Ren Xu

Conatumumab, a fully human agonist antibody to death receptor 5, induces apoptosis via caspase activation in multiple tumor types

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4 and 5 (DR4, DR5) to transduce apoptotic signals. Conatumumab (AMG 655) is an investigational, fully human monoclonal agonist antibody (IgG1) to human DR5, which induces apoptosis via caspase activation. In this study, we demonstrate that conatumumab binds to DR5, activating intracellular caspases in vitro in the presence of a cross-linker. We also show that conatumumab has activity in vivo and inhibits tumor growth in colon (Colo205 and HCT-15), lung (H2122), and pancreatic (MiaPaCa2/T2) xenograft models. Conatumumab also enhances the anti-tumor activity of chemotherapeutics in vivo. Caspase activation in Colo205 tumors is dose-dependent and correlated with serum concentrations of conatumumab. We demonstrate for the first time that increases in serum caspase-3/7 activity and levels of M30 (neoepitope of caspase-cleaved cytokeratin-18) are linked to activation of the extrinsic apoptotic pathway using conatumumab in a preclinical model. These data suggest that conatumumab has potential as a therapeutic agent for treating patients with multiple tumor types, and that serum caspase-3/7 and M30 levels may serve as biomarkers of conatumumab activity.

Anti-PCSK9 Antibody Pharmacokinetics and Low-Density Lipoprotein-Cholesterol Pharmacodynamics in Nonhuman Primates Are Antigen Affinity–Dependent and Exhibit Limited Sensitivity to Neonatal Fc Receptor–Binding Enhancement

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as an attractive therapeutic target for cardiovascular disease. Monoclonal antibodies (mAbs) that bind PCSK9 and prevent PCSK9:low-density lipoprotein receptor complex formation reduce serum low-density lipoprotein-cholesterol (LDL-C) in vivo. PCSK9-mediated lysosomal degradation of bound mAb, however, dramatically reduces mAb exposure and limits duration of effect. Administration of high-affinity mAb1:PCSK9 complex (1:2) to mice resulted in significantly lower mAb1 exposure compared with mAb1 dosed alone in normal mice or in PCSK9 knockout mice lacking antigen. To identify mAb-binding characteristics that minimize lysosomal disposition, the pharmacokinetic behavior of four mAbs representing a diverse range of PCSK9-binding affinities at neutral (serum) and acidic (endosomal) pH was evaluated in cynomolgus monkeys. Results revealed an inverse correlation between affinity and both mAb exposure and duration of LDL-C lowering. High-affinity mAb1 exhibited the lowest exposure and shortest duration of action (6 days), whereas mAb2 displayed prolonged exposure and LDL-C reduction (51 days) as a consequence of lower affinity and pH-sensitive PCSK9 binding. mAbs with shorter endosomal PCSK9:mAb complex dissociation half-lives (<20 seconds) produced optimal exposure-response profiles. Interestingly, incorporation of previously reported Fc-region amino acid substitutions or novel loop-insertion peptides that enhance in vitro neonatal Fc receptor binding, led to only modest pharmacokinetic improvements for mAbs with pH-dependent PCSK9 binding, with only limited augmentation of pharmacodynamic activity relative to native mAbs. A pivotal role for PCSK9 in mAb clearance was demonstrated, more broadly suggesting that therapeutic mAb-binding characteristics require optimization based on target pharmacology.

Effect of evolocumab on cholesterol synthesis and absorption.

The effects of cholesterol-lowering drugs, including those that reduce cholesterol synthesis (statins) and those that reduce cholesterol absorption (ezetimibe), on cholesterol absorption and synthesis are well understood. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are a novel class of cholesterol-lowering drugs that robustly reduce LDL-cholesterol (LDL-C), but little is known about their effects on cholesterol absorption and synthesis. We evaluated how treatment with evolocumab, a fully human monoclonal IgG2 antibody to PCSK9, affects markers of cholesterol synthesis and absorption by measuring these markers in patients from an evolocumab clinical trial. At 2 weeks, changes in β-sitosterol/total cholesterol (TC) from baseline were 4% for placebo, 10% for evolocumab 140 mg (nonsignificant vs. placebo), and 26% for evolocumab 420 mg (P < 0.001 vs. placebo). Changes in campesterol/TC at week 2, relative to baseline between placebo and evolocumab, were all nonsignificant. Evolocumab had a modest effect on markers of cholesterol synthesis. At 2 weeks, changes in desmosterol/TC were 1% for placebo, 7% for evolocumab 140 mg (nonsignificant vs. placebo), and 15% for evolocumab 420 mg (P < 0.01 vs. placebo). Changes from baseline in lathosterol/TC at week 2 between placebo and evolocumab were nonsignificant. These results suggest that evolocumab has a modest effect on cholesterol synthesis and absorption despite significant LDL-C lowering.

