ADMINISTRATION OF HER2 CHIMERIC RECEPTOR AND TGFBETA DOMINANT NEGATIVE RECEPTOR

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 摘要 复发/转移性肺癌患者的预后在过去20年中没有改变，大多数患者死于他们的疾病。需要新的抗肺癌疗法，而免疫疗法有望满足这一需求。 人体对癌症的免疫防御经常失败，因为癌症不会诱导免疫，也不会主动抑制免疫。我们将通过i)设计杀手T细胞或识别肺癌细胞上的结构，以及ii)抵抗癌细胞环境施加的防御来抵消这些限制。利用基因转移技术，有可能利用杀伤T细胞的肿瘤杀伤能力，使它们对肿瘤细胞分泌的抑制因子产生抗药性。我们建议将患者自己的T细胞放入两个基因中。第一个基因是人造结构(受体)，它将T细胞导向肿瘤，第二个基因是显性负性受体(DNR)，它使T细胞对肺癌细胞分泌的主要抑制因子之一具有抵抗力。 我们将把这两个基因放入T细胞中，T细胞是根据其识别爱泼斯坦-巴尔病毒的能力预先选择的，这种病毒在大多数人中长期存在。由于这些EBV特异性T细胞与病毒阳性细胞和肿瘤细胞相遇，它们会受到额外的刺激。我们将使用一种改良的病毒(Moloney逆转录病毒载体)将人造受体和DNR的基因注入患者EBV特异性T细胞。然后，这些经过修饰的T细胞将通过中心线或静脉直接进入患者的血流。患者将在诊所接受治疗，并将在输液后密切监测几个小时。我们会定期从外周血中采集血液样本。我们将寻找我们植入患者体内的细胞的安全性、持久性和功能。最终，我们希望获得证据，证明这些经过修饰的T细胞在对抗癌症方面是有效的。 一、假说 根据我们的经验，输注EBV-CTL在癌症患者中的持久性是有限的，很可能是由于癌症适应的免疫逃避策略。例如，肺癌表达高水平的转化生长因子，这是一种强大的免疫细胞负调节因子。在临床前研究中，我们已经证明EBV-CTL可以通过表达显性阴性的转化生长因子受体II(DNR)来抵抗转化生长因子的抑制作用。在临床前动物模型中，表达CTL的DNR保留了它们的效应功能，并具有增强的抗肿瘤活性。重要的是，表达CTL的DNR没有显示出任何自主(抗原非依赖性)CTL增殖的证据。这些结果为RAC和FDA批准的临床试验(RAC#0507-724)提供了理论基础，在该试验中，我们正在测试(转化生长因子)抗性CTL在淋巴瘤患者中的安全性和有效性。 在这项研究中，我们将产生自体的EBV特异性CTL，并使用逆转录病毒载体表达显性阴性的转化生长因子β受体II(DNRII)使其抵抗转化生长因子β的免疫抑制作用，并使用识别HER2的嵌合抗原受体对其进行基因修饰。将使用两个不同的载体来转移基因。因此，每个细胞产品将包含： 1)未修饰的CTL 2)HER2 CAR CTL 3)转化生长因子[DNR]CTL 4)HER2 CAR和转化生长因子β-DNR CTL 我们将过继地将这些CTL转移给晚期HER2阳性肺癌患者。我们将评估输液的安全性，并测量2、3和4亚组中基因修饰细胞的存活率。我们将通过定量聚合酶链式反应以及外周血和肿瘤组织/其他液体(如果有)的免疫染色来检测每个细胞群。我们预计，表达转化生长因子βDNR的CTL群体(亚组3和亚组4)将比没有这种修饰的群体(亚组2)存活得更好。我们还预计第3组和第4组的存活率相同，但如果CAR表达本身对CTL存活率有不利影响，那么第3亚组将占据主导地位。或者，如果同时表达转化生长因子和CAR的细胞在肿瘤中定位更好，那么我们预计在组织样本中将看到亚组4的优势。 二、具体目标 主要： 目的：探讨晚期HER2阳性肺癌患者一次静脉注射抗转化生长因子(TGF)抗性细胞毒性T淋巴细胞(CTL)的安全性。 次要： 比较移植的CAR-CTL中抗转化生长因子和非抗转化生长因子成分的存活率和免疫功能。 目的：评价CAR-CTL的抗肿瘤作用。

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 The outcome for patients with recurrent/metastatic lung cancer has not changed over the last 2 decades and most patients die of their disease. New anti-lung cancer therapies are needed and immunotherapy holds the promise to fulfill this need. The bodys immune defense against cancers often fails because cancers either do not induce or actively inhibit immunity. We will counteract these limitations by i) engineering killer T cells or recognize structures on lung cancer cells and ii) to resist the defenses imposed by the cancer cell environment. Using gene transfer technology, it may be possible to take advantage of the tumor killing ability of killer T cells and render them resistant to inhibitory factors secreted by tumor cells. We propose taking the patients own T cells and putting into them two genes. The first gene is for an artificial structure (receptor) that will direct the T cells to the tumor and the second gene is for a dominant negative receptor (DNR), which renders T cells resistant against one of the major inhibitory factors secreted by lung cancer cells. We will place the two genes into T cells that are pre-selected for their ability to recognize the Epstein Barr virus which is chronically present in most people. Because these EBV-specific T cells meet the virus positive cells as well as tumor cells, they receive extra stimulation. We will put the genes for the artificial receptor and the DNR into patients EBV-specific T cells using a modified virus (Moloney retroviral