Imaging T Cell Interactions in Adoptive Therapy of EBV-Associated Malignancies

项目1的重点是开发报告基因成像方法，可以转化为临床 应用.具体地说，我们希望测试是否连续成像过继转移的抗原特异性 T细胞可用于在转移后早期预测靶向肿瘤的应答，并识别T细胞受体。 影响抗肿瘤活性的相互作用。我们建议首先开发和测试一系列矢量编码 组成型和诱导型报告基因，并评估其区分亚群的能力 EBV特异性T细胞在其特异性抗原诱导的活化-增殖或凋亡期间的增殖（Aim 1）。我们 然后将评估转导的抗原特异性T细胞的不同亚群是否以及在何种程度上 可以通过共施用的T细胞的顺序成像在体内区分，过继转移到 携带人EBV淋巴瘤异种移植物的NOD/SCID小鼠。我们还将研究 不同功能的T细胞亚群，转导以表达可区分的报告基因，以靶向和 在EBV淋巴瘤异种移植物中积累，以在这些肿瘤中增殖和存活，并评估其 在过继转移后的连续时间间隔对靶向肿瘤细胞的杀肿瘤活性（Aim 2）。在Aim中 3，我们建议进行用新载体转导的EBV特异性T细胞的I期临床试验 编码两种人基因，突变型LNGFR和人碘同向转运体（hNIS），在治疗 EBV淋巴瘤并发异基因造血祖细胞移植或器官移植。 此后，我们将把这种载体纳入第二阶段试验测试和成像EB病毒特异性T细胞 不同的是体外选择的库和持续时间，以及它们的CD 4和CD 8 T细胞的含量， 将在它们用可区分的报道构建体转导后被鉴定。其中一个载体， 称为NIT，是编码人神经生长因子的生物学失活突变体的逆转录病毒载体 受体和IRES连接的HSV-胸苷激酶基因。这个载体正在进行临床试验 引入的新的双顺反子载体衍生自NIT载体，但用hNIS基因替代了NIT载体。 HSVItk，因为目前用作报道基因的HSVItk（和掺入HSVItk的融合基因）是有限的 这种新的双顺反子载体，它专门编码人的免疫原性。 用于体外选择和体内成像的基因，可能是免疫原性低得多的，因此不应该 损害过继转移后转导的T细胞的寿命。计划的第二阶段试验应 还提供了早期选择的抗原特异性T细胞的活性和持久性的直接比较， 在体外培养的后期。

Project 1 focuses on developing reporter gene imaging methods that can be translated into clinical applications. Specifically, we wish to test whether sequential imaging of adoptively transferred antigenspecific T cells can be used to predict responses of targeted tumors, early after transfer, and to identify T cell interactions affecting anti-tumor activity. We propose to initially develop and test a series of vectors encoding both a constitutive and an inducible reporter gene, and to assess their capacity to distinguish subpopulations of EBV-specific T cells during their specific antigen-induced activation-proliferation or apoptosis (Aim 1). We will then evaluate whether and to what degree different subpopulations of transduced antigen-specific T-cells can be distinguished in vivo by sequential imaging of co-administered T-cells, adoptively transferred into NOD/SCID mice bearing human EBV lymphoma xenografts. We will also examine the contributions of different functional subsets of T-cells, transduced to express distinguishable reporter genes, to target and accumulate in EBV lymphoma xenografts, to proliferate and survive in these tumors, and to assess their tumoricidal activity against targeted tumor cells at sequential intervals after adoptive transfer (Aim 2). In Aim 3, we propose to conduct a phase I clinical trial of EBV-specific T-cells transduced with a new vector encoding two human genes, a mutant LNGFR and the human iodide symporter (hNIS), in the treatment of EBV lymphomas complicating allogeneic hematopoietic progenitor cell transplants or organ allografts. Thereafter, we will incorporate this vector into a phase II trial testing and imaging EBV-specific T-cells differing in repertoire and duration of selection in vitro, as well as in their content of CD4 and CDS T-cells that will be identifiable following their transduction with distinguishable reporter constructs. One of these vectors, termed NIT, is a retroviral vector encoding a biologically inactive mutant of the human nerve growth factor receptor and an IRES-linked HSV-thymidine kinase gene. This vector being used in a current clinical trial. The new dicistronic vector being introduced, is derived from the NIT vector, but substitutes the hNIS gene for HSVItk because HSVItk (and fusion genes incorporating HSVItk) currently in use as reporters are limited by their potential immunogenicity in man. This new dicistronic vector, which exclusively encodes human genes for in vitro selection and in vivo imaging, is likely to be far less immunogenic, and should therefore not compromise the life span of transduced T-cells following adoptive transfer. The planned phase II trial should also provide a direct comparison of the activity and persistence of antigen-specific T-cells selected early or late in the course of in vitro culture.