WO2000063383A1

WO2000063383A1 - IgA BINDING POLYPEPTIDE

Info

Publication number: WO2000063383A1
Application number: PCT/GB2000/001539
Authority: WO
Inventors: Gunner Lindahl
Original assignee: Affitech As.
Priority date: 1999-04-19
Filing date: 2000-04-19
Publication date: 2000-10-26
Also published as: AU4131700A; GB9908920D0

Abstract

This invention provides polypeptides capable of binding IgA and polynucleotides which encode such polypeptides. The invention also provides expression vectors, host cells and methods for purification or isolation of IgA.

Description

IgA BINDING POLYPEPTIDE

Field of the invention

The invention relates to polypeptides having the ability to bind immunoglobulin A, IgA.

Background of the invention

Many bacteria and in particular pathogenic bacteria express surface proteins which bind to a variety of human plasma proteins. Surface proteins that bind to the Fc-part of human IgA are expressed by many strains of Streptococcus pyogenes, a major human pathogen. IgA-binding proteins expressed by this bacterium are members of the M protein family, a family of coiled-coil proteins that are important for virulence. However, characterisation of several of the IgA-binding proteins of S.pyogenes has shown that they not only bind IgA, but also bind other human plasma proteins, Stenberg et al J. Biol. Chem 1994 269, 13458-13464 and Thern et al J.

Immunol 1995 154 375-386. This property has complicated studies of the IgA- binding property of the protein. For structural analysis, it would be desirable to have access to an IgA-binding molecule of a smaller size. In addition, such peptides would be useful for purification or removal of IgA from a sample without also isolating additional plasma proteins.

Protein Arp is an IgA binding protein of S.pyogenes. The IgA binding region of this protein has been studied, Johnsson et al J. of Immunology. 1994 153 3557. Sir22 is a streptococcal surface protein which binds IgA, IgG and C4b-binding protein (C4BP). The regions in Sir 22 that bind these ligands have been partly identified (Stenberg et al).

Summary of the invention

A peptide derived from protein Sir22 which binds IgA but which does not bind other plasma proteins has been identified and can be used in isolation or purification of IgA as well as further studies into structural interactions of streptococcal protein. In a first aspect of the invention, there is provided a polypeptide capable of binding the Fc region of IgA consisting of the amino acid sequence of SEQ ID No. 2 or a variant thereof.

In a second aspect, the invention provides a chimeric protein capable of binding the Fc region of IgA comprising a first polypeptide having the amino acid sequence of SEQ ID No. 2 or a variant thereof and a second polypeptide which is not naturally contiguous to the first polypeptide.

In a further aspect, the invention relates to novel polynucleotides having the sequence selected from (i) the DNA sequence of SEQ ID No. 1 or the sequence complementary thereto, (ii) a sequence which selectively hybridises to a said sequence (i) or a fragment thereof, or (iii) the sequence which codes for a polypeptide having the same amino acid sequence as that encoded by said sequence (i) or

(ϋ), wherein the polynucleotide does not encode full length protein Sir 22 or a fragment thereof which is capable of binding IgG or C4BP and wherein the polynucleotide encodes for a polypeptide having the ability to bind the Fc region of IgA. The invention also relates to a recombinant vector such as an expression vector, comprising a polynucleotide of the invention operably linked to a regulatory sequence, for example a promoter; a host cell which is transformed with a polynucleotide of the invention; and a process for producing a polypeptide having the ability to bind IgA comprising maintaining a host cell transformed with a polynucleotide of the invention under conditions to provide expression of the polypeptide.

In a further aspect, the invention provides a method of isolating or purifying IgA from a sample comprising providing a support having attached thereto a polypeptide of the invention and bringing a sample into contact with said support such that any IgA present in the sample binds to said polypeptide.

The sample from which IgA has been removed can be collected for further use where it is desirable to use an IgA-free sample. Alternatively, or in addition IgA can be removed from the solid support such that IgA is purified.

