Search

Objectives: Neutrophils play an important role in regulating immune and inflammatory responses in rheumatoid arthritis (RA). We assessed whether baricitinib, a JAK1/JAK2 inhibitor, could reduce neutrophil activation, and whether a neutrophil activation score could predict treatment response., Methods: Markers of neutrophil activation, calprotectin, and neutrophil extracellular traps (NETs) were analyzed using ELISA in RA plasma (n=271) and healthy controls (n=39). For RA patients, neutrophil activation markers were measured at baseline, 12 weeks, and 24 weeks after treatment with placebo, 2 mg, and 4 mg baricitinib. Whole blood RNA analyses from multiple randomized baricitinib RA trials were performed to study neutrophil-related transcripts (n=1651)., Results: Baseline levels of plasma neutrophil markers were elevated in RA patients compared to healthy controls (p<0.001). Baricitinib reduced levels of soluble calprotectin at 12 and 24 weeks, especially in RA patients responding to treatment, as determined by ACR20. Whole blood RNA analysis revealed similar changes in the predominant neutrophil markers calprotectin and FcαRI upon treatment with baricitinib in three randomized clinical trials involving RA patients at various stages of disease modifying therapy. Clustering analysis of plasma activation markers showed elevated levels of calprotectin and NETs, e.g., a neutrophil activation score, at baseline, could predict treatment response to baricitinib. In contrast, CRP levels could not distinguish between responders and non-responders., Conclusions: Neutrophil activation markers may add clinical value in predicting treatment response to baricitinib and other drugs targeting RA. This study supports personalized medicine, in treating RA patients, not only based on symptoms, but also based on immunophenotyping., (This article is protected by copyright. All rights reserved.)

Motivation: On-target gene knockdown, using siRNA, ideally results from binding fully complementary regions in mRNA transcripts to induce direct cleavage. Off-target siRNA gene knockdown can occur through several modes, one being a seed-mediated mechanism mimicking miRNA gene regulation. Seed-mediated off-target effects occur when the ∼8 nucleotides at the 5' end of the guide strand, called a seed region, bind the 3' untranslated regions of mRNA, causing reduced translation. Experiments using siRNA knockdown paired with RNA-seq can be used to detect siRNA sequences with off-target effects driven by the seed region. However, there are limited computational tools designed specifically for detecting siRNA off-target effects mediated by the seed region in differential gene expression experiments., Results: SeedMatchR is an R package developed to provide users a single, unified resource for detecting and visualizing seed-mediated off-target effects of siRNA using RNA-seq experiments. SeedMatchR is designed to extend current differential expression analysis tools, such as DESeq2, by annotating results with predicted seed matches. Using publicly available data, we demonstrate the ability of SeedMatchR to detect cumulative changes in differential gene expression attributed to siRNA seed region activity., Availability: SeedMatchR is available on CRAN. Documentation and example workflows are available through the SeedMatchR GitHub page at https://github.com/tacazares/SeedMatchR., (© The Author(s) 2024. Published by Oxford University Press.)

Biologic drugs (therapeutic proteins or peptides) have become one of the most important therapeutic modalities over the past few decades. Drug-induced immunogenicity is a significant concern as it may affect safety, tolerability, and efficacy. With more sensitive and drug-tolerant screening assays in use today, reliable estimation of anti-drug-antibody (ADA) titer has become more important for understanding clinically relevant ADA levels. Titer is commonly defined as the dilution factor resulting in an assay signal equal to a pre-specified cut point factor. Given its influence on the resulting titer precision, the choice of a titer cut point factor warrants careful consideration. In this paper, we discuss the theoretical dilution model, investigate how titer variability depends on the cut point factor and propose a standardized cut point factor to increase precision of titer estimates. Additionally, we demonstrate that non-linear regression-based titer estimation provides both improved precision and implementation efficiency relative to commonly used estimation approaches., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Based on interim analyses and modeling data, lower doses of bamlanivimab and etesevimab together (700/1400 mg) were investigated to determine optimal dose and expand availability of treatment., Methods: This Phase 3 portion of the BLAZE-1 trial characterized the effect of bamlanivimab with etesevimab on overall patient clinical status and virologic outcomes in ambulatory patients ≥12 years old, with mild-to-moderate coronavirus disease 2019 (COVID-19), and ≥1 risk factor for progressing to severe COVID-19 and/or hospitalization. Bamlanivimab and etesevimab together (700/1400 mg) or placebo were infused intravenously within 3 days of patients' first positive COVID-19 test., Results: In total, 769 patients were infused (median age [range]; 56.0 years [12, 93], 30.3% of patients ≥65 years of age and median duration of symptoms; 4 days). By day 29, 4/511 patients (0.8%) in the antibody treatment group had a COVID-19-related hospitalization or any-cause death, as compared with 15/258 patients (5.8%) in the placebo group (Δ[95% confidence interval {CI}] = -5.0 [-8.0, -2.1], P < .001). No deaths occurred in the bamlanivimab and etesevimab group compared with 4 deaths (all COVID-19-related) in the placebo group. Patients receiving antibody treatment had a greater mean reduction in viral load from baseline to Day 7 (Δ[95% CI] = -0.99 [-1.33, -.66], P < .0001) compared with those receiving placebo. Persistently high viral load at Day 7 correlated with COVID-19-related hospitalization or any-cause death by Day 29 in all BLAZE-1 cohorts investigated., Conclusions: These data support the use of bamlanivimab and etesevimab (700/1400 mg) for ambulatory patients at high risk for severe COVID-19. Evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants will require continued monitoring to determine the applicability of this treatment., Clinical Trials Registration: NCT04427501., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

