1. Introduction
Most of the monoclonal antibodies being approved or in clinical trials are of the IgG isotype [
1,
2]. Production technologies and purification protocols are well established, and regulatory agencies are acquainted with relevant safety issues. However, new antibody formats such as IgA are gaining more and more interest [
3,
4,
5]. Out of all antibody classes, the most abundant isoform in the human body is IgA [
6]. Three molecular forms of IgA exist: monomeric, dimeric and secretory IgA, each with distinct characteristics and functions in the immune system. Secretory IgA constitutes a first line of mucosal defense against invading pathogens. It is a heavily glycosylated multimeric protein consisting of two monomeric IgA molecules covalently linked by the joining J-chain (JC) and the secretory component (SC), which wraps around the antibody complex and confers resistance to proteolytic degradation, along with protection in low pH environments [
7]. Being adapted to mucosal secretions means SIgA has an intrinsic advantage as a potential therapeutic targeting mucosal pathogens compared to other antibody classes. In airway infections, SIgA has been shown to neutralize influenza viruses and prevent virus-induced pathology in the upper respiratory tract, performing at times better than IgG [
8,
9]. This is likely to be due to its ability to bind antigens with high avidity on the mucosal surface, interact with mucins via the SC and prevent adherence to the epithelium, which is a process called immune exclusion [
7,
10].
Passive immunization would involve the topical administration of pathogen-specific SIgA directly to the oral, nasal, respiratory or gastro-intestinal mucosa to enhance the protective mucosal barrier. The respiratory application of SIgA therapy was highlighted against COVID-19 with monoclonal SIgAs showing higher neutralization against SARS-CoV-2 compared to IgG antibodies [
11,
12]. With functional efficacy and applications against a wide range of mucosal pathogens, it is understandable why SIgA emerges as an attractive candidate in the growing field of antibody therapy. However, several factors must be addressed before SIgA fulfills its therapeutic potential. An important step would be to identify an optimal IgA constant region scaffold on which to build SIgA antibodies with different specificities.
In nature, immunoglobulin A exists in several isotypes, namely IgA1 and IgA2, of which the latter exists in two common allotypes (IgA2m(1), IgA2m(2)). The major differences between IgA1 and IgA2 are located in the hinge region, with IgA2 lacking the 13-amino acid elongation with up to six O-glycosylation sites in IgA1 (
Figure 1). Furthermore, IgA1 has two N-glycosylation sites in the CH2 domain and the tailpiece. IgA2m(1) has additional N-glycosylation sites (Asn166 in the CH1 and Asn 337 in the CH2 domain). IgA2m(2) has an additional N-linked glycan at Asn211 in the CH1 domain [
13,
14]. These additional glycan sites create a greater proinflammatory response of neutrophils and macrophages [
15]. The IgA2m(1) allotype lacks a covalent disulfide bond between heavy and light chains, which is present in IgA1 and in the IgA2m(2) allotype. Previous studies have shown that the presence of P221 in IgA2m(1) interferes with the formation of a heavy and light chain (HL) disulfide bond in the absence of C133. In this allotype, only heavy-chain dimer (H2) molecules are being efficiently formed with only small quantities of HL and H2L2 molecules present in secretions [
16,
17].
In recombinant antibodies, IgA1 showed better bivalent binding of special separated epitopes due to its extended flexible hinge region and increased neutralization of pathogens, while IgA2 was more effective in inducing effector functions and is more stable in terms of bacterial degradation at mucosal surfaces. This is believed to be due to the lack of the extended hinge region, which is susceptible to proteolytic degradation [
15,
18,
19]. However, the unusual noncovalent linkage of the light chain and heavy chain pairing in the IgA2m(1) allotype may result in other production and stability concerns [
14]. Stability needs to be addressed for SIgAs during expression, formulation and in terms of proteolytic degradation at the site of application. Regarding the latter, aerosolization is an attractive means for delivery to the upper respiratory tract, so conformational and thermal stability are important considerations. Engineering the IgA heavy chain to reduce sensitivity to bacterial proteases and environmental pH is also necessary. A potential strategy in antibody engineering is the introduction of covalent bonds in the form of disulfide bridges. For example, the introduction of a P221R mutation was previously demonstrated in CHO cell-produced IgA2m(1) to sterically allow the formation of a new disulfide bond between the heavy (Cys-220) and the light chain cysteines (Cys-214) [
20].
