Internalization of the vedolizumab/α4β7 complex and kinetics of restoring functional activity

Vedolizumab (former versions known as MLN0002, MLN02, and LDP-02) is an investigational humanized monoclonal antibody in Phase 3 clinical development for ulcerative colitis and Crohn's disease. Vedolizumab binds to the α4β7 integrin, which is a transmembrane cell adhesion molecule, thereby selectively blocking the migration of inflammatory cells into the gastrointestinal tract. In clinical trials, vedolizumab saturated the α4β7 integrin on peripheral blood lymphocytes, and this effect persisted even when vedolizumab was no longer detectable in serum. Although the 50% effective concentration (EC50) (0.31nM) for binding is below the limits of drug assay detection (>0.83nM), we investigated whether this continued pharmacodynamic (PD) effect could have an alternative explanation. Understanding this mechanism could impact the dosing of patients with this gut-selective, anti-inflammatory biologic. We hypothesized that vedolizumab may alter expression of the α4β7 integrin on the surface of lymphocytes and examined the in vitro effects of vedolizumab binding to the α4β7 integrin on gut-homing, memory T helper lymphocytes. To investigate the subcellular localization of the α4β7 integrin following in vitro binding by vedolizumab, human peripheral blood was incubated for 24 hours at 4°C or 37°C with unlabeled or Alexa-647-labeled vedolizumab. After incubation, cells were washed with acid to remove extracellular vedolizumab, and internalized vedolizumab was examined by immunofluorescence and flow cytometry. Potential re-expression of the α4β7 integrin on the cell surface was determined by continuing incubation at 37°C. On days 0, 1 and 4, an aliquot of cells was stained with vedolizumab-Alexa-647 or control. The function of restored extracellular α4β7 integrin expression was examined by assessing the ability of restored α4β7 to bind soluble mucosal addressin cell adhesion molecule-1 (MAd-CAM-1), the primary physiologic ligand of α4β7 integrin. Upon binding of vedolizumab to α4β7, the vedolizumab/α4β7 complex was internalized within target cells. This internalization began within 4 hours and was complete by 24 hours. Upon complete removal of excess vedolizumab, partial restoration of extracellular α4β7 (50% to 58% of the initially detectable concentration) occurred within 24 hours; near complete restoration of α4β7 (90%) required at least 4 days. Importantly, the restored membrane-bound α4β7 was functional in that it had the ability to bind MAdCAM-1. These results demonstrate that extracellular expression of the α4β7 integrin complex decreases after binding of vedolizumab due to the internalization of the vedolizumab/α4β7 complex within lymphocytes. Upon removal of extracellular vedolizumab, a rapid return of the α4β7 integrin complex occurred, though at least 4 days are required to restore membrane expression to pre-exposure levels. This re-expressed α4β7 integrin is functional. These data indicate that the persistence of the clinical PD effect can be explained in part by the high affinity binding of vedolizumab-induced α4β7 integrin internalization, and that this effect is readily reversible after the removal of vedolizumab.