Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 70
Filtrar
Mais filtros

Bases de dados
Tipo de documento
Intervalo de ano de publicação
1.
Neurobiol Dis ; 179: 106050, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36809847

RESUMO

Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.


Assuntos
Doença de Alzheimer , Esclerose Lateral Amiotrófica , Demência Frontotemporal , Doença de Pick , Camundongos , Animais , Esclerose Lateral Amiotrófica/genética , Demência Frontotemporal/genética , Doença de Alzheimer/genética , Neuroproteção , Proteínas de Ligação a DNA/metabolismo , Imunoterapia
2.
Immunity ; 33(1): 84-95, 2010 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-20643339

RESUMO

Germinal centers (GCs) are specialized microenvironments where antigen-activated B cells undergo proliferation, immunoglobulin (Ig) class switch recombination, somatic hypermutation (SHM), and affinity maturation. Within GCs, follicular dendritic cells (FDCs) are key players in driving these events via direct interaction with GC B cells. Here, we provide in vivo evidence that FDCs express and upregulate Toll-like-receptor (TLR) 4 in situ during germinal center reactions, confirm that their maturation is driven by TLR4, and associate the role of FDC-expressed TLR4 with quantitative and qualitative affects of GC biology. In iterative cycles of predictions by in silico modeling subsequently verified by in vivo experiments, we demonstrated that TLR4 signaling modulates FDC activation, strongly impacting SHM and generation of Ig class-switched high-affinity plasma and memory B cells. Thus, our data place TLR4 in the heart of adaptive humoral immunity, providing further insight into mechanisms driving GCs arising in both health and disease.


Assuntos
Linfócitos B/metabolismo , Células Dendríticas Foliculares/metabolismo , Centro Germinativo/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Anticorpos Bloqueadores , Afinidade de Anticorpos , Antígenos de Diferenciação/biossíntese , Linfócitos B/imunologia , Linfócitos B/patologia , Transplante de Medula Óssea , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Dendríticas Foliculares/patologia , Centro Germinativo/patologia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Memória Imunológica , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Mutação/genética , Quimera por Radiação , Transdução de Sinais/imunologia , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia
3.
J Biol Chem ; 292(10): 4185-4197, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28154179

RESUMO

To improve our understanding of properties that confer successful inhibition of chemokines in vivo, we analyzed anti-murine CXCL10 monoclonal antibodies (mAb) having different characteristics. 1B6 displayed potent inhibition of cell recruitment in vitro with an IC50 of 0.5 nm but demonstrated little efficacy in various animal models of human disease. On the contrary, 1F11 showed efficacy in several models of inflammation yet was less potent at inhibiting chemotaxis in vitro with an IC50 of 21 nm Furthermore, we observed that 1B6 displayed a rapid dose-dependent clearance (t½ 10-60 h) in contrast to 1F11, which presented a dose-proportional pharmacokinetic profile and a half-life of 12 days. Moreover, 1B6 recognized glycosaminoglycan (GAG)-bound CXCL10, resulting in target-mediated clearance, which was corroborated using CXCL10-deficient mice. In contrast to 1B6, 1F11 inhibited the interaction of CXCL10 with GAGs, did not recognize GAG-bound CXCL10, and did not display target-mediated drug disposition. Confirming previous animal studies, 1B6 was poor at reversing glycemia in a model of type 1 diabetes, whereas 1F11 induced early and prolonged control of diabetes. Furthermore, when using 1A4, a subsequently generated anti-mCXCL10 mAb that shares the property with 1F11 of being unable to recognize CXCL10 immobilized on GAG, we observed a similar superior control of diabetes as compared with 1B6. We therefore concluded that targeting chemokines with antibodies such as 1B6 that recognize the more abundant GAG-bound form of the chemokine may not be the optimal strategy to achieve disease control.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Quimiocina CXCL10/antagonistas & inibidores , Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 1/prevenção & controle , Endotélio Vascular/metabolismo , Glicosaminoglicanos/metabolismo , Animais , Anticorpos Monoclonais/farmacocinética , Células Cultivadas , Quimiocina CXCL10/imunologia , Quimiocina CXCL10/metabolismo , Quimiotaxia de Leucócito/fisiologia , Cricetinae , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Tecidual
4.
Mol Ther ; 25(2): 523-533, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28153099