The complexity of recombinant production of secretory IgA will drive the cost, which determines their viability as therapeutic products. Due to the need to transcribe and assemble four components, SIgA production is already a complex multi-step process which has proved challenging in different protein production systems. Attempts to produce SIgA in mammalian cells have resulted in only modest success [
21,
22,
23]. Plants have emerged as an attractive platform for SIgA production, enabling complete assembly in planta without the need for in vitro processes. This method has achieved promising yields of up to 100 mg/kg of leaf fresh weight [
12]. However, none of the available sequences are optimal either due to expression yields, low thermal stability, or high susceptibility to bacterial proteases. Here, we compare four different IgA2 versions with IgA1 to further develop secretory IgA antibodies against SARS-CoV-2 for topical delivery to mucosal surfaces.
3. Discussion
While there are many possible advantages of IgA in antibody therapy, there are several issues that need to be overcome. These include the efficiency of expression, production, purification, and complete assembly of recombinant IgA monoclonal antibodies with appropriate homogeneity as well as stability during topical delivery. In IgA2, while the shorter hinge may restrict the movements of the Fab regions to access antigens [
27], it provides a functional advantage by being resistant to bacterial IgA1 proteases. This may explain why IgA2 is more abundant in the intestinal secretions where most of the bacteria reside. IgA1 may suffer stability issues in vivo, particularly at bacterially colonized sites. Additionally,
O-glycosylation is typically diverse and difficult to control during biomolecule production, which limits regulatory and safety experience [
28]. Importantly, if applied in serum, aberrantly hypogalactosylated natural IgA1 antibodies are critically involved in the development of IgA nephropathy, which is a common cause of renal failure [
29].
Traditionally, IgA2m(1) has been described as being an antibody lacking covalent bonds between H and L, while IgA1 and IgA2m(2) have covalent HL disulfides. Previous studies have shown that this is not strictly true [
16]. For IgA2m(1), some HL disulfide bonds do form, albeit only inefficiently, suggesting that in IgA2m(1), the cysteine residue involved in forming the HL disulfide is partially accessible, but the majority of the molecules fail to form this bond. Also, lambda light chain rather than kappa light chain is better at forming these disulfide bridges [
20]. Additionally, in IgA2m(1), a significant amount of the noncovalent L chains are dimers, suggesting that the noncovalent L chains assume different orientations in IgA2m(1) and IgA2m(2). Thus, IgA2m(2) with covalently linked H and L chains may be more stable than IgA2m(1) in the milieu of the mucosal secretions with varying pH and salt concentrations.
We therefore decided to modify IgA2m(1), using two well-characterized SARS-CoV-2 specific antibodies, COVA2-15 and 2E8, that express well in plants [
12]. We introduced a cysteine at position 133, which is found in the CH1 domain of IgA1 and is responsible for disulfide bonding with the LC. For another variant, a different mutation in the CH1 domain of the IgA2 heavy chain from proline to arginine at position 221 was made, which has already been described previously [
16,
20]. Finally, we introduced a further mutation in IgA2m(1)_P221R to introduce the
N-glycosylation site at position Asn-212 in the CH1 to generate the common IgA2m(2) allotype.
