Interleukin-2 (IL-2) is a T cell growth factor that is essential for the proliferation of T cells and the generation of effector and memory cells. The antitumor activity of high-dose IL-2 therapy requires maintaining the affinity between IL-2 and IL2-Rα, which can also bring serious toxic side effects. To address this issue, we designed ZGP-Cysteamine-IL-2-K64C and (ZGP-Cysteamine)2-IL-2-(K43C, K64C) based on the strategy of FAPα enzyme-activated prodrugs, and investigated their anti-tumour activity and side effects. In vitro FAPα enzyme cleavage results indicated that the side-chain modified ZGP-Cysteamine moiety could be precisely recognized and cleaved by FAPα, thereby restoring the activity of native IL-2 capable of binding to IL-2Rα in the tumour microenvironment, where it promotes the expansion of CD8+ T cells. Meanwhile, surface plasmon resonance analysis revealed that, compared to wt-IL-2, both ZGP-Cysteamine-IL-2-K64C and (ZGP-Cysteamine)2-IL-2-(K43C, K64C) exhibited significantly reduced affinity for IL-2Rα, while their affinity for IL-2Rβγ remained unchanged. Remarkably, ZGP-Cysteamine-IL-2-K64C and (ZGP-Cysteamine)2-IL-2-(K43C, K64C) almost completely eliminated the pulmonary edema and vascular permeability. Furthermore, the combination of ZGP-Cysteamine-IL-2-K64C and PD-1 blockade showed robust anti-tumour activity in mice tumour models. Our study provides new insights into the structural design of IL-2 prodrug with low side effect and robust anti-tumour efficacy.
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