Search

This post hoc analysis of the randomized, placebo-controlled N-MOmentum study (NCT02200770) of inebilizumab in neuromyelitis optica spectrum disorder (NMOSD) evaluated relationships between circulating B-cell subsets and aquaporin-4 immunoglobulin G (AQP4-lgG) titers and attacks. Among participants receiving placebo, CD20 + and CD27 + B-cell counts were modestly increased from the pre-attack visit to attack; plasmablast/plasma cell gene signature was increased from baseline to the pre-attack visit (p = 0.016) and from baseline to attack (p = 0.009). With inebilizumab treatment, B-cell subset counts decreased and did not increase with attacks. No difference in change of AQP4-IgG titers from baseline to time of attack was observed., (© 2024 The Author(s). Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Aberrant or dysfunctional cellular enzymes are responsible for a wide range of diseases including cancer, neurodegenerative conditions, and metabolic disorders. Deficiencies in enzyme level or biofunction may lead to intracellular accumulation of substrate to toxic levels and interfere with overall cellular function, ultimately leading to cell damage, disease, and death. Marketed therapeutic interventions for inherited monogenic enzyme deficiency disorders include enzyme replacement therapy and small molecule chaperones. Novel approaches of in vivo gene therapy and ex vivo cell therapy are under clinical evaluation and provide promising opportunities to expand the number of available disease-modifying treatments. To support the development of these different therapeutics, assays to quantify the functional activity of protein enzymes have gained importance in the diagnosis of disease, assessment of pharmacokinetics and pharmacodynamic response, and evaluation of drug efficacy. In this review, we discuss the technical aspects of enzyme activity assays in the bioanalytical context, including assay design and format as well as the unique challenges and considerations associated with assay development, validation, and life cycle management., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Immunogenicity testing and characterization is an important part of understanding the immune response to administration of a protein therapeutic. Neutralizing antibody (NAb) assays are used to characterize a positive anti-drug antibody (ADA) response. Harmonization of reporting of NAb assay performance and results enables efficient communication and expedient review by industry and health authorities. Herein, a cross-industry group of NAb assay experts have harmonized NAb assay reporting recommendations and provided a bioanalytical report (BAR) submission editable template developed to facilitate agency filings. This document addresses key bioanalytical reporting gaps and provides a report structure for documenting clinical NAb assay performance and results. This publication focuses on the content and presentation of the NAb sample analysis report including essential elements such as the method, critical reagents and equipment, data analysis, study samples, and results. The interpretation of immunogenicity data, including the evaluation of the impact of NAb on safety, exposure, and efficacy, is out of scope of this publication., (© 2024. The Author(s).)

Objective: To investigate relationships between serum neurofilament light chain (sNfL), ubiquitin C-terminal hydrolase L1 (sUCHL1), tau (sTau) and glial fibrillary acidic protein (sGFAP) levels and disease activity/disability in neuromyelitis optica spectrum disorder (NMOSD), and the effects of inebilizumab on these biomarkers in N-MOmentum., Methods: N-MOmentum randomised participants to receive inebilizumab or placebo with a randomised controlled period (RCP) of 28 weeks and an open-label follow-up period of ≥2 years. The sNfL, sUCHL1, sTau and sGFAP were measured using single-molecule arrays in 1260 scheduled and attack-related samples from N-MOmentum participants (immunoglobulin G (IgG) autoantibodies to aquaporin-4-positive, myelin oligodendrocyte glycoprotein-IgG-positive or double autoantibody-negative) and two control groups (healthy donors and patients with relapsing-remitting multiple sclerosis)., Results: The concentration of all four biomarkers increased during NMOSD attacks. At attack, sNfL had the strongest correlation with disability worsening during attacks (Spearman R 2 =0.40; p=0.01) and prediction of disability worsening after attacks (sNfL cut-off 32 pg/mL; area under the curve 0.71 (95% CI 0.51 to 0.89); p=0.02), but only sGFAP predicted upcoming attacks. At RCP end, fewer inebilizumab-treated than placebo-treated participants had sNfL>16 pg/mL (22% vs 45%; OR 0.36 (95% CI 0.17 to 0.76); p=0.004)., Conclusions: Compared with sGFAP, sTau and sUCHL1, sNfL at attack was the strongest predictor of disability worsening at attack and follow-up, suggesting a role for identifying participants with NMOSD at risk of limited post-relapse recovery. Treatment with inebilizumab was associated with lower levels of sGFAP and sNfL than placebo., Trial Registration Number: NCT02200770., Competing Interests: Competing interests: OA reports grants from the German Ministry of Education and Research (BMBF) and the German Research Foundation (DFG); grants and personal fees from Biogen and