RESUMO
In addition to their well characterized role in mediating IgE-dependent allergic diseases, aberrant accumulation and activation of mast cells (MCs) is associated with many non-allergic inflammatory diseases, whereby their activation is likely triggered by non-IgE stimuli (e.g., IL-33). Siglec-8 is an inhibitory receptor expressed on MCs and eosinophils that has been shown to inhibit IgE-mediated MC responses and reduce allergic inflammation upon ligation with a monoclonal antibody (mAb). Herein, we evaluated the effects of an anti-Siglec-8 mAb (anti-S8) in non-allergic disease models of experimental cigarette-smoke-induced chronic obstructive pulmonary disease and bleomycin-induced lung injury in Siglec-8 transgenic mice. Therapeutic treatment with anti-S8 inhibited MC activation and reduced recruitment of immune cells, airway inflammation, and lung fibrosis. Similarly, using a model of MC-dependent, IL-33-induced inflammation, anti-S8 treatment suppressed neutrophil influx, and cytokine production through MC inhibition. Transcriptomic profiling of MCs further demonstrated anti-S8-mediated downregulation of MC signaling pathways induced by IL-33, including TNF signaling via NF-κB. Collectively, these findings demonstrate that ligating Siglec-8 with an antibody reduces non-allergic inflammation and inhibits IgE-independent MC activation, supporting the evaluation of an anti-Siglec-8 mAb as a therapeutic approach in both allergic and non-allergic inflammatory diseases in which MCs play a role.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Lectinas/metabolismo , Mastócitos/imunologia , Pneumonia/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Sistema Respiratório/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Degranulação Celular , Fumar Cigarros , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Imunoglobulina E/metabolismo , Interleucina-33/metabolismo , Lectinas/genética , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Ativação de Neutrófilo , Transdução de SinaisRESUMO
Siglecs (sialic acid-binding immunoglobulin-like lectins) are single-pass cell surface receptors that have inhibitory activities on immune cells. Among these, Siglec-8 is a CD33-related family member selectively expressed on human mast cells and eosinophils, and at low levels on basophils. These cells can participate in inflammatory responses by releasing mediators that attract or activate other cells, contributing to the pathogenesis of allergic and non-allergic diseases. Since its discovery in 2000, initial in vitro studies have found that the engagement of Siglec-8 with a monoclonal antibody or with selective polyvalent sialoglycan ligands induced the cell death of eosinophils and inhibited mast cell degranulation. Anti-Siglec-8 antibody administration in vivo to humanized and transgenic mice selectively expressing Siglec-8 on mouse eosinophils and mast cells confirmed the in vitro findings, and identified additional anti-inflammatory effects. AK002 (lirentelimab) is a humanized non-fucosylated IgG1 antibody against Siglec-8 in clinical development for mast cell- and eosinophil-mediated diseases. AK002 administration has safely demonstrated the inhibition of mast cell activity and the depletion of eosinophils in several phase 1 and phase 2 trials. This article reviews the discovery and functions of Siglec-8, and strategies for its therapeutic targeting for the treatment of eosinophil- and mast cell-associated diseases.
