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BACKGROUND AND OBJECTIVES: Autoantibody discovery in complex autoimmune diseases is challenging. Diverse successful antigen identification strategies are available, but, so far, have often been unsuccessful, especially in the discovery of protein antigens in which conformational and post-translational modification are critical. Our study assesses the utility of a human membrane and secreted protein microarray technology to detect autoantibodies in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). METHODS: A cell microarray consisting of human embryonic kidney-293 cells expressing >5,000 human proteins was used. First, a validation step was performed with 4 serum samples from patients with autoimmune nodopathy (AN) to assess the ability of this technology to detect circulating known autoantibodies. The ability of the cell microarray technology to discover novel IgG autoantibodies was assessed incubating the array with 8 CIDP serum samples. Identified autoantibodies were subsequently validated using cell-based assays (CBAs), ELISA, and/or tissue immunohistochemistry and analyzed in a cohort of CIDP and AN (n = 96) and control (n = 100) samples. RESULTS: Serum anti-contactin-1 and anti-neurofascin-155 were detected by the human cell microarray technology. Nine potentially relevant antigens were found in patients with CIDP without other detectable antibodies; confirmation was possible in six of them: ephrin type-A receptor 7 (EPHA7); potassium-transporting ATPase alpha chain 1 and subunit beta (ATP4A/4B); leukemia-inhibitory factor (LIF); and interferon lambda 1, 2, and 3 (IFNL1, IFNL2, IFNL3). Anti-ATP4A/4B and anti-EPHA7 antibodies were detected in patients and controls and considered unrelated to CIDP. Both anti-LIF and anti-IFNL antibodies were found in the same 2 patients and were not detected in any control. Both patients showed the same staining pattern against myelinating fibers of peripheral nerve tissue and of myelinating neuron-Schwann cell cocultures. Clinically relevant correlations could not be established for anti-LIF and anti-IFNL3 antibodies. DISCUSSION: Our work demonstrates the utility of human cell microarray technology to detect known and discover unknown autoantibodies in human serum samples. Despite potential CIDP-associated autoantibodies (anti-LIF and anti-IFNL3) being identified, their clinical and pathogenic relevance needs to be elucidated in bigger cohorts.
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Enfermedades Autoinmunes , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante , Humanos , Autoanticuerpos , Proteoma , Neuronas/químicaRESUMEN
Rozanolixizumab is a humanized anti-neonatal Fc receptor (FcRn) monoclonal antibody (mAb) of the immunoglobulin G4 (IgG4) sub-class, currently in clinical development for the treatment of IgG autoantibody-driven diseases. This format is frequently used for therapeutic mAbs due to its intrinsic lower affinity for Fc gamma receptors (FcγR) and lack of C1q engagement. However, with growing evidence suggesting that no Fc-containing agent is truly "silent" in this respect, we explored the engagement of FcγRs and potential functional consequences with rozanolixizumab. In the study presented here, rozanolixizumab was shown to bind to FcγRs in both protein-protein and cell-based assays, and the kinetic data were broadly as expected based on published data for an IgG4 mAb. Rozanolixizumab was also able to mediate antibody bipolar bridging (ABB), a phenomenon that led to a reduction of labeled FcγRI from the surface of human macrophages in an FcRn-dependent manner. However, the presence of exogenous human IgG, even at low concentrations, was able to prevent both binding and ABB events. Furthermore, data from in vitro experiments using relevant human cell types that express both FcRn and FcγRI indicated no evidence for functional sequelae in relation to cellular activation events (e.g., intracellular signaling, cytokine production) upon either FcRn or FcγR binding of rozanolixizumab. These data raise important questions about whether therapeutic antagonistic mAbs like rozanolixizumab would necessarily engage FcγRs at doses typically administered to patients in the clinic, and hence challenge the relevance and interpretation of in vitro assays performed in the absence of competing IgG.