Description of the figures FIG. 1. Sequence and binding properties of the IgA-binding peptide. A,

Schematic representation of protein Sir22 and sequence of the derived peptide. The regions in Sir22 known to include binding sites for C4BP, IgA, and IgG are indicated. B, Specificity of binding. Microtiter wells coated with Sir22 (left) or peptide (right) were analyzed for ability to bind radiolabeled human proteins. C, Competitive inhibition. Microtiter wells were coated with the peptide. Binding of radiolabeled serum IgA (left) or S-IgA (right) was inhibited with unlabeled protein or peptide, as indicated. Inhibition by BSA was tested at one concentration. In the experiments in B and C, each value is the average of duplicate samples. All experiments were performed twice, with similar results. FIG. 2. Binding specificity analyzed with radioactive peptide. A, The preparations indicated were analyzed by Western blot under non-reducing conditions. B, Dot-blot analysis of normal human serum and serum from an IgA-deficient individual. C, Western blot analysis of serum IgA and IgG under reducing conditions. The probe used in A-C was radiolabeled peptide. These experiments were performed twice, with similar results.

FIG. 3. The peptide binds IgA-Fc and binds IgA of both subclasses. A, Dot-blot analysis of an IgAl protein and its Fab and Fc fragments. The membrane was probed with radiolabeled peptide. B, Dot-blot analysis of purified human monoclonal Igs. The indicated amounts of Ig were applied to two identical membranes, which were probed with radiolabeled Sir22 or peptide. Each vertical row corresponds to one monoclonal protein. These experiments were performed twice, with similar results.

FIG. 4. Western blot analysis of the peptide and effect of heating. A, Samples of the peptide were subjected to Western blot analysis, using a radiolabeled IgAl protein as the probe. B, Binding of radiolabeled serum IgA to peptide, immobilized in microtiter wells, was inhibited with untreated peptide (closed symbols), or with peptide that had been boiled in PBS for 5 min and kept at room temperature for 30 min (open symbols). These experiments were performed twice, with similar results.

FIG 5. Comparison of amino acid sequences of Arp4, Arp60, Enn4, ML2.2 and Sir22. Dots indicate gaps introduced to get the optimal alignment of sequences.

Description of the sequences

SEQ ID No. ID NO. 1 sets out the amino acid sequence which conesponds to residues 35 to 83 of the processed form of Sir 22. SEQ ED No.1 also sets out the polynucleotide encoding this peptide.

SEQ ID No.2 is the amino acid sequence alone for the region 35 to 83 of the processed form of Sir 22.

Detailed description of the invention The invention provides a polypeptide having the ability to bind to the Fc region of IgA. A polypeptide of the invention consists of the sequence SEQ ED No.2 or a variant thereof. Variants in accordance with the invention maintain the ability to bind IgA. References herein to binding of IgA relate to the property of the peptide to bind the Fc region of IgA. The ability to bind IgA can be assessed using radiolabelled human proteins such as ¹²⁵I-labelled IgA. For example, a peptide under investigation could be immobilized. Radiolabelled ligand can be added to the immobilized peptide and incubated for example at room temperature for two hours. After washing, radioactivity associated with the immobilized peptide can be determined. Competitive immunoassays can be carried out in a similar way. Preferably, a polypeptide of the invention will maintain the ability to bind

IgA, at a similar level to naturally occurring protein Sir 22. However, those skilled in the art will readily appreciate that polypeptides with a reduced capacity to bind IgA may also prove useful, as will polypeptides which have a greater capacity to bind IgA. Preferably the polypeptide specifically binds IgA. However, as will be described in more detail below, the invention also relates to a chimeric protein in which the first polypeptide has the ability to bind IgA and a second polypeptide which binds other proteins.

SEQ ED No: 2 is the amino acid sequence for the region 35 to 83 of the processed form of Sir22. Sir22 is a streptococcal surface protein of S.pyogenes. Sir22 is a member of the M-protein family. There are a number of related IgA binding proteins of S.pyogenes expressed by different M-serotypes such as protein

Arp4, Arp60, Enn4, ML2.2.

The region from amino acid 45 to amino acid 73 of Sir22 has been identified as an IgA binding domain. SEQ ID NO: 2 includes additional flanking regions of 10 amino acids in length which allow the binding region to adopt the conect conformation for IgA binding to occur. Preferably, a polypeptide of the invention consisting of the SEQ ED NO: 2 also includes a C-terminal cysteine to facilitate dimer formation.