Assuntos
Anticorpos Monoclonais Humanizados , Antígenos CD , Antígenos de Diferenciação de Linfócitos B , Eosinófilos/imunologia , Hipersensibilidade , Inflamação , Lectinas , Mastócitos/imunologia , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD/imunologia , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Ensaios Clínicos como Assunto , Eosinófilos/patologia , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Lectinas/imunologia , Lectinas/fisiologia , Mastócitos/patologia , Camundongos , Camundongos TransgênicosRESUMO
Aberrant accumulation and activation of eosinophils and potentially mast cells (MCs) contribute to the pathogenesis of eosinophilic gastrointestinal diseases (EGIDs), including eosinophilic esophagitis (EoE), gastritis (EG), and gastroenteritis (EGE). Current treatment options, such as diet restriction and corticosteroids, have limited efficacy and are often inappropriate for chronic use. One promising new approach is to deplete eosinophils and inhibit MCs with a monoclonal antibody (mAb) against sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8), an inhibitory receptor selectively expressed on MCs and eosinophils. Here, we characterize MCs and eosinophils from human EG and EoE biopsies using flow cytometry and evaluate the effects of an anti-Siglec-8 mAb using a potentially novel Siglec-8-transgenic mouse model in which EG/EGE was induced by ovalbumin sensitization and intragastric challenge. MCs and eosinophils were significantly increased and activated in human EG and EoE biopsies compared with healthy controls. Similar observations were made in EG/EGE mice. In Siglec-8-transgenic mice, anti-Siglec-8 mAb administration significantly reduced eosinophils and MCs in the stomach, small intestine, and mesenteric lymph nodes and decreased levels of inflammatory mediators. In summary, these findings suggest a role for both MCs and eosinophils in EGID pathogenesis and support the evaluation of anti-Siglec-8 as a therapeutic approach that targets both eosinophils and MCs.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Enterite/tratamento farmacológico , Eosinofilia/tratamento farmacológico , Eosinófilos/efeitos dos fármacos , Gastrite/tratamento farmacológico , Lectinas/efeitos dos fármacos , Mastócitos/imunologia , Animais , Modelos Animais de Doenças , Enterite/imunologia , Eosinofilia/imunologia , Esofagite Eosinofílica/tratamento farmacológico , Eosinófilos/imunologia , Feminino , Gastrite/imunologia , Gastroenterite , Humanos , Lectinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , OvalbuminaRESUMO
INTRODUCTION: Pathologic accumulation and activation of mast cells and eosinophils are implicated in allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is an inhibitory receptor selectively expressed on mast cells, eosinophils and, at a lower extent, basophils. When engaged with an antibody, Siglec-8 can induce apoptosis of activated eosinophils and inhibit mast cell activation. AK002 is a humanized, non-fucosylated IgG1 anti-Siglec-8 antibody undergoing clinical investigation for treatment of allergic, inflammatory, and proliferative diseases. Here we examine the human tissue selectivity of AK002 and evaluate the in vitro, ex vivo, and in vivo activity of AK002 on eosinophils and mast cells. METHODS: The affinity of AK002 for Siglec-8 and CD16 was determined by biolayer interferometry. Ex vivo activity of AK002 on human eosinophils from blood and dissociated human tissue was tested in apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The in vivo activity of a murine precursor of AK002 (mAK002) was tested in a passive systemic anaphylaxis (PSA) humanized mouse model. RESULTS: AK002 bound selectively to mast cells, eosinophils and, at a lower level, to basophils in human blood and tissue and not to other cell types examined. AK002 induced apoptosis of interleukin-5-activated blood eosinophils and demonstrated potent ADCC activity against blood eosinophils in the presence of natural killer cells. AK002 also significantly reduced eosinophils in dissociated human lung tissue. Furthermore, mAK002 prevented PSA in humanized mice through mast cell inhibition. CONCLUSION: AK002 selectively evokes potent apoptotic and ADCC activity against eosinophils and prevents systemic anaphylaxis through mast cell inhibition.
Assuntos
Anafilaxia/prevenção & controle , Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Eosinófilos/imunologia , Lectinas/imunologia , Mastócitos/imunologia , Anafilaxia/imunologia , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Basófilos/imunologia , Humanos , Camundongos , Ácido N-Acetilneuramínico/imunologia , Receptores de IgG/imunologiaRESUMO
BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 is selectively expressed on eosinophils, mast cells, and basophils and, when engaged on eosinophils, can cause cell death. OBJECTIVE: We sought to characterize surface and soluble Siglec-8 (sSiglec-8) levels in normal donors (NDs) and eosinophilic donors (EOs) and assess the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. METHODS: Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. Serum sSiglec-8 levels were measured by means of ELISA. Induction of eosinophil death by IgG4 (chimeric 2E2 IgG4) and afucosylated IgG1 (chimeric 2E2 IgG1 [c2E2 IgG1]) anti-Siglec-8 antibodies was evaluated in vitro by using flow cytometry and in vivo in humanized mice. RESULTS: Siglec-8 was consistently expressed on eosinophils from NDs and EOs and did not correlate with absolute eosinophil count or disease activity. sSiglec-8 levels were measurable in sera from most donors unrelated to absolute eosinophil counts or Siglec-8 surface expression. c2E2 IgG1 and chimeric 2E2 IgG4 were equally effective at inducing cell death (Annexin-V positivity) of purified eosinophils from NDs and EOs after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EOs, and natural killer cell-mediated eosinophil killing was seen only with c2E2 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. CONCLUSIONS: Siglec-8 is highly expressed on blood eosinophils from EOs and NDs and represents a potential therapeutic target for eosinophilic disorders. Enhanced killing of eosinophils in the presence of IL-5 might lead to increased efficacy in patients with IL-5-driven eosinophilia.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Eosinofilia/imunologia , Eosinófilos/imunologia , Células Matadoras Naturais/imunologia , Lectinas/metabolismo , Animais , Anticorpos Bloqueadores/genética , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Morte Celular , Células Cultivadas , Citotoxicidade Imunológica , Eosinofilia/terapia , Humanos , Imunoglobulina G/genética , Interleucina-5/metabolismo , Lectinas/genética , Lectinas/imunologia , Contagem de Leucócitos , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Proteínas Recombinantes de Fusão/genética , TranscriptomaRESUMO
BACKGROUND: Mast cells are a critical component of allergic responses in humans, and animal models that allow the in vivo investigation of their contribution to allergy and evaluation of new human-specific therapeutics are urgently needed. OBJECTIVE: To develop a new humanized mouse model that supports human mast cell engraftment and human IgE-dependent allergic responses. METHODS: This model is based on the NOD-scid IL2rg(null)SCF/GM-CSF/IL3 (NSG-SGM3) strain of mice engrafted with human thymus, liver, and hematopoietic stem cells (termed Bone marrow, Liver, Thymus [BLT]). RESULTS: Large numbers of human mast cells develop in NSG-SGM3 BLT mice and populate the immune system, peritoneal cavity, and peripheral tissues. The human mast cells in NSG-SGM3 BLT mice are phenotypically similar to primary human mast cells and express CD117, tryptase, and FcεRI. These mast cells undergo degranulation in an IgE-dependent and -independent manner, and can be readily cultured in vitro for additional studies. Intradermal priming of engrafted NSG-SGM3 mice with a chimeric IgE containing human constant regions resulted in the development of a robust passive cutaneous anaphylaxis response. Moreover, we describe the first report of a human mast cell antigen-dependent passive systemic anaphylaxis response in primed mice. CONCLUSIONS: NSG-SGM3 BLT mice provide a readily available source of human mast cells for investigation of mast cell biology and a preclinical model of passive cutaneous anaphylaxis and passive systemic anaphylaxis that can be used to investigate the pathogenesis of human allergic responses and to test new therapeutics before their advancement to the clinic.
Assuntos
Anafilaxia/imunologia , Modelos Animais de Doenças , Mastócitos/imunologia , Anafilaxia Cutânea Passiva/imunologia , Animais , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoglobulina E/imunologia , Transplante de Fígado , Camundongos , Timo/transplanteRESUMO
EphA3 is expressed in solid tumors and leukemias and is an attractive target for the therapy. We have generated a panel of Humaneered® antibodies to the ligand-binding domain using a Fab epitope-focused library that has the same specificity as monoclonal antibody mIIIA4. A high-affinity antibody was selected that competes with the mIIIA4 antibody for binding to EphA3 and has an improved affinity of â¼1 nM. In order to generate an antibody with potent cell-killing activity the variable regions were assembled with human IgG1k constant regions and expressed in a Chinese hamster ovary (CHO) cell line deficient in fucosyl transferase. Non-fucosylated antibodies have been reported to have enhanced binding affinity for the IgG receptor CD16a (FcγRIIIa). The affinity of the antibody for recombinant CD16a was enhanced approximately 10-fold. This resulted in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity against EphA3-expressing leukemic cells, providing a potent antibody for the evaluation as a therapeutic agent.