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1.
PLoS Med ; 17(10): e1003348, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33125391

RESUMEN

BACKGROUND: Biopharmaceutical products (BPs) are widely used to treat autoimmune diseases, but immunogenicity limits their efficacy for an important proportion of patients. Our knowledge of patient-related factors influencing the occurrence of antidrug antibodies (ADAs) is still limited. METHODS AND FINDINGS: The European consortium ABIRISK (Anti-Biopharmaceutical Immunization: prediction and analysis of clinical relevance to minimize the RISK) conducted a clinical and genomic multicohort prospective study of 560 patients with multiple sclerosis (MS, n = 147), rheumatoid arthritis (RA, n = 229), Crohn's disease (n = 148), or ulcerative colitis (n = 36) treated with 8 different biopharmaceuticals (etanercept, n = 84; infliximab, n = 101; adalimumab, n = 153; interferon [IFN]-beta-1a intramuscularly [IM], n = 38; IFN-beta-1a subcutaneously [SC], n = 68; IFN-beta-1b SC, n = 41; rituximab, n = 31; tocilizumab, n = 44) and followed during the first 12 months of therapy for time to ADA development. From the bioclinical data collected, we explored the relationships between patient-related factors and the occurrence of ADAs. Both baseline and time-dependent factors such as concomitant medications were analyzed using Cox proportional hazard regression models. Mean age and disease duration were 35.1 and 0.85 years, respectively, for MS; 54.2 and 3.17 years for RA; and 36.9 and 3.69 years for inflammatory bowel diseases (IBDs). In a multivariate Cox regression model including each of the clinical and genetic factors mentioned hereafter, among the clinical factors, immunosuppressants (adjusted hazard ratio [aHR] = 0.408 [95% confidence interval (CI) 0.253-0.657], p < 0.001) and antibiotics (aHR = 0.121 [0.0437-0.333], p < 0.0001) were independently negatively associated with time to ADA development, whereas infections during the study (aHR = 2.757 [1.616-4.704], p < 0.001) and tobacco smoking (aHR = 2.150 [1.319-3.503], p < 0.01) were positively associated. 351,824 Single-Nucleotide Polymorphisms (SNPs) and 38 imputed Human Leukocyte Antigen (HLA) alleles were analyzed through a genome-wide association study. We found that the HLA-DQA1*05 allele significantly increased the rate of immunogenicity (aHR = 3.9 [1.923-5.976], p < 0.0001 for the homozygotes). Among the 6 genetic variants selected at a 20% false discovery rate (FDR) threshold, the minor allele of rs10508884, which is situated in an intron of the CXCL12 gene, increased the rate of immunogenicity (aHR = 3.804 [2.139-6.764], p < 1 × 10-5 for patients homozygous for the minor allele) and was chosen for validation through a CXCL12 protein enzyme-linked immunosorbent assay (ELISA) on patient serum at baseline before therapy start. CXCL12 protein levels were higher for patients homozygous for the minor allele carrying higher ADA risk (mean: 2,693 pg/ml) than for the other genotypes (mean: 2,317 pg/ml; p = 0.014), and patients with CXCL12 levels above the median in serum were more prone to develop ADAs (aHR = 2.329 [1.106-4.90], p = 0.026). A limitation of the study is the lack of replication; therefore, other studies are required to confirm our findings. CONCLUSION: In our study, we found that immunosuppressants and antibiotics were associated with decreased risk of ADA development, whereas tobacco smoking and infections during the study were associated with increased risk. We found that the HLA-DQA1*05 allele was associated with an increased rate of immunogenicity. Moreover, our results suggest a relationship between CXCL12 production and ADA development independent of the disease, which is consistent with its known function in affinity maturation of antibodies and plasma cell survival. Our findings may help physicians in the management of patients receiving biotherapies.


Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/genética , Productos Biológicos/inmunología , Adalimumab/uso terapéutico , Adulto , Anticuerpos Monoclonales Humanizados/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Productos Biológicos/uso terapéutico , Terapia Biológica/métodos , Estudios de Cohortes , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/genética , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/genética , Femenino , Estudio de Asociación del Genoma Completo/métodos , Cadenas alfa de HLA-DQ/genética , Humanos , Inmunosupresores/uso terapéutico , Infliximab/uso terapéutico , Interferón beta-1a/uso terapéutico , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/genética , Estudios Prospectivos , Rituximab/uso terapéutico
2.
J Cell Biol ; 168(1): 35-40, 2005 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-15623583

RESUMEN

Cell cycle exit is required for proper differentiation in most cells and is critical for normal development, tissue homeostasis, and tumor suppression. However, the mechanisms that link cell cycle exit with differentiation remain poorly understood. Here, we show that the master melanocyte differentiation factor, microphthalmia transcription factor (MITF), regulates cell cycle exit by activating the cell cycle inhibitor INK4A, a tumor suppressor that frequently is mutated in melanomas. MITF binds the INK4A promoter, activates p16(Ink4a) mRNA and protein expression, and induces retinoblastoma protein hypophosphorylation, thereby triggering cell cycle arrest. This activation of INK4A was required for efficient melanocyte differentiation. Interestingly, MITF was also required for maintaining INK4A expression in mature melanocytes, creating a selective pressure to escape growth inhibition by inactivating INK4A. These findings demonstrate that INK4A can be regulated by a differentiation factor, establish a mechanistic link between melanocyte differentiation and cell cycle exit, and potentially explain the tissue-specific tendency for INK4A mutations to occur in melanoma.


Asunto(s)
Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Melanocitos/fisiología , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Línea Celular , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas de Unión al ADN/genética , Fibroblastos/citología , Fibroblastos/fisiología , Silenciador del Gen , Humanos , Melanocitos/citología , Ratones , Factor de Transcripción Asociado a Microftalmía , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/genética , Transcripción Genética
3.
AAPS J ; 20(3): 51, 2018 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-29549534

RESUMEN

Bridging immunoassays commonly used to detect and characterize immunogenicity during biologic development do not provide direct information on the presence or development of a memory anti-drug antibody (ADA) response. In this study, a B cell ELISPOT assay method was used to evaluate pre-existing ADA for anti-TNFR1 domain antibody, GSK1995057, an experimental biologic in treatment naive subjects. This assay utilized a 7-day activation of PBMCs by a combination of GSK1995057 (antigen) and polyclonal stimulator followed by GSK1995057-specific ELISPOT for the enumeration of memory B cells that have differentiated into antibody secreting cells (ASC) in vitro. We demonstrated that GSK1995057-specific ASC were detectable in treatment-naïve subjects with pre-existing ADA; the frequency of drug-specific ASC was low and ranged from 1 to 10 spot forming units (SFU) per million cells. Interestingly, the frequency of drug-specific ASC correlated with the ADA level measured using an in vitro ADA assay. We further confirmed that the ASC originated from CD27+ memory B cells, not from CD27--naïve B cells. Our data demonstrated the utility of the B cell ELISPOT method in therapeutic protein immunogenicity evaluation, providing a novel way to confirm and characterize the cell population producing pre-existing ADA. This novel application of a B cell ELISPOT assay informs and characterizes immune memory activity regarding incidence and magnitude associated with a pre-existing ADA response.


Asunto(s)
Anticuerpos/inmunología , Linfocitos B/inmunología , Productos Biológicos/inmunología , Inmunoensayo/métodos , Memoria Inmunológica , Anticuerpos/sangre , Células Productoras de Anticuerpos/inmunología , Linfocitos B/citología , Diferenciación Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Leucocitos Mononucleares/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología
4.
Methods Mol Biol ; 383: 81-99, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-18217680

RESUMEN

Squamous cell carcinomas and several other cancers have been found to exhibit microarray expression profiles that include genes related to nuclear factor (NF)-kappaB, a signal activated transcription factor that is evolutionarily important in regulating early response gene programs to injury and infection. Inhibition of NF-kappaB by expression of a dominant negative signal phosphorylation site mutant of inhibitor-kappaB, IkappaBalphaM, under a tetracycline inducible promoter, established the role of NF-kappaB as an essential molecular switch modulating multiple genes important in the malignant phenotype. Bioinfomatic analysis of the promoter and coding region of IkappaBalphaM-modulated genes has enabled identification of new candidates with and without known NF-kappaB related motifs for validation and functional studies of their relationship to NF-kappaB. These studies illustrate how microarray data can be used to generate a hypothesis regarding regulation of genes by a specific signal transcription factor, and how genetic mutants and bioinformatic analysis can be used to analyze the relative importance of the regulatory molecule to expression of genes involved in the malignant phenotype.


