IMAGING T CELLS BY POSITRON EMISSION TOMOGRAPHY

描述（由申请方提供）：对CD 19具有特异性的临床级T细胞已证明具有抗肿瘤活性。我们现在提出了一个翻译研究，以调查的时间-空间生物分布和微环境与过继转移的CD 19特异性T细胞，实现使用正电子发射断层扫描（PET）。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，以输注自体和同种异体T细胞，这些T细胞经过遗传修饰以表达CD 19特异性嵌合抗原受体（CAR），该受体识别细胞表面上的CD 19，不依赖于MHC。这项新的R 01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，与行业（CellSight Technologies，Inc.）研究通过PET对输注的CAR+ T细胞进行成像的平台。这将通过使用睡美人（SB）转座子/转座酶系统在T细胞中共表达单纯疱疹病毒-1胸苷激酶（sr 39 tk）的突变体与CD 19特异性CAR来实现，我们已经将该系统用于临床翻译。我们将同步电转移表达SB转座子（i）CAR和（ii）sr 39 tk的两个DNA质粒，使用一种我们称为双转座的新方法。目的#1试图确定非病毒基因转移是否会产生在组成型和条件性启动子控制下共表达CD 19特异性CAR和sr 39 tk的T细胞。sr 39 tk报告基因将与潮霉素磷酸转移酶（Hy）融合，因此CAR+ sr 39 tk + T细胞将在杀细胞浓度的潮霉素B存在下在共表达CD 19沿着期望的T细胞共刺激分子的照射的人工抗原呈递细胞上选择性增殖。目的#2试图在免疫受损小鼠中用报告探针[18 F]FHBG对输注的人CAR+ sr 39 tk + T细胞进行纵向PET成像，以评估生物分布和检测灵敏度。通过比较（i）NFAT启动子控制下sr 39 tk的条件表达与（ii）CellSight开发的新PET探针[18 F]F-AraG，对T细胞活化状态进行成像。通过引入氧分子传感器来评估T细胞缺氧，以测试sr 39 tk是否可以报告低氧张力。目的#3旨在将这些临床前数据转化为一项新的临床研究，在接受CD 19特异性T细胞基因治疗的患者中输注CAR+ sr 39 tk + T细胞。本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淋巴恶性肿瘤研究受试者输注CAR+ T细胞。根据IND #61880，由CellSight Technologies销售的PET探针[18 F]FHBG将在TMH生产用于临床成像。总的来说，这些研究将测试中心假设，即CD 19特异性CAR+ sr 39 tk + T细胞可以使用PET在人体中成像。这些研究将建立基于PET的CAR+ T细胞成像的原则和实践，并提供关于转基因T细胞生物分布的第一个人类成像数据。 公共卫生相关性：转基因T细胞正在被注入作为淋巴瘤的研究性靶向治疗。在这里，我们寻求建立在我们的基因治疗，使注入的T细胞使用正电子发射断层扫描成像。

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.