RESUMEN
The homeostasis of IgG is maintained by the neonatal Fc receptor, FcRn. Consequently, antagonism of FcRn to reduce endogenous IgG levels is an emerging strategy for treating antibody-mediated autoimmune disorders using either FcRn-specific antibodies or an engineered Fc fragment. For certain FcRn-specific antibodies, this approach has resulted in reductions in the levels of serum albumin, the other major ligand transported by FcRn. Cellular and molecular analyses of a panel of FcRn antagonists have been carried out to elucidate the mechanisms leading to their differential effects on albumin homeostasis. These analyses have identified 2 processes underlying decreases in albumin levels during FcRn blockade: increased degradation of FcRn and competition between antagonist and albumin for FcRn binding. These findings have potential implications for the design of drugs to modulate FcRn function.
Asunto(s)
Antígenos de Histocompatibilidad Clase I , Receptores Fc , Receptores Fc/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Animales , Transporte de Proteínas/efectos de los fármacos , Albúmina Sérica/metabolismo , Ratones , Unión ProteicaRESUMEN
Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT), but how this affects immune responses broadly remains unknown. Using a preclinical model of CMV reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells, resulting in sustained IFN-γ secretion, which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for IgG recycling, and rapid IgG loss. T cell-specific deletion of IL-6R led to persistence of recipient-derived, CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN-γ in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invoked IFN-γ-dependent EC injury and consequent IgG loss, leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury, thereby preserving humoral immunity after immunotherapy.
Asunto(s)
Trasplante de Médula Ósea , Infecciones por Citomegalovirus , Inmunidad Humoral , Interleucina-6 , Antivirales , Trasplante de Médula Ósea/efectos adversos , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/metabolismo , Inmunoglobulina G , Interleucina-6/metabolismo , Animales , RatonesRESUMEN
Serum albumin (SA), the most abundant soluble protein in the body, maintains plasma oncotic pressure and regulates the distribution of vascular fluid and has a range of other important functions. The goals of this review are to expand clinical knowledge regarding the functions of SA, elucidate effects of dysregulated SA concentration, and discuss the clinical relevance of hypoalbuminemia resulting from various diseases. We discuss potential repercussions of SA dysregulation on cholesterol levels, liver function, and other processes that rely on its homeostasis, as decreased SA concentration has been shown to be associated with increased risk for cardiovascular disease, hyperlipidemia, and mortality. We describe the anti-inflammatory and antioxidant properties of SA, as well as its ability to bind and transport a plethora of endogenous and exogenous molecules. SA is the primary serum protein involved in binding and transport of drugs and as such has the potential to affect, or be affected by, certain medications. Of current relevance are antibody-based inhibitors of the neonatal Fc receptor (FcRn), several of which are under clinical development to treat immunoglobulin G (IgG)-mediated autoimmune disorders; some have been shown to decrease SA concentration. FcRn acts as a homeostatic regulator of SA by rescuing it, as well as IgG, from intracellular degradation via a common cellular recycling mechanism. Greater clinical understanding of the multifunctional nature of SA and the potential clinical impact of decreased SA are needed; in particular, the potential for certain treatments to reduce SA concentration, which may affect efficacy and toxicity of medications and disease progression.
Asunto(s)
Enfermedades Autoinmunes , Inmunoglobulina G , Enfermedades Autoinmunes/tratamiento farmacológico , Homeostasis , Humanos , Inmunoglobulina G/metabolismo , Recién Nacido , Receptores Fc , Albúmina Sérica/metabolismoRESUMEN
The prolonged in vivo persistence of antibodies results in high background and poor contrast during their use as molecular imaging agents for positron emission tomography (PET). We have recently described a class of engineered Fc fusion proteins that selectively deplete antigen-specific antibodies without affecting the levels of antibodies of other specificities. Here, we demonstrate that these Fc fusions (called Seldegs, for selective degradation) can be used to clear circulating, radiolabeled HER2-specific antibody during diagnostic imaging of HER2-positive tumors in mice. The analyses show that Seldegs have considerable promise for the reduction of whole-body exposure to radiolabel and improvement of contrast during PET.
