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Abstract
Background Vasoactive intestinal peptide (VIP) is an immunosuppressive neuropeptide that significantly affect proliferation and anti-tumor properties of T cells.1-3 VIP overexpression is a potential mechanism of immune escape in solid tumors with paracrine VIP production. Our published work shows that inhibiting VIP receptor (VIP-R) signaling via VIPhyb, an antagonistic fusion peptide between neurotensin and VIP, improves T cell dependent anti-tumor response in mouse models of acute myeloid leukemia (AML) and T lymphoblastic leukemia (TLL).4 In this study, we developed novel VIP-R antagonists with enhanced efficacy when compared to VIPhyb, to generate a significantly more robust anti-tumor response in mouse models of AML.
Methods We created a combinatorial library of 300 peptide sequences that contain the six charged N-terminal residues of the neurotensin present in VIPhyb (first-generation VIP antagonist) with two or more amino acid substitutions within the C-terminal amino acid sequence of VIP (table 1). We performed in-silico screening to identify 10 novel VIP-R antagonists that were predicted to have increased binding affinity to VIP receptors VPAC1 and VPAC2 when compared to VIP or VIPhyb. The efficacy of these peptides where tested in-vitro using T cells from luciferase transgenic mice seeded and expanded on anti-CD3 monoclonal antibody coated plates for three days. Enhanced potency of the novel antagonists in vivo, was tested in a mouse AML model, by treating C1498-bearing mice with subcutaneous administration of VIP, VIPhyb, scrambled peptide or the second-generation VIP-R antagonists (labeled as ‘ANT’) from day 6-12 after tumor implantation.
Results T cell proliferation using 0.3 µM of a novel VIP-R antagonist was increased up to 216% + 20% of control cultures without added peptides versus 197% + 38% in cultures with VIPhyb at 1 uM (table 1). Furthermore, the novel VIP-R antagonists increased median survival times (MST) by up to 57 days and rendered 40% of mice leukemia-free at 60 days compared to MST of 34 days and 5% long-term survival with VIPhyb (figure 1).
Novel second generation VIP-R antagonistsPredicted binding affinity of VIP, VIPhyb and VIP antagonists to VPAC1 and VPAC2 based upon in silico screening is shown along with the proliferation of luciferase+ mouse T cells and their anti-leukemia activity in mice. The level of T cell bioluminescence vs. control T cells stimulated with anti-CD3 alone is shown along with the lowest peptide concentration that achieved the maximal effect on T cell proliferation. Median survival times and the fraction of mice alive at day 60 along with the numbers of animals tested with each VIP antagonist are shown.
Prolonged survival with second generation peptidesC57BL/6 mice were injected intravenously with 1 x 106 C1498 myeloid leukemia cells on day 0. The presence of engrafted was confirmed on day 6 by flow cytometry and leukemia-bearing mice were treated with daily subcutaneous injections of PBS, 10 µg VIP scrambled peptide (SCRAM), VIP, VIPhyb, or a second-generation VIP antagonist (ANT008, etc…) for 10 days. Survival of all groups treated with a VIP antagonist was significantly better than groups treated with PBS, VIP, or SCRAM at p < 0.001. Survival between mice treated with ANT300 was significantly better than those treated with VIPhyb (p<0.02)
Conclusions In this study, for the first time, we have identified novel and more potent VIP-R antagonists when compared to VIPhyb, with enhanced potency to activate and proliferate T cells and generate an effective anti-tumor response in mouse models of leukemia. These novel antagonists can lead to peptide-based immunotherapy for the treatment of various solid and liquid cancers, such as the cancer of the colon and pancreas, that overexpress VIP intratumorally.
References
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Anderson P, Gonzalez-Rey E. Vasoactive intestinal peptide induces cell cycle arrest and regulatory functions in human T cells at multiple levels. Mol Cell Biol 2010;30(10):2537-51.
Reubi JC, et al. Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin. Cancer Res 2000;60(11):3105-12.
Petersen CT, Li JM, Waller EK. Administration of a vasoactive intestinal peptide antagonist enhances the autologous anti-leukemia T cell response in murine models of acute leukemia. Oncoimmunology 2017;6(5):e1304336.
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