Novartis; and travel support and personal fees from Alexion, Almirall, MedImmune, Merck Serono, Roche, Sanofi, Viela Bio/Horizon Therapeutics and Zambon. OA is a member of the European Reference Network for Rare Eye Diseases (ERN-EYE), co-funded by the Health Program of the European Union under the Framework Partnership Agreement No 739534 'ERN-EYE. H-PH has received fees for consulting, speaking and serving on steering committees from Bayer Healthcare, Biogen Idec, Celgene Receptos, CSL Behring, GeNeuro, Genzyme, MedDay, MedImmune, Merck Serono, Novartis, Roche, Sanofi, TG Therapeutics and Viela Bio/Horizon Therapeutics, with approval by the Rector of Heinrich Heine University Düsseldorf. KF has received fees for consulting, speaking and serving on steering committees from AbbVie, Alexion, Asahi Kasei Medical, Biogen, Chugai/Roche, Eisai, Japan Tobacco, MedImmune/Viela Bio, Merck, Merck Biopharma, Mitsubishi-Tanabe, Novartis, Takeda, Teijin and UCB, and has received Grant-in-Aid for Scientific Research from the Ministry of Health, Welfare and Labor of Japan. FP has received research support, speaker fees and travel reimbursement from Bayer, Biogen Idec, Merck Serono, Novartis, Sanofi Genzyme and Teva; is supported by the German Competence Network for Multiple Sclerosis and the German Research Council (DFG Exc 257); has received travel reimbursement from the Guthy–Jackson Charitable Foundation; and serves on the steering committee of the OCTIMS study sponsored by Novartis. RM serves on scientific advisory boards for Alexion, Roche and Viela Bio/Horizon Therapeutics; and has received funding for travel and fees from Alexion, Biogen, Merck, Novartis, Roche and Viela Bio/Horizon Therapeutics. JLB reports payment for study design/consultation from MedImmune/Viela Bio/Horizon Therapeutics; reports personal fees from AbbVie, Alexion, Beigene, Chugai, Clene Nanomedicine, Genentech, Genzyme, Mitsubishi Tanabe Pharma, Reistone Biopharma and Roche; reports grants and personal fees from EMD Serono and Novartis; reports grants from Alexion, Mallinckrodt and the National Institutes of Health; and has a patent for Aquaporumab issued. HJK has received a grant from the National Research Foundation of Korea; consultancy/speaker fees or research support from Alexion, AprilBio, ALTOS Biologics, Biogen, Celltrion, Daewoong, Eisai, GC Pharma, HanAll BioPharma, Handok, Horizon Therapeutics (formerly Viela Bio), Kolon Life Science, MDimune, Mitsubishi Tanabe Pharma, Merck Serono, Novartis, Roche, Sanofi Genzyme, Teva-Handok and UCB; and is a co-editor for the Multiple Sclerosis Journal and an associated editor for the Journal of Clinical Neurology. BGW receives payments for serving as chair of attack adjudication committees for clinical trials in NMOSD for Alexion, MedImmune, UCB Bioscience and Viela Bio/Horizon Therapeutics; has consulted with Chugai, Genentech, Horizon Pharmaceuticals, Mitsubishi Tanabe Pharma and Roche; has received payments for speaking for Genentech and Roche; and has a patent for NMO-IgG for diagnosis of neuromyelitis optica, with royalties paid by Hospices Civils de Lyon, MVZ Labor PD Dr Volkmann und Kollegen GbR, RSR and the University of Oxford. SJP has received personal compensation for serving as a consultant for Astellas, Genentech and Sage Therapeutics; has received personal compensation for serving on scientific advisory boards or data safety monitoring boards for F. Hoffman-La Roche AG, Genentech and UCB; has received research support from Alexion, Roche/Genentech and Viela Bio/MedImmune/Horizon; has a Patent# 8,889,102 (Application#12-678350, Neuromyelitis Optica Autoantibodies as a Marker for Neoplasia)—issued; has a patent, Patent# 9,891,219B2 (Application#12-573942, Methods for Treating Neuromyelitis Optica [NMO] by Administration of Eculizumab to an individual that is Aquaporin-4 (AQP4)-IgG Autoantibody positive)—issued; and his institution has received compensation for serving as a consultant for Alexion, Astellas and Viela Bio/MedImmune/Horizon. DMW reports personal fees from Biogen, Genentech, Horizon, Mitsubishi Tanabe, Roche, UCB Pharma and Viela Bio; and research support paid to Mayo Clinic by Alexion Pharmaceuticals. GC has received personal fees for participation on data and safety monitoring boards from AI Therapeutics, AMO Pharma, AstraZeneca, Avexis Pharmaceuticals, Biolinerx, Brainstorm Cell Therapeutics, Bristol Meyers Squibb/Celgene, CSL Behring, Galmed Pharmaceuticals, Green Valley Pharma, Horizon Pharmaceuticals, Immunic, Karuna Therapeutics, Mapi Pharmaceuticals, Merck, Mitsubishi Tanabe Pharma Holdings, NHLBI (Protocol Review Committee), Novartis, Opko Biologics, Prothena Biosciences, Regeneron, Sanofi-Aventis, Reata Pharmaceuticals, University of Texas Southwestern, University of Pennsylvania and Visioneering Technologies; has received personal fees for consulting or advisory board participation from Alexion, Antisense Therapeutics, Biogen, Clinical Trial Solutions, Entelexo Biotherapeutics, Genentech, Genzyme, GW Pharmaceuticals, Immunic, Klein-Buendel Incorporated, Merck/Serono, Novartis, Osmotica Pharmaceuticals, Perception Neurosciences, Protalix Biotherapeutics, Recursion/Cerexis Pharmaceuticals, Regeneron, Roche, SAB Biotherapeutics; and is employed by the University of Alabama at Birmingham and President