Asunto(s)
Neoplasias , Tomografía de Emisión de Positrones , Animales , Anticuerpos , Línea Celular Tumoral , Ratones , Tomografía de Emisión de Positrones/métodos , Receptor ErbB-2RESUMEN
The neonatal Fc receptor (FcRn) is an MHC class I-like molecule that is widely distributed in mammalian organs, tissues, and cells. FcRn is critical to maintaining immunoglobulin G (IgG) and albumin levels through rescuing these molecules from lysosomal degradation. IgG autoantibodies are associated with many autoimmune diseases, including myasthenia gravis (MG), a rare neuromuscular autoimmune disease that causes debilitating and, in its generalized form (gMG), potentially life-threatening muscle weakness. IgG autoantibodies are directly pathogenic in MG and target neuromuscular junction proteins, causing neuromuscular transmission failure. Treatment approaches that reduce autoantibody levels, such as therapeutic plasma exchange and intravenous immunoglobulin, have been shown to be effective for gMG patients but are not indicated as ongoing maintenance therapies and can be associated with burdensome side effects. Agents that block FcRn-mediated recycling of IgG represent a rational and promising approach for the treatment of gMG. Blocking FcRn allows targeted reduction of all IgG subtypes without decreasing concentrations of other Ig isotypes; therefore, FcRn blocking could be a safe and effective treatment strategy for a broad population of gMG patients. Several FcRn-blocking antibodies and one antibody Fc fragment have been developed and are currently in various stages of clinical development. This article describes the mechanism of FcRn blockade as a novel approach for IgG-mediated disease therapy and reviews promising clinical data using such FcRn blockers for the treatment of gMG.
Asunto(s)
Inmunoglobulina G , Miastenia Gravis , Animales , Autoanticuerpos , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Miastenia Gravis/tratamiento farmacológicoRESUMEN
In a scientific career that spanned over three decades, Dr. Jeff Foote made seminal contributions to antibody humanization and the biophysical aspects of antibody recognition. In this Perspective, we discuss his life and work.
RESUMEN
Single-molecule microscopy allows for the investigation of the dynamics of individual molecules and the visualization of subcellular structures at high spatial resolution. For single-molecule imaging experiments, and particularly those that entail the acquisition of multicolor data, calibration of the microscope and its optical components therefore needs to be carried out at a high level of accuracy. We propose here a method for calibrating a microscope at the nanometer scale, in the sense of determining optical aberrations as revealed by point source localization errors on the order of nanometers. The method is based on the imaging of a standard sample to detect and evaluate the amount of geometric aberration introduced in the optical light path. To provide support for multicolor imaging, it also includes procedures for evaluating the geometric aberration caused by a dichroic filter and the axial chromatic aberration introduced by an objective lens.
RESUMEN
Current treatments for antibody-mediated autoimmunity are associated with lack of specificity, leading to immunosuppressive effects. To overcome this limitation, we have developed a class of antibody-based therapeutics for the treatment of autoimmunity involving antibodies that recognize the autoantigen, myelin oligodendrocyte glycoprotein (MOG). These agents ("Seldegs," for selective degradation) selectively eliminate antigen (MOG)-specific antibodies without affecting the levels of antibodies of other specificities. Seldeg treatment of mice during antibody-mediated exacerbation of experimental autoimmune encephalomyelitis by patient-derived MOG-specific antibodies results in disease amelioration. Consistent with their therapeutic effects, Seldegs deliver their targeted antibodies to Kupffer and liver sinusoidal endothelial cells that are known to have tolerogenic effects. Our results show that Seldegs can ameliorate disease mediated by MOG-specific antibodies and indicate that this approach also has the potential to treat other autoimmune diseases where the specific clearance of antibodies is required.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Esclerosis Múltiple/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Animales , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de IgG/metabolismoRESUMEN
As a versatile reaction for bioconjugation, Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has enormous potential in the synthesis of antibody-drug conjugates (ADCs). In order to optimize CuAAC-based ADC synthesis, we characterized kinetically different formulation processes by mimicking ADC synthesis using small molecules and subsequently revealed unique kinetic behaviors of different combinations of alkyne and azide conditions. Our results indicate that under ADC synthesis conditions, for an alkyne-containing drug, its concentration has minimal impact on the reaction rate when an antibody has a non-metal-chelating azide but is proportional to concentration when an antibody contains a metal-chelating azide; however, for an alkyne-containing antibody, the ADC synthesis rate is proportional to the concentration of a drug with a non-metal-chelating azide but displays almost no dependence on drug concentration with a metal-chelating azide. Based on our results, we designed and tested an optimal "click" formulation strategy that allowed rapid and cost-effective synthesis of a new ADC.