A variant of SEQ ID NO: 2 is accordance with the invention maintains the ability to bind IgA. A variant in accordance with the invention includes polypeptides in which the IgA binding domain identified above is varied, polypeptides in which the flanking sequences are varied or polypeptides in which both the IgA binding domain and the flanking sequences are varied. An IgA binding domain of approximately 29 amino acids in length may be derived from an IgA binding protein of S.pyogenes. The relevant sequence may be identified through alignment of the amino acid sequences of other IgA binding proteins. Figure 5 shows the alignment of the IgA binding regions of streptococcal M proteins, namely Arp4, Arp60, Enn4, ML2.2 and Sir22. Similar alignment could be made with other IgA binding proteins to derive the best possible alignment and thus identification of the IgA binding region and in particular the binding domain conesponding to the 29 amino acid domain of Sir22.

A variant in accordance with the invention will preferably be at least 50% homologous to SEQ ID NO: 2 within the 29 amino acid residue region based on amino acid identity. Preferably, the polypeptide is at least 60 or 70% or more preferably at least 80 or 90% and preferably at least 95, 97 or 99% homologous to SEQ JD NO: 2 over this 29 amino acid residue regions. All references to percentage homology are based on amino acid identity. A variant in accordance with the invention also comprises flanking sequences flanking the IgA domain. These flanking sequences allow the IgA binding domain to adopt the conect conformation to allow IgA binding to occur. Appropriate flanking sequences may be derived from other streptococcal IgA binding proteins. For example, SEQ ID NO: 2 comprises the flanking sequences of the IgA binding domain derived from Sir22. Alternatively, flanking sequences located on either side of the IgA binding domain identified in Figure 5 for proteins Arp4, ArpόO, Enn4 and ML2.2 could be used as flanking sequences for the IgA binding domain derived from Sir22 or another variant in accordance with the invention. The N-terminal, C-terminal or both flanking regions may be derived from another protein which forms a fusion protein or chimeric protein with the polypeptide of the invention such that the IgA binding domain adopts the conect conformation for IgA binding to occur.

Flanking regions which allow the polypeptide to adopt a coiled coil conformation are especially prefened. For example, the GCN4 leucine zipper could be used or other appropriate flanking sequences containing a parallel 2-stranded coiled coil motif.

A variant will preferably be at least 50% homologous to SEQ ID No.2. Preferably the polypeptide is at least 60 or 70% or more preferably at least 80 or 90% and preferably 95, 97 or 99% homologous to SEQ ID No.2 based on amino acid identity. Amino acid substitutions may be made to SEQ ID No.2 for example, from 1, 2 or 3 up to 7, 8, 9 or 10 up to 15, 20 or 25 substitutions. The modified polypeptide retains the ability to bind IgA. Prefened substitutions can be identified by reference to an IgA binding domains of streptococcal proteins, for example as shown in Figure 5. Residues which are conserved between three or more proteins are preferably not substituted. Residues which show no conservation between IgA binding proteins may preferably be substituted. Conservative substitutions may be made, for example, according to table 1 below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. Table 1

One or more amino acids may be alternatively or additionally deleted. From

1, 2 or 3 up to 4, 5 or 6 residues may be deleted.

One or more amino acids may be alternatively or additionally added to any one of the polypeptides described above in accordance with the invention. An extension may be provided at the N-terminus or C-terminus or both of the sequence of SEQ ID No.2 or a variant thereof. The, or each extension may be quite short, for example, from 1 to 10 amino acids in length. Alternatively, the extension may be longer. A carrier protein may be fused to an amino acid sequence according to the invention. A fusion protein or chimeric protein incorporating the polypeptides described above can thus be provided. In these embodiments, a carrier protein may be used to carry the specific polypeptide of SEQ ED No.2 or a variant thereof. Additional amino acid sequences may be introduced which bind other plasma proteins. This may be particularly useful where it is desired to remove two or more plasma proteins from a sample, one of said plasma proteins being IgA. However, the invention does not encompass the naturally occurring protein Sir 22 or fragments thereof which include binding domains for IgG or C4BP . Preferred chimeric proteins may bind IgE, IgM or IgG or fragments thereof, for example fragments conesponding to variable regions, light or heavy chains or Fc.

Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with caniers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99% by weight of the polypeptide in the preparation is a polypeptide of the invention.

Polypeptides of the invention may be modified for example by the addition of histidine residues, to assist their identification or purification or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. Preferably a cysteine residue is added at the C- terminal of SEQ ID No.2 which facilitates formation of peptide dimers.

A polypeptide of the invention above may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵1, ³⁵S, enzymes, antibodies, polynucleotides and linkers such as biotin. Labelled polypeptides of the invention may be used in diagnostic procedures such as immunoassays in order to determine the amount of a polypeptide of the invention in a sample. A polypeptide or labelled polypeptide of the invention or fragment thereof may also be fixed to a solid phase, for example the surface of an immunoassay well or dipstick. In particular, a polypeptide according to the invention may be provided attached to a solid support for use in a column. A sample containing IgA may then be applied to the column for use in the isolation or purification of IgA. Such labelled and/or immobilized polypeptides may be packaged into kits in a suitable container optionally including additional suitable reagents, controls or instructions in the light. The kits may be used to isolate or purify IgA.

Such methods of isolation and purification are well known in the art and will generally comprise: (a) providing a polypeptide according to the invention capable of binding IgA bound to a solid support; (b) incubating the biological sample with said polypeptide under conditions which allow for the binding of IgA to said polypeptide; and (c) collecting the samples from which IgA has been removed and/or collecting IgA from said solid support. In alternative aspects of the invention where a polypeptide is provided which has the ability to bind more than one plasma protein, a sample potentially containing said plasma proteins may be applied to a solid support and the plasma proteins which bind to the polypeptides of the invention can be removed from the sample. Polypeptides of the invention may be made by synthetic means or recombinantly, as described below.

The polypeptides of the invention may be introduced into a cell by in situ expression of the polypeptide form a recombinant expression vector. The expression vector optionally canies an inducible promoter to control the expression of the polypeptide. A polypeptide in the invention can be produced in large scale following purification by high pressure liquid chromatography (EEPLC) or other techniques after recombinant expression as described below.

Polynucleotides A polynucleotide of the invention is capable of hybridising selectively with the coding sequence of SEQ ID No.1 or to the sequence complementary to that coding sequence. Polynucleotides of the invention include variants of the coding sequence of SEQ ED No.1 which encode the amino acid sequence of SEQ ID No.2; and variants which bind Fc of IgA. Typically, a polynucleotide of the invention is a contiguous sequence of nucleotides which is capable of selectively hybridizing to the coding sequence of SEQ ID. No. 1 or to the complement of that coding sequence.

A polynucleotide of the invention hybridizing to the coding sequence of SEQ ID No.1 can hybridize at a level significantly above background. Background hybridization may occur, for example, because of other DNAs present in a DNA library. The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence of SEQ ID No.1 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID No.1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³²P. Selective hybridization is typically achieved using conditions of medium to high stringency

(for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C).

A nucleotide sequence capable of selectively hybridizing to the DNA coding sequence of SEQ ID NO: 1 or to the sequence complementary to that coding sequence will be generally at least 50% or 60%, preferably at least 70% or 80%, preferably at least 90% and more preferably at least 95%, homologous to the coding sequence of SEQ ED NO: 1 or its complement over a region of at least 100 and preferably at least 120, 130 or 140 or more contiguous nucleotides such as over the entire length of SEQ ID No: 1 or its complement. Methods of measuring polynucleotide homology are well known in the art. The UWGCG Package which provides the BESTFIT program can be used to calculate homology, e.g. on its default settings (Deveraux et al, Nucl. Acids. Res. 12. 387-395, 1984).

Any combination of the above mentioned degrees of homology and minimum size may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher homology over longer lengths) being prefened. Polynucleotides of the invention may comprise DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to polynucleotides are known in the art. These include methylphosphate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art.

Polynucleotides of the invention may be used to produce a primer, e.g a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by contential means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.

