RESUMO
Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Epitopos , Subunidade alfa1 de Receptor de Interleucina-13/química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação/imunologia , Cristalografia por Raios X , Dimerização , Humanos , Fragmentos Fab das Imunoglobulinas/química , Interleucina-13/imunologia , Interleucina-13/metabolismo , Subunidade alfa1 de Receptor de Interleucina-13/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Leucina/metabolismo , Camundongos , Camundongos Transgênicos , Estrutura Terciária de ProteínaRESUMO
Generating a cytotoxic CD8(+) T-cell response that can eradicate malignant cells is the primary objective of cancer vaccine strategies. In this study we have characterized the innate and adaptive immune response to the ISCOMATRIX adjuvant, and the ability of vaccine antigens formulated with this adjuvant to promote antitumor immunity. ISCOMATRIX adjuvant led to a rapid innate immune cell response at the injection site, followed by the activation of natural killer and dendritic cells (DC) in regional draining lymph nodes. Strikingly, major histocompatibility complex (MHC) class I cross-presentation by CD8α(+) and CD8α(-) DCs was enhanced by up to 100-fold when antigen was formulated with ISCOMATRIX adjuvant. These coordinated features enabled efficient CD8(+) T-cell cross-priming, which exhibited prophylactic and therapeutic tumoricidal activity. The therapeutic efficacy of an ISCOMATRIX vaccine was further improved when co-administered with an anti-CD40 agonist antibody, suggesting that ISCOMATRIX-based vaccines may combine favorably with other immune modifiers in clinical development to treat cancer. Finally, we identified a requirement for the myeloid differentiation primary response gene 88 (MyD88) adapter protein for both innate and adaptive immune responses to ISCOMATRIX vaccines in vivo. Taken together, our findings support the utility of the ISCOMATRIX adjuvant for use in the development of novel vaccines, particularly those requiring strong CD8(+) T-cell immune responses, such as therapeutic cancer vaccines.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Colesterol/imunologia , Fosfolipídeos/imunologia , Saponinas/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Antígenos de Neoplasias/imunologia , Antígenos CD40/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Vacinas Anticâncer/administração & dosagem , Colesterol/administração & dosagem , Apresentação Cruzada/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Combinação de Medicamentos , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Ovalbumina/imunologia , Fosfolipídeos/administração & dosagem , Receptor Cross-Talk/efeitos dos fármacos , Saponinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacosRESUMO
Cancer vaccines aim to induce antitumor CTL responses, which require cross-presentation of tumor Ag to CTLs by dendritic cells (DCs). Adjuvants that facilitate cross-presentation of vaccine Ag are therefore key for inducing antitumor immunity. We previously reported that human DCs could not efficiently cross-present the full-length cancer/testis Ag NY-ESO-1 to CTL unless formulated as either an immune complex (NY-ESO-1/IC) or with ISCOMATRIX adjuvant. We now demonstrate that NY-ESO-1/ICs induce cross-presentation of HLA-A2- and HLA-Cw3-restricted epitopes via a proteasome-dependent pathway. In contrast, cross-presentation of NY-ESO-1/ISCOMATRIX vaccine was proteasome independent and required the cytosolic protease tripeptidyl peptidase II. Trafficking studies revealed that uptake of ICs and ISCOMATRIX vaccine by DCs occurred via endocytosis with delivery to lysosomes. Interestingly, ICs were retained in lysosomes, whereas ISCOMATRIX adjuvant induced rapid Ag translocation into the cytosol. Ag translocation was dependent on endosomal acidification and IL-4-driven differentiation of monocytes into DCs. This study demonstrates that Ag formulation determines Ag processing and supports a role for tripeptidyl peptidase II in cross-presentation of CTL epitopes restricted to diverse HLA alleles.