IMAGING T CELLS BY POSITRON EMISSION TOMOGRAPHY

描述（由申请人提供）：临床级CD19特异性T细胞已被证明具有抗肿瘤活性。我们现在提出一项转化研究，利用正电子发射断层扫描（PET）来研究与过继性转移cd19特异性T细胞相关的时空生物分布和微环境。为了靶向侵袭性b细胞恶性肿瘤，我们已经启动了两项临床试验，注入经过基因修饰的自体和异体T细胞，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。这项新的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）团队，与工业界（CellSight Technologies, Inc.）合作，研究一个通过PET注入CAR+ T细胞成像的平台。这将通过使用睡美人（SB）转座子/转座酶系统在T细胞中与cd19特异性CAR共表达单纯疱疹病毒-1胸苷激酶（sr39tk）突变体来实现，我们已经适应了临床翻译。我们将使用一种我们称为“双转座”的新方法，同步电转移两个表达SB转座子(i) CAR和（ii） sr39tk的DNA质粒。Aim #1旨在确定非病毒基因转移是否会产生在组成启动子和条件启动子控制下共同表达cd19特异性CAR和sr39tk的T细胞。sr39tk报告基因将融合到潮霉素磷酸转移酶（Hy）中，因此CAR+sr39tk+ T细胞将在潮霉素B的细胞杀伤浓度下选择性地在&#947；辐照的人工抗原提呈细胞上繁殖，这些细胞与所需的T细胞共刺激分子共同表达CD19。Aim #2试图在免疫功能低下小鼠中使用报告探针[18F]FHBG对输注的人CAR+sr39tk+ T细胞进行纵向PET成像，以评估检测的生物分布和灵敏度。将通过比较(i) NFAT启动子控制下sr39tk的条件表达与（ii） CellSight开发的新型PET探针[18F]F-AraG来成像t细胞的激活状态。t细胞缺氧将通过引入氧气分子传感器来评估，以测试sr39tk是否可以报告低氧张力。Aim #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。该试验将是我们现有试验（IND# 14193）的伴随研究，该试验在自体造血干细胞移植后为晚期b淋巴细胞恶性肿瘤的研究参与者注入CAR+ T细胞。PET探针[18F]FHBG由CellSight Technologies销售，将根据IND #61880在TMH生产用于临床成像。总的来说，这些研究将验证cd19特异性CAR+sr39tk+ T细胞可以使用PET在人类中成像的中心假设。这些研究将建立基于pet的CAR+ T细胞翻译成像的原则和实践，并提供关于基因修饰T细胞生物分布的第一个人类成像数据。

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. PUBLIC HEALTH RELEVANCE: Genetically modified T cells are being infused as investigational targeted treatment for lymphomas. Here, we seek to build on our gene therapy to enable infused T cells to be imaged using positron emission tomography.