A Dual-Antigen NK Cell Engager to Overcome Ovarian Cancer Tumor Heterogeneity

ABSTRACT Ovarian cancer remains the deadliest gynecologic malignancy, with a five-year survival rate below 50% and a high likelihood of recurrence. Current treatment strategies, centered on surgical debulking and chemotherapy, have shown limited advancements over the past several decades, underscoring the urgent need for novel therapies. Immunotherapy holds immense promise in this context, as increased natural killer (NK) cell activity has been correlated with improved outcomes in ovarian cancer patients. Unlike T cells, NK cells are inherently non-antigen-specific, enabling them to respond to heterogeneous tumors without prior sensitization, while also avoiding complications like graft-versus-host disease. Like T cells, however, NK cells activity is hindered by the immunosuppressive tumor microenvironment. To overcome this, our lab has developed and rigorously tested the Tri-specific Killer Engager (TriKE) platform, which enhance NK cell activity by simultaneously targeting CD16 on NK cells, binding tumor-associated antigens, and delivering an interleukin-15 (IL-15) cytokine domain. Together, the TriKE promotes NK cell migration, activation, proliferation, and sustained cytotoxicity. However, as with any immunotherapy targeting only a single antigen, the therapeutic application of the TriKE molecules could be hindered by the risk of antigen escape, particularly in ovarian cancer’s highly heterogeneous tumor microenvironment (TME). To address this challenge, we have developed a dual-antigen targeting NK cell engager platform. A Poly-Antigen Cytokine Complex (PACC), which binds to the TriKE’S IL-15 domain, was made by linking IL-15 receptor subunit alpha (IL-15Rα) to a tumor-targeting antibody domain. This innovative system combines the well-established benefits of TriKEs with targeting of a second tumor-associated antigen. By simultaneously engaging B7H3 and CD133, the TriKE-PACC is designed to exert greater immunologic pressure, reducing the likelihood of antigen escape and improving therapeutic efficacy. Aim 1 will evaluate the ability of the TriKE-PACC to prevent antigen escape in vitro and in vivo using ovarian cancer cell lines with CRISPR-modulated antigen expression to model antigen escape. Aim 2 will assess the preclinical efficacy of the TriKE-PACC against patient-derived xenograft (PDX) models of ovarian cancer to capture the full heterogeneity of clinical tumors. Together, these studies will establish the therapeutic potential of the TriKE-PACC and provide a foundation for its clinical translation. By targeting a dual-antigen approach to enhance NK cell immunotherapy, this project seeks to provide a novel treatment avenue for ovarian cancer patients, particularly the unmet need of those with recurrent disease who currently lack effective therapeutic options.