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BACKGROUND: Increased vascular endothelial growth factor B (VEGF-B) expression in patients with diabetic kidney disease (DKD) is associated with increased lipid deposition in glomerular podocytes. Reducing VEGF-B activity in animal models of DKD using an anti-VEGF-B antibody improved histological evidence of glomerular injury and reduced albuminuria; effects attributed to prevention of ectopic lipid deposition in the kidney. CSL346 is a novel humanized monoclonal antibody that binds VEGF-B with high affinity. Targeting VEGF-B in patients with type 2 diabetes mellitus may improve DKD progression markers. METHODS: An international, randomized, double-blind, placebo-controlled, phase 2a study (NCT04419467) assessed CSL346 (8 mg/kg or 16 mg/kg subcutaneously every 4 weeks for 12 weeks) in participants with type 2 diabetes mellitus and a urinary albumin-to-creatinine ratio (UACR) ≥150 mg/g (17.0 mg/mmol), and eGFR >20 mL/min/1.73m2. Efficacy, safety/tolerability, pharmacokinetics, and pharmacodynamics of CSL346 were evaluated. The primary analysis compared the change from baseline in log-transformed UACR between the two CSL346 dose groups combined versus placebo at Week 16. RESULTS: In total, 114 participants were randomized. CSL346 did not significantly reduce UACR compared with placebo at Week 16 (combined CSL346 group difference from placebo 95% confidence interval]: 4.0% [-14.7, 26.8]). Furthermore, no effect was seen in participant subgroups (degree of kidney impairment or sodium-glucose cotransporter 2 [SGLT2] inhibitor use) or on urinary biomarkers reflecting proximal tubular injury. CSL346 was generally well tolerated; however, diastolic blood pressure was significantly higher with CSL346 16 mg/kg versus placebo from Week 2 onwards, with differences ranging from +3.8 to +5.3 mmHg (P = 0.002 at Week 16). CONCLUSIONS: CSL346 did not reduce UACR compared with placebo at 16 weeks in participants with type 2 diabetes mellitus and DKD, and was associated with an increase in diastolic blood pressure.
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BACKGROUND & AIMS: Hepatic steatosis is a hallmark of non-alcoholic fatty liver disease (NAFLD), a common comorbidity in type 2 diabetes mellitus (T2DM). The pathogenesis of NAFLD is complex and involves the crosstalk between the liver and the white adipose tissue (WAT). Vascular endothelial growth factor B (VEGF-B) has been shown to control tissue lipid accumulation by regulating the transport properties of the vasculature. The role of VEGF-B signaling and the contribution to hepatic steatosis and NAFLD in T2DM is currently not understood. METHODS: C57BL/6 J mice treated with a neutralizing antibody against VEGF-B, or mice with adipocyte-specific overexpression or under-expression of VEGF-B (AdipoqCre+/VEGF-BTG/+ mice and AdipoqCre+/Vegfbfl/+mice) were subjected to a 6-month high-fat diet (HFD), or chow-diet, whereafter NAFLD development was assessed. VEGF-B expression was analysed in WAT biopsies from patients with obesity and NAFLD in a pre-existing clinical cohort (n = 24 patients with NAFLD and n = 24 without NAFLD) and correlated to clinicopathological features. RESULTS: Pharmacological inhibition of VEGF-B signaling in diabetic mice reduced hepatic steatosis and NAFLD by blocking WAT lipolysis. Mechanistically we show, by using HFD-fed AdipoqCre+/VEGF-BTG/+ mice and HFD-fed AdipoqCre+/Vegfbfl/+mice, that inhibition of VEGF-B signaling targets lipolysis in adipocytes. Reducing VEGF-B signaling ameliorated NAFLD by decreasing WAT inflammation, resolving WAT insulin resistance, and lowering the activity of the hormone sensitive lipase. Analyses of human WAT biopsies from individuals with NAFLD provided evidence supporting the contribution of VEGF-B signaling to NAFLD development. VEGF-B expression levels in adipocytes from two WAT depots correlated with development of dysfunctional WAT and NAFLD in humans. CONCLUSIONS: Taken together, our data from mouse models and humans suggest that VEGF-B antagonism may represent an approach to combat NAFLD by targeting hepatic steatosis through suppression of lipolysis. IMPACT AND IMPLICATIONS: Non-alcoholic fatty liver disease (NAFLD) is a common comorbidity in type 2 diabetes mellitus (T2DM) and has a global prevalence of between 25-29%. There are currently no approved drugs for NAFLD, and given the scale of the ongoing diabetes epidemics, there is an urgent need to identify new treatment options. Our work suggests that VEGF-B antagonism may represent an approach to combat NAFLD by targeting hepatic steatosis through suppression of lipolysis. The neutralizing anti-VEGF-B antibody, which was used in this study, has already entered clinical trials for patients with diabetes. Therefore, we believe that our results are of great general interest to a broad audience, including patients and patient organizations, the medical community, academia, the life science industry and the public.