[Show abstract][Hide abstract] ABSTRACT: Objective:
Previous work has suggested that the granulocyte macrophage colony stimulating factor (GM-CSF)-GM-CSF receptor α axis (GM-CSFRα) may provide a new therapeutic target for the treatment of rheumatoid arthritis (RA). Therefore, we investigated the cellular expression of GM-CSFRα in RA synovial tissue and investigated the effects of anti-GM-CSFRα antibody treatment in vitro and in vivo in a preclinical model of RA.
We compared GM-CSFRα expression on macrophages positive for CD68 or CD163 on synovial biopsy samples from patients with RA or psoriatic arthritis (PsA) to disease controls. In addition, we studied the effects of CAM-3003, an anti-GM-CSFR antibody in a collagen induced arthritis model of RA in DBA/1 mice. The pharmacokinetic profile of CAM-3003 was studied in naïve CD1(ICR) mice (see online supplement) and used to interpret the results of the pharmacodynamic studies in BALB/c mice.
GM-CSFRα was expressed by CD68 positive and CD163 positive macrophages in the synovium, and there was a significant increase in GM-CSFRα positive cells in patients in patients with RA as well as patients with PsA compared with patients with osteoarthritis and healthy controls. In the collagen induced arthritis model there was a dose dependent reduction of clinical arthritis scores and the number of F4/80 positive macrophages in the inflamed synovium after CAM-3003 treatment. In BALB/c mice CAM-3003 inhibited recombinant GM-CSF mediated margination of peripheral blood monocytes and neutrophils.
The findings support the ongoing development of therapies aimed at interfering with GM-CSF or its receptor in various forms of arthritis, such as RA and PsA.
Annals of the Rheumatic Diseases 06/2014; 74(10). DOI:10.1136/annrheumdis-2014-205234 · 10.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The critical role played by IgE in allergic asthma is well-documented and clinically precedented, but some patients in whom IgE neutralization may still offer clinical benefit are excluded from treatment with the existing anti-IgE therapy, omalizumab, due to high total IgE levels or body mass. In this study, we sought to generate a novel high affinity anti-IgE antibody (MEDI4212) with potential to treat a broad severe asthma patient population. Analysis of body mass, total and allergen-specific IgE levels in a cohort of severe asthmatics was used to support the rationale for development of a high affinity IgE-targeted antibody therapeutic. Phage display technology was used to generate a human IgG1 lead antibody, MEDI4212, which was characterized in vitro using binding, signaling and functional assay systems. Protein crystallography was used to determine the details of the interaction between MEDI4212 and IgE. MEDI4212 bound human IgE with an affinity of 1.95 pM and was shown to target critical residues in the IgE Cε3 domain critical for interaction with FcεRI. MEDI4212 potently inhibited responses through FcεRI and also prevented the binding of IgE to CD23. When used ex vivo at identical concentration, MEDI4212 depleted free-IgE from human sera to levels ~1 log lower than omalizumab. Our results thus indicate that MEDI4212 is a novel, high affinity antibody that binds specifically to IgE and prevents IgE binding to its receptors. MEDI4212 effectively depleted free-IgE from human sera ex vivo to a level (1 IU/mL) anticipated to provide optimal IgE suppression in severe asthma patients.
[Show abstract][Hide abstract] ABSTRACT: How the immune system senses aeroallergens and triggers an aberrant inflammation is poorly understood. Dectin-2 is a house dust mite (HDM)-sensing pattern recognition receptor. In a 3-week mouse model of repeated intranasal HDM challenge, anti-Dectin-2 potently attenuated the characteristic allergic inflammation and airway hyper-responsiveness. Anti-Dectin-2 also prevented neutrophil influx following a single HDM challenge. Interestingly, cysteinyl leukotrienes, but not chemokine and cytokine levels were inhibited by anti-Dectin-2 in this acute model, and in ex vivo challenge of cultured alveolar macrophages with HDM. Furthermore in the single-challenge model, zileuton, an inhibitor of leukotriene production, produced a similar effect as Dectin-2 blockade. Together these data suggest alveolar macrophage sensing of HDM by Dectin-2 elicits the production of cysteinyl leukotrienes, and this axis is key for the initiation of airway inflammation to this aeroallergen. Finally, we found Dectin-2-positive infiltrating cells present in bronchial biopsies from asthmatic subjects.Mucosal Immunology advance online publication, 16 October 2013; doi:10.1038/mi.2013.74.
