RESUMEN
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 PNPLA3, 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. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.
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Aciltransferasas , Aparato de Golgi , Gotas Lipídicas , Fosfolipasas A2 Calcio-Independiente , Humanos , Aciltransferasas/metabolismo , Aparato de Golgi/metabolismo , Lipasa/metabolismo , Lipasa/genética , Gotas Lipídicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Fosfolipasas A2 Calcio-Independiente/metabolismoRESUMEN
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 PNPLA3, 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. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.
RESUMEN
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. Here we explore the use of a commercially available skin model to investigate potential immune activation in response to subcutaneously injected biotherapeutics. Exposure to high levels of a mixture of process-related impurities (that are known potent immune system activators) induced a robust immune response from the skin model, as indicated by enhanced metabolic activity and increased secretion of 19 cytokines and chemokines. The skin model also responded to aggregated antibodies (generated by extreme mechanical stirring and pH-jump stress, which resulted in orders of magnitude higher particle numbers than that found in products), as shown by the secretion of several signature cytokines (GM-CSF, RANTES, and MCP-1). However, the magnitude of the responses to the aggregates were significantly lower than the response to the impurities. These results highlight the promising utility of in vitro skin models for investigating the potential immune response to process-related impurities and biotherapeutic attributes in a subcutaneous environment. The use of skin models for assessing drug safety may provide new insights to help guide drug product and process development, and potentially mitigate the risk of injection site reactions and systemic immunogenic responses that may compromise the safety and efficacy of subcutaneously administered drugs.
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Citocinas , Piel , Anticuerpos , Citocinas/metabolismo , Humanos , Inmunoterapia , Inyecciones Subcutáneas , Piel/metabolismoRESUMEN
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. Additionally, in vitro synergy of ABP 798 or RP with chemotherapeutic agents, in vivo xenograft studies in mice, and toxicological assessments in cynomolgus monkeys (including B cell depletion and toxicokinetics) were also conducted. Results from these non-clinical assessments contribute to the TOE supporting the biosimilarity between ABP 798 and rituximab RP across a range of primary and secondary MOAs and support justification for extrapolation to all indications of use for ABP 798 for which the RP is approved.
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Antineoplásicos , Biosimilares Farmacéuticos , Rituximab , Animales , Antineoplásicos/farmacología , Biosimilares Farmacéuticos/farmacología , Humanos , Células Asesinas Naturales/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Estándares de Referencia , Rituximab/farmacologíaRESUMEN
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. CONCLUSIONS: These results also support the scientific justification of extrapolation to all approved indications of trastuzumab given the established functional similarity of the two products and the same mechanisms of action across all conditions of use.
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Antineoplásicos/química , Biosimilares Farmacéuticos/química , Trastuzumab/química , Animales , Unión Competitiva , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Cinética , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales , Unión Proteica , Receptor ErbB-2/química , Transducción de Señal , Neoplasias Gástricas/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
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. In addition, we demonstrate that retention at the injection site through aggregation is concentration-dependent and leads to macrophage association and germinal center localization. Although there was delayed disposition of the aggregated antibody to draining lymph nodes, no overall impact on the immune response in lymph nodes, systemic exposure of the antibody, or enhancement of the anti-drug antibody response was evident. Unexpectedly, retention of the precipitated antibody in the subcutaneous space delayed the onset of the immune response and led to an immune suppressive response. Thus, we conclude that precipitation due to poor solubility of high doses of antibody formulations delivered subcutaneously may not be of special concern in terms of exposure or immunogenicity.
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Anticuerpos Monoclonales/inmunología , Reacción en el Punto de Inyección/inmunología , Agregado de Proteínas/inmunología , Tejido Subcutáneo/efectos de los fármacos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Femenino , Centro Germinal/efectos de los fármacos , Centro Germinal/inmunología , Humanos , Reacción en el Punto de Inyección/sangre , Inyecciones Subcutáneas , Masculino , Ratones , Solubilidad , Tejido Subcutáneo/inmunología , Distribución TisularRESUMEN
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.g. surface immunophenotyping). In addition to typical considerations regarding stability of the cell type of interest, stability of the target-bound therapeutic agent and stability of the target receptor must be taken into account. Reagent selection is also challenging as reagents need to be evaluated for the potential to compete with the therapeutic agent and bind with comparable affinity. This article provides technical guidance for the development and validation of cytometry-based receptor occupancy assays.
