Abstract

Interleukin-15 (IL-15) holds great promise for cancer immunotherapy because it selectively activates cytotoxic immune cells without expanding regulatory T cells. It also has the potential to convert “cold” tumors into more immunologically active ones. However, its clinical utility is limited by systemic toxicity. Trophoblast cell surface antigen 2 (TROP2), overexpressed in many cancers and associated with poor prognosis, is a validated therapeutic target. Variable new antigen receptor (VNARs) are the smallest single-domain antibodies derived from cartilaginous fishes and are characterized by compact size, stability, and favorable tissue penetration. Here, we report a fusion protein that leverages a shark-derived VNAR to deliver an IL-15 super-agonist complex to TROP2-expressing tumors. To reduce systemic toxicity and enable localized cytokine signaling, we fused the low-internalizing, high-affinity VNAR vGY2 to an IL-15 super-agonist (IL-15N72D) and the IL-15Rα sushi domain, yielding vGY2-IL-15sa. The design rationale prioritizes tumor cell surface retention ( via low internalization) to support extracellular IL-15 signaling in the tumor microenvironment. vGY2-IL-15sa was produced in HEK293F cells with over 95% purity and displayed dual functionality: high affinity for TROP2 (K D  = 1.32 × 10 −8  M) and potent IL-15 bioactivity (EC 50  = 1.800 ± 0.008 nM in CTLL-2 assays). In safety studies, the fusion protein was well-tolerated with minimal changes in body weight. In a humanized NSCLC mouse model, the fusion protein achieved significant tumor growth inhibition and delayed tumor progression. Collectively, these findings highlight the potential of vGY2-IL-15sa for targeted cancer immunotherapy and establish a compact VNAR-based immunocytokine platform, offering a promising approach to enhancing the therapeutic effects of IL-15 while minimizing toxicity.