RESUMEN
The generation of anti-drug antibodies (ADAs) is one of the most serious problems in therapy using monoclonal antibodies (mAbs), because ADAs can impact the pharmacokinetics, efficacy, and safety of mAbs. It is therefore important to detect the generated ADAs in patients. For the appropriate detection of ADAs, methods that detect various types of ADAs (e.g., low- and high-affinity ADAs) are needed, but since there are no adequate reference preparations of ADAs relevant to human ADAs in most cases, it is difficult to determine whether or not the developed methods have enough analytical performance. Here, we developed human-rat chimeric ADA panels against the anti-TNF-α therapeutic antibodies infliximab and adalimumab. The developed ADA panels consist of 7 (for infliximab) and 11 (for adalimumab) ADAs with various binding characters, and most of the ADAs are neutralizing antibodies. Using these ADA panels, we compared the detectability of model methods, i.e., binding assays using SPR, BLI, and ECL, and a cell-based assay to detect neutralization activity. Since we obtained ADAs showing low and high responses with the various methods, the ADA panels we developed were shown to be useful for the development of ADA assays.
Asunto(s)
Adalimumab , Anticuerpos Neutralizantes , Infliximab , Adalimumab/química , Adalimumab/inmunología , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Células HEK293 , Humanos , Infliximab/química , Infliximab/inmunología , RatasRESUMEN
Cell therapy using endothelial progenitor cells (EPCs) is a promising strategy for the treatment of ischemic diseases. Two types of EPCs have been identified: early EPCs and late EPCs. Late EPCs are able to form tube structure by themselves, and have a high proliferative ability. The functional marker(s) of late EPCs, which relate to their therapeutic potential, have not been fully elucidated. Here we compared the gene expression profiles of several human cord blood derived late EPC lines which exhibit different tube formation activity, and we observed that the expression of occludin (OCLN) in these lines correlated with the tube formation ability, suggesting that OCLN is a candidate functional marker of late EPCs. When OCLN was knocked down by transfecting siRNA, the tube formation on Matrigel, the S phase + G2 /M phase in the cell cycle, and the spheroid-based sprouting of late EPCs were markedly reduced, suggesting the critical role of OCLN in tube formation, sprouting, and proliferation. These results indicated that OCLN plays a novel role in neovascularization and angiogenesis.
Asunto(s)
Células Progenitoras Endoteliales/metabolismo , Neovascularización Fisiológica , Ocludina/metabolismo , Línea Celular , Proliferación Celular , Sangre Fetal/citología , Puntos de Control de la Fase G2 del Ciclo Celular , Humanos , Neovascularización Fisiológica/genética , Ocludina/genética , Interferencia de ARN , Puntos de Control de la Fase S del Ciclo Celular , Transducción de Señal , Transcriptoma , TransfecciónRESUMEN
Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0-102.8% and 101.6-102.8%, respectively), good intermediate precision (1.9-2.1% and 2.4-4.2%, respectively) and good reproducibility (4.0-4.8% and 3.6-6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.
Asunto(s)
Inhibidores del Factor Xa , Heparina/farmacología , Protrombina/antagonistas & inhibidores , Anticoagulantes/metabolismo , Anticoagulantes/farmacología , Antitrombinas/metabolismo , Factor Xa/metabolismo , Heparina/metabolismo , Humanos , Oligopéptidos/metabolismo , Protrombina/metabolismo , Reproducibilidad de los Resultados , Tecnología Farmacéutica/métodosRESUMEN
Fcγ receptor IIa (FcγRIIa) plays an important role in antibody-dependent cellular cytotoxicity and inflammation. Changes in FcγRIIa expression levels or activity caused by genetic polymorphisms in FCGR2A, the gene encoding FcγRIIa, may lead to differences in disease progression as well as efficacy of antibody therapeutics between individuals. In this study, we sequenced the 5'-flanking region along with all exons and their flanking regions of FCGR2A from 111 Japanese subjects. Forty-eight genetic variations were found including 12 novel ones. Beside the well-known functional 497A > G (H166R) polymorphism, we detected 818T > C (L273P) at 0.005 frequency. Since the functional significance of this polymorphism has not been revealed, we next assessed the functions of the L273P substitution by expressing wild-type and the variant proteins in human Jurkat cells. The L273P variant protein showed similar cell surface expression and IgG-binding properties as the wild-type, but it exhibited a stronger signal transduction activity based on the approximately 2-fold enhancement of tyrosine phosphorylation of FcγRIIa itself. The current results suggest that L273P could have functional significance in the antibody-dependent clinical responses through FcγRIIa.
