Linker-specific monoclonal antibodies present a simple and reliable detection method for scFv-based CAR NK cells

Chimeric antigen receptor (CAR) T cell therapies have demonstrated significant successes in treating cancer. Currently, there are six approved CAR T cell products available on the market that target different malignancies of the B cell lineage. However, to overcome the limitations of CAR T cell therapies, other immune cells are being investigated for CAR-based cell therapies. CAR natural killer (NK) cells can be applied as allogeneic cell therapy, providing an economical, safe, and efficient alternative to autologous CAR T cells. To improve CAR research and future in-patient monitoring of cell therapeutics, a simple, reliable, and versatile CAR detection reagent is crucial. As most existing CARs contain a single-chain variable fragment (scFv) with either a Whitlow or a G4S linker site, linker-specific monoclonal antibodies (mAbs) can detect a broad range of CARs. This study demonstrates that these linker-specific mAbs can detect different CAR NK cells <i>in vitro</i>, spiked in whole blood, and within patient-derived tumor spheroids with high specificity and sensitivity, providing an effective and almost universal alternative for scFv-based CAR detection. Additionally, we confirm that linker-specific antibodies can be used for functional testing and enrichment of CAR NK cells, thereby providing a useful research tool to fast-track the development of novel CAR-based therapies.