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Receptores Fc , Receptores de IgG , Humanos , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales , Inmunoglobulina G , Antígenos de Histocompatibilidad Clase IRESUMEN
BACKGROUND AND OBJECTIVES: Myelin oligodendrocyte glycoprotein antibody-associated disorder (MOGAD) is a rare, autoimmune demyelinating CNS disorder, distinct from multiple sclerosis and neuromyelitis optica spectrum disorder. Characterized by pathogenic immunoglobulin G (IgG) antibodies against MOG, a potential treatment strategy for MOGAD is to reduce circulating IgG levels, e.g., by interference with the IgG recycling pathway mediated by the neonatal Fc receptor (FcRn). Although the optic nerve is often detrimentally involved in MOGAD, the effect of FcRn blockade on the visual pathway has not been assessed. Our objective was to investigate effects of a monoclonal anti-FcRn antibody in murine MOG-IgG-associated experimental autoimmune encephalomyelitis (EAE). METHODS: We induced active MOG35-55 EAE in C57Bl/6 mice followed by the application of a monoclonal MOG-IgG (8-18C5) 10 days postimmunization (dpi). Animals were treated with either a specific monoclonal antibody against FcRn (α-FcRn, 4470) or an isotype-matched control IgG on 7, 10, and 13 dpi. Neurologic disability was scored daily on a 10-point scale. Visual acuity was assessed by optomotor reflex. Histopathologic hallmarks of disease were assessed in the spinal cord, optic nerve, and retina. Immune cell infiltration was visualized by immunohistochemistry, demyelination by Luxol fast blue staining and complement deposition and number of retinal ganglion cells by immunofluorescence. RESULTS: In MOG-IgG-augmented MOG35-55 EAE, anti-FcRn treatment significantly attenuated neurologic disability over the course of disease (mean area under the curve and 95% confidence intervals (CIs): α-FcRn [n = 27], 46.02 [37.89-54.15]; isotype IgG [n = 24], 66.75 [59.54-73.96], 3 independent experiments), correlating with reduced amounts of demyelination and macrophage infiltration into the spinal cord. T- and B-cell infiltration and complement deposition remained unchanged. Compared with isotype, anti-FcRn treatment prevented reduction of visual acuity over the course of disease (median cycles/degree and interquartile range: α-FcRn [n = 16], 0.50 [0.48-0.55] to 0.50 [0.48-0.58]; isotype IgG [n = 17], 0.50 [0.49-0.54] to 0.45 [0.39-0.51]). DISCUSSION: We show preserved optomotor response and ameliorated course of disease after anti-FcRn treatment in an experimental model using a monoclonal MOG-IgG to mimic MOGAD. Selectively targeting FcRn might represent a promising therapeutic approach in MOGAD.
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Anticuerpos Monoclonales/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Receptores Fc/inmunología , Animales , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/etiología , Femenino , Ratones , Ratones Endogámicos C57BL , Trastornos de la VisiónRESUMEN
Rozanolixizumab is a fully humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (mAb) that accelerates the removal of circulating immunoglobulin G (IgG), including pathogenic IgG autoantibodies, via the natural lysosomal degradation pathway. The aim of this study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model characterizing the effect of rozanolixizumab on IgG levels in cynomolgus monkeys, translate it into humans to support the first-in-human (FIH) rozanolixizumab clinical trial study design, and, ultimately, develop a PK/PD model in humans. Simulations from the preclinical model were performed to predict IgG responses in humans and select clinically relevant doses in the FIH study. Good alignment was observed between predicted and observed reductions in IgG, which increased with increasing dose in the FIH study. The model successfully described the PK of the 4 and 7 mg/kg intravenous (i.v.) dose groups, although the PKs were underpredicted for the 1 mg/kg i.v. dose group. Updating the model with subsequent human data identified parameters that deviated from preclinical assumptions. The updated PK/PD model was able to effectively characterize the PK FcRn-IgG nonlinear system in response to rozanolixizumab in the FIH data.