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Receptor EphA3/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Células CHO , Cricetinae , Cricetulus , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Macaca mulatta , Dados de Sequência Molecular , Receptores de IgG/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Eph receptor tyrosine kinases are critical for cell-cell communication during normal and oncogenic tissue patterning and tumor growth. Somatic mutation profiles of several cancer genomes suggest EphA3 as a tumor suppressor, but its oncogenic expression pattern and role in tumorigenesis remain largely undefined. Here, we report unexpected EphA3 overexpression within the microenvironment of a range of human cancers and mouse tumor xenografts where its activation inhibits tumor growth. EphA3 is found on mouse bone marrow-derived cells with mesenchymal and myeloid phenotypes, and activation of EphA3(+)/CD90(+)/Sca1(+) mesenchymal/stromal cells with an EphA3 agonist leads to cell contraction, cell-cell segregation, and apoptosis. Treatment of mice with an agonistic α-EphA3 antibody inhibits tumor growth by severely disrupting the integrity and function of newly formed tumor stroma and microvasculature. Our data define EphA3 as a novel target for selective ablation of the tumor microenvironment and demonstrate the potential of EphA3 agonists for anticancer therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptores Proteína Tirosina Quinases/agonistas , Receptores Proteína Tirosina Quinases/biossíntese , Receptor EphA3/agonistas , Receptor EphA3/biossíntese , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA3/imunologia , Receptor EphA3/metabolismo , Transdução de Sinais , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in all subjects. Additional approaches are clearly needed. OBJECTIVE: We sought to explore the potential of the human eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1) as a therapeutic target for eosinophilic disorders. METHODS: EMR1 expression was assessed in blood and bone marrow specimens from eosinophilic and healthy subjects, cell lines, CD34(+) cells differentiated in vitro, and tissue biopsy specimens by using flow cytometry, quantitative PCR, and immunostaining. Eosinophil targeting by a novel, humanized, afucosylated anti-EMR1 IgG1 was evaluated in vitro by using a natural killer cell-mediated killing assay and in vivo in cynomolgus monkeys. RESULTS: Analysis of blood and bone marrow cells from healthy and eosinophilic donors and in vitro-differentiated CD34(+) cells confirmed restriction of human EMR1 surface and mRNA expression to mature eosinophils. Tissue eosinophils also expressed EMR1. Although EMR1 was highly expressed on eosinophils from all subjects, surface expression was negatively correlated with absolute eosinophil counts (r = -0.46, P < .001), and soluble plasma levels correlated positively with absolute eosinophil counts (r = 0.69, P < .001), suggesting modulation of EMR1 in vivo. Nevertheless, afucosylated anti-EMR1 mAb dramatically enhanced natural killer cell-mediated killing of eosinophils from healthy and eosinophilic donors and induced a rapid and sustained depletion of eosinophils in monkeys. CONCLUSION: EMR1 expression is restricted to mature blood and tissue eosinophils. Targeting of eosinophils with afucosylated anti-EMR1 antibody shows promise as a treatment for eosinophilic disorders.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Eosinofilia/tratamento farmacológico , Eosinófilos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina G/farmacologia , Glicoproteínas de Membrana/imunologia , Mucinas/imunologia , Receptores Acoplados a Proteínas G/imunologia , Anticorpos Monoclonais Murinos/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Proteínas de Ligação ao Cálcio , Eosinofilia/imunologia , Eosinofilia/patologia , Eosinófilos/patologia , Feminino , Humanos , Imunoglobulina G/imunologia , Células K562 , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Mucinas/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Células U937RESUMO
Granulocyte-macrophage-colony-stimulating factor (GM-CSF) hypersensitivity is a hallmark of juvenile myelomonocytic leukemia (JMML) but has not been systematically shown in the related human disease chronic myelomonocytic leukemia (CMML). We find that primary CMML samples demonstrate GM-CSF-dependent hypersensitivity by hematopoietic colony formation assays and phospho-STAT5 (pSTAT5) flow cytometry compared with healthy donors. Among CMML patients, the pSTAT5 hypersensitive response positively correlated with high-risk disease, peripheral leukocytes, monocytes, and signaling-associated mutations. When compared with IL-3 and G-CSF, GM-CSF hypersensitivity was cytokine specific and thus a possible target for intervention in CMML. To explore this possibility, we treated primary CMML cells with KB003, a novel monoclonal anti-GM-CSF antibody, and JAK2 inhibitors. We found that an elevated proportion of immature GM-CSF receptor-α(R) subunit-expressing cells were present in the bone marrow myeloid compartment of CMML. In survival assays, we found that myeloid and monocytic progenitors were sensitive to GM-CSF signal inhibition. Our data indicate that a committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML, consistent with results in JMML. These preclinical data indicate that GM-CSF signaling inhibitors merit further investigation in CMML and that GM-CSFR expression on myeloid progenitors may be a biomarker for this therapy.