As the coronavirus disease 2019 (COVID-19) pandemic evolves and vaccine rollout progresses, the availability and demand for monoclonal antibodies for the prevention and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are also accelerating. This longitudinal serological study evaluated the magnitude and potency of the endogenous antibody response to COVID-19 vaccination in participants who first received a COVID-19 monoclonal antibody in a prevention study. Over the course of 6 months, serum samples were collected from a population of nursing home residents and staff enrolled in a clinical trial who were randomized to either bamlanivimab treatment or placebo. In an unplanned component of this trial, a subset of these participants was subsequently fully vaccinated with two doses of either SpikeVax (Moderna) or Comirnaty (BioNTech/Pfizer) COVID-19 mRNA vaccines. This post hoc analysis assessed the immune response to vaccination for 135 participants without prior SARS-CoV-2 infection. Antibody titers and potency were assessed using three assays against SARS-CoV-2 proteins that bamlanivimab does not efficiently bind to, thereby reflecting the endogenous antibody response. All bamlanivimab and placebo recipients mounted a robust immune response to full COVID-19 vaccination, irrespective of age, risk category, and vaccine type with any observed differences of uncertain clinical importance. These findings are pertinent for informing public health policy with results that suggest that the benefit of receiving COVID-19 vaccination at the earliest opportunity outweighs the minimal effect on the endogenous immune response due to prior prophylactic COVID-19 monoclonal antibody infusion.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants., Competing Interests: Declaration of interests Eli Lilly and Company provided resources for this study. AbCellera Biologics Inc. received funding from the U.S. Department of Defense, Defense Advanced Research Projects Agency (DARPA) Pandemic Prevention Platform, agreement D18AC00002. This research was funded in part by the U.S. Government (the views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the U.S. Government). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357 (https://www.aps.anl.gov/Science/Publications/Acknowledgment-Statement-for-Publications). Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly and Company, which operates the facility (http://lrlcat.lilly.com/). This work was supported by the Intramural Program at the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Vaccine Research Center (to B.S.G. and J.R.M.). Operations support of the Galveston National Laboratory was supported by NIAID/NIH grant UC7AI094660. D.F., P.V., A.P., J.H., J.M.S., R.W.S., J.C., I. H., J.J.F., S.H., H.C.P., B.R., B.A.H., R.W.S., J.C., J.M.S., R.E.H., N.K., and B.E.J. are employees and/or stockholders of Eli Lilly and Company. K.W., S.Ž., M.W., E.L., L.K., Y.H., K.J., R.G., M.A.S., D.W.C., D.P., P.X., V.d.P., R.v.d.L., M.R., L.D., C.P., I.L., L.A., P.S., T.L.F., C.L.H., E.F., and B.C.B. are employees and stockholders of AbCellera Biologics Inc. AbCellera Biologics Inc. and the National Institutes of Health have filed patent applications related to the work described herein (US patent application 17/192243 and international patent application PCT/US21/20843, both titled “Anti-Coronavirus Antibodies and Methods of Use”)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: A thorough understanding of a patient's inflammatory response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is crucial to discerning the associated, underlying immunological processes and to the selection and implementation of treatment strategies. Defining peripheral blood biomarkers relevant to SARS-CoV-2 infection is fundamental to detecting and monitoring this systemic disease. This safety-focused study aims to monitor and characterize the immune response to SARS-CoV-2 infection via analysis of peripheral blood and nasopharyngeal swab samples obtained from patients hospitalized with Coronavirus disease 2019 (COVID-19), in the presence or absence of bamlanivimab treatment., Methods: 23 patients hospitalized with COVID-19 were randomized to receive a single dose of the neutralizing monoclonal antibody, bamlanivimab (700 mg, 2800 mg or 7000 mg) or placebo, at study initiation (Clinical Trial; NCT04411628). Serum samples and nasopharyngeal swabs were collected at multiple time points over 1 month. A Proximity Extension Array was used to detect inflammatory profiles from protein biomarkers in the serum of hospitalized COVID-19 patients relative to age/sex-matched healthy controls. RNA sequencing was performed on nasopharyngeal swabs. A Luminex serology assay and Elecsys® Anti-SARS-CoV-2 immunoassay were used to detect endogenous antibody formation and to monitor seroconversion in each cohort over time. A mixed model for repeated measures approach was used to analyze changes in serology and serum proteins over time., Results: Levels of IL-6, CXCL10, CXCL11, IFNγ and MCP-3 were > fourfold higher in the serum of patients with COVID-19 versus healthy controls and linked with observations of inflammatory and viral-induced interferon response genes detected in nasopharyngeal swab samples from the same patients. While IgA and IgM titers peaked around 7 days post-dose, IgG titers remained high, even after 28 days. Changes in biomarkers over time were not significantly different between the bamlanivimab and placebo groups., Conclusions: Similarities observed between nasopharyngeal gene expression patterns and peripheral blood biomarker profiles reveal a connection between the circulation and processes in the nasopharyngeal cavity, reinforcing the potential utility of systemic blood biomarker profiling for therapeutic monitoring of patient response. Serological antibody responses in patients correlated closely with reductions in the COVID-19 inflammatory protein biomarker signature. Bamlanivimab did not affect the biomarker dynamics in this hospitalized patient population., (© 2022. The Author(s).)

Background: Neutralizing monoclonal antibodies (mAbs) to SARS-CoV-2 are clinically efficacious when administered early, decreasing hospitalization and mortality in patients with mild or moderate COVID-19. We investigated the effects of receiving mAbs (bamlanivimab alone and bamlanivimab and etesevimab together) after SARS-CoV-2 infection on the endogenous immune response., Methods: Longitudinal serum samples were collected from patients with mild or moderate COVID-19 in the BLAZE-1 trial who received placebo (n=153), bamlanivimab alone [700 mg (n=100), 2800 mg (n=106), or 7000 mg (n=98)], or bamlanivimab (2800 mg) and etesevimab (2800 mg) together (n=111). A multiplex Luminex serology assay measured antibody titers against SARS-CoV-2 antigens, including SARS-CoV-2 protein variants that evade bamlanivimab or etesevimab binding, and SARS-CoV-2 pseudovirus neutralization assays were performed., Results: The antibody response in patients who received placebo or mAbs had a broad specificity. Titer change from baseline against a receptor-binding domain mutant (Spike-RBD E484Q), as well as N-terminal domain (Spike-NTD) and nucleocapsid protein (NCP) epitopes were 1.4 to 4.1 fold lower at day 15-85 in mAb recipients compared with placebo. Neutralizing activity of day 29 sera from bamlanivimab monotherapy cohorts against both spike E484Q and beta variant (B.1.351) were slightly reduced compared with placebo (by a factor of 3.1, p=0.001, and 2.9, p=0.002, respectively). Early viral load correlated with the subsequent antibody titers of the native, unmodified humoral response (p<0.0001 at Day 15, 29, 60 and 85 for full-length spike)., Conclusions: Patients with mild or moderate COVID-19 treated with mAbs develop a wide breadth of antigenic responses to SARS-CoV-2. Small reductions in titers and neutralizing activity, potentially due to a decrease in viral load following mAb treatment, suggest minimal impact of mAb treatment on the endogenous immune response., Competing Interests: Authors LZ, JP, SB, MDa, TNVT, MC, JF, PV, DP, AN, NK, RH, and RB are all employees and shareholders of Eli Lilly and Company. MDo reports receiving grants from Eli Lilly and Company, and Novartis; and receiving consulting fees from Moderna, Tillotts, SQZ, AzurRx, Partner Therapeutics, and ORIC Pharmaceuticals; and has Neoleukin Therapeutics stock. PC reports receiving consulting fees from Eli Lilly and Company and Gilead; and receiving payment or honoraria from Rockpointe, Frontier Collaborative, CME Outfitter, and Physician Education Resource. RG reports receiving consulting fees from Eli Lilly and Company, GSK Pharmaceuticals, and Gilead Sciences; and receiving payment or honoraria from Gilead Sciences. GH reports receiving grants from Eli Lilly and company, Gilead, Viiv, Janssen, and Proteus; receiving consulting fees from Gilead, Viiv, and Janssen; and receiving payment or honoraria from Rockpointe, CME Outfitter, Simply Speaking, and Clinical Care Options. The authors declare that this study received funding from Eli Lilly and Company. The funder had the following involvement with the study: study design, data analysis, decision to publish, and preparation of the manuscript., (Copyright © 2021 Zhang, Poorbaugh, Dougan, Chen, Gottlieb, Huhn, Beasley, Daniels, Ngoc Vy Trinh, Crisp, Freitas, Vaillancourt, Patel, Nirula, Kallewaard, Higgs and Benschop.)

