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BACKGROUND: Asthma is often accompanied by type 2 immunity rich in IL-4, IL-5 and IL-13 cytokines produced by TH2 lymphocytes or type 2 innate lymphoid cells (ILC2s). Interleukin-2 family cytokines play a key role in the differentiation, homeostasis and effector function of innate and adaptive lymphocytes. OBJECTIVE: IL-9 and IL-21 boost the activation and proliferation of TH2 and ILC2s, but the relative importance and potential synergism between these γc cytokines is currently unknown. METHODS: Using newly generated antibodies, we inhibited IL-9 and IL-21 alone or in combination, in various murine models of asthma. In a translational approach using segmental allergen challenge, we recently described elevated IL-9 levels in human allergic asthmatics in comparison to non-asthmatic controls. Here, we also measured IL-21 in both groups. RESULTS: IL-9 played a central role in controlling innate IL-33 induced lung inflammation by promoting proliferation and activation of ILC2s, in an IL-21 independent manner. Conversely, chronic house dust mite induced airway inflammation, mainly driven by adaptive immunity, was solely dependent on IL-21, that controlled TH2 activation, eosinophilia, total serum IgE and formation of tertiary lymphoid structures. In a model of innate on adaptive immunity driven by papain allergen, a clear synergy was found between both pathways, since combined anti-IL-9 or anti-IL-21 blockade was superior in reducing key asthma features. In human bronchoalveolar lavage (BAL) samples we measured elevated IL-21 protein within the allergic asthmatic group, compared with the allergic control group. We also found increased IL21R transcripts and predicted IL-21 ligand activity in various disease-associated cell subsets. CONCLUSION: IL-9 and IL-21 play important and non-redundant roles in allergic asthma by boosting ILC2s and TH2 cells, revealing a dual IL-9 and IL-21 targeting strategy as a new and testable approach.
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BACKGROUND: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. OBJECTIVE: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. METHODS: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. RESULTS: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. CONCLUSIONS: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.
Assuntos
Anticorpos Monoclonais/farmacologia , Complemento C2/antagonistas & inibidores , Inativadores do Complemento/farmacologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/farmacocinética , Cálcio , Ativação do Complemento/efeitos dos fármacos , Complemento C2/análise , Complemento C2/metabolismo , Inativadores do Complemento/sangue , Inativadores do Complemento/farmacocinética , Mapeamento de Epitopos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Macaca fascicularis , MasculinoRESUMO
BACKGROUND: Asthma is a chronic inflammatory airway disease in which innate and adaptive immune cells act together to cause eosinophilic inflammation, goblet cell metaplasia (GCM), and bronchial hyperreactivity (BHR). In clinical trials using biologicals against IL-4 receptor (IL-4R) α or IL-5, only a subset of patients with moderate-to-severe asthma responded favorably, suggesting that distinct pathophysiologic mechanisms are at play in subgroups of patients called endotypes. However, the effect of multiple cytokine blockade using bispecific antibodies has not been tested. OBJECTIVE: We sought to target simultaneously the IL-4, IL-13, and IL-5 signaling pathways with a novel IL-4Rα/IL-5-bispecific antibody in a murine house dust mite (HDM) model of asthma. METHODS: Two mAbs neutralizing IL-4Rα and IL-5 were generated by using a llama-based antibody platform. Their heavy and light chains were then cotransfected in mammalian cells, resulting in a heterogeneous antibody mixture from which the bispecific antibody was isolated by using a dual anti-idiotypic purification process. C57BL/6J mice were finally sensitized and challenged to HDM extracts and treated during challenge with the antibodies. RESULTS: We successfully generated and characterized the monospecific and bispecific antibodies targeting IL-4Rα and IL-5. The monospecific antibodies could suppress eosinophilia, IgE synthesis, or both, whereas only the IL-4Rα/IL-5-bispecific antibody and the combination of monospecific antibodies additionally inhibited GCM and BHR. CONCLUSION: Type 2 cytokines act synergistically to cause GCM and BHR in HDM-exposed mice. These preclinical results show the feasibility of generating bispecific antibodies that target multiple cytokine signaling pathways as superior inhibitors of asthma features, including the difficult-to-treat GCM.