of Pythagoras, a private consulting company located in Birmingham, Alabama. BAC reports personal compensation for consulting from Alexion, Atara Biotherapeutics, Autobahn, Avotres, Biogen, EMD Serono, Gossamer Bio, Horizon, Neuron23, Novartis, Sanofi, TG Therapeutics and Therini Bio; and has received research support from Genentech. MAS, WAR, DS and DC are employees of Horizon Therapeutics and own stock. MG and EK are former employees of Horizon Therapeutics and own stock., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: To evaluate the safety, efficacy and response duration of four different dosing regimens of dazodalibep (DAZ), a non-antibody biological antagonist of CD40L, in patients with rheumatoid arthritis (RA)., Methods: This double-blind study included adult patients with moderate-to-severe active RA with a positive test for serum rheumatoid factor and/or anticitrullinated protein antibodies, an inadequate response to methotrexate, other conventional disease-modifying antirheumatic drugs or tumour necrosis factor-α inhibitors, and no prior treatment with B-cell depleting agents. Eligible participants were randomised equally to five groups receiving intravenous infusions of DAZ or placebo. The primary endpoint was the change from baseline in the Disease Activity Score-28 with C reactive protein (DAS28-CRP) at day 113. Participants were followed through day 309., Results: The study randomised 78 eligible participants. The change from baseline in DAS28-CRP (least squares means±SE) at day 113 was significantly greater for all DAZ groups (-1.83±0.28 to -1.90±0.27; p<0.05) relative to PBO (-1.06±0.26); significant reductions in DAS28-CRP were also observed for all DAZ groups at day 309. The distribution of adverse events was generally balanced among DAZ and PBO groups (74% and 63%, respectively). There were four serious adverse events deemed by investigators to be unrelated to study medication., Conclusions: DAZ treatment for all dosage regimens significantly reduced DAS28-CRP at day 113 relative to PBO. The safety data suggest an acceptable safety and tolerability profile. Treatment effects at day 113 and the prolonged duration of responses after DAZ cessation support the use of longer dosing intervals., Trial Registration Number: NCT04163991., Competing Interests: Competing interests: AK has received study funding, medical writing support, and article processing charges from Amgen. He has received consulting fees from AXDEV Group, Pfizer, Janssen, Boehringer Ingelheim, AbbVie, Flexion, Gilead, Grunenthal, Orion, Regeneron, Sun Pharma Advance Research, and ECOR1. He has received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Merck & Co, Eli Lilly, Novartis, Pfizer, Flexion, AbbVie, Amgen, Genentech, Regeneron, UCB, Horizon, and GSK. He has participated in a data safety monitoring board for AbbVie and Amgen. He has been part of a board or advisory board for AbbVie, Bendcare, Boehringer Ingelheim, ChemoCentryx, Flexion, Gilead, Grunenthal, Horizon, Eli Lilly, Janssen, Pfizer, Regeneron, UCB, and Novartis. He has stock or stock options in Pfizer, GSK, Gilead, Novartis, and Amgen. LW, IA are current employees of Horizon Therapeutics and own stock. MG, JF and GI are former employees of Horizon Therapeutics and own stock. EWS has consulted for Horizon Therapeutics, Bristol Myers Squibb, CSL Behring, Resolve Therapeutics, and Sonoma Biotherapeutics, and receives royalties from UpToDate., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Inebilizumab is an anti-CD19 antibody approved for the treatment of neuromyelitis optica spectrum disorder (NMOSD) in adults with aquaporin-4 autoantibodies. The relationship between B-cell, plasma-cell (PC), and immunoglobulin depletion with longitudinal reductions in NMOSD activity after inebilizumab treatment was characterised post hoc in an exploratory analysis from the N-MOmentum study (NCT02200770)., Methods: Peripheral blood CD20+ B cells, PC gene signature, and immunoglobulin levels were assessed throughout N-MOmentum (follow-up ≥2.5 years); correlations with clinical metrics and magnetic resonance imaging (MRI) lesion activity were assessed., Findings: Inebilizumab induced durable B-cell and PC depletion within 1 week versus placebo. Although no association was observed between B-cell counts at time of attack and NMOSD activity, depth of B-cell depletion after the first dosing period correlated with clinical outcomes. All participants receiving inebilizumab demonstrated a robust long-term therapeutic response, and participants with ≤4 cells/μL after the first 6-month dosing interval had persistently deeper B-cell depletion, lower annualised attack rates (estimated rate [95% CI]: 0.034 [0.024-0.04] vs 0.086 [0.056-0.12]; p = 0.045), fewer new/enlarging T2 MRI lesions (0.49 [0.43-0.56] vs 1.36 [1.12-1.61]; p < 0.0001), and a trend towards decreased Expanded Disability Status Scale worsening (0.076 [0.06-0.10] vs 0.14 [0.10-0.18]; p = 0.093). Antibodies to inebilizumab, although present in a proportion of treated participants, did not alter outcomes., Interpretation: This analysis suggests that compared with placebo, inebilizumab can provide specific, rapid, and durable depletion of B cells in participants with NMOSD. Although deep and persistent CD20+ B-cell depletion correlates