[Show abstract][Hide abstract] ABSTRACT: Rationale: The relationship between airway inflammation and obesity in severe asthma is poorly understood. Objectives: We sought to determine the relationship between sputum mediator profiles, the distribution of eosinophilic inflammation and obesity in severe asthmatics. Methods: Clinical parameters and 8 mediators in sputum were assessed in 131 severe asthmatic subjects from a single centre categorised into lean, overweight and obese groups defined by their body mass index. In an independent group of severe asthmatics (n=45) and healthy controls (n=19) eosinophilic inflammation was enumerated in bronchial submucosa, blood and sputum and related to their body mass index. Measurements and Main Results: Sputum interleukin (IL)-5 geometric mean [95% confidence interval] (pg/ml) was elevated in the obese (1.5 [1.0-2.3]) compared to overweight (0.9 [0.7-1.1]; p=0.02) and lean (0.7 [0.5-1.0]; p=0.01) asthmatics and was correlated with the body mass index (r=0.29, p<0.001). There was no relationship between body mass index, the sputum cell count or other sputum mediators. In the bronchoscopy group the submucosal eosinophil number in the asthmatic subjects was correlated with body mass index (Spearman's rank correlation rs=0.38, p=0.013) and the median (interquartile range) number of submucosal eosinophils was increased in obese (19.4 [11.8-31.2]) cells/mm2 versus lean subjects (8.2 [5.4-14.6]) (p=0.006). There was no significant association between sputum or peripheral blood eosinophil counts and body mass index. Conclusion: Sputum IL-5 and submucosal eosinophils, but not sputum eosinophils are elevated in obese severe asthmatics. Whether specific anti-eosinophilic therapy is beneficial, or improved diet and lifestyle in obese asthma has anti-inflammatory effects beyond weight reduction, requires further study.
American Journal of Respiratory and Critical Care Medicine 04/2013; 188(6). DOI:10.1164/rccm.201208-1470OC · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background and purpose:
For antibody therapies against receptor targets, in vivo outcomes can be difficult to predict because of target-mediated clearance or antigen 'sink' effects. The purpose of this work was to engineer an antibody to the GM-CSF receptor α (GM-CSFRα) with pharmacological properties optimized for chronic, s.c. treatment of rheumatoid arthritis (RA) patients.
We used an in silico model of receptor occupancy to guide the target affinity and a combinatorial phage display approach for affinity maturation. Mechanism of action and internalization assays were performed on the optimized antibody in vitro before refining the modelling predictions of the eventual dosing in man. Finally, in vivo pharmacology studies in cynomolgus monkeys were carried out to inform the predictions and support future clinical development.
Antibody potency was improved 8600-fold, and the target affinity was reached. The refined model predicted pharmacodynamic effects at doses as low as 1 mg kg(-1) and a study in cynomolgus monkeys confirmed in vivo efficacy at 1 mg kg(-1) dosing.
Conclusions and implications:
This rational approach to antibody drug discovery enabled the isolation of a potent molecule compatible with chronic, s.c. self-administration by RA patients. We believe this general approach enables the development of optimal biopharmaceuticals.
British Journal of Pharmacology 08/2012; 168(1). DOI:10.1111/j.1476-5381.2012.02173.x · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IL-13 is a pleiotropic Th2 cytokine considered likely to play a pivotal role in asthma. Here we describe the preclinical in vitro and in vivo characterization of CAT-354, an IL-13-neutralizing IgG4 monoclonal antibody (mAb), currently in clinical development.
In vitro the potency, specificity and species selectivity of CAT-354 was assayed in TF-1 cells, human umbilical vein endothelial cells and HDLM-2 cells. The ability of CAT-354 to modulate disease-relevant mechanisms was tested in human cells measuring bronchial smooth muscle calcium flux induced by histamine, eotaxin generation by normal lung fibroblasts, CD23 upregulation in peripheral blood mononuclear cells and IgE production by B cells. In vivo CAT-354 was tested on human IL-13-induced air pouch inflammation in mice, ovalbumin-sensitization and challenge in IL-13 humanized mice and antigen challenge in cynomolgus monkeys.
CAT-354 has a 165 pM affinity for human IL-13 and functionally neutralized human, human variant associated with asthma and atopy (R130Q) and cynomolgus monkey, but not mouse, IL-13. CAT-354 did not neutralize human IL-4. In vitro CAT-354 functionally inhibited IL-13-induced eotaxin production, an analogue of smooth muscle airways hyperresponsiveness, CD23 upregulation and IgE production. In vivo in humanized mouse and cynomolgus monkey antigen challenge models CAT-354 inhibited airways hyperresponsiveness and bronchoalveolar lavage eosinophilia.
CAT-354 is a potent and selective IL-13-neutralizing IgG4 mAb. The preclinical data presented here support the trialling of this mAb in patients with moderate to severe uncontrolled asthma.
British Journal of Pharmacology 09/2011; 166(1):177-93. DOI:10.1111/j.1476-5381.2011.01659.x · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF.
miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts.
These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing.