Asunto(s)
Pueblo Asiatico/genética , Polimorfismo Genético , Receptores de IgG/genética , Línea Celular , Exones , Humanos , Células Jurkat , Leucina/genética , Fosforilación , Prolina/genética , Transducción de Señal/genética , Tirosina/metabolismoRESUMEN
The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos/inmunología , Antígenos de Histocompatibilidad Clase I/fisiología , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/uso terapéutico , Receptores Fc/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/uso terapéutico , Células CHO , Cricetinae , Cricetulus , Semivida , Antígenos de Histocompatibilidad Clase I/sangre , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/sangre , Inmunoglobulina G/sangre , Recién Nacido , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores Fc/sangre , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Therapeutic immunoglobulin G (IgG) antibodies have comparatively long half-lives because the neonatal Fc receptor (FcRn) binds to the IgG Fc at acidic pH in the endosome and protects IgG from degradation. To further prolong the half-lives, amino acid-substituted antibodies having high affinity to FcRn are being developed, and one such therapeutic antibody (ravulizumab) has been approved. In this study, we investigated the binding property to FcγR and the conformation of seven FcRn affinity-modulated adalimumab variants to clarify the impact of the amino acid substitutions on the function and conformation of IgG Fc. The amino acid substitutions in T254-P261 caused a change in deuterium uptake into some regions of Fc in HDX-MS analysis, but those at T311, M432 and N438 did not cause such a change. The conformations around F245-L255 (FLFPPKPKDTL) were particularly influenced by the amino acid substitution in M256-P261, and the conformational changes of this region were correlated with the decrease of the affinity to FcγRIIIa. Additionally, we investigated the conformational difference of Fc between a Fc fusion protein (etanercept) and a native IgG (adalimumab). Although the Fc fusion proteins were expected to have similar FcRn affinity to IgGs, the affinity of etanercept to FcRn was lower than that of adalimumab, and its half-life was shorter than those of the IgG antibodies. Differences in deuterium uptakes were observed in the two regions where they were also detected in the adalimumab variants, and the conformational differences appeared to be an important factor for the low FcRn affinity of etanercept.
Asunto(s)
Anticuerpos Monoclonales/química , Afinidad de Anticuerpos/fisiología , Antígenos de Histocompatibilidad Clase I/química , Conformación Molecular , Receptores Fc/química , Diseño de Fármacos/métodos , Humanos , Ingeniería de Proteínas/métodosRESUMEN
It is necessary to know the amount of food on dishes in order to encourage taking medicine after eating. Also, for health management, it is vital to record what and how much a person ate. Although there are research cases using weight sensors or color cameras, it has been difficult to estimate the food volume accurately and inexpensively at home. In previous works, the authors developed a technique for estimating volume based on a depth image acquired by a depth camera. In this paper, the authors propose a new point cloud processing method for a more accurate estimation. A point cloud is a set of coordinate points on objects and is suitable for processing objects three-dimensionally. The authors have developed a technique for recognizing dishes on the dining table based on a point cloud and constructing the dish space. Additionally, another technique was developed for estimating the volume of food in the dish space.
Asunto(s)
Nube Computacional , Alimentos , ColorRESUMEN
Human lactoferrin (hLF), a soluble factor of the innate immune system, exhibits various biological functions and therefore has potential as a therapeutic protein. However, the clinical applications of hLF are limited by its low stability in blood. We therefore attempted to resolve this by producing recombinant hLF fused to human serum albumin (HSA). Two HSA-fused hLFs with different fusion orientations (hLF-HSA and HSA-hLF) were produced in Chinese hamster ovary (CHO) DG44 cells. hLF-HSA revealed higher thermal stability, resistance to peptic degradation, and stability during the process of cellular uptake and release in an intestinal enterocyte model (Caco-2 cells) than HSA-hLF. The lower stability of HSA-hLF is presumably due to the steric hindrance imposed by HSA fusion to the N-terminus of hLF. Both HSA fusion proteins, especially HSA-hLF, displayed improved pharmacokinetic properties despite the lower protein stability of HSA-hLF. hLF-HSA and HSA-hLF exhibited approximately 3.3- and 20.7-fold longer half-lives (64.0 and 403.6 min), respectively, than holo-rhLF (19.5 min). Both HSA fusion proteins were found to exert enhanced growth inhibition effects on cancer cells in vitro, but not normal cells. Their enhanced growth inhibitory activities were considered to be due to the synergetic effects of hLF and HSA because hLF alone or HSA alone failed to exert such an effect. Altogether, Fusion of HSA to hLF yielded superior pharmacokinetics and anti-proliferative activities against cancer cells. HSA-fused hLF is a novel candidate for further application of hLF as biopharmaceuticals for intravenous administration.