Objectives: Heterogeneity of SLE patients in clinical trials remains a challenge for developing new therapies. This study used a combinatorial analysis of four molecular biomarkers to define key sources of heterogeneity., Methods: Combinations of IFN (high/low), anti-dsDNA (+/-) and C3 and C4 (low/normal) were used to subset n = 1747 patients from two randomized phase III trials. A dichotomous classification scheme defined SLE (+) as: IFN (high), anti-dsDNA (+), C3 (low) and/or C4 (low). SLE (-) required all of the following: IFN (low), anti-dsDNA (-), C3 (normal) and C4 (normal). Additional analyses subset the data further by IFN, anti-dsDNA and complement., Results: The trials enrolled n = 2262 patients of which n = 1747 patients had data for IFN, anti-dsDNA, C3 and C4 at baseline. There were n = 247 patients in the SLE (-) population and n = 1500 patients in the SLE (+) population. The SLE (-) population had more mucocutaneous and musculoskeletal disease at baseline, while SLE (+) had more haematological, renal and vascular involvement. There was lower concomitant medication use in the SLE (-) population for corticosteroids and immunosuppressants, except for MTX. Time to severe flare was significantly longer in SLE (-) vs SLE (+) (P < 0.0001) and SRI-4 response rate was significantly lower in SLE (-) vs SLE (+) (P = 0.00016). The USA had more SLE (-) patients (22%) than Mexico/Central America/South America (10%), Europe (7%) and the rest of the world (5%)., Conclusion: Combinatorial analysis of four molecular biomarkers revealed subsets of SLE patients that discriminated by disease manifestations, concomitant medication use, geography, time to severe flare and SRI-4 response. These data may be useful for designing clinical trials and identifying subsets of patients for analysis. Rheumatology key messages SLE patients from a P3 trial were categorized by IFN, anti-dsDNA, C3 and C4 status. Patients lacking molecular markers of SLE distinguished from biomarker positive patients on multiple clinical parameters. Biomarker negative patients have distinct disease characteristics that may impact clinical trial outcomes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Atopic dermatitis (AD) is a heterogeneous systemic inflammatory skin disease associated with dysregulated immune responses, barrier dysfunction and activated sensory nerves. To characterize circulating inflammatory profiles and underlying systemic disease heterogeneity within AD patients, blood samples from adult patients (N = 123) with moderate-to-severe AD in a phase 2 study of baricitinib (JAHG) were analysed. Baseline levels of 131 markers were evaluated using high-throughput and ultrasensitive proteomic platforms, patient clusters were generated based on these peripheral markers. We implemented a novel cluster reproducibility method to validate cluster outcomes within our study and used publicly available AD biomarker data set (73 markers, N = 58 patients) to validate our findings. Cluster reproducibility analysis demonstrated best consistency for 2 clusters by k-means, reproducibility of this clustering outcome was validated in an independent patient cohort. These unique JAHG patient subgroups either possessed elevated pro-inflammatory mediators, notably TNFβ, MCP-3 and IL-13, among a variety of immune responses (high inflammatory) or lower levels of inflammatory biomarkers (low inflammatory). The high inflammatory subgroup was associated with greater baseline disease severity, demonstrated by greater EASI, SCORAD Index, Itch NRS and DLQI scores, compared with low inflammatory subgroup. African-American patients were predominantly associated with the high inflammatory subgroup and increased baseline disease severity. In patients with moderate-to-severe AD, heterogeneity was identified by the detection of 2 disease subgroups, differential clustering amongst ethnic groups and elevated pro-inflammatory mediators extending beyond traditional polarized immune responses. Therapeutic strategies targeting multiple pro-inflammatory cytokines may be needed to address this heterogeneity., (© 2021 Eli Lilly & Company. Experimental Dermatology published by John Wiley & Sons Ltd.)

Background: Patients with underlying medical conditions are at increased risk for severe coronavirus disease 2019 (Covid-19). Whereas vaccine-derived immunity develops over time, neutralizing monoclonal-antibody treatment provides immediate, passive immunity and may limit disease progression and complications., Methods: In this phase 3 trial, we randomly assigned, in a 1:1 ratio, a cohort of ambulatory patients with mild or moderate Covid-19 who were at high risk for progression to severe disease to receive a single intravenous infusion of either a neutralizing monoclonal-antibody combination agent (2800 mg of bamlanivimab and 2800 mg of etesevimab, administered together) or placebo within 3 days after a laboratory diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The primary outcome was the overall clinical status of the patients, defined as Covid-19-related hospitalization or death from any cause by day 29., Results: A total of 1035 patients underwent randomization and received an infusion of bamlanivimab-etesevimab or placebo. The mean (±SD) age of the patients was 53.8±16.8 years, and 52.0% were adolescent girls or women. By day 29, a total of 11 of 518 patients (2.1%) in the bamlanivimab-etesevimab group had a Covid-19-related hospitalization or death from any cause, as compared with 36 of 517 patients (7.0%) in the placebo group (absolute risk difference, -4.8 percentage points; 95% confidence interval [CI], -7.4 to -2.3; relative risk difference, 70%; P<0.001). No deaths occurred in the bamlanivimab-etesevimab group; in the placebo group, 10 deaths occurred, 9 of which were designated by the trial investigators as Covid-19-related. At day 7, a greater reduction from baseline in the log viral load was observed among patients who received bamlanivimab plus etesevimab than among those who received placebo (difference from placebo in the change from baseline, -1.20; 95% CI, -1.46 to -0.94; P<0.001)., Conclusions: Among high-risk ambulatory patients, bamlanivimab plus etesevimab led to a lower incidence of Covid-19-related hospitalization and death than did placebo and accelerated the decline in the SARS-CoV-2 viral load. (Funded by Eli Lilly; BLAZE-1 ClinicalTrials.gov number, NCT04427501.)., (Copyright © 2021 Massachusetts Medical Society.)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour -1 kg -1 , consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Importance: Coronavirus disease 2019 (COVID-19) continues to spread rapidly worldwide. Neutralizing antibodies are a potential treatment for COVID-19., Objective: To determine the effect of bamlanivimab monotherapy and combination therapy with bamlanivimab and etesevimab on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in mild to moderate COVID-19., Design, Setting, and Participants: The BLAZE-1 study is a randomized phase 2/3 trial at 49 US centers including ambulatory patients (N = 613) who tested positive for SARS-CoV-2 infection and had 1 or more mild to moderate symptoms. Patients who received bamlanivimab monotherapy or placebo were enrolled first (June 17-August 21, 2020) followed by patients who received bamlanivimab and etesevimab or placebo (August 22-September 3). These are the final analyses and represent findings through October 6, 2020., Interventions: Patients were randomized to receive a single infusion of bamlanivimab (700 mg [n = 101], 2800 mg [n = 107], or 7000 mg [n = 101]), the combination treatment (2800 mg of bamlanivimab and 2800 mg of etesevimab [n = 112]), or placebo (n = 156)., Main Outcomes and Measures: The primary end point was change in SARS-CoV-2 log viral load at day 11 (±4 days). Nine prespecified secondary outcome measures were evaluated with comparisons between each treatment group and placebo, and included 3 other measures of viral load, 5 on symptoms, and 1 measure of clinical outcome (the proportion of patients with a COVID-19-related hospitalization, an emergency department [ED] visit, or death at day 29)., Results: Among the 577 patients who were randomized and received an infusion (mean age, 44.7 [SD, 15.7] years; 315 [54.6%] women), 533 (92.4%) completed the efficacy evaluation period (day 29). The change in log viral load from baseline at day 11 was -3.72 for 700 mg, -4.08 for 2800 mg, -3.49 for 7000 mg, -4.37 for combination treatment, and -3.80 for placebo. Compared with placebo, the differences in the change in log viral load at day 11 were 0.09 (95% CI, -0.35 to 0.52; P = .69) for 700 mg, -0.27 (95% CI, -0.71 to 0.16; P = .21) for 2800 mg, 0.31 (95% CI, -0.13 to 0.76; P = .16) for 7000 mg, and -0.57 (95% CI, -1.00 to -0.14; P = .01) for combination treatment. Among the secondary outcome measures, differences between each treatment group vs the placebo group were statistically significant for 10 of 84 end points. The proportion of patients with COVID-19-related hospitalizations or ED visits was 5.8% (9 events) for placebo, 1.0% (1 event) for 700 mg, 1.9% (2 events) for 2800 mg, 2.0% (2 events) for 7000 mg, and 0.9% (1 event) for combination treatment. Immediate hypersensitivity reactions were reported in 9 patients (6 bamlanivimab, 2 combination treatment, and 1 placebo). No deaths occurred during the study treatment., Conclusions and Relevance: Among nonhospitalized patients with mild to moderate COVID-19 illness, treatment with bamlanivimab and etesevimab, compared with placebo, was associated with a statistically significant reduction in SARS-CoV-2 viral load at day 11; no significant difference in viral load reduction was observed for bamlanivimab monotherapy. Further ongoing clinical trials will focus on assessing the clinical benefit of antispike neutralizing antibodies in patients with COVID-19 as a primary end point., Trial Registration: ClinicalTrials.gov Identifier: NCT04427501.

