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Principal Investigator: Jennifer Prescher
Organization: UNIVERSITY OF CALIFORNIA, SAN DIEGO
Fiscal Year: 2024
Award: $675,594
Funding agency: National Cancer Institute
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.
Terms: <Activities of Daily Living><Activities of everyday life><Adopted><Adoption><Antigens><Benchmarking><Best Practice Analysis><Bioavailability><Biochemical><Biological><Biological Availability><Bioluminescence><CAR T cell therapy><CAR T cells><CAR T therapy><CAR modified T cells><CAR-T><CAR-Ts><CD8 Cell><CD8 T cells><CD8 lymphocyte><CD8+ T cell><CD8+ T-Lymphocyte><CD8-Positive Lymphocytes><CD8-Positive T-Lymphocytes><CRISPR><CRISPR/Cas system><Calcium><Calcium Ion Signaling><Calcium Signaling><Cancer Relapse><Cancer Treatment><Cancers><Cell Body><Cell Communication and Signaling><Cell Function><Cell Physiology><Cell Process><Cell Signaling><Cell model><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular model><Chimera Protein><Chimeric Proteins><Clinical><Clinical Trials><Clone Cells><Clustered Regularly Interspaced Short Palindromic Repeats><Co-culture><Cocultivation><Coculture><Coculture Techniques><Coupled><DNA Alteration><DNA Sequence Alteration><DNA mutation><Dysfunction><Engineering><Event><Exposure to><FRET><Family><Fluorescence><Fluorescence Activated Cell Sorting Fractionation><Fluorescence Resonance Energy Transfer><Fluorescence-Activated Cell Sorting><Fluorescence-Activated Cell Sortings><Functional disorder><Fusion Protein><Future><Förster Resonance Energy Transfer><Genetic mutation><Habitats><Hematopoietic Cell Tumor><Hematopoietic Malignancies><Hematopoietic Neoplasms><Hematopoietic Neoplasms including Lymphomas><Hematopoietic Tumor><Hematopoietic and Lymphoid Cell Neoplasm><Hematopoietic and Lymphoid Neoplasms><Heterograft><Heterologous Transplantation><History><Image><Imaging Device><Imaging Instrument><Imaging Tool><Immune mediated therapy><Immunologically Directed Therapy><Immunotherapy><In Vitro><Individual><Intracellular Communication and Signaling><Investigators><Kinetics><Knock-out><Knockout><Libraries><Light><Luciferase Immunologic><Luciferases><Malignant Hematopoietic Neoplasm><Malignant Neoplasm Therapy><Malignant Neoplasm Treatment><Malignant Neoplasms><Malignant Tumor><Measures><Methods><Mice><Mice Mammals><Microscopy><Modeling><Monitor><Murine><Mus><Neurosciences><Optical reporter><Optics><Oranges><Organism><Performance><Photoradiation><Physiologic Availability><Physiopathology><Population><Proteins><Reagent><Recording of previous events><Reporter><Reporting><Research Personnel><Researchers><Resistance><Resolution><Role><Sequence Alteration><Signal Transduction><Signal Transduction Systems><Signaling><Solid Neoplasm><Solid Tumor><Source><Specificity><Subcellular Process><T cells for CAR><T memory cell><T-Cell Activation><T-Cell Receptor Interaction><T-Cells><T-Lymphocyte><T8 Cells><T8 Lymphocytes><TCR Activation><TCR Interaction><Technology><Therapeutic><Therapeutic Agents><Therapeutic Intervention><Time><Translations><Tumor Cell><Visualization><Work><Xenograft><Xenograft procedure><Xenotransplantation><activate T cells><anti-cancer therapy><benchmark><biologic><biological signal transduction><blood cancer><blood treatment><calcium indicator><cancer microenvironment><cancer of blood><cancer of the blood><cancer therapy><cancer-directed therapy><cell type><chimeric antigen T cell receptor><chimeric antigen receptor><chimeric antigen receptor (CAR) T cell therapy><chimeric antigen receptor (CAR) T cells><chimeric antigen receptor T cell therapy><chimeric antigen receptor T cells><chimeric antigen receptor T therapy><chimeric antigen receptor fusion protein T-cells><chimeric antigen receptor modified T cells><daily living function><daily living functionality><design><designing><disease model><disorder model><exhaust><exhaustion><experiment><experimental research><experimental study><experiments><functional ability><functional capacity><genomic alteration><histories><imaging><imaging platform><imaging probe><immune therapeutic approach><immune therapeutic interventions><immune therapeutic regimens><immune therapeutic strategy><immune therapy><immune-based therapies><immune-based treatments><immuno therapy><immunogen><improved><in vivo><insight><interest><intervention therapy><leukemia><light emission><living system><luciferin><malignancy><memory T lymphocyte><neoplasm/cancer><neoplastic cell><optical><pathophysiology><resistant><resolutions><sensor><small molecule><social role><subcutaneous><subdermal><success><therapeutic effectiveness><therapeutically effective><thymus derived lymphocyte><tool><translation><tumor><tumor microenvironment><xeno-transplant><xeno-transplantation>