Search

The discovery of multiple costimulatory cell surface molecules that influence the course of T cell activation has increased our appreciation of the complexity of the T cell response. It remains clear, however, that CD28 and cytotoxic T lymphocyte antigen 4 (CTLA-4) are the critical costimulatory receptors that determine the early outcome of stimulation through the T cell antigen receptor (TCR). Details of how the T cell integrates TCR stimulation with the costimulatory signals of CD28 and the inhibitory signals of CTLA-4 remain to be established, but unique features of the cell biology of CTLA-4 provide important insights into its function. We summarize here recent findings that suggest a previously unrecognized role for CTLA-4 in the regulation of T cell responses. We also describe preclinical and clinical results that indicate manipulation of CTLA-4 has considerable promise as a strategy for the immunotherapy of cancer.

Increased Expression of T Cell Associated Genes in Baseline Biopsies From TNF Antagonist Naive Patients That Achieve Remission With Etrolizumab Treatment in a Phase II Trial in Moderate to Severely Active Ulcerative Colitis Gaik Wei Tew, Jason Hackney, Deena L. Gibbons, Christopher A. Lamb, Diana Luca, Jackson G. Egen, Severine Vermeire, John C. Mansfield, Brian G. Feagan, Julian Panes, Daniel C. Baumgart, Stefan Schreiber, Iris Dotan, William Sandborn, John A. Kirby, Gert De Hertogh, Gert A. Van Assche, Paul J. Rutgeerts, Sharon O'Byrne, Adrian C. Hayday, Mary E. Keir