Asunto(s)
Carcinoma de Células Escamosas/genética , Biología Computacional , Regulación Neoplásica de la Expresión Génica , FN-kappa B/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Humanos , Ratones , Transducción de Señal/genética , Programas Informáticos , Tetraciclina/farmacología
5.
J Immunol Methods ; 441: 15-23, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27889561

RESUMEN

Mepolizumab, a humanized IgG1 monoclonal antibody that blocks native homodimeric interleukin-5 (IL-5) from binding to the IL-5 receptor, has recently been approved for treatment of severe eosinophilic asthma. Our initial immunogenicity assay method for phase I and II studies utilized a bridging electrochemiluminescence format with biotin and ruthenium-labelled mepolizumab linked by anti-drug antibodies (ADA). We discovered that IL-5 significantly increased in dosed subjects from a phase II study and that the increased IL-5 was in the form of a drug-bound complex. We demonstrated that the elevated drug-bound IL-5 produced false-positive response in the in vitro ADA assay, in which drug-bound IL-5 dissociated and then bridged mepolizumab conjugates to yield positive signal. To eliminate the IL-5 interference, we compared two strategies: a solid-phase immunodepletion of IL-5 and an in-solution IL-5 immunocompetition. We identified the best competitive antibody for each purpose. We found both methods demonstrated similar effectiveness in reducing the false positive signal in IL-5 spiked samples; however, the in-solution immunocompetition for IL-5 had fewer false positives in study samples. Additionally, the in-solution immunocompetition method was experimentally simpler to execute. We modified the ADA assay by adding a pre-treatment step with a mepolizumab competitive anti- IL-5 antibody. Using this new method, we retested clinical samples from two phase II studies (MEA112997 and MEA114092). The confirmed ADA positive incidence was reduced from 29% and 61% to 1% and 8% with the modified in-solution immune inhibition method. Target interference is a fairly common problem facing immunogenicity testing, and target-induced false positive cannot be distinguished from true ADA response by the commonly used drug competitive confirmation assay. The approach and method used here for resolving target interference in ADA detection will be useful for differentiating between a true ADA response and target induced false positive as well as similar challenges in other programs.


Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos/análisis , Técnicas Inmunológicas , Interleucina-5/inmunología , Interleucina-5/metabolismo , Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/tratamiento farmacológico , Ensayos Clínicos como Asunto , Propuestas de Licitación , Reacciones Falso Positivas , Humanos , Interleucina-5/antagonistas & inhibidores
6.
Cancer Res ; 64(18): 6511-23, 2004 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-15374962

RESUMEN

We reported previously that transcription factor nuclear factor (NF)-kappaB is constitutively activated in human and murine squamous cell carcinomas (SCCs). The role of NF-kappaB in the cumulative changes in gene expression with transformation and progression of the murine SCC Pam 212 and after switching off NF-kappaB by a dominant negative inhibitor kappaB mutant (IkappaBalphaM) was explored by profiling with a 15,000-element cDNA micoarrray. Remarkably, NF-kappaB modulated the expression of >60% of the 308 genes differentially expressed between normal keratinocytes and metastatic SCCs. NF-kappaB directly or indirectly modulated expression of programs of genes functionally linked to proliferation, apoptosis, adhesion, and angiogenesis. Among these, changes in expression of cyclin D1, inhibitor of apoptosis-1, mutant Trp53, and beta-catenin detected with modulation of NF-kappaB by microarray were confirmed by Western and Northern blot. NF-kappaB DNA binding motifs were detected in the promoter of approximately 63% of genes showing increased expression and 33% of the genes showing decreased expression. The ACTACAG motif implicated in the NF-kappaB-dependent down-regulation of mRNA expression of MyoD and Sox9 was detected in the coding portion of about 15% of genes showing increased or decreased expression. Inactivation of NF-kappaB inhibited malignant phenotypic features including proliferation, cell survival, migration, angiogenesis, and tumorigenesis. These results provide evidence that NF-kappaB is an important modulator of gene expression programs that contribute to the malignant phenotype of SCC.