PLoS ONE 06/2011; 6(6):e21253. DOI:10.1371/journal.pone.0021253 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Corresponding author's email: firstname.lastname@example.org Understanding the phenotypic heterogeneity of asthma is likely to shed light upon its immunopathogenesis. Introduction: To examine patterns of cytokine expression in different clinical phenotypes of severe asthma. Rationale: We performed k-means clustering on demographic, lung function, allergen testing and sputum induction data of 164 patients Methods: attending our Difficult Asthma Clinic. Sputum supernatant was analysed for 23 mediators using Meso Scale Discovery platform. The data obtained was reduced using principal component analysis. Factorial patterns of mediator expression were compared across clinical phenotypes. Using unbiased data reduction tools we were able to identify severe asthma clinical sub-phenotypes. Cluster analysis identified 4 Results: clinical phenotypes: A. Discordant high symptoms/non-eosinophilic, obese, normal FEV ‚; B. Late onset, concordant 1 symptoms/eosinophilia, normal FEV ‚; C. Early onset, discordant low symptoms/eosinophilia, low FEV ; D. Early onset, concordant 1 1 symptoms/eosinophilia, normal FEV ‚. The individual mediators did not differ significantly across clusters. Four cytokine factors were 1 identified 1. IL6R, IL8, TNFRI; 2. CXCL11, IL6, CCL5; 3. CCL26, IL13, IL5 and; 4. IL10, IL17, IL4. The factors loaded onto the clusters as shown in the figure below where data are shown as the mean factor loading ± SEM. Graph showing cytokine factor loading across clinical clusters Clinical phenotypes of severe asthma differ in their patterns of mediator expression. The biological basis of clinical disease Conclusion: expression requires further study. Clinical clustering is proving a useful tool at identifying underlying biology. This abstract is funded by: None
American Journal of Respiratory and Critical Care Medicine 05/2011; 183:A3179. DOI:10.1164/ajrccm-conference.2011.183.1_MeetingAbstracts.A3719 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a subchronic exposure model of cigarette smoke (CS)-induced inflammation using antibodies directed against GM-CSF or the GM-CSF receptor (GM-CSFR) α-chain. CS-induced mononuclear and neutrophilic inflammation following 4 days of CS exposure in BALB/c mice was assessed in bronchoalveolar lavage (BAL) fluid. An increase in mature dendritic cells (DCs) (CD11c+ and major histocompatibility complex II+) and Gr-1-high neutrophils was also observed by flow cytometric analysis of whole-lung tissue. Daily i.p. injection of 400 μg GM-CSF or GM-CSFR antibody prior to daily smoke exposure attenuated the accumulation of neutrophils within the BAL by 60%. A reduction in mature DCs was also observed. Anti-GM-CSFR antibody administration did not have an effect on the percentage of lung T-cells; however, a significant decrease in activated CD69+ CD8+ T-cells was observed. Anti-GM-CSFR antibody administration decreased the mRNA and protein expression of interleukin-12 p40 and matrix metalloproteinase 12. Taken together, intervention with this receptor antibody implicates the GM-CSF pathway as an important mediator of smoke-induced inflammation.
European Respiratory Journal 03/2011; 38(2):285-94. DOI:10.1183/09031936.00076210 · 7.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Asthma and COPD are characterized by airway dysfunction and inflammation. Neutrophilic airway inflammation is a common feature of COPD and is recognized in asthma, particularly in severe disease. The T helper (Th) 17 cytokines IL-17A and IL-17F have been implicated in the development of neutrophilic airway inflammation, but their expression in asthma and COPD is uncertain.
We assessed IL-17A and IL-17F expression in the bronchial submucosa from 30 subjects with asthma, 10 ex-smokers with mild to moderate COPD, and 27 nonsmoking and 14 smoking control subjects. Sputum IL-17 concentration was measured in 165 subjects with asthma and 27 with COPD.
The median (interquartile range) IL-17A cells/mm² submucosa was increased in mild to moderate asthma (2.1 [2.4]) compared with healthy control subjects (0.4 [2.8]) but not in severe asthma (P = .04). In COPD, IL-17A(+) cells/mm² submucosa were increased (0.5 [3.7]) compared with nonsmoking control subjects (0 ) but not compared with smoking control subjects (P = .046). IL-17F(+) cells/mm² submucosa were increased in severe asthma (2.7 [3.6]) and mild to moderate asthma (1.6 [1.0]) compared with healthy controls subjects (0.7 [1.4]) (P = .001) but was not increased in subjects with COPD. IL-17A and IL-17F were not associated with increased neutrophilic inflammation, but IL-17F was correlated with the submucosal eosinophil count (rs = 0.5, P = .005). The sputum IL-17 concentration in COPD was increased compared with asthma (2 [0-7] pg/mL vs 0 [0-2] pg/mL, P < .0001) and was correlated with post-bronchodilator FEV₁% predicted (r = -0.5, P = .008) and FEV(1)/FVC (r = -0.4, P = .04).
Our findings support a potential role for the Th17 cytokines IL-17A and IL-17F in asthma and COPD, but do not demonstrate a relationship with neutrophilic inflammation.