RESUMO
Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a ß-arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [3H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6+ peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases.
Assuntos
Quimiocinas CC , Interleucina-23 , Humanos , Animais , Camundongos , Quimiocinas CC/genética , Receptores CCR7 , Ligantes , Linfócitos T , Inflamação , Receptores CCR6RESUMO
Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton's tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.
Assuntos
Linfócitos B/imunologia , Glomerulonefrite/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Piperidinas/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/uso terapêutico , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Centro Germinativo/citologia , Glomerulonefrite/metabolismo , Glomerulonefrite/prevenção & controle , Rim/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/prevenção & controle , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NZB , Piperidinas/farmacologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Proteínas Tirosina Quinases/metabolismo , Pirazóis/farmacologia , Receptores Fc , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
PURPOSE: Study the impact of CXCL13 neutralization on germinal center (GC) response in vivo, and build quantitative relationship between target coverage and pharmacological effects at the target tissue. METHODS: An anti-CXCL13 neutralizing monoclonal antibody was dosed in vivo in a T-dependent mouse immunization (TDI) model. A quantitative site-of-action (SoA) model was developed to integrate antibody PK and total CXCL13 levels in serum and spleen towards estimating target coverage as a function of dose. To aid in the SoA model development, a radio-labeled study using [I(125)] CXCL13 was conducted in mice. Model estimated target coverage was linked to germinal center response using a sigmoidal inhibitory effect model. RESULTS: In vivo studies demonstrated that CXCL13 inhibition led to an architectural change in B-cell follicles, dislocation of GCs and a significant reduction in the GC absolute numbers per square area (GC/mm(2)). The SoA modeling analysis indicated that ~79% coverage in spleen was required to achieve 50% suppression of GC/mm(2). The 3 mg/kg dose with 52% spleen coverage resulted in no PD suppression, whereas 30 mg/kg with 93% coverage achieved close to maximum PD suppression, highlighting the steepness of PD response. CONCLUSIONS: This study showcases an application of SoA modeling towards a quantitative understanding of CXCL13 pharmacology.
Assuntos
Anticorpos Neutralizantes/farmacologia , Quimiocina CXCL13/imunologia , Centro Germinativo/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Feminino , Centro Germinativo/imunologia , Centro Germinativo/ultraestrutura , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ratos , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T/efeitos dos fármacosRESUMO
Development of long-term humoral immunity, characterized by the formation of long-lived plasma cells (PCs) in the bone marrow and memory B cells, is a critical component of protective immunity to pathogens, and as such it is the major goal of vaccination. However, the mechanisms involved in the generation of long-term humoral immunity remain poorly understood. In this study, we used IL-21R-deficient (IL-21R.KO) mice to examine the role of the IL-21 pathway in the development of the B cell memory response. Primary IgG serum Ab responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenylacetyl (NP) hapten conjugated to chicken γ globulin were delayed in IL-21R.KO mice, but reached normal titers within 3 to 4 wk of immunization. IL-21R.KO mice formed germinal centers and generated normal numbers of PCs in their bone marrow. Additionally, memory B cell formation was similar in wild-type and IL-21R.KO mice. However, NP-specific memory B cells and PCs failed to expand following secondary immunization of IL-21R.KO mice, and consequently, secondary IgG Ab responses to NP hapten conjugated to chicken γ globulin were significantly impaired. These results identify the IL-21 pathway as a critical component of the memory B cell response.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Memória Imunológica , Receptores de Interleucina-21/fisiologia , Animais , Antígenos de Superfície/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Subpopulações de Linfócitos B/citologia , Diferenciação Celular/genética , Galinhas/imunologia , Células Dendríticas Foliculares/imunologia , Células Dendríticas Foliculares/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Haptenos/administração & dosagem , Haptenos/imunologia , Imunização Secundária , Memória Imunológica/genética , Memória Imunológica/imunologia , Antígenos Comuns de Leucócito/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/administração & dosagem , Nitrofenóis/imunologia , Fenilacetatos/administração & dosagem , Fenilacetatos/imunologia , Receptor de Morte Celular Programada 1 , Receptores CXCR5/biossíntese , Receptores de Interleucina-21/deficiência , Receptores de Interleucina-21/genética , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , gama-Globulinas/administração & dosagem , gama-Globulinas/imunologiaRESUMO
C-C motif chemokine receptor-like 2 (CCRL2) is a non-signaling 7 transmembrane receptor that binds chemotactic ligands to shape leukocyte recruitment to sites of inflammation. However, there is a lack of consensus on the ligands that directly bind CCRL2 or their functional impact. Studies with CCRL2 knockout mice have demonstrated that neutrophils have impaired degranulation and migration in response to CXCL8, where the underlying molecular mechanism is proposed to be due to the formation of CCRL2 heterodimers with the chemokine receptor CXCR2. Herein, we characterized the ligands that bind directly to CCRL2 and interrogated the impact of CCRL2 neutralization on CXCL8 signaling in neutrophils using pharmacological antibody tools. Using flow cytometry and Surface Plasmon Resonance microscopy (SPRm) cell binding experiments, we confirmed that chemerin, but not previously reported C-C chemokines, binds CCRL2. Furthermore, we identified human and mouse CCRL2 antibodies that neutralized chemerin binding to CCRL2. Unexpectedly, we found that neutralization of CCRL2 with these antibodies did not attenuate CXCL8-induced human neutrophil degranulation nor CXCL8-induced murine neutrophil recruitment to the peritoneum. Based on the observed differences in modulating CCRL2 function with neutralizing antibodies compared to the reported CCRL2 deficient murine models, we hypothesize that the ligand binding function of CCRL2 is dispensable for CXCL8 signaling in neutrophils. Finally, extensive profiling of CCRL2 expression on peripheral blood leukocytes revealed monocytes, dendritic cells (DC), and subpopulations of natural killer T (NKT) cells as additional targets, highlighting potential roles for CCRL2 in human cell types beyond neutrophils that warrants future investigation.
