Blockage of FOXP3 transcription factor dimerization and FOXP3/AML1 interaction inhibits T regulatory cell activity: sequence optimization of a peptide inhibitor

Abstract

Although T regulatory cells (Treg) are essential for the prevention of autoimmune diseases, their immunoregulatory function restrains the induction of immune responses against cancer. Thus, development of inhibitors of FOXP3, a key transcription factor for the immunosuppressive activity of Treg, might give new therapeutic opportunities. In a previous work we identified a peptide (named P60) able to enter into the cells, bind to FOXP3, and impair Treg activity in vitro and in vivo. Here we show that P60 binds to the intermediate region of FOXP3 and inhibits its homodimerization as well as its interaction with the transcription factor AML1. Alanine-scanning of P60 revealed the relevance of each position on FOXP3 binding, homodimerization, association with AML1 and inhibition of Treg activity. Introduction of alanine at positions 2, 5 and 11 improved the activity of the original P60, whereas alanine mutations at positions 1, 7, 8, 9, 10 and 12 were detrimental. Multiple mutation experiments allowed us to identify peptides with higher FOXP3 binding affinity and stronger biological activity than the original P60. Head to tail macrocyclization of peptide P60-D2A-S5A improved Treg inhibition and enhanced anti-tumor activity of anti-PD1 antibodies in a model of hepatocellular carcinoma. Introduction of a D-aminoacid at position 2 augmented significantly microsomal stability while maintained FOXP3 binding capacity and Treg inhibition in vitro. In vivo, when combined with the cytotoxic T-cell epitope AH1, it induced protection against CT26 tumor implantation. This study provides important structure–function relationships essential for further drug design to inhibit Treg cells in cancer.

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character

“…For instance, amino acid substitution is a rational method to optimize peptide sequences that relates physiological properties and conformation of peptides. 9,10 The peptide array has been used for cyclic peptide screening. 11,12 The amino acids scanning of peptides using peptide array is common method to optimize the peptide sequences and to identify the essential amino acids.…”

Neoantigen vaccine: an emerging tumor immunotherapy

“…Klages et al [131] observed that tumor growth was retarded when a transgenic diphtheria toxin receptor was used to prostrate Tregs in a mouse model of melanoma, thereby significantly improving the anti-cancer effect of the tumor vaccine. Other two studies conducted by Casares et al [132,133] showed that FoxP3 inhibitory peptide P60 occupied the intermediate domain of FoxP3, inhibited its homologous dimerization and binding with transcription factors, attenuated the activity of Tregs in vivo and in vitro, and enhanced the efficacy of tumor vaccines in mouse models.…”

Peptide-based materials for cancer immunotherapy

“…found that a 15-mer synthetic peptide (P60) was able to bind to FOXP3, abrogate the FOXP3/AML1 interaction, and impede Treg cell activity, which caused significant anti-tumor activity both in vitro and in vivo . They further synthesized a macrocyclic peptide (P60-D2A-S5A) that had enhanced Treg cell inhibition and demonstrated that it could strengthen the anti-tumor activity of anti-PD1 antibodies against hepatocellular carcinoma 92.…”