Nanocapsule-based vaccination for inhibition of tumor escape mechanisms
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Abstract
Tumor-associated tolerance mechanisms promote tumor growth and greatly inhibit the success of anti-cancer therapies. Regulatory T cells (Tregs) critically contribute to tumor-associated tolerance and are involved in IL-10- and signal transducer and activator of transcription (STAT) 3-mediated cancer-induced immunosuppression. In this study, CD25+ T cells with focus on Tregs were cell-specifically addressed by using human IL-2 functionalized hydroxyethyl starch (HES-D-IL-2) nanocapsules (NC). After coupling to the NC surface, the targeting vector human IL-2 was still fully biological functional and only a negligible amount of non-attached IL-2 was present in the NC supernatant. Flow cytometry and laser scanning microscopy experiments revealed an enhanced uptake/binding and intracellular localization of HES-D-IL-2 by human CD4+CD25+ activated T cells compared to control NC. Additionally, HES NC with a twofold (HES-D-IL-2/2) and tenfold (HES-D-IL-2/10) reduced amount of surface IL-2 were generated. In all tested NC-coupled IL-2 concentrations, flow cytometric experiments highlighted a reduced uptake/binding of HES-D-IL-2 by human naïve CD4+CD25- T cells in comparison to human activated T cells. Furthermore, the uptake by naïve and activated T cells was dose- dependent on the amount of IL-2 that was present on the NC surface. In contrast, human CD4+CD25high Tregs demonstrated the highest uptake. Intriguingly, the uptake of HES-D-IL-2 by Tregs was IL-2 dose-independent, revealing a high IL-2 receptor affinity and preferably targeting of Tregs by low amounts of IL-2. Furthermore, CD25 staining and blockade by the monoclonal chimeric antibody basiliximab (anti-CD25-mAb) indicated a CD25-dependent uptake/binding of HES-D-IL-2. This was confirmed by competitive studies using naïve CD4+CD25- and activated CD4+CD25+ human T cells. Furthermore in vivo experiments using wild-type C57BL/6 mice, and immunodeficient RAG2-/-γ-/- mice reconstituted with human CD4+ T cell or human peripheral blood mononuclear cells, revealed a successful CD25-specific targeting of murine and human CD4+ and CD8+ T cells in vivo.
In order to inhibit STAT3-mediated immune tolerance in Tregs, ovalbumin protein NC with known high intracellular release capacities were generated and applied to HeLa cells to perform proof-of-principle experiments. After encapsulation of the specific STAT3 inhibitor S3I-201, an inhibition of STAT3 phosphorylation was observed in vitro.
Targeting of Tregs and cytotoxic T cells by HES-D-IL-2 in combination with inhibition of STAT3-mediated tolerance by the ovalbumin protein NC may inhibit tumor-associated immunosuppression thus leading to enhanced tumor rejection.