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Leucemia Mielomonocítica Crônica/metabolismo , Fator de Transcrição STAT5/metabolismo , Citometria de Fluxo , Humanos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Granulocyte macrophage colony-stimulating factor (GM-CSF) promotes infarct expansion and inappropriate collagen synthesis in a myocardial infarction (MI). This study was designed to determine if treatment with anti-GM-CSF will inhibit macrophage migration, preserve function, and limit left ventricular (LV) remodeling in the rat coronary artery ligation model. Treatment with a monoclonal antibody to GM-CSF (5 mg/kg) was initiated 24 hours before coronary artery ligation and continued every 3 days for 3 weeks. Left coronary arteries of rats were ligated, animals were recovered, and cardiac function was evaluated 3 weeks postligation. Tissue samples were processed for histochemistry. Anti-GM-CSF treatment increased LV ejection fraction (37 ± 3% vs 47 ± 5%) and decreased LV end systolic diameter (0.75 ± 0.12 vs 0.59 ± 0.05 cm) with no changes in LV systolic pressure (109 ± 4 vs 104 ± 5 mm Hg), LV end diastolic pressure (22 ± 4 vs 21 ± 2 mm Hg), LV end diastolic diameter (0.96 ± 0.04 vs 0.92 ± 0.05 cm), or the time constant of LV relaxation tau (25.4 ± +2.4 vs 22.7 ± 1.4 milliseconds) (P < 0.05). Significantly lower numbers of tissue macrophages and significant reductions in infarct size were found in the myocardium of antibody-treated animals (81 ± 21.24 vs 195 ± 31.7 positive cells per 0.105 mm, compared with controls. These findings suggest that inhibition of macrophage migration may be beneficial in the treatment of heart failure after MI.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/farmacologia , Contagem de Células , Movimento Celular/efeitos dos fármacos , Vasos Coronários/imunologia , Vasos Coronários/patologia , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Hemodinâmica/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/fisiopatologia , Ratos , Ratos Sprague-Dawley , Remodelação Ventricular/efeitos dos fármacosRESUMO
The objective of our study was to determine granulocyte-macrophage colony-stimulating factor (GM-CSF) activity in the brain following GM-CSF induction. We injected recombinant mouse GM-CSF into the brains of 8-month-old C57BL6 mice via intracerebroventricular injections and studied the activities of microglia, astrocytes, and neurons. We also sought to determine whether an anti-GM-CSF antibody could suppress endogenous microglial activity in the C57BL6 mice and could also suppress microglial activity induced by the recombinant mouse GM-CSF in another group of C57BL6 mice. Using quantitative real-time RT-PCR, we assessed microglial, astrocytic, and neuronal activity by measuring mRNA expression of pro-inflammatory cytokines, GFAP, and the neuronal marker NeuN in the cerebral cortex tissues from C57BL6 mice. We performed immunoblotting and immunohistochemistry of activated microglia in different regions of the brains from control (phosphate-buffered saline-injected C57BL6 mice) and experimental mice (recombinant GM-CSF-injected C57BL6 mice, GM-CSF antibody-injected C57BL6 mice, and recombinant mouse GM-CSF plus anti-GM-CSF antibody-injected C57BL6 mice). We found increased mRNA expression of CD40 (9.75-fold), tumor necrosis factor-alpha (2.1-fold), CD45 (1.73-fold), and CD11c (1.70-fold) in the cerebral cortex of C57BL6 mice that were induced with recombinant GM-CSF, compared with control mice. Further, the anti-GM-CSF antibody suppressed microglia in mice that were induced with recombinant GM-CSF. Our immunoblotting and immunohistochemistry findings of GM-CSF-associated cytokines in C57BL6 mice induced with recombinant GM-CSF, in C57BL6 mice injected with the anti-GM-CSF antibody, and in C57BL6 mice injected with recombinant mouse GM-CSF plus anti-GM-CSF antibody concurred with our real-time RT-PCR findings. These findings suggest that GM-CSF is critical for microglial activation and that anti-GM-CSF antibody suppresses microglial activity in the CNS. The findings from this study may have implications for anti-inflammatory effects of Alzheimer's disease and experimental autoimmune encephalomyelitis mice (a multiple sclerosis mouse model).
