Affinity ligand for purification of antibodies of isotype iga

Abstract

The present disclosure relates to a class of polypeptides derived from Staphylococcus Protein A (SpA) or any domain thereof, that exhibit an improved binding affinity for IgA. Moreover, IgA binding polypeptides that selectively bind IgA among other immunoglobulin isotypes are also provided. The present disclosure also relates to methods for isolating IgA, such as antibodies of isotype IgA, using said polypeptides as well as related products, such as separation matrices coupled to said IgA binding polypeptides and / or multimers thereof.

Description

[0001] AFFINITY LIGAND FOR PURIFICATION OF ANTIBODIES OF ISOTYPE IGA

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a class of polypeptides derived from Staphylococcus Protein A (SpA) or any domain thereof, that exhibit an improved binding affinity for IgA. Moreover, IgA binding polypeptides that selectively bind IgA among other immunoglobulin isotypes are also provided. The present disclosure also relates to methods for isolating IgA, such as antibodies of isotype IgA, using said polypeptides as well as related products, such as separation matrices coupled to said IgA binding polypeptides or multimers thereof.

[0004] BACKGROUND

[0005] Immunoglobulins represent the most prevalent biopharmaceutical products in either manufacture or development worldwide. The high commercial demand for and hence value of this particular therapeutic market has led to the emphasis being placed on pharmaceutical companies to maximize the productivity of their respective monoclonal antibodies (mAb) manufacturing processes whilst controlling the associated costs. Antibodies of isotype immunoglobulin A (IgA) offer a promising format for certain therapies, for example, beside IgG antibodies which dominate the therapeutic field, IgA mAbs are also effective in killing tumor cells. Therapeutic applications of IgA target receptors of the inflammatory signalling pathway and auto immune diseases, moreover IgA is used in IgA deficiency treatment (J. Leusen, Molecular Immunology, Volume 68, Issue 1, Pages 35-39 (2015); D. Sterlin and G. Gorochov, Pharmacology Volume 106, No. 1-2 (2021)). IgA (slgA for its secretory form) is important for the immune function of mucous membranes but is also present at high levels in plasma (Fagarasan S and T. Honjo, Nature Reviews, Immunology, 3(1): 63-72. (2003)). IgA has two isoforms, IgAl and lgA2, where IgAl is usually monomeric and predominantly found in serum, and lgA2 is mainly secreted as a dimer. Affinity chromatography is used in most cases as one of the key steps in the purification of immunoglobulin molecules, such as monoclonal or polyclonal antibodies. A particularly interesting class of affinity reagents is proteins capable of specific binding to invariable parts of an immunoglobulin molecule, such interaction being independent of the antigen-binding specificity of the antibody. Such reagents can be widely used for affinity chromatography recovery of immunoglobulins from different samples, such as but not limited to serum or plasma preparations or cell culture derived feed stocks. An example of such a protein is staphylococcal protein A (SpA), containing domains capable of binding to the Fc (fragment crystallizable) and Fab (fragment antigen-binding) portions of IgG immunoglobulins from different species These domains are commonly denoted as the E-, D-, A-, B- and C domains. SpA-based proteins have due to their high affinity and selectivity found a widespread use in the field of biotechnology, e.g. as ligands in affinity chromatography for capture and purification of antibodies as well as for detection or quantification. At present, SpA-based affinity medium is probably the most widely used affinity medium for isolation of monoclonal antibodies and their fragments from different samples including industrial cell culture supernatants. Accordingly, various matrices comprising protein A or protein A-derived ligands are commercially available, for example, in the form of MabSelect™ SuRe, MabSelect™ SuRe LX, MabSelect™ PrismA and HiScreen Fibro™ PrismA from Cytiva, Uppsala, Sweden. Purification of IgA is considered difficult. Lectin-based methods, e.g. jacalin-grafted agarose (Thermo Fisher Scientific), utilize interactions with O-linked glycans for IgA purification. These ligands bind IgAl and also have affinity for lgA2 and IgD, but do not bind IgG (P. Aucouturier et al., Journal of Immunological Methods, 113, pp. 185- 191, (1988)). LigaTrap markets resin for IgA purification but it is not recommended for serum purification due to cross-reactivity with other immunoglobulins. Thermo Fisher Scientific also offers CaptureSelect™ IgA-XL Affinity Matrix, which is an affinity ligand resin with a 13 kDa single domain fragment comprising 3 CDRs (complementarity-determining regions) that form the binding domain. Efficient and selective methods for IgA detection and purification are of major importance for a vast number of applications, however it is problematic due to the lack of affinity reagents that are capable of selective recognition of IgA, including IgAl and lgA2 subclasses, from different immunoglobulin isotypes. Affinity reagents that exhibit suboptimal binding characteristics for IgA and low selectivity hinder the development of IgA-based therapeutics.

[0006] SUMMARY

[0007] It is an object of the present disclosure to provide IgA binding SpA-derived ligands that allow for efficient isolation of IgA and / or fragments thereof while alleviating, at least in part, the abovementioned and other drawbacks of the prior art.

[0008] It is an object of the present disclosure to provide IgA binding SpA-derived ligands which exhibit improved binding affinity for IgA. Such ligands could for example be used in methods for isolating IgA and / or fragments thereof, such as antibodies of IgA isotype.

[0009] It is an object of the present disclosure to provide IgA binding SpA-derived ligands that selectively bind IgA among other immunoglobulin isotypes.

[0010] Another object of the present disclosure is to provide IgA binding SpA-derived ligands which decrease or prevent co-purification of IgA product-related impurities, such as non-lgA isotype immunoglobulins.

[0011] It is an object of the present disclosure to provide IgA binding SpA-derived ligands that are alkaline stable.

[0012] Another object of the present disclosure is to provide IgA binding SpA-derived ligands that are optimized for industrial production in recombinant systems.

[0013] These and other objects which are evident to the skilled person from the present disclosure are met by different aspects of the invention as claimed in the appended claims and as generally disclosed herein.

[0014] Thus, in a first aspect of the disclosure, there is provided an IgA binding polypeptide derived from SpA or any domain thereof, wherein said polypeptide has a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NO:9 for the same IgA polypeptide.

[0015] In a second aspect of the disclosure, there is provided an IgA binding polypeptide comprising an amino acid sequence comprising Sequence A, which Sequence A consists of an amino acid sequence selected from i), ii) and iii), wherein i), ii) and iii) are defined as follows: i) X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35 (SEQ ID NO:11), wherein X33 is selected from T, S, G, Q, A, E, H, R, P, D, K and N; ii) an amino acid sequence which has at least 81% identity to a sequence defined by i); iii) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 9-35 in SEQ ID NO:1, residues 9-35 in SEQ ID NO:2, residues 9-35 in SEQ ID NO:3, residues 9- 35 in SEQ ID NO:4, residues 9-35 in SEQ ID NO:5, residues 9-35 in SEQ ID NO:6, residues 12-38 in SEQ ID NO:7 and residues 2-28 in SEQ ID NO:8. wherein additionally, in each of i), ii) and iii), independently from each other, X9 is selected from T and S,

[0016] X10 is I,

[0017] Xu is selected from Q, E, V and L,

[0018] X13 is S,

[0019] X14 is selected from Q and R,

[0020] X17 is R;

[0021] Xis is selected from L, Q, E and V,

[0022] X24 is selected from R and H,

[0023] X25 is selected from R and Q,

[0024] X27is K,

[0025] X28 is L,

[0026] X32 is H, and

[0027] X35 is L. In certain embodiments, the IgA binding polypeptide according to the first aspect of the disclosure comprises the amino acid sequence comprising Sequence A according to the second aspect of the disclosure.

