An engineered IL-21 with half-life extension enhances anti-tumor immunity as a monotherapy or in combination with...

Interleukin-21 (IL-21) has exhibited anti-tumor activity in preclinical and clinical studies; however, its modest efficacy and short half-time has limited its therapeutic utility as a monotherapy. Therefore, we engineered a fusion protein (IL-21-αHSA) in which a nanobody targeting human serum albumin (HSA) was fused to the C-terminus of rhIL-21. The αHSA nanobody displayed broad species cross-reactivity and bound to a HSA epitope that does not overlap with the FcRn binding site, thus providing a strategic design for half-life extension. The IL-21-αHSA fusion protein showed increased stability compared to rhIL-21, while retaining its bioactivity in a liquid solution for at least 6 months. Moreover, IL-21-αHSA showed a dramatically extended half-life and prolonged exposure in cynomolgus monkeys, with the t_1/2 and AUC nearly 10 and 50 times greater than that of rhIL-21, respectively. Furthermore, IL-21-αHSA displayed enhanced anti-tumor efficacy in two syngeneic mouse models. Notably, IL-21-αHSA increased the anti-tumor effect of programmed cell death protein 1 (PD-1) and T cell immunoglobulin and ITIM domain (TIGIT) blockades when used in combination, with a protection against tumor rechallenge, suggesting the formation of long-term anti-tumor memory response. KEGG analysis identified significantly enriched pathways associated with anti-tumor immune response, with increased expression of genes associated with CD8⁺ T and NK cell cytotoxicity. Overall, these data support further clinical evaluation of IL-21-αHSA as a monotherapy or in combination with immune checkpoint blockades.