Background: Physicians treating patients with coronavirus disease 2019 (COVID-19) increasingly believe that the hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating biomarkers seen across the spectrum of COVID-19 have not been characterized compared with healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms., Objective: To identify and characterize the host inflammatory response to severe acute respiratory syndrome coronavirus 2 infection, we assessed levels of proteins related to immune responses and cardiovascular disease in patients stratified as mild, moderate, and severe versus matched healthy controls., Methods: Blood samples from adult patients hospitalized with COVID-19 were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of 185 markers., Results: Results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within patients with COVID-19, and strong biomarker association with patient response as measured by Ordinal Scale. As patients progress, we observe statistically significant dysregulation of IFN-γ, IL-1RA, IL-6, IL-10, IL-19, monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, CXCL9, CXCL10, CXCL5, ENRAGE, and poly (ADP-ribose) polymerase 1. Furthermore, in a limited series of patients who were sampled frequently, confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm., Conclusions: These wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. They also provide insights into mechanisms of severe acute respiratory syndrome coronavirus 2 pathogenesis and the host response., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To characterise the molecular pathways impacted by the pharmacologic effects of the Janus kinase (JAK) 1 and JAK2 inhibitor baricitinib in SLE., Methods: In a phase II, 24-week, randomised, placebo-controlled, double-blind study (JAHH), RNA was isolated from whole blood in 274 patients and analysed using Affymetrix HTA2.0 array. Serum cytokines were measured using ultrasensitive quantitative assays., Results: Gene expression profiling demonstrated an elevation of STAT1 , STAT2 and multiple interferon (IFN) responsive genes at baseline in patients with SLE. Statistical and gene network analyses demonstrated that baricitinib treatment reduced the mRNA expression of functionally interconnected genes involved in SLE including STAT1 -target, STAT2 -target and STAT4- target genes and multiple IFN responsive genes. At baseline, serum cytokines IFN-α, IFN-γ, interleukin (IL)-12p40 and IL-6 were measurable and elevated above healthy controls. Treatment with baricitinib significantly decreased serum IL-12p40 and IL-6 cytokine levels at week 12, which persisted through week 24., Conclusion: Baricitinib treatment induced significant reduction in the RNA expression of a network of genes associated with the JAK/STAT pathway, cytokine signalling and SLE pathogenesis. Baricitinib consistently reduced serum levels of two key cytokines implicated in SLE pathogenesis, IL-12p40 and IL-6., Competing Interests: Competing interests: TD has received grant support from Chugai, Janssen, Novartis and Sanofi. He has received consultancy support from AbbVie, Celgene, Eli Lilly and Company, Janssen, Novartis, Roche, Samsung and UCB, and speaker bureau fees from Eli Lilly and Company and Roche. YT has received grant support from AbbVie, Astellas, BMS, Chugai, Daiichi-Sankyo, Eisai, Mitsubishi-Tanabe, MSD, Ono, Taisho-Toyama and Takeda. He has received speaker bureau fees from AbbVie, Asahi-kasei, Astellas, BMS, Chugai, Daiichi-Sankyo, Eisai, Eli Lilly and Company, GSK, Janssen, Mitsubishi-Tanabe, Novartis, Pfizer, Sanofi, Takeda, UCB and YL Biologics. MP has received consultancy support from Eli Lilly and Company. JS has received grant support from AbbVie, Eli Lilly and Company, Janssen, MSD, Novartis, Pfizer and Roche. He has received consultancy support from AbbVie, Amgen, Astra-Zeneca, Astro, BMS, Celgene, Celltrion, Chugai, Eli Lilly and Company, Gilead, ILTOO, Janssen, Medimmune, MSD, Novartis, Pfizer, Roche, Samsung, Sanofi and UCB. DJW has received consulting support from Amgen, Eli Lilly and Company, EMD Merck Serono and Pfizer. ERD, REH, GR, BC, RJB, NLB, MES, SdB and RWH are employees and stockholders of Eli Lilly and Company., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID-19 infection via proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID-19 patients., (© 2020 Eli Lilly and Company Published under the terms of the CC BY 4.0 license.)

Systemic lupus erythematosus (SLE) is a chronic, remitting, and relapsing, inflammatory disease involving multiple organs, which exhibits abnormalities of both the innate and adaptive immune responses. A limited number of transcriptomic studies have characterized the gene pathways involved in SLE in an attempt to identify the key pathogenic drivers of the disease. In order to further advance our understanding of the pathogenesis of SLE, we used a novel Bayesian network algorithm to hybridize knowledge- and data-driven methods, and then applied the algorithm to build an SLE gene network using transcriptomic data from 1,760 SLE patients' RNA from the two tabalumab Phase III trials (ILLUMINATE-I & -II), the largest SLE RNA dataset to date. Further, based on the gene network, we carried out hub- and key driver-gene analyses for gene prioritization. Our analyses identified that the JAK-STAT pathway genes, including JAK2, STAT1, and STAT2, played essential roles in SLE pathogenesis, and reaffirmed the recent discovery of pathogenic relevance of JAK-STAT signaling in SLE. Additionally, we showed that other genes, such as IRF1, IRF7, PDIA4, FAM72C, TNFSF10, DHX58, SIGLEC1, and PML, may be also important in SLE and serve as potential therapeutic targets for SLE. In summary, using a hybridized network construction approach, we systematically investigated gene-gene interactions based on their transcriptomic profiles, prioritized genes based on their importance in the network structure, and revealed new insights into SLE activity., Competing Interests: Thomas Dörner has received grant support from Chugai, Janssen, Novartis, and Sanofi. He has received consultancy support from AbbVie, Celgene, Eli Lilly and Company, Janssen, Novartis, Roche, Samsung, and UCB, and speaker bureau fees from Eli Lilly and Company and Roche. Michelle Petri has received consultancy support from Eli Lilly and Company. Daniel Wallace has received consulting support from Amgen, Eli Lilly and Company, EMD Merck Serono, and Pfizer. Yupeng Li, Richard Higgs, Robert Hoffman, Ernst Dow, Xiong Liu, Brian Eastwood, Bradley Miller and Yushi Liu are employees and stockholders of Eli Lilly and Company. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level., Methods: Peripheral blood mononuclear cells from healthy donors were incubated with different JAKis, levels of phosphorylated signal transducer and activator of transcription (pSTAT) were measured following cytokine stimulation, and half maximum inhibitory concentration (IC 50 ) values were calculated in phenotypically gated leukocyte subpopulations. Therapeutic dose relevance of the in vitro analysis was assessed using calculated mean concentration-time profiles over 24 h obtained from JAKi-treated subjects. Time above IC 50 and average daily percent inhibition of pSTAT formation were calculated for each JAKi, cytokine, and cell type., Results: Distinct JAKis displayed different in vitro pharmacologic profiles. For example, tofacitinib and upadacitinib were the most potent inhibitors of the JAK1/3-dependent cytokines tested (interleukin [IL]-2, IL-4, IL-15, and IL-21) with lower IC 50 values and increased time above IC 50 translating to a greater overall inhibition of STAT signaling during the dosing interval. All JAKis tested inhibited JAK1/2-dependent cytokines (e.g., IL-6 and interferon [IFN]-γ), the JAK1/tyrosine kinase 2 (TYK2)-dependent cytokines IL-10 and IFN-α, the JAK2/2-dependent cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the JAK2/TYK2-dependent cytokine granulocyte colony-stimulating factor (G-CSF), but often to significantly differing degrees., Conclusions: Different JAKis modulated distinct cytokine pathways to varying degrees, and no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Notably, baricitinib inhibited JAK1/3 signaling to a lesser extent than upadacitinib and tofacitinib, while upadacitinib, baricitinib, and tofacitinib inhibited the signaling of JAK2/2-dependent cytokines, including GM-CSF and IL-3, as well as the signaling of the JAK2/TYK2-dependent cytokine G-CSF.

