Developments in the production of mucosal antibodies in plants

JBA-06990; No of Pages 11 Biotechnology Advances xxx (2015) xxx–xxx

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Developments in the production of mucosal antibodies in plants Nikolay Vasilev a, C. Mark Smales b, Stefan Schillberg a, Rainer Fischer a,c, Andreas Schiermeyer a,⁎ a b c

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Department of Plant Biotechnology, Forckenbeckstrasse 6, 52074 Aachen, Germany School of Biosciences, University of Kent, CT2 7NJ Kent, UK RWTH Aachen University, Institute for Molecular Biotechnology, Worringerweg 1, 52074 Aachen, Germany

a r t i c l e

i n f o

Article history: Received 2 September 2015 Received in revised form 17 November 2015 Accepted 24 November 2015 Available online xxxx Keywords: Biopharmaceuticals Immunoglobulin A Immunoglobulin M Plant glycoengineering Plant-made recombinant proteins Secretory antibodies Transgenic plants

a b s t r a c t Recombinant mucosal antibodies represent attractive target molecules for the development of next generation biopharmaceuticals for passive immunization against various infectious diseases and treatment of patients suffering from mucosal antibody deficiencies. As these polymeric antibodies require complex post-translational modifications and correct subunit assembly, they are considered as difficult-to-produce recombinant proteins. Beside the traditional, mammalian-based production platforms, plants are emerging as alternative expression hosts for this type of complex macromolecule. Plant cells are able to produce high-quality mucosal antibodies as shown by the successful expression of the secretory immunoglobulins A (IgA) and M (IgM) in various antibody formats in different plant species including tobacco and its close relative Nicotiana benthamiana, maize, tomato and Arabidopsis thaliana. Importantly for biotherapeutic application, transgenic plants are capable of synthesizing functional IgA and IgM molecules with biological activity and safety profiles comparable with their native mammalian counterparts. This article reviews the structure and function of mucosal IgA and IgM antibodies and summarizes the current knowledge of their production and processing in plant host systems. Specific emphasis is given to consideration of intracellular transport processes as these affect assembly of the mature immunoglobulins, their secretion rates, proteolysis/degradation and glycosylation patterns. Furthermore, this review provides an outline of glycoengineering efforts that have been undertaken so far to produce antibodies with homogenous human-like glycan decoration. We believe that the continued development of our understanding of the plant cellular machinery related to the heterologous expression of immunoglobulins will further improve the production levels, quality and control of post-translational modifications that are ‘human-like’ from plant systems and enhance the prospects for the regulatory approval of such molecules leading to the commercial exploitation of plant-derived mucosal antibodies. © 2015 Elsevier Inc. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IgA and IgM antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Structure and classification of IgA and IgM antibodies . . . . . . . . . . 2.2. Receptors for IgA and IgM molecules . . . . . . . . . . . . . . . . . . 2.3. Biological functions of IgA and IgM molecules . . . . . . . . . . . . . . Plant-based production of IgA variants and analysis of their biological characteristics 3.1. Proof-of- concept: the chimeric murine IgA/G inactivating Streptococcus mutans 3.2. Production of human IgAs in maize . . . . . . . . . . . . . . . . . . .

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Abbreviations: Ara, arabinose; CDR, complementary-determining region; CHO cells, Chinese hamster ovary cells; CMP-Neu5Ac, cytidine 5′-monophosphate N-acetylneuraminic acid; CRISPR/Cas9, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system; DC, dendritic cell; ΔXT/FT, a mutant plant line lacking β1,2xylosyltransferases and α1,3-fucosyltransferases; dIgA, dimeric IgA; ER, endoplasmic reticulum; ETEC, enterotoxigenic Escherichia coli; Fab′, fragment antigen-binding; Fc region, fragment crystallizable region; FcRL, Fc receptor-like proteins; FR1, framework region 1; fwt, fresh weight; Gal, galactose; Gb3, globotriaosylceramide receptor; GlcNAc, N-acetylglucosamine; GnTII, N-acetylglucosaminyltransferase II; GS, N-glycosylation site; HC, heavy chain; HRGP, hydroxyproline-rich glycoproteins; Hyp, hydroxyproline; Ig, immunoglobulin; IgAN, IgA nephropathy; IL, interleukin; J-chain, joining chain; LC, light chain; MALDI MS, matrix-assisted laser desorption/ionization mass spectrometry; pIgR, polymeric immunoglobulin receptor; Pro, proline; PSV, protein storage vacuoles; PTMs, post-translational modifications; SC, secretory component; sIgA, secretory immunoglobulin A; sIgM, secretory immunoglobulin M; SIGNR1 receptor, specific intracellular adhesion molecule-grabbing non-integrin receptor; Stx1, Shiga toxin 1; TALENs, transcription activator-like endonucleases; Thr, threonine; TNF, tumor necrosis factors; TSP, total soluble protein; VHH, variable domains of heavy chain-only antibodies; ZFNs, zinc-finger nucleases. ⁎ Corresponding author at: Fraunhofer IME, Forckenbeckstrasse 6, 52074 Aachen, Germany. E-mail address: [email protected] (A. Schiermeyer).

http://dx.doi.org/10.1016/j.biotechadv.2015.11.002 0734-9750/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Vasilev, N., et al., Developments in the production of mucosal antibodies in plants, Biotechnol Adv (2015), http:// dx.doi.org/10.1016/j.biotechadv.2015.11.002

