CD24 targeting with NK-CAR immunotherapy in testis, prostate, renal and (luminal-type) bladder cancer and identification of direct CD24 interaction partners.

Alternative therapeutic options targeting urologic malignancies, such as germ cell tumors, urothelial, renal and prostate carcinoma, are still urgently needed. The membrane protein CD24 represents a promising immunotherapeutical approach. This study aimed to decipher the molecular function of CD24 in vitro and evaluate the cytotoxic capacity of a third-generation NK cell CAR against CD24 in urologic tumor cell lines. Up to 20 urologic tumor cell lines and several non-malignant control cells were included. XTT assays and Annexin V / propidium iodide flow cytometry analyses were performed to measure cell viability and apoptosis rates, respectively. Co-immunoprecipitation followed by mass spectrometry analyses identified direct interaction partners of CD24. Luciferase reporter assays were used to functionally validate transactivation of CD24 expression by SOX2. N- and O-glycosylation of CD24 were evaluated by enzymatic digestion and mass spectrometry. This study demonstrated that SOX2 transactivates CD24 expression in embryonal carcinoma cells. In cells of different urological origins, CD24 interacted with proteins involved in cell adhesion, ATP binding, phosphoprotein binding, and post-translational modifications, such as histone acetylation and ubiquitination. Treatment of urological tumor cells with NK-CD24-CAR cells resulted in a decreased cell viability and apoptosis induction specifically in CD24 tumor cells. Limitations include the in vitro setting, which still has to be confirmed in vivo. In conclusion, here we identified CD24 as a promising novel target forimmune therapeutic approaches targeting urologic malignancies.