Dissecting the role of T cell subpopulations during CAR T cell immunotherapy using non-invasive in vivo imaging in murine models of solid cancer

• Applicant: Dr. Barbara Schörg
• Subject Area: Radiology ; Medical Physics, Biomedical Technology ; Nuclear Medicine, Radiotherapy, Radiobiology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 555230620

CAR T cell therapies have revolutionized cancer treatment, particularly for hematopoietic malignancies. Nevertheless, CAR T cells are not as effective against solid tumors, leading to efforts to boost their performance. Current clinical CAR T cell generation protocols yield very potent but short-lived therapeutic cells due to susceptibility to activation-induced cell death (AICD). Improved CAR T cell products show less AICD but still face physical and molecular barriers such as lung sequestration or restricted access to the tumor tissue which highlights the importance of understanding the migration and biodistribution of CAR T cells post-infusion. This is crucial to overcome these limitations and for predicting therapy response. Non-invasive imaging techniques like bioluminescent optical imaging (BLI) are powerful, cost-effective tools for studying CAR T cell kinetics in vivo. In this project, CAR T cells will be generated in presence of IL-2 +- N-acetylcysteine, elevated Potassium-Ion levels, or of IL-7/IL-15 to enhance cellular memory and stemness. This may result in better in vivo persistence and anti-tumor activity. The two CAR T cell products demonstrating the best and the worst in vivo performance in A549 lung cancer xenograft mice will be used to establish a novel multiplex BLI approach for non-invasive in vivo imaging of CAR T cell subpopulations with respect to their biodistribution in the tumor tissue and the lymphatic system. For this, tumor cells and three different CD4+ and CD8+ CAR T cell subpopulations of the respective product will be genetically engineered to express distinct combinations of fluorescent and luciferase proteins and subsequently tracked in vivo by multiplex BLI using the A549 lung cancer model. We will use spectral unmixing to separate the individual emission spectra of the bioluminescence signals derived from the four different cell populations and evaluate them qualitatively and quantitatively. Next, we will modify the CAR and BLI reporter constructs for expression in murine T cells (mCARs) to subsequently perform non-invasive multiplex BLI cell tracking studies in immunocompetent, MC38 colon-carcinoma-bearing mice to identify endogenous cellular barriers. Our project will establish a novel imaging "toolbox" non-invasively study solid tumor growth together with the dynamics of CAR T cell subpopulations for the first time in vivo. Exploring the interactions between different CAR T cell subpopulations and endogenous, cellular host factors will improve our understanding of endogenous antitumor immunity to ultimately optimize CAR T cell persistence as well as the ability to overcome an immunosuppressive TME. Ultimately, the project will contribute to improving the treatment of solid tumors and to the development of non-invasive, predictive cellular markers for monitoring the treatment of cancer patients.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Vladimir Ponomarev, Ph.D.