Asunto(s)
Lactoferrina , Neoplasias , Albúminas , Animales , Células CHO , Células CACO-2 , Cricetinae , Cricetulus , Humanos , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
During the development of monoclonal antibodies (mAbs) and other therapeutic proteins, immunogenicity, in particular the induction of anti-drug antibodies (ADAs), is an important concern, and thus immunogenicity assessment is a requirement for their approval. Establishment of appropriate methods for detecting and characterizing ADAs is necessary for immunogenicity assessment, but the lack of commonly available reference standards makes it difficult to compare and evaluate the methods. It is also difficult to compare the data with those obtained by other methods or facilities without reference standards. Here, we developed a panel of ADAs against anti-CD20 rituximab (Rituxan®, MabThera®); the panel consisted of eight clones of recombinant human-rat chimeric mAbs that target rituximab. The anti-rituximab mAbs showed different binding properties (specificity, epitope and affinity), and different neutralization potencies for CD20 binding, complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. The molecular size of the immune complex consisting of rituximab and the anti-rituximab mAb differed among the clones, and was well correlated with their level of Fcγ-receptor activation. These results suggest that the ADAs chosen for the newly developed panel are suitable surrogates for human ADAs, which exhibit different potential to affect the efficacy and safety of rituximab. Next, we used this panel to compare several ADA-detecting assays and revealed that the assays had different abilities to detect the ADAs with different binding characteristics. We conclude that our panel of ADAs against rituximab will be useful for the future development and characterization of assays for immunogenicity assessment.
Asunto(s)
Anticuerpos Monoclonales , Proteínas Recombinantes de Fusión , Rituximab/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Humanos , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Development of an appropriate assay to detect anti-drug antibody (ADA) is important for assessing immunogenicity to therapeutic protein products. However, characterizing ADA assay methods is difficult because human ADA as a reference standard is not available in most cases. We compared the analytical performance of three ligand-binding assay methods for ADA, namely, surface plasmon resonance (SPR), electrochemiluminescence (ECL), and biolayer interferometry (BLI) methods, by using the anti-erythropoietin (EPO) monoclonal antibody reference panel developed by the World Health Organization (WHO) in 2015. Dose-dependent binding responses were observed for all nine anti-EPO antibodies in the anti-EPO panel by the SPR and BLI methods. In contrast, the ECL method did not clearly detect binding of low-affinity anti-EPO antibodies. Regarding IgG2 and IgM antibodies derived from the same clone, IgG2 exhibited a higher binding response in the SPR assay, whereas the IgM binding response was higher than that of IgG2 in the ECL assay. In the case of the BLI method, there was no consistent pattern observed in the binding responses of IgG2 or IgM. Results of the anti-EPO antibody reference panel, which contains a variety of monoclonal antibodies, indicated that the ability to detect ADAs differed among these assay methods. Therefore, with ligand-binding assays, differences in assay platforms can affect the sensitivity and other characteristics of assays to detect ADAs. These results show that understanding the analytical performance of ADA assays is important for an appropriate assessment of immunogenicity. Our study also indicated the benefits of using the established human ADA reference panel to assess the assay methods for ADA detection.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Técnicas Electroquímicas , Eritropoyetina/inmunología , Humanos , Interferometría , Mediciones Luminiscentes , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie , Organización Mundial de la SaludRESUMEN
A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins.
Asunto(s)
Adalimumab/análisis , Complejo Antígeno-Anticuerpo/análisis , Fluorescencia , Infliximab/análisis , Factor de Necrosis Tumoral alfa/análisis , Adalimumab/química , Complejo Antígeno-Anticuerpo/química , Humanos , Infliximab/química , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Factor de Necrosis Tumoral alfa/química , UltracentrifugaciónRESUMEN
A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-alpha and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.