Asunto(s)
Química Clic , Inmunoconjugados/química , Alquinos/química , Anticuerpos Monoclonales Humanizados/química , Azidas/química , Catálisis , Cobre/química , Reacción de Cicloadición , Preparaciones Farmacéuticas/químicaRESUMEN
Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder, characterized by a low platelet count (<100 × 109 /L) in the absence of other causes associated with thrombocytopenia. In most patients, IgG autoantibodies directed against platelet receptors can be detected. They accelerate platelet clearance and destruction, inhibit platelet production, and impair platelet function, resulting in increased risk of bleeding and impaired quality of life. Efgartigimod is a human IgG1 antibody Fc-fragment, a natural ligand of the neonatal Fc receptor (FcRn), engineered for increased affinity to FcRn, while preserving its characteristic pH-dependent binding. Efgartigimod blocks FcRn, preventing IgG recycling, and causing targeted IgG degradation. In this Phase 2 study, 38 patients were randomized 1:1:1 to receive four weekly intravenous infusions of either placebo (N = 12) or efgartigimod at a dose of 5 mg/kg (N = 13) or 10 mg/kg (N = 13). This short treatment cycle of efgartigimod in patients with ITP, predominantly refractory to previous lines of therapy, was shown to be well tolerated, and demonstrated a favorable safety profile consistent with Phase 1 data. Efgartigimod induced a rapid reduction of total IgG levels (up to 63.7% mean change from baseline), which was associated with clinically relevant increases in platelet counts (46% patients on efgartigimod vs 25% on placebo achieved a platelet count of ≥50 × 109 /L on at least two occasions, and 38% vs 0% achieved ≥50 × 109 /L for at least 10 cumulative days), and a reduced proportion of patients with bleeding. Taken together, these data warrant further evaluation of FcRn antagonism as a novel therapeutic approach in ITP.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Inmunoglobulina G/uso terapéutico , Púrpura Trombocitopénica Idiopática , Receptores Fc/antagonistas & inhibidores , Adulto , Anciano , Método Doble Ciego , Femenino , Estudios de Seguimiento , Antígenos de Histocompatibilidad Clase I/sangre , Humanos , Masculino , Persona de Mediana Edad , Recuento de Plaquetas , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Receptores Fc/sangreRESUMEN
Endosomes are subcellular organelles which serve as important transport compartments in eukaryotic cells. Fluorescence microscopy is a widely applied technology to study endosomes at the subcellular level. In general, a microscopy image can contain a large number of organelles and endosomes in particular. Detecting and annotating endosomes in fluorescence microscopy images is a critical part in the study of subcellular trafficking processes. Such annotation is usually performed by human inspection, which is time-consuming and prone to inaccuracy if carried out by inexperienced analysts. This paper proposes a two-stage method for automated detection of ring-like endosomes. The method consists of a localization stage cascaded by an identification stage. Given a test microscopy image, the localization stage generates a voting-map by locally comparing the query endosome patches and the test image based on a bag-of-words model. Using the voting-map, a number of candidate patches of endosomes are determined. Subsequently, in the identification stage, a support vector machine (SVM) is trained using the endosome patches and the background pattern patches. Each of the candidate patches is classified by the SVM to rule out those patches of endosome-like background patterns. The performance of the proposed method is evaluated with real microscopy images of human myeloid endothelial cells. It is shown that the proposed method significantly outperforms several state-of-the-art competing methods using multiple performance metrics.
