RESUMEN
The objective of this study was to characterize the complement-inhibiting activity of SAR445088, a novel monoclonal antibody specific for the active form of C1s. Wieslab® and hemolytic assays were used to demonstrate that SAR445088 is a potent, selective inhibitor of the classical pathway of complement. Specificity for the active form of C1s was confirmed in a ligand binding assay. Finally, TNT010 (a precursor to SAR445088) was assessed in vitro for its ability to inhibit complement activation associated with cold agglutinin disease (CAD). TNT010 inhibited C3b/iC3b deposition on human red blood cells incubated with CAD patient serum and decreased their subsequent phagocytosis by THP-1 cells. In summary, this study identifies SAR445088 as a potential therapeutic for the treatment of classical pathway-driven diseases and supports its continued assessment in clinical trials.
Asunto(s)
Anemia Hemolítica Autoinmune , Complemento C1s , Humanos , Complemento C1s/metabolismo , Activación de Complemento , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Inactivadores del Complemento/uso terapéutico , Vía Clásica del ComplementoRESUMEN
Dysregulated activation of the complement system is implicated in the onset or progression of several diseases. Most clinical-stage complement inhibitors target the inactive complement proteins present at high concentrations in plasma, which increases target-mediated drug disposition and necessitates high drug levels to sustain therapeutic inhibition. Furthermore, many efforts are aimed at inhibiting only terminal pathway activity, which leaves opsonin-mediated effector functions intact. We describe the discovery of SAR443809, a specific inhibitor of the alternative pathway C3/C5 convertase (C3bBb). SAR443809 selectively binds to the activated form of factor B (factor Bb) and inhibits alternative pathway activity by blocking the cleavage of C3, leaving the initiation of classical and lectin complement pathways unaffected. Ex vivo experiments with patient-derived paroxysmal nocturnal hemoglobinuria erythrocytes show that, although terminal pathway inhibition via C5 blockade can effectively inhibit hemolysis, proximal complement inhibition with SAR443809 inhibits both hemolysis and C3b deposition, abrogating the propensity for extravascular hemolysis. Finally, intravenous and subcutaneous administration of the antibody in nonhuman primates demonstrated sustained inhibition of complement activity for several weeks after injection. Overall, SAR443809 shows strong potential for treatment of alternative pathway-mediated disorders.
Asunto(s)
Factor B del Complemento , Vía Alternativa del Complemento , Animales , Factor B del Complemento/antagonistas & inhibidores , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Convertasas de Complemento C3-C5/antagonistas & inhibidores , Vía Alternativa del Complemento/efectos de los fármacos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/enzimología , Humanos , Macaca fascicularis , Anticuerpos/administración & dosificación , Proteolisis/efectos de los fármacosRESUMEN
Chronic autoimmune demyelinating neuropathies are a group of rare neuromuscular disorders with complex, poorly characterized etiology. Here we describe a phenotypic, human-on-a-chip (HoaC) electrical conduction model of two rare autoimmune demyelinating neuropathies, chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN), and explore the efficacy of TNT005, a monoclonal antibody inhibitor of the classical complement pathway. Patient sera was shown to contain anti-GM1 IgM and IgG antibodies capable of binding to human primary Schwann cells and induced pluripotent stem cell derived motoneurons. Patient autoantibody binding was sufficient to activate the classical complement pathway resulting in detection of C3b and C5b-9 deposits. A HoaC model, using a microelectrode array with directed axonal outgrowth over the electrodes treated with patient sera, exhibited reductions in motoneuron action potential frequency and conduction velocity. TNT005 rescued the serum-induced complement deposition and functional deficits while treatment with an isotype control antibody had no rescue effect. These data indicate that complement activation by CIDP and MMN patient serum is sufficient to mimic neurophysiological features of each disease and that complement inhibition with TNT005 was sufficient to rescue these pathological effects and provide efficacy data included in an investigational new drug application, demonstrating the model's translational potential.