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Anticuerpos Monoclonales/inmunología , Descubrimiento de Drogas , Citometría de Flujo , Anticuerpos Monoclonales/uso terapéutico , Colorantes Fluorescentes/uso terapéutico , HumanosRESUMEN
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. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents.
Asunto(s)
Anticuerpos/inmunología , Descubrimiento de Drogas , Citometría de Flujo/métodos , Anticuerpos/uso terapéutico , Citometría de Flujo/tendencias , HumanosRESUMEN
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 (0.1, 0.3, 1, or 3 mg/kg subcutaneous [SC] or 1, 3, or 6 mg/kg intravenous [IV]) or placebo (phase 1a; N = 54), or four weekly doses of blisibimod (0.3, 1, or 3 mg/kg SC or 6 mg/kg IV) or placebo (phase 1b; N = 63). Safety and tolerability measures were collected, and B cell subset measurements and pharmacokinetic analyses were performed. RESULTS: All subjects (93 % female; mean age 43.7 years) carried the diagnosis of SLE for ≥ 1 year. Single- and multiple-dose treatment with blisibimod produced a decrease in the number of naïve B cells (24-76 %) and a transient relative increase in the memory B cell compartment, with the greatest effect on IgD(-)CD27+; there were no notable changes in T cells or natural killer cells. With time, memory B cells reverted to baseline, leading to a calculated 30 % reduction in total B cells by approximately 160 days after the first dose. In both the single- and multiple-dosing SC cohorts, the pharmacokinetic profile indicated slow absorption, dose-proportional exposure from 0.3 through 3.0 mg/kg SC and 1 through 6 mg/kg IV, linear pharmacokinetics across the dose range of 1.0-6.0 mg/kg, and accumulation ratios ranging from 2.21 to 2.76. The relative increase in memory B cells was not associated with safety signals, and the incidence of adverse events, anti-blisibimod antibodies, and clinical laboratory abnormalities were comparable between blisibimod- and placebo-treated subjects. CONCLUSIONS: Blisibimod changed the constituency of the B cell pool and single and multiple doses of blisibimod exhibited approximate dose-proportional pharmacokinetics across the dose range 1.0-6.0 mg/kg. The safety and tolerability profile of blisibimod in SLE was comparable with that of placebo. These findings support further studies of blisibimod in SLE and other B cell-mediated diseases. TRIAL REGISTRATION: Clinicaltrials.gov NCT02443506 . Registered 11 May 2015. NCT02411136 Registered 7 April 2015.
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Factor Activador de Células B/metabolismo , Subgrupos de Linfocitos B/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Adulto , Área Bajo la Curva , Factor Activador de Células B/antagonistas & inhibidores , Subgrupos de Linfocitos B/efectos de los fármacos , Mareo/inducido químicamente , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Esquema de Medicación , Femenino , Cefalea/inducido químicamente , Humanos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Lupus Eritematoso Sistémico/tratamiento farmacológico , Recuento de Linfocitos , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Náusea/inducido químicamente , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/efectos adversos , Resultado del Tratamiento , Adulto JovenRESUMEN
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. In control patients, females had higher levels of endotoxin neutralization than males, mirroring clinical outcomes from bacterial infection and sepsis. In addition to the total amount of neutralization, we used inactivation techniques to elucidate the nature of this activity and develop a system to compare early and late immune responses. Using this method to monitor patients with inflammatory bowel disease, we found a more robust total response that relies more on long-term, adaptive components of the immune system and less on early, innate components. Our results indicate that endotoxin neutralization is a valuable method to discern inflammatory bowel disease patients from a control population. Additionally, the nature of neutralization may be valuable in monitoring disease severity and/or the role of medication.
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Biomarcadores/metabolismo , Endotoxinas/metabolismo , Enfermedades Inflamatorias del Intestino/diagnóstico , Pruebas de Neutralización , Ácidos/farmacología , Adulto , Distribución por Edad , Anciano , Femenino , Calor , Humanos , Masculino , Persona de Mediana Edad , Índice de Severidad de la EnfermedadRESUMEN
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. Peripheral blood mononuclear cells were studied by B cell ELISPOT. These assays were built upon precedent assay development in cynomolgus monkeys, which pointed towards their utility in humans. Primary anti-KLH IgG responses rose to a mean of 65-93-fold above baseline for HV and SLE patients, respectively, and secondary responses rose to a mean of 260-170-fold above baseline. High levels of anti-tetanus IgG were detected in pre-immunization samples and their levels did not change over the course of study. Anti-KLH IgG1-4 subclasses were characterized by a predominant IgG1 response, with no significant differences in subclass magnitude or distribution between HV and SLE subjects. Anti-KLH IgM levels were detectable, although the overall response was lower. IgM was not detected in two SLE subjects whodid generate an IgG response. All subjects responded to KLH by B cell ELISPOT, with no significant differences observed between HV and SLE subjects. The CBA and B cell ELISPOT assays reliably measured anti-KLH B cell responses, supporting use of this approach and these assays to assess the pharmacodynamic and potential safety impact of marketed/investigational immune-therapeutics.
