Fluorescent Chimeric Antigen Receptor T Cell (CAR-T) Reporter Assay

Abstract The objective of this project is to develop a fluorescently activatable reporter Chimeric Antigen Receptor T cellCAR Tcell line for discovery of novel CAR T receptors and their corresponding ligandsSignificanceCAR T therapy has demonstrated long lasting treatment for hematological B cell malignancies but has yet to be effectively extended to other indicationse gsolid tumorsAdditional research tools are needed to expand the repertoire of available CAR T therapiesCharacterizing the performance of novel antigen CAR T combinations responsible for tumor clearance has proven to be a difficult challengeWe are developing a technologyPANACEAthat allows us to probe the behavior of a chimeric antigen receptor and its activation in a cellular contextInnovationThe core of the platform is an engineered cell line that contains an activatable synthetic reporter pathwayUpon CAR T binding and cell activationa reporter genee ga fluorescent proteinis expressedThis allows the researcher to detect activation events and use platforms such as cell sorting to isolate and study activated cellsBy changing input parametersPANACEA enables the study of variant libraries of CARs against single antigensindividual CARs vs libraries of antigensor even full CAR library to antigen library screensThis reporter tool would enable rapid discovery and engineering of CAR T constructs in a scientifically relevant contextPreliminary DataWe have constructed a novel cell line that fluorescently reports CAR activation with a library of inserted CARs and validated its ability to selectively trigger activation and find events as rare asMoreoverwe have developed a suite of microfluidic platforms that allow for generation and cloning of paired heavy light chain libraries into a CAR vectorSpecific AimsEngineer the system components and assay conditions to increase assay activation while maintaining sensitivity in our model systemDemonstrate the platform s performance against a suite of clinically relevant targetsUse the system to screen a library of variant CARs against a known target Project NarrativeRelevance to Public Health The objective of this project is to develop a biosensor platform for engineering novel and improved candidate CAR T therapiesGiven the positive patient response of patients with hematological cancers to CAR T therapywe expect that expanding the applicability of CAR T therapy to new classes of cancer and beyond will provide an alternative approach to therapy for a substantial fraction of the population who could benefit from immunotherapiesincluding andgtpatients who otherwise die from cancer each yearThis innovation will provide researchers with a new way of visualizing the behavior of CARs in a cellular contextallowing for rapid discovery and engineering of novel therapies