RESUMO
Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (~30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.
Assuntos
Anticorpos Neutralizantes/imunologia , Antígeno CD11b/imunologia , Antígenos CD18/imunologia , Anticorpos de Domínio Único/imunologia , Antitoxina Tetânica/imunologia , Animais , Afinidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domains (nanobodies) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS: Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct use for pull-down/MS target identification. RESULTS: The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS: This strategy streamlines the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE: This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.
Assuntos
Células Dendríticas/imunologia , Receptores de Superfície Celular/imunologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Biotina/imunologia , Antígeno CD11b/imunologia , Antígenos CD18/imunologia , Camelídeos Americanos , Linhagem Celular , Técnicas de Visualização da Superfície Celular/métodos , Células Cultivadas , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunização/métodos , Antígenos Comuns de Leucócito/imunologia , Camundongos Endogâmicos BALB C , Peptídeos/imunologia , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of worldwide significance. Infection with Hc produces a broad spectrum of diseases and may progress to a life-threatening systemic disease, particularly in individuals with HIV infection. Resolution of histoplasmosis is associated with the activation of cell-mediated immunity, and leukotriene B(4) plays an important role in this event. Lipid bodies (LBs) are increasingly being recognized as multifunctional organelles with roles in inflammation and infection. In this study, we investigated LB formation in histoplasmosis and its putative function in innate immunity. LB formation in leukocytes harvested from Hc-infected C57BL/6 mice peaks on day 2 postinfection and correlates with enhanced generation of lipid mediators, including leukotriene B(4) and PGE(2). Pretreatment of leukocytes with platelet-activating factor and BLT1 receptor antagonists showed that both lipid mediators are involved in cell signaling for LB formation. Alveolar leukocytes cultured with live or dead Hc also presented an increase in LB numbers. The yeast alkali-insoluble fraction 1, which contains mainly beta-glucan isolated from the Hc cell wall, induced a dose- and time-dependent increase in LB numbers, indicating that beta-glucan plays a signaling role in LB formation. In agreement with this hypothesis, beta-glucan-elicited LB formation was inhibited in leukocytes from 5-LO(-/-), CD18(low) and TLR2(-/-) mice, as well as in leukocytes pretreated with anti-Dectin-1 Ab. Interestingly, human monocytes from HIV-1-infected patients failed to produce LBs after beta-glucan stimulation. These results demonstrate that Hc induces LB formation, an event correlated with eicosanoid production, and suggest a role for these lipid-enriched organelles in host defense during fungal infection.
Assuntos
Antígenos CD18/metabolismo , Infecções por HIV/imunologia , Histoplasmose/imunologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organelas/imunologia , Receptor 2 Toll-Like/metabolismo , beta-Glucanas/imunologia , Adulto , Animais , Antígenos CD18/imunologia , Parede Celular/química , Parede Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , HIV-1 , Histoplasma/imunologia , Humanos , Lectinas Tipo C , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucotrieno B4/biossíntese , Leucotrieno B4/imunologia , Lipídeos , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/imunologia , Organelas/metabolismo , Receptor 2 Toll-Like/imunologiaRESUMO
Toward obtaining a more comprehensive understanding of factors governing activation and/or function during visceral leishmaniasis (VL), we have compared active disease (pre-treatment) versus post-chemotherapy immune response in VL patients by means of ex vivo staining with different cell markers. Our results show that during active disease, the frequency of T cells positive for CD25, CTLA-4 and CD45RO was significantly lower in VL patients compared with healthy controls, whereas cells staining positive for Annexin V and CD95 were significantly higher. In all cases, chemotherapy was able to restore these frequencies to normal levels. Interestingly, significant differences in the frequency of CD18 and in the frequency of CD45RO-positive cells were observed in the CD8+ T cell subset. These two frequencies were also significantly higher in bone marrow when compared with peripheral blood, suggesting a possible compartmentalization of certain CD8+ T cell populations during active disease. Given that CD8+ T cells have been shown to play an essential role in immunity to infection with Leishmania, our data indicate that the lower frequency of CD18+ and CD45RO+ lymphocytes in the bone marrow CD8+ T cell subset may be considered a biomarker of acute VL.