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3.3. Production of coccidia-specific chicken IgAs in Nicotiana benthamiana . . . . . . . . . 3.4. Production of virus-specific IgAs in tomato and in N. benthamiana . . . . . . . . . . . 3.5. Production of chimeric enterotoxigenic bacteria-specific IgAs in Arabidopsis thaliana seeds 3.6. Production of chimeric toxin-specific IgAs in A. thaliana plants . . . . . . . . . . . . . 3.7. Production of therapeutic IgA antibodies in N. benthamiana . . . . . . . . . . . . . . 4. Plant-based production of IgMs and analysis of their biological characteristics . . . . . . . . . 5. Purification of plant-produced IgAs and IgMs . . . . . . . . . . . . . . . . . . . . . . . . 6. Subcellular compartmentalization of plant-produced IgAs . . . . . . . . . . . . . . . . . . 7. Glycosylation and glycoengineering of IgA and IgM molecules in plants . . . . . . . . . . . . 8. Concluding remarks and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Plant expression systems are emerging as an attractive platform for the production of recombinant pharmaceutical proteins including enzymes, vaccines and antibodies (Fischer et al. 2013; Fischer et al. 2015; Schiermeyer and Schillberg 2012). The cost and difficulties of manufacturing some of the new biotherapeutic molecules in development are among the main driving forces for the increased acceptance of transgenic plants as production hosts of valuable and complex therapeutic proteins (Schiermeyer and Schillberg 2010). This is largely due to the fact that plants can be cultivated at large scale and low cost in the field or in the greenhouse. Although here we review the use of plant systems for the production of IgA and IgM molecules, plant cell culture systems are also now well established whereby cells can be cultured in simple, chemically defined media at a scale to produce recombinant material as an alternative to whole plant systems (Schillberg et al. 2013). Two molecules whose expression has been investigated in plant systems and that have potential commercial biopharmaceutical applications are IgA and IgM antibodies. IgA and IgM are considered difficultto-produce glycoproteins as recombinants (as opposed to IgG molecules where technology for their expression is well established), because they require complex post-translational modifications (PTM) and subunit assembly. IgA and IgM belong to the group of multimeric antibodies. IgA is the most abundant antibody class in humans in terms of the biosynthesis rate. The estimated biosynthetic rate of IgA is 66 mg/kg body weight per day, compared with 34 mg/kg/day and 7.9 mg/kg/day for IgG and IgM, respectively (Manz et al. 2005). However, IgG is the predominant class in serum, making up to 85% of total serum immunoglobulins, followed by monomeric IgA constituting 7–15% of the total and (mainly) pentameric IgM which constitutes approximately 5% of the total immunoglobulins (Manz et al. 2005). The high cumulative biosynthetic rates of IgA and IgM are explained by the large surface of mucosae where both antibody classes dominate. The mucosal surface comprises a vast area of approximately 400 m2 (compared with 1.8 m2 for skin) and represents the major site of attack by invading pathogens (Childers et al. 1989; Woof and Kerr 2006). While the mucosal linings producing IgAs and IgMs provide a physical barrier against infection, additional protection is provided by the mucosal immune system. IgA and IgM play an essential role in the first-line defense at the mucosal surfaces of the gastrointestinal, uro-genital and respiratory tracts and also in the fluids of tears, saliva and milk (Bakema and van Egmond 2011b; Norderhaug et al. 1999). The growing knowledge around the previously neglected polymeric IgA and IgM antibodies as potential biotherapeutics has opened up the possibility of developing these for applications such as mucosal vaccination, treatment of congenital disorders in the mucosal defense and design of a next generation of improved immunotherapeutics (Chintalacharuvu and Morrison 1999; Corthésy 2002; Corthésy 2003; Longet et al. 2013). Recombinant IgAs have now been produced successfully in several expression platforms including mammalian, plant and insect cells and

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transgenic animals (Yoo et al. 2007). The yields from these systems remain low (mg's/L), largely due to their complex assembly and PTM requirements. Plants have been considered as an economical and safe system for production of secretory antibodies due to their scale-up potential and the lack of contaminating mammalian viruses or prions (Chargelegue et al. 2004; Wycoff 2005). An IgM antibody was recently produced for the first time in plants, 20 years after the successful expression of sIgA antibodies in transgenic plants (Loos et al. 2014; Ma et al. 1994). Here, we summarize the advancements in the expression of mucosal antibodies by plant-based systems over this period of time. Specifically, various characteristics of IgA and IgM molecules and their heterogeneous expression are reviewed here, with a particular focus on the expression and assembly, biological activity, intracellular trafficking and glycosylation of both these mucosal antibodies in plants. 2. IgA and IgM antibodies 2.1. Structure and classification of IgA and IgM antibodies The basic monomer units of IgA (~160 kDa) and IgM (~180 kDa), in common with antibodies from other classes of immunoglobulins (Igs), consist of two paired heavy chains (α- and μ-chain for IgA and IgM, respectively) and two light (κ- or λ-) chains, each linked to one heavy chain (Fig. 1). The nature of these linkages is discussed in more detail below. The monomeric structures are arranged into two Fab regions (responsible for antigen recognition) and one Fc region, which mediates interactions with receptors and effector molecules (Woof and Russell 2011). The Fab arms are associated through a hinge region with the Fc region in IgA and IgM (Fig. 1A). IgA exists predominantly in serum as a monomer, whereas at the mucosal surfaces IgA is present mainly as dimeric (Fig. 1B) or polymeric macromolecule forms (Fig. 1C). IgM exists in pentameric or hexameric forms (Fig. 1D) in the secretions and blood circulation and in a monomeric form (Fig. 1A) as antigen receptor on Blymphocytes (Reth 1992; Woof and Kerr 2006; Woof and Mestecky 2005). Each IgA α-chain consists of four domains (starting from the Nterminus: Vα (variable domain), Cα1, Cα2 and Cα3 (constant domains)) while the IgM μ-chain comprises five domains (from Nterminus: Vμ (variable domain), Cμ1, Cμ2, Cμ3, and Cμ4 (constant domains)); each light chain (LC) contains two domains, the VL (variable light) and CL (constant light) domains (Arnold et al. 2005; Woof and Russell 2011). There are two subclasses of IgA in humans, IgA1 and IgA2, which differ in respect to the hinge region that separates the Cα1 and Cα2 domains and the glycosylation patterns. IgA1 possesses a hinge region which is 13 amino acids longer than that found in IgA2 molecules and this hinge insertion provides a flexible stretch and thus the potential for interactions with more distant antigens (Woof and Kerr 2006). The hinge region is rich in amino acid Pro, Ser and Thr residues, resulting in the decoration of this region with three to five, or occasionally six, O-linked oligosaccharides in IgA1 molecules (Tarelli et al. 2004). IgA1 predominates in human serum and airways, while IgA2 is

Please cite this article as: Vasilev, N., et al., Developments in the production of mucosal antibodies in plants, Biotechnol Adv (2015), http:// dx.doi.org/10.1016/j.biotechadv.2015.11.002

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Fig. 1. Schematic representation of the IgA and IgM structures (Arnold et al. 2005; Bakema and van Egmond 2011b; Klimovich 2011; Woof and Kerr 2006; Woof and Russell 2011). A. Monomer structure of IgA and IgM, representing variable and constant domains of the light (blue) and heavy chains (green and magenta for IgA and IgM, respectively). The diagram also shows the Fab and Fc fragments. B. DIgA with J-chain (yellow). C. SIgA with J-chain (yellow) and secretory component (red). D. Pentameric IgM with J-chain (yellow). The cysteines and their positions which are responsible for inter-chain disulfide bridges are depicted by arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

more abundant in the colon; both isoforms are found at similar levels in the small intestine (Pabst 2012; Woof and Russell 2011). IgA2 has been found in at least two allotypic forms, IgA2m(1) and IgA2m(2) (Bakema and van Egmond 2011b; Bonner et al. 2009). The major difference

between these allotypes lies in the nature of the linkage between the heavy and light chains; in IgA2m(2) this linkage is accomplished by disulfide bridges, while the heavy and light chains in IgA2m(1) are bound non-covalently.

