CliCAR: A non-genetic CAR engineering strategy to empower NK cells for targeted cancer immunotherapy.

Conventional CAR-based immunotherapy has demonstrated promising efficacy in hematological malignancies. However, the inherently low transduction efficiency of viral vectors in innate immune cells, particularly natural killer (NK) cells, represents a significant barrier to the broader application of CAR-based immunotherapies. To overcome these limitations, in this study, we developed a nonviral platform, recombinant chimeric antigen receptor fusion protein (CliCAR), that rapidly converts immune cells into CAR-expressing cells without gene editing. CliCAR protein was rapidly internalized and localized to the cell membrane of NK cells, allowing it to exhibit CAR-like functions without genetic modification. As a result, CliCAR-NK cells exhibited selective cytotoxicity against HER2-positive cancer cells and enhanced immune activation, including increased levels of IFN-γ, perforin, and granzyme B. In vivo experiments, CliCAR-NK cells significantly suppressed HER2-positive tumor growth without observable toxicity in a xenograft model, supporting its therapeutic potential. Through this study, we introduce the CliCAR system as a novel protein-based CAR expression strategy that eliminates the need for genetic engineering and viral vector-mediated delivery. The ability to confer CAR function without gene editing provides a safe, rapid, and customizable strategy to produce potent, off-the-shelf immune cell therapies applicable to a broad range of tumor types, including solid tumors.