RESUMO
The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50-68 or 86-102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a ß-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this ß-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Oxirredutases Intramoleculares/química , Fatores Inibidores da Migração de Macrófagos/química , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Dermatite de Contato/tratamento farmacológico , Dermatite de Contato/imunologia , Modelos Animais de Doenças , Humanos , Oxirredutases Intramoleculares/imunologia , Fatores Inibidores da Migração de Macrófagos/imunologia , Camundongos , Sepse/tratamento farmacológico , Sepse/imunologiaRESUMO
KLK1 (tissue kallikrein 1) is a member of the tissue kallikrein family of serine proteases and is the primary kinin-generating enzyme in human airways. DX-2300 is a fully human antibody that inhibits KLK1 via a competitive inhibition mechanism (Ki=0.13 nM). No binding of DX-2300 to KLK1 was observed in a surface-plasmon-resonance biosensor assay when KLK1 was complexed to known active-site inhibitors, suggesting that DX-2300 recognizes the KLK1 active site. DX-2300 did not inhibit any of the 21 serine proteases that were each tested at a concentration of 1 microM. We validated the use of DX-2300 for specific KLK1 inhibition by measuring the inhibition of KLK1-like activity in human urine, saliva and bronchoalveolar lavage fluid, which are known to contain active KLK1. In human tracheobronchial epithelial cells grown at the air/liquid interface, DX-2300 blocked oxidative-stress-induced epidermal-growth-factor receptor activation and downstream mucus cell proliferation and hypersecretion, which have been previously shown to be mediated by KLK1. In an allergic sheep model of asthma, DX-2300 inhibited both allergen-induced late-phase bronchoconstriction and airway hyper-responsiveness to carbachol. These studies demonstrate that DX-2300 is a potent and specific inhibitor of KLK1 that is efficacious in in vitro and in vivo models of airway disease.
Assuntos
Anticorpos Monoclonais/farmacologia , Asma/enzimologia , Asma/terapia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/terapia , Calicreínas Teciduais/antagonistas & inibidores , Calicreínas Teciduais/fisiologia , Animais , Anticorpos Monoclonais/metabolismo , Asma/imunologia , Sítios de Ligação de Anticorpos , Ligação Competitiva , Células Cultivadas , Humanos , Camundongos , Doença Pulmonar Obstrutiva Crônica/imunologia , Ovinos , Calicreínas Teciduais/imunologia , Calicreínas Teciduais/metabolismoRESUMO
As potential therapeutics, peptides offer several advantages over small molecules (increased specificity) and antibodies (small size). Nevertheless, a number of key issues have hampered their use as drug candidates. A series of new technologies have recently been developed that allow peptides to be viable drug candidates in areas usually restricted to protein therapeutics, such as monoclonal antibodies. These include the development of various types of peptide-conjugates that have lower rates of clearance and hence the potential to increase the exposure of peptide drug candidates in chronic diseases. Structural additions have also been made to peptides, including the use of unnatural amino acids, mainchain modifications and other novel substitutions, which have helped to improve peptide stability and further their therapeutic potential.
Assuntos
Biotecnologia/tendências , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/tendências , Desenho de Fármacos , Peptídeos/uso terapêutico , Engenharia de Proteínas/métodos , Tecnologia Farmacêutica/tendências , Estabilidade de MedicamentosRESUMO
LFA-1 (alpha(L)beta(2)) mediates cell-cell and cell-extracellular matrix adhesions essential for immune and inflammatory responses. One critical mechanism regulating LFA-1 activity is the conformational change of the ligand-binding alpha(L) I domain from low-affinity (LA), closed form, to the high-affinity (HA), open form. Most known integrin antagonists bind both forms. Antagonists specific for the HA alpha(L) I domain have not been described. Here, we report the identification and characterization of a human antibody AL-57, which binds to the alpha(L) I domain in a HA but not LA conformation. AL-57 was discovered by selection from a human Fab-displaying library using a locked-open HA I domain as target. AL-57 Fab-phage bound HA I domain-expressing K562 cells (HA cells) in a Mg(2+)-dependent manner. AL-57 IgG also bound HA cells and PBMCs, activated by Mg(2+)/EGTA, PMA, or DTT. The binding profile of AL-57 IgG on PBMCs was the same as that of ICAM-1, the main ligand of LFA-1. In contrast, an anti-alpha(L) murine mAb MHM24 did not distinguish between the HA and LA forms. Moreover, AL-57 IgG blocked ICAM-1 binding to HA cells with a potency greater than MHM24. It also inhibited ICAM-1 binding to PBMCs, blocked adhesion of HA cells to keratinocytes, and inhibited PHA-induced lymphocyte proliferation with potencies comparable with MHM24. These results indicate that specifically targeting the HA I domain is sufficient to inhibit LFA-1-mediated, adhesive functions. AL-57 represents a therapeutic candidate for treatment of inflammatory and autoimmune diseases.
Assuntos
Anticorpos Monoclonais/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Imunoglobulina G/farmacologia , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/imunologia , Dados de Sequência Molecular , Fito-Hemaglutininas/antagonistas & inibidores , Fito-Hemaglutininas/farmacologia , Relação Estrutura-AtividadeRESUMO
The control of proteolysis in an organism is achieved under normal circumstances through a balance of protease production, degradation and inactivation, via interaction with an endogenous inhibitor. When one of these mechanisms for control of proteolysis fails, it can result in the onset or progression of disease. Control of aberrant proteolysis is, therefore, a potetntial point of therapeutic intervention, and can be achieved either through the replacement of an absent endogenous inhibitor or by dosing with an inhibitor that is specific for a protease that is being over produced. Engineered protein inhibitors of proteases offer the potential to overcome the difficulties involved in identifying specific inhibitors via small-molecule-based approaches.