Hepatic proton density fat fraction correlates with histologic measures of steatosis and is responsive to change in those measures in a multi-center nonalcoholic steatohepatitis clinical trial

Michael S. Middleton,1 Eric J. Lawitz,2 Saumya Jayakumar,3 Parvez Mantry,4 Stephen Caldwell,5 Anna Mae Diehl,6 Matt Peach,7 Ren Xu,7 Guang Cheng,7 C. Stephen Djedjos,7 Robert P. Myers,7 G. Mani Subramanian,7 Zachary Goodman,8 Michael Charlton,9 Nezam H. Afdhal,10 Claude B. Sirlin,1 Rohit Loomba1 1UC San Diego, La Jolla, CA; 2Texas Liver Institute, UT Health, San Antonio, TX; 3University of Calgary, Alberta, Canada; 4The Liver Institute at Methodist Dallas, TX; 5University of Virginia, Charlottesville; 6Duke Clinical Research Institute, Durham, NC; 7Gilead Sciences, Inc., Foster City, CA; 8Inova Fairfax Hospital, Falls Church, VA; 9Intermountain Medical Center, Salt Lake City, UT; 10Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Longitudinal correlations between MRE, MRI-PDFF, and liver histology in patients with non-alcoholic steatohepatitis: Analysis of data from a phase II trial of selonsertib

BACKGROUND & AIMSnNon-invasive tools for monitoring treatment response and disease progression in non-alcoholic steatohepatitis (NASH) are needed. Our objective was to evaluate the utility of magnetic resonance (MR)-based hepatic imaging measures for the assessment of liver histology in patients with NASH.nnnMETHODSnWe analyzed data from patients with NASH and stage 2 or 3 fibrosis enrolled in a phase II study of selonsertib. Pre- and post-treatment assessments included centrally read MR elastography (MRE)-estimated liver stiffness, MR imaging-estimated proton density fat fraction (MRI-PDFF), and liver biopsies evaluated according to the NASH Clinical Research Network classification and the non-alcoholic fatty liver disease activity score (NAS).nnnRESULTSnAmong 54 patients with MRE and biopsies at baseline and week 24, 18 (33%) had fibrosis improvement (≥1-stage reduction) after undergoing 24u202fweeks of treatment with the study drug. The area under the receiver operating characteristic curve (AUROC) of MRE-stiffness to predict fibrosis improvement was 0.62 (95% CI 0.46-0.78) and the optimal threshold was a ≥0% relative reduction. At this threshold, MRE had 67% sensitivity, 64% specificity, 48% positive predictive value, 79% negative predictive value. Among 65 patients with MRI-PDFF and biopsies at baseline and week 24, a ≥1-grade reduction in steatosis was observed in 18 (28%). The AUROC of MRI-PDFF to predict steatosis response was 0.70 (95% CI 0.57-0.83) and the optimal threshold was a ≥0% relative reduction. At this threshold, MRI-PDFF had 89% sensitivity and 47% specificity, 39% positive predictive value, and 92% negative predictive value.nnnCONCLUSIONSnThese preliminary data support the further evaluation of MRE-stiffness and MRI-PDFF for the longitudinal assessment of histologic response in patients with NASH.nnnLAY SUMMARYnLiver biopsy is a potentially painful and risky method to assess damage to the liver due to non-alcoholic steatohepatitis (NASH). We analyzed data from a clinical trial to determine if 2 methods of magnetic resonance imaging - 1 to measure liver fat and 1 to measure liver fibrosis (scarring) - could potentially replace liver biopsy in evaluating NASH-related liver injury. Both imaging methods were correlated with biopsy in showing the effects of NASH on the liver.

DNA methylation signatures reflect aging in patients with nonalcoholic steatohepatitis

A DNA methylation (DNAm) signature (the Horvath clock) has been proposed as a measure of human chronological and biological age. We determined peripheral blood DNAm in patients with nonalcoholic steatohepatitis (NASH) and assessed whether accelerated aging occurs in these patients. DNAm signatures were obtained in patients with biopsy-proven NASH and stage 2-3 fibrosis. The DNAm profile from one test and two validation cohorts served as controls. Age acceleration was calculated as the difference between DNAm age and the predicted age based on the linear model derived from controls. Hepatic collagen content was assessed by quantitative morphometry. The Horvath clock accurately predicts the chronological age of the entire cohort. Age acceleration was observed among NASH subjects compared with control data sets and our test controls. Age acceleration in NASH subjects did not differ by fibrosis stage but correlated with hepatic collagen content. A set of 152 differentially methylated CpG islands between NASH subjects and controls identified gene set enrichment for transcription factors and developmental pathways. Patients with NASH exhibit epigenetic age acceleration that correlates with hepatic collagen content.