s of the 9th Congress of ECCO the European Crohn’s and Colitis Organisation S7 (biventricular heart dysfunction and sepsis) that occurred between wks 54 and 104. Conclusions: These data continue to support a positive benefit/ risk profile for GLM in the treatment of moderate to severe UC; GLM treatment for up to 2 y maintained clinical benefit including a reduction in corticosteroid use. No new safety signals were observed with continued GLM treatment through wk 104; safety profile was similar to wk 54. OP010 AlphaE integrin expression as a predictive biomarker for induction of clinical remission by etrolizumab: Analysis of a phase II trial in moderate-to-severely active ulcerative colitis M. Keir1 *, G.W. Tew1, D. Luca1, J. Eastham-Anderson1, L. Diehl1, J.G. Egen1, S. Vermeire2, J.C. Mansfield3, C.A. Lamb3, J. Panes4, D.C. Baumgart5, S. Schreiber6, I. Dotan7, W.J. Sandborn8, G. De Hertogh2, J.A. Kirby3, G. Van Assche2, P. Rutgeerts2, S. O’Byrne1. 1Genentech, Research and Early Development, South San Francisco, United States, 2KU Leuven, Division of Gastroenterology, Leuven, Belgium, 3Royal Victoria Infirmary, Gastroenterology, Newcastle upon Tyne, United Kingdom, 4Hospital Clinic Barcelona, Gastroenterology, Barcelona, Spain, 5HumboldtUniversity of Berlin, Charite Medical School, Berlin, Germany, 6Christian Albrechts University, Institute of Clinical Molecular Biology, Kiel, Germany, 7Tel Aviv University, Sackler Faculty of Medicine, Tel Aviv, Israel, 8University of California, San Diego, Gastroenterology, San Diego, United States Background: Etrolizumab is a humanized monoclonal antibody to the beta7 integrin subunit that blocks alpha4beta7: MAdCAM-1 and alphaEbeta7:E-cadherin interactions and has been shown to be effective at inducing remission in patients with moderate to severely active ulcerative colitis (UC). Methods: Patients enrolled in the Phase 2 study of etrolizumab in UC were assessed for baseline biopsy gene expression of MAdCAM1, alphaE and alpha4 integrin by qPCR (n = 106) as well as baseline alphaE+ cells per total cells in biopsies from the inflamed colon by immunohistochemistry (n = 76). Enrichment of baseline measures was evaluated using the primary outcome measure of the study, remission at week 10, defined as a total MCS of 2 with no individual subscore >1. Results: AlphaE gene expression and alphaE+ cells were lognormally distributed in baseline biopsies from UC patients. No difference in baseline characteristics was observed between alphaE high and alphaE low groups. Enrichment in response to treatment with 100mg/dose etrolizumab as measured by remission was observed in patients with higher than median levels of alphaE gene expression at screening (38% alphaE high vs. 13% alphaE low). No enrichment was observed in alpha4 high patients (27% alpha4 high vs. 24% alpha4 low) or MAdCAM1 high patients (27% MAdCAM1 high vs. 24% MAdCAM1 low). Enrichment in response to treatment as measured by remission was observed in TNF antagonist naive patients with high levels of alphaE gene expression at screening (67% alphaE high vs. 17% alphaE low). Similarly, enrichment of remission in response to treatment was observed in patients with higher than median levels of alphaE+ cells per total cells (50% alphaE high vs. 7% alphaE low). TNF antagonist naive patients with high levels of alphaE+ cells per total cells at screening had enriched response to treatment with etrolizumab as measured by remission (67% alphaE high vs. 25% alphaE low). Conclusions: These results suggest that higher than median levels of alphaE gene expression and/or alphaE+ cells per total cells in the inflamed colon are associated with increased clinical benefit of etrolizumab treatment in patients. Thus alphaE gene expression or alphaE+ cells in colonic biopsies may be predictive biomarkers to identify UC patients most likely to benefit from etrolizumab treatment. OP011 Tralokinumab (CAT-354), an interleukin 13 antibody, in moderate to severe ulcerative colitis: A phase 2 randomized placebo-controlled study S. Danese1, J. Rudzinski2, W. Brandt3, J.L. Dupas4, L. Peyrin-Biroulet5, Y. Bouhnik6, D. Kleczkowski7, P. Uebel8, M. Lukas9, M. Knutsson10, F. Erlandsson10, M. Berner Hansen10 *, S. Keshav11. 1Istituto Clinico Humanitas, IBD Center, Milan, Italy, 210 Wojskowy Szpital Kliniczny z Poliklinik?, Kliniczny Oddzia? Gastroenterologii, Warsaw, Poland, 3Facharzt fur Innere Medizin, Internal Medicine, Potsdam, Germany, 4CHU d’Amiens Hopital Nord, Hepato-Gastroenterology, Amiens, France, 5CHU Nancy Brabois, Gastrology, Vandoeuvre les Nancy, France, 6Hopital Beaujon APHP, Gastroenterologie, Clichy, France, 7Endoskopia Sp.z o.o., Gastroenterology, Sopot, Poland, 8Haus der Gesundheit, Internal Medicine, Ludwigshafen, Germany, 9Iscare, Gastroenterology, Prague, Czech Republic, 10AstraZeneca, Innovative Medicines, Molndal, Sweden, 11John Radcliffe Hospital, Translational Gastroenterology Unit, Oxford, United Kingdom Background: Interleukin 13 (IL-13) is a central cytokine effector in the Th2 immune response and potentially a key player in ulcerative colitis (UC) pathogenesis. Tralokinumab (CAT-354) is a humanized IgG4 antibody that binds and inhibits IL-13. The purpose of this study was to evaluate the efficacy and safety of tralokinumab in subjects with moderate to severe active UC. Methods: Subjects (18 75 yrs) with UC and total Mayo score 6 12 received tralokinumab or placebo as add-on therapy in this European multicenter, randomized, double-blind, placebocontrolled study sponsored by AstraZeneca (ClinicalTrials.gov #NCT01482884). Subjects received subcutaneous tralokinumab (300mg) or placebo every 2 wks during a 12-wk treatment phase followed by a 12-wk safety follow-up. The primary endpoint was clinical response at wk 8 defined as a reduction of 3 points and 30% or more from baseline total Mayo score. Secondary endpoints included clinical remission (Mayo score 2 with no subscore >1 point), mucosal healing (endoscopic Mayo score 0), improvement in Mayo score at wk 8, partial Mayo score, and safety. Allowed background medications included stable doses of 5-ASA, prednisolone ( 20mg daily), and thiopurines. Results: 111 subjects were randomized (tralokinumab n = 56; placebo n = 55). Baseline subject and disease characteristics were similar in both arms. The median baseline Mayo score was 8 in both arms; 23% and 31%, respectively, had received prior TNF-a therapies; 43 (77%) tralokinumab subjects and 37 (67%) placebo subjects, respectively, completed the study. Improvements in mean partial Mayo scores were greater in the tralokinumab subjects ( 1.8 vs. 0.8, p = 0.04). Results at week 8 Tralokinumab Placebo P value Clinical response (%) 38 33 0.41 Clinical remission (%) 18 6 0.03 Mucosal healing (%) 32 20 0.10 Mean improvement in Mayo Score 3.2 2.6 0.39 Serum eosinophil counts and total IL-13 levels increased during therapy with tralokinumab. Symptoms of UC were the most frequently reported adverse events (AEs). The number of subjects experiencing AEs and AEs leading to discontinuation of drug were similar in both groups. There were no clinically relevant changes in laboratory variables, vital signs, and electrocardiograms. No new safety signals were identified for tralokinumab. Conclusions: Multiple efficacy measures were numerically higher in the tralokinumab arm. However, the difference in clinical response at wk 8 (primary endpoint) between by gest on Feruary 5, 2016 http://eccoxfordjournals.org/ D ow nladed from

