Gastrointestinal stability of therapeutic anti-TNF α IgG1 monoclonal antibodies

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Abstract

Monoclonal antibodies (mAbs) are highly effective therapeutic agents, administered exclusively by the parenteral route owing to their previously-documented instability in the gastrointestinal (GI) tract when delivered orally. To investigate the extent of the validity of this assumption, the stability of the tumor necrosis factor alpha (TNF-α) neutralizing IgG1 mAbs, infliximab and adalimumab, was studied in human GI conditions. In gastric fluid, infliximab and adalimumab degraded rapidly, with complete degradation occurring within 1 min. In small intestinal fluid, the molecules were shown to be more stable, but nonetheless degraded within a short time frame of 30 min. Investigations into the mechanisms responsible for infliximab and adalimumab instability in the small intestine revealed that the proteolytic enzyme elastase, and to a lesser extent the enzymes trypsin and chymotrypsin, was responsible for their degradation. By contrast, in the human colon, 75% and 50% of the dose of infliximab and adalimumab, respectively, were intact after 60 min, with conversion of mAbs into F(ab’)2 Fab and Fc fragments detected in colonic conditions. These data indicate that therapeutic IgG1 antibodies are more stable in the colon than in the upper GI tract, therefore highlighting the potential for oral delivery of anti-TNF-α mAbs targeted to the colon.

Introduction

Monoclonal antibodies (mAbs) are one of the fastest growing classes of biotherapeutic agents. mAbs are used in the treatment and diagnosis of a wide range of diseases, including cancer, inflammatory and autoimmune disorders. These molecules are administered via the parenteral route, which include intravenous and subcutaneous injection. However, in conditions that afflict the gut, such as inflammatory bowel disease (ulcerative colitis and Crohn’s disease), serious adverse effects have been reported such as lymphoma and non-lymphoma cancers, infections, cardiovascular diseases and lupus, due to the high systemic exposure and immunosupressive effects from the parenteral administration of therapeutic anti-TNF α antibodies (Lin et al., 2008, Scheinfeld, 2004). High systemic exposure can also lead to the generation of anti-drug antibodies which may enhance drug clearance and reduce drug neutralizing ability causing serious hypersensitivity reactions and loss of response (Miheller et al., 2012). Therefore, oral administration may represent a viable therapeutic opportunity by maximizing the local intestinal antibody concentration and TNF-α neutralisation, thereby avoiding the high systemic exposure and immunosupression mediated adverse effects, along with improved patient compliance associated with the oral route of delivery. However, these molecules are considered unsuitable candidates for oral administration because the gastrointestinal (GI) tract presents a formidable array of physical and chemical barriers that are likely to inactivate the molecules (Wang et al., 2007). The first barrier is the stomach where acid and pepsin are responsible for protein breakdown. The small intestine presents various luminally secreted proteases and membrane-bound peptidases including trypsin, α-chymotrypsin and elastase; these enzymes cleave susceptible amino acids in proteins leading to the formation of smaller peptides and constituent amino acids (Smart et al., 2014). Moving into the large intestine, the colon is colonized with large numbers of bacteria (1011–1012 cfu/ml); the metabolic reactions performed by these bacteria and their enzymes have the ability to metabolize drugs far more extensively than any other part of the body (Sekirov et al., 2010, Sousa et al., 2008).

Currently 6 mAbs, which act at different molecular targets along the inflammatory process cascade, have been approved in moderate to severe inflammatory bowel disease (IBD); these molecules are all administered via the parenteral route: infliximab (Remicade®), adalimumab (Humira®), golimumab (Simponi®), certolizumab pegol (Cimzia®), natalizumab (Tysabri®) and vedolizumab (Entyvio®). Infliximab, adalimumab, golimumab and certolizumab pegol target tumor necrosis alpha (TNF-α), natalizumab inhibits α4β1 and α4β7 integrin binding with cell adhesion molecules, while vedolizumab specifically inhibits α4β7 interaction with mucosal addressin cell adhesion molecule protein 1 (MAdCAM-1).

In previous studies, the stability of general immunoglobulins (de Rham and Isliker, 1977, Roos et al., 1995) or IgY immunoglobulin (Hatta et al., 1993) or modified heavy chain antibody variable domains (VHHs) (Hussack et al., 2011), have been evaluated in the presence of individual proteolytic enzymes such as pepsin, trypsin or chymotrypsin. Differences in the stability of different forms of immunoglobulins such as IgA, IgY and IgG towards acidic conditions and proteolytic digestion have also been shown (Blum et al., 1981, Shimizu et al., 1988). IgA was more resistant to proteolysis than IgY and IgG, whereas IgG was more stable to pepsin digestion and less stable to trypsin and chymotrypsin digestion as compared to IgY. The aim of this study is to explore the stability of therapeutic anti-TNF α IgG1 mAbs, infliximab and adalimumab, in human GI fluids, with the aim of establishing their suitability for local delivery in the GI tract.

Section snippets

Materials

Infliximab (Remicade®) and adalimumab (Humira®) were obtained from University College London Hospital NHS foundation trust, London, UK. Infliximab (Remicade®) comes as a 100 mg lyophilized powder to be reconstituted with 10 ml sterile water for injection, while adalimumab (Humira®) comes as a solution for injection (80 mg/1.6 ml). Infliximab is a chimeric IgG1 kappa mAb (molecular weight  149 kDa) composed of human constant region and murine variable region, produced and secreted by mouse myeloma

Results

The stability of infliximab and adalimumab in human gastric fluid (HGF) is shown in Fig. 1. Degradation of both mAbs was rapid and extensive. In simulated gastric fluid (SGF without pepsin), infliximab and adalimumab were completely broken down, confirming that acid, and not pepsin, was responsible for mAb degradation in gastric conditions.

The stability of infliximab and adalimumab in human intestinal fluid (HIF) and simulated intestinal fluid (SIF) with and without pancreatin is shown in Fig. 2

Discussion

The mechanism for the degradation of the mAbs in stomach conditions can be explained by peptide bond hydrolysis: Nucleophilic attack of the Asp side chain to the carbonyl of the preceding residue, leading to cleavage at the N-terminal side of the Asp residue, resulting in a six-membered ring intermediate that is further broken down (Inglis, 1983). Acid-mediated peptide bond hydrolysis of mAbs can also occur by cleavage at the Asp-Lys bond, while the Asn residues which can undergo deamidation

Conclusion

Degradation of infliximab and adalimumab was rapid and extensive in human gastric fluid because of the highly acidic pH of the fluid. In the small intestine, proteolysis was the major cause of degradation leading to fragmentation into Fab and Fc fragments. The mechanism of proteolytic degradation can be attributed to the protease elastase, and to a lesser extent trypsin and chymotrypsin. In the human colonic model, both mAbs displayed high stability, and the observed loss of the intact IgG1

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