Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. After processing of the inactive precursor within monocytes and macrophages, mature IL18 can promote the expansion, survival, and cytotoxicity of T and NK cells expressing its receptor subunits IL-18Rα (IL18R1) and IL18Rβ (IL18RAP). Preclinical studies with recombinant IL18 have demonstrated anti-tumor activity in animal models, including impressive synergy with both immune checkpoint inhibitors and CAR-T therapy. However, clinical development exhibited poor pharmacokinetics and an overall lack of efficacy despite heavy dosing. IL18 participates in a negative feedback loop with a very high affinity natural inhibitor, IL18 binding protein (IL18BP), whose expression is induced by IFNγ. As IL18BP upregulation was observed in early phase clinical trials, it likely limited the efficacy of recombinant IL18. Here, we have engineered next generation IL18 cytokines that aim to improve on the poor PK of WT and contend with IL18BP induction.

Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Following that principle, we generated monovalent IL18-Fc fusions upon our XmAb® heterodimeric Fc platform and introduced substitutions that could modulate IL18 stability, affinity toward the IL18 heterodimeric receptor, and affinity toward IL18BP. Initially, IL18-Fc was difficult to produce but yields improved to typical bispecific antibody levels after stabilization. An engineered disulfide bridge increased the WT thermal denaturation temperature from 45 °C to 65 °C, and demonstrated a significant improvement in serum clearance in mouse PK experiments. Variants at IL18 positions along the receptor and IL18BP interfaces were explored in vitro by measuring PD-L1 induction on KG-1 cells, with and without a high concentration of IL18BP. Recombining a select number of hits generated a potency series with variants exhibiting over a 2,000-fold reduction in PD-L1 induction potency as compared to WT IL18-Fc. Importantly, we identified variants that no longer detectably bound IL18BP, relieving natural inhibition of our engineered IL18-Fc. In vivo immune-mediated inflammation was explored in human PBMC engrafted mouse models of graft versus host disease (GvHD). We observed potency-dependent exacerbation of GvHD, with corresponding dramatic increases in the numbers and activation of T and NK cells as compared to a human PBMC only control. Analysis of serum from the study revealed up to 100-fold increases in IFNγ levels. Our engineered, reduced-potency IL18-Fc cytokines demonstrate robust inflammation activity in vivo, improved pharmacokinetics in mice, and insensitivity to IL18BP inhibition.

Citation Format: Alex Nisthal, Sung-Hyung Lee, Christine Bonzon, Ruschelle Love, Kendra N. Avery, Rumana Rashid, Panida Lertkiatmongkol, Nicole Rodriguez, Hanh Nguyen, Araz Eivazi, Sher Karki, Norm Barlow, Seung Y. Chu, Greg L. Moore, John R. Desjarlais. Engineered IL18 heterodimeric Fc-fusions featuring improved stability, reduced potency, and insensitivity to IL18BP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3515.