Asunto(s)
Endosomas/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Algoritmos , Células Endoteliales/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Microscopía Fluorescente/estadística & datos numéricos , Máquina de Vectores de SoporteRESUMEN
OBJECTIVE: To investigate safety and explore efficacy of efgartigimod (ARGX-113), an anti-neonatal Fc receptor immunoglobulin G1 Fc fragment, in patients with generalized myasthenia gravis (gMG) with a history of anti-acetylcholine receptor (AChR) autoantibodies, who were on stable standard-of-care myasthenia gravis (MG) treatment. METHODS: A phase 2, exploratory, randomized, double-blind, placebo-controlled, 15-center study is described. Eligible patients were randomly assigned (1:1) to receive 4 doses over a 3-week period of either 10 mg/kg IV efgartigimod or matched placebo combined with their standard-of-care therapy. Primary endpoints were safety and tolerability. Secondary endpoints included efficacy (change from baseline to week 11 of Myasthenia Gravis Activities of Daily Living, Quantitative Myasthenia Gravis, and Myasthenia Gravis Composite disease severity scores, and of the revised 15-item Myasthenia Gravis Quality of Life scale), pharmacokinetics, pharmacodynamics, and immunogenicity. RESULTS: Of the 35 screened patients, 24 were enrolled and randomized: 12 received efgartigimod and 12 placebo. Efgartigimod was well-tolerated in all patients, with no serious or severe adverse events reported, no relevant changes in vital signs or ECG findings observed, and no difference in adverse events between efgartigimod and placebo treatment. All patients treated with efgartigimod showed a rapid decrease in total immunoglobulin G (IgG) and anti-AChR autoantibody levels, and assessment using all 4 efficacy scales consistently demonstrated that 75% showed a rapid and long-lasting disease improvement. CONCLUSIONS: Efgartigimod was safe and well-tolerated. The correlation between reduction of levels of pathogenic IgG autoantibodies and disease improvement suggests that reducing pathogenic autoantibodies with efgartigimod may offer an innovative approach to treat MG. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that efgartigimod is safe and well-tolerated in patients with gMG.
Asunto(s)
Factores Inmunológicos/uso terapéutico , Miastenia Gravis/tratamiento farmacológico , Receptores Fc/antagonistas & inhibidores , Actividades Cotidianas , Corticoesteroides/uso terapéutico , Adulto , Anciano , Autoanticuerpos/inmunología , Inhibidores de la Colinesterasa/uso terapéutico , Método Doble Ciego , Femenino , Antígenos de Histocompatibilidad Clase I , Humanos , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Inmunosupresores/uso terapéutico , Masculino , Persona de Mediana Edad , Miastenia Gravis/inmunología , Receptores Colinérgicos/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
The maintenance of the homeostasis of immunoglobulin G (IgG) represents a fundamental aspect of humoral immunity that has direct relevance to the successful delivery of antibody-based therapeutics. The ubiquitously expressed neonatal Fc receptor (FcRn) salvages IgG from cellular degradation following pinocytic uptake into cells, conferring prolonged in vivo persistence on IgG. However, the cellular sites of FcRn function are poorly defined. Pinocytic uptake is a prerequisite for FcRn-mediated IgG salvage, prompting us to investigate the consequences of IgG uptake and catabolism by macrophages, which represent both abundant and highly pinocytic cells in the body. Site-specific deletion of FcRn to generate mice harboring FcRn-deficient macrophages results in IgG hypercatabolism and ~threefold reductions in serum IgG levels, whereas these effects were not observed in mice that lack functional FcRn in B cells and dendritic cells. Consistent with the degradative activity of FcRn-deficient macrophages, depletion of these cells in FcRn-deficient mice leads to increased persistence and serum levels of IgG. These studies demonstrate a pivotal role for FcRn-mediated salvage in compensating for the high pinocytic and degradative activities of macrophages to maintain IgG homeostasis.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulina G/sangre , Macrófagos/inmunología , Receptores Fc/metabolismo , Animales , Linfocitos B , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Endoteliales , Antígenos de Histocompatibilidad Clase I/genética , Homeostasis/inmunología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pinocitosis/inmunología , Receptores Fc/genéticaRESUMEN
We improve the potency of antibody-drug conjugates (ADCs) containing the human epidermal growth factor receptor 2 (HER2)-specific antibody pertuzumab by substantially reducing their affinity for HER2 at acidic endosomal pH relative to near neutral pH. These engineered pertuzumab variants show increased lysosomal delivery and cytotoxicity towards tumor cells expressing intermediate HER2 levels. In HER2int xenograft tumor models in mice, the variants show higher therapeutic efficacy than the parent ADC and a clinically approved HER2-specific ADC.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Inmunoconjugados/uso terapéutico , Receptor ErbB-2/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Femenino , Humanos , Inmunoconjugados/inmunología , Lisosomas/inmunología , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The MHC class I-related receptor FcRn serves multiple roles ranging from the regulation of levels of IgG isotype antibodies and albumin throughout the body to the delivery of antigen into antigen loading compartments in specialized antigen-presenting cells. In parallel with studies directed towards understanding FcRn at the molecular and cellular levels, there has been an enormous expansion in the development of engineering strategies involving FcRn to modulate the dynamic behavior of antibodies, antigens, and albumin. In this review article, we focus on a discussion of FcRn-targeted approaches that have resulted in the production of novel antibody-based platforms with considerable potential for use in the clinic.
