Neil Gozzard

The discovery, engineering and characterisation of a highly potent anti-human IL-13 fab fragment designed for administration by inhalation.

We describe the discovery, engineering and characterisation of a highly potent anti-human interleukin (IL)-13 Fab fragment designed for administration by inhalation. The lead candidate molecule was generated via a novel antibody discovery process, and the selected IgG variable region genes were successfully humanised and reformatted as a human IgG γ1 Fab fragment. Evaluation of the biophysical properties of a selection of humanised Fab fragments in a number of assays allowed us to select the molecule with the optimal stability profile. The resulting lead candidate, CA652.g2 Fab, was shown to have comparable activity to the parental IgG molecule in a range of in vitro assays and was highly stable. Following nebulisation using a mesh nebuliser, CA652.g2 Fab retained full binding affinity, functional neutralisation potency and structural integrity. Epitope mapping using solution nuclear magnetic resonance confirmed that the antibody bound to the region of human IL-13 implicated in the interaction with IL-13Rα1 and IL-13Rα2. The work described here resulted in the discovery and design of CA652.g2 human γ1 Fab, a highly stable and potent anti-IL-13 molecule suitable for delivery via inhalation.

Double-stranded RNA evokes exacerbation in a mouse model of corticosteroid refractory asthma.

RNA viruses are a major cause of respiratory infections and are known to exacerbate asthma and other respiratory diseases. Our aim was to test the ability of poly(I:C) (polyinosinic:polycytidylic acid), a viral surrogate, to elicit exacerbation in a model of severe asthma driven by HDM (house dust mite) in FCA (Freunds complete adjuvant). Poly(I:C) was administered intranasally around the HDM challenge in FCA-HDM-sensitized animals. Changes in AHR (airway hyperresponsiveness), BALF (bronchoalveolar lavage fluid) inflammatory infiltrate, HDM-specific immunoglobulins and cytokine/chemokine release were evaluated at different points after the challenge. The effect of oral dexamethasone was also assessed. Exacerbation was achieved when poly(I:C) was administered 24 h before the HDM challenge and was characterized by enhanced AHR and an increase in the numbers of neutrophils, macrophages and lymphocytes in the BALF. Th1, Th2 and Th17 cytokines were also elevated at different time points after the challenge. Peribronchial and alveolar inflammation in lung tissue were also augmented. AHR and inflammatory infiltration showed reduced sensitivity to dexamethasone treatment. We have set up a model that mimics key aspects of viral exacerbation in a corticosteroid-refractory asthmatic phenotype which could be used to evaluate new therapies for this condition.

Efficacy of an Inhaled Interleukin-13 Antibody Fragment in a Model of Chronic Asthma.

Rationale: IL‐13 is an important cytokine implicated in the pathogenesis of allergic asthma and is an attractive target for an inhaled therapeutic. Objective: To investigate the efficacy of CDP7766, a nebulized inhaled anti‐IL‐13 monoclonal antibody Fab fragment, in a model of allergic asthma in cynomolgus macaques naturally sensitized to Ascaris suum. Methods: CDP7766 was nebulized using a vibrating‐membrane nebulizer on the basis of eFlow technology. The aerosol generated was analyzed to determine the particle size profile and the biophysical and functional properties of CDP7766. Nebulized CDP7766 (0.1‐60 mg/animal, once daily for 5 d) was delivered via the inhaled route. Measurements and Main Results: The investigational eFlow nebulizer used in this study generated a respirable aerosol of CDP7766 with no evidence of degradation, loss of potency, aggregation, or formation of particulates. Inhaled CDP7766 was well tolerated in the model (no adverse effects related to local irritation) and significantly inhibited BAL allergen‐induced cytokine and chemokine upregulation (60 mg vs. vehicle: eotaxin‐3, P < 0.0008; MIP [macrophage inflammatory protein]‐1&bgr;, IL‐8, IFN‐&ggr;, P ≤ 0.01). CDP7766 significantly inhibited the increase in pulmonary resistance stimulated by inhaled allergen, measured 15 minutes and 24 hours after allergen challenge. Conclusion: Inhaled CDP7766 potently inhibited the function of IL‐13 generated during the airway response to inhaled allergen in cynomolgus macaques, demonstrating the potential of inhaled anti‐IL‐13 therapeutics for the treatment of allergic asthma.

Poly I:C causes exacerbation in a murine allergic inflammation model driven by house dust mite in Freund's complete adjuvant

Background RNA viruses are major causes of respiratory infections and known to exacerbate asthma and other respiratory diseases. The objective of the study was to use poly I:C, a synthetic analogue dsRNA, to elicit exacerbation in a model of allergic inflammation driven by house dust mite (HDM) in Freund’s Complete Adjuvant (FCA). This model is characterized by airway hyperresponsiveness (AHR) and a mixed T-helper phenotype [1].

Immunological characterisation of an acute murine model of lung inflammation and airway hyperreactivity

Asthma is associated with chronic airway inflammation and airway hyperreactivity (AHR). Approximately 5-10% of the asthmatic patient population suffers from severe disease which remains uncontrolled by current therapies. An acute murine model that replicates aspects of severe asthma was established. Airway inflammation and AHR were elicited by s.c. sensitisation to house dust mite extract (HDM) in complete Freund’s adjuvant (CFA) and a subsequent HDM challenge into the airways (i.n.) 14 days later. The ensuing inflammation comprised of eosinophilia, a marked neutrophilia and a mixed T cell response with significant infiltration of Th1, Th2 and Th17 cells into airways. To investigate the role of these T-helper subsets in this model, the effects of neutralising the key T-helper cell cytokines IFNγ (Th1), IL-4 (Th2) and IL-17 (Th17) were evaluated. Neutralising antibodies to IFNγ, IL-4 and IL-17 were administered prophylactically (s.c), from one day prior to HDM immunisation. AHR to methacholine was assessed by Penh 48 hours post HDM challenge followed by collection of broncho-alveolar lavage (BAL) and serum for analysis of cellular inflammation, cytokines and HDM specific antibodies. IFNγ neutralisation profoundly inhibited AHR yet enhanced BAL eosinophilia, neutrophilia, IL-17 levels and HDM specific serum IgE. Conversely, anti-IL-4 treatment inhibited eosinophil accumulation, IL-13 production and HDM specific serum IgE and IgG1, without affecting AHR. Anti-IL-17 treatment reduced BAL neutrophilia and KC levels, however no significant modulation of AHR, eosinophilia or HDM specific serum IgE and IgG1 was observed. In summary, the T cell cytokines IFNγ, IL-4, and IL-17 each play distinct roles in the development of airway inflammation and AHR in this acute murine model. Furthermore, these data indicate there is dissociation between cellular inflammation and hyperreactivity of the airways in this model. In conclusion, this acute HDM driven model may be representative of the mixed inflammatory response (Th1, Th2 & Th17) reported to be present in the airways of severe asthmatics.