TCR-gene modified t-cells for adaptive immunotherapy

描述（由申请人提供）：已证明肿瘤反应性T细胞可诱导转移性黑色素瘤患者的肿瘤消退，并且已鉴定出一些靶抗原。尽管如此，目前通过使用细胞因子/生物反应调节剂、疫苗接种或使用过继免疫治疗的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CD 4肿瘤浸润淋巴细胞的T细胞受体（TCR），其具有MHC I类限制性识别和对其靶点酪氨酸酶的368-376肽的强亲和力。该提案的总体目标是开发使用TCR基因修饰的PBL的过继免疫治疗方法。在具体目标1中，我们将确定编码来自HLA-A2限制性、酪氨酸酶：368-376反应性T细胞克隆（TIL 13831）的TCR的逆转录病毒载体是否可以工程化来自正常供体和黑素瘤患者的正常外周血T细胞以高亲和力识别黑素瘤细胞。为此目的，我们将使用专门设计的具有SAMEN骨架的逆转录病毒载体，该载体适用于转移TCR α和β，并且在某些情况下还表达用于G418选择的neo（r）基因。在具体目标2中，我们将确定TCR基因修饰来自正常供体和黑色素瘤患者的人外周血T细胞用于患者治疗的最佳条件。对于这组实验，我们将使用先前产生的携带低亲和力TIL 5 TCR的A6和A7逆转录病毒载体。利用该模型，我们将优化正常PBL的转导和扩增方法。我们将评估2种转导方法，基于spinoculation和retronectin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释放测定）。预期在具体目标2下开发的方法将适用于用在具体目标1下生成的较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。为此目的，将设计TIL 13831的特异性引物以及克隆特异性抑制剂。测试的有效性及其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.