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Colesterol/administração & dosagem , Apresentação Cruzada/imunologia , Citosol/imunologia , Células Dendríticas/imunologia , Proteínas de Membrana/imunologia , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Serina Endopeptidases/metabolismo , Aminopeptidases , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/metabolismo , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Diferenciação Celular/imunologia , Citosol/enzimologia , Citosol/metabolismo , Células Dendríticas/enzimologia , Células Dendríticas/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases , Combinação de Medicamentos , Endocitose/imunologia , Endossomos/enzimologia , Endossomos/imunologia , Endossomos/metabolismo , Humanos , Hidrólise , Lisossomos/enzimologia , Lisossomos/imunologia , Lisossomos/metabolismo , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/metabolismo , Células Progenitoras Mieloides/enzimologia , Células Progenitoras Mieloides/imunologia , Células Progenitoras Mieloides/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Transporte Proteico/imunologia , Quillaja/imunologia , Transdução de Sinais/imunologiaRESUMO
We have cloned the mouse and human C-type lectin Clec12A, expressed both, and produced mAb recognizing both. Mouse Clec12A is highly expressed on splenic CD8(+) dendritic cells (DC) and plasmacytoid DC. A proportion of CD8(-)DC also expresses lower levels of Clec12A, as do monocytes, macrophages, and B cells. Human CLEC12A, like the mouse counterpart, is expressed on blood monocytes and DC, including pDC and BDCA-3(+)DC, the proposed equivalent of mouse CD8(+)DC. To determine whether Ag targeted to Clec12A could induce immune responses, mice were injected with a rat mAb recognizing Clec12A, or a control rat mAb, then production of anti-rat Ig was measured. Anti-Clec12A mAb alone produced only moderate responses, but these were amplified by coinjecting only small amounts of LPS as a DC activation agent. Furthermore, when OVA was conjugated to anti-Clec12A mAb, OVA-specific T cells were induced to proliferate. This Ag presentation to naive T cells was due to targeting conventional DC, because their ablation eliminated T cell activation. The potent Ab responses induced using microgram amounts of anti-Clec12A and minimal amounts of adjuvant demonstrate that this molecule can be used as an Ag-delivery target to enhance Ab responses to vaccines.
Assuntos
Formação de Anticorpos/imunologia , Antígenos/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Receptores Mitogênicos/imunologia , Animais , Apresentação de Antígeno/imunologia , Membrana Celular/imunologia , Células Cultivadas , Humanos , Leucócitos/imunologia , CamundongosRESUMO
A novel dendritic cell (DC)-restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8(+) conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.
Assuntos
Células Dendríticas/citologia , Lectinas Tipo C/química , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Vacinas/química , Vacinas/metabolismoRESUMO
Th1 effector CD4+ cells contribute to the pathogenesis of proliferative and crescentic glomerulonephritis, but whether effector Th17 cells also contribute is unknown. We compared the involvement of Th1 and Th17 cells in a mouse model of antigen-specific glomerulonephritis in which effector CD4+ cells are the only components of adaptive immunity that induce injury. We planted the antigen ovalbumin on the glomerular basement membrane of Rag1(-/-) mice using an ovalbumin-conjugated non-nephritogenic IgG1 monoclonal antibody against alpha3(IV) collagen. Subsequent injection of either Th1- or Th17-polarized ovalbumin-specific CD4+ effector cells induced proliferative glomerulonephritis. Mice injected with Th1 cells developed progressive albuminuria over 21 d, histologic injury including 5.5 +/- 0.9% crescent formation/segmental necrosis, elevated urinary nitrate, and increased renal NOS2, CCL2, and CCL5 mRNA. Mice injected with Th17 cells developed albuminuria by 3 d; compared with Th1-injected mice, their glomeruli contained more neutrophils and greater expression of renal CXCL1 mRNA. In conclusion, Th1 and Th17 effector cells can induce glomerular injury. Understanding how these two subsets mediate proliferative forms of glomerulonephritis may lead to targeted therapies.