[0028] In some embodiments of the first and second aspects, the IgA binding polypeptide comprises an amino acid selected from T, S, G, Q, A, E, H, R, P, D, K and N, such as from S, G, A and E, such as from A and S, in a position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; an amino acid selected from E, G, R, D, K, Q, N, H and S, such as from G, R, E and D, such as from R and G, in a position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; and an amino acid selected from L, V, S, I, R and G, such as from V, L, I and R, such as from I, V and R, in a position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3. In particular embodiments of the first and second aspects, the polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; and a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3.

[0029] In some embodiments of the first and second aspects, the amino acid sequence of the IgA binding polypeptide comprises an E in a position in the amino acid sequence thereof that corresponds to position X43 in SEQ ID NO:3.

[0030] In some embodiments of the first and second aspects, the amino acid sequence of the IgA binding polypeptide comprises a Y in a position in the amino acid sequence thereof that corresponds to position X5 in SEQ ID NO:3.

[0031] In a third aspect of the disclosure, there is provided an IgA binding polypeptide multimer, wherein each monomer of the multimer comprises an IgA binding polypeptide which is independently selected from any IgA binding polypeptide defined in any one of the embodiments of the first and second aspects of the present disclosure.

[0032] In a fourth aspect, there is provided a polynucleotide encoding the IgA binding polypeptide as defined in any one of the embodiments of the first and second aspects of the present disclosure, or the IgA binding polypeptide multimer as defined in any one of the embodiments of the third aspect of the present disclosure. In a fifth aspect, there is provided an expression vector comprising the polynucleotide as defined in the fourth aspect of the present disclosure.

[0033] In a sixth aspect, there is provided a host cell comprising the expression vector as defined in the fifth aspect of the present disclosure.

[0034] In a seventh aspect, there is provided an adsorbent material comprising the IgA binding polypeptide as defined in any one of the embodiments of the first and second aspects of the present disclosure, or the IgA binding polypeptide multimer as defined in any one of the embodiments of the third aspect of the present disclosure, coupled to a solid support.

[0035] In some embodiments, said solid support is a separation matrix.

[0036] In an eighth aspect, there is provided a method of isolating IgA or a fragment thereof comprising a) contacting a liquid sample comprising said IgA or said fragment thereof with the adsorbent material as defined in any one of the embodiments of the seventh aspect of the present disclosure.

[0037] DETAILED DESCRIPTION

[0038] Aspects of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings and Examples. The terminology used herein is for the purpose of describing particular aspects of the disclosure only, and is not intended to limit the disclosure.

[0039] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0040] The term "IgA", as used herein, refers to immunoglobulin A that is also known as slgA in its secretory form. The term IgA includes its two isoforms, IgAl and lgA2. IgA binding polypeptides of the present disclosure bind both IgA isoforms with improved binding affinity, as demonstrated in the appended Examples, for example in Example 1. "An IgA polypeptide", as used herein, may thus be any of the two isoforms, and it may be used with reference to an IgA polypeptide mixture of the two isoforms. Moreover, the IgA polypeptide may be polyclonal IgA or monoclonal IgA, such as polyclonal IgA. It may be human IgA. In particular embodiments, the IgA polypeptide is polyclonal human IgA.

[0041] The term "% identity", as used throughout the disclosure, may for example be calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al., Nucleic Acids Research, 22: 4673-4680 (1994)). A comparison is made over the window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some instances be the target sequence. In other instances, the query sequence may constitute the shortest of the aligned sequences. The amino acid residues at each position are compared and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % identity.

[0042] Herein, "Xn" and "Xm" are used to indicate amino acid residues in positions n and m corresponding to positions n and m in e.g. SEQ ID NO:3 as defined herein, wherein n and m are integers which indicate the position of an amino acid residue within said sequence as counted from the N-terminal end of said sequence. For example, X24 and X13 indicate the amino acid residue in positions that corresponds to positions twenty-four and thirteen, respectively, from the N-terminal end of e.g. SEQ ID NO:3 as defined herein, in an alignment. To clarify the X numbering as used herein is based on the full-length scaffold comprising 58 amino acid residues. Hence X24 is to be understood as being the 24thamino acid residue in the 58-mer. The skilled person is able to make alignments of sequences to determine the position of an amino acid in accordance with the above.

[0043] The terms "IgA binding" and "having binding affinity for IgA" as used in this disclosure refer to a property of a polypeptide which may be tested for example by ELISA or the use of surface plasmon resonance (SPR) technology, as demonstrated in the appended Examples. IgA binding affinity may be tested in an experiment in which IgA, such as polyclonal IgA, IgAl and lgA2; or an Fc fragment thereof, such as human polyclonal IgA or an Fc fragment thereof, is immobilized on a sensor chip of the SPR instrument, and the sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing IgA, such as polyclonal IgA, IgAl and lgA2; or an Fc fragment thereof, such as human polyclonal IgA or an Fc fragment thereof, is passed over the chip. The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide for IgA. For example, when the polypeptide is immobilized on the sensor chip and IgA is used as analyte, 1:1 binding affinity cannot be calculated as the analyte has two binding sites. Accordingly, binding affinity in the context of the present disclosure is to be understood as apparent binding affinity. Characteristics of polypeptides of the disclosure as well as comparison to known polypeptides are discussed with reference to apparent binding affinity. If a quantitative measure is desired, for example to determine a KD value for the interaction, surface plasmon resonance methods may be used, wherein the IgA binding polypeptides (in a monomeric form) are used as analyte. Binding values may for example be defined in a Biacore (Cytiva) or ProteOn XPR 36 (Bio-Rad) instrument. IgA is suitably immobilized on a sensor chip of the instrument, and samples of the polypeptide whose affinity is to be determined are prepared by serial dilution and injected in random order. KD values may then be calculated from the results using for example the 1:1 Langmuir binding model of the BIAevaluation 4.1 software, or other suitable software, provided by the instrument manufacturer.

[0044] As explained below in more detail, IgA binding polypeptides and IgA binding polypeptide multimers of the present disclosure are able to bind to IgA including IgAl and lgA2 with improved binding characteristics, such as binding affinity, capacity and selectivity. Thus, IgA, IgAl and lgA2, such as human IgA, IgAl and lgA2, may be used in SPR experiments to further characterize the interactions. A shown in Example 5, the Fc region of polyclonal IgA, IgAl and / or lgA2 may also be used. For comparison of relative binding affinities, SEQ ID NO:9 and / or SEQ ID NO:10 can for example be used as controls under the same experimental conditions. The skilled person will appreciate that it may be useful to compare binding affinity (e.g. apparent binding affinity and / or KD values) obtained using the same assay and while some variation may occur between different assays, intra-assay comparisons generally demonstrate the same trends independent of the assay employed. In some embodiments, the binding affinity of an IgA binding polypeptide according to the present disclosure for an IgA polypeptide is considered higher than the binding affinity of SEQ ID NO:9 and / or SEQ ID NO:10 for the same IgA polypeptide if said binding affinity of the evaluated IgA binding polypeptide is more than 100%, such as more than 110%, such as more than 120%, of the corresponding binding affinity of SEQ ID NO:9 and / or SEQ ID NQ:10. As discussed above, said binding affinity may be apparent binding affinity. The skilled person will understand higher binding affinity in the present context, at least in light of the data shown in Examples 4 and 5. The terms "IgA binding polypeptide" and "IgA binding protein" mean a polypeptide or protein respectively, capable of binding to an antibody or a fragment thereof of an IgA isotype. Said fragment comprises the Fc fragment of IgA. It includes e.g. SpA, or any fragment or fusion protein thereof that has maintained said binding property. As it will be appreciated, it also includes any polypeptide construct which comprises an IgA Fc region and has maintained said binding property.

