555 Inhibition of FOXP3 by stapled alpha-helical peptides alters Regulatory T Cell function

• Published in: Journal for immunotherapy of cancer 2020-11, Vol.8 (Suppl 3), p.A590-A590
• Main Authors: Hawley, Katrina, Eclov, Rachel, Fefferman, Marie, Bird, Gregory, Schnorenberg, Mathew, Samaeekia, Ravand, Walensky, Loren, Tirrell, Matthew, LaBelle, James
• Format: Article
• Language: English
• Subjects: Gene expression; Immunotherapy; Peptides; Proteins
• ISSN: 2051-1426
• DOI: 10.1136/jitc-2020-SITC2020.0555

BackgroundRegulatory T cells (Tregs) have been a therapeutic target of interest since early pre-clinical work revealed that their depletion led to enhanced tumor control. Despite continuing advances in the development of novel cellular-, antibody- and chemotherapeutic-based strategies to increase anti-tumor immunity, Treg presence and activity within the tumor microenvironment 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 to target FOXP3, the master regulator of Treg development, maintenance, and suppressive function using hydrocarbon stapled alpha-helical peptides (SAHs). We developed SAHs in the likeness of a portion of the native FOXP3 antiparallel coiled-coil homodimerization domain, in an effort to impede FOXP3 transcriptional function. SAHs overcome three major protein-protein interaction (PPI) therapeutic hurdles, namely: cellular penetrance, target specificity, and secondary structure stability. Our overall goal is to use these SAHs as investigatory drugs to demonstrate proof-of concept of the druggability of FOXP3. We aim to show their utility to further understand FOXP3 transcriptional dynamics and explore their potential use in altering the immune landscape in combination with other immune-focused therapies.MethodsUtilizing the FOXP3 crystal structure as a guide, we developed a number of single and double SAH peptides corresponding to the leucine zipper homodimerization domain (DD) of FOXP3 (SAH-FOXP3DDs). We tested the ability of SAH-FOXP3DDs to bind FOXP3, to access the intracellular compartment, to alter Treg transcriptional and phenotypic profiles, and to inhibit Treg-mediated immune suppression.ResultsSelect SAH-FOXP3DDs bound recombinant FOXP3ΔN and the FOXP3 leucine zipper (LZCC) domain with high affinity and dose-dependently inhibited binding of FOXP3 to cognate DNA in vitro. Lead SAH-FOXP3DDs were cell permeable and showed no non-specific toxicity to T cells at high concentrations. Flow cytometric and qRT-PCR analysis of treated Tregs revealed dose-dependent changes in protein and gene expression of several FOXP3 targets suggestive of FOXP3-specific transcriptional alteration. Treatment of Tregs with lead SAHs effectively inhibited in vitro Treg-mediated T cell suppression and induced global gene expression changes corresponding to loss of function Foxp3 in vivo.ConclusionsThis work supports the a