The mutant IgA2m(2)_D133C was able to form disulfide bridges with the light chain, which was probably due to the proximity of C133 in the constant HC and C214 in the constant LC region [
27,
30]. However, expression in
N. benthamiana plants was markedly reduced, and conformational and structural stability were significantly impaired. The mutant IgA2m(2)_P221R sterically enables the formation of an alternate disulfide bridge between the heavy and light chains in the IgA2m(1) allotype, which was similar to the IgA2m(2) allotype. Our results show that a single P221R amino acid exchange derived from the IgA2m(2) sequence is sufficient to prevent dissociation into heavy and light-chain homodimers in monomeric and secretory IgA. Introduction of the P221R mutation enhanced the expression levels of monomeric IgA reaching reported values of the respective IgG counterpart in plants (
Figure 2) [
12]. Furthermore, the mutation enhanced the stability of the protein and increased expression levels in plants. Surprisingly, adding an additional mutation to introduce the CH1 resident
N-glycosylation site to generate mIgA2m(2) resulted in a significant reduction in expression in plants. A similar poorer expression of IgA2m(2) as well as assembly into multimeric IgA compared to other allotypes in plants has been reported previously [
12,
31,
32,
33].
The expression of multimeric SIgA is more complex than monomeric IgA, but it has been successfully reported for SIgA1 and SIgA2m(1) in plants [
12]. Generally, a higher capacity for assembly into polymers in planta can be observed for IgA1 rather than IgA2m(1) [
12,
31]. The introduction of mutation P221R has positive effects on the assembly efficacy of kappa LC antibodies but not those with lambda light chains. However, the overall expression levels of fully assembled SIgA2m(1)_P221R increased compared to SIgA2m(1), reaching those of SIgA1, and the presence of free light chain dimers could not be detected.
In addition to achieving high accumulation levels, ensuring the stability of mAbs is paramount for the successful development of topical delivery systems in therapeutic applications [
12,
34]. Topical delivery to the respiratory tract through aerosolization, such as using the widely available Omron MicroAir nebulizer, has been explored [
12]. However, the complex multimeric structure of antibodies, especially SIgA, adds further intricacy to the formulation process. Concerns regarding thermal denaturation during aerosolization highlight the importance of assessing thermal stability during preclinical development to ensure the viability of mAbs under different conditions. Prior studies have demonstrated that monomeric IgA1 and IgA2m(2) exhibit notable thermal stability with the unfolding of fully assembled secretory IgA remaining unexplored. However, the thermal unfolding of mIgA2m(1) is distinguished by a broad endotherm, signifying the onset of unfolding at temperatures approximately 6 °C lower than those observed for other allotypes. While glycosylation status is important for thermal stability, the formation of disulfide bridges between the L and H chain is even more critical [
26]. This was reflected in the thermal stability determined here by differential scanning fluorometry, where κLC-mIgA2m(1) in particular showed a much reduced
Tm compared to IgA1. With the introduction of further disulfide bonds between the L and H chain, thermal stability of not only monomeric but also secretory IgA2 improved drastically.
The topical administration of mAbs via oral delivery also holds potential. Challenges include the susceptibility of mAbs to degradation by gastric acid and proteases [
35]. Higher robustness in acidic conditions combined with the reports of higher resistance of SIgA against gastric enzymes like pepsin due to its unique structure highlights why SIgA is well suited for mucosal applications [
36]. This was also observed here, where under highly acidic conditions, SIgA1 and particularly stability engineered SIgA2_P221R displayed much improved midpoint temperatures of unfolding compared to IgG mAbs.
The increased thermal stability of engineered IgA2m(1) also translated into a better recovery of fully functional IgA after aerosolization. When formulated in 1xPBS, only 40% of the total protein was recovered. This was associated with a significant loss of protein and/or activity except for stability engineered monomeric and secretory IgA2m(1)_P221R. However, no aggregates were detectable in the condensate. The loss could be mostly reversed by adding 0.05% Tween-20 (Polysorbate-20) to the antibody preparation. While formulation seems to be the key determinant for successful aerosolization, stabilizing mutations make IgA more robust for topical delivery using less complex formulations.
