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Fecal Loss of Infliximab As a Cause of Lack of Response in SevereInflammatory Bowel DiseaseJohannan F. Brandse, Manon Wildenberg, Jessica R. de Bruyn, Gert-Jan Wolbink, M.Lowenberg, Cyriel Ponsioen, Gijs R. van den Brink, Geert R. D'Haens

BACKGROUND Although Infliximab (IFX) has proven its efficacy both in Crohn's disease(CD) and Ulcerative Colitis (UC), still a considerable group of patients fails to respond toinduction therapy. One of the hypotheses behind this phenomenon is increased clearanceor loss of the drug. In severe CD and UC, the mucosa is diffusely denuded and ulcerated,leading to massive loss of proteins, electrolytes and other nutrients. We hypothesized that,even after intravenous administration, IFX may be lost through this ‘leaky gut'. Fecal lossof IFX in this context has not been investigated up to date and this was the focus of thecurrent study METHODS In this pilot study we prospectively collected repeated fecal samplesof IBD patients within the first 14 days after starting IFX therapy. Fecal samples were weighedand homogenized in PBS containing bovine serum albumin. Supernatant was collected aftercentrifugation and tested with a well established ELISA by Sanquin Laboratories to detectthe presence of Infliximab. Clinical response in these patients was assessed 3 months afterthe initiation of IFX therapy. RESULTS 9 IBD patients (3 CD, 6 UC) with different diseaselocalizations (5 Ulcerative Pancolitis, 1 Proctitis, 2 Ileocecal CD, 1 Crohn's Colitis) startingon IFX 5 mg/kg at week 0-2-6 were included. One patient (Crohn's colitis) received anextra infusion (5mg/kg) at day 4 because of initial non response. IFX could be detected inthe feces of all patients. The highest concentrations were measured in the first days afterinitiation of therapy, meaning in the most acute phase of the disease. In non responders(3/9) the amount of drug detected in stool at the first day after infusion was significantly(p: 0.024) higher than in patients who had clinical response. CONCLUSION IFX can bedetected in the feces of patients with severe IBD, especially short after infusion. This phenome-non may contribute to rapid disappearance of the drug out of the gut and the circulationleading to insufficient response.

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Innate IL-10 Receptor Signaling Regulates Intestinal Mucosal HomeostasisDror S. Shouval, Jeremy A. Goettel, Marc-Andre Wurbel, Aleixo M. Muise, Scott B.Snapper

Background: IL-10 receptor (IL-10R) mutations cause severe very early onset IBD in humans,and similarly, IL-10Rβ KO mice develop spontaneous colitis. While recent data show thatIL-10R signaling is important on T cell populations to prevent IBD, there is no data on therole of IL-10R signaling on innate immune cells in maintaining mucosal homeostasis. Wehypothesized that innate IL-10R signaling is a key regulator in the intestine of immuneresponses and prevention of colitis. Methods: IL-10Rβ KO and RAG KO mice were crossedto generate double KO mice (DKO), which lack an adaptive immune system and possessIL-10R deficient innate immune cells. For adoptive transfer experiments, RAG KO and DKOmice were injected with wild-type (WT) CD4+ T cells, either unfractionated, or fractionatedas CD45RBhigh, CD45RBlow alone or in combination. Since IL-10R β is expressed on bothhematopoietic and non-hematopoietic cells, bone marrow chimeras of DKO into RAG KOmice were generated. Colitis was assessed based on clinical, endoscopic and histologicalscores. FACS analysis to assess immune cell populations was performed, as well as RT-PCRfor various transcripts. To assess whether IL-10R signaling in dendritic cells (DCs) affectsproliferation and suppression in-vitro, WT CFSE-labeled T Naïve cells were cultured withWT or IL-10Rβ KO DCs and different concentrations of WT Tregs. In-vitro Treg generationwas assessed by defining the frequency of CD4+CD25+FoxP3+ T cells, following culture ofWT T Naïve cells with TGF-β, αCD3 and either WT or IL-10Rβ KO DCs. Results: DKO micewere viable, occasionally developed abscesses, but importantly, did not develop spontaneouscolitis. However, transfer of total WT CD4+ T cells into DKO mice caused severe intestinalinflammation after 4-5 weeks, associated with Th1-Th17 immune responses. Similarly, whenCD4+ T cells were transferred into RAG KO mice reconstituted with DKO bone marrow,severe colitis resulted, indicating that the loss of IL-10R signaling on innate immune cellswas responsible for the phenotype. CD4+CD45RBhigh transfer also elicited severe colitis inDKO mice, was not ameliorated by co-transfer of CD45RBlow cells, and was associated witha significant decrease in generation of inducible Tregs. Decreased Treg generation was alsoobserved in-vitro in the presence of IL-10Rβ KO DCs. Moreover, IL-10Rβ KO DCs increasedthe proliferation of WT T effector cells, and decreased the suppressive function of WT Tregs.Conclusions: IL-10R signaling on innate immune cells is a key regulator of intestinal mucosalhomeostasis. Aberrant signaling in innate immune cells enables WT T cells to becomecolitogenic, and impairs the suppressive functions of Tregs. Ongoing work will elucidatethe relevant downstream signaling pathways mediated by IL-10R activation in innate immunecells and how it modulates the cross talk with T cells.

