(519c) Graduate Student Award Session: Anchoring Interleukin-12 Onto Cell-Membrane Targeted Nanoparticles Enhances Metastatic Ovarian Cancer Immunotherapy

Introduction: In the past decade, cancer immunotherapy has become a promising therapeutic strategy for cancer treatment. While not all patients respond to therapy, modulating the tumor microenvironment (TME) through the delivery of cytokines may be key to improve therapeutic outcomes. However, other than select approvals of cytokines in the 1990s, clinical translation of these natural immune signaling molecules has been limited due to their dose limiting toxicities. One promising approach to improve treatment with cytokines are nanoparticles (NPs) which, when modified via the layer-by-layer (LbL) technique, can provide many of the desirable characteristics of cytokine-delivery vehicles including tumor cell targeting, subcellular localization, and improved pharmacokinetics.

Materials and Methods: In this work, the stability of linker chemistry used to conjugate interleukin-12 (IL-12) onto LbL liposomes coated with a bilayer poly-L-arginine (PLR) and poly-L-glutamate (PLE) was assessed for its effect on intraperitoneally disseminated metastatic ovarian cancer models. IL-12 conjugation was performed either via the interaction of nickel with a histidine tag on IL-12 or the reaction of maleimide with a terminal cysteine in IL-12. Particle size, charge and morphology was characterized via dynamic light scattering, laser doppler electrophoresis, and transmission electron microscopy, respectively. IL-12 concentration and total lipid content were assessed through enzyme-linked immunosorbent assay (ELISA) and the colorimetric Stewart Assay, respectively. For efficacy studies, mice were inoculated intraperitoneally with a highly metastatic syngeneic ovarian cancer cell line (OV-2944-HM-1) and treated either daily for five days with 10 ug/day of IL12 or with a single 50 ug dose of IL12. Anti-tumor immune cells were quantified on blood and splenocytes of surviving mice via interferon-γ enzyme-linked immune absorbent spot (ELISpot) against the tumor cells.

Results: The linker chemistry had indistinguishable effect on size, zeta potential, and particle morphology. While a single intraperitoneal dose of IL-12 increased median survival of dextrose treated mice from 17 days to 27 days, a single dose of maleimide-conjugated IL-12-LbL-NPs was extended median survival of mice to 67 days compared to 36 days of nickel-conjugated LbL-NPs. When dosed daily for five days, 100% of mice dosed with maleimide-conjugated LbL-NPs were cured compared to 80% for nickel-conjugated IL-12-LbL-NPs and 40% for free IL-12. Analysis of tumor-specific immune response via ELISpot revealed a significantly elevated anti-tumor response in maleimide-linked IL-12-LbL NPs compared to free IL-12 or unlayered IL-12-NPs. Moreover, when dosed weekly for two weeks, only maleimide-linked LbL-NPs demonstrated enhanced survival over the free IL-12 protein. This effect was found to synergize with immune checkpoints inhibitors enabling 100% cures of metastatic ovarian cancer in a mouse model.

Conclusion: Taken together, these results demonstrated that stable conjugation of IL-12 onto LbL-NPs enhances cancer immunotherapy of metastatic ovarian cancer and that LbL-mediated targeting of ovarian cancer cells promoted anti-tumor immunity.