- Top of page
- Materials and methods
Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. Characterization of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding, with dissociation constants (Kd) in the range between 0.5 and 3 µm. One variant (ZIgA1) showing the strongest binding affinity was further analyzed, and showed that human IgA subclasses (IgA1 and IgA2) as well as secretory IgA were recognized with similar efficiencies. No detectable cross-reactivity towards human IgG, IgM, IgD or IgE was observed. The potential use of the ZIgA1 affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate, using an affinity column containing a divalent head-to-tail ZIgA1 affibody dimer construct as a ligand. In addition, efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated.
Efficient and selective methods for immunoglobulin (Ig) detection and purification are of major importance for a vast number of applications within areas such as immunology, diagnostics and biotechnology. For these purposes, an often recruited class of reagents is derived from so-called receptin proteins, produced as surface-anchored or soluble proteins by some bacteria . Many of these proteins show binding to one or more mammalian serum or cell surface proteins, including for example Ig, serum albumin and fibrinogen . One of the most well-known Ig-binding receptins is staphylococcal protein A, widely used in many formats for its capability to bind a wide spectrum of Igs via Fc or VH region recognition [2–5]. Other examples of frequently used Ig-binding receptins include streptococcal protein G [6,7] and Peptostreptococcus magnus protein L [8,9]. The binding specificities displayed by different Ig-binding receptins differ significantly in terms of Ig isotype, subclass, species origin and subfragment type (Fc, Fab, scFv, etc.), which makes the choice of reagent for a particular application important.
IgA is the most abundant Ig isotype in humans where it is present in two subclasses, IgA1 and IgA2, differing mainly in their hinge region sequences . IgA is predominantly localized to mucosal surfaces but is also present at high levels in plasma  and believed to play an important role in defence mechanisms against infections . IgA is also implicated in several diseases including IgA nephropathy , coeliac disease , Henoch-Schönlein purpura  and neurological diseases , where IgA levels are monitored as a disease marker or investigated for a more direct involvement in disease progression. Interestingly, recent data suggest that IgA should be an interesting isotype to validate for development of antibodies for immunotherapy applications, including cancer therapy, due to its effective recruitment of neutrophils .
The identification of receptins or the development of other classes of ligands capable of selective IgA binding will therefore be important for biotechnology tools, facilitating IgA recovery and detection. Interestingly, receptin proteins shown to be capable of IgA binding have been described, including the Sir22 and Arp4 proteins isolated from Streptococcus pyogenes[18,19]. These proteins were recently shown to bind both IgA1 and IgA2 and mapped to recognize the CH2–CH3 interface on the IgA Fc fragment. The biotechnological use of this class of proteins has been recognized and initially investigated for immunodetection applications using the IgA binding protein B from group B streptococci .
In this work, we describe an alternative approach to obtain novel IgA binding ligands, using combinatorial protein engineering to change the binding specificity of an already existing receptin protein. Employing a 58-amino-acid domain derived from one of the immunoglobulin binding (IgG) domains of staphylococcal protein A as a scaffold, we have previously described the construction of combinatorial protein libraries from which novel affinity proteins (denoted Z-affibodies) have been selected for binding to desired target proteins using phage display technology [21,22]. In common with their protein A ancestor, several of the described affibodies have been shown to be useful as selective and robust ligands in affinity chromatography applications [23–25]. Here, we describe the use of the affibody technology platform for the identification of protein A-derived variants showing IgA- rather than IgG-binding, thus expanding the available repertoire of tools for detection and recovery of native or recombinant IgA from different sources. The selection procedure, biosensor binding affinity and specificity data of candidate ligands as well as the affinity chromatographic recovery of IgA from a bacterial lysate and unconditioned human plasma is described.
- Top of page
- Materials and methods
In this work, a combinatorial protein engineering approach was used to obtain novel IgA binding proteins. Using the well-characterized protein A-derived IgG-binding, three-helix bundle domain Z as scaffold for library constructions, five out of eight investigated variants selected using phage display technology showed IgA binding. The reasons leading to the selection of non-IgA binding variants were not investigated further, but could be due to background binding of such variants to components other than IgA present during selection, such as streptavidin beads and phages. In several of the confirmed binders, some substitutions were identical with a preference to the second variegated helix (Fig. 1). This could indicate that these variants binds to a common site on the IgA protein.