vector). These modified T-cells will then be given directly into the patients blood stream through a central line or a vein. Patients will be treated in the clinic and will be monitored closely for several hours after infusion. We will collect samples of blood from peripheral blood at regular intervals. We will look for the safety, the persistence and the function of the cells we put into the patients. Ultimately we hope to get evidence that these modified T cells are effective at fighting the cancer. I. HYPOTHESIS From our experience, the persistence of infused EBV-CTL in cancer patients is limited, most likely due to immune evasion strategies adapted by cancers. For example, lung cancers express high levels of TGF, a potent negative regulator of immune cells. In preclinical studies we have shown that EBV-CTL can be made resistant to the inhibitory effects of TGF by expressing a dominant negative TGF receptor II (DNR). DNR expressing CTL retained their effector function and had enhanced antitumor activity in preclinical animal models. Importantly, DNR expressing CTL did not show any evidence of autonomous (antigen-independent) CTL proliferation. These results provided the rationale for a RAC and FDA approved clinical trial (RAC #0507-724), in which we are testing the safety and efficacy of (TGF)-resistant CTL in lymphoma patients. In this study we will generate autologous EBV-specific CTL and genetically modify them using a retroviral vector expressing a dominant negative TGF beta receptor II (DNRII) to render them resistant to the immunosuppressive effects of TGF beta, and a chimeric antigen receptor recognizing HER2. Two separate vectors will be used to transfer the genes. Each cell product will thus contain: 1) Unmodified CTLs 2) HER2 CAR CTLs 3) TGF¿] DNR CTLs 4) HER2 CAR and TGF beta DNR CTLs We will adoptively transfer these CTLs to patients with advanced HER2-positive lung cancer. We will assess the safety of the infusion and measure the survival of the gene modified cells in subgroups 2, 3 and 4. We will detect each cell population by quantitative PCR and by immunostaining of peripheral blood and in tumor tissue/other fluids (when available). We anticipate that the CTL populations expressing TGF¿ DNR (subgroups 3 and 4) will survive better than the population lacking this modification (subgroup 2). We also anticipate survival of groups 3 and 4 will be equivalent, but if CAR expression itself has an adverse influence on CTL survival, then subgroup 3 will come to dominate. Alternatively, if cells expressing both TGF¿ DNR and the CAR localize better in tumors, then we would expect to see dominance of subgroup 4 in tissue samples. II. SPECIFIC AIMS Primary: To determine the safety of one intravenous injections of autologous Transforming Growth Factor (TGF)-resistant cytotoxic T lymphocytes (CTLs) directed to Epstein Barr virus (EBV) through their native receptor and HER2 through their chimeric antigen receptor (CAR) in patients with advanced HER2-positive lung cancers. Secondary: To compare the survival and the immune function of the TGF-resistant and non resistant components of the infused CAR-CTL. To assess the anti-tumor effects of the infused CAR-CTL.