Project Grant R44GM149102

HYDROGEL MICROPARTICLE TECHNOLOGY FOR HIGH-THROUGHOUT SCREENING OF CHIMERIC ANTIGEN RECEPTOR-T CELLS BASED ON SINGLE CELL EFFECTOR FUNCTION - ABSTRACT ENGINEERED CELL THERAPIES ARE BECOMING A PILLAR OF MEDICINE, ALONG WITH MOLECULAR AND GENETIC INTERVENTIONS. IN PARTICULAR, CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELL THERAPIES HAVE HAD A NUMBER OF FDA APPROVALS IN THE LAST 5 YEARS TARGETING HEMATOLOGIC MALIGNANCIES. TO EXTEND THE SUCCESS OF THESE THERAPIES, ESPECIALLY FOR THE TREATMENT OF SOLID TUMORS, A MORE THOROUGH UNDERSTANDING OF HOW CAR STRUCTURE IS LINKED TO CAR-T CELL FUNCTION IS NECESSARY. NOTABLY, WIDESPREAD TOOLS TO ANALYZE AND SELECT SINGLE CAR-T CELLS FROM A LARGE POPULATION BASED ON FUNCTIONAL PROPERTIES, SUCH AS SECRETED PRODUCTS OR CYTOLYTIC ACTIVITY, ARE CRITICALLY LACKING. SINGLE-CELL FUNCTIONAL ASSAYS CAN ENABLE SCREENING A LIBRARY OF CAR DESIGNS INTRODUCED INTO A POOL OF CELLS TO IDENTIFY RARE FUNCTIONAL CELLS AND SORT THESE CELLS TO RECOVER CAR DESIGNS ASSOCIATED WITH IMPORTANT EFFECTOR FUNCTIONS. IF CONDUCTED IN HIGH THROUGHPUT, THOUSANDS OF CONSTRUCTS CAN BE SCREENED WITH HUNDREDS OF INDIVIDUAL EVENTS PER CONSTRUCT TO HAVE ROBUST STATISTICAL ACCURACY IN THE LINKAGE BETWEEN FUNCTION AND SEQUENCE. IN ADDITION, IF SINGLE-CELL FUNCTIONAL INFORMATION CAN BE TIED TO TRANSCRIPTOMIC INFORMATION, PATHWAY ANALYSIS ASSOCIATED WITH STRONG EFFECTOR FUNCTIONS CAN BE PERFORMED, IDENTIFYING OTHER GENE TARGETS THAT IMPROVE FUNCTION, EVEN IN THE PRESENCE OF IMMUNOSUPPRESSIVE, EXHAUSTION-PRONE, OR OTHER MICROENVIRONMENTS ASSOCIATED WITH SOLID TUMORS. PARTILLION'S NANOVIAL TECHNOLOGY PROVIDES A NEW APPROACH TO MEASURE THE FUNCTION OF SINGLE CELLS USING WIDELY AVAILABLE FLUORESCENCE ACTIVATED CELL SORTERS (FACS) AND SINGLE-CELL SEQUENCING INSTRUMENTS, WHICH WE AIM TO APPLY TO CELL THERAPY DISCOVERY. HERE, WE PROPOSE TO DEVELOP SINGLE-CELL SECRETION AND CELL-KILLING ASSAYS COMPATIBLE WITH THE NANOVIALS TO INTRODUCE A NEW PRODUCT THAT WOULD ENABLE SCALED CELL THERAPY DISCOVERY WORKFLOWS FROM MILLIONS OF CELLS. WE AIM TO IDENTIFY OPTIMAL NANOVIAL FORMULATIONS AND PROCEDURES TO MEASURE BOTH CYTOKINE PRODUCTION AND CYTOLYTIC FUNCTIONS FROM THE SAME CELLS, AND THEN LINK SINGLE-CELL TRANSCRIPTOMIC INFORMATION WITH THIS FUNCTIONAL READOUT. WE ALSO WILL ENGINEER NANOVIALS TO BETTER RECAPITULATE THE TUMOR MICROENVIRONMENT, ACTING AS AN ARTIFICIAL ANTIGEN-PRESENTING TARGET CELL WITH COMBINATIONS OF ANTIGEN AND IMMUNOSUPPRESSIVE SIGNALS. THE APPROACH SHOULD BE APPLICABLE BROADLY BEYOND CAR-T CELL THERAPIES TO OTHER CHIMERIC RECEPTORS IN NATURAL KILLER CELLS, AND MACROPHAGES, OR IN FINDING ENGINEERED T CELL RECEPTORS. ULTIMATELY, MORE ACCESS TO SOPHISTICATED CELL SELECTION APPROACHES CAN LEAD TO THERAPIES THAT ARE BOTH LOWER IN PRICE AND MORE EFFECTIVE AS WELL AS EXPANDING THE SCOPE OF APPLICATIONS TO UN-EXPLORED THERAPEUTIC AREAS.