RESUMO

CD47 is a ubiquitously expressed immune checkpoint receptor that is often upregulated in cancer. CD47 interacts with its counter-receptor SIRPα on macrophages and other myeloid cells to inhibit cancer cell phagocytosis and drive immune evasion. To overcome tolerability and "antigen sink" issues arising from widespread CD47 expression, we generated dual-targeting bispecific antibodies that selectively block the CD47-SIRPα interaction on malignant cells expressing a specific tumor-associated antigen; e.g., CD19 or mesothelin. These bispecific κλ bodies are fully human, native IgG1 molecules, combining tumor targeting and selective CD47 blockade with immune activating mechanisms mediated by the Fc portion of the antibody. CD47-neutralizing κλ bodies efficiently kill cancer cells in vitro and in vivo but interact only weakly with healthy cells expressing physiological levels of CD47. Accordingly, a κλ body administered to non-human primates showed a typical IgG pharmacokinetic profile and was well tolerated. Importantly, κλ bodies preserve their tumoricidal capabilities in the presence of a CD47 antigen sink. Thus, dual-targeting κλ bodies allow for efficacious yet safe targeting of CD47 in cancer. Such a bispecific design could be applied to limit the extent of neutralization of other ubiquitously expressed therapeutic targets.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antígeno CD47/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Antígeno CD47/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Mesotelina , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Fagocitose/imunologia , Ligação Proteica/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Eur J Immunol ; 46(11): 2629-2638, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27510283

RESUMO

Increased expression of endogenous Toll-like receptor 4 (TLR4) ligands (e.g., Tenascin-C, S100A8/A9, citrullinated fibrinogen (cFb) immune complexes) has been observed in patients with rheumatoid arthritis (RA). However, their roles in RA pathogenesis are not well understood. Here, we investigated the expression kinetics and role of endogenous TLR4 ligands in the murine model of collagen-induced arthritis (CIA). Tenascin-C was upregulated in blood early in CIA, and correlated positively with the clinical score at day 56. Levels of S100A8/A9 increased starting from day 28, peaking at day 42, and correlated positively with joint inflammation. Levels of anti-cFb antibodies increased during the late phase of CIA and correlated positively with both joint inflammation and cartilage damage. Blockade of TLR4 activation at the time of the first TLR4 ligand upregulation prevented clinical and histological signs of arthritis. A TLR4-dependent role was also observed for Tenascin-C and cFb immune complexes in osteoclast differentiation in vitro. Taken together, our data suggests that the pathogenic contribution of TLR4 in promoting joint inflammation and bone erosion during CIA occurs via various TLR4 ligands arising at different stages of disease. The data also suggests that Blockade of TLR4 with monoclonal antibodies is a promising strategy in RA treatment.


Assuntos
Artrite Reumatoide/imunologia , Osso e Ossos/patologia , Calgranulina A/sangue , Tenascina/sangue , Receptor 4 Toll-Like/metabolismo , Animais , Complexo Antígeno-Anticorpo/sangue , Artrite Experimental/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Calgranulina A/genética , Diferenciação Celular , Colágeno , Modelos Animais de Doenças , Fibrinogênio/imunologia , Articulações/imunologia , Ligantes , Camundongos , Camundongos Endogâmicos DBA , Osteoclastos/metabolismo , Análise Espaço-Temporal , Tenascina/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia
6.
J Biol Chem ; 290(45): 26943-26953, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26363066

RESUMO

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.


Assuntos
Interleucina-6/química , Interleucina-6/metabolismo , Receptores de Interleucina-6/química , Receptores de Interleucina-6/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Receptor gp130 de Citocina/química , Receptor gp130 de Citocina/metabolismo , Feminino , Teste de Complementação Genética , Humanos , Interleucina-6/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Células NIH 3T3 , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Ratos , Receptores de Interleucina-6/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais
7.
Ann Rheum Dis ; 75(6): 1177-86, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26698852