Polynucleotides such as a DNA polynucleotide and primers according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. The polynucleotides are typically provided in isolated and/or purified form,

In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinant means, for example using PCR (polym erase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the Sir 22 gene which it is desired to clone, bringing the primers into contact with DNA obtained from a bacterial cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al, Molecular Cloning: A Laboratory Manual, 1989.

Polynucleotides or primers of the invention may carry a revealing label. Suitable labels include radioisotopes such as ³²P or ³⁵S, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers of the invention and may be detected using techniques known er se.

Polynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells as described below in connection with expression vectors.

Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. Such expression vectors can be used to express a polypeptide of the invention.

The term "operably linked" refers to a juxtapositions wherein the components described are in a relationship permitting them to function in their intended manner.

A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

Such vectors may be transformed into a suitable host cell as described above to provide for expression of a polypeptide or polypeptide fragment of the invention.

Thus, in a further aspect the invention provides a process for preparing a polypeptide according to the invention, which process comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression of the polypeptide, and recovering the expressed polypeptide.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid.

A further embodiment of the invention provides host cells transformed or transfected with the polynucleotides or vectors for the replication and expression of polynucleotides of the invention. The cells will be chosen to be compatible with the said vector and preferably will be bacterial. Host cells may also be cells of a non- human animal, or a plant transformed with a polynucleotide of the invention.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.

The invention will now be described in more detail by reference to the following examples. Examples

Methods and Materials used in the Examples

Synthetic peptide, purified proteins - The 50-residue synthetic peptide (Fig. 1A) corresponds to amino acid residues 35-83 of protein Sir22, and includes a COOH-terminal cysteine residue not present in Sir22. The peptide was >95% pure. Recombinant Sir22 was purified as described in Stenberg et al J.Biol Chem (1994) 269 13458-13464. Purified human C4BP was the kind gift of Dr. B. Dahlback (Malmό General Hospital).

Immunoglohulins, human serum - All immunoglobulins were of human origin. Polyclonal serum IgA was from Cappel-Organon Teknika (Turnhout, Belgium). S-IgA was purified from colostrum. Monoclonal IgA proteins were isolated from serum of patients with IgA multiple myeloma. Three of the monoclonal IgA proteins and all monoclonal IgG proteins were kindly provided by

Dr. F. Skvaril (Bern, Switzerland). Monoclonal IgM and IgD were from The Binding Site (Birmingham, Great Britain) and IgE was the gift of Dr. I. Olsson (Lund University). Polyclonal IgG was from Kabi (Stockholm, Sweden). Fab and Fc fragments of an IgAl protein were purified after cleavage with IgA protease from Haemophilus influenzae. Serum from an IgA-deficient individual was kindly provided by Dr. A. Sjoholm (Lund University).

Binding assays, competitive inhibition - Binding of radiolabeled human proteins was analyzed after immobilization of peptide or Sir22 in microtiter wells. The wells were coated overnight at 4°C with 50 μl of a solution of peptide or Sir22 (100 μg/ml in PBS). After washing and blocking with PBSAT (PBS with 0.02% sodium azide and 0.05% Tween-20), radiolabeled ligand (-25,000 cpm) was added to each well, and the plates were incubated for 2 h at room temperature. After washing with PBSAT, the radioactivity associated with each well was determined. Nonspecific binding (<0.7%) was determined for wells coated only with PBSAT. The Igs used were polyclonal.

For inhibition tests, wells of microtiter plates were coated with the peptide (5 μg/ml for tests with serum IgA and 50 μg/ml for tests with S-IgA). After blocking, radiolabeled serum IgA or S-IgA (-25,000 cpm) was added together with inhibitor, as indicated. The final concentration of radiolabeled IgA was -0.3 nM. Binding was analyzed as described above. Nonspecific binding was <0.25%.

Other methods - Western blot and dot-blot analysis on PVDF membranes were performed as described Stenberg et al supra. Protein concentrations were determined with the MicroBCA kit from Pierce (Rockford, IL), using BSA as a reference. Radiolabeling of peptides and proteins with ¹²⁵I was performed with the chloramine-T method.