Asunto(s)
Caspasas/metabolismo , Potenciales de la Membrana , Mitocondrias/enzimología , Mitocondrias/fisiología , Secuencia de Aminoácidos , Caspasas/genética , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Electrofisiología , Activación Enzimática , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Estaurosporina/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Liver X receptors (LXRs) alpha and beta share considerable sequence homology and several functions, respond to the same endogenous and synthetic ligands, and play critical roles in maintaining lipid homeostasis. In this study, liverwort-derived riccardin C (RC) and F (RF) were identified as an LXRalpha agonist/LXRbeta antagonist and an LXRalpha antagonist, respectively. RC and RF bound to LXRs, but had different abilities to recruit a coactivator and thereby induce transactivation. Despite its unique subtype-selective activity, RC enhanced ABCA1 and ABCG1 expression and cellular cholesterol efflux in THP-1 cells. RC may provide a novel tool for identifying subtype-function and drug development.
Asunto(s)
Productos Biológicos/farmacología , Proteínas de Unión al ADN/agonistas , Proteínas de Unión al ADN/antagonistas & inhibidores , Éteres Cíclicos/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Línea Celular , Humanos , Receptores X del Hígado , Receptores Nucleares Huérfanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Therapeutic monoclonal antibodies (mAbs) exert their effects via binding to specific target molecules, which is expected to show rare off-target adverse reactions. However, nonclinical evaluation of mAbs is difficult because they often lack reactivity toward orthologous targets in animals. During the nonclinical evaluation of mAbs, not only the target molecules but fragment crystallizable (Fc) receptors, which regulate the immune effector functions and pharmacokinetic properties of mAbs, should be considered. In this review, factors for extrapolating nonclinical study results to clinical settings are discussed by focusing on Fc receptors. The human Fcγ receptor family consists of FcγRI, IIa, IIb, IIIa, and IIIb; Fcγ receptors in laboratory animals are structurally and functionally different from those in humans. In addition, interactions between human IgG-Fc, a component of therapeutic mAbs, and animal Fcγ receptors are still not fully understood. With regard to neonatal Fc receptor (FcRn), related molecules comprising the FcRn family are not known; however, critical amino acid residues involved in IgG binding are different between human and mouse. In case of next-generation mAbs with a novel structure or mode of action, knowledge from related drugs is limited. To ensure safety of next-generation mAbs, a thorough understanding of the differences in Fc receptors among species and the interactions between mAbs and Fc receptors is required, and the appropriateness of the nonclinical study design should be carefully examined prior to conducting clinical studies.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Cristalización , Humanos , Inmunoglobulina G/inmunología , Unión Proteica , Receptores Fc/inmunología , Receptores de IgG/inmunología , SeguridadRESUMEN
Many monoclonal antibodies have been developed for therapy over the last 2 decades. In the development of therapeutic antibodies, the preclinical assessment of an antibody's biodistribution is important for the prediction of the antibody's efficacy and safety. For imaging analyses of such biodistributions, radioisotope (RI) labeling and fluorescence labeling methods are typically used, but the resulting data are limited because these methods cannot distinguish breakdown products from intact antibodies. To resolve this problem, we developed a novel method using fluorescent resonance energy transfer (FRET)-type labeling and a spectral unmixing tool. With FRET-type labeling (labeling with 2 species of fluorophore), different fluorescence properties of labeled intact antibodies and their breakdown products (the hydrolyzed/digested type of breakdown products) are made visible. With the spectral unmixing tool, the fluorescence of a solution containing the intact antibody and its breakdown products could be unmixed in proportion to their contents. Moreover, when labeled antibodies that targeted either human epidermal growth factor receptor-2 or epidermal growth factor receptor were injected into nude mice implanted subcutaneously with tumor cells, the accumulation of the injected labeled antibodies and their breakdown products in the tumor could be separately analyzed by both whole-mouse imaging and a tumor homogenate analysis. These results suggest that our method using FRET-type labeling and a spectral unmixing tool could be useful in distinguishing breakdown products from intact antibodies.
Asunto(s)
Antineoplásicos , Colorantes Fluorescentes , Neoplasias Experimentales , Receptor ErbB-2/antagonistas & inhibidores , Trastuzumab , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Colorantes Fluorescentes/farmacología , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Trastuzumab/química , Trastuzumab/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We developed an epoch-making portable intelligent medicine case for recipients who take medicine every day. The medicine case consists of a recipient's smartphone and a mirror-embedded pill organizer. The smartphone captures medicines in the storage compartments of the organizer by using its built-in camera. The positions and shapes of mirrors were optimized in order that the smartphone can confirm the whole spaces. The medicine case calculates changes caused by medications and approximates them by quadratic curves. If a fitted curve in a space was convex downward, the medicine case judged that there are medicines in the space.