[0045] The term "essentially no binding affinity" means reduced or abolished binding to a non-target molecule, such as IgM, IgD, IgE and IgG, and / or the Fc region thereof, in the context of the present disclosure. It is to be understood that IgA binding polypeptides of the present disclosure have essentially no binding affinity for the Fc region of IgM, IgD, IgE and / or IgG. In particular embodiments, the non-target molecule is a VH3 class immunoglobulin, such as VH3 class IgG. It is to be understood that essentially no binding affinity in this particular context includes reduced or abolished binding to a VH3 region of an immunoglobulin, such as IgG. As explained above, binding affinity refers to apparent binding affinity. This may be measured using the techniques described above for assessing IgA binding affinity and may be evaluated in comparison to the binding affinity of one or more control polypeptide(s), such as SEQ ID NO:1, SEQ ID NO:2 and / or SEQ ID NO:3, for the same non-target molecule. The most suitable control polypeptide(s) may be selected based on which non-target molecule is tested for binding. For example, the binding of SEQ ID NO:2 to a VH3 region of an immunoglobulin, such as IgG, is considered reduced or abolished in comparison to the binding of SEQ ID NO:1 and / or SEQ ID NO:3 to the same VH3 region. In some embodiments, as shown in the appended examples, the binding to certain non-target molecules, such as IgM, IgD, IgE and / or IgG, and / or the Fc region thereof, is considered reduced or abolished in comparison to the binding of SEQ ID NO:1, SEQ ID NO:2 and / or SEQ ID NO:3 to the same nontarget molecule(s), and / or the Fc region thereof. The binding is considered reduced or abolished according to the present disclosure if the binding affinity of the evaluated binding polypeptide or multimer for said non-target molecule is at most 1 % of the binding affinity of one or more of said control polypeptides for the same non-target molecule. As demonstrated in Example 4, polypeptides of the present disclosure show essentially no binding affinity for VHl class IgG. Some IgA binding polypeptides of the present disclosure show binding affinity or "minimal binding affinity" for the VH3 region of immunoglobulins, such ashuman IgG, which may be reduced or abolished by further amino acid substitutions as demonstrated in the same Example. The IgA binding polypeptides may thus show binding affinity, minimal binding affinity or essentially no binding affinity for a VH3 class Fab region of an immunoglobulin, such as human IgG. In some embodiments, the IgA binding polypeptides show minimal binding affinity or essentially no binding affinity for a VH3 class Fab region of an immunoglobulin, such as human IgG. In particular embodiments, the IgA binding polypeptides show essentially no binding affinity for a VH3 class Fab region of an immunoglobulin, such as human IgG. The term "minimal binding affinity" corresponds to a weak binding ability (i.e. weak apparent binding affinity) to a non-target molecule, such as to VH3 class IgG as shown e.g. in Fig. 6 and Example 4 for SEQ ID NQ:104. As demonstrated for example in Fig. 5, binding affinity or "minimal binding affinity" for VH3 class IgG of the IgA binding polypeptides (if retained) is lower than the binding affinity thereof to the target molecule, such as IgA, for example polyclonal IgA (being compared at the same analyte concentrations). The term "linker" herein means an element linking two polypeptide units, monomers or domains to each other in a multimer.

[0046] The term "spacer" herein means an element connecting a polypeptide or a polypeptide multimer to a support.

[0047] As discussed above, in a first aspect of the disclosure, there is provided an IgA binding polypeptide derived from SpA or any domain thereof, wherein said polypeptide has a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NO:9 for the same IgA polypeptide. As an alternative, IgA binding polypeptides as disclosed herein may be evaluated for IgA binding affinity in comparison to IgA binding affinity of SEQ ID NO:10. Thus, as an alternative, there is provided an IgA binding polypeptide derived from SpA or any domain thereof, wherein said polypeptide has a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NQ:10 for the same IgA polypeptide. Thus it will be understood that in some embodiments, there is provided an IgA binding polypeptide derived from SpA or any domain thereof, wherein said polypeptide has a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NO:9 and SEQ ID NQ:10 for the same IgA polypeptide.

[0048] As demonstrated in Example 5, IgA binding polypeptides of the present disclosure have binding affinity for the Fc region of IgA, such as human IgA. The IgA binding polypeptides may show binding affinity, minimal binding affinity or essentially no binding affinity for a VH3 class Fab region of IgA, such as human IgA, as discussed above and shown in Examples 5 and 4. In some embodiments, the IgA binding polypeptides show minimal binding affinity or essentially no binding affinity for a VH3 class Fab region of IgA, such as human IgA. In particular embodiments, the IgA binding polypeptides show essentially no binding affinity for a VH3 class Fab region of IgA, such as human IgA. The IgA binding polypeptides of the present disclosure have a higher binding affinity for an Fc region of the IgA polypeptide compared to the binding affinity of SEQ ID NO:9 and / or SEQ ID NQ:10 for the same Fc region. Moreover, it is to be understood that the IgA binding polypeptides bind IgA polypeptide fragments that comprise the Fc region of the IgA polypeptide. Thus ligands of the disclosure could for example be used in methods for isolating any antibody fragment, provided that it comprises at least an Fc region of IgA, such as human IgA. The inventors demonstrate binding of the disclosed polypeptides to polyclonal IgA, the Fc fragment thereof, IgAl and lgA2 but it is to be understood that the IgA binding polypeptides have affinity to additional molecules that comprises the Fc region of IgA, such as human IgA. These include for example bi- or multi-specific antibodies, and, as mentioned above, antibody fragments.

[0049] The results presented in the appended Examples also demonstrate that the IgA binding polypeptides have a higher binding capacity for the IgA polypeptide compared to SEQ ID NO:9 and / or SEQ ID NO:10 for the same IgA polypeptide. Thus in some embodiments, the IgA binding polypeptides have a higher binding capacity for the IgA polypeptide compared to SEQ ID NO:9 and / or SEQ ID NQ:10 for the same IgA polypeptide.

