An indirect labelling approach to track the therapeutic CAR T-cells with dual PET and fluorescence imaging

Proposed Research Question: To determine the viability and efficacy of a generic dual PET and fluorescence imaging reagent to indirectly label and track CAR T-cells in vivo

Cell-based therapies have shown great promise in cancer treatment. A fundamental challenge in the successful development and clinical application of cellular therapeutics is the need to better understand the in vivo behaviour of adoptively infused cell products. This project aims to develop a generic indirect labelling approach to track the persistence and proliferation of the c-myc tag-containing CAR T-cells with dual PET and fluorescence imaging. A dual reporter bio-conjugation reagent, 124I-Fluorescein, labelled anti-c-myc 9E10 antibody will be systematically evaluated for this purpose. This dual PET and fluorescence imaging approach would enable detection of the in vivo behaviour of the c-myc tag-containing CAR T-cells across cellular to macroscopic scales. PET would provide real-time images of the living CAR T-cells and quantitatively measure their migration and proliferation in the whole-body. Meanwhile, the high-resolution ex vivo fluorescence imaging will unveil precisely the distribution of these CAR T-cells in the target and non-target tissues. John Maher's A20-28z CAR (targeting AvB6 integrin) will be used.
After assessing the labelling reagents alone for efficacy, we will characterise their functionality when bound to the 9E10 antibody in vitro and evaluate the anti-tumour activity of the CAR prior to and post incubation with the labelling reagent. This can be achieved with ELISAs for cytokines such as interferon gamma, and IL-2, and by conducting MTT assays to assess the cytotoxic effect of labelled and unlabelled CAR T-cells. We can then examine the biodistribution of the antibody-labelling reagent complex. If the biodistribution is favourable then we will commence in vivo tracking studies of the A20-28z CAR T-cells