Assuntos
Glomerulonefrite Membranoproliferativa/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Células Th1/imunologia , Imunidade Adaptativa , Transferência Adotiva , Animais , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animais de Doenças , Genes RAG-1 , Glomerulonefrite Membranoproliferativa/etiologia , Glomerulonefrite Membranoproliferativa/genética , Imunoglobulina G/metabolismo , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Neutrophils are the most abundant WBCs and have an essential role in the clearance of pathogens. Tight regulation of neutrophil numbers and their recruitment to sites of inflammation is critical in maintaining a balanced immune response. In various inflammatory conditions, such as rheumatoid arthritis, vasculitis, cystic fibrosis, and inflammatory bowel disease, increased serum G-CSF correlates with neutrophilia and enhanced neutrophil infiltration into inflamed tissues. We describe a fully human therapeutic anti-G-CSFR antibody (CSL324) that is safe and well tolerated when administered via i.v. infusion to cynomolgus macaques. CSL324 was effective in controlling G-CSF-mediated neutrophilia when administered either before or after G-CSF. A single ascending-dose study showed CSL324 did not alter steady-state neutrophil numbers, even at doses sufficient to completely prevent G-CSF-mediated neutrophilia. Weekly infusions of CSL324 (≤10 mg/kg) for 3 wk completely neutralized G-CSF-mediated pSTAT3 phosphorylation without neutropenia. Moreover, repeat dosing up to 100 mg/kg for 12 wk did not result in neutropenia at any point, including the 12-wk follow-up after the last infusion. In addition, CSL324 had no observable effect on basic neutrophil functions, such as phagocytosis and oxidative burst. These data suggest that targeting G-CSFR may provide a safe and effective means of controlling G-CSF-mediated neutrophilia as observed in various inflammatory diseases.
Assuntos
Anticorpos Neutralizantes/farmacologia , Neutropenia , Neutrófilos/efeitos dos fármacos , Receptores de Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Macaca fascicularis , Ressonância de Plasmônio de SuperfícieRESUMO
The ß common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared ß common (ßc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human ßc receptor. The binding epitope of CSL311 on the ßc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human ßc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 ß common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human ßc receptor is central to pathogenesis. The coordinates for the ßc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).
Assuntos
Anticorpos Monoclonais Murinos , Subunidade beta Comum dos Receptores de Citocinas , Epitopos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Interleucina-3 , Interleucina-5 , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/uso terapêutico , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Asma/tratamento farmacológico , Asma/imunologia , Asma/patologia , Cristalografia por Raios X , Subunidade beta Comum dos Receptores de Citocinas/química , Subunidade beta Comum dos Receptores de Citocinas/imunologia , Eosinófilos/imunologia , Eosinófilos/patologia , Epitopos/química , Epitopos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Interleucina-3/antagonistas & inibidores , Interleucina-3/imunologia , Interleucina-5/antagonistas & inibidores , Interleucina-5/imunologia , CamundongosRESUMO
Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes.
Assuntos
Coagulação Sanguínea , Fator XII/metabolismo , Angioedema Hereditário Tipo III/metabolismo , Mutação de Sentido Incorreto , Adulto , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/farmacologia , Bradicinina/genética , Bradicinina/metabolismo , Modelos Animais de Doenças , Fator XII/genética , Feminino , Glicosilação/efeitos dos fármacos , Angioedema Hereditário Tipo III/tratamento farmacológico , Angioedema Hereditário Tipo III/genética , Angioedema Hereditário Tipo III/patologia , Humanos , Camundongos , Camundongos KnockoutRESUMO
Interleukin-3 (IL-3) is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα) in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD), a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-13 receptors, adopting unique "open" and classical "closed" conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas "open-like" IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a "double hit" cytokine receptor blockade.
Assuntos
Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Subunidade alfa de Receptor de Interleucina-3/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/imunologia , Antineoplásicos/metabolismo , Sítios de Ligação de Anticorpos , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Systemic delivery of Ag usually induces poor mucosal immunity. To improve the CD8 T cell response at mucosal sites, we targeted the Ag to MAdCAM-1, a mucosal addressin cell adhesion molecule expressed mainly by high endothelial venules (HEV) in mesenteric lymph nodes (MLN) and Peyer's patches of gut-associated lymphoid tissue. When chemical conjugates of anti-MAdCAM-1 Ab and model Ag OVA were injected i.v., a greatly enhanced proliferative response of Ag-specific OT-I CD8 T cells was detected in MLN. This was preceded by prolonged accumulation, up to 2 wk, of the anti-MAdCAM OVA conjugate on HEV of Peyer's patches and MLN. In contrast, nontargeted OVA conjugate was very inefficient in inducing OT-I CD8 T cell proliferation in MLN and required at least 20-fold more Ag to induce a comparable response. In addition, MAdCAM targeting elicits an endogenous OVA-specific CD8 T cell response, evident by IFN-gamma production and target killing. Induced response offers protection against an OVA-expressing B cell lymphoma. We propose that the augmentation of gut CD8 T cell responses by MAdCAM targeting is due to both accumulation of Ag in the HEV and conversion of a soluble Ag to a cell-associated one, allowing cross-presentation by DCs.