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Anticuerpos Monoclonales Humanizados/farmacología , Inmunoglobulina G/efectos de los fármacos , Receptores Fc/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/farmacocinética , Simulación por Computador , Relación Dosis-Respuesta a Droga , Antígenos de Histocompatibilidad Clase I , Humanos , Macaca fascicularis , Modelos BiológicosRESUMEN
OBJECTIVE: To determine whether blocking the neonatal Fc receptor (FcRn) during gestation with an anti-FcRn monoclonal antibody (mAb) reduces transfer of pathogenic maternal antibodies in utero and decreases the likelihood of maternal antibody-mediated neonatal disease in the offspring. METHODS: Using a previously established maternal-to-fetal transfer mouse model of arthrogryposis multiplex congenita (AMC), we assessed the effect of 4470, an anti-FcRn mAb, on the transfer of total human immunoglobulin G (IgG) and specific acetylcholine receptor (AChR)-antibodies from mother to fetus, as well as its effect on the prevention of neurodevelopmental abnormalities in the offspring. RESULTS: Offspring of pregnant dams treated with 4470 during gestation showed a substantial reduction in total human IgG and AChR antibody levels compared with those treated with the isotype mAb control. Treatment with 4470 was also associated with a significant reduction in AMC-IgG-induced deformities (limb or spinal curve malformations) when compared with mAb control-exposed embryos and a nonsignificant increase in the percentage of fetuses showing spontaneous movements. 4470 exposure during pregnancy was not associated with changes in general parameters of maternal well-being or fetal development; indeed, male neonates showed faster weight gain and shorter time to reach developmental milestones. CONCLUSIONS: FcRn blockade is a promising therapeutic strategy to prevent the occurrence of AMC and other human maternal autoantibody-related diseases in the offspring.
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Anticuerpos Bloqueadores/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Artrogriposis/prevención & control , Autoanticuerpos/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/efectos de los fármacos , Intercambio Materno-Fetal , Receptores Fc/efectos de los fármacos , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina G , Ratones , Ratones Endogámicos ICR , Embarazo , Receptores Colinérgicos/inmunologíaRESUMEN
Since the first description of the administration of high doses of pooled serum IgG, also referred to as intravenous IgG (IVIg) therapy, as being able to ameliorate various autoimmune diseases, researchers have been investigating which molecular and cellular pathways underlie IVIg activity. Apart from trying to understand the obvious conundrum that IgG can trigger both autoimmune pathology and resolution of inflammation, the rapidly expanding use of IVIg has led to a lack of availability of this primary blood product, providing a strong rationale for developing recombinant alternatives. During the last decade, a tremendous number of novel insights into IVIg activity brought the goal of replacing IVIg within reach, at least in select indications, and has led to the initiation of several clinical trials. At the forefront of this effort is the modulation of autoantibody half-life and blocking access of autoantibodies to fragment cystallizable γ receptors (Fcγ receptors). In this rostrum article, we will briefly discuss current models of IVIg activity, followed by a more specific focus on novel therapeutic avenues that are entering the clinic and may replace IVIg in the future.
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Enfermedades Autoinmunes/terapia , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulinas Intravenosas/uso terapéutico , Inflamación/terapia , Receptores Fc/metabolismo , Receptores de IgG/metabolismo , Animales , Autoanticuerpos/metabolismo , Autoinmunidad , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Terapia Molecular Dirigida , Receptores Fc/inmunologíaRESUMEN
The delta isoform of phosphoinositide 3-kinase (PI3Kδ) regulates various lymphocyte functions. Considering the key pro-inflammatory role of IL-17A and IL-17F cytokines in psoriasis and spondyloarthritis (SpA), we investigated the potential of PI3Kδ blockade to suppress IL-17A, IL-17F and associated pro-inflammatory cytokines that could synergize with IL-17A and IL-17F. Using in vitro studies with primary human cells and ex vivo studies with inflamed target tissues, we assessed if seletalisib, a selective PI3Kδ inhibitor, suppresses cytokine production by T cells and innate-like lymphocytes, and if seletalisib modulates the inflammatory responses in stromal cell populations in psoriasis (human dermal fibroblasts (HDF)) and SpA (fibroblast-like synoviocytes (FLS)). In vitro, seletalisib inhibited the production of pro-inflammatory cytokines, including IL-17A and IL-17F, from peripheral blood mononuclear cells (PBMCs), T helper 17 (Th17) cells as well as γδ-T cells and mucosal-associated invariant T cells. This inhibition resulted in decreased inflammatory activation of HDF in co-culture systems. Seletalisib was also efficacious in inhibiting SpA PBMCs and synovial fluid mononuclear cells (SFMCs) from producing pro-inflammatory cytokines. Furthermore, supernatant derived from cultured seletalisib-treated Th17 cells showed reduced potency for activating inflammatory responses from cultured SpA FLS and decreased their osteogenic differentiation capacity. Finally, analysis of inflamed SpA synovial tissue biopsies revealed activation of the PI3K-Akt-mTOR pathway. We observed that ex vivo seletalisib treatment of inflamed synovial tissue reduced IL-17A and IL-17F expression. Collectively, inhibition of PI3Kδ reduces the production of pro-inflammatory cytokines from IL-17-producing adaptive and innate-like lymphocytes and thereby inhibits downstream inflammatory and tissue remodeling responses. PI3Kδ-targeting may therefore represent a novel therapeutic avenue for the treatment of IL-17-mediated chronic inflammatory diseases such as psoriasis and SpA.