RESUMEN
Terminal complement pathway inhibition at the level of C5 alleviates symptoms of several diseases associated with complement overactivation. However, C5 blockade is associated with an increased risk of invasive meningococcal disease despite immunization. Targeting specific complement pathways proximal to C5 provides the theoretical advantage of leaving the other pathways (including the terminal pathway) intact for immune surveillance. We aimed to address the risk of Neisseria meningitidis and Streptococcus pneumoniae infections when inhibiting the classical pathway (CP) using a specific C1s inhibitor (TNT005). Addition of TNT005 to 20% normal human serum that contained anti-meningococcal capsular Ab decreased C4 deposition 8-fold and abrogated killing of N. meningitidis, despite leaving C3 deposition intact. TNT005 impaired killing of N. meningitidis in 78% nonimmune human plasma and 78% whole blood but permitted killing in both when specific anti-capsular Ab was added. Simultaneously inhibiting both the CP and alternative pathway (AP) blocked killing of Ab-coated N. meningitidis in whole blood. Blocking the AP alone abrogated C3 deposition, whereas TNT005 only partially inhibited (â¼40% decrease) C3 deposition on S. pneumoniae coated with anti-capsular Ab. Blocking either the CP or AP alone did not impair killing of pneumococci in whole blood containing specific Ab (<10% survival at 3 h); however, blocking both pathways resulted in â¼35% bacterial survival. These data suggest that killing of N. meningitidis or S. pneumoniae in whole blood containing specific anti-capsular Abs is unimpeded by TNT005. Meningococcal and pneumococcal capsular conjugate vaccines may mitigate risk of these infections in patients receiving C1s inhibitors.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Cápsulas Bacterianas/inmunología , Complemento C1/antagonistas & inhibidores , Neisseria meningitidis/inmunología , Streptococcus pneumoniae/inmunología , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/inmunología , Complemento C4/inmunología , Vía Clásica del Complemento/efectos de los fármacos , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunologíaRESUMEN
Deposition of autoantibodies (α-BP180 and BP230) and complement along the dermal-epidermal-junction is a hallmark of bullous pemphigoid and was shown to be important for pathogenesis. Given the adverse effects of standard treatment (glucocorticoids, immunosuppressants), there is an unmet need for safe and effective therapies. In this phase 1 trial, we evaluated the safety and activity of BIVV009 (sutimlimab, previously TNT009), a targeted C1s inhibitor, in 10 subjects with active or past bullous pemphigoid (NCT02502903). Four weekly 60 mg/kg infusions of BIVV009 proved sufficient for inhibition of the classical complement pathway in all patients, as measured by CH50. C3c deposition along the dermal-epidermal junction was partially or completely abrogated in 4 of 5 patients, where it was present at baseline. BIVV009 was found to be safe and tolerable in this elderly population, with only mild to moderate adverse events reported (e.g., headache, fatigue). One serious adverse event (i.e., fatal cardiac decompensation) occurred at the end of the post-treatment observation period in an 84-year-old patient with a history of diabetes and heart failure, but was deemed unlikely to be related to the study drug. This trial provides the first results with a complement-targeting therapy in bullous pemphigoid, to our knowledge, and supports further studies on BIVV009's efficacy and safety in this population.
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Autoantígenos/administración & dosificación , Complemento C3/metabolismo , Vía Clásica del Complemento/efectos de los fármacos , Dermis/patología , Distonina/administración & dosificación , Epidermis/patología , Colágenos no Fibrilares/administración & dosificación , Penfigoide Ampolloso/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Dermis/metabolismo , Epidermis/metabolismo , Femenino , Humanos , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Penfigoide Ampolloso/metabolismo , Penfigoide Ampolloso/patología , Colágeno Tipo XVIIRESUMEN
The classical pathway of complement (CP) can mediate C3 opsonization of Ags responsible for the costimulation and activation of cognate B lymphocytes. In this manner, the complement system acts as a bridge between the innate and adaptive immune systems critical for establishing a humoral response. However, aberrant complement activation is often observed in autoimmune diseases in which C3 deposition on self-antigens may serve to activate self-reactive B cell clones. In this study, we use BIVV009 (Sutimlimab), a clinical stage, humanized mAb that specifically inhibits the CP-specific serine protease C1s to evaluate the impact of upstream CP antagonism on activation and proliferation of normal and autoimmune human B cells. We report that BIVV009 significantly inhibited complement-mediated activation and proliferation of primary human B cells. Strikingly, CP antagonism suppressed human Ig-induced activation of B cells derived from patients with rheumatoid arthritis. These results suggest that clinical use of CP inhibitors in autoimmune patients may not only block complement-mediated tissue damage, but may also prevent the long-term activation of autoimmune B cells and the production of autoantibodies that contribute to the underlying pathologic condition of these diseases.
