Using fluorescently labeled vedolizumab to visualize local drug distribution during colonoscopy and identify mucosal target cells in patients with inflammatory bowel disease

Background Improving patient selection and development of biological therapies such as vedolizumab in inflammatory bowel disease (IBD) requires a thorough understanding of the mechanism of action and target binding, thereby providing individualized treatment strategies. Our goal was to visualize the macroscopic and microscopic distribution of intravenous injected fluorescently labeled vedolizumab, vedo-800CW, and identify its target cells using fluorescence molecular imaging (FMI). Methods In total 43 FMI procedures were performed in 37 IBD patients. FMI procedures consisted of macroscopic in vivo assessment during endoscopy, followed by macroscopic and microscopic ex vivo imaging. In phase A patients received a dose of 4·5 mg or 15 mg vedo-800CW or no tracer prior to endoscopy. In phase B patients received 15 mg vedo-800CW preceded by an unlabelled (sub)therapeutic dose of vedolizumab. Findings FMI quantification showed a significant dose-dependent increase in vedo-800CW fluorescence intensity in inflamed tissues, with 15 mg (153·7 a.u. [132·3-163·7]) as most suitable tracer dose compared to 4·5 mg (55·3 a.u. [33·6-78·2]) in naïve patients (p=0·0002). Moreover, the fluorescence signal decreased by 61% when vedo-800CW was administered after a therapeutic dose of unlabeled vedolizumab, suggesting target saturation in the inflamed tissue. Fluorescence microscopy and immunostaining showed that vedolizumab penetrated the inflamed mucosa and was associated with several immune cell types. Finally, surface binding of vedo-800CW was most prominent in plasma cells, whereas intracellular localization was observed primarily in macrophages and eosinophils. Interpretation These results indicate the potential of FMI to macroscopically determine the local distribution of drugs in the inflamed target tissue and identify drug target cells, providing new insights into targeted agents for their use in IBD. Regarding vedolizumab, we provide valuable information about its main target cells, contributing to our understanding of the underlying mechanism of action. Funding This work received funding from the EU/EFPIA/IMI2 JU Immune-Image grant no831514. Evidence before this study Combining fluorescence molecular imaging (FMI) with fluorescently labeled drugs holds high potential for providing detailed insights into the drug’s mechanism of action by allowing researchers to visualize its distribution and its target cells. Strikingly, with respect to inflammatory bowel disease (IBD) our lack of understanding regarding the mechanism of action of therapeutic compounds such as vedolizumab remains a major hurdle to improving prognosis and quality of life. Vedolizumab inhibits α4β7 integrin and was developed to prevent the migration of α4β7-expressing gut-homing T cells from vessels into the mucosa, thereby preventing inflammation. However, recent studies have speculated that the anti-inflammatory effect of vedolizumab is mediated by a wide range of α4β7-expressing immune cells, not just T cells. Unfortunately, a literature search revealed that drug distribution studies on vedolizumab in IBD that examined the mucosal distribution of vedolizumab or its target cells are lacking. Added value of this study Here, we show for the first time that intravenous administration of a fluorescently labelled drug can be used to visualize both the macroscopic and microscopic tissue distribution using FMI. Importantly, we combined fluorescently labeled vedolizumab with FMI in 43 procedures in patients with IBD, revealing valuable information regarding the drug’s distribution. We performed both in vivo and ex vivo FMI in order to quantify vedolizumab levels in inflamed mucosal tissues and found that vedolizumab targets a variety of immune cell types. We examined subcellular localization in these immune cells in more detail and found that vedolizumab binds to the surface of plasma cells, but is taken up into the cytoplasm in macrophages and eosinophils. These findings provide proof-of-concept to support the notion that FMI can be used to determine the distribution of a drug in the target tissue and identify the drug’s cellular target. Using this novel imaging technique will additionally provide valuable new insights regarding a drug’s ideal dose and the target saturation of specific drugs used to treat inflammatory disease. Implications of all the available evidence The ability to localize a drug’s distribution and identify its target cells is an essential step towards improving treatment options for IBD and other inflammatory diseases, thereby eventually improving outcome and increasing quality of life. Our step-by-step FMI approach consisting of in vivo macroscopic fluorescence imaging, ex vivo fluorescence tissue analysis, and fluorescence microscopy can be used to increase our understanding of drug distribution at the target levels and thereby help understanding the underlying mechanism of action for a wide range of drugs. Ultimately, these findings may help minimize the economic and social impacts of chronic inflammatory diseases. ### Competing Interest Statement GD received research grants from Royal DSM, Takeda and Janssen Pharmaceuticals and speaker fees from AbbVie, Pfizer, Takeda and Janssen Pharmaceuticals. EAMF is supported by a ZonMW Clinical Fellowship grant (projectnr 90719075) and has received an unrestricted research Grant from Takeda. MD & SJ are employees and shareholders of Regeneron Pharmaceuticals Inc. ### Clinical Trial NCT04112212 ### Funding Statement This work received funding from the EU/EFPIA/IMI2 JU Immune-Image grant no831514.  ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Medical Research Ethics Committee University Medical Center Groningen gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes * a.u. : arbitrary units GMP : good manufacturing practice CD : Crohn’s disease FFPE : formalin-fixed, paraffin-embedded FI : fluorescence intensity FMI : fluorescence molecular imaging H&E : hematoxylin and eosin IHC : immunohistochemistry UC : ulcerative colitis UMCG : University Medical Center Groningen.