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Adyuvantes Inmunológicos/administración & dosificación , Linfocitos B/inmunología , Hemocianinas/administración & dosificación , Lupus Eritematoso Sistémico/prevención & control , Vacunación , Linfocitos B/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Hemocianinas/inmunología , Humanos , Inmunoglobulina G/sangre , Lupus Eritematoso Sistémico/inmunologíaRESUMEN
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. Some technical considerations are also addressed.
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Cartílago/anatomía & histología , Colágenos Fibrilares , Imagenología Tridimensional/métodos , Animales , Crioultramicrotomía , Humanos , Microscopía de Fluorescencia por Excitación MultifotónicaRESUMEN
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. Furthermore, important instrument parameters should be considered including the maintenance and operation of the cytometer in a controlled state to ensure aspects like data traceability, reliability, and integrity. In the present chapter we describe a method to evaluate caspase activation in Colo205 cells and fine needle aspirate tumors by slide-based, laser scanning cytometry. This approach can be applied to cell cultures, preclinical and clinical fine needle aspirate material.
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Biomarcadores de Tumor/metabolismo , Caspasa 3/metabolismo , Citometría de Barrido por Láser/métodos , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Biopsia con Aguja Fina , Línea Celular Tumoral , Neoplasias Colorrectales , Citotoxinas/farmacología , Activación Enzimática , Humanos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Coloración y Etiquetado/métodosRESUMEN
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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Citometría de Flujo/métodos , Citometría de Flujo/normas , Ensayos Clínicos como Asunto , Humanos , Manejo de EspecímenesRESUMEN
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. Whole blood was collected for flow cytometric analysis on days 1, 8, 15, 105, 195, 285, and 375 and held overnight for analysis. A subset of samples was additionally analyzed on the day of collection. RESULTS: Variability arising from overnight storage of whole blood was found to be within 20% for most lymphocyte subsets. There was greater between rather than within subject variability over a 1-year period. As anticipated, the data showed higher CD38 and lower CD19 densities on B cells from patients with SLE compared to HV. Although a higher percentage of cells with markers of plasmablasts/cells were observed in the blood of patients with SLE relative to HV, these measurements were found to be among the least reliable (i.e., most variable). CONCLUSIONS: This study provides technical perspectives for those conducting immunophenotypic analyses of B-cells in patients with SLE. We envision that our data, which addresses sample stability issues and presents a method to describe the relative reliability of one measure over another, holds value for clinical assessments of B-cells in SLE and the evaluation of investigational agents designed to modify the B-cell compartment.
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Subgrupos de Linfocitos B/citología , Conservación de la Sangre , Lupus Eritematoso Sistémico , Adolescente , Adulto , Subgrupos de Linfocitos B/inmunología , Errores Diagnósticos , Femenino , Citometría de Flujo , Humanos , Inmunofenotipificación , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/patología , Masculino , Persona de Mediana Edad , Estándares de Referencia , Factores de TiempoRESUMEN
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. The median fluorescence intensity of phosphorylated ribosomal protein S6 in lymphocytes was assessed on a BD FACSCalibur. RESULTS: We demonstrate here that tubes pre-loaded with lyophilized versions of the liquid reagents can provide equivalent stimulation in healthy volunteer specimens. CONCLUSIONS: The value of this approach is that it safeguards against omission or erroneous addition of bulk liquid formulations of PMA and ionomycin to the reaction vessel (i.e., plate or tube) and also lends itself to extended stability/shelf-life of these reagents. On the basis of this initial success, we plan to expand our evaluation of lyophilized reagents so that they can be incorporated into our clinical biomarker campaigns as appropriate.