The T cell compartment of adaptive immunity provides vertebrates with the potential to survey for and respond specifically to an incredible diversity of antigens. The T cell repertoire must be carefully regulated to prevent unwanted responses to self. In the periphery, one important level of regulation is the action of costimulatory signals in concert with T cell antigen-receptor (TCR) signals to promote full T cell activation. The past few years have revealed that costimulation is quite complex, involving an integration of activating signals and inhibitory signals from CD28 and CTLA-4 molecules, respectively, with TCR signals to determine the outcome of a T cell's encounter with antigen. Newly emerging data suggest that inhibitory signals mediated by CTLA-4 not only can determine whether T cells become activated, but also can play a role in regulating the clonal representation in a polyclonal response. This review primarily focuses on the cellular and molecular mechanisms of regulation by CTLA-4 and its manipulation as a strategy for tumor immunotherapy.

Abstract 3586 Poster Board III-523 Shortly following G-CSF administration there is a dramatic yet transient disappearance of neutrophils from the circulation. To evaluate this disappearance, C57BL/6 LysM-EGFP knock-in male mice between 6-12 weeks of age were used. The LysM promoter/enhancer is specifically expressed in neutrophils and monocytes permitting evaluation of tissue distribution using EGFP marker localization by confocal microscopy. The ratio of EGFP+ leukocytes to background cellularity was determined in fixed volumes of tissue collected from the lung, liver, spleen, and kidney of mice either receiving saline (n=4) or at 15 minutes (n=6), 30 minutes (n=6), or 240 minutes (n=4) following 50ug/kg SQ rmG-CSF administration. No changes in ratio were observed in triplicate images of the liver or kidney of mice following G-CSF administration; however, there was a significant increase in EGFP+ leukocytes in the spleen and lung shortly following G-CSF administration. It increased from a control value of 6.3(2.6)% (n=12) to 15.9 (6.8)% (n=18) at 30 minutes in the lung and from 1.6 (0.7)% (n=12) to 8.3 (4.2)% (n=18) at 30 minutes in the spleen. While the value for the pulmonary images was less at 240 minutes (9.2 (5.1)%) the level of EGFP+ cells in the spleen continued to rise (12.3 (7.2)%). The increases were in neutrophils based on nuclear morphology. Thus the transient loss of neutrophils from the circulation following G-CSF administration appeared to be due to an accumulation of neutrophils within the pulmonary and splenic tissues. To confirm this observation, tissues were collected from three rhesus macaques 30 minutes following 10ug/kg SQ G-CSF. These tissues were compared to those of a control. Neutrophils were labeled with CD18 and a secondary antibody conjugated to FITC for quantification by confocal microscopy. As before, ratios of fluorescence were determined. The mean threshold intensity for CD18+ cells for the lung lobes for those animals receiving G-CSF (3.8 (3.0)%, n=54) was greater than control specimen lungs (1.5(1.1)%, n=18) and slightly increased for the spleen( from 4.1(3.7)% to 8.5 (6.3)%, n=9). Detailed confocal microscopy using a myeloid/histiocyte antigen clone Mac387 which labels neutrophils and monocytes, an endothelial marker anti-caveolin-1-Cy3 antibody, and a nuclear stain (To-Pro3) showed (see below, stained green, red, and blue, respectively) that the neutrophils were elongated, appeared activated, and were tightly adhered to the endothelium of the pulmonary vasculature. These results suggest that the transient neutropenia following G-CSF administration is associated with an accumulation of neutrophils within the pulmonary and splenic tissues. Disclosures: No relevant conflicts of interest to declare.