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Anticorpos Biespecíficos/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Asma/tratamento farmacológico , Citocinas/antagonistas & inibidores , Eosinofilia/tratamento farmacológico , Animais , Anticorpos Monoclonais/uso terapêutico , Asma/imunologia , Asma/patologia , Asma/fisiopatologia , Camelídeos Americanos , Linhagem Celular , Citocinas/imunologia , Eosinofilia/imunologia , Eosinofilia/patologia , Eosinofilia/fisiopatologia , Escherichia coli , Feminino , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Humanos , Camundongos Endogâmicos C57BL , Pyroglyphidae/imunologiaRESUMO
Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe(229) and Phe(279) of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe(279) Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe(279) In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe(279), whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe(229).
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Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/química , Receptores de Interleucina-6/imunologia , Animais , Camelus , Humanos , Interleucina-6/química , Interleucina-6/imunologia , Camundongos , Estrutura Quaternária de ProteínaRESUMO
Chemokine receptors and their ligands play a prominent role in immune regulation but many have also been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, allograft rejection after transplantation, and also in cancer metastasis. Most approaches to therapeutically target the chemokine system involve targeting of chemokine receptors with low molecular weight antagonists. Here we describe the selection and characterization of an unprecedented large and diverse panel of neutralizing Nanobodies (single domain camelid antibodies fragment) directed against several chemokines. We show that the Nanobodies directed against CCL2 (MCP-1), CCL5 (RANTES), CXCL11 (I-TAC), and CXCL12 (SDF-1α) bind the chemokines with high affinity (at nanomolar concentration), thereby blocking receptor binding, inhibiting chemokine-induced receptor activation as well as chemotaxis. Together, we show that neutralizing Nanobodies can be selected efficiently for effective and specific therapeutic treatment against a wide range of immune and inflammatory diseases.
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Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Quimiocinas/metabolismo , Anticorpos de Domínio Único/farmacologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Camelídeos Americanos , Quimiocinas/química , Quimiocinas/genética , Quimiocinas/imunologia , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Doenças do Sistema Imunitário/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Camundongos , Células NIH 3T3 , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologiaRESUMO
BACKGROUND: Direct cell-cell spread of HIV-1 is a very efficient mode of viral dissemination, with increasing evidence suggesting that it may pose a considerable challenge to controlling viral replication in vivo. Much current vaccine research involves the study of broadly neutralising antibodies (bNabs) that arise during natural infection with the aims of eliciting such antibodies by vaccination or incorporating them into novel therapeutics. However, whether cell-cell spread of HIV-1 can be effectively targeted by bNabs remains unclear, and there is much interest in identifying antibodies capable of efficiently neutralising virus transmitted by cell-cell contact. RESULTS: In this study we have tested a panel of bNAbs for inhibition of cell-cell spread, including some not previously evaluated for inhibition of this mode of HIV-1 transmission. We found that three CD4 binding site antibodies, one from an immunised llama (J3) and two isolated from HIV-1-positive patients (VRC01 and HJ16) neutralised cell-cell spread between T cells, while antibodies specific for glycan moieties (2G12, PG9, PG16) and the MPER (2F5) displayed variable efficacy. Notably, while J3 displayed a high level of potency during cell-cell spread we found that the small size of the llama heavy chain-only variable region (VHH) J3 is not required for efficient neutralisation since recombinant J3 containing a full-length human heavy chain Fc domain was significantly more potent. J3 and J3-Fc also neutralised cell-cell spread of HIV-1 from primary macrophages to CD4+ T cells. CONCLUSIONS: In conclusion, while bNabs display variable efficacy at preventing cell-cell spread of HIV-1, we find that some CD4 binding site antibodies can inhibit this mode of HIV-1 dissemination and identify the recently described llama antibody J3 as a particularly potent inhibitor. Effective neutralisation of cell-cell spread between physiologically relevant cell types by J3 and J3-Fc supports the development of VHH J3 nanobodies for therapeutic or prophylactic applications.