Asunto(s)
Teléfono Celular , Cumplimiento de la Medicación , Sistemas Recordatorios/instrumentación , Almacenaje de Medicamentos , Humanos , Procesamiento de Imagen Asistido por Computador , Proyectos PilotoRESUMEN
Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Bioensayo/métodos , Receptores de IgG/metabolismo , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Línea Celular , Cetuximab , Citometría de Flujo , Humanos , Células Jurkat , Panitumumab , RituximabRESUMEN
Heterozygous mutations in the JAG1 gene, encoding Notch ligand Jagged1, cause Alagille syndrome (ALGS). As most of the mutations are nonsense or frameshift mutations producing inactive truncated proteins, haplo-insufficiency is considered the major pathogenic mechanism of ALGS. However, the molecular mechanisms by which the missense mutations cause ALGS remain unclear. Here we analyzed the functional properties of four ALGS missense mutant proteins, P163L, R184H, G386R and C714Y, using transfected mammalian cells. P163L and R184H showed Notch-binding activities similar to that of the wild-type when assessed by immunoprecipitation. However, their trans-activation and cis-inhibition activities were almost completely impaired. These mutant proteins localized mainly to the endoplasmic reticulum (ER), suggesting that the mutations induced improper protein folding. Furthermore, the mutant proteins bound more strongly to the ER chaperone proteins calnexin and calreticulin than the wild-type did. C714Y also localized to the ER, but possessed significant trans-activation activity and lacked enhanced binding to the chaperones, indicating a less severe phenotype. The properties of G386R were the same as those of the wild-type. Dominant-negative effects were not detected for any mutant protein. These results indicate that accumulation in the ER and binding to the chaperones correlate with the impaired signal-transduction activities of the missense mutant proteins, which may contribute to the pathogenic mechanism of ALGS. Our findings, which suggest the requirement for cell-surface localization of Jagged1 for cis-inhibition activities, also provide important information for understanding the molecular basis of Notch-signaling pathways.
Asunto(s)
Síndrome de Alagille/metabolismo , Proteínas de Unión al Calcio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Mutación Missense/genética , Síndrome de Alagille/genética , Western Blotting , Proteínas de Unión al Calcio/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Proteínas de la Membrana/genética , Unión Proteica , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
New technologies are needed to deliver medicines safely and effectively. Polymeric nanoparticulate carriers are one such technology under investigation. We examined the intracellular trafficking of doxorubicin-bound block copolymers quantitatively and by imaging doxorubicin-derived fluorescence using confocal microscopy. The polymers were internalized by endocytosis and distributed in endosomal/lysosomal compartments and the endoplasmic reticulum; unlike free doxorubicin, the polymers were not found in the nucleus. Moreover, the ATP-binding cassette protein B1 (ABCB1) transporter may be involved in the efflux of the polymer from cells. This drug delivery system is attractive because the endogenous transport system is used for the uptake and delivery of the artificial drug carrier to the target as well as for its efflux from cells to medium. Our results show that a drug delivery system strategy targeting this endogenous transport pathway may be useful for affecting specific molecular targets.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Doxorrubicina/metabolismo , Portadores de Fármacos , Polietilenglicoles/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antibióticos Antineoplásicos/química , Transporte Biológico , Supervivencia Celular/efectos de los fármacos , Química Farmacéutica , Doxorrubicina/química , Composición de Medicamentos , Endocitosis , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Femenino , Células HeLa , Humanos , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Lisosomas/metabolismo , Micelas , Microscopía Confocal , Polietilenglicoles/química , Polietilenglicoles/toxicidad , Interferencia de ARN , Tecnología Farmacéutica/métodos , Factores de Tiempo , Transfección , Neoplasias del Cuello Uterino/genéticaRESUMEN
In this paper, we propose a new intelligent medicine case with a home medical interface (iMec G2) to assist an elderly recipient in taking his/her medicine adequately. A touchscreen was equipped in the front surface of the iMec which was tilted backward. The home medical interface for self-medication support was displayed on the touchscreen and handled by the recipient's fingers. The elderly recipient could confirm the information related to his/her prescription by touching a virtual medicine and could contact a recipient's caregiver by clicking an icon if necessary. The new iMec recognized medicine in its storage cartridge by an improved image processing method and evaluated the adequacy of dosage and dosing timing. By an experiment, we verified that the iMec G2 was able to create the home medical interface correctly and indicate a particular medicine which should be taken.