Publications by authors named "John Ferbas"

Article Synopsis
  • Small interfering RNAs (siRNAs) have the potential to target and silence difficult disease-related genes, providing new ways to treat diseases.* -
  • While delivering siRNAs to the liver via N-acetylgalactosamine (GalNAc) has proven effective, delivering siRNAs to other cell types remains a challenge.* -
  • Research shows that certain cellular mechanisms, including retrograde transport and lipid droplets, can enhance the effectiveness of siRNA delivery and gene silencing in both liver and non-liver cell types.*
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Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene , encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear.

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Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene , encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear.

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Subcutaneous (SubQ) injection is a common administration route for biotherapeutics. However, limited tools are available for understanding the dynamic relationships between drug products and resident cells following injection. Advances in tissue engineering have enabled the production of in vitro skin models that recapitulate the morphological structure and functional activity of human skin.

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ABP 798 is a biosimilar to Rituxan® (rituximab reference product [RP]). Non-clinical assessments relevant to the primary and secondary mechanisms of action (MOA) contribute to the totality of the evidence (TOE) in supporting biosimilarity and are critical in providing scientific evidence for extrapolation of indications. Similarity of ABP 798 with rituximab RP was investigated across a range of biological activities which have potential impact on pharmacokinetics and clinical efficacy with non-clinical assessments relevant to MOA such as CD20 internalization, trogocytosis, binding to primary human natural killer (NK) cells as well as the ability to induce antibody-dependent cellular phagocytosis (ADCP) in peripheral blood mononuclear cells.

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Purpose: The in vitro and in vivo pharmacologic assessment of ABP 980 similarity to its reference product is intended to compare the activity of ABP 980 and trastuzumab and support the overall conclusion of similarity based on a comprehensive analytical and functional evaluation.

Methods: This work complements the primary assessment of functional similarity with additional in vitro assays, binding studies, and non-clinical studies including human epidermal growth factor receptor-2 (HER2) kinetic binding, HER2 signaling, HER2 internalization, synergy with docetaxel chemotherapy, FcγR kinetic binding, primary natural killer and monocyte cell binding, antibody-dependent cellular phagocytosis activity, in vivo xenograft studies, and toxicokinetic parameters.

Results: The results contribute to the totality of evidence with respect to functional similarity and support that ABP 980 is similar to trastuzumab in all primary and secondary mechanisms of action.

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Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody.

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The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent.

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Receptor occupancy measurements demonstrate the binding of a biotherapeutic agent to its extra-cellular target and represent an integral component of the pharmacodynamic (PD) portfolio utilized to advance the development and commercialization of a therapeutic agent. Coupled with traditional pharmacokinetic (PK) assessments derived from serum drug concentration, receptor occupancy data can be used to model PK/PD relationships and validate dose selection decisions throughout the drug development lifecycle. Receptor occupancy assays can be even more challenging to develop than other flow cytometric methods (e.

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Introduction: Blisibimod is a potent B cell-activating factor (BAFF) antagonist that binds to both cell membrane-expressed and soluble BAFF. The goal of these first-in-human studies was to characterize the safety, tolerability, and pharmacokinetic and pharmacodynamic profiles of blisibimod in subjects with systemic lupus erythematosus (SLE).

Methods: SLE subjects with mild disease that was stable/inactive at baseline received either a single dose of blisibimod (0.

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Gram-negative bacterial endotoxin is a potent immunostimulant implicated in the development and/or progression of a variety of diseases. The mammalian immune system has both innate and adaptive immune responses to neutralize endotoxin. In this study, a system was developed to monitor bacterial exposure by measuring the extent and nature of endotoxin neutralization in plasma.

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The aim of the study was to characterize performance of a complementary set of assays to measure antigen-specific immune responses in subjects immunized with a neoantigen. Healthy volunteers (HV) (n = 8) and patients with systemic lupus erythematosus (SLE) (n = 6) were immunized with keyhole limpet haemocyanin (KLH) on days 1 and 29. Serum antibodies were detected using a flow cytometric bead array (CBA) that multiplexed the KLH response alongside pre-existing anti-tetanus antibodies.

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Multiphoton-induced second-harmonic generation (SHG) has developed into a very powerful approach for in depth visualization of some biological structures with high specificity. In this unit, we describe the basic principles of three-dimensional SHG microscopy. In addition, we illustrate how SHG imaging can be utilized to assess collagen fibrils in biological tissues.