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Antiinflamatorios/farmacología , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fibroblastos/fisiología , Linfocitos/inmunología , Psoriasis/inmunología , Piridinas/farmacología , Quinolinas/farmacología , Espondilitis Anquilosante/inmunología , Sinoviocitos/fisiología , Células Th17/inmunología , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Femenino , Humanos , Inmunidad Innata , Interleucina-17/metabolismo , Masculino , Persona de Mediana Edad , OsteogénesisRESUMEN
BACKGROUND AND PURPOSE: Pemphigus and pemphigoid diseases are characterized and caused predominantly by IgG autoantibodies targeting structural proteins of the skin. Their current treatment relies on general and prolonged immunosuppression that causes severe adverse events, including death. Hence, novel safe and more effective treatments are urgently needed. Due to its' physiological functions, the neonatal Fc receptor (FcRn) has emerged as a potential therapeutic target for pemphigus and pemphigoid, primarily because IgG is protected from proteolysis after uptake into endothelial cells. Thus, blockade of FcRn would reduce circulating autoantibody concentrations. However, long-term effects of pharmacological FcRn inhibition in therapeutic settings of autoimmune diseases are unknown. EXPERIMENTAL APPROACH: Therapeutic effects of FcRn blockade were investigated in a murine model of the prototypical autoantibody-mediated pemphigoid disease, epidermolysis bullosa acquisita (EBA). B6.SJL-H2s C3c/1CyJ mice with clinically active disease were randomized to receive either an anti-FcRn monoclonal antibody (4470) or an isotype control over 4 weeks. KEY RESULTS: While clinical disease continued to worsen in isotype control-treated mice, overall disease severity continuously decreased in mice injected with 4470, leading to almost complete remission in over 25% of treated mice. These clinical findings were paralleled by a reduction of autoantibody concentrations. Reduction of autoantibody concentrations, rather than modulating neutrophil activation, was responsible for the observed therapeutic effects. CONCLUSION AND IMPLICATIONS: The clinical efficacy of anti-FcRn treatment in this prototypical autoantibody-mediated disease encourages further development of anti-FcRn antibodies for clinical use in pemphigoid diseases and potentially in other autoantibody mediated diseases.
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Epidermólisis Ampollosa Adquirida , Animales , Autoanticuerpos , Células Endoteliales , Epidermólisis Ampollosa Adquirida/tratamiento farmacológico , Antígenos de Histocompatibilidad Clase I , Ratones , Receptores FcRESUMEN
Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by pathogenic immunoglobulin G (IgG) autoantibodies that bind to platelets, causing their phagocytic removal and leading to reductions in platelet number. The neonatal Fc receptor (FcRn) selectively salvages and recycles IgG, including pathogenic IgG, thereby extending the half-life of IgG in plasma. Two anti-mouse FcRn monoclonal antibodies (mAb) (4470 and 4464) were generated to evaluate the effect of inhibiting IgG recycling. Statistically significant reductions in plasma IgG concentration were observed upon administration of 4470 (10, 30 and 100â¯mg/kg) in wild-type mice. In a passive mouse model of ITP, 4464 alleviated the reduction in platelet number and/or preserved newly produced platelets when dosed prophylactically as well as in a therapeutic dosing regimen once platelet numbers had already been reduced. These results support the investigation of anti-FcRn therapy as a potential treatment for ITP.