Please cite this article as: Vasilev, N., et al., Developments in the production of mucosal antibodies in plants, Biotechnol Adv (2015), http:// dx.doi.org/10.1016/j.biotechadv.2015.11.002

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Dimerization of IgA and pentamerization of IgM is achieved by means of an additional single polypeptide chain (~ 16 kDa), named a joining chain (J-chain). The heavy chains of IgA and IgM possess an 18-amino acid extension in the C-terminus known as the tailpiece, which contains a conserved penultimate Cys residue: Cys471 (Cα) and Cys575 (Cμ). Polymerization is accomplished via a linkage of the IgA or IgM penultimate amino acid from the tailpiece with Cys14 and Cys68 from the J-chain (Chapuis and Koshland 1974; Frutiger et al. 1992; Johansen et al. 2000; Mestecky and Schrohen 1974). N-linked carbohydrates account for 5–8% of the total mass of IgA1 and 6–11% of IgA2 (Tomana et al. 1976). This difference arises because IgA2 has additional sites for N-glycosylation in the Fc and Fab compared with those sites found in IgA1 molecules. Similar to the IgA molecules, the carbohydrate component represents 7–12% of the mass of IgM molecules (Klimovich 2011).

by means of regulatory T cell expansion. The interaction between the sIgA and the DC-SIGN/SIGNR1 receptors is dependent on glycosylation, specifically the presence of mannose residues and of the SC (Diana et al. 2013; Mkaddem et al. 2014). FcRL4 receptors are predominantly expressed by B cells and bind efficiently to IgA. Although, the biological functions of FcRL4 receptors remain unclear, it is suggested that they are involved in the regulation of B cell responses due to their signaling effects and binding circulating antibodies (Wilson et al. 2012). Finally, the recent discovery of a new receptor, FcμR, for IgM has been identified that represents a transmembrane sialoglycoprotein (~ 60 kDa) with an extracellular Ig-like domain which is homologous to two other IgM-binding receptors (pIgR and Fcα/μR) but which demonstrates an exclusive Fcμ-binding specificity (Kubagawa et al. 2009). The role of this binding is yet to be elucidated but it is thought to be involved in promoting B cell survival by an as yet unknown mechanism (Ouchida et al. 2015).

2.2. Receptors for IgA and IgM molecules 2.3. Biological functions of IgA and IgM molecules IgA has been shown to interact with several cellular receptors. Three IgA receptors have been characterized in detail with respect to structure and functionality: pIgR, FcαRI and Fcα/μR. Additional IgA receptors are not so well characterized and include asialoglycoprotein receptors, transferrin receptors (CD71), SC receptors, M-cell receptors, DC-SIGN/ SIGNR1 (CD209) receptors and FcRL4 receptors (Bakema and van Egmond 2011a; Mkaddem et al. 2014; Woof and Russell 2011). The polymeric immunoglobulin receptor (pIgR) is expressed basolaterally on epithelial cells and specifically transports IgA and IgM across the mucosal epithelial cell to the apical (luminal) surface. The Jchain is essential not only for the polymerization of the IgA and IgM molecules, but necessary for binding to pIgR as only polymeric antibodies encompassing a J-chain can bind to the pIgR and thus be transported to the mucosal surfaces (Brandtzaeg and Prydz 1984; Cattaneo and Neuberger 1987; Davis et al. 1988). The pIgR portion, which is located extracellularly and binds to IgA and IgM, consists of five domains. The sixth pIgR extracellular domain contains a cleavage site that leads to the release of the extracellular fragment (~80 kDa), named as the secretory component, either free or bound to sIgA and sIgM (Kaetzel and Bruno 2007). One of the biological roles of the SC is to protect the secretory antibodies against proteolysis and in this manner to extend the half-life of sIgA and sIgM in the enzymatically hostile environments of the mucosal surfaces (Crottet and Corthesy 1998; Russell 2007). FcαRI (also known as CD89) is present on neutrophils, monocytes, eosinophils and on some macrophages and dendritic cells. CD89 binds to the IgA Fc and as a result can induce phagocytosis, antibodydependent cell-mediated cytotoxicity, synthesis and release of cytokines and other inflammatory mediators e.g. activated oxygen species (Morton 2007; Woof and Kerr 2006). Another receptor, Fcα/μR, is a receptor for both IgA and IgM (Shibuya et al. 2000). It has been suggested that Fcα/μR is involved in microbial neutralization (Shibuya et al. 2000) and in the pathologic kidney deposition of IgA-containing immune complexes in IgA nephropathy (IgAN) (Morton 2007). The hepatocyte asialoglycoprotein receptor binds the terminal glycans (Gal or N-acetylgalactosamine residues) of desialylated IgA proteins and delivers its cargo for degradation into the lysosomes of hepatocytes (Stockert et al. 1982). The transferrin receptor (also CD71) selectively binds IgA1 and associates with monomeric forms better than polymeric forms. It is thought that CD71 also plays a role in the pathogenesis of IgAN (Moura et al. 2001). The eosinophil receptor specific for SC mediates the preferential degranulation elicited by sIgA, when compared with serum IgA (Lamkhioued et al. 1995). M-cell receptors are expressed on the apical surfaces of human Peyer's patch M cells and these selectively bind IgA, with or without SC, but not IgG or IgM. M-cell receptors may mediate the trans-cytosis of sIgA and sIgAantigen complexes (Mantis et al. 2002). Secretory IgA can also elicit anti-inflammatory properties through the interaction with DC-SIGN/ SIGNR1 receptors on dendritic cells, which results in immune tolerance

IgA is characterized by heterogeneity regarding its effector functions due to the various molecular forms, subclasses, allotypes, and glycoforms (Russell 2007). The functions of sIgA at mucosal surfaces include suppression of pathogen adherence, neutralization of toxins, enzymes, and viruses, inhibition of antigen penetration and interaction with innate defense factors. In relation to IgA biological activities within the tissues and circulation, the following functions are described: interactions of IgA with the complement system, leukocytes (neutrophils, macrophages, eosinophils, basophils, lymphocytes and dendritic cells) and epithelial cells (Russell 2007). This wide range of potential activities makes these attractive molecules as potential protein based biotherapeutic medicines. IgM is the first antibody isotype that appears during ontogenetic development. In contrast to serum IgA, which is a poor activator of the complement system, the complement activation is considered to be the main effector mechanism of IgM (Klimovich 2011; Nielsen et al. 2000). There are two populations of IgMs: 1) natural antibodies, which appear without external antigen stimuli during prenatal development; these antibodies possess diverse anti-bacterial, anti-viral, anti-tumor activities and contribute to the maintenance of immunological tolerance; 2) IgMs, synthesized by follicle B2-cells after a contact with antigen and T-helpers during the post-natal period, are responsible for the transformation of B2-lymphocytes into mature plasma cells or memory cells with many IgM and IgD expressed on their surfaces (Klimovich 2011). Furthermore, sIgM may compensate IgA deficiency, which for example is a common genetic disorder in the Caucasian population with a prevalence of around ~ 1:500 to 1:700 individuals (Murphy 2012). 3. Plant-based production of IgA variants and analysis of their biological characteristics 3.1. Proof-of- concept: the chimeric murine IgA/G inactivating Streptococcus mutans The first report of the expression of IgA in plants relates to the murine Guy's 13 immunoglobulin that neutralizes S. mutans, the causative microorganism of tooth decay. The plant expression vector contained nucleotide sequences encoding the signal peptide, variable region, Cγ1/Cγ2 constant domains of the heavy chain from the Guy's 13 IgG antibody linked to Cα2 and Cα3 constant domains from the murine IgA (MOPC 315) (Ma et al. 1994). Using this expression system, the following two antibody formats were generated: plant G1/A, consisting of hybrid HCs with var.-Cγ1–Cα2–Cα3 domains and plant G2/A, containing var.-Cγ1–Cγ2–Cα2–Cα3 domains. The hybrid IgA/G antibodies have the ability to assemble J-chain and secretory component due to the presence of Cα2 and Cα3 domains. Furthermore, the plantibody G2/A can be purified by protein A affinity chromatography mediated by

