FY25 LDRD Project Accomplishments

There is a critical need for new approaches to effectively counter emerging pathogens, especially those which do not respond to antibodies or antibiotics.T cells represent an essential element of native immune response in many of the deadliest pathogens: controlling T cell reactivity and behavior would allow for countermeasures for currently untreatable diseases from cancer to coronaviruses, especially if using a patient's own T cells (autologous) where no host rejection will occur.However, current methods for modifying T cells, e.g., FDA-approved chimeric antigen receptor T cell (CAR) approaches, require genetic manipulation and expansion which can take weeks to generate.Instead of genetic engineering, we sought to determine if a rapid method of membrane protein delivery could generate functional CAR-T cells in a rapid, safer, cost-effective manner.Using cell free protein synthesis, we generated CAR proteins embedded in a nanodisc, and thus effectively solubilized the protein.We demonstrated the first functional CAR proteins outside of a cellular context.We next tested uptake into immune cells and found extremely high uptake into T cells and multiple other populations of immune cells.We found some evidence of cytotoxicity in vitro conferred by the nanodisc delivered protein.