Assuntos
Anticorpos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Microglia/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Antígeno CD11c/genética , Antígenos CD40/genética , Antígenos CD40/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Injeções Intraventriculares/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/metabolismoRESUMO
The purpose of our study was to investigate microglia and astrocytes that are associated with human mutant amyloid precursor protein and amyloid beta (Abeta). We investigated whether the anti-granulocyte-macrophage-colony stimulating factor (GM-CSF) antibody can suppress microglial activity and decrease Abeta production in Alzheimer's disease transgenic mice (Tg2576 line). An antibody to mouse GM-CSF was introduced by intracerebroventricular (ICV) injections into the brains of 10-month-old Tg2576 male mice. We assessed the effect of several GM-CSF-associated cytokines on microglial activities and their association with Abeta using quantitative real-time RT-PCR, immunoblotting, immunohistochemistry analyses in anti-GM-CSF antibody-injected Tg2576 mice. Using sandwich ELISA technique, we measured intraneuronal Abeta in Tg2576 mice injected with GM-CSF antibody and PBS vehicle-injected control Tg2576 mice. Using double-labeling immunofluorescence analysis of intraneuronal Abeta, Abeta deposits and pro-inflammatory cytokines, we assessed the relationship between Abeta deposits and microglial markers in the Tg2576 mice, and also in the anti-GM-CSF antibody-injected Tg2576 mice. Our real-time RT-PCR analysis showed an increase in the mRNA expression of IL6, CD11c, IL1beta, CD40 and CD11b in the cerebral cortices of the Tg2576 mice compared with their littermate non-transgenic controls. Immunohistochemistry findings of microglial markers agreed with our real-time RT-PCR results. Interestingly, we found significantly decreased levels of activated microglia and Abeta deposits in anti-GM-CSF antibody-injected Tg2576 mice compared with PBS vehicle-injected Tg2576 mice. Findings from our real-time RT-PCR and immunoblotting analysis agreed with immunohistochemistry results. Our double-labeling analyses of intraneuronal Abeta and CD40 revealed that intraneuronal Abeta is associated with neuronal expression of CD40 in Tg2576 mice. Our quantitative sandwich ELISA analysis revealed decreased levels of soluble Abeta1-42 and increased levels of Abeta1-40 in Tg2576 mice injected with the anti-GM-CSF antibody, suggesting that anti-GM-CSF antibody alone decreases soluble Abeta1-42 production and suppresses microglial activity in Tg2576 mice. These findings indicating the ability of the anti-GM-CSF antibody to reduce Abeta1-42 and microglial activity in Tg2576 mice may have therapeutic implications for Alzheimer's disease.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Anticorpos/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Microglia/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/genética , Animais , Anticorpos/imunologia , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Encéfalo/metabolismo , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microglia/imunologia , Microglia/metabolismo , Fragmentos de Peptídeos/genéticaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.
