Imaging Infused CD19 Specific T Cells in the Tumor

描述（由申请人提供）：抗原特异性T细胞过继转移可以增强免疫反应，预防和治疗机会性感染和恶性肿瘤。基因疗法可以提高过继免疫疗法的治疗潜力，正如最近发表的（科学2006年8月31日），输注具有重定向特异性的黑色素瘤基因修饰T细胞可以发挥抗肿瘤作用。基于这一临床经验，我们已经启动了一项首次人体试验，利用基因修饰的T细胞表达嵌合抗原受体（CAR）靶向CD19治疗b细胞恶性肿瘤（BB-IND 11411，作者LJN Cooper）。在这项由NCI赞助的试验中（R21CA105824）， cd19特异性T细胞通过嵌合CD3-？利用第一代技术将car与CD19结合，导致抗原依赖性激活并杀死恶性B细胞。胸苷激酶（TK）的共表达允许在体内条件更昔洛韦（GCV）介导的严重毒性消融。虽然这项首次人体临床试验确定了CAR+ cd19特异性T细胞过继转移的安全性和可行性，但还需要进一步评估注入T细胞的存活和功能。特别是，我们寻求遗传工具来成像T细胞，这有助于移植物抗肿瘤（GVT）效应。因此，我们提出非侵入性成像来确定注入的转基因T细胞的持久性、分布和激活，我们假设激活的cd19特异性T细胞与静止的cd19特异性T细胞的时空分布与治疗效果相关。成像将结合荧光素酶依赖的生物光子学与tk依赖的微正电子发射断层扫描（PET），后者是一种可以很容易地转化为md安德森癌症中心临床的方法。这些研究将对来自外周血（PB）和脐带血（UCB）的T细胞进行，以产生cd19特异性效应细胞，这些效应细胞可以输注以增强gvt效应，特别是在同种异体造血干细胞移植后，特别是在UCB移植后。体内分析将用于确定这些表达第二代CAR的cd19特异性T细胞是否指定CD19RCD28，该细胞可以通过嵌合CD28和CD3-？在分布、活化电位和治疗效果上都优于表达第一代CAR，即CD19R的T细胞。为了实现这一目标，Aim #1将开发新的基于睡美人转座子/转座酶的DNA质粒，分别在组成型启动子和激活依赖性启动子下，在T细胞CD19R或CD19RCD28 CARs以及Photinus pyralis和Renilla reniformis衍生的荧光素酶和人类和HSV TK报告基因中共表达。Aim #2将过继性地将表达荧光素酶和TK报告基因的基因修饰的第一代和第二代PB和ucb衍生的CD19特异性T细胞转移到携带CD19+肿瘤的小鼠中，通过CD19R和CD19RCD28 car特异性激活T细胞，进行无创图像分布和激活。该项目开发了非侵入性测量输注肿瘤特异性T细胞分布和激活状态的工具。这将有助于研究人员评估t细胞免疫疗法的疗效。

DESCRIPTION (provided by applicant): Adoptive transfer of antigen-specific T cells can augment the immune response to prevent and treat opportunistic infections and malignancies. Gene therapy can improve the therapeutic potential of adoptive immunotherapy and as recently published (Science 2006 Aug 31), infusion of genetically modified T cells with redirected specificity for melanoma can treat exert an anti-tumor effect. Building up this clinical experience, we have initiated a first-in-human trial using T cells genetically modified to express a chimeric antigen receptor (CAR) to target CD19 on B-cell malignancies (BB-IND 11411, author LJN Cooper). In this trial sponsored by the NCI (R21CA105824), the CD19-specific T cells are activated via chimeric CD3-??using 1st-generation technology upon CAR-binding to CD19, resulting in antigen-dependent activation and killing of malignant B cells. Co-expression of thymidine kinase (TK) permits in vivo conditional ganciclovir (GCV)-mediated ablation in event of serious toxicity. While this first-in-human clinical trial establishes the safety and feasibility of adoptive transfer of CAR+ CD19-specific T cells, further insight is needed to evaluate the survival and function of infused T cells. In particular, we seek the genetic tools to image T cells which are contributing to the graft- versus-tumor (GVT)-effect. Therefore, we propose non-invasive imaging to determine persistence, distribution, and activation of infused genetically modified T cells and we hypothesize that the temporal-spatial distribution of activated versus quiescent CD19-specific T cells will correlate therapeutic efficacy. The imaging will combine luciferase-dependent biophotonics with TK-dependent micro-positron-emission tomography (PET), the latter being a methodology which can be readily translated to the clinic at the M.D. Anderson Cancer Center. These studies will be undertaken on T cells derived from peripheral blood (PB) and umbilical cord blood (UCB) to generate CD19-specific effector cells which can be infused to augment the GVT-effect, especially after allogeneic hematopoietic stem-cell transplantation in general and UCB transplantation in particular. An in vivo analysis will be used to determine if these CD19-specific T cells, expressing the 2nd- generation CAR, designated CD19RCD28, which can signal through chimeric CD28 and CD3-? are superior in distribution, activation potential and therapeutic efficacy, to T cells expressing the 1st-generation CAR, designated CD19R. To accomplish this, Aim #1 will develop new DNA plasmids based on Sleeping Beauty transposon/transposase to co-express in T cells CD19R or CD19RCD28 CARs as well as Photinus pyralis and Renilla reniformis derived luciferases and human and HSV TK reporter transgenes, under constitutive and activation-dependent promoters, respectively. Aim #2 will adoptively transfer genetically modified 1st- and 2nd- generation PB- and UCB-derived CD19-specific T cells expressing the luciferase and TK reporter genes into mice bearing CD19+ tumor to noninvasively image distribution and activation of T cells specifically activated through CD19R and CD19RCD28 CARs. This project develops the tools to non-invasively measure the distribution and activation status of infused tumor-specific T cells. This will help investigators gauge the efficacy of T-cell immunotherapies.