Example 1

Design of a synthetic peptide including the IgA-binding region of protein Sir 22

Previous studies of the S. pyogenes protein Arp4 demonstrated that the IgA- binding region of this protein is localized in a 29-residue region in the N-terminal part of the molecule. An attempt was made to demonstrate IgA-binding for a 33- residue synthetic peptide including this region from Arp4, but no binding was observed (data not shown). A longer peptide was therefore tested, as described below. This new peptide was derived from the Sir22 protein, which is closely related to Arp4 and is expressed by a bacterial strain that is suitable for analysis of pathogenetic mechanisms.

Protein Sir22 is a dimeric coiled-coil molecule that binds at least three different human plasma proteins, IgA, IgG, and C4BP (C4b-binding protein) (Fig. 1 A). The regions in Sir22 that bind these ligands have been partly identified. The 29- residue IgA-binding region (defined by homology with Arp4) overlaps with an upstream region that includes a C4BP-binding site. The IgA-binding region also overlaps with a downstream region including an IgG-binding site. The bindings of IgA and IgG to Sir22 are mutually exclusive, indicating shared or contiguous binding sites. In addition to these ligands, serum albumin may bind to the central C-repeat region of Sir22. The synthetic 50-residue peptide covers the predicted IgA-binding region of

Sir22 and also includes 10 residues on either side, added to enhance the probability for conect folding. Since the Sir22 protein must form a coiled-coil dimer to bind IgA, a COOH-terminal cysteine residue was included, to ensure that dimerization could occur. However, it also seemed possible that the peptide would be able to form a coiled-coil by itself.

Example 2

The peptide specifically binds IgA

Wells of microtiter plates were coated with the peptide or with Sir22 and analyzed for ability to bind radiolabeled ligands (Fig. IB). As expected, immobilized Sir22 bound all four ligands. For unknown reasons, the binding of IgG was lower than for serum IgA in this test, although Sir22 has similar affinity for these two ligands. The immobilized peptide bound serum IgA and S-IgA, but not C4BP or IgG, indicating that it specifically binds IgA. The binding of serum IgA and S-IgA to the immobilized peptide could be completely inhibited by unlabeled IgA, but not by BSA, confirming that the binding of IgA was specific and showing that the binding was not due to the radiolabeiing (Fig. IC). Moreover, binding of serum IgA or S-IgA to immobilized peptide could be inhibited by free peptide, indicating that the ability to bind IgA was not due to a conformational change in the peptide upon immobilization (Fig. IC). Finally, the binding of serum IgA and S-IgA to immobilized peptide could be inhibited by a purified monoclonal IgAl protein, showing that the binding of IgA was independent of the antigen binding site (data not shown).

The binding properties of the peptide were further studied by Western blot analysis under non-reducing conditions (Fig. 2A). Radiolabeled peptide, used as the probe, bound to serum IgA but not to IgG. Thus, the peptide in solution retains its binding ability after radiolabeiing. Striking evidence for the specificity of the peptide came from analysis of whole normal serum and serum from an IgA-deficient individual. The peptide lacked reactivity with any of the many proteins present in IgA-deficient serum, but bound to IgA present in normal serum. Similar results were obtained in a dot-blot analysis (Fig. 2B). In saliva, the peptide recognized a high molecular weight protein with the mobility of S-IgA, and also detected some monomeric IgA (Fig. 2 A), giving further support for the specificity of the binding. The peptide even recognized separated α chains, as shown by Western blot analysis of IgA under reducing conditions (Fig. 2C).

Example 3

The peptide binds IgA-Fc and binds IgA of both subclasses

As expected, the peptide binds to the Fc-part of IgA (Fig. 3 A). To analyze whether the peptide binds IgA of both subclasses, a_dot-blot analysis was performed with purified monoclonal proteins (Fig. 3B). The analysis also included analysis of monoclonal IgG, IgM, IgD, and IgE proteins, and the binding properties of the peptide were compared with those of the parental Sir22 molecule. Sir22 was found to bind most of the IgA and IgG proteins. The peptide only bound IgA proteins, and binding was observed for 8 out of 9 IgAl proteins and for 10 out of 11 IgA2 proteins. However, this semi-quantitative analysis indicated that the affinity varied considerably for different IgA proteins. Possible reasons for this variation are differences in glycosylation and loss of binding ability during purification of monoclonal proteins. The ability of the peptide to bind different IgA proteins was similar to that of protein Sir22, supporting the hypothesis that the peptide conesponds to an IgA-binding domain in Sir22.