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Hepatopatia Gordurosa não Alcoólica , Humanos , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/metabolismo , Lipólise , Fator B de Crescimento do Endotélio Vascular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Camundongos Endogâmicos C57BL , Fígado/patologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Dieta Hiperlipídica/efeitos adversos , Tecido Adiposo/metabolismoRESUMO
BACKGROUND: Preclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1-1 versus Hp 2-2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200-900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions. RESULTS: In the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies. CONCLUSIONS: We present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions.
Assuntos
Haptoglobinas/genética , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Engenharia de Proteínas/métodos , Serina Endopeptidases/genética , Animais , Vasos Coronários/efeitos dos fármacos , Cobaias , Haptoglobinas/farmacologia , Heme/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Óxido Nítrico/metabolismo , Fenótipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Serina Endopeptidases/metabolismo , SuínosRESUMO
Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-µTP-L309C) generated by fusion of the IgM µ-tailpiece to the C terminus of human IgG1 Fc. Fc-µTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-µTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-µTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-µTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-µTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.
Assuntos
Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Doenças Autoimunes/imunologia , Proteínas do Sistema Complemento/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Receptores Fc/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular , Ativação do Complemento/imunologia , Humanos , Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/imunologia , Receptores de IgG/imunologiaRESUMO
Purpose: We tested the ability of an antibody fragment with specificity for vascular endothelial growth factor-B (VEGF-B) to regress nascent and established corneal blood vessels in the rat. Methods: A single chain variable antibody fragment (scFv) with specificity for VEGF-B was engineered from the 2H10 hybridoma. Binding to rat, mouse, and human VEGF-B was confirmed by surface plasmon resonance. Activity of the anti-VEGF-B scFv on developing and established corneal blood vessels was assessed following unilateral superficial cautery in male and female outbred Sprague Dawley rats. Groups (untreated, control scFv-treated, or anti-VEGF-B scFv-treated) comprised 6 to 22 rats. Treatment consisted of 5 µL scFv, 1 mg/mL, applied topically five times per day for 14 days, or two subconjunctival injections, 50 µg scFv each, applied 7 days apart, or combined topical and subconjunctival treatment. Corneal vessel area was quantified on hematoxylin-stained corneal flat-mounts, and groups were compared using the Mann-Whitney U test, with post hoc Bonferroni correction. Immunohistochemistry for cleaved caspase-3 was performed. Results: Topical anti-VEGF-B scFv therapy alone did not regress corneal blood vessels significantly (P > 0.05). Subconjunctival injection and combined treatment regressed 14-day established corneal blood vessels (25% reduction in vessel area [P = 0.04] and 37% reduction in vessel area [P < 0.001], respectively, compared to results in untreated controls). Cleaved caspase-3 was identified in vascular endothelial cells of anti-VEGF-B scFv-treated corneas. In scFv-treated rats, corneal endothelial cell function was maintained to 12 weeks after treatment and a normal blink reflex was present. Conclusions: The anti-VEGF-B scFv significantly regressed established but not developing corneal blood vessels in rats.