Please cite this article as: Vasilev, N., et al., Developments in the production of mucosal antibodies in plants, Biotechnol Adv (2015), http:// dx.doi.org/10.1016/j.biotechadv.2015.11.002

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Cγ2. Both G1/A and G2/A plantibodies were produced successfully in Nicotiana tabacum (Ma et al. 1994). The transgenic tobacco plants expressing the murine hybrid var.Cγ1–Cγ2–Cα2–Cα3 HCs were first crossed with plants that expressed antibody LCs and then with plants synthesizing a murine J-chain to form a dimeric antibody (dIgA/G). As a next step, the mature dIgA/G plants were crossed with homozygous plants expressing a rabbit SC to yield secretory IgA/G (sIgA/G). The quantitative results showed that the sIgA/G accumulated in transgenic tobacco plants to levels of up to 200–500 μg per g fresh weight (fwt) (Ma et al. 1995), which is comparable with IgG yields obtained in transgenic plants. Importantly, the plant-derived IgAs were shown to be functionally active. G1/A and G2/A plantibodies were able to bind the cell-surface protein (SA I/II) of S. mutans, which is required for the attachment of bacteria to teeth. Single antibody LCs or HCs cannot form a CDR and thus were not able to recognize the surface adhesion antigen. The plant G1/A and G2/A antibodies exerted the same inhibitory potential as the original murine Guy's antibody, which is indicative of competition for the same streptococcal epitope. Both plantibodies led to aggregation of S. mutans which further confirmed their functionality (Ma et al. 1994). The assembly of SC to dIgA/G did not interfere with the antigen recognition. The sIgA/G showed efficient binding to the purified and native SA I/II antigen and the titration curve resembled the pattern of the native murine antibody. The synthesis of the sIgA/G molecule gave higher yields than the plant monomeric IgA/G antibody, which is in agreement with the hypothesis that the secretory antibodies are more resistant to proteolytic degradation (Ma et al. 1995). An additional functional comparison was performed between the sIgA/G originating from transgenic plants and the native murine IgG Guy's 13 using surface plasmon spectrometry to examine the binding to streptococcal SA I/II antigen. The functional analysis demonstrated that there was no significant difference between the affinity and dissociation constants of plant- and hybridoma-derived antibodies. This result implies that the binding activity of the plantibody was not affected by the different glycosylation patterns. However, the sIgA/G from plants showed higher avidity determined by a competition ELISA in comparison with the original Guy's 13 antibody. The murine IgG required approximately four times higher IC50 concentrations in order to inhibit binding to the streptococcal antigen. The difference in functional avidity was attributed to the tetravalent sIgA/G format compared with the IgG bivalence as the dissociation constants were similar (Ma et al. 1998). Plant-produced secretory antibodies were characterized further in vivo. The sIgA/G antibody produced in plants demonstrated longer half-life in the human oral cavity in comparison with the murine IgG Guy's 13: 72 h compared with 24 h, respectively. The plant secretory antibody provided specific protection against oral S. mutans colonization for at least four months, following a treatment course of six applications on days 1, 4, 8, 11, 15 and 18. Further, plant-derived and affinity purified sIgA/G did not manifest in any observable immunogenicity when applied orally. There was no major difference in IgG, IgA or IgM titers of pre- and post-applications of the chimeric sIgA/G. With the dosing/ treatment scheme utilized, topical passive immunization required the antibody yield from 10 to 15 mature transgenic tobacco plants per patient (Ma et al. 1998). 3.2. Production of human IgAs in maize The first human IgAs (hIgA) made in plants were produced in maize. However, these immunoglobulins were lacking a J-chain and SC and thus were not assembled into dimers or sIgAs. Two maize-expressed monomeric IgA1 antibodies, targeted against glycoprotein-D of herpes simplex virus (HX8 hIgA1) and against the sperm agglutination antigen-1 (SAGA-1 antigen, H6-3C4 hIgA1 or “sperm-immobilizing” antibody) were designed to explore the O-glycosylation in the hinge region of the antibody HCs (Karnoup et al. 2005). The analysis

