Targeting Inhibition of Foxp3 by MMP2/9 Sensitive Short Peptide Linked P60 Fusion Protein 6(P60‐MMPs) to Enhance Antitumor Immunity

Abstract Regulatory T‐cells (Tregs) play an important role in tumor immunosuppressive network, thus Tregs‐targeted strategy is expected to enhance antitumor immunity and improve the effect of immunotherapy. Short peptide P60 can bind to the forkhead box protein P3 (Foxp3), a crucial transcriptional regulator for the development and inhibitory function of Tregs, and inhibit Foxp3 nuclear translocation in Tregs. However, its treatment effect in cancer is limited due to nonspecificity. Therefore, realizing the specific delivery of P60 in tumor microenvironment will greatly facilitate its Treg‐suppressing effect for tumor therapeutics. Herein, utilizing the unique matrix metallase protease 2/9 (MMP2/9) overexpressing feature in tumor tissues, a fusion protein 6(P60‐MMPs) containing six segments of P60 linked by MMP2/9‐sensitive peptides is constructed for antitumor targeting immunotherapy. The fusion protein 6(P60‐MMPs) specifically degrades into short peptide P60 in tumor, and then binds to Foxp3 to inhibit Foxp3 nuclear translocation in Tregs, thus impairing Tregs’ activity. This fusion protein efficiently inhibits murine breast cancer 4T1 transplanted tumor growth and decreases lung metastasis through down‐regulating tumor‐infiltrated Tregs and up‐regulating CD8 + T cells in tumor tissue. The study develops a Treg‐targeted anticancer fusion protein with effective therapeutic activity, suggesting its potential in clinical translation.