RESUMO

BACKGROUND: Interferon γ (IFNγ) is considered a seminal cytokine in the pathogenesis of giant cell arteritis (GCA), but its functional role has not been investigated. We explored changes in infiltrating cells and biomarkers elicited by blocking IFNγ with a neutralising monoclonal antibody, A6, in temporal arteries from patients with GCA. METHODS: Temporal arteries from 34 patients with GCA (positive histology) and 21 controls were cultured on 3D matrix (Matrigel) and exposed to A6 or recombinant IFNγ. Changes in gene/protein expression were measured by qRT-PCR/western blot or immunoassay. Changes in infiltrating cells were assessed by immunohistochemistry/immunofluorescence. Chemotaxis/adhesion assays were performed with temporal artery-derived vascular smooth muscle cells (VSMCs) and peripheral blood mononuclear cells (PBMCs). RESULTS: Blocking endogenous IFNγ with A6 abrogated STAT-1 phosphorylation in cultured GCA arteries. Furthermore, selective reduction in CXCL9, CXCL10 and CXCL11 chemokine expression was observed along with reduction in infiltrating CD68 macrophages. Adding IFNγ elicited consistent opposite effects. IFNγ induced CXCL9, CXCL10, CXCL11, CCL2 and intracellular adhesion molecule-1 expression by cultured VSMC, resulting in increased PBMC chemotaxis/adhesion. Spontaneous expression of chemokines was higher in VSMC isolated from GCA-involved arteries than in those obtained from controls. Incubation of IFNγ-treated control arteries with PBMC resulted in adhesion/infiltration by CD68 macrophages, which did not occur in untreated arteries. CONCLUSIONS: Our ex vivo system suggests that IFNγ may play an important role in the recruitment of macrophages in GCA by inducing production of specific chemokines and adhesion molecules. Vascular wall components (ie, VSMC) are mediators of these functions and may facilitate progression of inflammatory infiltrates through the vessel wall.


Assuntos
Quimiocinas CXC/metabolismo , Arterite de Células Gigantes/imunologia , Interferon gama/antagonistas & inibidores , Macrófagos/imunologia , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/genética , Quimiocina CXCL11/metabolismo , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Quimiocinas CXC/genética , Quimiotaxia/imunologia , Regulação para Baixo/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/biossíntese , Interferon gama/farmacologia , Masculino , Músculo Liso Vascular/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Artérias Temporais/imunologia , Técnicas de Cultura de Tecidos
8.
J Immunol ; 192(4): 1641-50, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24442438

RESUMO

B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell-targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell-targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell-targeted approach predicated on the induction of B cell anergy.


Assuntos
Doenças Autoimunes/prevenção & controle , Linfócitos B/imunologia , Antígenos CD79/imunologia , Anergia Clonal/imunologia , Animais , Anticorpos Monoclonais/imunologia , Autoimunidade/imunologia , Feminino , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout
9.
J Biol Chem ; 289(22): 15309-18, 2014 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-24737331

RESUMO

Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.


Assuntos
Inflamação/imunologia , Macrófagos/imunologia , Receptores de IgG/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetulus , Dimerização , Feminino , Humanos , Inflamação/metabolismo , Macrófagos/citologia , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de IgG/metabolismo , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/metabolismo , Células U937
10.
Nat Rev Immunol ; 3(9): 764-9, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12949500

RESUMO

Follicular dendritic cells (FDCs), which reside in the primary B-cell follicles and germinal centres of lymphoid tissues, can sequester antigen in the form of immune complexes and are thought to be pivotal to the germinal-centre reaction and the maintenance of immunological memory. But, many recent studies question the importance of FDCs and their bound immune complexes in B-cell responses. This article asks whether we can truly rule out a requirement for these cells in host defence.


Assuntos
Células Dendríticas Foliculares/imunologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Apoptose/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Células Dendríticas Foliculares/citologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , Memória Imunológica/imunologia , Receptores de Antígenos de Linfócitos B/imunologia
11.
Methods ; 60(1): 99-110, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23500657

RESUMO

The use of next generation sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.


Assuntos
Anticorpos/química , Bacteriófagos/genética , Descoberta de Drogas , Sequenciamento de Nucleotídeos em Larga Escala , Biblioteca de Peptídeos
12.
J Biol Chem ; 287(2): 1458-67, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22041899

RESUMO

Dual-specific antibodies are characterized by an antigen-combining site mediating specific interactions with two different antigens. We have generated five dual-specific single chain variable fragments (scFv) that neutralize the activity of the two chemokines, CXCL9 and CXCL10, to bind to their receptor CXCR3. To better understand how these dual-specific scFvs bind these two chemokines that only share a 37% sequence identity, we mapped their epitopes on human CXCL9 and CXCL10 and identified serine 13 (Ser(13)) as a critical residue. It is conserved between the two chemokines but not in the third ligand for CXCR3, CXCL11. Furthermore, Ser(13) is exposed in the tetrameric structure of CXCL10, which is consistent with our finding that the scFvs are able to bind to CXCL9 and CXCL10 immobilized on glycosaminoglycans. Overall, the data indicate that these dual-specific scFvs bind to a conserved surface involved in CXCR3 receptor interaction for CXCL10 and CXCL9. Thus, structural mimicry between the two targets is likely to be responsible for the observed dual specificity of these antibody fragments.