Assuntos
Ativação de Neutrófilo , Receptores CCR , Humanos , Animais , Camundongos , Receptores CCR/metabolismo , Anticorpos Neutralizantes/farmacologia , Ligantes , Transdução de Sinais , Interleucina-8 , Neutrófilos/metabolismoRESUMO
OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.
Assuntos
Artrite Experimental/tratamento farmacológico , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Isoquinolinas/uso terapêutico , Lactamas/uso terapêutico , Macrófagos/efeitos dos fármacos , Doenças Reumáticas/tratamento farmacológico , Sinoviócitos/efeitos dos fármacos , Animais , Artrite Experimental/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Isoquinolinas/farmacologia , Lactamas/farmacologia , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Camundongos , Ratos , Doenças Reumáticas/imunologia , Sinoviócitos/imunologiaRESUMO
Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in V(L) CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Fas(lpr) mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC(0-infinity)) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2-3-fold or 4-7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Fas(lpr) mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.
Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Lúpus Eritematoso Sistêmico/terapia , Receptores de Interleucina-21/antagonistas & inibidores , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/imunologia , Células Cultivadas , Regiões Determinantes de Complementaridade/genética , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Injeções Subcutâneas , Interleucinas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Endogâmicos , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-21/imunologiaRESUMO
Development of long-term humoral immunity is a major goal of vaccination, but the mechanisms involved in the formation of long-term Ab responses are still being determined. In this study, we identify a previously unknown requirement for MyD88, an adaptor molecule that mediates signals at most TLRs, for the generation of long-term humoral immunity during live virus infection. Polyoma virus-infected MyD88 knockout mice generated strong acute T cell-dependent antiviral IgM and IgG responses and developed germinal centers. Activation-induced cytidine deaminase, an enzyme required for isotype switching and somatic hypermutation, was also induced in germinal center B cells, similar to wild-type mice. However, MyD88 knockout mice failed to develop bone marrow plasma cells and did not maintain long-term serum antiviral Ab responses. The isotype distribution of antiviral IgG responses was also altered; serum IgG2a and IgG2b levels were diminished, whereas IgG1 responses were not affected. The requirement for MyD88 for the formation of long-term humoral immunity to polyoma virus was intrinsic to B cells and was independent of IL-1R and IL-18R, cytokine receptors that also signal through MyD88. Our findings show that MyD88-dependent signaling pathways in B cells are essential for effectively generating long-term Ab responses and implicate a role for TLR in the formation of long-term humoral immunity.
Assuntos
Anticorpos Antivirais/sangue , Fator 88 de Diferenciação Mieloide/fisiologia , Infecções por Polyomavirus/imunologia , Animais , Linfócitos B/imunologia , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-1/fisiologia , Receptores de Interleucina-18/fisiologia , Transdução de Sinais , Linfócitos T/imunologia , Receptores Toll-Like/fisiologia , Carga ViralRESUMO
Pollen tube growth is a polarized growth process whereby the tip-growing tubes elongate within the female reproductive tissues to deliver sperm cells to the ovules for fertilization. Efficient and regulated membrane trafficking activity incorporates membrane and deposits cell wall molecules at the tube apex and is believed to underlie rapid and focused growth at the pollen tube tip. Rab GTPases, key regulators of membrane trafficking, are candidates for important roles in regulating pollen tube growth. We show that a green fluorescent protein-tagged Nicotiana tabacum pollen-expressed Rab11b is localized predominantly to an inverted cone-shaped region in the pollen tube tip that is almost exclusively occupied by transport vesicles. Altering Rab11 activity by expressing either a constitutive active or a dominant negative variant of Rab11b in pollen resulted in reduced tube growth rate, meandering pollen tubes, and reduced male fertility. These mutant GTPases also inhibited targeting of exocytic and recycled vesicles to the pollen tube inverted cone region and compromised the delivery of secretory and cell wall proteins to the extracellular matrix. Properly regulated Rab11 GTPase activity is therefore essential for tip-focused membrane trafficking and growth at the pollen tube apex and is pivotal to reproductive success.
Assuntos
Flores/crescimento & desenvolvimento , Nicotiana/citologia , Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Parede Celular/metabolismo , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nicotiana/fisiologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Plant Rac-like GTPases have been classified phylogenetically into two major groups-class I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen- and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP-AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction.