IgA as a potential candidate for enteric monoclonal antibody therapeutics with improved gastrointestinal stability

Abstract

Mucosal surfaces of the gastrointestinal tract play an important role in immune homeostasis and defense and may be compromised by enteric disorders or infection. Therapeutic intervention using monoclonal antibody (mAb) offers the potential for treatment with minimal off-target effects as well as the possibility of limited systemic exposure when administered orally. Critically, to achieve efficacy at luminal surfaces, mAb must remain stable and functionally active in the gastrointestinal environment. To better understand the impact of isotype, class, and molecular structure on the intestinal stability of recombinant antibodies, we used an in vitro simulated intestinal fluid (SIF) assay to evaluate a panel of antibody candidates for enteric mAb-based therapeutics. Recombinant IgG1 was the least stable following SIF incubation, while the stability of IgA generally increased upon polymerization, with subtle differences between subclasses. Notably, patterns of variability within and between mAbs suggest that variable regions contribute to mAb stability and potentially mediate mAb susceptibility to proteases. Despite relatively rapid degradation in SIF, mAbs targeting Enterotoxigenic Escherichia coli (ETEC) displayed functional activity following SIF treatment, with SIgA1 showing improved function compared to SIgA2. The results of this study have implications for the design of enteric therapeutics and subsequent selection of lead candidates based upon in vitro intestinal stability assessments.

Keywords: Immunoglobulin A; Intestinal stability; dIgA; sIgA.

Fig. 1

Stability of antibodies in SIF is mediated by isotype and isomerization. IgG and monomeric, dimeric, and secretory IgA were incubated with SIF and tested for antigen-specific binding over multiple timepoints by ELISA, with representative results for each antibody shown.

Fig. 2

Antibodies expressed as SIgA demonstrate improved stability in SIF compared to IgG. SIF ½max concentrations mAbs1-3 IgG and mAbs1-4 SIgA are shown. Samples with ½ Max concentrations >1 μg/mL (shown by a dotted line) were outside of the assay limits and are not displayed, however these samples were assigned a value of 1 μg/mL for purposes of analysis.

Fig. 3

Antibody half-life is differentially impacted by isotype and multimerization state. Normalized T50% for each mAb was determined as the mean time in minutes for a 50% reduction in immunoreactivity relative to a positive control. Antibodies that displayed >50% immunoreactivity over the course of the assay were assigned a T50% of 120 min for purposes of analysis and are shown on the graphs for clarity. Significance across mAbs for a given construct is shown at the top of each graph. Due to the small sample size for mAb3 dIgA2 (N = 2), dIgA2 was not analyzed.