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INTRODUCTION: Immunotherapy targeting amyloid-ß peptide is under active clinical investigation for treatment of Alzheimer's disease (AD). Among the hypotheses being investigated for impact on clinical outcome are the preferred epitope or conformation of amyloid-ß to target for treatment, and the mechanism of action underlying immunotherapy. Bapineuzumab (humanized 3D6), a neo-epitope specific antibody recognizing amyloid-ß1-5 with strong preference for an exposed Asp residue at the N-terminus of the peptide, has undergone advanced clinical testing for treatment of AD. METHODS: To gain further insight into the epitope conformation, we interrogated structural details of amino-terminal epitopes in amyloid-ß using x-ray crystallography of 3D6Fab:amyloid-ß complexes. Humanization of 3D6 was carried out using standard procedures integrating recombinant methods, sequence informatics, and homology modeling predictions to identify important mouse framework residues for retention in the finished humanized product. RESULTS: Here we report the crystal structure of a recombinant Fab fragment of 3D6 in complex with amyloid-ß1-7 solved at 2.0 Å resolution. The N-terminus of amyloid-ß is bound to 3D6 as a 310 helix. The amino-terminal Asp residue is buried deepest in the antibody binding pocket, with the Cß atom of residue 6 visible at the entrance to the binding pocket near the surface of the antibody. We further evaluate homology model based predictions used to guide humanization of 3D6 to bapineuzumab, with actual structure of the Fab. The structure of the Fab:amyloid-ß complex validates design of the humanized antibody, and confirms the amyloid-ß epitope recognized by 3D6 as previously mapped by ELISA. CONCLUSIONS: The conformation of amyloid-ß antigen recognized by 3D6 is novel and distinct from other antibodies recognizing N-terminal epitopes. Our result provides the first report demonstrating structural conservation of antigen contact residues, and conformation of antigen recognized, between the parent murine antibody and its humanized version.
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Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in V(L) CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Fas(lpr) mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC(0-infinity)) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2-3-fold or 4-7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Fas(lpr) mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.
Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Lúpus Eritematoso Sistêmico/terapia , Receptores de Interleucina-21/antagonistas & inibidores , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/imunologia , Células Cultivadas , Regiões Determinantes de Complementaridade/genética , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Injeções Subcutâneas , Interleucinas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Endogâmicos , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-21/imunologiaRESUMO
Immunotherapy targeting of amyloid beta (Abeta) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Abeta-targeted immunotherapy has also been demonstrated to reverse Abeta-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Abeta, Abeta(3-7), differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Abeta in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Abeta. The conformation of the Abeta peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Abeta:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Abeta species postulated to underlie cognitive deficits in AD.
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Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Animais , Comportamento Animal , Reagentes de Ligações Cruzadas/farmacologia , Cristalografia por Raios X/métodos , Modelos Animais de Doenças , Epitopos/química , Heterozigoto , Humanos , Cinética , Masculino , Camundongos , Conformação Molecular , Proteínas Recombinantes/químicaRESUMO
Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.
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Anticorpos Monoclonais/isolamento & purificação , Região Variável de Imunoglobulina/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Escherichia coli/genética , Escherichia coli/imunologia , Feminino , Humanos , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/imunologia , Regiões Constantes de Imunoglobulina/isolamento & purificação , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Masculino , Periplasma/genética , Periplasma/imunologia , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/imunologia , Proteínas Periplásmicas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Ralpha1/IL-4Ralpha) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Ralpha, and Ab02 blocks IL-13 interaction with IL-13Ralpha1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Ralpha-binding epitope or the IL-13Ralpha1-binding epitope.
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Antígenos de Helmintos/imunologia , Ascaris/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/imunologia , Inflamação/imunologia , Interleucina-13/imunologia , Pulmão/imunologia , Receptores de Interleucina-13/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Basófilos/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Epitopos/imunologia , Liberação de Histamina/imunologia , Humanos , Macaca fascicularis , MasculinoRESUMO
BACKGROUND: Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates. OBJECTIVE: We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation. METHODS: Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count. RESULTS: Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13-treated animals compared with levels seen in control animals. CONCLUSION: These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates. CLINICAL IMPLICATIONS: IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.