with long-term clinical stability, early, deep B-cell depletion correlates with improved disease activity metrics in the first 2 years., Funding: Horizon Therapeutics (formerly from Viela Bio/MedImmune)., Competing Interests: Declaration of interests J.L.Bennett reports payment for study design/consultation from MedImmune; personal fees from AbbVie, Alexion, Antigenomycs, BeiGene, Chugai, Clene Nanomedicine, Genentech, Genzyme, Reistone Bio, Roche, and TG; grants from Alexion, the National Institutes of Health, and Novartis. In addition, Dr Bennett has a patent ‘Compositions and methods for the treatment of neuromyelitis optica’. O.Aktas reports grants from the German Research Foundation (DFG) and the German Ministry of Education and Research (BMBF); grants and personal fees from Bayer HealthCare, Biogen, Genzyme, Horizon Therapeutics (formerly from Viela Bio), Novartis, and Teva; and personal fees from Almirall, MedImmune, Merck Serono, and Roche. W.A.Rees, M.A.Smith, D.She, and D.Cimbora are employees of Horizon Therapeutics (formerly Viela Bio) and own stock. L.Yan, M.Gunsior, and E.Katz are former employees of Horizon Therapeutics. S.J.Pittock reports grants, personal fees, and non-financial support from Alexion Pharmaceuticals, Inc.; grants from Autoimmune Encephalitis Alliance and Grifols; grants, personal fees, non-financial support, and other from Horizon Therapeutics (formerly from Viela Bio) and MedImmune; personal fees for consulting services from Astellas; grants, personal fees, non-financial support and other from Roche/Genentech; personal fees for consulting services from UCB; and has a patent #9,891,219 (Application#12-573942) ’Methods for Treating Neuromyelitis Optica (NMO) by Administration of Eculizumab to an individual that is Aquaporin-4 (AQP4)-IgG Autoantibody Positive’. B.G.Weinshenker received payments for serving as chair of attack adjudication committees for clinical trials in NMOSD for Alexion, MedImmune, and Viela Bio/Horizon Therapeutics; has consulted with Chugai, Genentech, Horizon Therapeutics, Mitsubishi Tanabe Pharma, and Roche Pharmaceuticals; and has a patent for NMO-IgG for diagnosis of neuromyelitis optica, with royalties paid by Hospices Civils de Lyon, MVZ Labor PD Dr. Volkmann und Kollegen GbR, University of Oxford, and RSR. F.Paul has received research support, speaker honoraria, and travel reimbursement from Bayer, Biogen Idec, Merck Serono, Novartis, Sanofi Genzyme, and Teva; is supported by the German Research Council (DFG Exc 257) and the German Competence Network for Multiple Sclerosis; has received travel reimbursement from the Guthy-Jackson Charitable Foundation; and serves on the steering committee of the OCTIMS study, sponsored by Novartis. R.Marignier reports personal fees for consulting from Alexion, Horizon Therapeutics (formerly Viela Bio), Roche, and UCB. D.Wingerchuk reports personal fees from Biogen, Celgene, Genentech, MedImmune, Mitsubishi Tanabe, Novartis, Reistone Biopharma, and TG Therapeutics; research support paid to the Mayo Clinic by Alexion and Terumo BCT; and has served on a clinical trial adjudication committee for Horizon Therapeutics (formerly Viela Bio) and MedImmune. G.Cutter has received personal fees for participation on Data and Safety Monitoring Boards from AstraZeneca, Avexis Pharmaceuticals, BioLineRx, Brainstorm Cell Therapeutics, Bristol Myers Squibb/Celgene, CSL Behring, the Eunice Kennedy Shriver National Institute of Child Health and Human Development (Obesity Policy Research Unit oversight committee), Galmed Pharmaceuticals, Hisun Pharmaceutical, Horizon Pharmaceuticals (formerly Viela Bio), Mapi Pharma, Merck, Merck/Pfizer, the National Heart, Lung, and Blood Institute (Protocol Review Committee), Neurim Pharmaceuticals, Novartis, OncoImmune, OPKO Biologics, Orphazyme, Reata Pharmaceuticals, Sanofi-Aventis, Teva Pharmaceuticals, and Vivus; personal fees for consulting or advisory board participation from BioDelivery Sciences International, Biogen, Click Therapeutics, Genentech, Genzyme, GW Pharmaceuticals, Immunic, Klein Buendel, MedDay, MedImmune, NeuroGenesis, Novartis, Osmotica Pharmaceuticals, Perception Neurosciences, Recursion/Cerexis Pharmaceuticals, Roche, and TG Therapeutics; is employed by the University of Alabama at Birmingham, AL, USA; and is President of Pythagoras, Inc., a private consulting company based in Birmingham, AL, USA. A.Green reports grants from the Conrad N. Hilton Foundation and the Tom Sherak MS Hope Foundation; other financial relationships (for activities as expert witness, associate editor, advisory board/steering committee participation, and endpoint adjudication) with Bionure, Inception Sciences, JAMA Neurology, MedImmune/Horizon Therapeutics (formerly Viela Bio), Mylan, Synthon, and Trims Pharma; and personal fees from and other financial relationships with Pipeline Therapeutics. H.-P.Hartung has received fees for consulting, speaking, and serving on steering committees from Bayer HealthCare, Biogen Idec, Celgene Receptos, CSL Behring, GeNeuro, Genzyme, Horizon Therapeutics (formerly Viela Bio), MedDay, MedImmune, Merck Serono, Novartis, Roche, Sanofi, and TG Therapeutics with approval by the Rector of Heinrich Heine University Düsseldorf. H.J.Kim has received a grant from the National Research Foundation of Korea; consultancy/speaker fees or research support from Alexion, AprilBio, Celltrion, Daewoong Pharmaceutical, Eisai, GC Pharma, HanAll Biopharma, Horizon Therapeutics (formerly Viela Bio), Kolon Life Science, MedImmune, Merck Serono, Mitsubishi Tanabe Pharma, Novartis, Sanofi Genzyme, Teva-Handok, and UCB; and is a co-editor for the Multiple Sclerosis Journal and an associate editor for the Journal of Clinical Neurology. K.Fujihara has received fees for consulting, speaking, and serving on steering committees from AbbVie, Alexion, Asahi Kasei Kuraray Medical Co., Biogen, Chugai/Roche, Eisai, Japan Tobacco, MedImmune/Viela Bio, Merck, Merck Biopharma, Mitsubishi Tanabe Pharma, Novartis, Teijin, Takeda Pharmaceutical Company, and UCB; and a grant-in-aid for scientific research from the Ministry of Health, Labour and Welfare of Japan. M.Levy currently receives research support from Acorda Therapeutics, Alexion, Alnylam Pharmaceuticals, ApoPharma, Maryland Technology Development Corporation, the National Institutes of Health, Sanofi Genzyme, and Shire/Takeda; has received personal compensation for consultation with Acorda Therapeutics, Alexion, and Genzyme; and serves on the scientific advisory boards for Acorda Therapeutics, Alexion, and Quest Diagnostics. B.A.C.Cree reports personal compensation for consulting from Alexion, Atara Biotherapeutics, Autobahn Therapeutics, Avotres Inc., Biogen, Boston Pharma, EMD Serono, Gossamer Bio, Hexal/Sandoz, Horizon Therapeutics, Neuron23, Novartis, Sanofi, Siemens, TG Therapeutics, and Therini Bio; and has received research support from Genentech., (Copyright © 2022 Horizon Therapeutics plc. Published by Elsevier B.V. All rights reserved.)

A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication., (© 2022. The Author(s).)

Plasmacytoid dendritic cells (pDCs) not only are specialized in their capacity to secrete large amounts of type I interferon (IFN) but also serve to enable both innate and adaptive immune responses through expression of additional proinflammatory cytokines, chemokines, and costimulatory molecules. Persistent activation of pDCs has been demonstrated in a number of autoimmune diseases. To evaluate the potential benefit of depleting pDCs in autoimmunity, a monoclonal antibody targeting the pDC-specific marker immunoglobulin-like transcript 7 was generated. This antibody, known as VIB7734, which was engineered for enhanced effector function, mediated rapid and potent depletion of pDCs through antibody-dependent cellular cytotoxicity. In cynomolgus monkeys, treatment with VIB7734 reduced pDCs in blood below the lower limit of normal by day 1 after the first dose. In two phase 1 studies in patients with autoimmune diseases, VIB7734 demonstrated an acceptable safety profile, comparable to that of placebo. In individuals with cutaneous lupus, VIB7734 profoundly reduced both circulating and tissue-resident pDCs, with a 97.6% median reduction in skin pDCs at study day 85 in VIB7734-treated participants. Reductions in pDCs in the skin correlated with a decrease in local type I IFN activity as well as improvements in clinical disease activity. Biomarker analysis suggests that responsiveness to pDC depletion therapy may be greater among individuals with high baseline type I IFN activity, supporting a central role for pDCs in type I IFN production in autoimmunity and further development of VIB7734 in IFN-associated diseases., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background and Purpose: Inhibition of the T- and B-cell interaction through the CD40/CD40 ligand (L) axis is a favourable approach for inflammatory disease treatment. Clinical studies of anti-CD40L molecules in autoimmune diseases have met challenges because of thromboembolic events and adverse haemostasis. VIB4920 (formerly MEDI4920) is a novel CD40L antagonist and Tn3 fusion protein designed to prevent adverse haemostasis and immunopharmacology. We evaluated the pharmacokinetics, activity and toxicity of VIB4920 in monkeys., Experimental Approach: Cynomolgus monkeys received i.v. or s.c. 5-300 mg·kg -1 VIB4920 or vehicle, once weekly for 1 month (Studies 1 and 2) or 28 weeks (Study 3). VIB4920 exposure and bioavailability were determined using pharmacokinetic analyses, and immune cell population changes via flow cytometry. Pharmacological activity was evaluated by measuring the animals' capacity to elicit an immune response to keyhole limpet haemocyanin (KLH) and tetanus toxoid (TT)., Key Results: VIB4920 demonstrated linear pharmacokinetics at multiple doses. Lymphocyte, monocyte, cytotoxic T-cell and NK cell counts were not significantly different between treatment groups. B-cell counts reduced dose-dependently and the T-cell dependent antibody response to KLH was suppressed by VIB4920 dose-dependently. The recall response to TT was similar across treatment groups. No thromboembolic events or symptoms of immune system dysfunctionality were observed., Conclusions and Implications: VIB4920 demonstrated an acceptable safety profile in monkeys. VIB4920 showed favourable pharmacokinetics, dose-dependent inhibition of a neoantigen-specific immune response and no adverse effects on immune function following long-term use. Our data support the use of VIB4920 in clinical trials., (© 2019 The British Pharmacological Society.)