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Anticuerpos Monoclonales/uso terapéutico , Plaquetas/inmunología , Inmunoglobulina G/sangre , Inmunoglobulinas Intravenosas/uso terapéutico , Inmunoterapia/métodos , Anticuerpos de Cadena Única/uso terapéutico , Trombocitopenia/terapia , Animales , Anticuerpos Monoclonales/genética , Autoanticuerpos/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Femenino , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunidad Humoral , Ratones , Ratones Endogámicos C57BL , Recuento de Plaquetas , Receptores Fc/inmunología , Anticuerpos de Cadena Única/genética , Trombocitopenia/inmunologíaRESUMEN
OBJECTIVE: EMBODY 1 (ClinicalTrials.gov identifier: NCT01262365) and EMBODY 2 (ClinicalTrials.gov identifier: NCT01261793) investigated the efficacy and safety of epratuzumab, a CD22-targeted humanized monoclonal IgG antibody, in patients with systemic lupus erythematosus (SLE). The studies showed no significant difference from placebo in primary or secondary clinical outcome measures but did demonstrate B cell-specific immunologic activity. The aim of this post hoc analysis was to determine whether epratuzumab had a different clinical efficacy profile in SLE patients with versus those without an associated diagnosis of Sjögren's syndrome (SS). METHODS: The efficacy and safety of epratuzumab were compared between 2 patient subpopulations randomized in EMBODY 1 and 2: SLE patients with and those without a diagnosis of associated SS. British Isles Lupus Assessment Group (BILAG) total score, BILAG-based Combined Lupus Assessment (BICLA) clinical response to treatment, biologic markers (including B cells, IgG, IgM, and IgA), and safety were assessed. RESULTS: A total of 1,584 patients were randomized in the EMBODY 1 and EMBODY 2 trials; 113 patients were anti-SSA positive and had a diagnosis of associated SS, and 1,375 patients (86.8%) had no diagnosis of associated SS (918 patients were randomized to receive epratuzumab and 457 to receive placebo). For patients with associated SS, but not those without associated SS, a higher proportion of patients receiving epratuzumab achieved a BICLA response and a reduction from baseline in BILAG total score. B cell reduction was faster in patients with associated SS. The sensitivity of B cells to epratuzumab as measured by the mean concentration producing 50% of the maximum B cell count depletion was lower for patients with associated SS (9.5 µg/ml) versus the total EMBODY population (87.1 µg/ml). No difference in the frequency of adverse events in those receiving placebo was reported. CONCLUSION: Patients with SLE and associated SS treated with epratuzumab showed improvement in SLE disease activity, which was associated with bioactivity, such as decreases in B cell number and IgM level.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Lupus Eritematoso Sistémico/tratamiento farmacológico , Síndrome de Sjögren/tratamiento farmacológico , Adulto , Linfocitos B/inmunología , Estudios de Casos y Controles , Ensayos Clínicos Fase III como Asunto , Femenino , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Ensayos Clínicos Controlados Aleatorios como Asunto , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Síndrome de Sjögren/complicaciones , Síndrome de Sjögren/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: Abnormal B-cell activation is implicated in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). The B-cell surface molecule CD22, which regulates activation through the B-cell receptor (BCR), is a potential target for inhibiting pathogenic B cells; however, the regulatory functions of CD22 remain poorly understood. In this study, we determined how targeting of CD22 with epratuzumab (Emab), a humanized anti-CD22 IgG1 monoclonal antibody, affects the activation of human B-cell subsets in response to Toll-like receptor 7 (TLR7) and BCR engagement. METHODS: B-cell subsets were isolated from human tonsils and stimulated with F(ab')2 anti-human IgM and/or the TLR7 agonist R848 in the presence of Emab or a human IgG1 isotype control. Changes in mRNA levels of genes associated with B-cell activation and differentiation were analyzed by quantitative PCR. Cytokine production was measured by ELISA. Cell