[0050] The skilled person will appreciate that the IgA binding polypeptide derived from SpA or any domain thereof as disclosed herein may for example, but not necessarily, be derived from any one of domains A (SEQ ID NO:6), B (SEQ ID NO:5), C (SEQ ID NO:4), D (SEQ ID NO:7) and E (SEQ ID NO:8) of SpA or derivatives thereof, such as domain Z (SEQ ID NO:3) or variants thereof, such as SEQ ID NO:1 or SEQ ID NO:2. The IgA binding polypeptide derived from an SpA domain may thus be a derivative, a mutant, a variant or a fragment of an SpA domain as defined above. In certain embodiments, the IgA binding polypeptide comprises an amino acid sequence that has at least 70% identity to a sequence selected from the group consisting of SEQ ID NO:l-8. In some embodiments, the IgA binding polypeptide comprises an amino acid sequence that has at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, identity to a sequence selected from the group consisting of SEQ ID NO:l-8. In some embodiments, said amino acid sequence has at least 70% identity to SEQ ID NO:1 and / or SEQ ID NO:2, such as SEQ ID NO:2. In some embodiments, said amino acid sequence has at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, identity to SEQ ID NO:1 and / or SEQ ID NO:2, such as SEQ ID NO:2. IgA binding polypeptides according to the present disclosure exhibit binding preference for IgA among other immunoglobulin isotypes, such as IgM, IgD, IgE and IgG. Thus, these IgA binding polypeptides are particularly advantageous for applications wherein IgA is to be purified from a mixture of immunoglobulin isotypes. In addition, the present inventors have surprisingly found that introducing amino acid mutations in positions X33, X40 and X51 of the IgA binding polypeptides derived from SpA or domains thereof further improves selectivity of the polypeptides for IgA molecules. Thus, in one embodiment the IgA binding polypeptide has binding affinity to an Fc region of an immunoglobulin, and has lower binding affinity for a VH3 region of trastuzumab compared to the binding affinity of SEQ ID NO:3 for the same VH3 region. The skilled person will appreciate in the context of the present disclosure that said Fc region of the immunoglobulin may be an Fc region of IgA. As shown in the appended Examples, binding affinity for VH3 class IgG may also be tested using Denosumab. Thus, in some embodiments, the IgA binding polypeptide has binding affinity to an Fc region of an immunoglobulin, and has lower binding affinity for a VH3 region of Denosumab compared to the binding affinity of SEQ ID NO:3 for the same VH3 region. As demonstrated by experimental data in Example 4, said lower binding affinity corresponds to essentially no binding affinity for a VH3 region of Denosumab and / or trastuzumab. Such mutations, which further improve selectivity of the IgA binding polypeptides for IgA molecules, lead to reduced or abolished ability of the polypeptides to bind the VH3 region of immunoglobulins, and are described in detail in WQ2023 / 046886. For the sake of brevity, WQ2023 / 046886 is included hereby by reference in its entirety and these amino acid mutations as described in WQ2023 / 046886 are only briefly mentioned herein in the context of the present disclosure. It is to be understood that any amino acid mutation or a combination of amino acid mutations that leads to reduced or abolished ability of the polypeptides to bind the VH3 region of immunoglobulins is beneficial for improved selectivity of the IgA binding polypeptides according to the present disclosure. In some embodiments, the IgA binding polypeptide according to the first aspect comprises an amino acid selected from T, S, G, Q, A, E, H, R, P, D, K and N, such as from S, G, A and E, such as from A and S, in a position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; an amino acid selected from E, G, R, D, K, Q, N, H and S, such as from G, R, E and D, such as from R and G, in a position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; and an amino acid selected from L, V, S, I, R and G, such as from V, L, I and R, such as from I, V and R, in a position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3. In some embodiments, when the amino acid in the position corresponding to position X51 in SEQ ID NO:3 is L then the amino acids in positions that correspond to positions X33 and X40in SEQ ID NO:3 are selected from AD, HK, EG, ER, GR, AK, AR, PK, RR and KK, respectively, and when the amino acid in the position corresponding to position X51 in SEQ ID NO:3 is G then the amino acids in positions that correspond to positions X33 and X40 in SEQ ID NO:3 are TK, respectively. In some embodiments, the IgA binding polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3. In some embodiments, the IgA binding polypeptide comprises a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3. In some embodiments, the IgA binding polypeptide comprises a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3. In a particular embodiment, the IgA binding polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; and a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3.

[0051] The present inventors have found that certain amino acid mutations increase solubility of the herein disclosed IgA binding polypeptides at neutral pH (pH 7). This may be particularly beneficial for enabling purification of recombinantly produced IgA binding polypeptides according to the present disclosure under optimal conditions. Such mutations are designed for lowering the pl of the IgA binding polypeptides without altering the binding affinity thereof for IgA. Thus, in some embodiments, the IgA binding polypeptide comprises an E in a position in the amino acid sequence thereof that corresponds to position X43 in SEQ ID NO:3. In a particular embodiment, the IgA binding polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3; and an E in a position in the amino acid sequence thereof that corresponds to position X43 in SEQ ID NO:3.

[0052] In addition to the above, the present inventors have found that certain amino acid substitutions may be beneficial for increasing the extinction coefficient of the IgA binding polypeptides according to the present disclosure, which is useful to improve detectability of these in chromatography applications. Thus, in certain embodiments, the IgA binding polypeptide comprises a Y in a position in the amino acid sequence thereof that corresponds to position X5 in SEQ ID NO:3. In a particular embodiment, the IgA binding polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3; an E in a position in the amino acid sequence thereof that corresponds to position X43 in SEQ ID NO:3; and a Y in a position in the amino acid sequence thereof that corresponds to position X5 in SEQ ID NO:3.

[0053] In certain embodiments of the first aspect of the present disclosure, the IgA binding polypeptide comprises an amino acid sequence as defined in any one of the embodiments of the second aspect of the present disclosure. In other words, it is to be understood that that embodiments discussed below in relation to the second aspect of the disclosure are equally relevant for the first aspect of the disclosure. For the sake of brevity, these are not repeated herein or just briefly mentioned. The present inventors have surprisingly found that IgA binding polypeptides according to the present disclosure can be obtained with particularly advantageous binding characteristics for IgA by introducing certain amino acid mutations in positions Xg, Xio, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35 of polypeptides derived from SpA or any domain thereof. The presently identified mutations in positions X9, Xio, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35 of the IgA binding polypeptides as defined herein improve IgA binding affinity and selectivity for IgA among other immunoglobulin isotypes. Accordingly, in a second aspect of the present disclosure, there is provided an IgA binding polypeptide comprising an amino acid sequence comprising Sequence A, which Sequence A consists of an amino acid sequence selected from i), ii) and iii), wherein i), ii) and iii) are defined as follows: i) X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35 (SEQ ID NO:11), wherein X33 is selected from T, S, G, Q, A, E, H, R, P, D, K and N; ii) an amino acid sequence which has at least 81% identity to a sequence defined by i); iii) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 9-35 in SEQ ID NO:1, residues 9-35 in SEQ ID NO:2, residues 9-35 in SEQ ID NO:3, residues 9- 35 in SEQ ID NO:4, residues 9-35 in SEQ ID NO:5, residues 9-35 in SEQ ID NO:6, residues 12-38 in SEQ ID NO:7 and residues 2-28 in SEQ ID NO:8; wherein additionally, in each of i), ii) and iii), independently from each other, X9 is selected from T and S, Xio is I,

[0054] Xu is selected from Q, E, V and L,

[0055] X13 is S,

[0056] X14 is selected from Q and R,

[0057] X17 is R;

[0058] Xis is selected from L, Q, E and V,

[0059] X24 is selected from R and H,

[0060] X25 is selected from R and Q,

[0061] X27is K, X28 is L, X32 is H, and

[0062] X35 is L.

[0063] It is to be understood that positions X9, X10, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35 refer to positions 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32 and 35 in an SpA domain, such as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. The skilled person will appreciate that in certain SpA domains or derivatives thereof, positions X9, X10, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35 may refer to positions which have altered numbering but correspond to positions 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32 and 35 in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in alignment. Thus, Sequence A may be comprised in the amino acid sequence of the IgA binding polypeptide in an amino acid region that corresponds to positions X9 to X35 in SEQ ID NO:3.

[0064] To clarify, it will be appreciated that the % identity in ii) and iii) does not relate to X9, X10, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35. Thus, the amino residues in positions X9, X10, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35 are as defined above in IgA binding polypeptides encompassed by the definition according to ii) and iii). As the skilled person will realize, the function of any polypeptide is dependent on the tertiary structure of the polypeptide. It is therefore possible to make minor changes to the sequence of amino acids in a polypeptide without affecting the function thereof. Thus, the disclosure encompasses modified variants of the IgA binding polypeptides, which are such that the IgA binding characteristics are retained.