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Vasos Sanguíneos/efeitos dos fármacos , Córnea/irrigação sanguínea , Neovascularização da Córnea/tratamento farmacológico , Fragmentos de Imunoglobulinas/farmacologia , Fator B de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Caspase 3/metabolismo , Córnea/efeitos dos fármacos , Córnea/metabolismo , Neovascularização da Córnea/metabolismo , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Diabetic kidney disease (DKD) is the most common cause of severe renal disease, and few treatment options are available today that prevent the progressive loss of renal function. DKD is characterized by altered glomerular filtration and proteinuria. A common observation in DKD is the presence of renal steatosis, but the mechanism(s) underlying this observation and to what extent they contribute to disease progression are unknown. Vascular endothelial growth factor B (VEGF-B) controls muscle lipid accumulation through regulation of endothelial fatty acid transport. Here, we demonstrate in experimental mouse models of DKD that renal VEGF-B expression correlates with the severity of disease. Inhibiting VEGF-B signaling in DKD mouse models reduces renal lipotoxicity, re-sensitizes podocytes to insulin signaling, inhibits the development of DKD-associated pathologies, and prevents renal dysfunction. Further, we show that elevated VEGF-B levels are found in patients with DKD, suggesting that VEGF-B antagonism represents a novel approach to treat DKD.
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Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/prevenção & controle , Rim/patologia , Lipídeos/toxicidade , Transdução de Sinais , Fator B de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Albuminúria/complicações , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Progressão da Doença , Dislipidemias/complicações , Dislipidemias/metabolismo , Dislipidemias/patologia , Proteínas de Transporte de Ácido Graxo/metabolismo , Feminino , Deleção de Genes , Humanos , Insulina/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Adulto JovemRESUMO
The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface. IMPORTANCE Hepatitis C virus (HCV) infection affects â¼2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context of a recombinant soluble ectodomain. Our data demonstrate the variable motifs modulate binding of the E2 ectodomain to the major host cell receptor CD81 and have an impact on the formation of an E2 homodimer with high-affinity binding to CD81.
Assuntos
Hepacivirus/fisiologia , Proteínas do Envelope Viral/química , Internalização do Vírus , Regulação Alostérica , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Linhagem Celular Tumoral , Epitopos/química , Epitopos/imunologia , Células HEK293 , Hepatócitos/virologia , Humanos , Cinética , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Tetraspanina 28/química , Proteínas do Envelope Viral/fisiologiaRESUMO
Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant.
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Anticorpos Monoclonais/isolamento & purificação , Robótica , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Automação , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Miniaturização , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismoRESUMO
PURPOSE: The species cross-reactivity of the monoclonal antibodies infliximab, bevacizumab, and an anti-VEGF-B antibody, 2H10, in humans and rodents was determined. METHODS: The binding of infliximab to human, mouse, and rat TNF-α, of bevacizumab to human, mouse, and rat VEGF-A, and of the 2H10 antibody to human, mouse, and rat VEGF-B was evaluated by ELISA. The sequence of human, mouse, and rat TNF-α and VEGF-A at the binding sites for infliximab and bevacizumab were compared. RESULTS: Infliximab bound to human TNF-α, but no binding to mouse or rat TNF-α was detected between 10 pg/mL and 10 µg/ml. Sequence comparison of the binding site revealed four changes in mouse and five in rat TNF-α compared with human. Bevacizumab bound strongly to human VEGF-A, but showed 5-log weaker binding to both mouse and rat VEGF-A. There was a single amino acid substitution in mouse and rat VEGF-A at the bevacizumab binding site. The 2H10 antibody displayed a similar binding profile to human, mouse, and rat VEGF-B. CONCLUSIONS: The species cross-reactivity of monoclonal antibodies should be determined prior to their use in preclinical animal models. The 2H10 antibody binds to human, mouse, and rat VEGF-B making it suitable for testing in rodent models of human disease.
