Journal of Molecular Biology
Volume 425, Issue 3, 8 February 2013, Pages 577-593
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The Discovery, Engineering and Characterisation of a Highly Potent Anti-Human IL-13 Fab Fragment Designed for Administration by Inhalation

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Abstract

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.

Graphical Abstract

Highlights

► Generation of a highly potent anti-IL-13 antibody designed for inhalation. ► Application of a novel antibody discovery platform. ► Biophysical profiling enabled selection of a robust and stable molecule. ► NMR enabled elucidation of the epitope and the mode of action of the lead candidate.

Introduction

Interleukin (IL)-13 is proposed to be a key mediator of asthma pathogenesis and has been proposed as a therapeutic target for the treatment of this disease.1 IL-13 is implicated in many aspects of asthma pathology. This includes goblet cell metaplasia and mucus hypersecretion,2 IgE production,3 airway fibrosis,4 activation of airway smooth muscle5 and airway hyperreactivity to inhaled spasmogens.6 The majority of the biological effects of IL-13 are thought to be driven through a heterodimeric receptor complex composed of IL-13Rα1 and IL-4Rα subunits. IL-13 initially binds to IL-13R α1 with relatively low affinity; an IL-4Rα subunit is then recruited to form a high-affinity receptor complex capable of signalling through signal transducer and activator of transcription (STAT)-6.[7], [8], [9], [10], [11], [12] IL-13 also binds to a second receptor, IL-13Rα2.13 Although IL-13Rα2 was originally believed to be a decoy receptor, signalling via AP-1 (activator protein-1) in macrophages and induction of transforming growth factor (TGF)-β-mediated fibrosis in vivo have been demonstrated.14 In a recent phase 2 study in moderate to severe asthma, the anti-IL-13 monoclonal antibody lebrikizumab administered subcutaneously was shown to significantly improve lung function forced expiratory volume in one second in patients with elevated serum periostin at baseline.15

We have previously shown that an antibody that directly blocks binding of IL-13 to IL-13Rα1 and IL-13Rα2 (described here as a “bin A” monoclonal antibody) efficiently neutralised the effects of IL-13 in a murine allergic lung inflammation model stimulated by endogenous IL-13.[16], [17] Here, we have used a novel antibody discovery platform combined with functional and stability screening to generate a high-quality anti-human IL-13 bin A monoclonal antibody suitable for therapeutic development. In order to support the potential for local administration via inhaled delivery, we reformatted the antibody as a Fab fragment. The choice of Fab rather than IgG was based on the observation that IgG are more susceptible to degradation during nebulisation in a vibrating membrane nebuliser whereas Fab molecules are highly stable and compatible with such processes (R.P., personal communication).

Although the hybridoma technique described by Kohler and Milstein18 has revolutionised the use of antibodies, due to its reliance on a suitable myeloma fusion partner, only a very small percentage of splenocytes from an immunised animal are immortalised (5 × 10 6 efficiency with conventional polyethylene glycol fusion).19 As a result, the vast majority of B cells are not sampled, and it is possible that rare antibodies, with desirable properties, will not be identified. In this study, we used a novel method whereby large numbers of B cells were cultured directly from immunised animals to induce clonal expansion and antibody secretion,[20], [21] thereby providing a far more effective means to interrogate the immune B cell repertoire. Following screening of B cell culture supernatants for IL-13-reactive antibodies, we applied a fluorescence-based deconvolution method, termed the “fluorescent foci method”, to identify the antigen-specific B cell clones.22 The fluorescent foci method enabled us to identify and micromanipulate single antigen-specific B cells. Antibody variable region genes were then recovered from single cells by reverse transcription (RT)-PCR and subsequently cloned and expressed as recombinant antibody.

The screening and V-region recovery process facilitates the efficient mining of the immune B cell repertoire from an animal. As a result, large and diverse panels of very high affinity antibodies can be readily identified. This then allows further stringent selection criteria to be applied during the screening phase of therapeutic antibody discovery, resulting in the selection of a therapeutic antibody molecule with optimal characteristics.

We applied this novel platform to efficiently interrogate the immune B cell repertoire from a group of immunised rats and generate a large panel of functional anti-human IL-13 antibodies from which to select a lead candidate. Binding to wild-type and human R130Q variant IL-13 (a mutant associated with atopy),23 neutralisation through direct inhibition of receptor binding (bin A), cynomolgus IL-13 cross-reactivity and sub-100 pM affinity were all selection criteria that were applied during the antibody screening process. Twenty-seven unique anti-hIL-13 antibody families were generated, from which a lead antibody candidate, CA652, was selected and successfully humanised and reformatted as a human IgG1 Fab fragment. We screened a number of humanised CA652 Fab grafts, which differed in their variable region amino acid sequences, in a number of biophysical screens in order to select a candidate with the optimal stability profile. The resulting Fab molecule was shown to have retained full activity as compared to the parental molecule in a range of in vitro assays. In addition, Biacore studies demonstrated the ability of the Fab to bind to IL-13 with high affinity (Kd = 8.7 pM) and directly prevent the association of IL-13 with its two receptors, confirming the bin A classification. Epitope mapping using solution nuclear magnetic resonance (NMR) spectroscopy confirmed that the antibody bound to the region of human IL-13 responsible for binding IL-13Rα1 and IL-13Rα2. In particular, it was demonstrated that CA652 binds IL-13 in the region believed to interact with site II of the receptor molecules. This article describes a novel process of antibody discovery, screening, engineering and functional characterisation in order to identify a high-quality antibody Fab fragment that is suitable for therapeutic development and delivery via inhalation.

Section snippets

Immunisations

Sprague–Dawley or Lister hooded rats were immunised with combinations of human IL-13 protein and human IL-13-transfected rat RFL6 fibroblasts. Sera were monitored for binding to antibody-captured rat fibroblast-derived human IL-13 in ELISA and shown to titrate to 1:10,000–1:50,000. Sera were also screened in a STAT-6 reporter assay to determine neutralising titres. All rats demonstrated 100% neutralisation of hIL-13 (Escherichia coli-derived material) at a 1:100 dilution of sera, and

Discussion

In this report, we describe the use of a novel antibody discovery platform in the discovery of a high-quality neutralising anti-human IL-13 antibody. Over a 3-month period, 2700 × 96-well B cell culture plates were screened, equating to over 1 billion B cells from a number of immunised rats. This was possible through the application of a number of automation devices and liquid handlers to facilitate culture setup and screening.22 Following a primary screen to identify binders, we subjected B

Immunisations

Sprague–Dawley or Lister hooded rats were immunised with combinations of 100 μg human IL-13 (PeproTech) and 5 × 107 human IL-13-transfected rat RFL6 fibroblasts. Complete Freund's adjuvant (Sigma Aldrich) was co-administered with the first dose and subsequent protein boosts included incomplete Freund's adjuvant. All animal work complied with the Animals (Scientific Procedures) Act 1986. Sera were monitored for binding to antibody-captured human IL-13 derived from rat RFL6 fibroblasts via ELISA.

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