Assuntos
Antígenos de Protozoários/metabolismo , Antígenos CD18/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Leishmania infantum , Leishmaniose Visceral/imunologia , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Doença Aguda , Adolescente , Adulto , Animais , Antígenos de Protozoários/imunologia , Biomarcadores/sangue , Linfócitos T CD8-Positivos/patologia , Separação Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Leishmaniose Visceral/sangue , Antígenos Comuns de Leucócito/genética , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/patologiaRESUMO
Leishmania promastigotes interact with macrophages through the association of multiple membrane surface receptors. Macrophage complement receptor CR3 (CD11b/CD18 or Mac-1) has been implicated in the interaction of both human and murine macrophages with serum-opsonized promastigotes. The aim of this study was to determine CR3 expression in the livers and spleens of dogs naturally infected with Leishmania (Leishmania) chagasi. CR3 expression in liver was higher in asymptomatic than in symptomatic animals. Moreover, the hepatic parasitism load determined by immunocytochemical analysis was lower in parallel with higher numbers of granulomas. In contrast, in spleens, CR3 expression was higher in symptomatic animals than in asymptomatic ones. However, the tissue parasite load was greater in spleens of symptomatic dogs. There was a strict correlation between the parasite load and cellular CR3 expression in the spleens of dogs naturally infected with L. chagasi. CR3 macrophage integrins could be essential receptors for Leishmania survival. Considering that the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their spleens, we can conclude that these splenic cells (monocyte-macrophages) might serve to perpetuate intracellular infection.
Assuntos
Doenças do Cão/parasitologia , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Hepatopatias Parasitárias/veterinária , Antígeno de Macrófago 1/imunologia , Esplenopatias/veterinária , Animais , Antígeno CD11b/imunologia , Antígenos CD18/imunologia , Doenças do Cão/imunologia , Cães , Imuno-Histoquímica/veterinária , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Hepatopatias Parasitárias/imunologia , Hepatopatias Parasitárias/parasitologia , Esplenopatias/imunologia , Esplenopatias/parasitologiaRESUMO
Macrophage migration and adhesion are important for the control of mycobacterial infection and are critically dependent on the reorganization of the cytoskeleton. Mycobacteria elicit rapid morphological changes, such as cell spreading, a process relevant to in vivo changes of macrophage shape during extravasation and migration. In this study, we investigated the BCG mycobacteria-induced signaling events leading to macrophage cytoskeletal rearrangements employing specific pharmacological inhibitors to suppress distinct kinase pathways known to be elicited by infection. Viable or lysed mycobacteria, as well as purified cell wall lipoprotein p19, TLR2 agonist, induced RAW264.7 cells to extend actin-rich pseudopods, which impart radial spreading within 3 h, leading later to persistent cell polarization. BCG induced rapid activation of phosphatidylinositol 3-kinase, PI3K, activation that was recruited to the activated TLR2 receptor. TLR2- neutralizing antibody inhibited macrophage spreading and PI3K activation induced by p19. Additionally, BCG induced spreading and polarization of bone marrow-derived macrophages from TLR2- expressing mice in contrast to their TLR2-knockout counterparts. Neither MEK1/ERK, p38 MAPK, nor NF-kappaB activation were important for the early cytoskeletal rearrangements observed, although suppression of these pathways is known to inhibit chemokine secretion by activated macrophages. Beta2-integrins blockade with a corresponding antibody inhibited macrophage spreading and polarization but had no effect on pseudopodia protrusions demonstrating the downstream position of integrin-mediated adhesion in PI3K- dependent signaling pathway leading to the motility phenotype. The obtained data demonstrate that the direct effect of mycobacteria on macrophage shape might be mediated through TLR2-dependent PI3K activation.
Assuntos
Citoesqueleto/imunologia , Macrófagos/imunologia , Mycobacterium bovis/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/farmacologia , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Polaridade Celular/imunologia , Quimiocinas/imunologia , Quimiocinas/metabolismo , Citoesqueleto/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , MAP Quinase Quinase 1/imunologia , MAP Quinase Quinase 1/metabolismo , Macrófagos/enzimologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Pseudópodes/imunologia , Pseudópodes/metabolismo , Pseudópodes/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/deficiência , Tuberculose/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cellular infiltration to renal tissues is an important feature during acute puromycin aminonucleoside nephrosis (PAN) in rats. The mechanisms responsible for this infiltration are poorly understood. To elucidate the participation of adhesion molecules in PAN, nephrosis was induced in rats by intraperitoneal puromycin aminonucleoside injection. Controls represent animals injected with a 0.9% saline solution. ICAM-1 (intercellular adhesion molecule 1), CD18 (beta chain of lymphocyte-function-associated antigen), LCA (leukocyte common antigen), ED1 (monocyte/macrophage marker), and proliferating cell nuclear antigen expressions were evaluated in renal tissues 1, 2, and 7 weeks after injection. Frozen sections from PAN rat kidneys showed increased expressions of ICAM-1 and its ligand, and these findings were associated with increased levels of LCA+ and ED1+ cells in glomerulus and interstitium. The kinetics of leukocyte infiltration was similar to the kinetics of ICAM-1 expression: high values at week 2 which returned to normal values at week 7. Increased glomerular and interstitial proliferative activities (proliferating cell nulear antigen positive cells) were also found at week 2 of nephrosis. There was a correlation between ICAM-1 expression and numbers of LCA+ and ED1+ cells and between numbers of LCA+ cells and proliferating cells in glomerulus and interstitium. Correlations between glomerular and tubular ICAM-1 expression, interstitial leukocyte infiltration, and glomerular, interstitial, and tubular proliferative activities with the proteinuria were also observed during the nephrotic phase. In addition, increased lymphocyte binding to PAN renal tissues was observed, and this binding was diminished by anti-LFA-1beta monoclonal antibody pretreatment of lymphocytes. A similar result was found with anti-ICAM-1 monoclonal antibody pretreatment of renal tissues. Our results suggest that increased expression of ICAM-1 and proliferative activity could be important determinants in the renal hypercellularity found in this experimental model.