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Anticorpos Monoclonais/uso terapêutico , Ascaríase/imunologia , Interleucina-13/antagonistas & inibidores , Pneumonia/imunologia , Pneumonia/prevenção & controle , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/uso terapêutico , Antígenos de Helmintos/imunologia , Ascaris suum , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interleucina-13/genética , Interleucina-13/imunologia , Macaca fascicularis , Masculino , Dados de Sequência Molecular , Pneumonia/metabolismo , Homologia de Sequência de AminoácidosRESUMO
IL-13 contributes to airway hyperresponsiveness, mucus secretion, inflammation, and fibrosis, suggesting that it plays a central role in asthma pathogenesis. Neutralization of IL-13 with sIL-13Ralpha2-Fc (sIL-13R) reduces allergen-induced airway responses in rodent models of respiratory disease, but its efficacy in a large animal model has not been previously reported. In this study, we determined whether two different strategies for IL-13 neutralization modified experimental asthma in sheep. Sheep with natural airway hypersensitivity to Ascaris suum antigen were treated intravenously either with sIL-13R, a strong antagonist of sheep IL-13 bioactivity in vitro, or with IMA-638 (IgG1, kappa), a humanized antibody to human IL-13. Higher doses of IMA-638 were used because, although it is a potent antagonist of human IL-13, this antibody has 20 to 30 times lower binding and neutralization activity against sheep IL-13. Control animals received human IgG of irrelevant specificity. Sheep were treated 24 h before inhalation challenge with nebulized A. suum. The effects on antigen-induced early and late bronchial responses, and antigen-induced hyperresponsiveness, were assessed. Both sIL-13R and IMA-638 provided dose-dependent inhibition of the antigen-induced late responses and airway hyperresponsiveness. The highest dose of IMA-638 also reduced the early phase response. These findings suggest that IL-13 contributes to allergen-induced airway responses in this sheep model of asthma, and that neutralization of IL-13 is an effective strategy for blocking these A. suum-induced effects.
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Asma/tratamento farmacológico , Asma/imunologia , Modelos Animais de Doenças , Interleucina-13/antagonistas & inibidores , Interleucina-13/imunologia , Carneiro Doméstico/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Ascaris suum/fisiologia , Asma/induzido quimicamente , Asma/fisiopatologia , Sequência de Bases , Hiper-Reatividade Brônquica/parasitologia , Hiper-Reatividade Brônquica/patologia , Broncoconstrição/efeitos dos fármacos , Broncoconstrição/imunologia , Carbacol/farmacologia , Feminino , Células HT29 , Humanos , Interleucina-13/química , Interleucina-13/genética , Cinética , Dados de Sequência Molecular , Testes de Neutralização , Receptores de Interleucina-13/metabolismo , Carneiro Doméstico/parasitologia , Solubilidade/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
We have characterized the IgG form of a previously isolated and engineered single-chain Fv (scFv), named RR2r3s4-1, that binds to human PSGL-1. This fully human IgG was determined to have a Kd of 1.8+/-0.7 nM by fluorescence quenching titration. It better inhibits P-selectin-PSGL-1 interactions than a commercially available murine monoclonal antibody KPL1 and better inhibits neutrophil rolling than KPL1. Thus, RR2r3s4-1 is the most effective antibody at inhibiting P-selectin-PSGL-1 interactions known. Specificity analysis reveals that RR2r3s4-1 does not cross react with murine PSGL-1 and thus requires more than tyrosine sulfate for binding to human PSGL-1. This evidence demonstrates the therapeutic potential of this antibody as a potent anti-inflammatory therapeutic.
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Movimento Celular/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Selectina-P/química , Selectina-P/imunologia , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , HumanosRESUMO
To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.
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Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Técnicas Biossensoriais/métodos , Antígeno Prostático Específico/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/normas , Humanos , Cinética , Ligantes , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/imunologia , Ligação Proteica , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/métodos , Ressonância de Plasmônio de Superfície/normas , Fatores de TempoRESUMO
High plasma levels of soluble P-selectin are associated with thrombotic disorders and may predict future cardiovascular events. Mice with high levels of soluble P-selectin have more microparticles in their plasma than do normal mice. Here we show that chimeras of P-selectin and immunoglobulin (P-sel-Ig) induced formation of procoagulant microparticles in human blood through P-selectin glycoprotein ligand-1 (PSGL-1; encoded by the Psgl1 gene, officially known as Selpl). In addition, Psgl1-/- mice produced fewer microparticles after P-sel-Ig infusion and did not spontaneously increase their microparticle count in old age as do wild-type mice. Injected microparticles specifically bound to thrombi and thus could be involved in thrombin generation at sites of injury. Infusion of P-sel-Ig into hemophilia A mice produced a 20-fold increase over control immunoglobulin in microparticles containing tissue factor. This significantly improved the kinetics of fibrin formation in the hemophilia A mice and normalized their tail-bleeding time. P-sel-Ig treatment could become a new approach to sustained control of bleeding in hemophilia.
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Hemofilia A/terapia , Hemostasia/fisiologia , Glicoproteínas de Membrana/metabolismo , Selectina-P/metabolismo , Adulto , Animais , Coagulação Sanguínea/fisiologia , Modelos Animais de Doenças , Fator VIII/metabolismo , Hemofilia A/metabolismo , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Selectina-P/genética , Fenótipo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.