proliferation, survival, and differentiation were assessed by flow cytometry. RESULTS: Pretreatment of phenotypically naïve CD19+CD10-CD27- cells with Emab led to a significant increase in IL-10 expression, and in some but not all patient samples to a reduction of IL-6 production in response to TLR7 stimulation alone or in combination with anti-IgM. Emab selectively inhibited the expression of PRDM1, the gene encoding B-lymphocyte-induced maturation protein 1 (Blimp-1) in activated CD10-CD27- B cells. CD10-CD27-IgD- cells were highly responsive to stimulation through TLR7 as evidenced by the appearance of blasting CD27hiCD38hi cells. Emab significantly inhibited the activation and differentiation of CD10-CD27-IgD- B cells into plasma cells. CONCLUSIONS: Emab can both regulate cytokine expression and block Blimp1-dependent B-cell differentiation, although the effects of Emab may depend on the stage of B-cell development or activation. In addition, Emab inhibits the activation of CD27-IgD- tonsillar cells, which correspond to so-called double-negative memory B cells, known to be increased in SLE patients with more active disease. These data may be relevant to the therapeutic effect of Emab in vivo via modulation of the production of pro-inflammatory and anti-inflammatory cytokines by B cells. Because Blimp-1 is required by B cells to mature into antibody-producing cells, inhibition of Blimp1 may reduce autoantibody production.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Linfocitos B/metabolismo , Citocinas/biosíntesis , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Receptor Toll-Like 7/metabolismo , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/metabolismo , Linfocitos B/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Sistemas de Liberación de Medicamentos/métodos , Humanos , Factor 1 de Unión al Dominio 1 de Regulación Positiva/antagonistas & inhibidores , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Receptor Toll-Like 7/antagonistas & inhibidoresRESUMEN
Treatment of systemic lupus erythematosus patients with epratuzumab (Emab), a humanized monoclonal antibody targeting CD22, leads to moderately reduced B-cell numbers but does not completely deplete B cells. Emab appears to induce immunomodulation of B cells, but the exact mode of action has not been defined. In the present study, we aimed to understand the effects of Emab on B cells using a humanized mouse model (Huki CD22), in which the B cells express human instead of murine CD22. Emab administration to Huki CD22 mice results in rapid and long-lasting CD22 internalization. There was no influence on B-cell turnover, but B-cell apoptosis ex vivo was increased. Emab administration to Huki CD22 mice had no effect on B-cell numbers in several lymphatic organs, nor in blood. In vitro exposure of B cells from Huki CD22 mice to Emab resulted in decreased B-cell receptor (BCR) induced Ca(2+) mobilization, whereas B-cell proliferation after Toll-like receptor (TLR) stimulation was not affected. In addition, IL-10 production was slightly increased after TLR and anti-CD40 stimulation, whereas IL-6 production was unchanged. In conclusion, Emab appears to inhibit BCR signaling in a CD22-dependent fashion without strong influence on B-cell development and B-cell populations.
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Anticuerpos Monoclonales Humanizados/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Recuento de Linfocitos , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Biomarcadores , Calcio/metabolismo , Citocinas/biosíntesis , Endocitosis , Humanos , Inmunomodulación/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Fosforilación , Receptores de Antígenos de Linfocitos B/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismoRESUMEN
The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.
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INTRODUCTION: CD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG1 antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8. METHODS: The potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE. RESULTS: CDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria. CONCLUSIONS: These findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.