The CD40/CD40L axis plays a central role in the generation of humoral immune responses and is an attractive target for treating autoimmune diseases in the clinic. Here, we report the generation and clinical results of a CD40L binding protein, VIB4920, which lacks an Fc domain, therefore avoiding platelet-related safety issues observed with earlier monoclonal antibody therapeutics that targeted CD40L. VIB4920 blocked downstream CD40 signaling events, resulting in inhibition of human B cell activation and plasma cell differentiation, and did not induce platelet aggregation in preclinical studies. In a phase 1 study in healthy volunteers, VIB4920 suppressed antigen-specific IgG in a dose-dependent fashion after priming and boosting with the T-dependent antigen, KLH. Furthermore, VIB4920 significantly reduced circulating Ki67 + dividing B cells, class-switched memory B cells, and a plasma cell gene signature after immunization. In a phase 1b proof-of-concept study in patients with rheumatoid arthritis, VIB4920 significantly decreased disease activity, achieving low disease activity or clinical remission in more than 50% of patients in the two higher-dose groups. Dose-dependent decreases in rheumatoid factor autoantibodies and Vectra DA biomarker score provide additional evidence that VIB4920 effectively blocked the CD40/CD40L pathway. VIB4920 demonstrated a good overall safety profile in both clinical studies. Together, these data demonstrate the potential of VIB4920 to significantly affect autoimmune disease and humoral immune activation and to support further evaluation of this molecule in inflammatory conditions., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose: To use preclinical models to identify a dosing schedule that improves tolerability of highly potent pyrrolobenzodiazepine dimers (PBDs) antibody drug conjugates (ADCs) without compromising antitumor activity. Experimental Design: A series of dose-fractionation studies were conducted to investigate the pharmacokinetic drivers of safety and efficacy of PBD ADCs in animal models. The exposure-activity relationship was investigated in mouse xenograft models of human prostate cancer, breast cancer, and gastric cancer by comparing antitumor activity after single and fractionated dosing with tumor-targeting ADCs conjugated to SG3249, a potent PBD dimer. The exposure-tolerability relationship was similarly investigated in rat and monkey toxicology studies by comparing tolerability, as assessed by survival, body weight, and organ-specific toxicities, after single and fractionated dosing with ADCs conjugated to SG3249 (rats) or SG3400, a structurally related PBD (monkeys). Results: Observations of similar antitumor activity in mice treated with single or fractionated dosing suggests that antitumor activity of PBD ADCs is more closely related to total exposure (AUC) than peak drug concentrations ( C max ). In contrast, improved survival and reduced toxicity in rats and monkeys treated with a fractionated dosing schedule suggests that tolerability of PBD ADCs is more closely associated with C max than AUC. Conclusions: We provide the first evidence that fractionated dosing can improve preclinical tolerability of at least some PBD ADCs without compromising efficacy. These findings suggest that preclinical exploration of dosing schedule could be an important clinical strategy to improve the therapeutic window of highly potent ADCs and should be investigated further. Clin Cancer Res; 23(19); 5858-68. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Human S100A7 (psoriasin) is considered a marker for specific stages of breast cancer. hS100A15 is almost identical to hS100A7 and difficult to discriminate. We developed specific probes to distinguish hS100A7 and hS100A15, and demonstrate their differential distribution in normal breast tissue. Further, hS100A7 and S100A15 transcripts are elevated in ER/PR negative breast cancers, but hS100A15 protein is detected in all cancer specimens while hS100A7 protein is sporadically expressed. The differential regulation, expression and distribution of hS100A7 and hS100A15 and their reported distinct functions are compelling reasons to discriminate among these proteins in normal breast and breast cancers.

We recently used RNA interference to show that a negative correlation of L-asparaginase (L-ASP) chemotherapeutic activity with asparagine synthetase (ASNS) expression in the ovarian subset of the NCI-60 cell line panel is causal. To determine whether that relationship would be sustained in a larger, more diverse set of ovarian cell lines, we have now measured ASNS mRNA expression using microarrays and a branched-DNA RNA assay, ASNS protein expression using an electrochemiluminescent immunoassay, and L-ASP activity using an MTS assay on 19 human ovarian cancer cell lines. Contrary to our previous findings, L-ASP activity was only weakly correlated with ASNS mRNA expression; Pearson's correlation coefficients were r = -0.21 for microarray data and r = -0.39 for the branched-DNA RNA assay, with just the latter being marginally statistically significant (P = 0.047, one-tailed). ASNS protein expression measured by liquid-phase immunoassay exhibited a much stronger correlation (r = -0.65; P = 0.0014, one-tailed). We conclude that ASNS protein expression measured by immunoassay is a strong univariate predictor of L-ASP activity in ovarian cancer cell lines. These findings provide rationale for evaluation of ASNS protein expression as a predictive biomarker of clinical L-ASP activity in ovarian cancer.