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demonstrated that proline residues in the hinge region were converted to hydroxyproline and subsequently decorated with arabinose to different degrees. 3.3. Production of coccidia-specific chicken IgAs in Nicotiana benthamiana The first transient expression of IgA in plants was achieved in N. benthamiana. Ten individual phage display clones were successfully expressed in plants to produce antibodies against Eimeria acervulina (Wieland et al. 2006), the causative agent of coccidiosis, which leads to significant economic losses in poultry farming. To test the ability of plant cells to assemble chicken secretory IgA antibodies, combinations of Agrobacterium tumefaciens cultures harboring chicken light chains and α-HCs were infiltrated with and without J-chain and/or secretory component. This study demonstrated that plants can serve as a suitable heterologous expression system of fully-assembled avian antibodies, reaching about 1.5% of the TSP content (Wieland et al. 2006). 3.4. Production of virus-specific IgAs in tomato and in N. benthamiana Transgenic edible fruits may represent an interesting strategy for the development of passive immunization although some open issues with respect to batch to batch variations, precise analytical control and exact dosing schemes still need to be addressed (Arntzen 2015). Furthermore, to avoid the contamination of the natural food supply with transgenic fruits, Antirrhinum majus Rosea1 and Delila genes, responsible for anthocyanin biosynthesis, were expressed in addition to the heterologous proteins. Thus, purple-colored and therefore transgenically labelled tomato fruits were grown, expressing a recombinant human IgA1 against the VP8* peptide of the rotavirus VP4 spike protein (strain SA11). The amount of IgA antibody reached 3.6 ± 0.8% of TSP in the fruit of the transformed plants. Consequently, the resulting purple-colored extracts from the transgenic fruits demonstrated anti-rotavirus prophylactic potential in combination with increased amounts of health-promoting (antioxidant) activity due to the anthocyanin content (Juarez et al. 2012). The binding activity of the plant-produced IgA1 against rotavirus SA11 was assayed by ELISA plates coated with the VP8* peptide. Endpoint antigen–ELISA titration showed that anti-VP8* activity was maintained in late ripening fruits as well as in tomatoes after minimal processing, although the formation of Fab′ fragments derived by proteolysis of the IgA molecule within the hinge region has been observed. The rotavirus neutralization of tomato-produced IgA was characterized further by immunofluorescent focus reduction assays on monolayers of MA104 cells, which were infected by the rotavirus SA11 strain (Asensi et al. 2006). Though the IC50 values of purified and unpurified plantderived IgA were very similar, crude preparations, when applied at low dilutions, were more effective in rotavirus SA11 inhibition than equivalent concentrations of purified IgA. Purple tomato fruit samples, expressing IgA and anthocyanins (Rosea1 and Delila transgenes) also neutralized rotavirus potently, with a calculated IC50 value of 1.98 μg/ml of IgA (Juarez et al. 2012). A combinatorial approach was followed to optimize sIgA production in plants, using the GoldenBraid multigene assembly system to generate combinations of α1 and α2 HC and λ and κ LC with or without ER retention motifs. All 16 versions of a human sIgA against the VP8* rotavirus antigen were tested for accumulation in N. benthamiana. It was observed that those sIgA versions carrying HCα1 and λ-LC chains and a KDEL motif for ER retention attached to the SC yielded the highest expression levels of 32.5 μg IgA/g fwt (Juarez et al. 2013). 3.5. Production of chimeric enterotoxigenic bacteria-specific IgAs in Arabidopsis thaliana seeds IgA antibodies have also been accumulated successfully in the seeds of A. thaliana. Passive mucosal immunization may prevent the development of enteric infections. Therefore, anti- enterotoxigenic Escherichia

Please cite this article as: Vasilev, N., et al., Developments in the production of mucosal antibodies in plants, Biotechnol Adv (2015), http:// dx.doi.org/10.1016/j.biotechadv.2015.11.002

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coli-(ETEC)-antibodies were created by fusing variable domains of llama heavy chain-only antibodies against ETEC to the Fc part of a porcine immunoglobulin IgA. VHH–IgAs were coexpressed with a porcine J-chain and SC to produce monomeric (bivalent), dimeric (tetravalent) and secretory (tetravalent) LC-devoid IgA formats. Furthermore, the hinge region in porcine IgA consisted of two amino acids (Asp-Pro) and thus were resistant to protease degradation after peroral administration. The VHH–IgA yield reached approximately 0.2% of seed weight (Virdi et al. 2013). Young mammalian animals become vulnerable to gastrointestinal infections after weaning because their passive mucosal gastrointestinal immunity gained by lacteal uptake is diminished or lost (Hurley and Theil 2011). Post-weaning diarrhea is a common gastrointestinal infection in piglets caused by ETEC strains, carrying F4 fimbriae (F4+ ETEC). It has been shown that the simplified LC-devoid VHH–immunoglobulins can agglutinate all F4+ ETEC bacterial strains in multivalent format. In vitro analysis of antibody-containing seed extracts showed that inseed-made anti-F4+ ETEC antibodies can render 50% or more specific inhibition of bacterial adhesion to porcine gut villous enterocytes. Monomeric, dimeric and secretory VHH–IgA formats exerted 59%, 66% and 61% inhibition, respectively. A 20 piglet group treated with 20 mg VHH–IgA per day per pig showed an elimination of F4+ ETEC in only three days post-challenge in comparison with 8-days post-challenge bacterial shedding within the control group (fed with wild-type Arabidopsis seeds). Successful passive immunization was further demonstrated by reduced levels of serum anti-F4+ ETEC antibodies and an increased rate of weight gain in the VHH–IgA piglet group after bacterial challenge (Virdi et al. 2013). Overall, these experimental results highlighted the potential application of recombinant antibody expression in crop seeds for oral passive immunization of economically important livestock animals, thus preventing the development of antibiotic resistance due to the intensive application of antibiotics in animal husbandry (Adjiri-Awere and Van Lunen 2005). 3.6. Production of chimeric toxin-specific IgAs in A. thaliana plants A hybrid IgG/IgA antibody has been expressed in A. thaliana plants against Shiga toxin (Stx) from the enterohemorrhagic E. coli O157:H7 strain, which causes a serious clinical condition characterized by watery diarrhea, hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome. The hybrid plantibody has variable regions of a Stx1Bspecific IgG antibody fused to IgA heavy chain constant regions to enable the production of dimeric IgA molecules (Nakanishi et al. 2013). The plant-produced dimeric IgA form neutralized Shiga toxin 1 in a dosedependent manner. The dimeric form of the hybrid IgG/IgA antibody was produced in A. thaliana leaves and binds to the B subunit of Stx1 and thus inhibited the toxin adhesion to the cell surface through the Gb3 receptor. This plantibody was also able to prevent Stx1-induced cytotoxicity through inhibition of apoptosis. The hybrid IgG/IgA produced in plants appeared to prevent Vero cell death via two anti-apoptotic mechanisms: caspase-3 suppression and inhibition of phosphatidyl serine translocation (annexin V binding) (Nakanishi et al. 2013). 3.7. Production of therapeutic IgA antibodies in N. benthamiana Monomeric IgA1κ variants of three commercial therapeutic antibodies, Infliximab (anti-TNFα), Adalimumab (anti-TNFα) and Ustekinumab (anti-IL12) have now also been successfully expressed transiently in N. benthamiana. The production in planta of the IgA1κ antibodies was compared with the production of their IgG1κ counterparts. This comparison did not reveal a wide heterogeneity between the IgA1κ and IgG1κ antibodies as stated in previous reports, concerning antibody quantity and quality between isotypes and idiotypes. The quantitative analysis showed that the expression of all six antibodies was in the range from 3.5% to 9% of total soluble protein. However, the IgG