Asunto(s)
Antígenos de Histocompatibilidad Clase I , Inmunoglobulina G , Receptores Fc , Animales , Complejo Antígeno-Anticuerpo , Humanos , Transporte de ProteínasRESUMEN
The toll-like receptor (TLR) and interleukin (IL)-1 family of receptors share several signaling components, including the most upstream adapter, MyD88. We previously reported the discovery of B cell adapter for phosphoinositide 3-kinase (BCAP) as a novel toll-IL-1 receptor homology domain-containing adapter that regulates inflammatory responses downstream of TLR signaling. Here we find that BCAP plays a critical role downstream of both IL-1 and IL-18 receptors to regulate T helper (Th) 17 and Th1 cell differentiation, respectively. Absence of T cell intrinsic BCAP did not alter development of naturally arising Th1 and Th17 lineages but led to defects in differentiation to pathogenic Th17 lineage cells. Consequently, mice that lack BCAP in T cells had reduced susceptibility to experimental autoimmune encephalomyelitis. More importantly, we found that BCAP is critical for IL-1R-induced phosphoinositide 3-kinase-Akt-mechanistic target of rapamycin (mTOR) activation, and minimal inhibition of mTOR completely abrogated IL-1ß-induced differentiation of pathogenic Th17 cells, mimicking BCAP deficiency. This study establishes BCAP as a critical link between IL-1R and the metabolic status of activated T cells that ultimately regulates the differentiation of inflammatory Th17 cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Diferenciación Celular/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Receptores de Interleucina-1/inmunología , Transducción de Señal/inmunología , Serina-Treonina Quinasas TOR/inmunología , Células Th17/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular/genética , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Interleucina-1/genética , Interleucina-1/inmunología , Interleucina-18/genética , Interleucina-18/inmunología , Activación de Linfocitos , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores de Interleucina-1/genética , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Células TH1/inmunología , Células TH1/patología , Células Th17/patologíaRESUMEN
BACKGROUND: Intravenous Ig (IVIg), plasma exchange, and immunoadsorption are frequently used in the management of severe autoimmune diseases mediated by pathogenic IgG autoantibodies. These approaches modulating IgG levels can, however, be associated with some severe adverse reactions and a substantial burden to patients. Targeting the neonatal Fc receptor (FcRn) presents an innovative and potentially more effective, safer, and more convenient alternative for clearing pathogenic IgGs. METHODS: A randomized, double-blind, placebo-controlled first-in-human study was conducted in 62 healthy volunteers to explore single and multiple ascending intravenous doses of the FcRn antagonist efgartigimod. The study objectives were to assess safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity. The findings of this study were compared with the pharmacodynamics profile elicited by efgartigimod in cynomolgus monkeys. RESULTS: Efgartigimod treatment resulted in a rapid and specific clearance of serum IgG levels in both cynomolgus monkeys and healthy volunteers. In humans, single administration of efgartigimod reduced IgG levels up to 50%, while multiple dosing further lowered IgGs on average by 75% of baseline levels. Approximately 8 weeks following the last administration, IgG levels returned to baseline. Efgartigimod did not alter the homeostasis of albumin or Igs other than IgG, and no serious adverse events related to efgartigimod infusion were observed. CONCLUSION: Antagonizing FcRn using efgartigimod is safe and results in a specific, profound, and sustained reduction of serum IgG levels. These results warrant further evaluation of this therapeutic approach in IgG-driven autoimmune diseases. TRIAL REGISTRATION: Clinicaltrials.gov NCT03457649. FUNDING: argenx BVBA.
