Foxp3 inhibitory peptide encapsulated in a novel CD25-targeted nanoliposome promotes efficient tumor regression in mice.

P60, a Foxp3 inhibitory peptide, can hinder the regulatory T cell (Treg) activity and impair tumor proliferation. However, low systemic stability and poor specificity have led to daily dosing to achieve therapeutic effect. Therefore, this study aims to improve P60 stability and specific delivery through its encapsulation in liposomes targeting CD25, constitutively expressed in Tregs. P60 liposomes formulated with DSPE-PEG₇₅₀ or DSPE-PEG₂₀₀₀ were incubated with DSPE-PEG₂₀₀₀-Maleimide micelles conjugated to Fab' fragments of anti-CD25 to develop two targeted formulations or immunoliposomes (IL): IL-P60₂₀₀₀ (DSPE-PEG₂₀₀₀ only) and IL-P60₇₅₀ (combining DSPE-PEG₇₅₀ and DSPE-PEG₂₀₀₀). P60 encapsulation efficiency was 50%-60% irrespective of PEG chain length. Treg uptake was 2.5 and 14 times higher for IL-PEG₇₅₀ compared with IL-PEG₂₀₀₀ and non-targeted liposomes, respectively, in in-vitro assays. In fact, IL-P60₇₅₀ allowed CD8⁺  T cells ex-vivo proliferation in presence of Treg at doses 10-20 times lower than for free P60. Antitumor response of P60 and IL-P60₇₅₀ in monotherapy and combined with anti-PD-1 was evaluated in MC38 and LLCOVA tumor bearing mice. In MC38 model, IL-P60₇₅₀ monotherapy induced total tumor regression in 40% of mice reaching 100% for anti-PD-1 combination. This effect was associated with a significant increase in activated CD8⁺ T cells in tumors. Notably, IL-P60₇₅₀ also inhibited human Treg in ex-vivo assay, showing the translational capability of this formulation. In conclusion, IL-P60₇₅₀ formulated with different PEG chain lengths, has demonstrated antitumor efficacy by selective inhibition of Treg activity and enhances the effect of anti-PD1. Altogether, this novel IL represents a promising nanoplatform for cancer immunotherapies.