[0065] Also encompassed by the present disclosure is an IgA binding polypeptide comprising Sequence A which consists of an amino acid sequence with at least 81% identity to a polypeptide as defined in i). In some embodiments, the polypeptide comprises Sequence A which consists of an amino acid sequence with at least 87%, such as at least 90%, such as at least 93%, such as at least 96% identity to a polypeptide as defined in i). For example, it is possible that an amino acid residue belonging to a certain functional grouping of amino acid residues (e.g. hydrophobic, hydrophilic, polar etc.) could be exchanged for another amino acid residue from the same functional group. In some embodiments, such changes may be made in any position except for X9, X10, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35 of the sequence of the IgA binding polypeptide as disclosed herein. In other embodiments, such changes may be made only in the non-variable positions, also denoted scaffold amino acid residues. In such cases, changes are not allowed in the variable positions, i.e. positions denoted with an "X" in sequence i).

[0066] Also encompassed by the present disclosure is an IgA binding polypeptide comprising Sequence A which consists of an amino acid sequence with at least 70% identity to any sequence selected from the group consisting of: residues 9-35 in SEQ ID NO:1, residues 9-35 in SEQ ID NO:2, residues 9-35 in SEQ ID NO:3, residues 9-35 in SEQ ID NO:4, residues 9-35 in SEQ ID NO:5, residues 9-35 in SEQ ID NO:6, residues 12-38 in SEQ ID NO:7 and residues 2-28 in SEQ ID NO:8. In some embodiments, Sequence A consists of an amino acid sequence with at least 74%, such as at least 77%, such as at least 80%, such as at least 83%, such as at least 87%, such as at least 90%, such as at least 93%, such as at least 96%, identity to any sequence selected from the group consisting of: residues 9-35 in SEQ ID NO:1, residues 9-35 in SEQ ID NO:2, residues 9-35 in SEQ ID NO:3, residues 9-35 in SEQ ID NO:4, residues 9-35 in SEQ ID NO:5, residues 9-35 in SEQ ID NO:6, residues 12-38 in SEQ ID NO:7 and residues 2-28 in SEQ ID NO:8.

[0067] In one embodiment of the IgA binding polypeptides as disclosed herein, said Sequence A fulfills the criteria i) and ii), or the criteria i) and iii), or the criteria ii) and iii). In one embodiment, said Sequence A fulfills all criteria i), ii) and iii).

[0068] In some embodiments of the second aspect of the disclosure, X9 is S. In some embodiments, Xu is selected from E and V. In some particular embodiments, Xu is E. In some embodiments, X14 is R. In certain embodiments, Xis is selected from L and Q. In some embodiments, Xis is Q. In some embodiments, X24 is H. In some embodiments, X25 is Q.

[0069] As discussed above in relation to the first aspect of the disclosure, certain amino acid residues in position X33 of the IgA binding polypeptides as disclosed herein may be particularly advantageous for IgA selectivity. Thus, in some embodiments, X33 in sequence i), ii) and / or iii) is selected from T, S, G, Q, A, E, H, R, P, D, K and N, such as from S, G, A and E. In some embodiments, X33 in sequence i), ii) and / or iii) is selected from A and S. In one particular embodiment, X33 in sequence i), ii) and / or iii) is S. In some embodiments of the second aspect of the disclosure, sequence i) corresponds to the sequence from position X9 to position X35 in a sequence selected from the group consisting of SEQ ID NO:14-29, such as the group consisting of SEQ ID NO:26-29. In a particular embodiment, sequence i) corresponds to the sequence from position X9 to position X35 in SEQ ID NO:29. Beneficial binding characteristics of such IgA binding polypeptides, such as the 16 exemplary IgA binding polypeptides according to SEQ ID NO:14-29, are described in the appended Examples.

[0070] As discussed above, the herein described amino acid substitutions in positions X9, X10, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35 are demonstrated to be advantageous for IgA binding affinity and selectivity. Thus, in one embodiment of the second aspect of the disclosure, the IgA binding polypeptide comprising the amino acid sequence which comprises Sequence A has a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NO:9 for the same IgA polypeptide. Moreover, in some embodiments, IgA binding polypeptide has binding affinity to an Fc region of an immunoglobulin, and has lower binding affinity for a VH3 region of trastuzumab compared to the binding affinity of SEQ ID NO:3 for the same VH3 region. Accordingly, in some embodiments, the IgA binding polypeptide comprises an amino acid selected from T, S, G, Q, A, E, H, R, P, D, K and N, such as from S, G, A and E, such as from A and S, in a position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; an amino acid selected from E, G, R, D, K, Q, N, H and S, such as from G, R, E and D, such as from R and G, in a position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; and an amino acid selected from L, V, S, I, R and G, such as from V, L, I and R, such as from I, V and R, in a position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3. In some embodiments, when the amino acid in the position corresponding to position X51 in SEQ ID NO:3 is L then the amino acids in positions that correspond to positions X33 and X40 in SEQ ID NO:3 are selected from AD, HK, EG, ER, GR, AK, AR, PK, RR and KK, respectively, and when the amino acid in the position corresponding to position X51 in SEQ ID NO:3 is G then the amino acids in positions that correspond to positions X33 and X40 in SEQ ID NO:3 are TK, respectively. In some embodiments, the IgA binding polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3. In some embodiments, the IgA binding polypeptide comprises a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3. In some embodiments, the IgA binding polypeptide comprises a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3. In a particular embodiment, the IgA binding polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; and a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3.

[0071] As discussed in relation to the first aspect of the disclosure, in some embodiments, the IgA binding polypeptide according to the second aspect comprises an E in a position in the amino acid sequence thereof that corresponds to position X43 in SEQ ID NO:3. In a particular embodiment, the IgA binding polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3; and an E in a position in the amino acid sequence thereof that corresponds to position X43 in SEQ ID NO:3. Moreover, in certain embodiments, the IgA binding polypeptide according to the second aspect comprises a Y in a position in the amino acid sequence thereof that corresponds to position X5 in SEQ ID NO:3. In a particular embodiment, the IgA binding polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3; an E in a position in the amino acid sequence thereof that corresponds to position X43 in SEQ ID NO:3; and a Y in a position in the amino acid sequence thereof that corresponds to position X5 in SEQ ID NO:3.

[0072] The modifications for improved IgA binding affinity identified by the present inventors is applicable to polypeptides based on the different three-helical domains of SpA, such as any one of the domains A (SEQ ID NO:6), B (SEQ ID NO:5), C (SEQ ID NO:4), D (SEQ ID NO:7) and E (SEQ ID NO:8), in particular domain B, and derivatives thereof. In particular, said modifications may be applicable to polypeptides based on the three-helical bundle protein domain Z (SEQ ID NO:3), which domain Z is derived from domain B of SpA or to the variant SEQ ID NO:1. Such polypeptides have been described in: WQ2003080655, EP230869A2, Sjodahl, Eur J Biochem 1977 Sep;78(2):471-90, WQ2008039141, WQ2015005859, US10308690, WQ2016079033, WO2017194596, US8859726, US9187555, US9683013, US10189891, US9663558, WQ2016079034, JP2006304633A, WQ2007097361, EP1992692A1, EP2202310A2, WQ2012083425, WQ2012086660, US20120208234, EP2495254A1, WO2012133342, W02013109302A2, W02015034000, W02015034056, CN105481954A,

[0073] WO2016152946A1, WQ2017009421, WQ2017014261, WQ2017014260, WQ2018009006, WQ2018029158, WQ2018029157, WQ2019030156, CN109721645A, WQ2019093439, WQ2020040307and WQ2020157281, which are hereby incorporated by reference. In addition, said modifications may be applicable to the variant SEQ ID NO:2 for improved selectivity, as discussed above.