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Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Linfócitos T/virologia , Animais , Antígenos CD4/metabolismo , Camelídeos Americanos , Infecções por HIV/transmissão , Humanos , Macrófagos/virologia , Reação em Cadeia da Polimerase em Tempo RealAssuntos
Urticária Crônica/diagnóstico , Urticária Crônica/etiologia , Expressão Gênica , Interleucinas/genética , Autoanticorpos/imunologia , Urticária Crônica/metabolismo , Humanos , Imunoglobulina E/imunologia , Interleucinas/imunologia , Interleucinas/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , RNA Mensageiro/metabolismoRESUMO
The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.
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Anticorpos/farmacologia , Quimiotaxia/efeitos dos fármacos , HIV-1 , Receptores CXCR4/imunologia , Replicação Viral/efeitos dos fármacos , Animais , Anticorpos/isolamento & purificação , Antígenos CD34 , Benzilaminas , Sítios de Ligação/genética , Células COS , Chlorocebus aethiops , Ciclamos , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Células HEK293 , Mobilização de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby neuromuscular junction (NMJ) function. NMJ dysfunction is a hallmark of several neuromuscular diseases, including MuSK myasthenia gravis. Aiming to restore NMJ function, we generated several agonist monoclonal antibodies targeting the MuSK Ig-like 1 domain. These activated MuSK and induced AChR clustering in cultured myotubes. The most potent agonists partially rescued myasthenic effects of MuSK myasthenia gravis patient IgG autoantibodies in vitro. In an IgG4 passive transfer MuSK myasthenia model in NOD/SCID mice, MuSK agonists caused accelerated weight loss and no rescue of myasthenic features. The MuSK Ig-like 1 domain agonists unexpectedly caused sudden death in a large proportion of male C57BL/6 mice (but not female or NOD/SCID mice), likely caused by a urologic syndrome. In conclusion, these agonists rescued pathogenic effects in myasthenia models in vitro, but not in vivo. The sudden death in male mice of one of the tested mouse strains revealed an unexpected and unexplained role for MuSK outside skeletal muscle, thereby hampering further (pre-) clinical development of these clones. Future research should investigate whether other Ig-like 1 domain MuSK antibodies, binding different epitopes, do hold a safe therapeutic promise.
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Miastenia Gravis , Receptores Proteína Tirosina Quinases , Masculino , Animais , Camundongos , Camundongos SCID , Receptores Proteína Tirosina Quinases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Miastenia Gravis/metabolismo , Receptores Colinérgicos/metabolismo , Autoanticorpos , Debilidade Muscular , AcetilcolinaRESUMO
BACKGROUND AND OBJECTIVES: To determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN. METHODS: iPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed. RESULTS: iPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs. DISCUSSION: Binding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.
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Anticorpos Monoclonais Humanizados/farmacologia , Ativação do Complemento/imunologia , Complemento C2/efeitos dos fármacos , Neurônios Motores , Polineuropatias/tratamento farmacológico , Polineuropatias/imunologia , Células Cultivadas , Humanos , Imunoglobulina M , Células-Tronco Pluripotentes InduzidasRESUMO
Exacerbated IL-22 activity induces tissue inflammation and immune disorders such as psoriasis. However, because IL-22 is also essential for tissue repair and defense at barrier interfaces, targeting IL-22 activity to treat psoriasis bears the risk of deleterious effects at mucosal sites such as the gut. We previously showed in vitro that IL-22 signaling relies on IL-22 receptor alpha (IL-22Rα) Y-dependent and -independent pathways. The second depends on the C-terminal Y-less region of IL-22Rα and leads to a massive signal transducer and activator of transcription 3 (STAT3) activation. Because STAT3 activation is associated with the development of psoriasis, we hypothesized that the specific inhibition of the noncanonical STAT3 activation by the Y-less region of IL-22Rα could reduce psoriasis-like disease while leaving intact its tissue defense functions in the gut. We show that mice expressing a C-terminally truncated version of IL-22Rα (ΔCtermut/mut mice) are protected from the development of psoriasis-like dermatitis lesions induced by imiquimod to a lesser extent than Il22ra-/- mice. In contrast, only Il22ra-/- mice lose weight after Citrobacter rodentium infection. Altogether, our data suggest that specific targeting of the noncanonical STAT3 activation by IL-22 could serve to treat psoriasis-like skin inflammation without affecting IL-22âdependent tissue repair or barrier defense at other sites.