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Anticuerpos Monoclonales Humanizados/farmacología , Artritis Reumatoide/tratamiento farmacológico , Linfocitos B/efectos de los fármacos , Complemento C1s/antagonistas & inhibidores , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Linfocitos B/inmunología , Linfocitos B/patología , Proliferación Celular/efectos de los fármacos , Activación de Complemento/efectos de los fármacos , Complemento C1s/inmunología , HumanosRESUMEN
Cold agglutinin disease is a difficult-to-treat autoimmune hemolytic anemia in which immunoglobulin M antibodies bind to erythrocytes and fix complement, resulting in predominantly extravascular hemolysis. This trial tested the hypothesis that the anti-C1s antibody sutimlimab would ameliorate hemolytic anemia. Ten patients with cold agglutinin disease participated in the phase 1b component of a first-in-human trial. Patients received a test dose of 10-mg/kg sutimlimab followed by a full dose of 60 mg/kg 1 to 4 days later and 3 additional weekly doses of 60 mg/kg. All infusions were well tolerated without premedication. No drug-related serious adverse events were observed. Seven of 10 patients with cold agglutinin disease responded with a hemoglobin increase >2 g/dL. Sutimlimab rapidly increased hemoglobin levels by a median of 1.6 g/dL within the first week, and by a median of 3.9 g/dL (interquartile range, 1.3-4.5 g/dL; 95% confidence interval, 2.1-4.5) within 6 weeks (P = .005). Sutimlimab rapidly abrogated extravascular hemolysis, normalizing bilirubin levels within 24 hours in most patients and normalizing haptoglobin levels in 4 patients within 1 week. Hemolytic anemia recurred when drug levels were cleared from the circulation 3 to 4 weeks after the last dose of sutimlimab. Reexposure to sutimlimab in a named patient program recapitulated the control of hemolytic anemia. All 6 previously transfused patients became transfusion-free during treatment. Sutimlimab was safe, well tolerated, and rapidly stopped C1s complement-mediated hemolysis in patients with cold agglutinin disease, significantly increasing hemoglobin levels and precluding the need for transfusions. This trial was registered at www.clinicaltrials.gov as #NCT02502903.