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Anticuerpos Monoclonales/inmunología , Ligando de CD40/inmunología , Inmunidad Humoral/inmunología , Trombosis/inmunología , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Formación de Anticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/prevención & control , Modelos Animales de Enfermedad , Humanos , Inmunidad Humoral/efectos de los fármacos , Fragmentos Fab de Inmunoglobulinas/inmunología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/prevención & control , Macaca fascicularis , Macaca mulatta , Ratones Endogámicos NZB , Polietilenglicoles/química , Toxoide Tetánico/inmunología , Trombosis/inducido químicamenteRESUMEN
Epratuzumab is a B-cell-directed non-depleting monoclonal antibody that targets CD22. It is currently being evaluated in two phase 3 clinical trials in patients with systemic lupus erythematosus (SLE), a disease associated with abnormalities in B-cell function and activation. The mechanism of action of epratuzumab involves perturbation of the B-cell receptor (BCR) signalling complex and intensification of the normal inhibitory role of CD22 on the BCR, leading to reduced signalling and diminished activation of B cells. Such effects may result from down-modulation of CD22 upon binding by epratuzumab, as well as decreased expression of other proteins involved in amplifying BCR signalling capability, notably CD19. The net result is blunting the capacity of antigen engagement to induce B-cell activation. The functional consequences of epratuzumab binding to CD22 include diminished B-cell proliferation, effects on adhesion molecule expression, and B-cell migration, as well as reduced production of pro-inflammatory cytokines, such as IL-6 and TNF. Studies in patients treated with epratuzumab have revealed a number of pharmacodynamic effects that are linked to the mechanism of action (i.e., a loss of the target molecule CD22 from the B-cell surface followed by a modest reduction in peripheral B-cell numbers after prolonged therapy). Together, these data indicate that epratuzumab therapy affords a unique means to modulate BCR complex expression and signalling.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Linfocitos B/inmunología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Humanos , Lupus Eritematoso Sistémico/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal/inmunologíaRESUMEN
INTRODUCTION: Cytokines produced by B cells are believed to play important roles in autoimmune diseases. CD22 targeting by epratuzumab has been demonstrated to inhibit phosphorylation of B cell receptor (BCR) downstream signaling in B cells. It has been shown that other sialoadhesin molecules related to CD22 have immunoregulatory functions; therefore, in the present study, we addressed the role of epratuzumab on the production of key cytokines by B cells of patients with systemic lupus erythematosus (SLE) and of healthy donors (HD). METHODS: Peripheral blood B cells were purified and activated by BCR with or without Toll-like receptor 9 (TLR9) stimulation in the presence or absence of epratuzumab. Cytokine production by B cells (interleukin [IL]-6, tumor necrosis factor [TNF]-α and IL-10) in the supernatant and the induction of IL-10(+) B cells from patients with SLE and HD were analyzed. RESULTS: The secretion of the proinflammatory cytokines TNF-α and IL-6 by anti-BCR and BCR- and/or TLR9-activated B cells from HD and patients with SLE was inhibited by epratuzumab. In contrast, the production of IL-10 by B cells was not affected by epratuzumab under either stimulation condition. Consistently, the induction of IL-10-producing B cells in culture was not affected by epratuzumab. CONCLUSIONS: Epratuzumab, by targeting CD22, was able to inhibit the production of the proinflammatory cytokines IL-6 and TNF-α by B cells, in contrast to IL-10, in vitro. These data suggest that targeting CD22 alters the balance between proinflammatory cytokines (TNF-α, IL-6) and the regulatory cytokine IL-10 as another B cell effector mechanism.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Linfocitos B/metabolismo , Interleucina-10 , Interleucina-6/antagonistas & inhibidores , Lupus Eritematoso Sistémico/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto , Linfocitos B/efectos de los fármacos , Donantes de Sangre , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Femenino , Humanos , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Interleucina-10/biosíntesis , Interleucina-6/biosíntesis , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/biosíntesis , Adulto JovenRESUMEN
CD22 is a 140-kDa member of the Siglec family of cell surface proteins that is expressed by most mature B-cell lineages. As a co-receptor of the B-cell receptor (BCR), it is known to contribute to the sensitive control of the B-cell response to antigen. Cross-linking of CD22 and the BCR by antigen triggers the phosphorylation of CD22, which leads to activation of signaling molecules such as phosphatases. Signal transduction pathways involving CD22 have been explored in a number of mouse models, some of which have provided evidence that in the absence of functional CD22, B cells have a "hyperactivated" phenotype, and suggest that loss of CD22 function could contribute to the pathogenesis of autoimmune diseases. Modulating CD22 activity has therefore been suggested as a possible therapeutic approach to such diseases. For example, the novel CD22-targeting monoclonal antibody epratuzumab is currently under investigation as a treatment for the connective tissue disorder systemic lupus erythematosus (SLE).
