With the goal of developing specific Treg inhibitors to improve antitumor immunity, Hawley et al. used the FOXP3 homodimer crystal structure to design and compare single- and double-hydrocarbon stapled alpha-helical (SAH-FOXP3) peptides that effectively blocked a key FOXP3 protein–protein interaction. Lead SAH-FOXP3s bound FOXP3, were non-toxic to T cells, localized intracellularly, induced dose-dependent RNA and protein changes in Tregs, and decreased Treg cell-mediated immune suppression ex vivo. In normal animals, the lead SAH-FOXP3 altered Treg gene expression consistent with loss of FOXP3 function, providing proof of concept.
Contributed by Katherine Turner
ABSTRACT: Despite continuing advances in the development of novel cellular-, antibody-, and chemotherapeutic-based strategies to enhance immune reactivity, the presence of regulatory T cells (Treg cells) remains a complicating factor for their clinical efficacy. To overcome dosing limitations and off-target effects from antibody-based Treg cell deletional strategies or small molecule drugging, we investigated the ability of hydrocarbon stapled alpha-helical (SAH) peptides to target FOXP3, the master transcription factor regulator of Treg cell 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 describe the design, synthesis, and biochemical evaluation of single- and double-stapled SAHs covering the entire coiled-coil expanse. We show that lead SAH-FOXP3s bind FOXP3, are cell permeable and nontoxic to T cells, induce dose-dependent transcript and protein level alterations of FOXP3 target genes, impede Treg cell function, and lead to Treg cell gene expression changes in vivo consistent with FOXP3 dysfunction. These results demonstrate a proof of concept for rationally designed FOXP3-directed peptide therapeutics that could be used as approaches to amplify endogenous immune responsiveness.
Author Info: (1) Department of Pediatrics, Section of Hematology/Oncology, The University of Chicago, Chicago, IL 60637. (2) Department of Pediatrics, Section of Hematology/Oncology, The Univer
Author Info: (1) Department of Pediatrics, Section of Hematology/Oncology, The University of Chicago, Chicago, IL 60637. (2) Department of Pediatrics, Section of Hematology/Oncology, The University of Chicago, Chicago, IL 60637. (3) Department of Pediatrics, Section of Hematology/Oncology, The University of Chicago, Chicago, IL 60637. Medical Scientist Training Program, The University of Chicago, Chicago, IL 60637. Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL (4) Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL (5) Department of Pediatrics, Section of Hematology/Oncology, The University of Chicago, Chicago, IL 60637. (6) Department of Pediatrics, Section of Hematology/Oncology, The University of Chicago, Chicago, IL 60637. (7) Department of Pediatrics, Section of Hematology/Oncology, The University of Chicago, Chicago, IL 60637. (8) Department of Pediatric Oncology, Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215. (9) Department of Pediatric Oncology, Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215. (10) Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL Argonne National Laboratory, Lemont, IL 60439. (11) Department of Pediatrics, Section of Hematology/Oncology, The University of Chicago, Chicago, IL 60637.