Asunto(s)
Anemia Hemolítica Autoinmune/tratamiento farmacológico , Anemia Hemolítica/prevención & control , Anticuerpos Monoclonales Humanizados/uso terapéutico , Complemento C1s/antagonistas & inhibidores , Hemólisis/efectos de los fármacos , Índice de Severidad de la Enfermedad , Anciano , Anemia Hemolítica/etiología , Anemia Hemolítica Autoinmune/complicaciones , Complemento C1s/inmunología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios ProspectivosRESUMEN
BACKGROUND: Complement may play a key role in antibody-mediated rejection. A promising therapeutic approach may be classical pathway (CP) inhibition at the level of early component C1. METHODS: In this first-in-human, double-blind, randomized placebo-controlled phase 1 trial, we evaluated the safety and complement inhibitory effect of TNT009, a humanized monoclonal anti-C1s antibody. Sixty-four adult healthy volunteers received either single (n = 48; 7 consecutive cohorts, 0.3-100 mg/kg) or 4 weekly infusions (n = 16; 2 consecutive cohorts, 30 and 60 mg/kg per infusion) of TNT009 or placebo. To assess the effect of treatment on complement activity, sera from dosed subjects were analyzed in a CP activation assay evaluating C3d deposition on HLA-coated microbeads spiked with alloantibodies. RESULTS: Single doses of TNT009 at 3 to 100 mg/kg uniformly and profoundly inhibited HLA antibody-mediated C3d deposition (≥86% after 60 minutes), whereby the duration of CP inhibition (2-14 days) was dose-dependent. Four weekly doses persistently blocked complement for 5 to 6 weeks. Ex vivo serum CP activity was profoundly inhibited when TNT009 concentrations exceeded 20 µg/mL. Infusions were well tolerated without serious or severe adverse events. CONCLUSIONS: Treatment with TNT009 was safe and potently inhibited CP activity. Future studies in patients are required to assess the potential of TNT009 for preventing or treating antibody-mediated rejection.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Activación de Complemento/efectos de los fármacos , Complemento C1s/antagonistas & inhibidores , Inactivadores del Complemento/administración & dosificación , Antígenos HLA/inmunología , Adulto , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/sangre , Anticuerpos Monoclonales Humanizados/uso terapéutico , Complemento C1s/inmunología , Inactivadores del Complemento/efectos adversos , Inactivadores del Complemento/sangre , Método Doble Ciego , Esquema de Medicación , Femenino , Voluntarios Sanos , Humanos , Infusiones Intravenosas , Masculino , Factores de Tiempo , Resultado del TratamientoRESUMEN
BACKGROUND: We aimed to evaluate metabolic outcomes in overweight/obese nulliparous and multiparous women and their offspring. STUDY DESIGN: Seventy-two overweight and obese women who participated in a randomized controlled trial of exercise in pregnancy were included in the study, comparing 18 nulliparous and 54 multiparous women and their singleton offspring. Women were assessed at 19 and 36 weeks of gestation. Fetal growth was measured using standard obstetric ultrasound techniques. Cord blood was collected at birth. Maternal and offspring body composition was assessed using DXA ~2 weeks after delivery. RESULTS: Nulliparous women had higher HbA1c in the third trimester of pregnancy than multiparous women (5.48% vs 5.29%; P=.002) and were more insulin-resistant based on the surrogate marker sex hormone-binding globulin (354 vs 408 nmol/L; P=.047). Nulliparous women also had higher levels of the inflammatory marker tumour necrosis factor-alpha (4.74 vs 3.62 pg/mL; P=.025). At birth, the offspring of nulliparous women were on average 340 g (P=.013) and 0.69 standard deviation scores (P=.026) lighter than those born of multiparous women. Cord blood data showed lower insulin-like growth factor-II (P=.026) and higher IGF binding protein-1 (P=.002) levels in the offspring of nulliparous women. In addition, a less favourable metabolic profile was observed in the offspring of nulliparous women, as indicated by higher triglyceride (P<.001) and interleukin-6 (P=.039) concentrations. CONCLUSIONS: Infants born of nulliparous overweight and obese women appear to be exposed to a less favourable metabolic environment in utero, with evidence of subtle adverse metabolic outcomes at birth compared to infants of overweight/obese multiparous women.
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Metaboloma/fisiología , Obesidad/complicaciones , Sobrepeso/complicaciones , Paridad/fisiología , Adulto , Peso al Nacer , Femenino , Desarrollo Fetal , Humanos , Recién Nacido , Masculino , Madres , Obesidad/metabolismo , Sobrepeso/metabolismo , Embarazo , Complicaciones del Embarazo/etiología , Adulto JovenRESUMEN
BACKGROUND: Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. METHODS: Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). RESULTS: Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. CONCLUSIONS: Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR.