antibodies were expressed at a higher, albeit a small increase, expression level in plants. Furthermore, the monomeric IgAs appeared to be more prone to proteolysis compared with the IgG counterparts (Westerhof et al. 2014). The antigen-binding capacity of the plant-produced IgA variants of Infliximab, Adalimumab and Ustekinumab antibodies was also assessed by two cell-based assays. The viability of fibroblast L929 cells was determined when exposed to TNFα in combination with various concentrations of plant-produced Infliximab and Adalimumab. L929 cell viability was enhanced in a dose-dependent manner as a result of the suppressed TNFα-related apoptosis by the plant IgA and IgG versions of Infliximab and Adalimumab. Interestingly, no major difference was reported in terms of TNFα-induced apoptosis between antibodies produced in murine myeloma cell (SP2/0) and plant-made Infliximab and Adalimumab IgA and IgG antibodies, despite the fact that plant-derived antibodies possess enriched oligomannose-type N-glycosylation. The ability of Ustekinumab to inhibit the p40 subunit of IL-12 and IL-23 was also examined by measuring the IL-23-induced production of IL-17 by murine splenocytes. IL-17 production was reduced dose dependently when exposed to plant-made IgG and IgA Ustekinumab. Additionally, no significant difference was observed regarding the IL-17 inhibition between Ustekinumab IgA and IgG forms with murine or plant origin (Westerhof et al. 2014). The broadly neutralizing anti-HIV human 2G12 sIgA was successfully produced in both stably transformed N. tabacum plants and transiently transformed N. benthamiana plants. The 2G12 sIgA complexes were not secreted into the apoplast but retained in intracellular compartments, including the vacuole. Maximum antibody yields were reported with 15.2 μg/g leaf fwt for transgenic plants and 25 μg/g leaf fwt for transient expression, whereas the assembly of sIgA complexes was superior in transgenic tobacco. Interestingly, plant-produced 2G12 sIgA, but not IgG, effectively aggregated HIV virions in vitro. Binding of 2G12 sIgA to the DC-SIGN receptor was observed. However, sIgA did not interact with FcαRI on a monocyte cell line indicating that the secretory component prevents the sIgA molecule from binding. Furthermore, 2G12 sIgA demonstrated improved stability in mucosal secretions in comparison with its IgG counterpart (Paul et al. 2014). 4. Plant-based production of IgMs and analysis of their biological characteristics IgM is among the most challenging human protein targets for recombinant expression due to its large size, complex assembly requirements and post-translational modifications. The anti-cancer PAT-SM6 antibody was reported as the first multimeric IgM produced in plants (Loos et al. 2014). This research focused upon investigating the impact of glycosylation on heteromultimeric IgM oligomerization and on the biological properties of IgMs. The model IgM was expressed transiently in N. benthamiana in three different plant lines: 1) wild-type plants; 2) ΔXT/FT plant line, lacking the plant-specific α1,3-fucose and β1,2xylose residues in the N-glycan part; and 3) plant line conferring human-type N-glycosylation with terminal sialic acid residues. Overall, the antibody yield reached up to 84 μg purified IgM per gram of infiltrated leaf. Various glycoforms (SM6wt, SM6ΔXF or SM6sia) of the anti-tumor IgM PAT-SM6 produced in plants and in PER.C6 cells were examined for antigen binding by flow cytometry using the human lung cancer cell line A549 as a target. Fluorescence-cell sorting analysis showed identical antigen-binding properties between mammalian and all three plant-derived IgM versions (Loos et al. 2014). 5. Purification of plant-produced IgAs and IgMs Alongside the expression of the complex IgA and IgM molecules different purification procedures have been investigated for their recovery. Initially, purification methods developed for microbial and mammalian protein production hosts were investigated to establish novel plant-

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specific purification approaches (Wilken and Nikolov 2012; Yoo et al. 2007). However, the purification of IgAs and IgMs is hampered by the inability to purify such molecules using conventional protein A or protein G affinity chromatography. Therefore, a new, one-step purification process was developed, using protein L affinity chromatography, to purify fullyassembled recombinant sIgA macromolecules from CHO cells (Moldt et al. 2014). Protein L is an Ig-binding protein from Peptostreptococcus magnus that interacts with framework region 1 (FR1) present in the variable region of certain kappa light chain subtypes (κ-LC subtypes) and therefore binds to representatives of all antibody classes, including IgG, IgM, IgA, IgE, and IgD (Nilson et al., 1992). Since protein L does not bind to lambda light chains (LC(λ)) nor to certain human and murine κ-LC, a grafting approach was employed for the purification of plantderived IgA by the replacement of the FR1 residues in the κ-LC variable domain with corresponding residues that can bind protein L (Boes et al. 2011). The grafted κ-LC chains were assembled with the IgA2 HCs to form a mouse/human chimeric antibody, which recognizes human chorionic gonadotropin. This grafting method did not affect antigen-binding characteristics, as shown by surface plasmon resonance spectrometry, or the yields of the chimeric plantibody in transiently transformed tobacco plants. The approach facilitated downstream processing of plant-made IgA through protein L purification resulting in the recovery of more than 50% of highly pure antibodies demonstrating that grafting could be used as a generic strategy to enable the purification of IgA molecules from plants (Boes et al. 2011; Paul et al. 2014). Other affinity reagents derived from microbes with affinity to IgA molecules comprise peptide M from Streptococcus pyogenes (Sandin et al. 2002) and the SSL7 (staphylococcal superantigen-like) protein from Staphylococcus aureus (Langley et al. 2005). Furthermore, small (14 kDa) single domain [VHH] camelid-derived antibodies (CaptureSelect®) have been developed for the specific interaction with the Fc part of IgA or IgM molecules (Hermans et al. 2014; Reinhart et al. 2012). These reagents have been used for purification of human IgA from recombinant and plasma sources but should be adaptable for plant-derived dimeric and secretory IgA molecules as well. 6. Subcellular compartmentalization of plant-produced IgAs Plant cells tend to deliver the heterologously expressed glycoproteins extracellularly. However, aside from the presence of the signal peptide that directs the polypeptides to the secretory pathway, the secretory route can be diverted and the synthesized proteins are then stored in the vacuoles, particularly if the target proteins possess vacuolar sorting signals. Indeed, a study of sIgA/G assembly in plant cells established that the secretion of the target recombinant antibody was slow and the secreted fraction represented only approximately 10% of all newly synthesized immunoglobulin molecules after 24 h. The majority of the sIgA/G and its breakdown products were transported to the vacuolar compartment whereas the IgG tetrameric molecule with native γ-HCs was secreted rapidly and efficiently (Frigerio et al. 2000). The observation that sIgA/G is delivered to/stored in vacuoles raises questions around the mechanisms and recognition events leading to vacuolar transport, instead of following the default secretory pathway. The C-terminal 18 amino acids of the additional constant Cα3 domain in the HC of IgA/G are required for binding of the J-chain. The truncation of this tailpiece led to the abolishment of vacuolar targeting. The unassembled LCs were efficiently transported extracellularly as monomers. Therefore, it was concluded that the cryptic vacuolar sorting signal is located in the tailpiece of the hybrid γ/α chain (Hadlington et al. 2003). The storage cells of transgenic rice endosperm were also used to investigate the intracellular fate of Guy's 13 secretory IgA with respect to the assembly and subcellular localization of heteromultimeric protein complexes. The non-assembled HC, LC and SC were accumulated within the ER-derived protein bodies and protein storage vacuoles (PSVs) where their content varied significantly. The assembled antibody was retained predominantly in the PSVs (Nicholson et al. 2005). It was

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shown that the secretion levels of IgG1κ-based Adalimumab and Ustekinumab were approximately three-fold higher than the secretion levels for the IgA counterparts while Infliximab was poorly secreted irrespectively of isotype. An IgA Ustekinumab variant without the αmotif (IgA-UΔT) resembling the murine IgA tailpiece was created, resulting in a two-fold increase in the secretion efficiency. The IgAbased Adalimumab (IgA-A) and Ustekinumab (IgA-U) idiotypes displayed higher secretion yields than IgA- Infliximab (IgA-I) (Westerhof et al. 2014). Finally, although there is little known about the secretion of plant-derived IgMs produced in plant cells, such information and understanding can now be gathered due to the landmark work describing the first expression of functionally active heteromultimeric IgM in plants (Loos et al. 2014).