One of the confirmed IgA-binders (clone ZIgA1) was characterized further and showed to be selective for human IgA in a series of biosensor binding studies, where the specificity was challenged with all other human isotypes, including polyclonal IgG. Further studies showed that both IgA1 and IgA2 subclasses, as well as secretory IgA, was efficiently recognized by the ZIgA1 variant. In comparison to three described natural bacterial IgA binding proteins, this gives the ZIgA1 affibody an IgA binding profile closer to those of the Arp4 and Sir22 proteins from Streptococcus pyogenes than that of the serologically distinct IgA-binding receptins from group B streptococci, showing only weak binding to secretory IgA . However, no obvious sequence similarity can be seen between the ZIgA1 affibody sequence and a 29-residue motif that shows sequence similarity between the Arp4 and Sir22 proteins and has been postulated to contain the IgA binding regions in these proteins. Interestingly, a 50-residue peptide derived from Sir22 containing this motif as central element was found to bind IgA with high specificity , but was described to be dependent on the formation of coiled-coiled dimers for IgA binding.
The binding sites for the three natural streptococcal IgA binding receptins were recently mapped to the CH2–CH3 region interface of the Fc fragment, overlapping with the binding site for the human CD89 IgA receptor , suggesting biological significance. It is worth noting that in the interactions between IgG and protein A or protein G, the corresponding domain interface is involved in the binding . However, the IgA binding proteins described in this work were selected in vitro, in the absence of any obvious biological selection pressure, suggesting that more or less any site on the IgA heavy chain not protected by the secretory component could contain the binding epitope. The recognition of both the IgA1 and IgA2 subclasses suggests that the IgA hinge region, which differs in length and sequence between the two forms of IgA, is not significantly involved in the binding.
In the affinity chromatography experiments, head-to-tail dimeric constructs of the ZIgA1 affibody were used, resulting in divalent ligands. Although the primary reason for the dimerization was to increase the likelihood that at least one moiety in each dimeric ligand was biologically active after the amine coupling procedure, a divalent capturing ligand could potentially also result in adventageous avidity effects in the binding between the ligand and the target as well as to provide a beneficial effect on the presentation of the immobilized ligand to the surrounding solution related to the use of a larger ligand (spacer effect).
The efficient recovery of IgA from the unconditioned human plasma suggests that the described ligand could be of interest to evaluate for use in extracorporeal IgA-removal applications, of potential relevance for investigation and treatment of IgA nephropathy. Interestingly, whereas the biosensor binding data showed that the ZIgA1 affibody was highly selective for IgA, small amounts of IgM and IgG could be detected by Western blotting in the eluate from the recovery of IgA from human plasma. This could possibly be explained by the presence of natural immunoglobulin-specific autoantibodies in healthy individuals, resulting in the formation of circulating immune complexes (CIC). Such natural autoantibodies have been described to be of IgM, IgG and IgA isotypes . In addition, anti-(protein A) Ig, recognizing the nonvariegated parts of protein A-derived affibody ligand structure, could also be present in the plasma, as a result of previous bacterial infections of the plasma donors.
A growing number of antibodies intended for therapeutic use are currently under investigation in clinical trials . The majority of those are of IgG isotype, which can effectively activate complement- and antibody-dependent cellular cytotoxicity pathways. In addition, antibodies of the IgG isotype are effectively purified by protein A affinity chromatography, which is also implemented at industrial scale processes . However, the IgA isotype has also been suggested for immunotherapy applications, owing its strong association with neutrophils and the possibility to direct antibodies to luminal surfaces. For example, Streptococcus mutans-specific IgA has been investigated for administration to the oral cavity for the prevention of caries . In analogy to the efficient protein A-based purification of IgG, the development of corresponding affinity chromatography media containing a robust affinity ligand selective for IgA should facilitate the purification of recombinant antibodies of this isotype. Based on the stable structure of a single protein A domain, the ligands of the type described in this work could constitute interesting candidates for such applications.