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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-SBIB-X (57))
Program Officer
Baker, Houston
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Texas MD Anderson Cancer Center
Other Domestic Higher Education
United States
Zip Code
Singh, Harjeet; Huls, Helen; Kebriaei, Partow et al. (2014) A new approach to gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells to target CD19. Immunol Rev 257:181-90
Rushworth, David; Jena, Bipulendu; Olivares, Simon et al. (2014) Universal artificial antigen presenting cells to selectively propagate T cells expressing chimeric antigen receptor independent of specificity. J Immunother 37:204-13
Maiti, Sourindra N; Huls, Helen; Singh, Harjeet et al. (2013) Sleeping beauty system to redirect T-cell specificity for human applications. J Immunother 36:112-23
Singh, Harjeet; Figliola, Matthew J; Dawson, Margaret J et al. (2013) Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells. PLoS One 8:e64138