Asunto(s)
Enfermedades Autoinmunes , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Inmunoglobulina G/sangre , Receptores Fc/antagonistas & inhibidores , Adulto , Animales , Autoanticuerpos/sangre , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/tratamiento farmacológico , Células CHO , Cricetulus , Método Doble Ciego , Femenino , Antígenos de Histocompatibilidad Clase I , Humanos , Fragmentos Fc de Inmunoglobulinas/efectos adversos , Macaca fascicularis , MasculinoRESUMEN
Despite the rapidly expanding use of antibody-based therapeutics to treat cancer, knowledge of the cellular processes following phagocytosis of antibody-opsonized tumor cells is limited. Here we report the formation of a phagosome-associated vacuole that is observed in macrophages as these degradative compartments mature following phagocytosis of HER2-positive cancer cells in the presence of the HER2-specific antibody, trastuzumab. We demonstrate that this vacuole is a distinct organelle that is closely apposed to the phagosome. Furthermore, the size of the phagosome-associated vacuole is increased by inhibition of the mTOR pathway. Collectively, the identification of this vacuolar compartment has implications for understanding the subcellular trafficking processes leading to the destruction of phagocytosed, antibody-opsonized cancer cells by macrophages.
Asunto(s)
Macrófagos/metabolismo , Fagocitosis/inmunología , Fagosomas/metabolismo , Vacuolas/metabolismo , Animales , Anticuerpos/inmunología , Humanos , Lisosomas/metabolismo , Fusión de Membrana/fisiología , Ratones , Neoplasias/inmunología , Neoplasias/metabolismo , Fagocitosis/fisiología , Receptores de IgG/metabolismoRESUMEN
In response to cellular stress, phosphatidylserine is exposed on the outer membrane leaflet of tumor blood vessels and cancer cells, motivating the development of phosphatidylserine-specific therapies. The generation of drug-conjugated phosphatidylserine-targeting agents represents an unexplored therapeutic approach, for which antitumor effects are critically dependent on efficient internalization and lysosomal delivery of the cytotoxic drug. In the current study, we have generated phosphatidylserine-targeting agents by fusing phosphatidylserine-binding domains to a human IgG1-derived Fc fragment. The tumor localization and pharmacokinetics of several phosphatidylserine-specific Fc fusions have been analyzed in mice and demonstrate that Fc-Syt1, a fusion containing the synaptotagmin 1 C2A domain, effectively targets tumor tissue. Conjugation of Fc-Syt1 to the cytotoxic drug monomethyl auristatin E results in a protein-drug conjugate (PDC) that is internalized into target cells and, due to the Ca2+ dependence of phosphatidylserine binding, dissociates from phosphatidylserine in early endosomes. The released PDC is efficiently delivered to lysosomes and has potent antitumor effects in mouse xenograft tumor models. Interestingly, although an engineered, tetravalent Fc-Syt1 fusion shows increased binding to target cells, this higher avidity variant demonstrates reduced persistence and therapeutic effects compared with bivalent Fc-Syt1. Collectively, these studies show that finely tuned, Ca2+-switched phosphatidylserine-targeting agents can be therapeutically efficacious. Mol Cancer Ther; 17(1); 169-82. ©2017 AACR.
Asunto(s)
Calcio/metabolismo , Inmunoconjugados/metabolismo , Neoplasias/tratamiento farmacológico , Fosfatidilserinas/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Ratones SCIDRESUMEN
Myelin oligodendrocyte glycoprotein (MOG) is exposed on the outer surface of the myelin sheath, and as such, represents a possible target antigen for antibodies in multiple sclerosis (MS) and other demyelinating diseases. However, despite extensive analyses, whether MOG-specific antibodies contribute to pathogenesis in human MS remains an area of uncertainty. In the current study we demonstrate that antibodies derived from adult MS patients exacerbate experimental autoimmune encephalomyelitis (EAE) in 'humanized' mice that transgenically express human FcγRs (hFcγRs). Importantly, this exacerbation is dependent on MOG recognition by the human-derived antibodies. The use of mice that express hFcγRs has allowed us to also investigate the contribution of these receptors to disease in the absence of confounding effects of cross-species differences. Specifically, by engineering the Fc region of MOG-specific antibodies to modulate FcγR and complement (C1q) binding, we reveal that FcγRs but not complement activation contribute to EAE pathogenesis. Importantly, selective enhancement of the affinities of these antibodies for specific FcγRs reveals that FcγRIIA is more important than FcγRIIIA in mediating disease exacerbation. These studies not only provide definitive evidence for the contribution of MOG-specific antibodies to MS, but also reveal mechanistic insight that could lead to new therapeutic targets.