New tools for quantitative non-invasive recording of biochemical signals - ABSTRACT Chimeric antigen receptor (CAR) T-cells are a revolutionary cancer treatment, with the particular benefit of generating memory T-cells that can last for years and suppress cancer relapse. CAR-expressing CD8+ T-cells have been shown to cure leukemia and other cancers in clinical trials with great success. However, many challenges remain before CAR-based immunotherapy can become widely adopted, particularly for solid tumors. A major problem is that CAR T-cells become “exhausted” or “dysfunctional” and display decreased therapeutic effectiveness when subjected to prolonged antigen stimulation in the tumor microenvironment. Elevated cytosolic calcium is associated with T-cell activation, and as such, calcium signaling activity can be used as a quantitative measure of T-cell function or dysfunction. The history of calcium signaling over short and long periods of time encodes information about the current functional capacity of the T-cell and the number of tumor cells encountered in the past, and can be used both to evaluate populations or individual clones and to select for clones resistant to exhaustion. However, imaging calcium signaling activity in vivo using traditional genetically encoded fluorescent indicators presents unique challenges with T-cells and other highly mobile cell types, especially when medium- to long-term activity tracking is required. There is currently no facile method to image calcium signals at the single-cell level over long time periods or to track calcium activity history in such a highly mobile cell population, either in vitro or in vivo. In this project, we will use several distinct varieties of photoactive fluorescent proteins coupled with a family of high-contrast bioluminescent calcium sensors to generate Optical Recorders for Calcium (ORCas) capable of reporting short-, medium-, and long-term calcium signaling histories in CAR T-cells upon repeated exposure to target tumor cells. We will additionally engineer the small molecule substrates used to time-gate history recording for improved bioavailability and cell specificity, and to diversify the wavelengths of light emitted by the bioluminescent sensor domains of these probes. The engineered ORCas will ultimately be used to (1) track the exhaustion status of CAR T-cells over long periods of repeated exposure to target tumor cells, (2) enrich exhaustion-resistant populations of CAR T-cells from CRISPR knockout libraries, and (3) quantitatively benchmark the exhaustion resistance of CAR T-cell clones. These probes will additionally be validated in CAR T-cells in vivo in a subcutaneous mouse tumor model. Ultimately, we anticipate that ORCas will be the first of a broad new class of genetically-encoded probes capable of recording specific biochemical signal history non-invasively in many disease models and therapeutic interventions.
