Background: Antibody-dependent cell-mediated cytotoxicity (ADCC), mediated by natural killer (NK) cells, plays an important role in the efficacy of monoclonal antibodies (mAb)s. CD137 is a costimulatory molecule expressed on immune cells following activation, including NK cells. We hypothesize that as the antitumor efficacy of mAbs is due to ADCC, their activity can be enhanced by stimulation of NK cells with an anti-CD137 agonistic mAb. Methods: Upregulation of CD137 on NK cells was assessed using CD20+lymphoma, HER2+breast, and EGFR+head and neck cell lines and primary patient samples. NK cell degranulation, cytokine release and cytotoxicity were assessed by CD107a mobilization, IFN-γ secretion, and chromium release. Mechanism of synergy was explored by cell depletion in an immune competent mouse model. Xenotransplanted models were used to demonstrate anti-tumor activity and sufficiency of an innate immune response. Results: NK cells in human primary patient samples do not express CD137 at baseline, however CD137 is highly upregulated when encountering mAb-coated tumor cells. MAb-induced NK cell degranulation and cytotoxicity are enhanced by anti-CD137 agonistic mAb. In a murine lymphoma model, anti-CD137 mAb significantly enhances anti-tumor activity of anti-CD20 mAb leading to complete tumor resolution and prolonged survival. NK cell depletion completely abrogates the therapeutic effect. In seven xenotransplant models, sequential administration of rituximab, trastuzumab or cetuximab plus anti-CD137 mAb provided superior reduction in tumor burden and prolonged overall survival. In a phase 0 biomarker study, level of CD137 expression on circulating and intratumoral NK cells was influenced by disease burden, prior treatment, FcγRIII polymorphism, and time since mAb therapy. Conclusions: Our results demonstrate the synergy of anti-CD137 mAb and a tumor-targeting mAb by stimulation of mAb-activated NK cells with anti-CD137 mAb to enhance ADCC. These results support a novel, sequential antibody approach against CD20+B cell, HER2+breast, and EGFR+head and neck malignancies by targeting first the tumor and then the host immune system.