7. Glycosylation and glycoengineering of IgA and IgM molecules in plants The rough ER, Golgi apparatus, secretory vesicles and vacuoles are intracellular organelles that form the endomembrane system of the eukaryotic cell. The proteins transported through the endomembrane system undergo elaborate PTMs, which may influence the physicochemical stability, pharmacokinetic properties and biological activity of recombinant therapeutic proteins (Liu et al. 2006; Sola and Griebenow 2009; Yoo et al. 2014). Protein glycosylation is among the most abundant of PTMs and glycoproteins produced by other host organisms may act as epitopes in the human body leading to undesired immune reactions (Altmann 2007; Bardor et al. 2003; Gomord et al. 2010; Jin et al. 2008; Yoo et al. 2014). Therefore, glycoengineering approaches to tailor glycosylation profiles present an opportunity to minimize the immunogenic potential of plant-derived glycoproteins (Gomord and Faye 2004; Gomord et al. 2010; Kaulfurst-Soboll et al. 2011; Paulus et al. 2011). Mucosal antibodies produced in plants undergo similar glycosylation steps and processing to the majority of endogenous proteins. Protein glycosylation in plants can be classified into two types: N- or Oglycosylation (Fig. 2). Both N- and O-linked types of glycosylation are reported in the literature for plant-derived IgA and IgM antibodies (Karnoup et al. 2005; Loos et al. 2014; Westerhof et al. 2014). Nlinked glycosylation occurs at asparagine (Asn) residues in the consensus sequence motif Asn-X-Ser/Thr, where X is any amino acid except Pro. O-linked glycosylation is less studied in plants because of the absence of a common consensus sequence for O-linked glycosylation and due to the lack of a clear understanding of O-glycan biological function (Karnoup et al. 2005; Van den Steen et al. 1998; Yoo et al. 2014). Nglycosylation is initiated co-translationally in the ER with subsequent processing steps being carried out in the Golgi apparatus (Castilho and Steinkellner 2012). The hydroxylation of proline represents the initial O-glycosylation step, which is likely to take place in the ER and/or in the Golgi apparatus and then the overall process of O-glycan synthesis is continued and completed in the Golgi system (Karnoup et al. 2005; Nguema-Ona et al. 2014; Shpak et al. 2001). N-glycan analysis of IgAs produced in N. benthamiana shows the glycan structures to consist of mainly complex and a small fraction of oligomannose-type glycans (Westerhof et al. 2014). IgAs expressed in plants lack one terminal N-acetylglucosamine (GlcNAc) and core α1,3-fucose compared with plant-derived IgGs, which possess typical biantennary GnGnXF3 structures for secreted proteins. Therefore, plant-made IgA-A, IgA-U and IgA-UΔT display prevailingly two Nglycan types: MGnX (or GnMX) and GnGnX (for detail on N-glycan nomenclature and abbreviations, refer to http://www.proglycan.com). IgA-I produced in plant cells displayed predominantly Man7 and Man8 types of N-glycan. IgA-based Infliximab also had lower secretion levels compared with the IgA versions of Adalimumab and Ustekinumab, a further indication of ER retention of the heterologously expressed protein that may explain the presence of oligomannosidic type N-glycosylation IgA-I. Consequently, these data show that the N-

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glycosylation of plant-produced antibodies can also be influenced by their idiotype (Westerhof et al. 2014). The glycan analysis of 2G12 sIgA showed predominantly high mannose structures present on most of the N-glycosylation sites of 2G12 sIgA with limited evidence for complex glycosylation or paucimannosidic forms (Paul et al. 2014). O-glycan structures were not revealed in this study. N-glycans bearing α1,3-fucose and β1,2-xylose residues represent important plant-specific carbohydrate epitopes which may provoke unwanted immunogenicity (Altmann 2007; Jin et al. 2008; Loos and Steinkellner 2014; van Ree et al. 2000; Yoo et al. 2014). Furthermore, plants lack terminal sialylation, which for human-like N-glycosylation is required for the optimal therapeutic effect of recombinant glycoprotein drugs (Castilho et al. 2010). A combined glycoengineering strategy was applied to the expression of the first plant-produced IgM antibody (SM6) (Loos et al. 2014). IgM produced in wild-type plants (SM6wt) showed a similar glycosylation pattern to human derived counterparts purified from serum (IgMhs) or produced in PER.C6 cells (SM6PER): the glycosylation sites GS 1-3 (Asn171, Asn332 and Asn395) carried complex type N-glycans (GnGnXF and MGnXF) and the C-terminal GS4 (Asn402) and GS5 (Asn563) were decorated with oligomannosidic structures (Loos et al. 2014). An RNA interference approach was applied to knock down the mRNA of the unwanted endogenous glycosyltransferases in N. benthamiana: β1,2-xylosyltransferase and α1,3-fucosyltransferase (ΔXT/FT plant line) (Strasser et al. 2008). The transient expression of IgM in a ΔXT/FT plant line (SM6ΔXF) led to the detection of glycan structures with terminal GlcNAc but lacking β1,2-xylose and core α1,3-fucose residues (GnGn). Furthermore, human glycosyltransferase GnTII was coexpressed to achieve elongation of truncated mannosidic arms (MGn(XF)) with GlcNAc residues, resulting in more glycan species with fully processed arms i.e. GnGn(XF) (Loos et al. 2014; Schneider et al. 2014). Additional steps were undertaken in order to produce IgM in N. benthamiana with human-like glycosylation: (i) expression of the entire pathway for the biosynthesis of the sialic acid precursor CMPNeu5Ac, (ii) transportation of CMP-Neu5Ac to the trans Golgi apparatus, and (iii) overexpression of a modified version of human β1,4galactosyltransferase (GalT) to produce terminal Gal in plants which is recognized as an acceptor substrate for the subsequent sialylation. The final sialylation step i.e. the transfer of CMP-Neu5Ac for β1,4-linkage with terminal Gal, was achieved by the introduction of the sialyltransferase enzyme (Castilho et al. 2010; Loos et al. 2014). The overall glycoengineering work resulted in the synthesis of disialylated (NaNa) and monosialylated (MNa and ANa) glycans on GS1-3 of plant-made IgM (Loos et al. 2014). Approximately 90% of the conserved hinge region of maizeexpressed IgA1 antibody was modified by either Pro/Hyp conversion(s), which may be explained by the remarkable amino acid similarity to the Pro-rich repetitive regions of the extensin-family of HRGPs in maize. Hence, it is likely that the conserved hinge region of maize-produced IgA1 may be a substrate for Pro/Hyp conversion by maize prolylhydroxylase(s) with the successive arabinosylation of the Hyp monosaccharide units. Up to six Pro/Hyp conversions and up to 10 arabinose units bound to each Hyp residue were detected in the hinge region of maize-expressed IgA1 HC. However, it was not possible to determine

