IMAGING T CELLS BY POSITRON EMISSION TOMOGRAPHY

描述(由申请人提供)：针对CD19的临床级T细胞已显示出抗肿瘤活性。我们现在提议进行一项翻译研究，通过正电子发射断层扫描(PET)来研究过继转移的CD19特异性T细胞的时空生物分布和微环境。为了针对侵袭性B细胞恶性肿瘤，我们启动了两项临床试验，以注入自体和同种异体T细胞，这些T细胞已经经过基因改造，表达CD19特异性嵌合抗原受体(CAR)，该受体识别细胞表面的CD19，独立于MHC。这一新的R01拨款申请建立了一个跨学科(化学、成像、生物统计学、生物信息学、核医学、基因治疗和免疫学)和多机构(MDACC和TMH)的团队，与业界(CellSight Technologies，Inc.)合作。目的：探索一种利用正电子发射计算机断层扫描(PET)对输注的CAR+T细胞进行成像的平台。这将通过使用我们已经适应于临床翻译的睡美人(SB)转座子/转座酶系统，在T细胞中共表达单纯疱疹病毒1型胸苷激酶(Sr39tk)的突变体和CD19特异性CAR来实现。我们将使用一种称为双转座子的新方法，同步电转移表达SB转座子(I)car和(Ii)sr39tk的两个DNA质粒。目的#1试图确定非病毒基因转移是否会在结构性启动子和条件性启动子的控制下产生共表达CD19特异性Car和sr39tk的T细胞。Sr39tk报告基因将与潮霉素磷酸转移酶(Hy)融合，从而CAR+sr39tk+T细胞将在细胞毒浓度潮霉素B存在下选择性地在照射的人工抗原提呈细胞上增殖，这些细胞共表达CD19和所需的T细胞共刺激分子。目的#2试图用报告探针[18F]FHBG在免疫低下小鼠体内对人CAR+sr39tk+T细胞进行纵向PET成像，以评估生物分布和检测的敏感性。T细胞的激活状态将通过比较(I)在NFAT启动子控制下的sr39tk的条件表达和(Ii)CellSight开发的新的PET探针[18F]F-Arag来成像。T细胞缺氧的评估将通过引入氧气分子传感器来测试sr39tk是否可以报告低氧分压。目的#3试图将这些临床前数据转化为一项新的临床研究，在接受CD19特异性T细胞基因治疗的患者中注入CAR+sr39tk+T细胞。这项试验将是我们现有试验(IND#14193)的配对研究，该试验是在为患有晚期B淋巴样恶性肿瘤的研究参与者进行自体造血干细胞移植后注入CAR+T细胞。由CellSight技术公司销售的PET探针[18F]FHBG将在TMH制造用于临床成像，根据IND#61880。总而言之，这些研究将检验核心假设，即CD19特异性CAR+sr39tk+T细胞可以使用PET在人类体内进行成像。这些研究将建立转换基于PET的CAR+T细胞成像的原则和实践，并提供第一批关于转基因T细胞生物分布的人体成像数据。

DESCRIPTION (provided by applicant): Clinical-grade T cells rendered specific for CD19 have demonstrated anti-tumor activity. We are now proposing a translational study to investigate the temporal-spatial biodistribution and microenvironment associated with adoptively transferred CD19-specific T cells as achieved using positron emission tomography (PET). To target aggressive B-cell malignancies, we have initiated two clinical trials to infuse autologous and allogeneic T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) which recognizes CD19 on the cell surface, independent of MHC. This new R01 grant application establishes an inter-disciplinary (chemistry, imaging, biostatistics, bioinformatics, nuclear medicine, gene therapy, and immunology) and multi-institution (MDACC and TMH) team, partnering with industry (CellSight Technologies, Inc.) to investigate a platform for imaging infused CAR+ T cells by PET. This will be accomplished by coexpressing a mutant of herpes simplex virus-1 thymidine kinase (sr39tk) with the CD19-specific CAR in T cells using the Sleeping Beauty (SB) transposon/transposase system which we have adapted for clinical translation. We will synchronously electro-transfer two DNA plasmids expressing the SB transposons (i) CAR and (ii) sr39tk, using a new method we dub ¿double transposition¿. Aim #1 seeks to determine if non-viral gene transfer will produce T cells that co-express CD19-specific CAR and sr39tk under control of constitutive and conditional promoters. The sr39tk reporter gene will be fused to hygromycin phosphotransferase (Hy) and thus CAR+sr39tk+ T cells will be selectively propagated in presence of cytocidal concentration of hygromycin B on γ- irradiated artificial antigen presenting cells that co-express CD19 along with desired T-cell co-stimulatory molecules. Aim #2 seeks to undertake longitudinal μPET imaging of infused human CAR+sr39tk+ T cells with the reporter probe [18F]FHBG in immunocompromised mice to assess biodistribution and sensitivity of detection. T-cell activation status will be imaged by comparing (i) conditional expression of sr39tk under control of NFAT promoter with (ii) the new PET probe [18F]F-AraG developed at CellSight. T-cell hypoxia will be assessed by introducing a molecular sensor for oxygen to test whether sr39tk can report low oxygen tension. Aim #3 seeks to translate these pre-clinical data to a new clinical study infusing CAR+sr39tk+ T cells in patients undergoing gene therapy with CD19-specific T cells. This trial will be a companion study to our existing trial (IND# 14193) infusing CAR+ T cells after autologous hematopoietic stem-cell transplantation for research participants with advanced B-lymphoid malignancies. The PET probe [18F]FHBG, marketed by CellSight Technologies, will be manufactured for clinical imaging at TMH, per IND #61880. In aggregate, these studies will test the central hypothesis that CD19-specific CAR+sr39tk+ T cells can be imaged in humans using PET. These studies will establish principles and practices for translating PET-based imaging of CAR+ T cells and provide the first human imaging data on the biodistribution of genetically modified T cells.