Assuntos
Anticorpos Monoclonais/farmacologia , Bevacizumab/farmacologia , Sítios de Ligação de Anticorpos/imunologia , Infliximab/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Corpo Vítreo/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Bevacizumab/imunologia , Reações Cruzadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Infliximab/imunologia , Camundongos , Ratos , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/imunologia , Doenças Retinianas/patologia , Corpo Vítreo/citologia , Corpo Vítreo/efeitos dos fármacosRESUMO
The development of therapeutic vaccines for treatment of established cancer has proven challenging. Cancer vaccines not only need to induce a robust tumor Ag-specific immune response but also need to overcome the tolerogenic and immunosuppressive microenvironments that exist within many solid cancers. ISCOMATRIX adjuvant (ISCOMATRIX) is able to induce both tumor Ag-specific cellular and Ab responses to protect mice against tumor challenge, but this is insufficient to result in regression of established solid tumors. In the current study, we have used B16-OVA melanoma, Panc-OVA pancreatic, and TRAMP-C1 prostate cancer mouse tumor models to test therapeutic efficacy of ISCOMATRIX vaccines combined with other immune modulators. The coadministration of an ISCOMATRIX vaccine with the TLR3 agonist, polyinosinic-polycytidylic acid, and TLR9 agonist, CpG, reduced tumor growth in all tumor models and the presence of ISCOMATRIX in the formulation was critical for the therapeutic efficacy of the vaccine. This vaccine combination induced a robust and multifunctional CD8(+) T cell response. Therapeutic protection required IFN-γ and CD8(+) T cells, whereas NK and CD4(+) T cells were found to be redundant. ISCOMATRIX vaccines combined with TLR3 and TLR9 agonists represent a promising cancer immunotherapy strategy.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Vacinas Anticâncer/administração & dosagem , Colesterol/administração & dosagem , Melanoma Experimental/terapia , Neoplasias Pancreáticas/terapia , Fosfolipídeos/administração & dosagem , Neoplasias da Próstata/terapia , Saponinas/administração & dosagem , Neoplasias Cutâneas/terapia , Adjuvantes Imunológicos/administração & dosagem , Animais , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Combinação de Medicamentos , Humanos , Imunoterapia/métodos , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/mortalidade , Camundongos , Camundongos Knockout , Transplante de Neoplasias , Oligodesoxirribonucleotídeos/farmacologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/mortalidade , Poli I-C/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/mortalidade , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Análise de Sobrevida , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Carga Tumoral/efeitos dos fármacosRESUMO
Interleukin-3 (IL-3) is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα) in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD), a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-13 receptors, adopting unique "open" and classical "closed" conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas "open-like" IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a "double hit" cytokine receptor blockade.
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Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Subunidade alfa de Receptor de Interleucina-3/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/imunologia , Antineoplásicos/metabolismo , Sítios de Ligação de Anticorpos , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Interleukin-13 (IL-13) is a cytokine implicated in airway diseases such as asthma and idiopathic pulmonary fibrosis. IL-13 signals through a heterodimeric receptor complex consisting of IL-13Rα1 and IL-4Rα, known as the type II IL-4R. IL-4 also signals through this receptor and as such many of the biological effects of IL-13 and IL-4 are similar. Here we describe the development of two sensitive bioassays to determine the potency of antagonists of the mouse type II IL-4R. Both IL-13 and IL-4 dose-dependently induce CCL17 production from J774 mouse monocytic cells and CCL11 production from NIH3T3 mouse fibroblasts in the presence of TNFα. The assays were optimized to minimize TNFα concentration, cell number and incubation time whilst retaining a suitable signal-to-background ratio. Anti-cytokine antibodies or recombinant soluble receptors completely neutralized IL-13 or IL-4 activity in these bioassays. The J774 assay was used to screen a panel of anti-mIL-13Rα1 antibodies for neutralizing activity against this receptor. We report the identification of the first monoclonal antibodies that bind mouse IL-13Rα1 and neutralize both IL-13-induced and IL-4-induced cellular function. These antibodies should prove useful for determining the effects of neutralizing IL-13Rα1 in mouse models of disease. In addition, these bioassays may be used for measuring the bioactivity of mouse IL-13 and IL-4 and for the discovery of additional antagonists of the mouse IL-13Rα1/IL-4Rα complex.
