Inhibition of FOXP3 by Stapled Alpha-Helical Peptides Dampens Regulatory T Cell Function

Despite continuing advances in the development of novel cellular-, antibody-, and chemotherapeutic-based strategies to enhance immune reactivity, the presence of regulatory T cells (Tregs) remains a complicating factor to their clinical efficacy. To overcome dosing limitations and off-target effects from antibody-based Treg deletional strategies, we investigated the ability of hydrocarbon stapled alpha-helical (SAH) peptides to target FOXP3, the master transcription factor regulator of Treg development, maintenance, and suppressive function. Using the crystal structure of the FOXP3 homodimer as a guide, we developed SAHs in the likeness of a portion of the native FOXP3 antiparallel coiled-coil homodimerization domain (SAH-FOXP3) to block this key FOXP3 protein-protein interaction (PPI) through molecular mimicry. We show that lead SAH-FOXP3s bind FOXP3, are cell permeable, non-toxic to T cells, induce dose-dependent transcript and protein level alterations of FOXP3 target genes, impede Treg function, and lead to Treg gene expression changes in vivo consistent with Foxp3 dysfunction. Comparative gene expression profiling analysis of RNA-seq data for naïve CD4+ Tcons and CD4+CD25+FOXP3+ Tregs following 2 days of daily treatment with either SAH(229-259)C or vehicle. There are 12 samples total (6 Tcon samples, 6 Treg samples). Within each set of 6 Tcon or Treg samples, there are 2 vehicle-treated controls and 4 SAH-treated controls representing cells from either the lymph nodes or spleen.