Human S100A7 (psoriasin) is overexpressed in inflammatory diseases. The recently discovered, co-evolved hS100A15 is almost identical in sequence and up-regulated with hS100A7 during cutaneous inflammation. The functional role of these closely related proteins for inflammation remains undefined. By generating specific Abs, we demonstrate that hS100A7 and hS100A15 proteins are differentially expressed by specific cell types in the skin. Although highly homologous, both proteins are chemoattractants with distinct chemotactic activity for leukocyte subsets. We define RAGE (receptor for advanced glycation end products) as the hS100A7 receptor, whereas hS100A15 functions through a Gi protein-coupled receptor. hS100A7-RAGE binding, signaling, and chemotaxis are zinc-dependent in vitro, reflecting the previously reported zinc-mediated changes in the hS100A7 dimer structure. When combined, hS100A7 and hS100A15 potentiate inflammation in vivo. Thus, proinflammatory synergism in disease may be driven by the diverse biology of these almost identical proteins that have just recently evolved. The identified S100A7 interaction with RAGE may provide a novel therapeutic target for inflammation.

L-Asparaginase (l-ASP), a bacterial enzyme used since the 1970s to treat acute lymphoblastic leukemia, selectively starves cells that cannot synthesize sufficient asparagine for their own needs. Molecular profiling of the NCI-60 cancer cell lines using five different microarray platforms showed strong negative correlations of asparagine synthetase (ASNS) expression and DNA copy number with sensitivity to l-ASP in the leukemia and ovarian cancer cell subsets. To assess whether the ovarian relationship is causal, we used RNA interference to silence ASNS in three ovarian lines and observed 4- to 5-fold potentiation of sensitivity to l-ASP with two of the lines. For OVCAR-8, the line that expresses the least ASNS, the potentiation was >500-fold. Significantly, that potentiation was >700-fold in the multidrug-resistant derivative OVCAR-8/ADR, showing that the causal relationship between ASNS expression and l-ASP activity survives development of classical multidrug resistance. Tissue microarrays confirmed low ASNS expression in a subset of clinical ovarian cancers as well as other tumor types. Overall, this pharmacogenomic/pharmacoproteomic study suggests the use of l-ASP for treatment of a subset of ovarian cancers (and perhaps other tumor types), with ASNS as a biomarker for patient selection.

The calcium-binding proteins of the human S100A7/A15 (hS100A7/A15) subfamily are differentially expressed in normal and pathological epidermis. The hS100A7 (psoriasin) and S100A15 reside in a chromosomal cluster of highly similar paralogs. To exploit the power of mouse models for determining functions of gene products, the corresponding S100A7/A15 ortholog was cloned and examined in murine skin. The single mouse S100A15 (mS100A15) gene encodes a protein of 104 amino acids with a predicted molecular weight of 12,870 Da and two EF-hand calcium binding sites. Using gene-specific primers and specific antibodies, expression of mS100A15 in both skin and isolated keratinocytes is confined to differentiating granular and cornified epidermal cells. Immunoblotting of epidermal extracts revealed a series of high molecular weight bands that are also recognized by an antibody for transglutaminase-mediated protein crosslinks. mS100A15 expression is upregulated in cultured keratinocytes induced to differentiate by calcium or phorbol esters. Maximal induction occurs concordantly with expression of late differentiation markers. Induction is enhanced in keratinocytes overexpressing protein kinase Calpha and is dependent on activator protein-1 transcription factors. The regulation, expression pattern and crosslinking of mS100A15 are consistent with the characteristics of the human orthologs, providing a valid surrogate model to study changes in these proteins associated with cutaneous pathologies.

D-phenylglycine (D-Phg) is an important side chain building block for semi-synthetic penicillins and cephalosporins such as ampicillin and cephalexin. To produce d-Phg ultimately from glucose, metabolic engineering was applied. Starting from phenylpyruvate, which is the direct precursor of L-phenylalanine, an artificial D-Phg biosynthesis pathway was created. This three-step route is composed of the enzymes hydroxymandelate synthase (HmaS), hydroxymandelate oxidase (Hmo), and the stereoinverting hydroxyphenylglycine aminotransferase (HpgAT). Together they catalyse the conversion of phenylpyruvate via mandelate and phenylglyoxylate to D-Phg. The corresponding genes were obtained from Amycolatopsis orientalis, Streptomyces coelicolor, and Pseudomonas putida. Combined expression of these activities in E. coli strains optimized for the production of L-phenylalanine resulted in the first completely fermentative production of D-Phg.