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Imiquimode/toxicidade , Psoríase/induzido quimicamente , Receptores de Interleucina/fisiologia , Fator de Transcrição STAT3/fisiologia , Animais , Citrobacter rodentium , Infecções por Enterobacteriaceae/imunologia , Interleucinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Interleucina 22RESUMO
Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6-12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy.
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The protein tyrosine phosphatase PEST (PTP-PEST) is involved in the regulation of the actin cytoskeleton. Despite the emerging functions attributed to both PTPs and the actin cytoskeleton in apoptosis, the involvement of PTP-PEST in apoptotic cell death remains to be established. Using several cell-based assays, we showed that PTP-PEST participates in the regulation of apoptosis. As apoptosis progressed, a pool of PTP-PEST localized to the edge of retracting lamellipodia. Expression of PTP-PEST also sensitized cells to receptor-mediated apoptosis. Concertedly, specific degradation of PTP-PEST was observed during apoptosis. Pharmacological inhibitors, immunodepletion experiments, and in vitro cleavage assays identified caspase-3 as the primary regulator of PTP-PEST processing during apoptosis. Caspase-3 specifically cleaved PTP-PEST at the (549)DSPD motif and generated fragments, some of which displayed increased catalytic activity. Moreover, caspase-3 regulated PTP-PEST interactions with paxillin, leupaxin, Shc, and PSTPIP. PTP-PEST acted as a scaffolding molecule connecting PSTPIP to additional partners: paxillin, Shc, Csk, and activation of caspase-3 correlated with the modulation of the PTP-PEST adaptor function. In addition, cleavage of PTP-PEST facilitated cellular detachment during apoptosis. Together, our data demonstrate that PTP-PEST actively contributes to the cellular apoptotic response and reveal the importance of caspases as regulators of PTPs in apoptosis.
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Apoptose , Caspase 3/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Caspase 3/farmacologia , Catálise/efeitos dos fármacos , Extensões da Superfície Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 12 , Proteínas Tirosina Fosfatases/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato/efeitos dos fármacosRESUMO
Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.
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Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Asma/terapia , Glicoproteínas/química , Glicoproteínas/farmacologia , Imunidade Inata/efeitos dos fármacos , Lisofosfolipase/química , Lisofosfolipase/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Asma/imunologia , Asma/patologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/terapia , Cristalização , Modelos Animais de Doenças , Glicoproteínas/administração & dosagem , Glicoproteínas/imunologia , Células Caliciformes/imunologia , Células Caliciformes/patologia , Humanos , Epitopos Imunodominantes/imunologia , Imunoglobulina E/imunologia , Lisofosfolipase/administração & dosagem , Lisofosfolipase/imunologia , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Muco/imunologiaRESUMO
Initially viewed as dangerous byproducts of aerobic life, reactive oxygen species (ROS) nowadays appear to be essential secondary messengers of many signaling cascades and cellular functions. The establishment of ROS as important signaling molecules has been confirmed by the existence of specialized ROS producing complexes expressed in nonphagocytic cells, the NADPH oxidase complex (NOX). Because of the diversity of their proteic targets (besides lipids and DNA), ROS have multiple and sometimes contradictory functions. In the present review, we focus on several different signaling pathways influenced by ROS and NOX in tumorigenesis, focusing on proliferation and angiogenesis. We review the ROS targets regulating proliferation, including cellular signaling (phosphatases, AP1, and nuclear factor-kappa B [NF-kappaB]) and cell cycle targets (CDC25, cyclin D, and forkhead proteins), and the role of NOX during proliferation. Finally, we review the direct and indirect involvement of ROS and NOX in (tumor) angiogenesis through the regulation of different biologic systems such as vascular endothelial growth factor, angiotensin II, hypoxia-inducible factor, AP1, and inflammation.
