P004 PN-943, an oral α4β7 integrin antagonist, inhibits MAdCAM1-mediated proliferation and cytokine release from CD4+ T cells independent of trafficking

Abstract Background PN-943, an oral gastrointestinal (GI) -restricted peptide antagonist of α4β7 integrin, is being developed for the treatment of inflammatory bowel disease (IBD). Blockade of α4β7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM1) is thought to treat IBD by preventing extravascular migration of blood T cells into the inflamed GI mucosa. Recent evidence suggests the involvement of other mechanisms. Saturation of circulating T-cell α4β7 by vedolizumab is not sufficient for optimal efficacy; and PTG-100, a PN-943 analogue, showed evidence of clinical and histological remission in ulcerative colitis patients at sub-saturating blood receptor occupancy (%RO). Here, we assessed the potential of a local GI-acting function of α4β7 by evaluating the ability of PN-943 to inhibit MAdCAM1-mediated CD4+ T-cell proliferation and cytokine production. Methods Primary CD4+ T cells were labelled fluorescently and incubated with plate-bound anti-CD3 alone or together with MAdCAM1 with or without inhibitors for three days. Phenotype, distribution of T-helper (Th) subsets and %RO analyses were conducted by flow cytometry. Supernatant cytokine levels were quantified by multiplex assays. Results MAdCAM1 combined with anti-CD3 markedly enhanced proliferation of CD4+ T cells compared with anti-CD3 alone (n = 7, 12–64%). Immune phenotyping revealed that proliferation occurred in both CD45RO- naïve and CD45RO+ memory T cells and was restricted to the β7+ population with successive cycles of proliferation showing increased β7 expression. Amongst the proliferated memory T cells, the percentage of the IFNγ-producing Th1 subset was higher than IL-17A-producing Th17 and IL-4-producing Th2 subsets. Multiplex profiling identified several cytokines, including but not limited to IL-6, IL-13, GM-CSF, and TNFα, whose release were promoted by MAdCAM1. PN-943 abolished MAdCAM1-mediated stimulation (n = 4, IC50 4.6 nM). The inhibitory effects were associated with lowered β7 expression indicative of internalisation by PN-943. Blockade was not observed with an inactive analogue indicating dependency on PN-943 binding to α4β7. Consistent with the requirement for a locally acting drug, 30 mg/kg oral dosing in mice demonstrated high exposure (n = 6, 39 nM) and occupancy of T-cell α4β7 (n = 6, 92%) in the GI compared within the blood. Conclusion The α4β7-MAdCAM1 interaction promoted β7+CD4+ T-cell proliferation and cytokine release, which may contribute to chronic inflammatory responses occurring in the diseased gut of IBD patients independent of T-cell trafficking. PN-943 inhibition of MAdCAM1-mediated signalling through α4β7 supports the potential therapeutic advantages for an oral GI-restricted approach, whereby PN-943 is delivered locally and directly blocks α4β7 function in the GI.