A homodimeric IL-15 superagonist F4RLI with easy preparation, improved half-life, and potent antitumor activities.

Interleukin-15 (IL-15) is a promising candidate for cancer immunotherapy due to its potent immune-activating effects. There are several IL-15 molecules currently in clinical trials but facing shortages of poor half-life, circulation instability, or complicated production and quality control processes. The aim of this study is to design a novel IL-15 superagonist to set out the above difficulties, and we constructed F4RLI consisting of the GS-linker spaced IgG4 Fc fragment, soluble IL-15 Rα (sIL-15Rα), and IL-15(N72D). Using a single plasmid transient transfection in HEK293E cells, the matured F4RLI was secreted in the form of homodimer and got purified by an easy step of protein A affinity chromatography. The F4RLI product can significantly stimulate the proliferation of human CD3+CD8+ T cells and NK cells in vitro. Meanwhile, F4RLI greatly extended the half-life and prolonged the exposure of IL-15 in mice nearly by 28- and 200-fold, respectively, in comparison with that of the IL-15 monomer. In vivo, F4RLI vastly expanded mouse splenic CD8+ T lymphocytes, illustrating its potential in tumor immunotherapy. Further studies showed that the combination of F4RLI with the immune checkpoint blocker atezolizumab played a synergistic effect in treating MC38 mouse tumor by increasing the percentage of CD8+ T cells in tumor tissue. Moreover, the combination therapy of F4RLI with the angiogenesis inhibitor bevacizumab resulted in significant tumor growth suppression in a xenograft human HT-29 mouse model. Overall, our results demonstrate a homodimeric IL-15 superagonist F4RLI with advances in manufacturing processes and biopharmaceutical applications for cancer immunotherapy. KEY POINTS: • The homodimeric structure of F4RLI facilitates its easy production processes and quality control. • The fusion with Fc and sIL-15Rα extends the plasma half-life of IL-15 by about 28-fold. • F4RLI can play synergistic antitumor activity with the PD-1/PD-L1 checkpoint inhibitor or angiogenesis inhibitor.