Transient Expression of Secretory IgA In Planta is Optimal Using a Multi-Gene Vector and may be Further Enhanced by Improving Joining Chain Incorporation.

Lotte B Westerhof,Ruud H P Wilbers,Debbie R Van Raaij,Arjen Schots,Aska Goverse,Jaap Bakker,Christina Z Van Wijk

doi:10.3389/fpls.2015.01200

Abstract

Secretory IgA (sIgA) is a crucial antibody in host defense at mucosal surfaces. It is a promising antibody isotype in a variety of therapeutic settings such as passive vaccination and treatment of inflammatory disorders. However, heterologous production of this heteromultimeric protein complex is still suboptimal. The challenge is the coordinate expression of the four required polypeptides; the alpha heavy chain, the light chain, the joining chain, and part of the polymeric-Ig-receptor called the secretory component, in a 4:4:1:1 ratio. We evaluated the transient expression of three sIgAκ variants, harboring the heavy chain isotype α1, α2m1, or α2m2, of the clinical antibody Ustekinumab in planta. Ustekinumab is directed against the p40 subunit that is shared by the pro-inflammatory cytokines interleukin (IL)-12 and IL-23. A sIgA variant of this antibody may enable localized treatment of inflammatory bowel disease. Of the three different sIgA variants we obtained the highest yield with sIgA1κ reaching up to 373 μg sIgA/mg total soluble protein. The use of a multi-cassette vector containing all four expression cassettes was most efficient. However, not the expression strategy, but the incorporation of the joining chain turned out to be the limiting step for sIgA production. Our data demonstrate that transient expression in planta is suitable for the economic production of heteromultimeric protein complexes such as sIgA.

Highlights

Secretory IgA is the predominant antibody type in mucosal secretions of the human body and plays a key role in the first line of defense against mucosal pathogens
In order to achieve in vivo assembly of a heteromultimeric protein complex all required genes should be expressed simultaneously in the same cell and preferably at the right stoichiometry
We have studied the plant-based expression and assembly of three Secretory IgA (sIgA) variants of the clinical antibody Ustekinumab (CNTO1275)

Summary

Introduction

Secretory IgA (sIgA) is the predominant antibody type in mucosal secretions of the human body and plays a key role in the first line of defense against mucosal pathogens. The receptor is cleaved and a part of the receptor called the secretory component stays associated with the protein complex that is referred to as secretory (s)IgA Both dIgA and sIgA have immunological roles without development. On the luminal side of epithelial cells glycans on the secretory component facilitate binding to the mucus thereby enabling clearance of sIgAantigen/pathogen complexes. This concept of antigen/pathogen binding and clearance that does not lead to inflammation is referred to as immune exclusion and is believed to play a role in combating mucosal pathogens, and in controlling commensal bacteria. In order to fulfill these roles the human body secretes 40–60 mg sIgA per kg body weight each day (Conley and Delacroix, 1987)

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