Assuntos
Especificidade de Anticorpos , Quimiocina CXCL10/química , Quimiocina CXCL9/química , Mimetismo Molecular , Anticorpos de Cadeia Única/química , Animais , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL11/química , Quimiocina CXCL11/genética , Quimiocina CXCL11/imunologia , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Humanos , Macaca fascicularis , Macaca mulatta , Camundongos , Coelhos , Receptores CXCR3/química , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
13.
Biotechnol Bioeng ; 110(4): 1153-63, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23096947

RESUMO

The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL-17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL-17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL-17F expression improves the efficiency of cell line subcloning processes. IL-17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame.


Assuntos
Divisão Celular , Interleucina-17/genética , Animais , Células CHO , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Vetores Genéticos , Humanos , Proteínas Recombinantes/biossíntese
14.
Arthritis Rheum ; 64(10): 3189-98, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22508436

RESUMO

OBJECTIVE: The goal of rheumatoid arthritis (RA) treatment is to achieve clinical remission in order to limit structural damage and physical disability. To this end, recent emphasis has been placed on aggressive treatment early in the course of disease with drugs such as anti-tumor necrosis factor (anti-TNF) agents. As T cells are also thought to play an important role in the initiation of RA, we hypothesized that targeting both TNF and T cells would result in better outcomes. The aim of this study was to examine the efficacy of combined therapy with anti-CD3 and anti-TNF in experimental RA. METHODS: Two anti-mouse antibodies were developed as surrogate reagents for anti-TNF and anti-CD3 therapies. Collagen-induced arthritis (CIA) was induced in DBA/1 mice, and antibodies were injected intraperitoneally, either alone on in combination, at predetermined subtherapeutic doses. The frequency and number of pathogenic and regulatory CD4+ T cell subsets in the draining lymph nodes were determined in order to investigate the mechanisms of action. RESULTS: Strikingly, the combination of the two antibodies demonstrated a potent synergy in established CIA, with long-term inhibition of disease progression and protection from joint destruction. The results did not demonstrate any enhancement of CD25+FoxP3+ regulatory T cells, but a profound depletion of pathogenic T cells from the draining lymph nodes was associated with reduced numbers of T cells in the joints. CONCLUSION: A short course of combination therapy with anti-CD3 and anti-TNF efficiently depletes pathogenic T cells from the draining lymph nodes, reducing the numbers of T cells in the joints and affording long-lasting inhibition of established CIA.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/tratamento farmacológico , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Articulações/efeitos dos fármacos , Articulações/patologia , Camundongos , Camundongos Endogâmicos DBA , Resultado do Tratamento
15.
Nature ; 446(7131): 83-7, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17268470

RESUMO

Germinal centres are specialized structures wherein B lymphocytes undergo clonal expansion, class switch recombination, antibody gene diversification and affinity maturation. Three to four antigen-specific B cells colonize a follicle to establish a germinal centre and become rapidly dividing germinal-centre centroblasts that give rise to dark zones. Centroblasts produce non-proliferating centrocytes that are thought to migrate to the light zone of the germinal centre, which is rich in antigen-trapping follicular dendritic cells and CD4+ T cells. It has been proposed that centrocytes are selected in the light zone on the basis of their ability to bind cognate antigen. However, there have been no studies of germinal-centre dynamics or the migratory behaviour of germinal-centre cells in vivo. Here we report the direct visualization of B cells in lymph node germinal centres by two-photon laser-scanning microscopy in mice. Nearly all antigen-specific B cells participating in a germinal-centre reaction were motile and physically restricted to the germinal centre but migrated bi-directionally between dark and light zones. Notably, follicular B cells were frequent visitors to the germinal-centre compartment, suggesting that all B cells scan antigen trapped in germinal centres. Consistent with this observation, we found that high-affinity antigen-specific B cells can be recruited to an ongoing germinal-centre reaction. We conclude that the open structure of germinal centres enhances competition and ensures that rare high-affinity B cells can participate in antibody responses.


