Abstract
Introduction: Vasoactive intestinal peptide (VIP) is an immunosuppressive neuropeptide signaling through VPAC1/VPAC2 receptors on T cells, limiting activation, proliferation, and cytokine production cells (Delgado, J Mol Med. 2002 Jan;80(1):16–24). While protective in inflammation, this suppression impairs anti-tumor immunity (Ganea, Acta Physiol. 2015 Feb;213(2):442–52). Acute myeloid leukemia (AML) often expresses VIP, potentially exploiting this pathway to evade immune surveillance (Lavallée, Blood. 2015 Jan; 125(1), 140–143). Prior studies showed the VIP antagonist VIPhyb enhances T-cell activation and delays leukemia progression, but its low potency and a predicted short half-life limit its clinical potential (Petersen, OncoImmunology. 2017 May 4;6(5):e1304336). We designed ANT308 as a competitive VPAC peptide antagonist to have a longer half-life and enhanced potency. ANT308 potently promotes T-cell activation and proliferation in preclinical studies. These findings suggest a direct role for ANT308 in modulating T-cell responses, but the underlying mechanism remains unclear. We hypothesized that ANT308 preferentially binds to activated T-cells and enhances activation marker expression by competing with endogenous VIP for receptor engagement.
Methods: T cells from naïve C57BL/6 were isolated from splenocytes, activated overnight with anti-CD3/CD28 and 30 IU/mL IL-2, and treated with 3 µM His-tagged ANT308, His-tagged VIP, or His-tagged scramble peptide. Anti-His antibodies were used to localize peptide binding. Binding and activation marker expression (CD69, 4-1BB, Ki67) was quantified by flow cytometry; receptor localization was examined by confocal microscopy. Binding to T cells from VPAC1- and VPAC2-knockout mice confirmed receptor specificity. AlphaFold modeling and free-energy calculations predicted interaction strengths between peptides and VPAC1 and VPAC2 receptors.
Results: AlphaFold generates binding scores for peptide-protein interactions, with lower numbers predicting higher affinity. ANT308 had high predicted affinity for human VPAC1 > VPAC2, with calculated binding scores of 16.98 and 19.67, respectively compared with 18.7 and 18.45 for VIP. Binding scores for ANT308 and VIP to VPAC1 correspond to predicted ΔG of -71.6 kcal/mol vs. -65.8 kcal/mol, suggesting competitive engagement of ANT308 to VPAC1 with endogenous VIP. Confocal imaging revealed ANT308 co-localized with VPAC1/2 in activated mouse T cells, displaying punctate receptor distribution distinct from uniform CD3/CD8 staining. No co-localization occurred with the His-tagged scramble control peptide. Flow cytometry confirmed preferential ANT308 binding to activated vs. resting T cells. ANT308 treatment increased CD69, 4-1BB, and Ki67 expression in activated T cells relative to scrambled peptide or VIP treatment.
Conclusions: ANT308 binds VPAC1/2 on activated T cells, reorganizing receptors and enhancing activation markers. These findings indicate ANT308 competitively engages VIP receptors with high affinity and may effectively counter VIP-mediated immunosuppression. Mechanistic studies will define ANT308-induced VPAC reorganization within the membrane and downstream signaling pathways, assess competitive binding kinetics with endogenous VIP, and test in vivo efficacy in AML and other cancers exploiting VIP signaling.
