Abstract B060: Fusokine IL-6/1, a novel immunotherapeutic agent against ovarian cancer

Abstract We have previously developed chimeras of GM-CSF and various γ-c interleukin (fusokines) into a single, bifunctional polypeptide. Fusokines confer immune cells with a gain of function that cannot be explained by the mere sum of their constituent moieties by serving as bispecific ligands to drive unique downstream signaling events. The synergy that arises from these fusions has shown great promise in their ability to modulate the immune response and overcome maladaptive biological processes that underlie diseases such as cancer and autoimmune conditions. Extending these studies, we have now developed IL-6/1, a chimera of IL-6 and IL-1β. Here, we report our initial studies demonstrating the immune activating role of IL-6/1 and develop a cell therapy approach for treatment of high grade serous ovarian cancer (HGSOC). Human and murine IL-6 and IL-1β gene sequences were combined in a single open reading frame either in IL-6/1β or IL-1β/6 (N-C terminal) format downstream of EF1A promoter in a second-generation lentiviral vector. Fluorescent protein markers NeonGreen for human and TurboRFP for mouse IL-6/1 were included in the vector downstream of a T2A cleavage site, respectively, and puromycin antibiotic resistance used for selection. The predicted ∼42 kDa MW of the IL-6/1 fusion protein was confirmed by Western blotting. ELISA and Western blotting experiments demonstrated that both the anti-IL6 and anti-IL-1β antibodies detected the IL-6/1 fusokine but not the IL-1β/6 chimera. In silico modeling using I-TASSER showed no steric hindrance in the presentation of the IL-6 and IL-1β functional domains in IL-6/1 fusokine. In response to anti-CD3/CD28, increased proliferation and decreased apoptosis of CD4 and CD8 human T cells was observed upon treatment with IL-6/1. Further analysis showed that the CD45Ra positive memory T cells were proliferating in response to IL-6/1. Next, we developed ID8-p53-/- cells that expressed murine IL-6/1 (ID8-6/1). Luciferase-expressing parent ID8 cells (ID8-Luc) were intraperitoneally implanted in female 6-8 week old mice and their growth was monitored by bioluminescence imaging. Approximately one week after ID8-Luc administration, when tumors were established, the mice were randomly divided into control and test groups (N=5/cohort). The control groups received a single bolus of ID8 cells transduced with sham control and the test animals received ID8-6/1. Weekly bioluminescence monitoring showed significantly slower growth of the ID8-Luc tumors with all of the mice in the control group dying between 3-5 weeks after initiation of treatment. In contrast, three mice from the IL-6/1 treatment group succumbed to high tumor burden by weeks 8, 10 and 12 after the start of treatment, respectively. The remaining two animals continue to show stable disease at week 18 after initiation of treatment. The positive results from these studies suggest that delivery of IL-6/1 by autologous tumor cells can serve as a novel immunotherapeutic strategy against HGSOC. Citation Format: Sejal Sharma, Rahul S. Das, Lisa Barroilhet, Manish S. Patankar, Jacques S. Galipeau. Fusokine IL-6/1, a novel immunotherapeutic agent against ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B060.