RESUMO
IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL36 cytokines in human atopic dermatitis skin and in IL36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL36 responses represent a key mechanism and potential therapeutic target against allergic diseases.
Assuntos
Dermatite Atópica/imunologia , Imunoglobulina E/imunologia , Interleucina-1/imunologia , Queratinócitos/imunologia , Plasmócitos/imunologia , Staphylococcus aureus/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Dermatite Atópica/genética , Dermatite Atópica/microbiologia , Humanos , Switching de Imunoglobulina , Imunoglobulina E/genética , Interleucina-1/genética , Interleucina-4/genética , Interleucina-4/imunologia , Queratinócitos/microbiologia , Camundongos , Camundongos Knockout , Plasmócitos/patologiaRESUMO
Accurate prediction of the human pharmacokinetics (PK) of a candidate monoclonal antibody from nonclinical data is critical to maximize the success of clinical trials. However, for monoclonal antibodies exhibiting nonlinear clearance due to target-mediated drug disposition, PK predictions are particularly challenging. That challenge is further compounded for molecules lacking cross-reactivity in a nonhuman primate, in which case a surrogate antibody selective for the target in rodent may be required. For these cases, prediction of human PK must account for any interspecies differences in binding kinetics, target expression, target turnover, and potentially epitope. We present here a model-based method for predicting the human PK of MAB92 (also known as BI 655130), a humanized IgG1 κ monoclonal antibody directed against human IL-36R. Preclinical PK was generated in the mouse with a chimeric rat anti-mouse IgG2a surrogate antibody cross-reactive against mouse IL-36R. Target-specific parameters such as antibody binding affinity (KD), internalization rate of the drug target complex (kint), target degradation rate (kdeg), and target abundance (R0) were integrated into the model. Two different methods of assigning human R0 were evaluated: the first assumed comparable expression between human and mouse and the second used high-resolution mRNA transcriptome data (FANTOM5) as a surrogate for expression. Utilizing the mouse R0 to predict human PK, AUC0-∞ was substantially underpredicted for nonsaturating doses; however, after correcting for differences in RNA transcriptome between species, AUC0-∞ was predicted largely within 1.5-fold of observations in first-in-human studies, demonstrating the validity of the modeling approach. Our results suggest that semi-mechanistic models incorporating RNA transcriptome data and target-specific parameters may improve the predictivity of first-in-human PK.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacocinética , Receptores de Interleucina-1/imunologia , Animais , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ratos , Receptores de Interleucina-1/metabolismo , Estudos Retrospectivos , TranscriptomaRESUMO
Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R.
Assuntos
Inflamação/imunologia , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Lisossomos/metabolismo , Psoríase/imunologia , Receptores de Interleucina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Endocitose , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-18/genética , Mutação/genética , Transporte Proteico , RNA Interferente Pequeno/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, ß and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.
Assuntos
Anticorpos Monoclonais/imunologia , Receptores de Interleucina/antagonistas & inibidores , Animais , Especificidade de Anticorpos , Humanos , Camundongos , Psoríase/imunologiaRESUMO
Compound 1 ((4-amino-3,5-dichlorophenyl)-1-(4-methylpiperidin-1-yl)-4-(2-nitroimidazol-1-yl)-1-oxobutane-2-sulfonamido) was discovered to be a 690nM antagonist of human CCR10 Ca2+ flux. Optimization delivered (2R)-4-(2-cyanopyrrol-1-yl)-S-(1H-indol-4-yl)-1-(4-methylpiperidin-1-yl)-1-oxobutane-2-sulfonamido (eut-22) that is 300 fold more potent a CCR10 antagonist than 1 and eliminates potential toxicity, mutagenicity, and drug-drug-interaction liabilities often associated with nitroaryls and anilines. eut-22 is highly selective over other GPCR's, including a number of other chemokine receptors. Finally, eut-22 is efficacious in the murine DNFB model of contact hypersensitivity. The efficacy of this compound provides further evidence for the role of CCR10 in dermatological inflammatory conditions.
