Authors / Presenters
Parijat Bhatnagar 1,Mian Alauddin 2,Payam Seifi 3,James A Bankson 4,Dickson Kirui 5,Helen Huls 6,Dean A Lee 6 7,Aydin Babakhani 3,Mauro Ferrari 5,King C Li 8,Laurence JN Cooper 6 7
Primary human immune T cells isolated and frozen at an early healthier stage can be restored, genetically modified for specificity, selectively expanded, and administered to the patient at a later morbid stage to exert the anti-tumor response. The response to this therapy during the clinical trials, Adoptive T Cell Therapy, remains inconsistent at best. In order to improve the reliability, efficacy and safety of the clinical trials it is important to image the biodistribution of these genetically modified T cells. This can determine if the T cells are homing to their tumor targets and can be used to modulate the T-cell-based drug load. Current methods to assess biodistribution of infused T cells include serial sampling from various tissues followed by quantitative PCR (Q-PCR) or flow cytometry; which is invasive, painful, and does not provide whole-body distribution. Magnetic Resonance Imaging (MRI) has been used to image cells in vivo, it requires the approximate location of cells to be already known. We hypothesized that if T cells can be labeled with PET-MRI imaging agents, this initial positioning of cells can be provided by Positron Emission Tomography (PET) at high-sensitivity. MRI can then be used to scan these areas and report on anatomically correlated biodistribution of adoptively transferred T cells at high-resolution.
Previously frozen peripheral blood mononuclear cells were thawed and genetically modified with Sleeping Beauty transposon/transposase system to express CD19-specific chimeric antigen receptor (CAR) and firefly luciferase (ffLuc) enzyme. CD19-specific-CAR+ffLuc+ T cells were selectively expanded on artificial antigen presenting cells and labeled with super paramagnetic iron oxide nanoparticles conjugated to fluorescent probe and positron emitter (SPION-FL-64Cu). SPION retention in T cells was studied using flow cytometry and the internalization into the cytoplasm was verified using confocal microscopy. Iron content in a single T cell was determined by inductively coupled plasma mass spectrometer. Effect of SPION and 64Cu on cell viability was assessed by the ffLuc enzymatic activity. MRI signal was obtained from the series of diluted and homogenously suspended T cell phantoms. Chromium release assay and live-cell time-lapse imaging was used to assess the in vitro tumor targeting capability of CD19-specific-CAR+ffLuc+SPION+ T cells.
Our approach builds upon ongoing clinical trials for CD19+ B-cell malignancies and uses an approach that can be readily undertaken in compliance with current good manufacturing practice for Phase I/II trials.