In conclusion, while IgA holds promise for monoclonal antibody therapy, overcoming challenges in expression, purification, and stability is imperative. The distinctive structural features of IgA, such as its hinge region and glycosylation patterns, present both advantages and complexities in therapeutic development. Our study highlights the importance of understanding the structural nuances of different IgA allotypes, such as IgA2m(1) and IgA2m(2), in order to engineer antibodies with improved stability and functionality. Through targeted mutations, such as the P221R substitution, we have demonstrated enhanced stability and assembly efficacy, particularly in the context of secretory IgA. Furthermore, our results underscore the importance of formulation optimization in facilitating successful aerosolization for topical delivery with stabilizing mutations proving to be instrumental in enhancing the resilience of IgA antibodies under varying conditions. Ultimately, our study contributes to advancing the understanding and development of IgA-based therapeutics for mucosal applications, offering promising avenues for combating infectious diseases and other mucosal disorders.
4. Materials and Methods
4.1. Construct Design and Cloning
Heavy-chain sequences of human gamma-1 (AAA02914.1), alpha-1 (AAT74070.1) or alpha-2m(1) (AAT74071.1) constant regions were cloned together with a human Ig heavy chain leader sequence (‘MDMRVPAQLLGLLLLWLPGARC’) separated by a BsaI type II restriction site into pDONR-based plasmids and have been previously described [
12]. Similarly, constant human lambda and kappa light chain were cloned together with the human light chain leader sequence (‘MDMRVPAQLLGLLLLWLPGARC’) also separated by a BsaI type II restriction site into pDONR. Site-directed mutagenesis of the pDONR-alpha-2m(1) scaffold to generate pDONR-alpha-2m(1)_D133C, pDONR-alpha-2m(1)_P221R and pDONR-alpha-2m(2) was performed with the primers described in
Table S8 using the QuickChange kit II XL Site-Directed Mutagenesis Kit (Agilent, Santa lara, CA, USA).
Nicotiana benthamiana codon-optimized sequences for the heavy and light-chain variable regions of COVA2-15 (QKQ15273.1, QKQ15189.1), COVA1-22 (QKQ15169.1, QKQ15253.1), 2-15 (PDB: 7L57_H, 7L57_L) and 2E8 IgG mAbs were costume synthesized by GeneArt (Thermo Fisher Scientific, Waltham, MA, USA) and flanked with BsaI type II restriction sites as previously described [
12,
37,
38]. Using Golden Gate assembly, the variable heavy-chain sequences were cloned into the pDONR-based heavy chain scaffold plasmids. Variable light-chain fragments of COVA2-15 were inserted into human kappa constant region pDONR scaffolds (AAA58989.1) and COVA1-22, 2-15 and 2E8 were inserted into lambda constant region pDONR scaffolds (CAA40940.1) [
23]. Full-length heavy and light-chain genes were separately subcloned into the binary high expression vector pEAQ-HT-DEST3 using gateway cloning [
39]. Human secretory component (SC) and joining chain (JC) constructs cloned separately into pEAQ-HT have been described previously [
23]. The pEAQ-HT plant expression vectors containing the gamma and alpha heavy chains as well as the kappa and lambda light chains were transformed into
Agrobacterium tumefaciens strain GV3101 (Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, DSM 12364) by electroporation.
The construct for expression of the receptor-binding domain (RBD) of the SARS-CoV-2 spike (PDB: 6VYB, R319-F541) with a C-terminal 6xHis-tag cloned into pCAGGS mammalian expression vector has been described previously [
12].
4.2. Transient Expression of IgG and IgA Variants in N. benthamiana
Agrobacterium strains carrying the relevant constructs were cultured overnight at 28 °C in Lysogeny Broth (LB) supplemented with 25 µg/mL rifampicin and 50 µg/mL kanamycin. For the expression of IgG or monomeric IgA1 and IgA2 variants, the overnight cultures containing the respective constructs for the heavy and light chains were diluted in infiltration buffer (10 mM MES, 10 mM MgSO
4, and 0.1 mM acetosyringone) to achieve an optical density at 600 nm (OD
600) of 0.1. For secretory IgA variants, the heavy and light-chain constructs were diluted to an OD
600 of 0.05. They were then mixed with the joining chain construct at an OD
600 of 0.2 and the secretory component construct at an OD
600 of 0.1. Subsequently, the Agrobacterium solutions were introduced into 6–8-week-old glycoengineered
Nicotiana benthamiana ΔXT/FT that are almost completely deficient in β1,2-xylosylation and core α1,3-fucosylation, resulting in glycoproteins carrying human-like
N-glycosylation, as previously described, by vacuum infiltration [
25,
40]. The plants were cultivated in a controlled environment room at 25 °C under a 16/8 h light/dark cycle. After 5 days, the infiltrated leaf material was harvested, and crude leaf extract was prepared by blending with three volumes of ice-cold phosphate-buffered saline (PBS) pH 7.4 containing 0.1% (
v/
v) Tween 20. The homogenized leaf material was filtered through a Miracloth filter (MilliporeSigma, Burlington, MA, USA) and centrifuged at 20,000×
g for 1 h, which was followed by filtration through 0.45 µm pore-size filters (Durapore membrane filter, MilliporeSigma, Burlington, MA, USA).
