1077 A novel dual-targeting engager strategy to amplify natural killer cell activity against ovarian cancer

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Background Ovarian cancer (OC) is the 5th deadliest cancer among women and the most lethal gynecologic malignancy. Despite recent advancements in maintenance therapy, 70–80% of patients experience recurrence, with an average post-recurrence survival duration of under two years. Immunotherapy holds promise for therapeutic development, but potent immunosuppression and high heterogeneity of OC pose significant barriers to effective treatment. Tri-specific Killer Engager molecules (TriKEs), comprising an anti-CD16 nanobody, an interleukin (IL)-15 moiety, and a tumor-targeting antibody (Ab) domain, have demonstrated promising preclinical data. In several tumor types, including ovarian, TriKEs enhance natural killer (NK) cell activation, cytotoxic ability, and expansion despite tumor-induced immunosuppression. However, as observed by other targeted immunotherapies for OC, the high heterogeneity and resulting antigen escape may limit TriKE efficacy. To address this, the TriKE platform was built upon to add a Poly-Antigen Cytokine-receptor Complex (PACC), linking IL-15 receptor-alpha (IL-15Rα) to a secondary tumor-targeting Ab domain. The TriKE-PACC (figure 1) targets B7H3 and CD133, differentially expressed antigens with functional significance in OC. B7H3 is a novel target expressed on several solid tumors and associated with OC progression. CD133, a marker of stem cells in some tumor settings, is associated with cells driving expansion and tumorigenesis in OC. Methods The anti-CD133/anti-B7H3 TriKE-PACC was synthesized using a mammalian Expi293 expression system, with expression and construct formation verified by bio-layer interferometry (BLI), SDS-PAGE gel electrophoresis, and flow cytometry-based binding assays. Functional assays were then conducted to gauge the TriKE-PACC’s ability to activate NK cells, measuring degranulation through CD107a externalization and cytokine (IFNγ and TNFα) production. Results BLI and gel electrophoresis confirmed TriKE-PACC expression, and binding assays confirmed complete formation of the construct. NK cell activation, demonstrated by IFNγ production (figure 2), is enhanced by the TriKE-PACC and occurs in an antigen-specific manner. Additionally, we are currently assessing cytotoxic activity mediated by the TriKE-PACC in heterogeneous tumor populations in vitro against CRISPR-Cas9 edited cell lines. Patient-derived OC samples are also being used to assess expression of B7H3 and CD133 and TriKE-PACC activity in a clinical setting. Future experiments will assess the extent to which dual-targeting limits antigen escape compared to single-targeting TriKEs, and test the TriKE-PACC in patient-derived xenografts. Conclusions Our findings confirm expression, complex formation, binding specificity, and NK-activation of a novel CD133 and B7H3 dual-targeting engager molecule. Ongoing and future studies will continue preclinical testing and could lead to clinical translation of this novel strategy in OC.

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