Despite recent advances in the development of tools to predict immunogenicity risk of biotherapeutic molecules, the ability of a protein to elicit the formation of anti-drug antibodies (ADA) remains one of the most common causes for termination of clinical development programs. In this study, we use ADA assays to detect and measure pre-existing reactivity or the ability of a molecule to produce an ADA-like response in serum from treatment-naïve, healthy donors. We report herein that the magnitude of pre-existing reactivity evaluated pre-clinically and expressed as the 90th percentile of Tier 2 inhibition correlates with the subsequent rate of ADA emergence in the clinic. Furthermore, a multi-domain biotherapeutic (IgG-scFv bispecific antibody) showed the highest pre-existing reactivity and incidence of treatment-emergent ADA (TE-ADA) (57% and 93%, respectively). Using the components of the multidomain molecule in the Tier 2 step of the ADA assay, we were able to identify the scFv as the target of the serum pre-existing reactivity. Most importantly, the domain specificity of pre-existing ADA was the same as that of the TE-ADA from patients treated with the molecule. Based on these data, we propose the evaluation of the magnitude and of the domain specificity of pre-existing reactivity as a powerful tool to understand the immunogenic potential of novel biotherapeutics.

Because development of reliable biomarkers in psoriasis and atopic dermatitis has lagged behind therapeutic progress, we created a blood-based test to fill the void in objective methods available for dermatological assessments. Our novel interleukin-19 (IL-19) immunoassay was initially tested to determine concentrations of IL-19 serum levels, then correlated with the psoriasis activity and severity index (PASI) in psoriasis, and the eczema area and severity index (EASI) in atopic dermatitis. Not only was IL-19 increased in psoriasis and correlated to PASI, but ixekizumab administration led to rapid, sustained IL-19 decreases to normal levels, with decreases at 2-weeks correlating with PASI improvement at 16-weeks. IL-19 increased upon ixekizumab withdraw, prior to relapse, and decreased following re-treatment. In baricitinib- and etanercept-treated psoriasis patients, IL-19 decreases also correlated with improvement. Many patients with limited skin disease, including genital psoriasis and psoriatic arthritis patients, also had increased IL-19, which was reduced to normal levels upon ixekizumab treatment, correlating with PASI improvement. We also measured IL-19 in baricitinib-treated atopic dermatitis patients. In atopic dermatitis, IL-19 was significantly elevated, correlated with EASI scores, and decreased with skin improvement. Therefore, measurement of serum IL-19 provides clinicians with an objective disease-activity assessment tool for psoriasis and atopic dermatitis patients.

Objective: To characterize baseline gene expression and pharmacodynamically induced changes in whole blood gene expression in 1,760 systemic lupus erythematosus (SLE) patients from 2 phase III, 52-week, randomized, placebo-controlled, double-blind studies in which patients were treated with the BAFF-blocking IgG4 monoclonal antibody tabalumab., Methods: Patient samples were obtained from SLE patients from the ILLUMINATE-1 and ILLUMINATE-2 studies, and control samples were obtained from healthy donors. Blood was collected in Tempus tubes at baseline, week 16, and week 52. RNA was analyzed using Affymetrix Human Transcriptome Array 2.0 and NanoString., Results: At baseline, expression of the interferon (IFN) response gene was elevated in patients compared with controls, with 75% of patients being positive for this IFN response gene signature. There was, however, substantial heterogeneity of IFN response gene expression and complex relationships among gene networks. The IFN response gene signature was a predictor of time to disease flare, independent of anti-double-stranded DNA (anti-dsDNA) antibody and C3 and C4 levels, and overall disease activity. Pharmacodynamically induced changes in gene expression following tabalumab treatment were extensive, occurring predominantly in B cell-related and immunoglobulin genes, and were consistent with other pharmacodynamic changes including anti-dsDNA antibody, C3, and immunoglobulin levels., Conclusion: SLE patients demonstrated increased expression of an IFN response gene signature (75% of patients had an elevated IFN response gene signature) at baseline in ILLUMINATE-1 and ILLUMINATE-2. Substantial heterogeneity of gene expression was detected among individual patients and in gene networks. The IFN response gene signature was an independent risk factor for future disease flares. Pharmacodynamic changes in gene expression were consistent with the mechanism of BAFF blockade by tabalumab., (© 2016, American College of Rheumatology.)

Background: Conventional antidepressants lack efficacy for many patients (treatmentresistant depression or TRD) and generally take weeks to produce full therapeutic response in others. Emerging data has identified certain drugs such as ketamine as rapidly-acting antidepressants for major depressive disorder and TRD. Scopolamine, a drug used to treat motion sickness and nausea, has also been demonstrated to function as a rapidly-acting antidepressant. The mechanisms associated with efficacy in TRD patients and rapid onset of action have been suggested to involve a-Amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mammalian target of rapamycin (mTOR) signaling. Since the work on these mechanisms with scopolamine has been limited, the present set of experiments was designed to further explore these mechanisms of action., Method: Male, NIH Swiss mice demonstrated a robust and immediate antidepressant signature with ketamine or scopolamine when studied under the forced-swim test., Results: The AMPA receptor antagonist NBQX prevented this antidepressant-like effect of scopolamine and ketamine. An orally-bioavilable mTOR inhibitor (AZD8055) also attenuated the antidepressant- like effects of scopolamine and ketamine. Scopolamine was also shown to augment the antidepressant- like effect of the selective serotonin reuptake inhibitor citalopram. When given in combination, scopolamine and ketamine acted synergistically to produce antidepressant-like effects. Although drug interaction data suggested that additional mechanisms might be at play, metabolomic analysis of frontal cortex and plasma from muscarinic M1+/+ and M1 -/- mice given scopolamine or vehicle did not reveal any hints as to the nature of these additional mechanisms of action., Conclusion: Overall, the data substantiate and extend the idea that AMPA and mTOR signaling pathways are necessary for the antidepressant-like effects of scopolamine and ketamine, mechanisms that appear to be of general significance for TRD therapeutic agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Aim: Immunogenicity testing of biotherapeutic drugs is a regulatory requirement. Herein, we describe a drug-tolerant assay for detecting neutralizing antibodies against a therapeutic antibody., Results: Excess target of the therapeutic antibody was incorporated into the detection step of an affinity capture elution assay. Signal generated from binding of antidrug antibody (ADA) to the therapeutic antibody was compared with signal from binding of ADA to the therapeutic antibody preincubated with its target. The results demonstrated that the target blocked binding of the therapeutic antibody to neutralizing monkey ADA and to two anti-idiotypic antibodies., Conclusion: This highly drug-tolerant novel approach enables the detection of neutralizing antibodies and allows for one basic assay format to achieve complete characterization of ADA responses.

