Anti-MAdCAM Antibody Increases ß7+ T Cells and CCR9 Gene Expression in the Peripheral Blood of Patients With Crohn's Disease.

OBJECTIVE: To define pharmacodynamic biomarkers in the peripheral blood of patients with Crohn's disease [CD] after treatment with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody. METHODS: In this Phase 2, randomised, double-blind, controlled study [OPERA], blood samples were analysed from patients with moderate to severe active CD who received placebo or 22.5 mg, 75 mg, or 225 mg of PF-00547659 subcutaneously at baseline and at Weeks 4 and 8, with follow-up at Week 12. Soluble MAdCAM [sMAdCAM] was measured by mass spectrometry, ß7-expressing T cells by flow cytometry, and gene transcriptome by RNA sequencing. RESULTS: A slight increase in sMAdCAM was measured in the placebo group from baseline to Week 12 [6%], compared with significant decreases in all PF-00547659 groups [-87% to -98%]. A slight increase from baseline to Week 12 was observed in frequency and molecules of equivalent soluble fluorochrome for ß7+ central memory T cells in the placebo group [4%], versus statistically significant increases in the active treatment groups [48% to 81%]. Similar trends were seen for ß7+ effector memory T cells [placebo, 8%; PF-00547659, 84-138%] and ß7+ naïve T cells [8%; 13-50%]. CCR9 gene expression had statistically significant up-regulation [p = 1.09e-06; false discovery rate < 0.1] with PF-00547659 treatment, and was associated with an increase in ß7+ T cells. CONCLUSIONS: Results of the OPERA study demonstrate positive pharmacology and dose-dependent changes in pharmacodynamic biomarker measurements in blood, including changes in cellular composition of lymphocytes and corresponding CCR9 gene expression changes.

Phase II evaluation of anti-MAdCAM antibody PF-00547659 in the treatment of Crohn's disease: report of the OPERA study.

OBJECTIVE: This phase II, randomised, double-blind, placebo-controlled clinical trial was designed to evaluate the efficacy and safety of PF-00547659, a fully human monoclonal antibody that binds to human mucosal addressin cell adhesion molecule (MAdCAM) to selectively reduce lymphocyte homing to the intestinal tract, in patients with moderate-to-severe Crohn's disease (CD). DESIGN: Eligible adults were aged 18-75 years, with active moderate-to-severe CD (Crohn's Disease Activity Index (CDAI) 220-450), a history of failure or intolerance to antitumour necrosis factor and/or immunosuppressive agents, high-sensitivity C reactive protein >3.0 mg/L and ulcers on colonoscopy. Patients were randomised to PF-00547659 22.5 mg, 75 mg or 225 mg or placebo. The primary endpoint was CDAI 70-point decrease from baseline (CDAI-70) at week 8 or 12. RESULTS: In all, 265 patients were eligible for study entry. Although CDAI-70 response was not significantly different with placebo versus PF-00547659 treatment at weeks 8 or 12, remission rate was greater in patients with higher baseline C reactive protein (>5 mg/L vs >18.8 mg/L, respectively). Soluble MAdCAM decreased significantly from baseline to week 2 in a dose-related manner and remained low during the study in PF-00547659-treated patients. Circulating ß7+ CD4+ central memory T-lymphocytes increased at weeks 8 and 12 with PF-00547659 treatment. No safety signal was seen. CONCLUSIONS: Clinical endpoint differences between PF-00547659 and placebo did not reach statistical significance in patients with moderate-to-severe CD. PF-00547659 was pharmacologically active, as shown by a sustained dose-related decrease in soluble MAdCAM and a dose-related increase in circulating ß7+ central memory T cells. TRIAL REGISTRATION NUMBER: NCT01276509; Results.

Validation of a standardized method for enumerating circulating endothelial cells and progenitors: flow cytometry and molecular and ultrastructural analyses.

PURPOSE: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. EXPERIMENTAL DESIGN: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability. RESULTS: Sorted DNA/Syto16(+)CD45(-)CD31(+)CD146(+) CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 +/- 171/mL in healthy subjects (n = 37) and 951 +/- 1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 +/- 14% in healthy subjects and 43 +/- 23% in cancer patients (P < 0.0001). CEPs were 181 +/- 167/mL in healthy donors and 429 +/- 507/mL in patients (P = 0.00019). Coefficients of variation were 4 +/- 4% (intrareader), 17 +/- 4% (interreader), and 17 +/- 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 +/- 8% (intrareader), 16 +/- 10% (interreader), and 26 +/- 16% (variability over 0-14 days of frozen storage), respectively. CONCLUSIONS: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.