑制作用具有抗性。 然后，我们将这些TGFB抗性LMP 2-CTL过继转移到患有复发性EBV阳性霍奇金淋巴瘤或非霍奇金淋巴瘤的患者。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ABSTRACT Improvements in radiochemotherapy have correspondingly improved the prognosis of patients with Hodgkin's Disease (HD) and non-Hodgkin's lymphoma (NHL). However, patients resistant to the standard therapeutic approaches have an extremely poor outcome. Moreover, life expectancy and quality of life of patients cured of lymphoma are both significantly reduced by treatment related mortality and morbidity. These limitations of current treatment protocols illustrate the need for more effective therpeutic approaches for patients who relapse. In HD and NHL, up to 49% of specimens have been shown to carry EBV-DNA and express EBV-genes. Our group has successfully generated EBV-specific CTL in patients with EBV-positive Hodgkin's lymphoma. After infusion these CTL home to the tumor sites persist in the circulation for up to 12 months, and produce transient clinical benefits. In that protocol, lymphoblastoid cell lines (LCL) were used as EBV-antigen presenting cells (APC). LCL activate polyclonal CTL populations that are preferentially directed against the immunodominant EBNA3A, 3B and 3C EBV-proteins. These immunogenic proteins are not expressed in HD/NHL tumor cells. Instead, the EBV-antigens on HD/NHL tumor cells are restricted to the expression of a subset of latent proteins such as LMP2. LCL have limited efficacy in stimulating CTL directed against these subdominant proteins. LMP2 epitopes were shown to be conserved among Lymphoma biopsy samples displaying little heterogeneity between viral strains. Hence we thought that LMP2 could be targeted by CTL inpatients with Lymphoma and in an ongoing study, our group is successfully generating CTL targeting LMP2 from the peripheral blood of patients with EBV +ev HD and NHL. In this current protocol, LMP2A -specific CTL were able to elimiante some EBV-positive lymphoma's and the LMP2-specific CTL accumulated at tumor sites, increased in the peripheral blood and produced transient clinical benefits. The CTL were however least effective in patients with bulky disease. It is therefore possible that LMP2A-specific CTL have good cytotoxicity in vitro but that when they are subjected to the highly immunosuppressive activity of Transforming Growth Factor-beta (TGFB) secreted by the tumor environment, this precludes effective tumor elimination. We therefore want to render the CTL resistant to TGFB, to overcome the problem of effective tumor kill. In preclinical studies we have cloned a truncated dominant negative TGFB receptor (DNR) into the retrovirus vector, SFG and transduced CTL. Under normal circumstances TGFB has profound effects on the proliferation, and cytolytic activity of established CTL, and the continued presence of TGFB in vitro leads to the death of cultures within two to three weeks. In contrast, when CTL transduced with DNR are cultured in the presence of TGFB, they retain apparently normal intracellular signaling, grow normally, continued to secrete GM-CSF and yIFN, and retained their cytolytic function. Thus, the transdominant-negative TGFB receptor mutant is capable of protecting CTL from the inhibitory effects of TGFB in vitro. Importantly, for the feasibility of using this approach clinically, culture of CTL in the absence of antigenic stimulation and growth factors, led to cell death as rapidly as in non-transduced T cells. We also developed and completed an in vivo safety study in a murine model to confirm the suitaility of the approach for clinical use. In this study we will generate LMP2A-specific cytotoxic T-cells using the same methodology as on our current study and genetically modify them using a retroviral vector expressing a dominant negative TGFB receptor II (DNRII) to render them resistant to the immunosuppressive effects of TGFB. We will then adoptively transfer these TGFB resistant LMP2-CTL to patients with relapsed EBV-positive Hodgkin's or non-Hodgkins Lymphoma.