Novel skeletal muscle (SKM) injury biomarkers that have recently been identified may outperform or add value to the conventional SKM injury biomarkers aspartate transaminase (AST) and creatine kinase (CK). The relative performance of these novel biomarkers of SKM injury including skeletal troponin I (sTnI), myosin light chain 3 (Myl3), CK M Isoform (Ckm), and fatty acid binding protein 3 (Fabp3) was assessed in 34 rat studies including both SKM toxicants and compounds with toxicities in tissues other than SKM. sTnI, Myl3, Ckm, and Fabp3 all outperformed CK or AST and/or added value for the diagnosis of drug-induced SKM injury (ie, myocyte degeneration/necrosis). In addition, when used in conjunction with CK and AST, sTnI, Myl3, CKm, and Fabp3 individually and collectively improved diagnostic sensitivity and specificity, as well as diagnostic certainty, for SKM injury and responded in a sensitive manner to low levels of SKM degeneration/necrosis in rats. These findings support the proposal that sTnI, Myl3, Ckm, and Fabp3 are suitable for voluntary use, in conjunction with CK and AST, in regulatory safety studies in rats to monitor drug-induced SKM injury and the potential translational use of these exploratory biomarkers in early clinical trials to ensure patient safety., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Scopolamine produces rapid and significant symptom improvement in patients with depression, and most notably in patients who do not respond to current antidepressant treatments. Scopolamine is a nonselective muscarinic acetylcholine receptor antagonist, and it is not known which one or more of the five receptor subtypes in the muscarinic family are mediating these therapeutic effects. We used the mouse forced-swim test, an antidepressant detecting assay, in wild-type and transgenic mice in which each muscarinic receptor subtype had been genetically deleted to define the relevant receptor subtypes. Only the M1 and M2 knockout (KO) mice had a blunted response to scopolamine in the forced-swim assay. In contrast, the effects of the tricyclic antidepressant imipramine were not significantly altered by gene deletion of any of the five muscarinic receptors. The muscarinic antagonists biperiden, pirenzepine, and VU0255035 (N-[3-oxo-3-[4-(4-pyridinyl)-1-piper azinyl]propyl]-2,1,3-benzothiadiazole-4-sulfonamide) with selectivity for M1 over M2 receptors also demonstrated activity in the forced-swim test, which was attenuated in M1 but not M2 receptor KO mice. An antagonist with selectivity of M2 over M1 receptors (SCH226206 [(2-amino-3-methyl-phenyl)-[4-[4-[[4-(3 chlorophenyl)sulfonylphenyl]methyl]-1-piperidyl]-1-piperidyl]methanone]) was also active in the forced-swim assay, and the effects were deleted in M2 (-/-) mice. Brain exposure and locomotor activity in the KO mice demonstrated that these behavioral effects of scopolamine are pharmacodynamic in nature. These data establish muscarinic M1 and M2 receptors as sufficient to generate behavioral effects consistent with an antidepressant phenotype and therefore as potential targets in the antidepressant effects of scopolamine., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.

Apelin is emerging as an important hormone regulator of cardiovascular homoeostasis and an important biomarker for heart failure. Apelin concentrations have historically been measured by immunoassays; however, reported apelin concentrations measured in healthy volunteers show a large disparity from a few picograms per milliliter (pg/ml) to several nanograms per milliliter (ng/ml). Apelin exists in several isoforms ranging in size from 12 to 36 residues, and immunoassays generally cannot distinguish the specific forms present. In this study, an optimized method for enriching apelin peptides with cation-exchange beads followed with mass spectrometry analysis is presented. Apelin peptides are labile in plasma at physiological conditions; however, by lowering the plasma pH to 4.5, the recovery of apelin peptides can be increased significantly. Through optimizing the cation-exchange extraction process, we improved the lower limit of detection for most of the apelin peptides monitored to a few pg/ml. Using the improved method, we detected pyroglutamyl apelin-13 [(pyr)apelin-13] as the major apelin isoform present in plasma from several healthy volunteers at concentrations ranging from 7.7 to 23.3pg/ml., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recent improvements in the mass accuracy and resolution of mass spectrometers have led to renewed interest in label-free quantification using data from the primary mass spectrum (MS1) acquired from data-dependent proteomics experiments. The capacity for higher specificity quantification of peptides from samples enriched for proteins of biological interest offers distinct advantages for hypothesis generating experiments relative to immunoassay detection methods or prespecified peptide ions measured by multiple reaction monitoring (MRM) approaches. Here we describe an evaluation of different methods to post-process peptide level quantification information to support protein level inference. We characterize the methods by examining their ability to recover a known dilution of a standard protein in background matrices of varying complexity. Additionally, the MS1 quantification results are compared to a standard, targeted, MRM approach on the same samples under equivalent instrument conditions. We show the existence of multiple peptides with MS1 quantification sensitivity similar to the best MRM peptides for each of the background matrices studied. Based on these results we provide recommendations on preferred approaches to leveraging quantitative measurements of multiple peptides to improve protein level inference.

The hormone ghrelin is a unique signaling peptide with powerful metabolic effects, mediated by its acylated forms. The acyl modification of ghrelin is unique in that it takes place via a susceptible ester linkage in the conserved serine-3 of ghrelin and is composed principally of octanoyl and, to lesser extent, decanoyl fatty acids. The nature of this ester linkage makes it susceptible to esterases, which convert it to its des-acyl forms, and, if not adequately inhibited, the conversion to des-acyl ghrelin, particularly post sample collection, can lead to artifactual and misleading results. Here, we describe sample processing and mass spectrometric methodologies for the accurate and simultaneous quantification of acylated and des-acylated forms of ghrelin. We exploited these methodologies (1) to characterize circulating and tissue-specific forms of acyl and des-acyl ghrelin, (2) to optimize a cell system for acyl ghrelin production and search for the enzyme responsible for ghrelin's acylation, and (3) to demonstrate that GOAT is ghrelin's O-acyl transferase., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cuprizone intoxication is a commonly used model of demyelination that allows the temporal separation of demyelination and remyelination. The underlying biochemical alterations leading to demyelination, using this model, remain unclear and may be multifold. Analysis of proteomic changes within the brains of cuprizone-exposed animals may help elucidate key cellular processes. In the current study, we report the results of the liquid chromatography tandem mass spectrometry analysis of total protein from the brain hemispheres of control and toxin-exposed mice at 6 weeks of exposure and after 3 and 6 weeks of recovery to identify protein changes during the remyelination phase. We found that at 6 weeks of cuprizone exposure, myelin proteins were reduced compared to controls and increased throughout the course of recovery, as expected. In contrast, other protein groups, such as proteins related to mitochondrial function, were increased at 6 weeks of treatment compared to untreated controls and returned toward control levels following withdrawal of toxin. These results suggest that a global proteomic analysis of the brain tissue of cuprizone-treated mice can identify changes related to the demyelination/remyelination process.