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unambiguously the positions of all Pro/Hyp conversions (Karnoup et al. 2005). 8. Concluding remarks and future perspectives Substantial progress has been made in recent years towards the production of mucosal antibodies in plants. For instance, a human-like Nglycosylation pattern has been achieved via glycoengineering although mucin-type O-glycosylation still remains a challenge for the plant cell. However, the manufacturing of recombinant antibodies in intact plants or plant cell cultures is still in its infancy. Further development of our understanding of the limitations in plants to produce commercially viable amounts of human-like IgA and IgM molecules needs to be undertaken to generate a system that can provide IgA and IgM products for the therapeutic market. Potential commercialization requires consolidation of production platforms and further increases and optimization of the production yield and improvement of downstream processing to deliver a process with competitive costs to other systems (Fischer et al. 2015). Regulatory considerations are a further important issue because plant-produced recombinant proteins are referred to as “biosimilars” i.e. they differ from the original pharmaceutical product in terms of the host organism and the manufacturing process (Fischer et al. 2012). Therefore, additional clinical studies may be required to address some specific aspects such as immunogenicity (Zuniga and Calvo 2009), resulting from modified PTMs in the new host or due to the presence of host-cell proteins from the plant in purified products. N-type glycoengineering of the plant cell machinery may help achieve similar-to-human IgAs and IgMs and thus reduce possible immunogenic responses. The recent advancements in genome editing tools such as ZFNs, TALENs or the CRISPR/Cas9 system (Lozano-Juste and Cutler 2014) may enable the design of plant-derived mucosal antibodies with improved physicochemical, pharmacokinetic and pharmacodynamic properties, termed “biobetters”, comprising also proline hydroxylation, Hyp-O-glycosylation and lipidation modifications (Webster and Thomas 2012). Acknowledgements The funding from the 4-th ERA-NET ERA-IB2 framework project INNOVATE (Project Nos. 031A339 (to AS) and BB/M000699/1 (to CMS)) is gratefully acknowledged. References Adjiri-Awere, A., Van Lunen, T.A., 2005. Subtherapeutic use of antibiotics in pork production: risks and alternatives. Can. J. Anim. Sci. 85, 117–130. Altmann, F., 2007. The role of protein glycosylation in allergy. Int. Arch. Allergy Immunol. 142, 99–115. Arnold, J.N., Wormald, M.R., Suter, D.M., Radcliffe, C.M., Harvey, D.J., Dwek, R.A., et al., 2005. Human serum IgM glycosylation: identification of glycoforms that can bind to mannon-binding lectin. J. Biol. Chem. 280, 29080–29087. Arntzen, C., 2015. Plant-made pharmaceuticals: from ‘edible vaccines’ to Ebola therapeutics. Plant Biotechnol. J. 13, 1013–1016. Asensi, M.T., Martinez-Costa, C., Buesa, J., 2006. Anti-rotavirus antibodies in human milk: quantification and neutralizing activity. J. Pediatr. Gastroenterol. Nutr. 42, 560–567. Bakema, J.E., van Egmond, M., 2011a. The human immunoglobulin A Fc receptor FcαRI: a multifaceted regulator of mucosal immunity. Mucosal Immunol. 4, 612–624.

Fig. 2. Glycosylation of mucosal antibodies produced by plants along the secretory pathway, based on currently available literature (Castilho and Steinkellner 2012; Castilho et al. 2010; Frigerio et al. 2000; Karnoup et al. 2005; Loos et al. 2014; Nicholson et al. 2005; Westerhof et al. 2014; Yoo et al. 2014). Dashed arrows depict hypothetical biosynthetic steps; the arrow thickness reflects the flux intensity. Detected N-type glycans in the plant-made mucosal antibodies: 1 — (M6M2–3)M2–2; 2 — (M6M3)M2–2; 3 — (M2–6M2–3)M2–2; 4 — MGnX; 5 — MGnXF; 6 — GnGnF; 7 — GnGn; 8 — GnGnX; 9 — GnGnXF; 10 — ANa; and 11 — NaNa. Used abbreviations in the legend: Dol-PP: dolichol diphosphate; OST: oligosaccharyltransferase complex; GCSI: α1,2-glucosidase; GCSII: α1,3-glucosidase; MNS3: α1,2-mannosidase; EDEM: ER degradation-enhancing alpha-mannosidase-like protein; GMI: Golgi α1,2-mannosidase; GMII: Golgi α1,2/α1,6-mannosidase; XylT: β1,2-xylosyltransferase; GnTII: β1,2-N-acetylglucosaminyltransferase; GnTII*: overexpressed GnTII from A. thaliana; FucTa: α1,3-fucosyltransferase; FucTb: α1,3-fucosyltransferase; GalTa: β1,3-galactosyltransferase; GalTb: β1,4-galactosyltransferase; Hexo 1 and Hexo3: β-N-acetylhexosaminidases; ST: α2,6-sialyltransferase; Δ — silenced gene; UDP-GlcNAc: uridine diphosphate-N-acetylglucosamine; ManNAc-6-P: N-acetylmannosamine-6-phosphate; ATP: adenosine triphosphate; NeuAc-9-P: N-acetylneuraminate 9-phosphate; PEP: phosphoenolpyruvate; CTP: cytidine triphosphate; CMP-Neu5Ac: cytidine 5′-monophosphate N-acetylneuraminic acid; GNE: UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine kinase; NANS: N-acetylneuraminic acid phosphate synthase; NANP: N-acetylneuraminic acid phosphate phosphatase; CMAS: CMP-N-acetylneuraminic acid synthetase; CST: CMP-Neu5Ac trans-Golgi transporter; PHL: prolyl-hydroxylase and HGT: Hyp-glycosyltranserases, needed for O-type glycosylation of extensin-like glycoproteins (i.e. the hinge region of IgA1).

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