Spatial and Temporal control of gene therapy in CAR-T cells with ionizing radiation to improve local and distant control of solid tumors

Preclinical and clinical evidence suggest that radiation therapy (RT) and CAR-T therapy have complementary and/or synergistic effects through multiple possible mechanisms including increased CART infiltration, induction of target protein expression, and activation of innate immunity. Our novel radioinducible plug-and-play CAR-T system could potentially transform the clinical use of radiation, gene therapy, and CAR-T therapy in solid cancers by improving local tumor control and treatment of metastatic disease. RT has the potential to increase the efficacy of CAR-T therapy by debulking the tumor, restricting the need for toxic systemic therapies, promoting tumor infiltration by CAR-T, increasing the expression of target molecules by cancer cells, and amplifying the native immune response. All these mechanisms address current obstacles in CAR-T therapy of solid tumors. In Aim 1 we will generate synthetic radioinducible promoters, CAR-T production protocols, and RT/drug combinations that result in optimized transgene induction and CAR-T function in vitro. In Aim 2 we will use irradiation of tumors (s.c. and orthotopic) in mice to activate cytokine production by tumor-infiltrating radioinducible (Rad-I) GA1CAR-T cells, and to increase antigen expression, taking advantage of the ability to switch targets in a plug-and-play fashion provided by the GA1CAR. We will also leverage IR-induced changes in protein expression to explore novel IR-induced targets. In Aim 3 we will study the antitumoral mechanisms of radioinducible and switchable Rad-I GA1CAR-T therapy combined with IR, with a special focus on the prevention of tumor antigen loss variant (ALV) escape , using both xenograft and immunocompetent mouse models. We will also image the destruction of antigen-positive and negative cells, by longitudinal in vivo microscopy using fluorescently labeled cancer cells, CAR-T cells, and tumor vasculature, in tumors treated with the Rad-I plug-and-play GA1CAR-T system and IR. The effects of anti-cancer therapies must be tested in animal models because in vitro models are inadequate for translation to the clinic; mouse models are the primary source of pre-clinical data for human phase I trials. Discovery of novel therapeutic targets in our proposal depends on the positive or negative selection of genetically modified cancer cells in an immune-dependent fashion. Selection is influenced by the complex and dynamic interactions between modified cancer cells with mouse immune system that can only be elicited in vivo. These so-called “tumor microenvironments” are impossible to recreate in vitro or in silico with current technology. The proposed studies will require the use of both immunocompetent and immunodeficient mice. C57BL/6 mice are chosen because GL26, SB28 and LLC tumors grow in this mouse strain. NSG are selected because human cancer cell lines (SKBR-3, HCC1954, SKOV-3, SH-SY5Y, A549 and U87) grow in them. NSG-A2 mice are selected because they express HLAA2 human MHC-I molecules and therefore can be used to assess graft-vs-host responses. Project Number: 1R01CA304126-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: RALPH WEICHSELBAUM (+1 co-PI) | Institution: UNIVERSITY OF CHICAGO, CHICAGO, IL | Award Amount: $651,674 | Activity Code: R01 | Study Section: Radiation Therapeutics and Biology Study Section[RTB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11449605