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Anticuerpos Monoclonales/farmacología , Adhesión Celular/efectos de los fármacos , Inactivadores del Complemento/farmacología , Vía Clásica del Complemento/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Antígenos HLA-A/inmunología , Inmunosupresores/farmacología , Monocitos/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Complemento C3a/farmacología , Complemento C5a/farmacología , Relación Dosis-Respuesta a Droga , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Exocitosis/efectos de los fármacos , Antígenos HLA-A/metabolismo , Humanos , Antígeno de Macrófago-1/inmunología , Antígeno de Macrófago-1/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Selectina-P/inmunología , Selectina-P/metabolismoRESUMEN
BACKGROUND: Policies and protocols vary widely for fetal surveillance in a pregnancy where the fetus is suspected to be large-for-gestational-age (LGA). All ultimately culminate in decisions about the mode and timing of birth. LGA is known to be associated with increased risks to both the mother and baby. Interventions based on surveillance regimen findings may be associated with risks to the mother and baby. OBJECTIVES: To assess the effectiveness or efficacy of different antenatal surveillance methods for the suspected LGA fetus on important health outcomes for the mother and baby. SEARCH METHODS: We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (30 August 2015), ClinicalTrials.gov and the WHO International Clinical Trials Registry Platform (ICTRP) (21 August 2015). SELECTION CRITERIA: Published and unpublished randomised, quasi-randomised and cluster-randomised trials comparing the effects of described antenatal fetal surveillance regimens for women with suspected LGA infants. DATA COLLECTION AND ANALYSIS: We identified no studies that met the inclusion criteria for this review. MAIN RESULTS: There are no included trials. AUTHORS' CONCLUSIONS: We found no randomised controlled trials that assessed the effect of antenatal fetal surveillance regimens of a suspected LGA fetus on important health outcomes for the mother and baby.There has been a rise in the prevalence of LGA babies over the past few decades in many countries. Research is therefore required on regimens of antenatal surveillance of suspected LGA infants, in order to guide practice and improve the health outcomes for the mother and infant. In particular, randomised control trials to investigate whether serial antenatal clinic and ultrasound assessments of suspected LGA infants (including liquor volume and markers of fetal adiposity) would be useful, to assess whether surveillance methods improve health outcomes. In addition, as there are concerns that identifying suspected LGA fetuses may lead to unnecessary maternal anxiety, investigations and interventions, any such trial would need to assess the risks as well as benefits of regimens of fetal surveillance for suspected LGA fetuses.
Asunto(s)
Macrosomía Fetal/diagnóstico , Monitoreo Fetal/métodos , Femenino , Humanos , EmbarazoRESUMEN
Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Activación de Complemento/efectos de los fármacos , Complemento C1q/efectos de los fármacos , Complemento C1s/antagonistas & inhibidores , Citocinas/efectos de los fármacos , Citofagocitosis/efectos de los fármacos , Tolerancia Inmunológica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Apoptosis/inmunología , Activación de Complemento/inmunología , Complemento C1q/inmunología , Complemento C1q/metabolismo , Complemento C1s/metabolismo , Complemento C3b/efectos de los fármacos , Complemento C3b/inmunología , Complemento C3b/metabolismo , Citocinas/inmunología , Citofagocitosis/inmunología , Humanos , Técnicas In Vitro , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Células Jurkat , Macrófagos/inmunología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The interaction of amyloid-beta (Aß) and tau in the pathogenesis of Alzheimer's disease is a subject of intense inquiry, with the bulk of evidence indicating that changes in tau are downstream of Aß. It has been shown however, that human tau overexpression in amyloid precursor protein transgenic mice increases Aß plaque deposition. Here, we confirm that human tau increases Aß levels. To determine if the observed changes in Aß levels were because of intracellular or extracellular secreted tau (eTau for extracellular tau), we affinity purified secreted tau from Alzheimer's disease patient-derived cortical neuron conditioned media and analyzed it by liquid chromatography-mass spectrometry. We found the extracellular species to be composed predominantly of a series of N-terminal fragments of tau, with no evidence of C-terminal tau fragments. We characterized a subset of high affinity tau antibodies, each capable of engaging and neutralizing eTau. We found that neutralizing eTau reduces Aß levels in vitro in primary human cortical neurons where exogenously adding eTau increases Aß levels. In vivo, neutralizing human tau in 2 human tau transgenic models also reduced Aß levels. We show that the human tau insert sequence is sufficient to cause the observed increase in Aß levels. Our data furthermore suggest that neuronal hyperactivity may be the mechanism by which this regulation occurs. We show that neuronal hyperactivity regulates both eTau secretion and Aß production. Electrophysiological analysis shows for the first time that secreted eTau causes neuronal hyperactivity. Its induction of hyperactivity may be the mechanism by which eTau regulates Aß production. Together with previous findings, these data posit a novel connection between tau and Aß, suggesting a dynamic mechanism of positive feed forward regulation. Aß drives the disease pathway through tau, with eTau further increasing Aß levels, perpetuating a destructive cycle.