[0074] Thus, the present inventors envision that the mutations in to X9, X10, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35 as disclosed herein may be introduced into any one of the previously known polypeptides derived from SpA or a domain thereof to achieve the effect of improved binding characteristics to IgA or a fragment thereof. Thus, in certain embodiments, the IgA binding polypeptide according to the second aspect of the disclosure comprises an amino acid sequence that has at least 70% identity to a sequence selected from the group consisting of SEQ ID NO:l-8. In some embodiments, the IgA binding polypeptide according to the second aspect of the disclosure comprises an amino acid sequence that has at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, identity to a sequence selected from the group consisting of SEQ ID NO:l-8. In some embodiments, said amino acid sequence has at least 70% identity to SEQ ID NO:1 and / or SEQ ID NO:2, such as SEQ ID NO:2. In some embodiments, said amino acid sequence has at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, identity to SEQ ID NO:1 and / or SEQ ID NO:2, such as SEQ ID NO:2.

[0075] In certain embodiments of the second aspect of the present disclosure, the amino acid sequence of the IgA binding polypeptide comprises Sequence B, which Sequence B consists of an amino acid sequence selected from iv), v) and vi), wherein iv), v) and vi) are defined as follows: iv) X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35DDPSX40SX42X43X4 4LX46X47AX49KX51 (SEQ ID NO:12); v) an amino acid sequence which has at least 81% identity to a sequence defined by iv); vi) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 9-51 in SEQ ID NO:1, residues 9-51 in SEQ ID NO:2, residues 9-51 in SEQ ID NO:3, residues 9- 51 in SEQ ID NO:4, residues 9-51 in SEQ ID NO:5, residues 9-51 in SEQ ID NO:6, residues 12-54 in SEQ ID NO:7 and residues 2-44 in SEQ ID NO:8; wherein the amino acid sequence from position X9 to position X35 is Sequence A is as defined above; and wherein additionally, in each of iv), v) and vi), independently from each other,

[0076] X40 is selected from V, E, G, R, D, K, Q, N, H and S, such as from E, G, R, D, K, Q, N, H and S, such as from G, R, E and D, such as from R and G, X42 is selected from K, A and T, X43 is selected from A, E and N, X44 is selected from I, L and V, X46 is selected from A, S and G, X47 is selected from E and K,

[0077] X49 is selected from K and Q, and

[0078] X51 is selected from L, V, S, I, R and G, such as from V, L, I and R, such as from I, V and R.

[0079] Also encompassed by the present disclosure is an IgA binding polypeptide comprising Sequence B which consists of an amino acid sequence with at least 81% identity to a polypeptide as defined in iv). In some embodiments, the polypeptide comprises Sequence B which consists of an amino acid sequence at least 87%, such as at least 90%, such as at least 93%, such as at least 96% identical to a polypeptide as defined in iv). Also encompassed by the present disclosure is an IgA binding polypeptide comprising Sequence B which consists of an amino acid sequence with at least 70% identity to any sequence selected from the group consisting of: residues 9-51 in SEQ ID NO:1, residues 9-51 in SEQ ID NO:2, residues 9-51 in SEQ ID NO:3, residues 9-51 in SEQ ID NO:4, residues 9-51 in SEQ ID NO:5, residues 9-51 in SEQ ID NO:6, residues 12-54 in SEQ ID NO:7 and residues 2-44 in SEQ ID NO:8. In some embodiments, Sequence B consists of an amino acid sequence with at least 74%, such as at least 77%, such as at least 80%, such as at least 83%, such as at least 87%, such as at least 90%, such as at least 93%, such as at least 96%, identity to any sequence selected from the group consisting of: residues 9-51 in SEQ ID NO:1, residues 9-51 in SEQ ID NO:2, residues 9-51 in SEQ ID NO:3, residues 9-51 in SEQ ID NO:4, residues 9-51 in SEQ ID NO:5, residues 9-51 in SEQ ID NO:6, residues 12-54 in SEQ ID NO:7 and residues 2-44 in SEQ ID NO:8.

[0080] In some embodiments of the second aspect of the present disclosure wherein the amino acid sequence of the IgA binding polypeptide comprises Sequence B, when the amino acid in position X51 is L then the amino acids in positions X33 and X40 are selected from SV, SQ, AD, HK, EG, ER, GR, AK, AR, PK, RR and KK, such as from AD, HK, EG, ER, GR, AK, AR, PK, RR and KK, respectively, and when the amino acid in position X51 is G then the amino acids in positions X33 and X40 is TK, respectively. In some embodiments, X40 is G. In some embodiments, X42 is K. In some embodiments, X43 is selected from A and E. In some embodiments, X43 is E. In some embodiments, X44 is I. In some embodiments, X46 is A. In some embodiments, X47 is E. In some embodiments, X49 is K. In some embodiments, X51 is V.

[0081] As demonstrated in the appended Examples, in some embodiments of the second aspect of the present disclosure wherein the amino acid sequence of the IgA binding polypeptide comprises Sequence B, sequence iv) corresponds to the sequence from position X9 to position X51 in a sequence selected from the group consisting of SEQ ID NO:14-61, such as the group consisting of SEQ ID NO:30-61, such as the group consisting of SEQ ID NO:46-61. In some embodiments, sequence iv) corresponds to the sequence from position X9 to position X51 in a sequence selected from the group consisting of SEQ ID NO:26-29, SEQ ID NO:42-45 and SEQ ID NO:58-61; such as the group consisting of SEQ ID NO:42-45 and SEQ ID NO:58-61; such as the group consisting of SEQ ID NO:58-61. In some embodiments, sequence iv) corresponds to the sequence from position X9 to position X51 in a sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:45 and SEQ ID NO:61; such as the group consisting of SEQ ID NO:45 and SEQ ID NO:61; such as the sequence from position X9 to position X51 in SEQ ID NO:61.

[0082] In certain embodiments of the second aspect of the present disclosure, the amino acid sequence of the IgA binding polypeptide comprises Sequence C, which Sequence C consists of an amino acid sequence selected from vii), viii) and ix), wherein vii), viii) and ix) are defined as follows: vii) X5X6KX8X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35DDPSX40S X42X43X44LX46X47AX49KX51 (SEQ ID NO:13); viii) an amino acid sequence which has at least 81% identity to a sequence defined by vii); ix) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 5-51 in SEQ ID NO:1, residues 5-51 in SEQ ID NO:2, residues 5-51 in SEQ ID NO:3, residues 5- 51 in SEQ ID NO:4, residues 5-51 in SEQ ID NO:5, residues 5-51 in SEQ ID NO:6, residues 8-54 in SEQ ID NO:7; wherein the amino acid sequence from position Xg to position X51 is Sequence B is as defined above; and wherein additionally, in each of vii), viii) and ix), independently from each other,

[0083] X5 is selected from F and Y,

[0084] Xe is selected from D and N, and Xs is selected from E, D and A.