Assuntos
Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/imunologia , Proteínas Recombinantes/imunologia , ADP Ribose Transferases/imunologia , Animais , Especificidade de Anticorpos , Citotoxicidade Imunológica/imunologia , Ensaio de Imunoadsorção Enzimática , Exotoxinas/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/uso terapêutico , Fatores de Virulência/imunologia , Exotoxina A de Pseudomonas aeruginosaRESUMO
Antibodies can be used for the prevention and treatment of bacterial infections in animal models of disease. Current antibody technology allows the generation of high affinity human/humanized antibodies that can be optimized for antibacterial activity and in vivo biodistribution and pharmacokinetics. Such antibodies have exquisite selectivity for their bacterial target antigen and promise efficacy and safety. Why are there no monoclonal antibody products approved for the treatment or prevention of bacterial infections? Can antibodies succeed where antibiotics are failing? Some antibody therapies are currently being evaluated in clinical trials but several have failed despite positive data in animal disease models. This review will discuss the pros and cons of antibody therapeutics targeted at bacterial infections.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Infecções Bacterianas/terapia , Antígenos de Bactérias/imunologia , Bactérias/imunologia , Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Testes de NeutralizaçãoRESUMO
Type I interferons (IFNs) are potent regulators of both innate and adaptive immunity. All type I IFNs bind to the same heterodimeric cell surface receptor composed of IFN-alpha receptor (IFNAR-1) and IFN-alpha/beta receptor (IFNAR-2) polypeptides. This study revealed that type I IFN receptor levels vary considerably on hematopoietic cells, with monocytes and B cells expressing the highest levels. Overnight treatment of peripheral blood mononuclear cells (PBMCs) with IFN-alpha2b or IFN-beta led to increased expression on monocytes and B cells of surface markers commonly associated with activated antigen-presenting cells (APCs), such as CD38, CD86, MHC class I, and MHC class II. Five-day exposure of adherent monocytes to granulocyte-macrophage colony-stimulating factor (GM-CSF) plus IFN-alpha or IFN-beta caused the development of potent allostimulatory cells with morphology similar to that of myeloid dendritic cells (DCs) obtained from culture with GM-CSF and interleukin-4 (IL-4) but with distinct cell surface marker profiles and activity. In contrast to IL-4-derived DCs, IFN-alpha-derived DCs were CD14+, CD1a-, CD123+, CD32+, and CD38+ and expressed high levels of CD86 and MHC class II. Development of these cells was completely blocked by an antibody to IFNAR-1. Furthermore, activity of the type I IFN-derived DC in a mixed lymphocyte reaction (MLR) was consistently more potent than that of IL-4-derived DCs, especially at high responder/stimulator ratios. This MLR activity was abrogated by the addition of anti-IFNAR-1 antibody at the start of the DC culture. In contrast, there was no effect of anti-IFNAR-1 on IL-4-derived DCs, indicating that this is a distinct pathway of DC differentiation. These results suggest a potential role for anti-IFNAR-1 immunotherapy in autoimmune diseases, such as systemic lupus erythematosus (SLE), in which the action of excessive type I IFN on B cells and myeloid DCs may play a role in disease pathology.
Assuntos
Linfócitos B/imunologia , Interferon Tipo I/genética , Ativação Linfocitária/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunoglobulina G/análise , Proteínas de Membrana , Camundongos , Monócitos/citologia , Monócitos/imunologia , Neutrófilos/imunologia , Receptor de Interferon alfa e beta , Receptores de Interferon/genética , Linfócitos T/imunologiaRESUMO
Traditional chemotherapeutic drugs are often restricted by severe side effects and lack of tumor specificity. Peptide prodrugs cleavable by peptidases present in the tumor environment have been explored to improve the therapeutic index of cytotoxic drugs. One such prodrug of doxorubicin (Dox), CPI-0004Na [N-succinyl-beta-alanyl-L-leucyl-L-alanyl-L-leucyl-Dox (sALAL-Dox)] has been shown to have an improved antitumor efficacy profile with reduced toxicity compared with Dox in tumor xenograft models (V. Dubois et al., Cancer Res., 62: 2327-2331, 2002). In this study, we demonstrate that CD10, a cell surface metalloprotease expressed on a variety of tumor cell types, is capable of cleaving CPI-0004Na and related peptide prodrugs such as N-succinyl-beta-alanyl-L-isoleucyl-L-alanyl-L-leucyl-Dox (sAIAL-Dox). This proteolytic cleavage generates leucyl-Dox, which is capable of entering cells and generating intracellular Dox. In a [(3)H]thymidine proliferation assay, analogues of CPI-0004Na showed a 100-300-fold increase in potency on CD10(+) cells compared with CD10(-) cells. Cytotoxicity of CPI-0004Na was inhibited by phosphoramidon, a known inhibitor of CD10 enzymatic activity. Furthermore, Chinese hamster ovary CHO-S cells, which are resistant to CPI-0004Na, could be sensitized to the cytotoxic effect of the prodrug by transfection of a CD10 cDNA. Tumor xenograft studies using LNCaP prostate tumor cells support the important role of CD10 in the antitumor efficacy of these prodrugs against tumors expressing CD10. CPI-0004Na and sAIAL-Dox achieved statistically significant 70% tumor growth inhibition at day 22. CD10 is expressed on many types of human tumors including B-cell lymphoma, leukemia, and prostate, breast, colorectal, and lung carcinomas; therefore, CD10-cleavable prodrugs may be effective in a range of different tumor types.