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Péptidos beta-Amiloides/metabolismo , Neuronas/metabolismo , Proteínas tau/fisiología , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Cromatografía Liquida , Humanos , Espectrometría de Masas , Ratones Transgénicos , Neuronas/fisiología , Proteínas tau/química , Proteínas tau/aislamiento & purificaciónRESUMEN
BACKGROUND: Obesity during pregnancy is associated with adverse outcomes for the offspring and mother. Lifestyle interventions in pregnancy such as antenatal exercise, are proposed to improve both short- and long-term health of mother and child. We hypothesise that regular moderate-intensity exercise during the second half of pregnancy will result in improved maternal and offspring outcomes, including a reduction in birth weight and adiposity in the offspring, which may be protective against obesity in later life. METHODS/DESIGN: The IMPROVE (Improving Maternal and Progeny Risks of Obesity Via Exercise) study is a two-arm parallel randomised controlled clinical trial being conducted in Auckland, New Zealand. Overweight and obese women (BMI ≥25 kg/m2) aged 18-40 years, with a singleton pregnancy of <20 weeks of gestation, from the Auckland region, are eligible for the trial. Exclusion criteria are ongoing smoking or medical contra-indications to antenatal exercise.Participants are randomised with 1:1 allocation ratio to either intervention or control group, using computer-generated randomisation sequences in variable block sizes, stratified on ethnicity and parity, after completion of baseline assessments. The intervention consists of a 16-week structured home-based moderate-intensity exercise programme utilising stationary cycles and heart rate monitors, commencing at 20 weeks of gestation. The control group do not receive any exercise intervention. Both groups undergo regular fetal ultrasonography and receive standard antenatal care. Due to the nature of the intervention, participants are un-blinded to group assignment during the trial.The primary outcome is offspring birth weight. Secondary offspring outcomes include fetal and neonatal body composition and anthropometry, neonatal complications and cord blood metabolic markers. Maternal outcomes include weight gain, pregnancy and delivery complications, aerobic fitness, quality of life, metabolic markers and post-partum body composition. DISCUSSION: The results of this trial will provide valuable insights on the effects of antenatal exercise on health outcomes in overweight and obese mothers and their offspring. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12612000932864.
Asunto(s)
Peso al Nacer , Ejercicio Físico , Obesidad/terapia , Atención Prenatal/métodos , Adiposidad , Adolescente , Adulto , Antropometría , Femenino , Humanos , Recién Nacido , Bienestar Materno , Obesidad/sangre , Sobrepeso/sangre , Sobrepeso/terapia , Aptitud Física/fisiología , Embarazo , Complicaciones del Embarazo , Segundo Trimestre del Embarazo/sangre , Tercer Trimestre del Embarazo/sangre , Calidad de Vida , Proyectos de Investigación , Ultrasonografía Prenatal , Aumento de Peso , Adulto JovenRESUMEN
Activation of the classical pathway (CP) of complement is often associated with autoimmune disorders in which disease pathology is linked to the presence of an autoantibody. One such disorder is cold agglutinin disease (CAD), an autoimmune hemolytic anemia in which autoantibodies (cold agglutinins) bind to red blood cells (RBCs) at low temperatures. Anemia occurs as a result of autoantibody-mediated CP activation on the surface of the erythrocyte, leading to the deposition of complement opsonins that drive extravascular hemolysis in the liver. Here we test the effects of TNT003, a mouse monoclonal antibody targeting the CP-specific serine protease C1s, on CP activity induced by cold agglutinins on human RBCs. We collected 40 individual CAD patient samples and showed that TNT003 prevented cold agglutinin-mediated deposition of complement opsonins that promote phagocytosis of RBCs. Furthermore, we show that by preventing CP activation, TNT003 also prevents cold agglutinin-driven generation of anaphylatoxins. Finally, we provide evidence that CP activity in CAD patients terminates prior to activation of the terminal cascade, supporting the hypothesis that the primary route of RBC destruction in these patients occurs via extravascular hemolysis. Our results support the development of a CP inhibitor for the treatment of CAD.
