FL115, a novel IL-15 superagonist rationally designed to minimize safety risks for cancer immunotherapy

Background: Interleukin(IL)-15 is a potent cytokine that activates NK and T cells but has been hampered by a short half-life, systemic toxicity, and dependence on IL-15Ralpha Sushi domain (IL-15RalphaSu) trans-presentation. ALT803, an IgG1 Fc-fused IL-15RalphaSu and IL-15, has been FDA-approved for bladder cancer via intravesical instillation. Nonetheless, no IL-15 agonist has yet been approved for systemic use due to safety concerns and challenging therapeutic index. We developed a novel superagonist, FL115, by fusing IL-15RalphaSu and IL-15 with a single-chain Fc (sFc). This sFc is half size of human IgG1 Fc and lacks FcgammaRs binding while retaining FcRn binding, conferring FL115 a distinct and potentially improved profile compared with ALT803 in vivo . Methods: Different mouse models were used to compare the efficacy and safety of FL115 and ALT803. The immunomodulatory mechanism of FL115 was analyzed via single-cell RNA sequencing (scRNA-seq), immune cell depletion, qPCR and flow cytometry. The structure of FL115/FcRn/beta2M were elucidated through cryo-electron microscopy (Cryo-EM). Results: FL115 was rationally designed to eliminate FcgammaR binding while retaining FcRn interaction. Cryo-EM analysis of FL115/FcRn/beta2M complex revealed that sFc aligns with only one chain of Fc but deviated from the other, disrupting the key interface required for FcgammaR engagement and preventing FcgammaR-mediated immune activation. Consistent with structural insight, FL115 exhibited markedly improved in vivo safety compared with ALT803. At 20 mg/kg, ALT803 caused 100% mortality accompanied by severe liver injury and pro-inflammatory cytokines such as IL-6. In contrast, all FL115-treated mice survived without detectable toxicity. The maximum tolerated dose of FL115 was over 10 times than ALT803. scRNA-seq of liver tissue revealed that ALT803 promotes the activation of neutrophils and macrophages through FcgammaR signaling, leading to cytokine release and systemic toxicity. FL115, lacking FcgammaR-binding capacity, did not trigger these inflammatory pathways. Importantly, FL115 showed comparable efficacy to ALT803 in CT26 and B16-F10 tumor models. Single-cell transcriptomics revealed FL115 reshapes the tumor microenvironment by enhancing NK and T cells infiltration, pro-inflammatory macrophage polarization, and dendritic cell expansion.. Immune cell depletion confirmed that FL115's antitumor effect is exclusively NK cell-dependent. Deep analysis further showed FL115 promotes NK cell maturation, cytotoxicity, and intratumoral recruitment. Conclusion: Our study revealed the mechanism underlying the superior safety of FL115 over ALT803 while exhibiting comparable anti-tumor activities. These findings highlight the rational design strategy of FL115 as next-generation IL-15-based superagonist, supporting its favorable safety and preliminary clinical responses observed in multiple Phase 1 clinical studies.