Assuntos
Anticorpos Neutralizantes/análise , Subunidade alfa1 de Receptor de Interleucina-13/antagonistas & inibidores , Animais , Anticorpos Neutralizantes/isolamento & purificação , Quimiocina CCL11/metabolismo , Citocinas/metabolismo , Fibroblastos/imunologia , Imunoensaio , Interleucina-13/metabolismo , Subunidade alfa1 de Receptor de Interleucina-13/imunologia , Interleucina-4/metabolismo , Camundongos , Monócitos/imunologia , Células NIH 3T3 , Receptores de Superfície Celular/imunologia , Transdução de SinaisRESUMO
Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined.
Assuntos
Anticorpos Monoclonais Humanizados/química , Subunidade alfa de Receptor de Interleucina-3/química , Sequência de Aminoácidos , Cristalização , Humanos , Dados de Sequência Molecular , Ligação Proteica , Difração de Raios XRESUMO
The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved ß-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Resistência à Insulina , Terapia de Alvo Molecular , Fator B de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator B de Crescimento do Endotélio Vascular/metabolismo , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , Dislipidemias/tratamento farmacológico , Dislipidemias/metabolismo , Endotélio Vascular/metabolismo , Feminino , Glucose/metabolismo , Teste de Tolerância a Glucose , Ilhotas Pancreáticas/anatomia & histologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/patologia , Metabolismo dos Lipídeos , Masculino , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator B de Crescimento do Endotélio Vascular/deficiência , Fator B de Crescimento do Endotélio Vascular/genéticaRESUMO
VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGF-B is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGF-B targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGF-B is achieved by regulating the expression of many vascular prosurvival genes via both NP-1 and VEGFR-1. Our work thus indicates that the function of VEGF-B in the vascular system is to act as a "survival," rather than an "angiogenic" factor and that VEGF-B inhibition may offer new therapeutic opportunities to treat neovascular diseases.
Assuntos
Neovascularização Patológica , Fator B de Crescimento do Endotélio Vascular/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genoma , Membro Posterior/irrigação sanguínea , Isquemia/genética , Isquemia/metabolismo , Camundongos , Camundongos Knockout , Ratos , Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Regulação para Cima , Fator B de Crescimento do Endotélio Vascular/deficiência , Fator B de Crescimento do Endotélio Vascular/genéticaRESUMO
Vascular endothelial growth factor (VEGF) B effects blood vessel formation by binding to VEGF receptor 1. To study the specifics of the biological profile of VEGF-B in both physiological and pathological angiogenesis, a neutralising anti-VEGF-B antibody (2H10) that functions by inhibiting the binding of VEGF-B to VEGF receptor 1 was developed. Here, we present the structural features of the 'highly ordered' interaction of the Fab fragment of this antibody (Fab-2H10) with VEGF-B. Two molecules of Fab-2H10 bind to symmetrical binding sites located at each pole of the VEGF-B homodimer, giving a unique U-shaped topology to the complex that has not been previously observed in the VEGF family. VEGF-B residues essential for binding to the antibody are contributed by both monomers of the cytokine. Our detailed analysis reveals that the neutralising effect of the antibody occurs by virtue of the steric hindrance of the receptor-binding interface. These findings suggest that functional complementarity between VEGF-B and 2H10 can be harnessed both in analysing the therapeutic potential of VEGF-B and as an antagonist of receptor activation.
Assuntos
Anticorpos/química , Fragmentos Fab das Imunoglobulinas/química , Fator B de Crescimento do Endotélio Vascular/química , Sequência de Aminoácidos , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Estrutura Secundária de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
IL-11 and IL-11 receptor (R)alpha are induced by Th2 cytokines. However, the role(s) of endogenous IL-11 in antigen-induced Th2 inflammation has not been fully defined. We hypothesized that IL-11, signaling via IL-11Ralpha, plays an important role in aeroallergen-induced Th2 inflammation and mucus metaplasia. To test this hypothesis, we compared the responses induced by the aeroallergen ovalbumin (OVA) in wild-type (WT) and IL-11Ralpha-null mutant mice. We also generated and defined the effects of an antagonistic IL-11 mutein on pulmonary Th2 responses. Increased levels of IgE, eosinophilic tissue and bronchoalveolar lavage (BAL) inflammation, IL-13 production, and increased mucus production and secretion were noted in OVA-sensitized and -challenged WT mice. These responses were at least partially IL-11 dependent because each was decreased in mice with null mutations of IL-11Ralpha. Importantly, the administration of the IL-11 mutein to OVA-sensitized mice before aerosol antigen challenge also caused a significant decrease in OVA-induced inflammation, mucus responses, and IL-13 production. Intraperitoneal administration of the mutein to lung-specific IL-13-overexpressing transgenic mice also reduced BAL inflammation and airway mucus elaboration. These studies demonstrate that endogenous IL-11R signaling plays an important role in antigen-induced sensitization, eosinophilic inflammation, and airway mucus production. They also demonstrate that Th2 and IL-13 responses can be regulated by interventions that manipulate IL-11 signaling in the murine lung.