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Anemia Hemolítica Autoinmune/tratamiento farmacológico , Anticuerpos Monoclonales de Origen Murino/farmacología , Activación de Complemento/efectos de los fármacos , Complemento C1s/antagonistas & inhibidores , Serina Proteasas/sangre , Inhibidores de Serina Proteinasa/farmacología , Anciano , Anafilatoxinas/metabolismo , Anemia Hemolítica Autoinmune/enzimología , Anemia Hemolítica Autoinmune/patología , Animales , Línea Celular Tumoral , Complemento C1s/metabolismo , Femenino , Hemólisis/efectos de los fármacos , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268-275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.
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Anticuerpos Monoclonales/química , Antígeno CD11b/química , Epítopos/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Antígeno CD11b/inmunología , Línea Celular Tumoral , Chlorocebus aethiops , Perros , Drosophila melanogaster , Mapeo Epitopo , Epítopos/inmunología , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica/efectos de los fármacos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Homología de Secuencia de Aminoácido , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/inmunología , Vitronectina/química , Vitronectina/inmunologíaRESUMEN
LD22-4, an 86-amino acid fragment of the basic fibroblast growth factor, is an inhibitor of cell migration. LD22-4 inhibits the migration of various tumor cells, endothelial cells, and fibroblasts in vitro and suppresses tumor growth and angiogenesis in vivo. LD22-4 is effective in the presence of multiple growth factors, either alone or in combination, as well as haptotactic factors. LD22-4 inhibits the rate of malignant gliomas prepared from U87MG cells in an orthotopic mouse model by 90% compared with untreated mice. Using U87MG cells, we identified the LD22-4 membrane receptor as neuropilin 1 (NRP1). The identification of NRP1 as the LD22-4 receptor was based upon mass spectrometric analysis of proteins that bind to LD22-4, immunoprecipitation of an NRP1-LD22-4 complex formed during incubation of LD22-4 with U87MG cells, LD22-4-NRP1 coimmunoprecipitation studies, and binding of LD22-4 to HEK293 cells expressing NRP1. In contrast, NRP1 binding of an inactive mutant of LD22-4 was substantially reduced. As is typical of NRP1-binding proteins, LD22-4 itself binds to heparin and requires heparan sulfate for binding to cells. The addition of heparin to migration assays increased the inhibitory activity of LD22-4. In addition to a heparin-binding region, LD22-4 contains a 5-amino acid C-terminus that matches an NRP1 consensus binding sequence. Thus, direct binding experiments, dependence on heparan sulfate, and the presence of a NRP1 consensus binding sequence indicate that NRP1 is the binding site of LD22-4 and mediates inhibition of cell migration.
