Ultrasound signaling cascade with calcium positive feedback loop as a signal amplifier for transient activation of chimeric antigen receptors for cancer immunotherapy

Cancer is one of the main causes of disease-related deaths in the world. Chimeric antigen receptors (CARs) cancer immunotherapy has emerged as an effective therapeutic strategy for hematological malignancies. One of the biggest concerns of CAR T cell therapy is safety. Therapeutic responses of CAR T cells are challenged by the adverse effects, such as a systemic release of a high level of cytokines, and toxicities resulting from specific interactions between the CAR receptor and its target antigen expressed by non-malignant cells. The challenge addressing toxicities in CAR T immunotherapy of solid tumors is to develop therapeutic cells that can be transiently activated at confined tissue space. Focused ultrasound has a unique advantage in providing non-invasive temporal control of cellular activity in deep tissue with spatial precision. We will use exposure to focused ultrasound, insonation, for noninvasive temporal activation of an engineered therapeutic device. The focused ultrasound activation of therapeutic cells in cancer immunotherapy presents several challenges; in the project, we aim to address two of them. A shortened insonation time will be achieved with the incorporation of a synthetic Ca2+ positive feedback loop based on Ca2+ release-activated Ca2+ channels (CRAC), which underline sustained Ca2+ signaling. Interaction between STIM1 and Orai1 will be exploited to build a synthetic Ca2+ signal amplifier. Furthermore, we aim to decode ultrasound parameters defining acoustic wave frequency and amplitude to regulate the opening of mechanosensitive Ca2+ ion channels and Ca2+ oscillation. To regulate the expression of autologous genes and CARs, the ultrasound-responsive transcription factors based on CRISPR DNA-binding domain, being able to bind diverse genes, will be synthesized. For proof-of-concept, a spatiotemporal non-invasive activation of the ultrasound-responsive CAR T cells inducing cytotoxicity and tumor eradication will be tested. Our concept introduces some known building blocks and processes into a new technological platform. Besides direct and indirect applications, the project will also provide an insight into the mechanisms of mechanosensing of cells. The main novelty of our proposal is an augmentation of cell sensitivity to the ultrasound by introducing a Ca2+ positive feedback loop amplifying the ultrasound signal. The ultrasound immunomodulation platform will provide a unique approach to interrogate CAR-mediated anti-solid tumor activity and can serve as an efficient tool for non-invasive spatiotemporal gene activation. The expected results will support critical progress on the development of ultrasound-responsive molecular devices that could influence a wide range of areas of biotechnology and medicine.