Assuntos
Inflamação/metabolismo , Interleucina-11/metabolismo , Interleucina-13/metabolismo , Muco/metabolismo , Transdução de Sinais , Células Th2/metabolismo , Alérgenos/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Regulação da Expressão Gênica/efeitos dos fármacos , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Mucina-5AC/genética , Mucina-5AC/metabolismo , Ovalbumina/imunologia , Fenótipo , Receptores de Interleucina-11/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.
Assuntos
Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator B de Crescimento do Endotélio Vascular/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Retina/metabolismoRESUMO
The development of blood vessels (angiogenesis) is critical throughout embryogenesis and in some normal postnatal physiological processes. Pathological angiogenesis has a pivotal role in sustaining tumour growth and chronic inflammation. Vascular endothelial growth factor-B (VEGF-B) is a member of the VEGF family of growth factors that regulate blood vessel and lymphatic angiogenesis. VEGF-B is closely related to VEGF-A and placenta growth factor (PlGF), but unlike VEGF-A, which binds to two receptor tyrosine kinases VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), VEGF-B and PlGF bind to VEGFR-1 and not VEGFR-2. There is growing evidence of a role for VEGF-B in physiological and pathological blood vessel angiogenesis. VEGF-B may provide novel therapeutic strategies for the treatment of vascular disease and be a potential therapeutic target in aberrant vessel formation. To help understand at the molecular level the differential receptor binding profile of the VEGF family of growth factors we have determined the crystal structure of human VEGF-B(10-108) at 2.48 Angstroms resolution. The overall structure is very similar to that of the previously determined cysteine-knot motif growth factors: VEGF-A, PlGF and platelet-derived growth factor-B (PDGF-B). We also present a predicted model for the association of VEGF-B with the second domain of its receptor, VEGFR-1. Based on this interaction and the present structural data of the native protein, we have identified several putative residues that could play an important role in receptor recognition and specificity.
Assuntos
Aminoácidos , Estrutura Terciária de Proteína , Fator B de Crescimento do Endotélio Vascular/química , Fator B de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/química , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Alinhamento de Sequência , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The formation of new blood vessels (angiogenesis) is critical for both embryonic development and a variety of normal postnatal physiological processes. Various pathological processes, most notably tumour growth and chronic inflammation, are also known to be dependent on the new vessel formation. Amongst the variety of factors that contribute to the regulation of this complex process, vascular endothelial growth factor (VEGF or VEGF-A) is arguably the most well characterised. The VEGF family of growth factors is now known to comprise of VEGF-A plus four additional members, including VEGF-B. In contrast to VEGF-A, surprisingly little is known about the precise biological role of VEGF-B. Unlike VEGF-A, which binds to the two receptor tyrosine kinases VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), VEGF-B binds only to VEGFR-1 and the functional significance of VEGFR-1 signalling has remained problematic. More recently, however, evidence has emerged suggesting a key role for VEGFR-1 signalling in pathological angiogenesis and this has raised the possibility that, like VEGF-A, VEGFR-1 specific ligands such as VEGF-B may provide for novel therapeutic strategies and/or represent new therapeutic targets. Here we review current knowledge of the biology of VEGF-B. We note that although analysis to date, including expression profiling and the generation of gene targetted mice, has provided only limited insights, future studies using recently generated recombinant proteins and antagonist monoclonal antibodies should provide for a more comprehensive understanding.