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Ionomicina/farmacología , Linfocitos/citología , Linfocitos/efectos de los fármacos , Proteína S6 Ribosómica/análisis , Acetato de Tetradecanoilforbol/farmacología , Biomarcadores/análisis , Recuento de Células Sanguíneas , Dimetilsulfóxido/farmacología , Citometría de Flujo , Humanos , Inmunoensayo/métodos , Activación de Linfocitos , Linfocitos/químicaRESUMEN
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. The binding of serum antibodies to the immobilized proteins are detected and recorded in real time based on the principles of SPR. Furthermore, antibody binding is confirmed with a secondary anti-human immunoglobulin antibody. Positive samples are further characterized to determine the relative concentration of the antibodies using an affinity-purified, rabbit anti-epoetin alfa antibody as a reference control. The assay can detect 80ng/ml and 100ng/ml of antibody to epoetin alfa and darbepoetin alfa, respectively. The dynamic range of the assay is from 0.078microg/ml to 10microg/ml using a rabbit antibody with demonstrated accuracy and intra- and inter-assay precision. Approximately 80 serum samples can be analyzed on each sensor chip while maintaining a stable baseline and consistent immunological reactivity. The analysis of serum samples from subjects administered with epoetin alfa or darbepoetin alfa provided evidence that the assay can detect varying concentrations of antibodies of different off rates, isotypes, and IgG subclasses.
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Anticuerpos/sangre , Eritropoyetina/análogos & derivados , Eritropoyetina/sangre , Hematínicos/sangre , Resonancia por Plasmón de Superficie/métodos , Animales , Anticuerpos/metabolismo , Técnicas Biosensibles/métodos , Darbepoetina alfa , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Epoetina alfa , Congelación , Humanos , Inmunoensayo/métodos , Unión Proteica , Conejos , Proteínas Recombinantes , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie/instrumentaciónRESUMEN
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. The number of tryptase-positive MCs approximately doubled 14 days after cutaneous injury in nonhuman primates. Treatment of animals with anti-CD117 or imatinib mesylate (Gleevec) reduced MC accumulation at the edge of healing wounds in mice and nonhuman primates, respectively. In translating this MC assay to become a biomarker for human studies, no differences in dermal MC numbers were evident between genders, ages or body mass index from 20 healthy donors. These data suggest that skin is a practical and useful tissue for tracking pharmacodynamic effects of MC-directed therapies.
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Mastocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Piel/inmunología , Cicatrización de Heridas/inmunología , Animales , Benzamidas , Chlorocebus aethiops , Humanos , Mesilato de Imatinib , Citometría de Barrido por Láser , Mastocitos/inmunología , Ratones , Ratones Endogámicos C3H , Piperazinas/farmacología , Pirimidinas/farmacología , Transducción de Señal , Triptasas/metabolismoRESUMEN
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. Moreover, cells of interest can be inadvertently excluded from the gate, and relationships between collected variables may go unappreciated because they were not included in the original analysis plan. A multivariate non-gating technique was developed and implemented that accomplished the same goal as traditional gating while eliminating many weaknesses. The procedure was validated against traditional gating for analysis of circulating B cells in normal donors (n = 20) and persons with Systemic Lupus Erythematosus (n = 42). The method recapitulated relationships in the dataset while providing for an automated and objective assessment of the data. Flow cytometry analyses are amenable to automated analytical techniques that are not predicated on discrete operator-generated gates. Such alternative approaches can remove subjectivity in data analysis, improve efficiency and may ultimately enable construction of large bioinformatics data systems for more sophisticated approaches to hypothesis testing.
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Citometría de Flujo/estadística & datos numéricos , Algoritmos , Subgrupos de Linfocitos B/clasificación , Subgrupos de Linfocitos B/inmunología , Estudios de Casos y Controles , Biología Computacional , Interpretación Estadística de Datos , Humanos , Inmunofenotipificación/estadística & datos numéricos , Lupus Eritematoso Sistémico/inmunología , Modelos EstadísticosRESUMEN
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. Our data show that this assay is capable of detecting anti-epoetin alfa antibodies with a relative antibody concentration of 50 ng/ml, where 25 of 25 sera spiked with antibodies at this concentration scored positive. Moreover, the assay was designed to include positive and negative control beads for each specimen that is processed to ensure the specificity of the signal when detected. Measurement of interassay precision supports quantitative estimates of relative antibody concentrations in the range of 313 to 5,000 ng/ml, where the percent coefficient of variation did not exceed 20%. With respect to clinical specimens, antibodies with specificity for epoetin alfa could be easily detected in a set of specimens from persons with pure red blood cell aplasia that had prior exposure to the EPREX brand of recombinant epoetin alfa. Further development and validation of this approach may facilitate successful widespread application of the method for detection of anti-epoetin alfa antibodies, as well as antibodies directed against other recombinant therapeutic proteins.