Example 4

Western blot analysis and effect of heating

Western blot analysis of the peptide under non-reducing conditions showed that it bound radiolabeled IgA (Fig. 4A). This analysis included an initial step, in which the peptide was boiled in SDS-containing buffer, a step that most likely caused denaturation. Thus, these results suggest that at least some peptide molecules renatured during the analysis. The peptide, probably a dimer, moved more slowly than expected, a common property in streptococcal surface proteins. Under reducing conditions, when the peptide migrated as a monomer, it also bound IgA after blotting. In this regard, the peptide behaved like protein Sir22, which also binds IgA in Western blot analysis, after having migrated as a monomer. Since Sir22 must form a coiled-coil dimer to bind IgA, it seems likely that dimerization of Sir22 can occur on the blotting membrane, allowing binding of IgA. Similarly, the ability of the peptide to bind IgA in Western blot analysis during reducing conditions might have been due to formation of coiled-coil dimers on the blotting membrane. The COOH-terminal part of the peptide includes a sequence (LEEKEKNLEKK) that is similar to a consensus "trigger" sequence implicated in the formation of coiled-coils Kammerer et al Proc Natl Acad Sci (1998) 95, 13419-13424.

The ability of the peptide to bind IgA after heating was further analyzed in an inhibition test (Fig. 4B). In this test, the ability of radiolabeled IgA to bind to immobilized peptide was inhibited by free peptide. Boiling of the peptide for 5 min, which most likely caused denaturation, had no effect on the ability of the peptide to subsequently inhibit binding, implying that most peptide molecules refolded into the native form after denaturation. Taken together, the data in Fig. 4 indicate that the peptide is a very stable molecule. The radiolabeled form of the peptide was also stable, since it could be kept frozen for four months without loosing the ability to specifically bind IgA (data not shown).

Claims

1. A polypeptide capable of binding IgA consisting of the amino acid sequence of SEQ ID No.2 or a variant thereof.

2. A chimeric protein capable of binding IgA comprising a first polypeptide having the amino acid sequence of SEQ ID No.2 or a variant thereof and a second polypeptide which is not naturally contiguous to said first polypeptide.

3. A polypeptide according to claim 1 or 2 wherein the variant has at least 80% amino acid identity to SEQ ID No.2 and retains the ability to bind IgA.

4. A polypeptide according to any one of the preceding claims wherein the variant comprises:

(a) amino acids 11 to 39 of SEQ ID No.2 or a variant sequence thereof ; and (b) additional amino acid residues flanking the sequence (a) such that the sequence (a) adopts the correct conformation for IgA binding to occur.

5. A polypeptide according to claim 4 wherein the variant to sequence (a) has at least 80% amino acid identity to amino acids 11 to 39 of SEQ ID No.2.

6. A polynucleotide having a sequence selected from (i) the DNA sequence of SEQ ED No.1 or the sequence complementary thereto, (ii) a sequence which selectively hybridises to a said sequence (i) or a fragment thereof; or (iii) a sequence which codes for a polypeptide having the same amino acid sequence as that encoded by a said sequence (i) or

(ϋ), wherein the polypeptide encoded by said polynucleotide has the ability to bind IgA and does not comprise full length Sir 22 or a fragment thereof capable of binding IgG or C4BP.

7. A recombinant expression or cloning vector comprising a polynucleotide according to claim 6.

8. A host cell transformed with a polynucleotide according to claim 3 or a vector according to claim 7.

9. A method of purification or isolation of IgA comprising

(a) providing a polypeptide according to any one of claims 1 to 5 bound to a solid support.

(b) incubating a sample with said solid support under conditions such that IgA present in said sample can bind to said polypeptide.

10. A method according to claim 9 for removing IgA from a sample, further comprising step (c) of collecting the sample from which IgA has been removed.

11. A method of purifying IgA according to claim 9 further comprising step (c) of eluting the bound IgA from the solid support.