Abstract LB-023: Dynamic real time in vivo CAR T cell tracking: Clinical and preclinical studies using a novel dual PET-MR imaging agent
Authors: Singla Ritu , Wall Dominic , Anderson Samuel , Zia Nicholas , Korte James C. , Kravets Lucy , McKiernan Gerard , Butler Jeanne , Gammilonghi Amanda , Arora Jyoti , Solomon Ben , Hicks Rodney , Cain Timothy , Darcy Phillip , Cullinane Carleen , Neeson Paul , Ramanathan Rajesh , Shukla Ravi , Bansal Vipul , Harrison Simon 
Published: September 2020

Abstract
Objective: The aim is to demonstrate dynamic in-vivo tracking of CAR T cell therapy for treatment of solid tumors using Cu-64 labeled superparamagnetic iron oxide nanoparticles (SPION) as novel dual PET-MR imaging agent.
Methodology: Cu-64 SPION: Cu-64 radioisotope is bound to silica coated SPION using enhanced electrolysis plating techniques with tin and palladium seeding. Preclinical Model: Mouse splenic T cells were activated with anti-CD3, anti-CD28 & cultured with IL-2 and IL-7, prior to being transduced with second generation anti-Her-2 CAR (scFv-CD28-CD3ζ). 5 x 105 E0771-hHER2 breast tumor cells were implanted subcutaneously into male C57Bl/6-human HER2 transgenic mice. 107 labeled CAR T or control T cells (Cu-64 5-8 MBq) were injected into tail vein. Clinical Model: Activated T cells using antibody CD3 (OKT3) & IL-2 are transduced with retroviral vector constructs encoding for chimeric T-cell receptor specific for Lewis Y antigen. Modified T-cells are further expanded ex-vivo and reinfused. 3 x 108 CAR T cells were labeled with Cu-64 (200 - 300 MBq). Labeling of CAR T cells with Cu-64 SPION: Transfecting agent protamine sulphate facilitated cellular uptake of Cu-64 SPION within cells. Functional assays: 51Chromium release, cytometric bead array and cell viability showed that labeling process did not affect CAR T cell cytotoxicity, cytokine secretion (TNFα and IFN-γ) and viability. CAR T Cell Tracking: Scanning was performed using clinical grade dual PET-MR scanner.
Preliminary Data: In this clinical trial (HREC/16/PMCC/30) patients are being enrolled for first in human in vivo study to determine how many cells distribute to solid tumor sites within first few days of CAR T cell therapy. This is first data that demonstrates that CAR-T cells can be consistently and efficiently labeled (≤60%) with cell viability (≥85%) and at sensitivity comparable to detecting at least z cells at tumor site using clinical grade dual PET-MR scanner. SUVmean values provides insight into individual response to therapy. The observed increase in SUVmax over time specifies localization of CAR T cells at tumor sites.
Clinical data at early time point showed moderate uptake in lungs posterior basal segments without increased activity over next few days, thus suggesting transient process. Mild, diffuse bone marrow and relatively intense uptake in the liver and spleen suggests margination of cells to the reticulo-endothelial system. Distinct PET signal suggests localization of labeled cells in the secondary tumor sites. Little background uptake in important organs such as brain and heart indicate the safety profile of imaging agent. Absence of signal in bladder indicates hepatobiliary excretion, which may allow re-absorption from GI tract and re-circulation.
Distinct PET signal within tumor in preclinical studies confirms trafficking of CAR T cells to tumor site as compared to controls. A negative contrast in the liver on T2 weighted MRI in both the preclinical and clinical studies. Preliminary Conclusion:This is first in human in vivo study to show CAR T cell distribution in real time and provides insight into individual responses of tumors to therapy. CAR T cell functionality largely remain unchanged due to labeling process. The preliminary findings indicate that labeled cells traffic to tumor sites in first few hours of infusion and remain persistent for extended period.
Citation Format: Ritu Singla, Dominic Wall, Samuel Anderson, Nicholas Zia, James C. Korte, Lucy Kravets, Gerard McKiernan, Jeanne Butler, Amanda Gammilonghi, Jyoti Arora, Ben Solomon, Rodney Hicks, Timothy Cain, Phillip Darcy, Carleen Cullinane, Paul Neeson, Rajesh Ramanathan, Ravi Shukla, Vipul Bansal, Simon Harrison. Dynamic real time in vivo CAR T cell tracking: Clinical and preclinical studies using a novel dual PET-MR imaging agent [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-023.