Asunto(s)
Carcinoma de Células Escamosas/genética , FN-kappa B/genética , Neoplasias Cutáneas/genética , Animales , Secuencia de Bases , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Ciclina D1/biosíntesis , Ciclina D1/genética , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/genética , Doxiciclina/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas I-kappa B/genética , Proteínas Inhibidoras de la Apoptosis , Queratinocitos/metabolismo , Queratinocitos/fisiología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/antagonistas & inhibidores , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/efectos de los fármacos , Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Homología de Secuencia de Ácido Nucleico , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Transactivadores/biosíntesis , Transactivadores/genética , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , beta Catenina
7.
J Interferon Cytokine Res ; 36(12): 667-670, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27918711

RESUMEN

Previous epidemiologic studies showed an increased risk of neutralizing antibody (NAb) development against Interferon beta in multiple sclerosis patients who smoke. Cotinine is an easily detectable metabolite of nicotine and, therefore, can be used as an objective surrogate marker for smoking status. We measured cotinine levels in NAb-positive and NAb-negative patients to find a potential association of nicotine consumption and NAb development. Cotinine was measured in 37 patients with known smoking status and in 123 patients with unknown smoking status, all of whom were routinely tested for NAb. Cotinine was detected by an enzyme-linked immunosorbent assay, inhibition assay. We compared cotinine levels by NAb status and tested for the strength of association between cotinine and NAb status. We found a discrepancy between smoking status stated by patients and status defined by cotinine levels in 7 of 37 patients. In both cohorts, together with and without previously known smoking status (n = 160), we found 34% and 39% smokers, respectively, as defined by cotinine levels in NAb-negative and NAb-positive patients (P = 0.511). In our analysis, smoking was not associated with higher risk of NAb development. Moreover, smoking habits stated by patients do not always correlate with cotinine levels.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Autoanticuerpos/inmunología , Cotinina/sangre , Interferón beta/inmunología , Esclerosis Múltiple/sangre , Esclerosis Múltiple/inmunología , Adulto , Anticuerpos Neutralizantes/sangre , Austria , Autoanticuerpos/sangre , Biomarcadores , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/diagnóstico , Pronóstico , Fumar , Adulto Joven
8.
Bioanalysis ; 7(10): 1253-60, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26045004

RESUMEN

BACKGROUND: Adalimumab is a therapeutic antibody used for treating inflammatory diseases. To understand interindividual PK variability, there is a need to develop and validate an assay to measure serum adalimumab concentrations. METHODS: An ELISA was developed on microtiter plates coated with TNF-α. Seven nonzero adalimumab standards ranging from 0.05 to 50 mg/l and three quality controls (0.2, 2.5 and 7 mg/l) were tested for their intra and interday precision on six occasions. RESULTS: The LOD, LLOQ and ULOQ of the assay were 0.022, 0.073 and 9 mg/l, respectively. CONCLUSION: This method is accurate, reproducible and may be useful for PK studies and for therapeutic drug monitoring of adalimumab.


Asunto(s)
Adalimumab/sangre , Antiinflamatorios/sangre , Monitoreo de Drogas/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Límite de Detección , Reproducibilidad de los Resultados
9.
PLoS One ; 10(3): e0098153, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25789616

