Research Papers:

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

Teresa Lozano, Marta Gorraiz, Aritz Lasarte-Cía, Marta Ruiz, Obdulia Rabal, Julen Oyarzabal, Sandra Hervás-Stubbs, Diana Llopiz, Pablo Sarobe, Jesús Prieto, Noelia Casares and Juan José Lasarte _

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Oncotarget. 2017; 8:71709-71724. https://doi.org/10.18632/oncotarget.17845

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Teresa Lozano1, Marta Gorraiz1, Aritz Lasarte-Cía1, Marta Ruiz1, Obdulia Rabal2, Julen Oyarzabal2, Sandra Hervás-Stubbs1, Diana Llopiz1, Pablo Sarobe1, Jesús Prieto1, Noelia Casares1 and Juan José Lasarte1

1Immunology and Immunotherapy Program, University of Navarra, 31008, IDISNA, Pamplona, Spain

2Small Molecule Discovery Platform, Molecular Therapeutics Program, Center for Applied Medical Research (CIMA), University of Navarra, 31008, IDISNA, Pamplona, Spain

Correspondence to:

Juan José Lasarte, email: [email protected]

Noelia Casares, email: [email protected]

Keywords: Foxp3, Treg, peptide inhibitor, cancer, immunomodulation

Received: November 10, 2016     Accepted: April 27, 2017     Published: May 13, 2017


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.

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