Assuntos
Inibidores de Proteases/farmacologia , Engenharia de Proteínas , Animais , Biblioteca Gênica , Humanos , Inibidores de Proteases/química , Inibidores de Proteases/uso terapêutico , Engenharia de Proteínas/tendênciasRESUMO
The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach - depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases.
RESUMO
Engagement of the immunoinhibitory receptor, programmed death-1 (PD-1) attenuates T-cell receptor (TCR)-mediated activation of IL-2 production and T-cell proliferation. Here, we demonstrate that PD-1 modulation of T-cell function involves inhibition of TCR-mediated phosphorylation of ZAP70 and association with CD3zeta. In addition, PD-1 signaling attenuates PKCtheta activation loop phosphorylation in a cognate TCR signal. PKCtheta has been shown to be required for T-cell IL-2 production. A phosphorylated PD-1 peptide, corresponding to the C-terminal immunoreceptor tyrosine-switch motif (ITSM), acts as a docking site in vitro for both SHP-2 and SHP-1, while the phosphorylated peptide containing the N-terminal PD-1 immunoreceptor tyrosine based inhibitory motif (ITIM) associates only with SHP-2.
Assuntos
Antígenos de Superfície/fisiologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Antígenos CD , Antígenos de Superfície/química , Proteínas Reguladoras de Apoptose , Humanos , Células Jurkat , Dados de Sequência Molecular , Fosforilação , Receptor de Morte Celular Programada 1 , Proteína Quinase C-theta , Homologia de Sequência de Aminoácidos , Proteína-Tirosina Quinase ZAP-70RESUMO
Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.
Assuntos
Anticorpos Monoclonais , Antineoplásicos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Inibidores de Metaloproteinases de Matriz , Neovascularização Patológica/prevenção & controle , Animais , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Genes Reporter , Humanos , Imuno-Histoquímica , Camundongos , Invasividade Neoplásica/patologia , Transfecção , Transplante Heterólogo , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacosRESUMO
Programmed death-1 (PD-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor expressed upon T cell activation. PD-1(-/-) animals develop autoimmune diseases, suggesting an inhibitory role for PD-1 in immune responses. Members of the B7 family, PD-L1 and PD-L2, are ligands for PD-1. This study examines the functional consequences of PD-1:PD-L engagement on murine CD4 and CD8 T cells and shows that these interactions result in inhibition of proliferation and cytokine production. T cells stimulated with anti-CD3/PD-L1.Fc-coated beads display dramatically decreased proliferation and IL-2 production, while CSFE analysis shows fewer cells cycling and a slower division rate. Costimulation with soluble anti-CD28 mAb can overcome PD-1-mediated inhibition by augmenting IL-2 production. However, PD-1:PD-L interactions inhibit IL-2 production even in the presence of costimulation and, thus, after prolonged activation, the PD-1:PD-L inhibitory pathway dominates. Exogenous IL-2 is able to overcome PD-L1-mediated inhibition at all times, indicating that cells maintain IL-2 responsiveness. Experiments using TCR transgenic CD4(+) or CD8(+) T cells stimulated with antigen-presenting cells expressing PD-L1 show that both T cell subsets are susceptible to this inhibitory pathway. However, CD8(+) T cells may be more sensitive to modulation by the PD-1:PD-L pathway because of their intrinsic inability to produce significant levels of IL-2.
Assuntos
Antígenos de Superfície , Apoptose/fisiologia , Antígeno B7-1 , Proteínas Sanguíneas , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/fisiologia , Peptídeos/fisiologia , Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Apresentação de Antígeno , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Antígeno B7-H1 , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Feminino , Fragmentos Fc das Imunoglobulinas/imunologia , Integrina beta1/imunologia , Interleucina-2/farmacologia , Cinética , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Microesferas , Dados de Sequência Molecular , Proteína 2 Ligante de Morte Celular Programada 1 , Receptor de Morte Celular Programada 1 , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
Programmed death-1 ligand (PD-L)1 and PD-L2 are ligands for programmed death-1 (PD-1), a member of the CD28/CTLA4 family expressed on activated lymphoid cells. PD-1 contains an immunoreceptor tyrosine-based inhibitory motif and mice deficient in PD-1 develop autoimmune disorders suggesting a defect in peripheral tolerance. Human PD-L1 and PD-L2 are expressed on immature dendritic cells (iDC) and mature dendritic cells (mDC), IFN-gamma-treated monocytes, and follicular dendritic cells. Using mAbs, we show that blockade of PD-L2 on dendritic cells results in enhanced T cell proliferation and cytokine production, including that of IFN-gamma and IL-10, while blockade of PD-L1 results in similar, more modest, effects. Blockade of both PD-L1 and PD-L2 showed an additive effect. Both whole mAb and Fab enhanced T cell activation, showing that PD-L1 and PD-L2 function to inhibit T cell activation. Enhancement of T cell activation was most pronounced with weak APC, such as iDCs and IL-10-pretreated mDCs, and less pronounced with strong APC such as mDCs. These data are consistent with the hypothesis that iDC have a balance of stimulatory vs inhibitory molecules that favors inhibition, and indicate that PD-L1 and PD-L2 contribute to the poor stimulatory capacity of iDC. PD-L1 expression differs from PD-L2 in that PD-L1 is expressed on activated T cells, placental trophoblasts, myocardial endothelium, and cortical thymic epithelial cells. In contrast, PD-L2 is expressed on placental endothelium and medullary thymic epithelial cells. PD-L1 is also highly expressed on most carcinomas but minimally expressed on adjacent normal tissue suggesting a role in attenuating antitumor immune responses.