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Movimiento Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Glioma/tratamiento farmacológico , Neuropilina-1/metabolismo , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Procesos de Crecimiento Celular/efectos de los fármacos , Femenino , Factor 2 de Crecimiento de Fibroblastos/química , Glioma/metabolismo , Glioma/patología , Células HEK293 , Heparina/química , Heparina/metabolismo , Humanos , Ratones , Ratones Desnudos , Neuropilina-1/antagonistas & inhibidores , Fragmentos de Péptidos/química , Unión Proteica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The urokinase receptor (uPAR) can recognize several ligands. The structural basis for this multiple ligand recognition by uPAR is unknown. This study reports the crystal structures of uPAR in complex with both urokinase (uPA) and vitronectin and reveal that uPA occupies the central cavity of the receptor, whereas vitronectin binds at the outer side of the receptor. These results provide a structural understanding of one receptor binding to two ligands.
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Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Vitronectina/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores de Superficie Celular/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Activador de Plasminógeno de Tipo Uroquinasa/química , Vitronectina/químicaRESUMEN
PURPOSE: ATN-161 (Ac-PHSCN-NH(2)) is an integrin-binding peptide that is currently in phase II trials in cancer patients. This peptide has been shown to have antitumor activity in a number of different preclinical models. EXPERIMENTAL DESIGN: In this study, we examined the binding, biodistribution, and dose and biomarker response of ATN-161 in several animal models. RESULTS: ATN-161 bound to the beta subunit of a number of different integrins implicated in tumor growth and progression, which depended on its cysteine thiol. The peptide had antiangiogenic activity in the Matrigel plug model, and this activity could be reversed by inhibitors of protein kinase A, an effector of alpha(5)beta(1)-dependent angiogenesis. A labeled analogue of ATN-161, ATN-453, localized to neovessels but not to preexisting vasculature in vivo. The half-life of the peptide when localized to a tumor was much longer than in plasma. Dose-response studies in the Matrigel plug model of angiogenesis or a Lewis lung carcinoma model of tumor growth showed a U-shaped dose-response curve with 1 to 10 mg/kg given thrice a week, being the optimal dose range of ATN-161. Two additional pharmacodynamic models of angiogenesis (dynamic contrast-enhanced magnetic resonance imaging and measurement of endothelial cell progenitors) also revealed U-shaped dose-response curves. CONCLUSIONS: The presence of a U-shaped dose-response curve presents a significant challenge to identifying a biologically active dose of ATN-161. However, the identification of biomarkers of angiogenesis that also exhibit this same U-shaped response should allow the translation of those biomarkers to the clinic, allowing them to be used to identify the active dose of ATN-161 in phase II studies.
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Inhibidores de la Angiogénesis/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Oligopéptidos/farmacología , Animales , Biomarcadores de Tumor/análisis , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Neovascularización Patológica/tratamiento farmacológicoRESUMEN
Human urokinase-type plasminogen activator receptor (uPAR/CD87) is expressed at the invasive interface of the tumor-stromal microenvironment in many human cancers and interacts with a wide array of extracellular molecules. An anti-uPAR antibody (ATN615) was prepared using hybridoma technology. This antibody binds to uPAR in vitro with high affinity (K(d) approximately 1 nM) and does not interfere with uPA binding to uPAR. Here we report the crystal structure of the Fab fragment of ATN615 at 1.77 A and the analysis of ATN615-suPAR-ATF structure that was previously determined, emphasizing the ATN615-suPAR interaction. The complementarity determining regions (CDRs) of ATN615 consist of a high percentage of aromatic residues, and form a relatively flat and undulating surface. The ATN615 Fab fragment recognizes domain 3 of suPAR. The antibody-antigen recognition involves 11 suPAR residues and 12 Fab residues from five CDRs. Structural data suggest that Pro188, Asn190, Gly191, and Arg192 residues of uPAR are the key residues for the antibody recognition, while Pro189 and Arg192 render specificity of ATN615 for human uPAR. Interestingly, this antibody-antigen interface has a small contact area, mainly polar interaction with little hydrophobic character, yet has high binding strength. Furthermore, several solvent molecules (assigned as polyethylene glycols) were clearly visible in the binding interface between antibody and antigen, suggesting that solvent molecules may be important for the maximal binding between suPAR and ATN615 Fab. ATN615 undergoes small but noticeable changes in its CDR region upon antigen binding.