Assuntos
Linfócitos B/citologia , Movimento Celular , Centro Germinativo/citologia , Centro Germinativo/imunologia , Animais , Linfócitos B/imunologia , Adesão Celular , Comunicação Celular , Camundongos , Camundongos Endogâmicos C57BL
16.
Eur Heart J ; 33(15): 1964-74, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21606075

RESUMO

AIMS: The chemokine CCL5 plays a critical role as neutrophil and macrophage activator do in atherosclerosis and myocardial infarction. Thus, we investigated whether the treatment with a neutralizing monoclonal antibody (mAb) to mouse CCL5 would provide therapeutic benefit when provoking a coronary-associated ischaemic event. METHODS AND RESULTS: C57Bl/6 mice were submitted to left coronary artery permanent ligature. Then, various parameters were monitored for up to 21 days. At5 min and 3 days after coronary occlusion, mice received one intravenous injection of the rat anti-mouse CCL5 mAb or isotype IgG control. Infarct size was assessed histologically and by measuring serum cardiac troponin I levels. Kinetics of CCL5 tissue expression, leucocyte infiltration, matrix metalloproteinase (MMP) levels, and collagen deposition were histologically assessed. Serum chemokine levels were measured by enzyme-linked immunosorbent assay. Cardiac function and dimensions were assessed by magnetic resonance imaging (MRI). Chronic ischaemia increased both circulating and intracardiac levels of CCL5. At 24 h, treatment with the anti-CCL5 mAb resulted in a smaller infarct size and reduced circulating levels of chemokines. This effect was associated with reduction of neutrophil and macrophage infiltration within the infarcted myocardium. After 3 days of chronic ischaemia, anti-CCL5 mAb treatment reduced cardiac MMP-9. At 7 days, collagen content was significantly lower. At 21 days, neutralizing CCL5 improved mouse survival, cardiac myocyte size, and cardiac function. CONCLUSION: Treatment with anti-CCL5 mAb significantly reduced both infarct size and post-infarction heart failure in a mouse model of chronic cardiac ischaemia. Cardioprotective effects were associated with the reduction of leucocyte recruitment within infarcted hearts.


Assuntos
Anticorpos Monoclonais/farmacologia , Quimiocina CCL5/fisiologia , Insuficiência Cardíaca/patologia , Infarto do Miocárdio/patologia , Animais , Peso Corporal/fisiologia , Quimiocina CCL5/antagonistas & inibidores , Quimiocina CCL5/imunologia , Quimiotaxia de Leucócito/fisiologia , Doença Crônica , Colágeno/metabolismo , Ligadura , Macrófagos/fisiologia , Metaloproteinase 8 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Isquemia Miocárdica/metabolismo , Reperfusão Miocárdica/métodos , Infiltração de Neutrófilos/fisiologia , Neutrófilos/fisiologia , Tamanho do Órgão/fisiologia , Taxa de Sobrevida
17.
MAbs ; 15(1): 2232087, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37408314

RESUMO

Optimal pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) are essential to achieve the desired pharmacological benefits in patients. To accomplish this, we followed an approach comprising structure-based mAb charge engineering in conjunction with the use of relevant preclinical models to screen and select humanized candidates with PK suitable for clinical development. Murine mAb targeting TDP-43, ACI-5891, was humanized on a framework (VH1-3/VK2-30) selected based on the highest sequence homology. Since the initial humanized mAb (ACI-5891.1) presented a fast clearance in non-human primates (NHPs), reiteration of humanization on a less basic human framework (VH1-69-2/VK2-28) while retaining high sequence homology was performed. The resulting humanized variant, ACI-5891.9, presented a six-fold reduction in clearance in NHPs resulting in a significant increase in half-life. The observed reduced clearance of ACI-5891.9 was attributed not only to the overall reduction in isoelectric point (pI) by 2 units, but importantly to a more even surface potential. These data confirm the importance and contribution of surface charges to mAb disposition in vivo. Consistent low clearance of ACI-5891.9 in Tg32 mice, a human FcRn transgenic mouse model, further confirmed its utility for early assessment and prediction of human PK. These data demonstrate that mAb surface charge is an important parameter for consideration during the selection and screening of humanized candidates in addition to maintaining the other key physiochemical and target binding characteristics.