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Autoinmunidad/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcr/genética , Proteínas Proto-Oncogénicas c-bcr/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética , Transducción de Señal/efectos de los fármacosRESUMEN
LFA-1 (lymphocyte function-associated antigen-1) plays a role in intercellular adhesion and lymphocyte trafficking and activation and is an attractive anti-inflammatory drug target. The alpha-subunit of LFA-1, in common with several other integrins, has an N-terminally inserted domain (I-domain) of approximately 200 amino acids that plays a central role in regulating ligand binding to LFA-1. An additional region, termed the I-domain allosteric site (IDAS), has been identified exclusively within the LFA-1 I-domain and shown to regulate the function of this protein. The IDAS is occupied by small molecule LFA-1 inhibitors when cocrystallized or analyzed by (15)N-(1)H HSQC (heteronuclear single-quantum coherence) NMR (nuclear magnetic resonance) titration experiments. We report here a novel arylthio inhibitor that binds the I-domain with a K(d) of 18.3 nM as determined by isothermal titration calorimetry (ITC). This value is in close agreement with the IC(50) (10.9 nM) derived from a biochemical competition assay (DELFIA) that measures the level of inhibition of binding of whole LFA-1 to its ligand, ICAM-1. Having established the strong affinity of the arylthio inhibitor for the isolated I-domain, we have used a range of techniques to further characterize the binding, including ITC, NMR, and X-ray crystallography. We have first developed an effective ITC binding assay for use with low-solubility inhibitors that avoids the need for ELISA-based assays. In addition, we utilized a fast NMR-based assay for the generation of I-domain-inhibitor models. This is based around the collection of HCCH-TOCSY spectra of LFA-1 in the bound form and the identification of a subset of side chain methyl groups that give chemical shift changes upon binding of LFA-1 inhibitors. This subset was used in two-dimensional (13)C-(15)N and (15)N-filtered and -edited two-dimensional NMR experiments to identify a minimal set of intraligand and ligand-protein NOEs, respectively (nuclear Overhauser enhancements). Models from the NMR data were assessed by comparison to an X-ray crystallographic structure of the complex, confirming that the method correctly predicted the essential features of the bound ligand.
Asunto(s)
Antígeno-1 Asociado a Función de Linfocito/química , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Sitio Alostérico , Amidas/química , Unión Competitiva , Calorimetría , Cinamatos/química , Cinamatos/metabolismo , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/químicaRESUMEN
We describe a series of dehydrophenylalanine derivatives where the Z isomers are potent VLA-4 antagonists but are subject to rapid biliary clearance and the E isomers have poor activity but have a slower rate of clearance. These configurationally constrained molecules have led to the design of a novel class of benzodiazepine VLA-4 antagonists.
Asunto(s)
Integrina alfa4beta1/antagonistas & inhibidores , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/farmacología , Animales , Benzodiazepinas/química , Benzodiazepinas/farmacología , Sistema Biliar/metabolismo , Diseño de Fármacos , Concentración 50 Inhibidora , Isomerismo , Fenilalanina/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
Macrophage clearance is essential for the resolution of inflammation. Much is known about how monocytes enter the inflammatory site but little is known about how resultant macro-phages are cleared. We have previously demonstrated that macrophage clearance from resolving peritonitis occurs by emigration into draining lymphatics rather than local apoptosis. We now examine mechanisms for this process, in particular by evaluating the hypothesis that modulation of adhesion interactions between macrophages and cells lining the lymphatics regulates the rate of macrophage clearance. We demonstrate in vivo that macrophages adhere specifically to mesothelium overlying draining lymphatics and that their emigration rate is regulated by the state of macrophage activation. We observed that macrophage-mesothelial adhesion is Arg-Gly-Asp (RGD) sensitive and partially mediated by very late antigen (VLA)-4 and VLA-5 but not alpha(v) or beta(2) integrins. Moreover, macrophage clearance into lymphatics can be blocked in vivo by RGD peptides and VLA-4 and VLA-5 but not beta(2) blocking antibodies. This is the first evidence that macrophage emigration from the inflamed site is controlled and demonstrates that this is exerted through specific adhesion molecule regulation of macrophage-mesothelial interactions. It highlights the importance of adhesion molecules governing entry of cells into the lymphatic circulation, thus opening a new avenue for manipulating the resolution of inflammation.