Lirilumab and Avelumab Enhance Anti-HPV+ Cervical Cancer Activity of Natural Killer Cells via Vav1-Dependent NF-κB Disinhibition

BACKGROUND: We investigated the efficacy and mechanism of the anti-KIR immunotherapy lirilumab and anti-PD-L1 immunotherapy avelumab on natural killer (NK) cell activity against HPV+ cervical cancer. METHODS: NK cell-mediated lysis of autologous biopsy-derived malignant cervical squamous cells and normal cervical squamous cells were measured by europium-release cytotoxicity assays. Cytokine and granzyme B release were measured by ELISPOT effector-cell-based assays and ELISA. Murine cervical cancer tumor models were constructed to assess implanted tumor volumes over time and intratumoral immune cell infiltration. Receptor-crosslinking and plate-immobilized antibody stimulation studies, with or without p65 and Vav1 silencing, were used to investigate NF-κB pathway disinhibition in NK cells. RESULTS: Lirilumab and avelumab each enhanced NK cell disinhibition and NK cell-mediated lysis of autologous cervical cancer cells in vitro while reducing HPV+ tumor volumes and increasing intratumoral NK cell infiltration and cytolysis in vivo. Moreover, lirilumab and avelumab each promoted NK cell NF-κB disinhibition as well as stimulated cytokine and granzyme B expression in a NF-κB-dependent manner. Lirilumab+avelumab enhanced all aforementioned effects compared to either monotherapy. Vav1 silencing eliminated disinhibition of NF-κB signaling by lirilumab and avelumab, indicating their disinhibiting effects are Vav1-dependent. CONCLUSIONS: This study supports a novel approach to enhancing NK cell lysis against HPV+ cervical cancer cells through combining lirilumab and avelumab.