4.3. Purification of IgG and IgA Variants from Crude Leaf Extract
The clarified leaf extracts underwent purification using columns packed with either Pierce™ Protein A resin for the isolation of IgG and COVA2-15 IgA variants or a CaptureSelect™ IgA affinity matrix (both from Thermo Fisher Scientific, Waltham, MA, USA) for the purification of 2E8 IgA variants, which were pre-equilibrated with PBS. Proteins were eluted using 0.1 M glycine at pH 2.7, which was followed immediately by the addition of 10% (v/v) 1 M Tris-HCl at pH 9.0 to neutralize the pH. Fractions containing the protein of interest were combined and dialyzed against PBS at 4 °C overnight using a dialysis cassette with a molecular weight cut-off (MWCO) of 10 kDa (Slide-A-Lyzer, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the pooled and dialyzed protein fractions were concentrated using Amicon centrifugal filters with an MWCO of 100 kDa (MilliporeSigma, Burlington, MA, USA) and subjected to size-exclusion chromatography (SEC) on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare, Chicago, IL, USA) pre-equilibrated with PBS at pH 7.4. The SEC was performed using an ÄKTA pure FPLC system (GE Healthcare, Chicago, IL, USA).
4.4. ELISA
For the quantification of IgA mAbs in clarified crude extract of infiltrated N. benthamiana plants, ELISA plates were coated with 250 ng/well goat polyclonal antibody to human anti-alpha chain (ab97211, Abcam, Cambridge, UK) in PBS pH 7.4 at 4 °C overnight. After blocking with PBS containing 2% (w/v) BSA and 0.1% Tween 20 (v/v), clarified crude plant extracts were added to the wells in normalized concentrations and incubated for 1.5 h at 37 °C. As standards, purified human IgA (P80-102, Bethyl Laboratory, Montgomery, TX, USA) and IgA from human colostrum (I2363, Sigma, USA) were used. The detection of secretory IgA variants was carried out with a mouse anti-secretory component antibody (SAB4200787, Sigma, USA), which was followed by an HRP-labeled anti-mouse antibody (SAB5300168, Sigma, USA). For monomeric IgA variants, HRP-labeled anti-kappa (A18853, Invitrogen, Waltham, MA, USA) or anti-lambda light-chain (ab200966, Abcam, Cambridge, UK) antisera were used. After incubation for 1 h at 37 °C, plates were developed using TMB (Thermo Fisher, USA) substrate, the reaction was stopped with 2 M H2SO4 and the read-out was performed on an Infinite F200 Pro plate reader (Tecan, Männedorf, CH, Switzerland) at 450 nm.
The ratio of functional and fully assembled SIgA to total IgA in each size-exclusion fraction was determined by similar ELISA assays. Capture was with 100 ng/well purified recombinant RBD-His. Purified mAbs were diluted to 2 µg/mL in blocking solution, added to RBD-coated plates in normalized concentrations and incubated for 1.5 h at 37 °C. The detection of secretory component or antibody kappa or lambda chains was carried out as described above.
