Identification and characterization of vasoactive intestinal peptide receptor antagonists with high-affinity and potent anti-leukemia activity.

Vasoactive intestinal peptide (VIP) is a neuropeptide involved in cancer proliferation and immune suppression. The limited potency of the VIP antagonist peptide VIPhyb in T-cell activation and murine anti-leukemia models prompted the development of a more potent antagonist. We screened a combinatorial library of VIPhyb C-terminal peptide sequence variants to identify a higher-affinity VIP-receptor (VIP-R) antagonist, hypothesizing that specific amino acid substitutions could improve receptor binding and/or plasma stability. In silico screening analyses identified sequences with docking scores predicting increased binding affinity to human VIP receptors VPAC1 and VPAC2. 15 peptides were synthesized and tested for their ability to potentiate activation of purified mouse and human T cells and enhance T cell-dependent anti-leukemia responses in murine acute myeloid leukemia models. Treating C57Bl/6 mice engrafted with a C1498 leukemia cell line with daily subcutaneous injections of VIP-R antagonist peptides induced anti-leukemia responses. Strikingly, the predicted binding of the VIP-R antagonists to VIP receptors correlated positively with their ability to augment mouse T-cell proliferation and anti-leukemia activity. ANT308 and ANT195 emerged as top candidates due to high predicted VIP-R binding, low EC50 for in vitro T cell activation, and potent anti-leukemia activities. ANT308 decreased CREB phosphorylation, a downstream signaling pathway of the VIP receptor, and stimulated granzyme B and perforin expression in CD8+ T cells from AML patients. Combining in silico modeling, in vitro T cell activation properties, and in vivo anti-leukemia activity has identified promising VIP-R antagonist candidates for further development as novel immunotherapies for patients with AML having relapsed disease.