Summary Background Etrolizumab is a humanised monoclonal antibody that selectively binds the β7 subunit of the heterodimeric integrins α4β7 and αEβ7. We aimed to assess etrolizumab in patients with moderately-to-severely active ulcerative colitis. Methods In this double-blind, placebo-controlled, randomised, phase 2 study, patients with moderately-to-severely active ulcerative colitis who had not responded to conventional therapy were recruited from 40 referral centres in 11 countries. Eligible patients (aged 18–75 years; Mayo Clinic Score [MCS] of 5 of higher [or ≥6 in USA]; and disease extending 25 cm or more from anal verge) were randomised (1:1:1) to one of two dose levels of subcutaneous etrolizumab (100 mg at weeks 0, 4, and 8, with placebo at week 2; or 420 mg loading dose [LD] at week 0 followed by 300 mg at weeks 2, 4, and 8), or matching placebo. The primary endpoint was clinical remission at week 10, defined as MCS of 2 or less (with no individual subscore of >1), analysed in the modified intention-to-treat population (mITT; all randomly assigned patients who had received at least one dose of study drug, had at least one post-baseline disease-activity assessment, and had a centrally read screening endoscopic subscore of ≥2). This study is registered with ClinicalTrials.gov, number NCT01336465. Findings Between Sept 2, 2011, and July 11, 2012, 124 patients were randomly assigned, of whom five had a endoscopic subscore of 0 or 1 and were excluded from the mITT population, leaving 39 patients in the etrolizumab 100 mg group, 39 in the etrolizumab 300 mg plus LD group, and 41 in the placebo group for the primary analyses. No patients in the placebo group had clinical remission at week 10, compared with eight (21% [95% CI 7–36]) patients in the etrolizumab 100 mg group (p=0·0040) and four (10% [0·2–24]) patients in the 300 mg plus LD group (p=0·048). Adverse events occurred in 25 (61%) of 41 patients in the etrolizumab 100 mg group (five [12%] of which were regarded as serious), 19 (48%) of 40 patients in the etrolizumab 300 mg plus LD group (two [5%] serious), and 31 (72%) of 43 patients in the placebo group (five [12%] serious). Interpretation Etrolizumab was more likely to lead to clinical remission at week 10 than was placebo. Therefore, blockade of both α4β7 and αEβ7 might provide a unique therapeutic approach for the treatment of ulcerative colitis, and phase 3 studies have been planned. Funding Genentech.

The reported affinity differences between CD28 and CTLA-4 binding to B7-1 and B7-2 may serve to selectively regulate CD28 and CTLA-4 function by differentially recruiting and/or stabilizing these molecules at the immunological synapse. Here we show that ligand binding is important for the accumulation of both CD28 and CTLA-4 at the synapse. While CD28 is recruited to the synapse in the absence of B7-1 and B7-2 binding, it is not effectively stabilized there, as its localization can be disrupted by CTLA-4. In the case of CTLA-4, ligand binding is critical for its concentration at the synapse. We also demonstrate that the affinity and avidity differences in ligand binding translate into selective recruitment of CD28 or CTLA-4 to the immunological synapse—B7-1 is the major ligand mediating CTLA-4 localization, while B7-2 is the main ligand for CD28 concentration at the synapse.

Dectin-1 is a fungal pattern recognition receptor that binds to β-glucans and triggers cytokine production by facilitating interaction with TLR2 or by directly activating spleen tyrosine kinase (Syk). To assess the possible role of Dectin-1 in the innate response to mycobacteria, we used an in vitro system in which IL-12p40 production is measured in splenic dendritic cells (SpDC) following exposure to live Mycobacterium tuberculosis bacilli. Treatment of SpDC with laminarin or glucan phosphate, two molecules known to block Dectin-1-dependent activity, led to a reduction in M. tuberculosis-induced IL-12p40 as well as IL-12p70 production. Moreover, SpDC from Dectin-1−/− chimeric mice displayed reduced IL-12p40 production in response to mycobacteria when compared with Dectin-sufficient DC. Laminarin treatment also inhibited mycobacterial-induced IL-12p40 production in DC from TLR2−/− mice, arguing that Dectin-1 functions independently of TLR2 signaling in this system. Importantly, a Dectin-1 fusion protein was found to directly bind to live mycobacteria in a laminarin-inhibitable manner indicating the presence of ligands for the receptor in the bacterium and laminarin pretreatment resulted in reduced association of mycobacteria to SpDC. In additional experiments, mycobacterial stimulation was shown to be associated with increased phosphorylation of Syk and this response was inhibited by laminarin. Furthermore, pharmacologic inhibition of Syk reduced the M. tuberculosis-induced IL-12p40 response. Together, these findings support a role for Dectin-1 in promoting M. tuberculosis-induced IL-12p40 production by DC in which the receptor augments bacterial-host cell interaction and enhances the subsequent cytokine response through an unknown mechanism involving Syk signaling.