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Quantitation of activated caspases in xenograft models by laser scanning cytometry has demonstrated mechanism-specific biological activity of Anti-Trail Receptor immunoglobulin therapies in situ. These preclinical data confirmed that caspase activation is an early event that precedes tumor regression. To apply this platform for clinical monitoring of caspase activation using fine needle aspirate (FNA) biopsies, additional assay feasibility and validation experiments need be addressed.

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Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

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Background: The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay set-up. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry.

Methods: Five reagents lyophilized from ethanol or CHAPS buffer stock solution of phorbol 12-myristate 13-acetate (PMA) and ionomycin were benchmarked against standard DMSO liquid formulation for their stimulation equivalency.

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Objective: Characterization of peripheral leukocytes is an important aspect of monitoring the effect of immunotherapeutic interventions in systemic lupus erythematosus (SLE). We analyzed cell surface markers commonly used to assess patients with SLE, focusing on the effect of holding blood prior to processing/analysis and the relative reliability of the measurements that were conducted.

Methods: Healthy volunteers (HV; n = 20) and patients with SLE (n = 42) were studied.

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A surface plasmon resonance (SPR)-based biosensor immunoassay was developed and validated using the Biacore 3000 instrument to detect, semi-quantitate, and characterize serum antibodies against darbepoetin alfa (Aranesp) and epoetin alfa (EPOGEN). In this sensitive, dual-flow cell assay, epoetin alfa and darbepoetin alfa are covalently immobilized onto consecutive flow cells of a carboxymethyl dextran-coated sensor chip. Diluted human serum samples are injected sequentially over both surfaces.

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Mast cells (MCs) have important functional roles in leukocyte recruitment, pain, and wound healing, and increased tissue resident MC function has been associated with several fibrotic diseases. Consequently, the study of MCs in situ can be a direct approach to studying the pharmacodynamic impact of MC-directed therapeutics in tissues. Here we describe an automated laser scanning cytometry assay that was used to characterize the kinetics of MC accumulation in healing skin wounds and to study the effect of inhibiting CD117 (cKit) signaling.

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Flow Cytometry has become a mainstay technique for measuring fluorescent and physical attributes of single cells in a suspended mixture. These data are reduced during analysis using a manual or semiautomated process of gating. Despite the need to gate data for traditional analyses, it is well recognized that analyst-to-analyst variability can impact the dataset.

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Immunogenicity profiles of recombinant therapeutic proteins are important to understand because antibodies raised against these molecules may have important clinical sequelae. The purpose of the present study was to demonstrate that a flow cytometric bead array could be used to detect clinically relevant antibodies with specificity to such therapeutics. We chose to evaluate well-characterized specimens from persons treated with epoetin alfa that developed antibody-mediated pure red blood cell aplasia as a means to demonstrate the utility of this platform.

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Most immunopathogenesis studies of HIV-1 use peripheral blood. Most lymphocytes reside in lymphoid tissues, however, and the extent to which blood mirrors tissues is unclear. Here, we analyze lymphocytes in blood and lymph nodes of HIV-1-uninfected and -infected persons.

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Background: From May 1998 to November 2000, 13 European patients developed antibody-mediated pure red cell aplasia during treatment with recombinant human erythropoietin (rHuEPO), reinforcing the need for analytical testing for antibodies against erythropoietic agents. Specimens from 8 patients were provided for further antibody testing and characterization.

Methods: We evaluated 4 analytical methods with these sera: radioimmune precipitation (RIP), enzyme-linked immunosorbent assay (ELISA), biosensor immunoassay, and a bioassay for identification of neutralizing antibodies.

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Objective: To develop a validated BIACORE immunoassay for the detection and characterization of serum antibodies with specificity for erythropoietic molecules (e.g. darbepoetin alfa).

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Purpose: Epratuzumab is a novel humanized antihuman CD22 IgG1 antibody that has recently shown promising clinical activity, both as a single agent and in combination with rituximab, in patients with non-Hodgkin's lymphomas (NHL). In an attempt to better understand the mode of action of epratuzumab, the antibody was tested in vitro in a variety of cell-based assays similar to those used to evaluate the biological activity of other therapeutic monoclonal antibodies, including rituximab. In this report, we present epratuzumab activities as they relate to binding, signaling, and internalization of the receptor CD22.

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