Comparative analysis of second and third-generation nanobody-based CAR-NK cells targeting PSMA in prostate cancer immunotherapy: In silico and in vitro studies.

OBJECTIVES: The use of CAR technology in the treatment of cancer cells is progressing, as well as the use of CAR T-cell in the treatment of blood malignancies is in the clinical trial stage. In this study, have compared the second and third generation CAR expressed in NK cells in dealing with prostate cancer cells. MATERIALS AND METHODS: To optimize the CAR design for detection of NK-92 cells, an in silico approach to select between two tags (Flag and c-Myc tags) was initially employed, ultimately favoring the Flag tag. By using lentiviral particles, transduced NK-92 cells with second- and third-generation CAR constructs were subsequently tested as effector cells against prostate cancer cells expressing the Prostate-Specific Membrane Antigen (PSMA). Then assessed both cytokine secretion and cytotoxic responses. RESULTS: Computational analysis indicated that the NB/Flag/hinge configuration would provide superior PSMA recognition. Flow cytometry confirmed successful CAR expression in 47% of second-generation and 49% of third-generation NK-92 cells. Upon co-culture with PSMA-positive LNCaP cells and PSMA-negative PC-3 cells, the third-generation CAR NK cells demonstrated a noticeably stronger cytotoxic effect. Furthermore, higher levels of IL-2, TNF-α, and IFN-γ secretion were observed in the third-generation CAR group compared to the second. CONCLUSION: According to the computational studies, NB/Flag/hinge interacts with PSMA primarily through electrostatic forces. More importantly, third-generation CAR NK cells targeting PSMA displayed enhanced cytotoxicity and cytokine production relative to second-generation counterparts, pointing to their greater potential for therapeutic application.