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P019. Internalization of the vedolizumab/α4β7 complex and kinetics of restoring functional activity

P019. Internalization of the vedolizumab/α4β7 complex and kinetics of restoring functional activity


L.‑L. Yang1, E. Fedyk2, T. Wyant1

1Millennium Pharmaceuticals, Inc./Takeda Pharmaceuticals, Department of Molecular Medicine, Cambridge, United States; 2Millennium Pharmaceuticals, Inc./Takeda Pharmaceuticals, Department of Drug Safety Evaluation, Cambridge, United States



Background: Vedolizumab (VDZ) (former versions MLN0002, MLN02, LDP-02) is a gut-selective, anti-inflammatory biologic in Phase 3 development for ulcerative colitis and Crohn's disease. VDZ binds to α4β7 integrin, a transmembrane cell adhesion molecule, selectively blocking migration of inflammatory cells into the gastrointestinal tract. In clinical trials, VDZ saturated the α4β7 integrin on peripheral blood lymphocytes; this effect persisted when VDZ was no longer detectable in serum. Understanding this continued pharmacodynamic (PD) effect of VDZ could impact patient dosing. Hypothesizing that VDZ may alter expression of α4β7 integrin on the lymphocyte surface, we examined in vitro effects of VDZ binding to α4β7 integrin on gut-homing memory T helper lymphocytes.

Methods: To study subcellular localization of α4β7 integrin after in vitro binding by VDZ, human peripheral blood was incubated for 24 h at 4°C or 37°C with unlabeled or Alexa-647-labeled VDZ. After incubation, cells were washed with acid to remove extracellular VDZ, and internalized VDZ was examined by immunofluorescence and flow cytometry. Potential cell surface re-expression of α4β7 integrin was determined by continuing incubation at 37°C. On days 0, 1 and 4, an aliquot of cells was stained with VDZ-Alexa-647 or control. The function of restored extracellular α4β7 integrin was examined by testing its ability to bind soluble mucosal addressin cell adhesion molecule‑1 (MAdCAM‑1, primary ligand of α4β7).

Results: The bound VDZ/α4β7 complex was internalized within target cells. Internalization began within 4 h and was complete by 24 h. Upon complete removal of excess VDZ, expression of extracellular α4β7 was partially restored (50% to 58% of initially detectable concentration) within 24 h. Near complete (90%) restoration of α4β7 expression required at least 4 days, and this α4β7 was functional (ie, could bind MAdCAM‑1).

Conclusions: Extracellular expression of α4β7 integrin complex decreased after VDZ binding due to internalization of the VDZ/α4β7 complex within lymphocytes. Upon removal of extracellular VDZ, the α4β7 integrin complex returned rapidly. At least 4 days are needed to restore membrane expression to pre-exposure levels. The re-expressed α4β7 integrin is functional. These data indicate that persistence of the clinical PD effect can be explained in part by high affinity binding of VDZ-induced α4β7 integrin internalization, and that this effect is readily reversible after removal of VDZ.