The objective of this project is to develop a fluorescently-activatable reporter Chimeric Antigen Receptor T-cell (CAR-T) cell line for discovery of novel CAR-T receptors and their corresponding ligands. Significance: CAR-T therapy has demonstrated long-lasting treatment for hematological B cell malignancies but has yet to be effectively extended to other indications, e.g. solid tumors. Additional research tools are needed to expand the repertoire of available CAR-T therapies. Characterizing the performance of novel antigen/CAR-T combinations responsible for tumor clearance has proven to be a difficult challenge. We are developing a technology, PANACEA, that allows us to probe the behavior of a chimeric antigen receptor and its activation in a cellular context. Innovation: The core of the platform is an engineered cell line that contains an activatable synthetic reporter pathway. Upon CAR-T binding and cell activation, a reporter gene (e.g. a fluorescent protein) is expressed. This allows the researcher to detect activation events and use platforms such as cell sorting to isolate and study activated cells. By changing input parameters, PANACEA enables the study of variant libraries of CARs against single antigens, individual CARs vs libraries of antigens, or even full CAR-library-to-antigen-library screens. This reporter tool would enable rapid discovery and engineering of CAR-T constructs in a scientifically relevant context. Preliminary Data: We 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 as 1:2000. Moreover, we have developed a suite of microfluidic platforms that allow for generation and cloning of paired heavy/light chain libraries into a CAR vector. Specific Aims: (1) Engineer the system components and assay conditions to increase assay activation while maintaining sensitivity in our model system (2) Demonstrate the platformâs performance against a suite of clinically-relevant targets (3) Use the system to screen a library of variant CARs against a known target.
Public Health Relevance Statement: Project Narrative - Relevance to Public Health The objective of this project is to develop a biosensor platform for engineering novel and improved candidate CAR-T therapies. Given the positive patient response of patients with hematological cancers to CAR-T therapy, we 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 immunotherapies, including >500,000 patients who otherwise die from cancer each year. This innovation will provide researchers with a new way of visualizing the behavior of CARs in a cellular context, allowing for rapid discovery and engineering of novel therapies.
Project Terms: antigen binding; Antigens; B lymphoid malignancy; Behavior; Binding; Biological; Biological Assay; Biological Models; Biosensor; CAR T cell therapy; Categories; Cell Line; Cell Separation; Cells; Characteristics; chimeric antigen receptor; chimeric antigen receptor T cells; clinically relevant; Cloning; Coupled; Data; design; Engineering; Event; Exposure to; Generations; Hematologic Neoplasms; Hematology; Immunotherapy; improved; Individual; innovation; Libraries; Ligand Binding; Ligands; malignant breast neoplasm; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Malignant Neoplasms; Measures; mesothelin; Microfluidics; Modification; Multiple Myeloma; Nature; novel; novel therapeutics; Pathway interactions; patient response; Patients; Performance; Population; preservation; Property; Proteins; Public Health; receptor; Reporter; Reporter Genes; Reporting; Research; Research Personnel; screening; Signal Transduction; Solid Neoplasm; Specificity; System; T-Cell Activation; T-Cell Receptor; Technology; tool; tumor; Variant; vecto