The most common cause of primary autosomal recessive microcephaly (MCPH) appears to be mutations in the ASPM gene which is involved in the regulation of neurogenesis. The predicted gene product contains two putative N-terminal calponin-homology (CH) domains and a block of putative calmodulin-binding IQ domains common in actin binding cytoskeletal and signaling proteins. Previous studies in mouse suggest that ASPM is preferentially expressed in the developing brain. Our analyses reveal that ASPM is widely expressed in fetal and adult tissues and upregulated in malignant cells. Several alternatively spliced variants encoding putative ASPM isoforms with different numbers of IQ motifs were identified. The major ASPM transcript contains 81 IQ domains, most of which are organized into a higher order repeat (HOR) structure. Another prominent spliced form contains an in-frame deletion of exon 18 and encodes 14 IQ domains not organized into a HOR. This variant is conserved in mouse. Other spliced variants lacking both CH domains and a part of the IQ motifs were also detected, suggesting the existence of isoforms with potentially different functions. To elucidate the biochemical function of human ASPM, we developed peptide specific antibodies to the N- and C-termini of ASPM. In a western analysis of proteins from cultured human and mouse cells, the antibodies detected bands with mobilities corresponding to the predicted ASPM isoforms. Immunostaining of cultured human cells with antibodies revealed that ASPM is localized in the spindle poles during mitosis. This finding suggests that MCPH is the consequence of an impairment in mitotic spindle regulation in cortical progenitors due to mutations in ASPM.

The monocyclic beta-lactam antibiotic nocardicin A is related structurally and biologically to the bicyclic beta-lactams comprised of penicillins/cephalosporins, clavams, and carbapenems. Biosynthetic gene clusters are known for each of the latter, but not for monocyclic beta-lactams. A previously cloned gene encoding an enzyme specific to the biosynthetic pathway was used to isolate the nocardicin A cluster from Nocardia uniformis. Sequence analysis revealed the presence of 14 open reading frames involved in antibiotic production, resistance, and export. Among these are a two-protein nonribosomal peptide synthetase system, p-hydroxyphenylglycine biosynthetic genes, an S-adenosylmethionine-dependent 3-amino-3-carboxypropyl transferase (Nat), and a cytochrome P450. Gene disruption mutants of Nat, as well as an activation domain of the NRPS system, led to loss of nocardicin A formation. Several enzymes involved in antibiotic biosynthesis were heterologously overproduced, and biochemical characterization confirmed their proposed activities.

p-Hydroxyphenylpyruvate dioxygenase (HPD) plays a key role in the normal catabolism of tyrosine. An Fe2+/oxygen-dependent enzyme, it converts p-hydroxyphenylpyruvate into homogentisate and is part of the superfamily of alpha-ketoglutarate-dependent enzymes that couples oxidative decarboxylation of an alpha-ketoacid cofactor to oxidative modification of its substrate. In this case, the alpha-ketoacid is part of the substrate side chain. HPD shows strong homology to p-hydroxymandelate synthase (HMS), an enzyme that catalyzes the formation of p-hydroxymandelate from p-hydroxyphenylpyruvate, an early step in the biosynthesis of p-hydroxyphenylglycine, which is a nonproteinogenic amino acid incorporated into several biologically active secondary metabolites. Sequence alignment between the HPD and the HMS enzyme families and analysis of the Pseudomonas fluorescens HPD crystal structure highlighted four residues within each active site that may play roles in catalytic differentiation between the two products. We attempted to convert Streptomyces avermitilis HPD into an engineered S. avermitilis HMS by site-directed mutagenesis of these four residues individually and in combination. HPLC assay analysis of each His6-tagged mutant indicated that F337I successfully produced p-hydroxymandelate, along with homogentisate and an unknown compound. The structure of the latter was determined to be an oxepinone derived from the benzene-oxide intermediate long hypothesized in HPD catalysis.

Using a single ferrous active site, clavaminate synthase 2 (CS2) activates O(2) and catalyzes the hydroxylation of deoxyguanidinoproclavaminic acid (DGPC), the oxidative ring closure of proclavaminic acid (PC), and the desaturation of dihydroclavaminic acid (and a substrate analogue, deoxyproclavaminic acid (DPC)), each coupled to the oxidative decarboxylation of cosubstrate, alpha-ketoglutarate (alpha-KG). CS2 can also catalyze an uncoupled decarboxylation of alpha-KG both in the absence and in the presence of substrate, which results in enzyme deactivation. Resting CS2/Fe(II) has a six-coordinate Fe(II) site, and alpha-KG binds to the iron in a bidentate mode. The active site becomes five-coordinate only when both substrate and alpha-KG are bound, the latter still in a bidentate mode. Absorption, CD, MCD, and VTVH MCD studies of the interaction of CS2 with DGPC, PC, and DPC provide significant molecular level insight into the structure/function correlations of this multifunctional enzyme. There are varying amounts of six-coordinate ferrous species in the substrate complexes, which correlate to the uncoupled reaction. Five-coordinate ferrous species with similar geometric and electronic structures are present for all three substrate/alpha-KG complexes. Coordinative unsaturation of the Fe(II) in the presence of both cosubstrate and substrate appears to be critical for the coupling of the oxidative decarboxylation of alpha-KG to the different substrate oxidation reactions. In addition to the substrate orientation relative to the open coordination position on the iron site, it is hypothesized that the enzyme can affect the nature of the reactivity by further regulating the binding energy of the water to the ferrous species in the enzyme/succinate/product complex.