To determine the binding of the purified recombinant mAbs to SARS-CoV-2 RBD, ELISA plates were coated with 100 ng/well purified RBD-His. The purified mAbs were added to the wells in normalized concentration. For detection, HRP-labeled anti-human kappa or lambda light chain antibodies were used as above. Half-maximal concentration (EC50) was calculated in GraphPad Prism 9.0.
4.5. SDS-PAGE
First, 5 µg of purified mAbs was resolved on a NuPage 4–12% Bis/Tris gel (Life Technologies, Carlsbad, CA, USA) and stained with InstantBlue (Expedeon, Harston, UK).
4.6. Immunoblotting
Diluted crude leaf extracts were resolved on a NuPage 4–12% Bis/Tris gel (Life Technologies, UK) and then blotted on nitrocellulose membrane by semi-dry transfer, and bands were visualized using HRP-labeled anti-alpha HC antibody (ab97215, Abcam, Cambridge, UK).
4.7. Differential Scanning Fluorimetry (DSF)
Differential scanning fluorimetry (DSF) was conducted using a CFX real-time PCR instrument (Bio-Rad Laboratories, Hercules, CA, USA) in 1×PBS buffer at pH 7.4. Each sample was analyzed in triplicate, utilizing 96-well MicroAmp Fast reaction plates with 25 µL of sample per well. Monoclonal antibodies (mAbs) were diluted to a concentration of 1 mg/mL in the formulation buffer. SYPRO Orange Fluorescent Dye (Thermo Fisher Scientific, Waltham, MA, USA) was diluted 1000-fold from a 5000× g concentrated stock to prepare the working dye solution in the formulation buffer before addition to the mAb samples. Thermal denaturation was initiated by gradually increasing the temperature from 25 to 95 °C at a rate of 0.05 °C/s. Fluorescence intensity measurements were recorded using the FRET channel. Automated data processing of thermal denaturation curves involved truncating the dataset to eliminate post-peak quenching effects. The first derivative approach to calculate Tm was used. In this method, Tm is the temperature corresponding to the maximum value of the first derivative of the DSF melting curve.
4.8. Aerosolization of Monoclonal Antibodies
COVA2-15 and 2E8 SIgA1 were aerosolized using a commercially available Omron Micro Air U22 electronic mesh nebulizer (Omron Healthcare, Milton Keynes, UK) as previously described [
41].
4.9. Dynamic Light Scattering
DLS measurements were performed as described previously with protein concentrations of 500 µg/mL in 1×PBS pH 7.4 supplemented with 0.05% Tween on a Malvern Zetasizer nano-ZS (Malvern Panalytical, Malvern, UK) in a 12 mL quartz cuvette [
12]. Samples were measured at 25.0 °C, and the LS was detected at 173° and collected in automatic mode. The mean values and SEs of the number weighted diameter were calculated from three measurements for each sample, and each reported value is an average.
4.10. SEC-LS
SEC-LS was used to characterize the recombinant expressed proteins in solutions relating to their purity, native oligomers or aggregates, and molecular weights as previously described [
12]. Analyses were performed on an OMNISEC multidetector gel permeation chromatography (GPC)/SEC system equipped with a refractive index detector, a right-angle LS detector, a low-angle LS detector and a UV/visible light photodiode array detector (Malvern Panalytical, Malvern, UK). A Superdex 200 Increase 10/300 GL column (Cytiva, Marlborough, MA, USA) was used and equilibrated with Dulbecco’s PBS without Ca and Mg, P04-361000 (PAN-Biotech, Germany), as running buffer. Experiments were performed at a flow rate of 0.5 mL min
−1 at 25 °C and analyzed using OMNISEC software version 11.40 (Malvern Panalytical, Malvern, UK). Proper performance of the instrument was ensured by calibration and verification using the 200 mg Pierce BSA standard (Thermo Fisher Scientific). Before analysis, samples were centrifuged (16,000×
g, 10 min) and filtered through 0.2 mm Durapore PVDF centrifugal filter(s) (MilliporeSigma, Burlington, MA, USA). A 100 mL volume of each sample was injected, having different concentrations between 0.1 and 0.5 mg/mL.