TCR-gene modified t-cells for adaptive immunotherapy

描述（由申请人提供）：已经证明肿瘤反应性t细胞可以诱导转移性黑色素瘤患者的肿瘤消退，并且已经确定了一些靶抗原。尽管如此，目前使用细胞因子/生物反应调节剂、疫苗接种或过继性免疫治疗等免疫调节方法的临床影响仍然有限。我们最近鉴定并克隆了一种CD4肿瘤浸润淋巴细胞的t细胞受体（TCR），该细胞具有MHC I类限制性识别和对其靶点酪氨酸酶368-376肽的强亲和力。本提案的总体目标是开发一种使用tcr基因修饰的PBL的过继性免疫治疗方法。在特定目标1中，我们将确定逆转录病毒载体编码来自HLA-A2限制性，tryosinase:368-376反应性T细胞克隆（TIL 13831）的TCR是否可以改造来自正常供体和黑色素瘤患者的正常外周血T细胞，以识别高亲和力的黑色素瘤细胞。为此，我们将使用专门设计的带有SAMEN主干的逆转录病毒载体，这些逆转录病毒载体适用于TCR α和β的转移，在某些情况下也表达用于G418选择的neo(r)基因。在具体目标2中，我们将确定TCR基因修饰正常供体和黑色素瘤患者外周血T细胞的最佳条件。对于这组实验，我们将使用先前生产的A6和A7逆转录病毒载体，它们携带低亲和力的TIL 5 TCR。利用该模型，我们将优化正常PBL的传导和扩展方法。我们将评估两种转导方法，脊髓定位和基于回缩蛋白的转导。我们还将优化体外扩增转导t细胞群的方法，并研究TCR转导（通过RT-PCR）表达（通过四聚体染色）以及功能活性（通过细胞因子释放和Cr释放试验）。预计在特定目标2下开发的方法将适用于在特定目标1下产生的新载体转导的细胞群。在具体目标3中，我们将确定是否可以使用RTP-CR和竞争性RT-PCR检测和定量TIL 13831 TCR基因修饰的T细胞。为此，将设计TIL 13831的特异性引物以及克隆特异性抑制剂。测试的有效性和准确性将被广泛评估。

DESCRIPTION (provided by applicant): It has been demonstrated that tumor reactive T-cells can induce tumor regression in patients with metastatic melanoma and some of the target antigens have been identified. Still, the clinical impact of current immunomodulatory approaches by use of cytokines/biologic response modifiers, vaccination or the use of adoptive immunotherapy remains limited. We have recently identified and cloned the T-cell receptor (TCR) of a CD4 Tumor infiltrating lymphocyte with MHC class I restricted recognition and strong affinity for its target, the 368-376 peptide of tyrosinase. The overall aim of this proposal is to develop an adoptive immunotherapy approach using TCR-gene modified PBL. In specific aim 1 we will determine if the retroviral vector encoding the TCR from an HLA-A2 restricted, tryosinase:368-376 reactive T cell clone (TIL 13831) can engineer normal peripheral blood T cells from normal donors and melanoma patients to recognize melanoma cells with high affinity. For this purpose we will use specifically designed retroviral vectors with a SAMEN backbone that are adapted for the transfer of TCR alpha and beta and that in some cases also express the neo(r) gene for G418 selection. In specific aim 2 we will determine the optimal conditions to TCR gene modify human peripheral blood T cells from normal donors and melanoma patients for patient treatment. For this set of experiments, we will use the previously produced A6 and A7 retroviral vectors that carry the low affinity TIL 5 TCR. Using this model, we will optimize methods of transducing and expanding normal PBL. We will evaluate 2 transduction methodologies, spinoculation and retronectin based transduction. We will also optimize methods for in vitro expansion of transduced T-cell populations and study TCR transduction (by RT-PCR) expression (by tetramer staining) as well as functional activity (by Cytokine release and Cr release assay). It is anticipated that methods developed under specific aim 2, will be applicable for cell populations transduced with newer vectors generated under specific aim 1. In specific aim 3 we will determine if TIL 13831 TCR gene modified T cells can be detected and quantified using RTP-CR and competitive RT-PCR. For this purpose specific primere for TIL 13831 will be designed, as well as a clone specific inhibitor. The validity of the test and its accuracy will be extensively evaluated.