RESUMEN

OBJECTIVE: To assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the Fc-inactivated anti-ß amyloid (Aß) monoclonal antibody (mAb) GSK933776 in patients with mild Alzheimer's disease (AD) or mild cognitive impairment (MCI). METHODS: This was a two-part, single blind, placebo-controlled, first-time-in-human (FTIH) study of single (n = 18) and repeat dose (n = 32) intravenous GSK933776 0.001-6 mg/kg (ClinicalTrials.gov: NCT00459550). Additional safety data from an open-label, uncontrolled, single dose study of intravenous GSK933776 1-6 mg/kg (n = 18) are included (ClinicalTrials.gov: NCT01424436). RESULTS: There were no cases of amyloid-related imaging abnormalities-edema (ARIA-E) or -hemorrhage (ARIA-H) after GSK933776 administration in both studies. Three patients across the two studies developed anti-GSK933776 antibodies. Plasma GSK933776 half-life (t1/2) was 10-15 days after repeat dosing. After each of three administrations of GSK933776, plasma levels of total Aß42 and Aß increased whereas plasma levels of free Aß decreased dose dependently; no changes were observed for placebo. For total Aß42 the peak:trough ratio was ≤2 at doses ≥3 mg/kg; for total Aß the ratio was ≤2 at 6 mg/kg. CSF concentrations of Aß showed increases from baseline to week 12 for Aß X-38 (week 12:baseline ratio: 1.65; 95%CI: 1.38, 1.93) and Aß X-42 (week 12:baseline ratio: 1.18; 95%CI: 1.06, 1.30) for values pooled across doses. CONCLUSION: In this FTIH study the Fc-inactivated anti-Aß mAb GSK933776 engaged its target in plasma and CSF without causing brain ARIA-E/H in patients with mild AD or MCI. TRIAL REGISTRATION: ClinicalTrials.gov NCT00459550.


Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Anticuerpos Monoclonales/administración & dosificación , Fragmentos Fc de Inmunoglobulinas , Fragmentos de Péptidos/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/sangre , Péptidos beta-Amiloides/sangre , Anticuerpos Monoclonales/efectos adversos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Isoanticuerpos/sangre , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/sangre
10.
Curr Eye Res ; 27(2): 69-74, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-14632157

RESUMEN

In the past decade, there have been impressive advances in our understanding of chromosomal, genetic and molecular alterations that occur in uveal melanoma. Nevertheless, a coherent picture of the molecular pathogenesis of this eye cancer is yet to emerge. Herein, we review the findings to date, discuss the insights they provide, and suggest future directions for molecular research in uveal melanoma.


Asunto(s)
Melanoma/genética , Neoplasias de la Úvea/genética , Humanos , Pérdida de Heterocigocidad , Biología Molecular , Proteínas de Neoplasias/genética , Hibridación de Ácido Nucleico , Cariotipificación Espectral
11.
Alzheimers Res Ther ; 6(2): 19, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24716469

RESUMEN

INTRODUCTION: In this study, we evaluated the safety and pharmacodynamic effects of the Fc-inactivated anti-ß-amyloid (anti-Aß) monoclonal antibody GSK933776 in patients with mild Alzheimer's disease and mild cognitive impairment. Aß and tau levels were investigated in cerebrospinal fluid (CSF), and the relationship between Aß levels and Aß modulation in plasma was explored. The feasibility of a continuous sampling method using a lumbar catheter was assessed. METHODS: This trial was a phase I, open-label, uncontrolled, single-dose, exploratory experimental medicine study of intravenous GSK933776 at doses of 1 mg/kg, 3 mg/kg or 6 mg/kg (n = 6/group). The time course of plasma and CSF concentrations of GSK933776 and Aß was assessed. Sample size was based on feasibility, and no formal statistical analyses were performed. RESULTS: Following administration of GSK933776 at doses of 1 mg/kg, 3 mg/kg and 6 mg/kg, there were decreases from baseline in CSF Aß1-42 (from 0 to 12 hours) by 22.8 pg/ml (6.2%), 43.5 pg/ml (9.2%) and 60.5 pg/ml (12.5%), respectively. Plasma concentrations of total Aß18-35 and Aß4228-42 increased immediately after infusion and CSF tau concentration increased slightly, but did not significantly change, following administration of all doses of GSK933776. Pharmacokinetics confirmed the presence of GSK933776 in the CSF, which exhibited a dose-response relationship. One patient underwent minor surgery without sequelae following a ruptured lumbar catheter. CONCLUSION: GSK933776 demonstrated pharmacological activity and target engagement in CSF and plasma, and the continuous sampling method via a catheter successfully assessed the Aß changes following single-dose administration of GSK933776. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01424436. Registered 4 August 2011.

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