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Differentiation Between Etrolizumab (Rhumab Beta7) and Placebo in theEucalyptus Phase II Randomized Double-Blind Placebo-Controlled InductionStudy to Evaluate Efficacy and Safety in Patients With Refractory Moderate-to-Severely Active Ulcerative ColitisSeverine Vermeire, Sharon O'Byrne, Marna Williams, John C. Mansfield, Brian G. Feagan,Julian Panes, Daniel C. Baumgart, Stefan Schreiber, Iris Dotan, William Sandborn, MaryE. Keir, Diana Luca, Paul J. Rutgeerts

Introduction: Selective inhibition of leucocyte trafficking is a promising treatment for IBD.We evaluated a humanized monoclonal antibody to the β7 subunit of the heterodimericintegrins α4β7 and αEβ7 in patients with moderate-to-severely active UC. Methods: Thisparallel, randomized, double-blind, placebo-controlled study enrolled outpatients with atotal Mayo Clinic Score (MCS) ≥ 5, an endoscopy subscore ≥ 2 by central reading and arectal bleeding subscore ≥ 1. The primary efficacy endpoint was the proportion of patientsin clinical remission at Wk 10 (defined as a total MCS of ≤ 2 with no individual subscore. 1). The secondary endpoint atWk 10 of endoscopic remission was defined as an endoscopy

S-36AGA Abstracts

score of 0. Patients (n=124) were randomized in a 1:1:1 ratio to either 2 dose levels ofetrolizumab (100mg monthly SC or 300mg monthly SC + loading dose [LD] of 420mg SCbetween Wk 0 & 2) or placebo for 3 doses. Concomitant therapy for UC remained stablethrough to the primary endpoint at Wk 10. MCS was evaluated at Wks 0, 6 & 10. Fivepatients were excluded from the primary efficacy analysis due to a screening endoscopysubscore of , 2 by central read. Results: Patient demographics and baseline characteristicsare outlined in Table 1. Primary and secondary efficacy endpoints are summarized in Table2. Etrolizumab showed significantly higher rates of clinical remission vs Pbo at Wk 10:100mg dose 20.5% and 300mg + LD dose 10.3% versus Pbo 0% (p=0.004 and 0.049respectively). In the anti-TNF Naïve subgroup, the rates of clinical remission at Wk 10 weresignificantly higher in the 100mg dose group compared with Pbo (43.8% vs 0%, p=0.007).Etrolizumab achieved endoscopic remission in the 100mg dose of 10.3% and in the 300mg+ LD dose of 7.7% vs Pbo of 0%. In the anti-TNF Naïve subgroup, endoscopic remissionwas 25% and 16.7% vs 0% respectively (p=0.058 vs Pbo for the 100mg dose group).Etrolizumab demonstrated apparent full occupancy of beta7 receptors on CD4 and CD8 Tcells in both colonic tissue (α4β7 and αEβ7) and peripheral blood (α4β7), and of the α4β7receptor on B cells in peripheral blood, in both etrolizumab dose groups. Rates of AEs werecomparable for the 3 groups. One etrolizumab treated patient suffered a rash & headacheafter the first dose and was admitted to hospital for observation. This patient was negativefor ATAs. There were a total of 4 actively treated patients with mild (Grade 1) injection sitereactions all of whom were in the 300mg + LD dose group. Conclusion: Etrolizumabadministered SC is well tolerated and has shown clinically meaningful efficacy comparedwith Pbo in patients with moderate-to-severely active UC. These promising results warrantfurther Phase III studies.Table 1: Patient demographics and baseline characteristics

Table 2: Summary of Primary and Secondary Efficacy Results at Week 10 in Eucalyptus

a4 patients distributed across the treatment groups are anti-TNF exposed but are not anti-TNF-inadequate responders (IR) * p,0.05, ** p,0.01, *** p,0.005 vs placebo