Assuntos
Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Neprilisina/metabolismo , Oligopeptídeos/farmacocinética , Pró-Fármacos/farmacocinética , Animais , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Doxorrubicina/efeitos adversos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Nus , Neprilisina/antagonistas & inibidores , Neprilisina/biossíntese , Neprilisina/genética , Oligopeptídeos/efeitos adversos , Pró-Fármacos/efeitos adversos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CD20 is a B-cell-specific cell surface protein expressed on mature B lymphocytes and is a target for monoclonal antibody therapy for non-Hodgkin's lymphoma (NHL). Though clear clinical efficacy has been demonstrated with several anti-CD20 antibodies, the mechanisms by which the antibodies activate CD20 and kill cells remain unclear. Proposed mechanisms of action include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and induction of apoptosis. In this report we compared the activity of two anti-CD20 antibodies, Anti-B1 Antibody (tositumomab) and rituximab (C2B8), in a variety of cellular assays using a panel of B-cell lines. Anti-B1 Antibody showed a low level of activity in a CDC assay against complement-sensitive B-cell lines, Ramos and Daudi. We found that there is an inverse correlation between the expression of CD55 and CD59 and CDC mediated by either Anti-B1 Antibody or rituximab. Rituximab was more potent at inducing CDC when compared to Anti-B1 Antibody. Using Raji cells as target cells and human peripheral blood leukocytes as effector cells, Anti-B1 Antibody was a potent inducer of ADCC. The activities of Anti-B1 Antibody and rituximab were nearly identical in the ADCC assay. In addition, Anti-B1 Antibody showed direct induction of apoptosis in all B-cell lines tested. In general, crosslinking Anti-B1 Antibody with a goat anti-mouse Ig did not further enhance the percentage of cells undergoing apoptosis. Importantly, a F(ab')(2) fragment of Anti-B1 Antibody induced apoptosis, while the Fab fragment did not, indicating that the Fc region was not required and dimerization of CD20 may be sufficient for induction of apoptosis. In contrast, rituximab, which binds to an overlapping epitope on CD20 with a three-fold lower affinity than Anti-B1 Antibody, did not efficiently induce apoptosis in the cell lines tested in the absence of crosslinking. In conclusion, these two anti-CD20 antibodies have overlapping, but distinct mechanisms of action on B-cell lines.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD20/fisiologia , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/farmacologia , Animais , Anexina A5/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos , Especificidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos CD20/imunologia , Antígenos de Neoplasias/análise , Apoptose/imunologia , Apoptose/fisiologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Antígenos CD55/análise , Antígenos CD59/análise , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Dimerização , Cabras , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Camundongos , Proteínas de Neoplasias/análise , Rituximab , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/imunologiaRESUMO
UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in anintegration independent manner. Efficient expression can bederived from a single copy of an integrated gene site resulting ina higher percentage of cells expressing the marker gene in theselected pool in comparison to standard non-UCOE containingvectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapidproduction of large quantities of protein in a short period oftime. Utilizing this system more than 300 mg of a recombinantantibody has been produced in less than 1 month from transfectionpools in shake flask. Selected subclones have been scaled intosmall bioreactors in less than 2 months, producing significantquantities of monoclonal antibody using a protocol generic for theparent cell line. The increased efficiency obtained with the UCOEvector reduces the number of transfectants which need to bescreened in order to obtain high productivity subclones.Transfection of a standard host cell line, preadapted to grow in alarge-scale setting, allows for rapid cell line developmentdecreasing the transition time from research into development andmanufacturing. Alternatively, the traditional approach of using aparent cell line which requires serum-free and suspensionadaptation after transfection further increases the need forscreening a large number of subclones, because many of thesubclones will not be able to grow under conditions that allowlarge-scale protein production. The use of a preadapted cell linecan reduce the time required to develop a cell line from months toweeks.