Assuntos
Amidas/farmacologia , Dermatite de Contato/tratamento farmacológico , Dinitrofluorbenzeno/toxicidade , Modelos Animais de Doenças , Receptores CCR10/antagonistas & inibidores , Amidas/química , Amidas/uso terapêutico , Animais , Ácidos Carboxílicos/química , Linhagem Celular , Humanos , Camundongos , Relação Estrutura-AtividadeRESUMO
Signal transduction by the IL-36 receptor (IL-36R) is linked to several human diseases. However, the structure and function of the IL-36R is not well understood. A molecular model of the IL-36R complex was generated and a cell-based reporter assay was established to assess the signal transduction of recombinant subunits of the IL-36R. Mutational analyses and functional assays have identified residues of the receptor subunit IL-1Rrp2 needed for cytokine recognition, stable protein expression, disulfide bond formation and glycosylation that are critical for signal transduction. We also observed that, overexpression of ectodomain (ECD) of Il-1Rrp2 or IL-1RAcP exhibited dominant-negative effect on IL-36R signaling. The presence of IL-36 cytokine significantly increased the interaction of IL-1Rrp2 ECD with the co-receptor IL-1RAcP. Finally, we found that single nucleotide polymorphism A471T in the Toll-interleukin 1 receptor domain (TIR) of the IL-1Rrp2 that is present in â¼2% of the human population, down-regulated IL-36R signaling by a decrease of interaction with IL-1RAcP.
Assuntos
Proteína Acessória do Receptor de Interleucina-1 , Subunidade alfa de Receptor de Interleucina-18 , Polimorfismo Genético , Células HEK293 , Humanos , Proteína Acessória do Receptor de Interleucina-1/química , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Subunidade alfa de Receptor de Interleucina-18/química , Subunidade alfa de Receptor de Interleucina-18/genética , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Domínios Proteicos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.
Assuntos
Endocitose/fisiologia , Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Humanos , Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/genética , Transporte Proteico/fisiologia , Receptores de Interleucina/genéticaRESUMO
Molecular K(d) and k(off) parameters are often used to define the molecular potency of drugs. These constants, however, are determined on purified target proteins, and their relationship to in vivo binding phenomena is poorly understood. Herein, we report two novel assays to determine the off-rates of allosteric antagonists from lymphocyte function-associated antigen 1 (LFA-1). The SPR assay involves using the non-blocking mAb TS2/4 to immobilize full-length LFA-1 on a hydrophilic chip surface, and the soluble, native ligand sICAM-1 to probe the fraction of free LFA-1. To determine the fraction of free LFA-1 on cell surfaces, a flow cytometry assay was developed utilizing the fluorophore-labeled Fab R3.1. The R3.1 antibody has been previously demonstrated to block the ability of both ICAM-1 and antagonists to bind to purified and cell-surface LFA-1. The molecular and ex vivo cellular parameters were determined for a set of nine structurally-related LFA-1 allosteric antagonists. The relationships between the parameters determined in the ELISA (K(d)), SPR (k(off)), and flow cytometry (k(off)) assays were shown to be linear with slopes approximately equal to 1, and a correlation analysis showed that the three assay datasets were equivalent at the alpha=0.05 level. These results were unexpected, as the ELISA and SPR assays involve high affinity LFA-1, and the flow cytometry assays involve cell surface LFA-1 in whole-blood, in which a distribution of affinity states would be expected. Nevertheless, the results presented herein show that the K(d) and k(off)'s determined in molecular assays can be used as predictors of LFA-1 receptor occupancy in ex vivo assays.
Assuntos
Moléculas de Adesão Celular/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Imidazolidinas/metabolismo , Integrinas/antagonistas & inibidores , Antígeno-1 Associado à Função Linfocitária/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/fisiologia , Anticorpos Monoclonais/metabolismo , Moléculas de Adesão Celular/química , Citometria de Fluxo , Hidantoínas/química , Hidantoínas/metabolismo , Hidantoínas/farmacologia , Imidazolidinas/química , Imidazolidinas/farmacologia , Integrinas/imunologia , Molécula 1 de Adesão Intercelular/farmacologia , Cinética , Antígeno-1 Associado à Função Linfocitária/química , Reprodutibilidade dos TestesRESUMO
The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.