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NADPH Oxidases/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Animais , Ciclo Celular , Proliferação de Células , Humanos , Inflamação/metabolismo , Modelos Biológicos , NADPH Oxidases/genética , Neoplasias/enzimologia , Neovascularização Patológica/enzimologia , Neovascularização Patológica/genética , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
INTRODUCTION: Bispecific antibodies have become increasingly of interest by enabling new therapeutic applications such as retargeting cellular immunity towards tumor cells. About 23 bispecific antibody platforms have therefore been developed, generating about 62 molecules which are currently being evaluated for potential treatment of a variety of indications, such as cancer and inflammatory diseases, among which three molecules were approved. This class of drugs will represent a multi-million-dollar market over the coming years. Many companies have consequently invested in the development of bispecific antibody platforms, creating an important patent activity in this field. AREAS COVERED: The present review gives an overview of the patent literature over the period 1994-2017 of different immunoglobulin gamma-based bispecific antibody platforms and the molecules approved or in clinical trials. EXPERT OPINION: Bispecific antibodies are progressively accepted as potentially superior therapeutic molecules in a broad range of diseases. This frantic activity creates a maze of hundreds of patents that pose considerable legal risks for both newcomers and established companies. It can consecutively be anticipated that the number of patent conflicts will increase. Nevertheless, it can be expected that patents related to the use of a bispecific antibody will have tremendous commercial value.
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Anticorpos Biespecíficos/administração & dosagem , Desenho de Fármacos , Imunoglobulina G/imunologia , Anticorpos Biespecíficos/imunologia , Antineoplásicos/administração & dosagem , Antineoplásicos/imunologia , Indústria Farmacêutica , Humanos , Imunidade Celular/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Patentes como AssuntoRESUMO
Asthma affects more than 300 million people worldwide and poses a large socioeconomic burden, particularly in the 5% to 10% of severe asthmatics. So far, each entry of new biologics in clinical trials has led to high expectations for treating all severe asthma forms, but the outcome has only been successful if the biologic, as add-on treatment, targeted specific patient subgroups. Indeed, we now realize that asthma is a heterogeneous disease with multiple phenotypes, based on distinct pathophysiological mechanisms, called endotypes. Thus, asthma therapy is gradually moving to a personalized medicine approach, tailored to individual's asthma endotypes identified through biomarkers. Here, we review the clinical efficacy of antibody-related therapeutics undergoing clinical trials, or those already approved, for the treatment of severe type 2 asthma. Biologics targeting type 2 cytokines have shown consistent efficacy, especially in patients with evidence of type 2 inflammation, suggesting that the future of asthma biologics is promising.
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Antiasmáticos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Produtos Biológicos/uso terapêutico , Citocinas/antagonistas & inibidores , Mediadores da Inflamação/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Medicina de Precisão/métodos , Animais , Antiasmáticos/efeitos adversos , Anti-Inflamatórios/efeitos adversos , Asma/diagnóstico , Asma/imunologia , Asma/metabolismo , Produtos Biológicos/efeitos adversos , Tomada de Decisão Clínica , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/fisiopatologia , Seleção de Pacientes , Transdução de Sinais/efeitos dos fármacosRESUMO
The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.
Assuntos
Anticorpos Monoclonais/imunologia , Imunização , Epitopos Imunodominantes/imunologia , Receptores de Glucagon/antagonistas & inibidores , Anticorpos de Cadeia Única/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Peptídeos Catiônicos Antimicrobianos , Células CHO , Camelídeos Americanos/imunologia , Catelicidinas/imunologia , Técnicas de Visualização da Superfície Celular , Células Cultivadas , Cricetulus , Fibroblastos , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas de Membrana , Plasmídeos/genética , Plasmídeos/imunologia , Receptores de Glucagon/genética , Receptores de Glucagon/imunologia , Anticorpos de Cadeia Única/sangueRESUMO
Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.
Assuntos
Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Mutação/genética , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Camelídeos Americanos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Interleucina-6/imunologia , Modelos Imunológicos , Modelos Moleculares , Proteínas Recombinantes/química , Alinhamento de SequênciaRESUMO
Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.