This work outlines a new de novo design process for the creation of novel kinase inhibitor libraries. It relies on a profiling paradigm that generates a substantial amount of kinase inhibitor data from which highly predictive QSAR models can be constructed. In addition, a broad diversity of X-ray structure information is needed for binding mode prediction. This is important for scaffold and substituent site selection. Borrowing from FBDD, the process involves fragmentation of known actives, proposition of binding mode hypotheses for the fragments, and model-driven recombination using a pharmacophore derived from known kinase inhibitor structures. The support vector machine method, using Merck atom pair derived fingerprint descriptors, was used to build models from activity from 6 kinase assays. These models were qualified prospectively by selecting and testing compounds from the internal compound collection. Overall hit and enrichment rates of 82% and 2.5%, respectively, qualified the models for use in library design. Using the process, 7 novel libraries were designed, synthesized and tested against these same 6 kinases. The results showed excellent results, yielding a 92% hit rate for the 179 compounds that made up the 7 libraries. The results of one library designed to include known literature compounds, as well as an analysis of overall substituent frequency, are discussed., (Copyright 2009 Elsevier B.V. All rights reserved.)

Recently we have described the development of an Immuno-chemo-proteomics method for drug target deconvolution and profiling the toxicity of known drugs ( Saxena , C. ; Zhen , E. ; Higgs , R. E. ; Hale , J. E. J. Proteome Res. 2008, 8 , 3490 - 3497 ). The orthogonal nature and advantage of the newly developed method over existing ones were presented. Most commonly, a small molecule was coupled to an epitope and used as an affinity probe to bind targets and later antibody against the epitope was used to isolate the probe-protein complex. However, such studies performed using cell lysates are prone to false positive identification because the protein source is not in its native physiological condition. Here we describe the development and application of a multipurpose soluble probe where a small molecule was coupled to a fluorophore-tagged cell-permeable peptide epitope, which was used to affinity isolate binding proteins from live cells. Fluorophore coupling allowed direct visualization of the compound in the cells, and cell permeability of the probe provided opportunity to capture the targets from the live cell. The GSK3-beta inhibitor Bisindolylmaleimide-III was coupled to a peptide containing the fluorescein-tagged TAT epitope. Following incubation with the live cells, the compound and associated proteins were affinity isolated using antifluorescein antibody beads. Using this approach, we captured the known Bisindolylmaleimide-III target GSK3-beta and previously unidentified targets from live cells. Dose-dependent inhibition of target binding to probe in the presence of uncoupled compound validated the approach. This method was directly compared with the one where cell lysate was used as the protein source providing an advanced strategy to aid in target deconvolution and help to eliminate false positives originating from non-native protein source.

Background: Current drug discovery organizations have renewed interest in phenotypic/function based screening for the identification of novel small-molecule drug candidates. Phenotypic screening faces the challenge of deconvoluting the identity of molecular targets of small-molecules through which they exert their biological effect. The identity of the target is crucial for understanding the mechanism of drug action, rational drug design, interpretation of any toxicological findings and patient stratification. Several methods are available to deconvolute the targets of small-molecules., Objective: This review describes successful examples, limitations and advances of drug target deconvolution using small-molecule affinity chromatography coupled mass spectrometry based methods. A brief discussion of other target deconvolution methods is also presented for comparative appreciation of mass spectrometry based methods., Conclusion: The use of small-molecule affinity chromatography coupled mass spectrometry based methods is gaining popularity as a technique for target identification. Mass spectrometry based methods provide fast, reliable and high-content information on the target. They can be used with relatively intact biological systems to develop a system-wide understanding of the drug-target interaction.

Quantitative structure-retention relationship (QSRR) models were studied for two databases: one with 151 compounds and the other with 1719 compounds. In both cases, the three modeling methods employed (multiple linear regression, partial least squares, and random forests) provided similar prediction results with regard to root-mean-square error of prediction. The reversed-phase retention related seven molecular descriptors provided better models for the smaller dataset, while the use of over 2000 molecular descriptors generated better models for the larger dataset. The QSRR models were then validated with a mixture of an active pharmaceutical ingredient and its four process/degradation impurities. Finally, classification of compounds based on similar logD profiles before QSRR modeling improved chromatographic predictability for the models used. The results showed that database composition had a desirable effect on prediction accuracy for certain input molecules.

High-throughput HPLC-mass spectrometry (HPLC-MS) is routinely used to profile biological samples for potential protein markers of disease, drug efficacy and toxicity. The discovery technology has advanced to the point where translating hypotheses from proteomic profiling studies into clinical use is the bottleneck to realizing the full potential of these approaches. The first step in this translation is the development and analytical validation of a higher throughput assay with improved sensitivity and selectivity relative to typical profiling assays. Multiple reaction monitoring (MRM) assays are an attractive approach for this stage of biomarker development given their improved sensitivity and specificity, the speed at which the assays can be developed and the quantitative nature of the assay. While the profiling assays are performed with ion trap mass spectrometers, MRM assays are traditionally developed in quadrupole-based mass spectrometers. Development of MRM assays from the same instrument used in the profiling analysis enables a seamless and rapid transition from hypothesis generation to validation. This report provides guidelines for rapidly developing an MRM assay using the same mass spectrometry platform used for profiling experiments (typically ion traps) and reviews methodological and analytical validation considerations. The analytical validation guidelines presented are drawn from existing practices on immunological assays and are applicable to any mass spectrometry platform technology.

Chemical proteomics is an emerging technique for drug target deconvolution and profiling the toxicity of known drugs. With the use of this technique, the specificity of a small molecule inhibitor toward its potential targets can be characterized and information thus obtained can be used in optimizing lead compounds. Most commonly, small molecules are immobilized on solid supports and used as affinity chromatography resins to bind targets. However, it is difficult to evaluate the effect of immobilization on the affinity of the compounds to their targets. Here, we describe the development and application of a soluble probe where a small molecule was coupled with a peptide epitope which was used to affinity isolate binding proteins from cell lysate. The soluble probe allowed direct verification that the compound after coupling with peptide epitope retained its binding characteristics. The PKC-alpha inhibitor Bisindolylmaleimide-III was coupled with a peptide containing the FLAG epitope. Following incubation with cellular lysates, the compound and associated proteins were affinity isolated using anti-FLAG antibody beads. Using this approach, we identified the known Bisindolylmaleimide-III targets, PKC-alpha, GSK3-beta, CaMKII, adenosine kinase, CDK2, and quinine reductase type 2, as well as previously unidentified targets PKAC-alpha, prohibitin, VDAC and heme binding proteins. This method was directly compared to the solid-phase method (small molecule was immobilized to a solid support) providing an orthogonal strategy to aid in target deconvolution and help to eliminate false positives originating from nonspecific binding of the proteins to the matrix.

The use of small inhibitors' fragment frequencies for understanding kinase potency and selectivity is described. By quantification of differences in the frequency of occurrence of fragments, similarities between small molecules and their targets can be determined. Naive Bayes models employing fragments provide highly interpretable and reliable means for predicting potency in individual kinases, as demonstrated in retrospective tests and prospective selections that were subsequently screened. Statistical corrections for prospective validation allowed us to accurately estimate success rates in the prospective experiment. Selectivity relationships between kinase targets are substantially explained by differences in the fragment composition of actives. By application of fragment similarities to the broader proteome, it is shown that targets related by sequence exhibit similar fragment preferences in small molecules. Of greater interest, certain targets unrelated by sequence are shown to have similar fragment preferences, even when the chemical similarity of ligands active at each target is low.