Assuntos
Anticorpos Monoclonais , Receptores Fc , Camundongos , Humanos , Animais , Camundongos Transgênicos , Taxa de Depuração Metabólica , Ponto Isoelétrico , Antígenos de Histocompatibilidade Classe I
18.
Front Neurosci ; 17: 1202208, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37449271

RESUMO

Introduction: People with DS are highly predisposed to Alzheimer's disease (AD) and demonstrate very similar clinical and pathological features. Ts65Dn mice are widely used and serve as the best-characterized animal model of DS. Methods: We undertook studies to characterize age-related changes for AD-relevant markers linked to Aß, Tau, and phospho-Tau, axonal structure, inflammation, and behavior. Results: We found age related changes in both Ts65Dn and 2N mice. Relative to 2N mice, Ts65Dn mice showed consistent increases in Aß40, insoluble phospho-Tau, and neurofilament light protein. These changes were correlated with deficits in learning and memory. Discussion: These data have implications for planning future experiments aimed at preventing disease-related phenotypes and biomarkers. Interventions should be planned to address specific manifestations using treatments and treatment durations adequate to engage targets to prevent the emergence of phenotypes.

19.
Nat Commun ; 14(1): 6750, 2023 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-37891183

RESUMO

A positron emission tomography (PET) tracer detecting α-synuclein pathology will improve the diagnosis, and ultimately the treatment of α-synuclein-related diseases. Here we show that the PET ligand, [18F]ACI-12589, displays good in vitro affinity and specificity for pathological α-synuclein in tissues from patients with different α-synuclein-related disorders including Parkinson's disease (PD) and Multiple-System Atrophy (MSA) using autoradiography and radiobinding techniques. In the initial clinical evaluation we include 23 participants with α-synuclein related disorders, 11 with other neurodegenerative disorders and eight controls. In vivo [18F]ACI-12589 demonstrates clear binding in the cerebellar white matter and middle cerebellar peduncles of MSA patients, regions known to be highly affected by α-synuclein pathology, but shows limited binding in PD. The binding statistically separates MSA patients from healthy controls and subjects with other neurodegenerative disorders, including other synucleinopathies. Our results indicate that α-synuclein pathology in MSA can be identified using [18F]ACI-12589 PET imaging, potentially improving the diagnostic work-up of MSA and allowing for detection of drug target engagement in vivo of novel α-synuclein targeting therapies.


Assuntos
Atrofia de Múltiplos Sistemas , Doença de Parkinson , Humanos , alfa-Sinucleína/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , Doença de Parkinson/metabolismo , Tomografia por Emissão de Pósitrons
20.
Brain Commun ; 5(6): fcad306, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38025276

RESUMO

In amyotrophic lateral sclerosis, a disease driven by abnormal transactive response DNA-binding protein of 43 kDa aggregation, CSF may contain pathological species of transactive response DNA-binding protein of 43 kDa contributing to the propagation of pathology and neuronal toxicity. These species, released in part by degenerating neurons, would act as a template for the aggregation of physiological protein contributing to the spread of pathology in the brain and spinal cord. In this study, a robust seed amplification assay was established to assess the presence of seeding-competent transactive response DNA-binding protein of 43 kDa species in CSF of apparently sporadic amyotrophic lateral sclerosis patients. These samples resulted in a significant acceleration of substrate aggregation differentiating the kinetics from healthy controls. In parallel, a second assay was developed to determine the level of target engagement that would be necessary to neutralize such species in human CSF by a therapeutic monoclonal antibody targeting transactive response DNA-binding protein of 43 kDa. For this, evaluation of the pharmacokinetic/pharmacodynamic effect for the monoclonal antibody, ACI-5891.9, in vivo and in vitro confirmed that a CSF concentration of ≍1100 ng/mL would be sufficient for sustained target saturation. Using this concentration in the seed amplification assay, ACI-5891.9 was able to neutralize the transactive response DNA-binding protein of 43 kDa pathogenic seeds derived from amyotrophic lateral sclerosis patient CSF. This translational work adds to the evidence of transmission of transactive response DNA-binding protein of 43 kDa pathology via CSF that could contribute to the non-contiguous pattern of clinical manifestations observed in amyotrophic lateral sclerosis and demonstrates the ability of a therapeutic monoclonal antibody to neutralize the toxic, extracellular seeding-competent transactive response DNA-binding protein of 43 kDa species in the CSF of apparently sporadic amyotrophic lateral sclerosis patients.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA