Targeting NKG2DLs with an ADCC enhanced fusion protein for induction of NK cell reactivity against ovarian cancer

Ligands for the activating immune receptor NKG2D (NKG2DLs) are frequently overexpressed on malignant cells while largely absent in healthy tissues. Elevated expression of various NKG2DLs has been reported in ovarian cancer, one of the most lethal gynecologic malignancies due to chemoresistance and relapse. Despite the advent of monoclonal antibodies (mAbs) in cancer therapy, patients with ovarian cancer have yet to benefit from this treatment modality. Natural killer (NK) cells play a crucial role in the efficacy of antitumor mAbs by mediating antibody-dependent cellular cytotoxicity (ADCC). Strategies to enhance ADCC often involve Fc region modifications to improve CD16 binding and NK cell activation.The tumor-associated expression of NKG2DLs was exploited by developing an Fc-optimized NKG2D-Ig fusion protein (NKG2D-ADCC) to trigger NK cell ADCC against ovarian cancer cells. The NKG2D-Ig fusion protein with a wildtype Fc version (NKG2D-WT) was modified in the Fc part by introducing the S239D/I332E mutations, thereby enhancing the binding affinity to the Fc receptor CD16. Flow cytometric analysis revealed a diverse range of expression patterns for NKG2DLs across ovarian cancer cell lines. Functionally, NKG2D-ADCC induced stronger NK cell activation than NKG2D-WT, promoting degranulation and secretion of effector molecules, including IFN-γ and granzymes. The unrelated control protein lacked such effects, confirming specificity. Notably, NKG2D-ADCC induced robust NK cell-mediated lysis of ovarian cancer cells in both short- and long-term cytotoxicity assays.Together, these findings demonstrate that the NKG2D-ADCC fusion protein potently enhances NK cell responses against ovarian cancer cells, supporting its promise as a novel immunotherapeutic strategy.