Assuntos
Antígenos CD18/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Molécula 1 de Adesão Intercelular/biossíntese , Nefrose/induzido quimicamente , Nefrose/metabolismo , Antígeno Nuclear de Célula em Proliferação/biossíntese , Puromicina Aminonucleosídeo/farmacologia , Doença Aguda , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/fisiopatologia , Antígenos Comuns de Leucócito/biossíntese , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos CD18/imunologia , Região Variável de Imunoglobulina/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Antígenos/imunologia , Humanos , Região de Junção de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologiaRESUMO
In the current study, we analyzed whether immunoglobulin A (IgA) is able to modulate neutrophil apoptosis. We found that culture of neutrophils on immobilized plasma IgA (iIgAp) or secretory IgA (iIgAs) induced a marked increase in apoptotic rates. By contrast, soluble IgAp, IgAs, or aggregated IgAp exerted no effect. Promotion of apoptosis by iIgA was almost completely prevented by blocking antibodies directed to CD18 or CD11b and was shown to be dependent on the activation of the respiratory burst as suggested by the ability of catalase to prevent apoptosis stimulation; the effect of azide, an heme enzyme inhibitor that significantly increased promotion of apoptosis by iIgA; and the inability of iIgA to stimulate apoptosis of neutrophils isolated from chronic granulomatous disease patients. Stimulation of neutrophil apoptosis by IgA might contribute to the control of inflammatory processes in certain autoimmune diseases such as IgA nephropathy in which tissue deposits of IgA or IgA containing immune complexes are found.
Assuntos
Apoptose/imunologia , Antígeno CD11b/imunologia , Antígenos CD18/imunologia , Imunoglobulina A/imunologia , Neutrófilos/imunologia , Animais , Células Cultivadas , Proteína Ligante Fas , Humanos , Imunoglobulina A/farmacologia , Glicoproteínas de Membrana/imunologia , Camundongos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Explosão Respiratória/imunologia , Receptor fas/imunologiaRESUMO
Integrin-mediated signals play an important but poorly understood role in regulating many leucocyte functions. In monocytes and macrophages, integrins of the beta2 subfamily are involved in cell-cell interactions that are important for migration of the cells through the endothelium and also for phagocytosis. On the other hand, in the same cells, beta1 integrin-mediated adhesion to extracellular matrix proteins results in a strong induction of immediate early genes that are important in inflammation. To investigate the signalling pathways from these two types of integrin in monocytic cells, THP-1 cells were selectively stimulated via beta1 or beta2 integrins by cross-linking each type of receptor with specific monoclonal antibodies or their natural ligands. The involvement of extracellular signal-regulated kinase (ERK), Syk and phosphoinositide 3-kinase (PI-3K) was then analysed. Nuclear factor kappaB (NF-kappaB) activation was also detected in THP-1 cells transiently transfected with an NF-kappaB-driven luciferase reporter gene. We found that binding of both types of integrin to their natural ligands activated ERK in a Syk- and PI-3K-dependent manner. Yet, cross-linking of integrins by anti-beta1 antibodies caused activation of ERK while that by anti-beta2 antibodies did not. Also both types of integrin activated NF-kappaB. However, PI-3K was required for beta1 integrin-, but not beta2 integrin-, mediated NF-kappaB activation. In addition, inhibition of PI-3K with wortmannin and LY294002 blocked beta1 integrin-mediated NF-kappaB activation, but did not affect that mediated by beta2 integrin. These data suggest that distinct integrins activate different signalling pathways in monocytic cells.