BACKGROUND: Current drug therapy of atherosclerosis is focused on treatment of major risk factors, e.g. hypercholesterolemia while in the future direct disease modification might provide additional benefits. However, development of medicines targeting vascular wall disease is complicated by the lack of reliable biomarkers. In this study, we took a novel approach to identify circulating biomarkers indicative of drug efficacy by reducing the complexity of the in vivo system to the level where neither disease progression nor drug treatment was associated with the changes in plasma cholesterol. RESULTS: ApoE-/- mice were treated with an ACE inhibitor ramipril and HMG-CoA reductase inhibitor simvastatin. Ramipril significantly reduced the size of atherosclerotic plaques in brachiocephalic arteries, however simvastatin paradoxically stimulated atherogenesis. Both effects occurred without changes in plasma cholesterol. Blood and vascular samples were obtained from the same animals. In the whole blood RNA samples, expression of MMP9, CD14 and IL-1RN reflected pro-and anti-atherogenic drug effects. In the plasma, several proteins, e.g. IL-1beta, IL-18 and MMP9 followed similar trends while protein readout was less sensitive than RNA analysis. CONCLUSION: In this study, we have identified inflammation-related whole blood RNA and plasma protein markers reflecting anti-atherogenic effects of ramipril and pro-atherogenic effects of simwastatin in a mouse model of atherosclerosis. This opens an opportunity for early, non-invasive detection of direct drug effects on atherosclerotic plaques in complex in vivo systems.

Pharmaceutical companies and regulatory agencies are pursuing biomarkers as a means to increase the productivity of drug development. Quantifying differential levels of proteins from complex biological samples like plasma or cerebrospinal fluid is one specific approach being used to identify markers of drug action, efficacy, toxicity, etc. Academic investigators are also interested in markers that are diagnostic or prognostic of disease states. We report a comprehensive, fully automated, and label-free approach to relative protein quantification including: sample preparation, proteolytic protein digestion, LCMS/MS data acquisition, de-noising, mass and charge state estimation, chromatographic alignment, and peptide quantification via integration of extracted ion chromatograms. Additionally, we describe methods for transformation and normalization of the quantitative peptide levels in multiplexed measurements to improve precision for statistical analysis. Lastly, we outline how the described methods can be used to design and power biomarker discovery studies.

Parathyroid hormone (PTH) and glycogen synthase kinase-3 (GSK-3) inhibitor 603281-31-8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281-31-8 in rat osteopenia model. Female 6-month-old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1-38) at 10 microg/kg/day s.c. or 603281-31-8 at 3 mg/kg/day p.o. for 60 days. Twenty-four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (P<0.05) were subjected to pathway analysis to delineate relevant bio-processes involved in skeletal biology. Genes involved in morphogenesis, cell growth/differentiation, and apoptosis were significantly altered by Ovx and the treatments. Analysis of morphogenesis genes showed an overrepresentation of genes involved in osteogenesis, chondrogenesis, and adipogenesis. A striking finding was that Ovx decreased several markers of osteogenesis/chondrogenesis and increased markers of adipogenesis/lipid metabolism. Treatment with either PTH or the GSK-3 inhibitor reversed these effects, albeit at different levels. Histological analysis confirmed that osteopenia in Ovx animals was associated with three-fold increase in marrow adiposity. PTH and GSK-3 inhibitor restored bone volume, and reversed or normalized marrow adiposity. Ex vivo studies showed that PTH and GSK-3 inhibitor increased the ratio of colony forming marrow stromal progenitors (CFU-fs) that were alkaline phosphatase positive (putative osteoblasts). Our results suggest that the bone anabolic actions of PTH and GSK-3 inhibitor in vivo involve concerted effects on mesenchymal lineages; osteoblasts, chondrocytes, and adipocytes., (Copyright (c) 2007 Wiley-Liss, Inc.)

Immunoprecipitation (IP) combined with matrix-assisted laser desorption ionization (MALDI) time of flight (Tof) mass spectrometry has been used to develop quantitative assays for amyloid-beta (Abeta) peptides in cerebrospinal fluid (CSF). Inclusion of (15)N labelled standard peptides allows for absolute quantification of multiple Abeta isoforms in individual samples. Characterization of variability associated with all steps of the assay indicated that the IP step is the single largest contributor to overall variability. Optimization of the assay resulted in overall coefficient of variation

The non-mineral component of bone matrix consists of 90% collagenous, 10% non-collagenous proteins. These proteins regulate mineralization, growth, cell signaling and differentiation, and provide bone with its tensile strength. Expression of bone matrix proteins have historically been studied individually or in small numbers owing to limitations in analytical technologies. Current mass-spectrometric and separations technologies allow a global view of protein expression patterns in complex samples. To our knowledge, no proteome profile of bone matrix has yet been reported. Therefore, we have used mass spectrometry as a tool to generate a profile of proteins present in the extracellular matrix of adult rat bone. Overall, 108 and 25 proteins were identified with high confidence in the metaphysis and diaphysis, respectively, using a bottom up proteomic technique. Twenty-one of these proteins were present in both the metaphysis and diaphysis including the bone specific proteins, osteocalcin, type I collagen, osteopontin, osteoregulin, and bone sialoprotein. Interestingly, type II collagen, a protein thought to be exclusively expressed in cartilage, was identified in both the metaphysis and diaphysis. This observation was validated by Western blot. Additionally, the presence of aggrecan, another protein expressed in cartilage was identified in the bone matrix extracts by Western blot. The proteome profile generated using this technology represents an initial survey of the acid soluble proteins of bone matrix which provides a reference for the analysis of deviations from the normal composition due to perturbations or disease states.

We present a wrapper-based approach to estimate and control the false discovery rate for peptide identifications using the outputs from multiple commercially available MS/MS search engines. Features of the approach include the flexibility to combine output from multiple search engines with sequence and spectral derived features in a flexible classification model to produce a score associated with correct peptide identifications. This classification model score from a reversed database search is taken as the null distribution for estimating p-values and false discovery rates using a simple and established statistical procedure. Results from 10 analyses of rat sera on an LTQ-FT mass spectrometer indicate that the method is well calibrated for controlling the proportion of false positives in a set of reported peptide identifications while correctly identifying more peptides than rule-based methods using one search engine alone.

Recognizing specific protein changes in response to drug administration in humans has the potential for significant utility in clinical research. In spite of this, many methodological and practical questions related to assessing such changes are unanswered. We conducted a series of clinical studies to assess the feasibility of measuring changes in proteins related to drug administration using a mass-spectrometry proteomics technique capable of quantifying hundreds of proteins simultaneously in cerebrospinal fluid (CSF) and plasma. Initially, the normal variability of proteins in these compartments was characterized in 16 healthy volunteers over a 2-week period. Drug-associated changes were subsequently assessed in the plasma and CSF proteomes of 11 subjects given atomoxetine, which served as a selective, centrally active probe to test the model. Protein levels in the CSF and plasma were unchanged between visits in the normal variability study. In contrast, statistically significant changes were detected in the CSF protein pattern after drug treatment. These studies suggest that identification of changes in the CSF proteome associated with the administration of centrally active drugs is feasible, and may be of value in the development of new drugs, as well as broader clinical research.