[0085] Also encompassed by the present disclosure is an IgA binding polypeptide comprising Sequence C which consists of an amino acid sequence with at least 81% identity to a polypeptide as defined in vii). In some embodiments, the polypeptide comprises Sequence C which consists of an amino acid sequence at least 87%, such as at least 90%, such as at least 93%, such as at least 96% identical to a polypeptide as defined in vii). Also encompassed by the present disclosure is an IgA binding polypeptide comprising Sequence C which consists of an amino acid sequence with at least 70% identity to any sequence selected from the group consisting of: residues 5-51 in SEQ ID NO:1, residues 5-51 in SEQ ID NO:2, residues 5-51 in SEQ ID NO:3, residues 5-51 in SEQ ID NO:4, residues 5-51 in SEQ ID NO:5, residues 5-51 in SEQ ID NO:6, residues 8-54 in SEQ ID NO:7. In some embodiments, Sequence C consists of an amino acid sequence with at least 74%, such as at least 77%, such as at least 80%, such as at least 83%, such as at least 87%, such as at least 90%, such as at least 93%, such as at least 96%, identity to any sequence selected from the group consisting of: residues 5-51 in SEQ ID NO:1, residues 5-51 in SEQ ID NO:2, residues 5-51 in SEQ ID NO:3, residues 5-51 in SEQ ID NO:4, residues 5-51 in SEQ ID NO:5, residues 5-51 in SEQ ID NO:6, residues 8-54 in SEQ ID NO:7.

[0086] In some embodiments, X5 is Y. In some embodiments, Xe is D. In some embodiments, X8is E.

[0087] As demonstrated in the appended Examples, in some embodiments of the second aspect of the present disclosure wherein the amino acid sequence of the IgA binding polypeptide comprises Sequence C, sequence vii) corresponds to the sequence from position X5 to position X51 in a sequence selected from the group consisting of SEQ ID NO:14-77, such as the group consisting of SEQ ID NO:30-77, such as the group consisting of SEQ ID NO:46-77, such as the group consisting of SEQ ID NO:62-77. In some embodiments, sequence vii) corresponds to the sequence from position Xs to position X51 in a sequence selected from the group consisting of SEQ ID NO:26-29, SEQ ID NO:42-45, SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:42-45, SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:74-77. In some embodiments, sequence vii) corresponds to the sequence from position X5 to position X51 in a sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:45, SEQ ID NO:61 and SEQ ID NO:77; such as the group consisting of SEQ ID NO:45, SEQ ID NO:61 and SEQ ID NO:77; such as the group consisting SEQ ID NO:61 and SEQ ID NO:77; such as the sequence from position X5 to position X51 in SEQ ID NO:77.

[0088] In some embodiments of the second aspect of the present disclosure, the amino acid sequence comprised in the IgA binding polypeptide is a sequence selected from the group consisting of SEQ ID NO:14-77, such as the group consisting of SEQ ID NQ:30-77, such as the group consisting of SEQ ID NO:46-77, such as the group consisting of SEQ ID NO:62-77. In some embodiments, the amino acid sequence comprised in the IgA binding polypeptide is a sequence selected from the group consisting of SEQ ID NO:26-29, SEQ ID NO:42-45, SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:42-45, SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:74-77. In some embodiments, the amino acid sequence comprised in the IgA binding polypeptide is a sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:45, SEQ ID NO:61 and SEQ ID NO:77; such as the group consisting of SEQ ID NO:45, SEQ ID NO:61 and SEQ ID NO:77; such as the group consisting of SEQ ID NO:61 and SEQ ID NO:77. In a particular embodiment, the amino acid sequence comprised in the IgA binding polypeptide is SEQ ID NO:77.

[0089] In some embodiments of the present disclosure, the polypeptide derived from SpA, said Sequence A, said Sequence B or said Sequence C as defined above "forms part of" a three-helix bundle protein domain. This is understood to mean that the sequence of the SpA derived polypeptide, said Sequence A, said Sequence B or said Sequence C is "inserted" into or "grafted" onto the sequence of the original three- helix bundle domain, such that the grafted sequence replaces a similar structural motif in the original domain. For example, without wishing to be bound by theory, the Sequence A is thought to constitute two of the three helices of a three-helix bundle and can therefore replace such a two-helix motif within any three-helix bundle. As the skilled person will realize, the replacement of two helices of the three-helix bundle domain by two Sequence A helices has to be performed so as not to affect the basic structure of the polypeptide. That is, the overall folding of the Ca backbone of the polypeptide according to this embodiment of the invention is substantially the same as that of the three-helix bundle protein domain of which it forms a part, e.g. having the same elements of secondary structure in the same order etc. Thus, for example, a Sequence A according to the disclosure "forms part" of a three-helix bundle domain if the polypeptide according to this embodiment has the same fold as the original domain, implying that the basic structural properties are shared, those properties e.g. resulting in similar CD spectra. The skilled person is aware of other parameters that are relevant.

[0090] Thus, in some embodiments of the first and the second aspects of the present disclosure, said polypeptide derived from SpA or any domain thereof, said Sequence A, said Sequence B or said Sequence C forms part of a three-helix bundle protein domain. In certain embodiments, said polypeptide derived from SpA or any domain thereof, said Sequence A, said Sequence B or said Sequence C forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain. In some embodiments, said three-helix bundle protein domain is selected from bacterial receptor domains. In some embodiments, said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof. As discussed above, the IgA binding polypeptide has essentially no binding affinity for an Fc fragment of an immunoglobulin selected from the group consisting of IgM, IgD, IgE and IgG. The IgA binding polypeptide may exhibit binding affinity or a minimal binding affinity for VH3 class immunoglobulins, such as VH3 class IgG, as demonstrated in the appended Examples. In some particular embodiments however, the IgA binding polypeptide has essentially no binding affinity for VH3 class immunoglobulins, such as VH3 class IgG. In some embodiments, the IgA binding polypeptide has thus essentially no binding affinity for an immunoglobulin selected from the group consisting of IgM, IgD, IgE and IgG. This is also shown and further explained in the Examples.

[0091] A further polypeptide domain with the same functionality may be attached to the IgA binding polypeptide as defined herein, for example any IgA binding polypeptide as defined in any one of the embodiments according to the first and the second aspects of the present disclosure, thus achieving an IgA binding polypeptide multimer. Such multimers may in particular be useful to increase the efficiency of binding IgA compared to the monomer variants thereof (in other words the IgA binding polypeptides). Thus, in a third aspect of the present disclosure, there is provided an IgA binding polypeptide multimer, wherein each monomer of the multimer comprises an IgA binding polypeptide which is independently selected from any IgA binding polypeptide defined in any one of the embodiments discussed in relation to the first and the second aspects of the present disclosure. Said multimer is understood to comprise at least two IgA binding polypeptides as disclosed herein as monomer units, the amino acid sequences of which may be the same or different. Said multimer may thus be selected from the group consisting of dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer and decamer. In some embodiments, said multimer is selected from the group consisting of tetramer and hexamer. In particular embodiments, said multimer is a tetramer. These monomers within the multimer may have the same amino acid sequence, but alternatively, they may have different amino acid sequences. Said multimer may thus be a heteromer or a homomer. In certain embodiments, said multimer is a homomer.

[0092] It will be appreciated that monomers in the multimers discussed herein may be directly coupled to each other or spaced apart by linker sequences. The skilled person appreciates that the presence or absence of linker sequences may be different between different monomer moieties in a multimer, and if linkers are present, the sequence of each individual linker may be the same or different. As the skilled person understands, the construction of a multimer, for example as a fusion protein, often involves use of linkers between the monomer moieties to be fused. The skilled person is aware of different kinds of linkers with different properties, such as flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers. Linkers may be used in order to for example increase stability or improve folding of fusion proteins, to increase expression or to improve activity, affinity and / or binding capacity. Thus, in some embodiments of the third aspect of the disclosure, the IgA binding polypeptide multimer further comprises at least one linker. The linker may for example be selected from the group consisting of flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers. Alternatively, the linker may be a non-peptidic linker. For example, two or more monomers, in other words monomer units or moieties, within the multimer can be linked by elements comprising oligomeric or polymeric species, such as elements comprising up to 15 or 30 amino acids, such as 1-5, 1-10 or 5-10 amino acids. In certain embodiments, said linker comprises up to 15 amino acid residues. The nature of such a linker should preferably not destabilize the spatial conformation of the protein units, that is of the IgA binding polypeptide monomers within the multimer. This can e.g. be achieved by avoiding the presence of proline in the linkers. Furthermore, said linkers should preferably also be sufficiently stable in alkaline environments not to impair the properties of the mutated protein units. For this purpose, it is advantageous if the linkers do not contain asparagine. It can additionally be advantageous if the linker does not contain glutamine. The IgA binding polypeptides disclosed herein may be linked to each other directly by peptide bonds between the C-terminal and N-terminal ends of the polypeptides. In some embodiments, said multimer thus comprises an amino acid sequence selected from the group consisting of SEQ ID NO:78-93, such as the group consisting of SEQ ID NO:82-93, such as the group consisting of SEQ ID NO:86-93, such as the group consisting of SEQ ID NQ:90-93. In yet other embodiments, said multimer comprises an amino acid sequence selected from the group consisting of SEQ ID NO:81, SEQ ID NO:85, SEQ ID NO:89 and SEQ ID NO:93; such as the group consisting of SEQ ID NO:85, SEQ ID NO:89 and SEQ ID NO:93; such as the group consisting of SEQ ID NO:89 and SEQ ID NO:93. In a particular embodiment, said multimer comprises SEQ ID NO:93.

[0093] The skilled person will understand that various modifications and / or additions can be made to an IgA binding polypeptide or to an IgA binding polypeptide multimer according to any aspect disclosed herein in order to tailor the polypeptide or multimer to a specific application without departing from the scope of the present disclosure. For example, in one embodiment there is provided an IgA binding polypeptide or an IgA binding polypeptide multimer as described herein, which polypeptide has been extended by and / or comprises additional amino acids at the C terminus and / or N terminus. Such a polypeptide or multimer should be understood as a polypeptide or multimer having one or more additional amino acid residues at the very first and / or the very last position in the polypeptide chain, i.e. at the N- and / or C-terminus of the polypeptide or multimer. In some embodiments of the first, the second and the third aspects of the present disclosure, the IgA binding polypeptide or the IgA binding polypeptide multimer thus comprises additional amino acids at the C-terminal and / or N-terminal end thereof. For example, the IgA binding polypeptide or the IgA binding polypeptide multimer may further at the N- terminal end comprise a plurality of amino acid residues originating from the cloning process or constituting a residue from a cleaved off signalling sequence. The number of additional amino acid residues may e.g. be 15 or less, such as 10 or less or 5 or less. The number of additional amino acid residues may for example be one, two, three, four, five, six, seven, eight, nine, ten or more. Said additional amino acid(s) at the C-terminal and / or N-terminal end of the IgA binding polypeptides or the IgA binding polypeptide multimers may improve production, purification, stabilization and / or in vitro coupling of the polypeptide or the polypeptide multimer. Nonlimiting exemplary N-terminal amino acid sequences for improved production yields are SEQ ID NO:101 and 103. Moreover, in vitro coupling may be performed using a C-terminal amino acid sequence according to SEQ ID NO:102, as shown and explained in Example 1 below. In some embodiments, said additional amino acid(s) improve(s) coupling of the polypeptides or multimers and are selected from the group consisting of one or more cysteine residues, a plurality of lysine residues and a plurality of histidine residues. The coupling element may e.g. be a single cysteine at the C-terminal end or a plurality of histidine residues. An advantage of having a C- terminal cysteine is that endpoint coupling of the protein can be achieved through reaction of the cysteine thiol with an electrophilic group on a support. This provides excellent mobility of the coupled protein which is important for binding capacity. A plurality of histidine residues may be six histidine residues, i.e. a His6-tag. Thus, in a particular embodiment, the IgA binding multimer according to the present disclosure comprises SEQ ID NO:99.

[0094] IgA binding polypeptides and IgA binding polypeptide multimers according to the present disclosure exhibit optimal alkali stability. In embodiments where the IgA binding polypeptide or the IgA binding polypeptide multimer is used for separation or isolation of e.g. antibodies / immunoglobulins having an IgA isotype, high alkali stability will allow for use of highly alkaline conditions during cleaning, essential for long-term repeated use in a bioprocess separation setting. Thus, in some embodiments of the first, the second and the third aspects of the present disclosure, the IgA binding polypeptide or the IgA binding polypeptide multimer exhibits an alkali stability of at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 89%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% of the alkali stability of SEQ ID NO:1 and / or SEQ ID NO:2 after incubation in 0.5 M NaOH. In one embodiment, said incubation is at least for 12 hours, such as at least for 24 hours or more. Said incubation may be for example at 22 + / - 2°C. In one embodiment, the IgA binding polypeptide or the IgA binding polypeptide multimer exhibits at least 80% residual binding capacity after about 80 cleaning cycles with 0.1 M NaOH. Said cleaning cycle may be performed at 22 + / - 2°C.

[0095] In a fourth aspect of the present disclosure, there is provided a polynucleotide encoding the IgA binding polypeptide as defined in any one of the embodiments of the first and second aspects of the present disclosure, or the IgA binding polypeptide multimer as defined in any one of the embodiments of the third aspect of the present disclosure.

[0096] In a fifth aspect, there is provided an expression vector comprising the polynucleotide as defined in the fourth aspect of the present disclosure.

[0097] In a sixth aspect, there is provided a host cell comprising the expression vector as defined in the fifth aspect of the present disclosure.

[0098] In a seventh aspect, there is provided an adsorbent material comprising the IgA binding polypeptide as defined in any one of the embodiments of the first and second aspects of the present disclosure, or the IgA binding polypeptide multimer as defined in any one of the embodiments of the third aspect of the present disclosure, coupled to a solid support. As the skilled person will understand, the expressed IgA binding polypeptide, in monomeric or multimeric form, should be purified to an appropriate extent before being immobilized to a support. Such purification methods are well known in the field, and the immobilization of protein-based ligands to supports is easily carried out using standard methods. Suitable methods and supports will be discussed below in more detail and are disclosed e.g. in W016079033 which is incorporated herein by reference in its entirety.

[0099] The support may be a solid support and may optionally be a porous material. The support may be or comprise a surface onto which the IgA binding polypeptide or the IgA binding polypeptide multimer is coupled. Examples of such support materials include conventional protein-binding support and surfaces such as a chip, a plate, a well, and a sheet. The support may optionally be provided in other forms such as a fiber, a membrane, a fibrous matrix, a filter, a porous monolith, a particle or a bead, such as a gel bead as used in chromatography resins. In some embodiments, the solid support material may be selected from a particle, a bead, a fiber, a fibrous membrane, a filter, a sheet, a porous monolith, a chip, a plate, and a well. Particles or beads can be porous or non-porous. Particles or beads may include magnetic beads. Supports in the form of beads or particles can be used as a packed bed or in a suspended form. Suspended forms include those known as expanded beds and pure suspensions, in which the particles or beads are free to move. In case of monoliths, packed bed and expanded beds, a separation procedure commonly follows conventional chromatography with a concentration gradient. In case of pure suspension, batch-wise mode will be used.

[0100] The support may be made of any suitable material, as outlined in more detail below. As a non-limiting example, a conventional affinity separation matrix is often of organic nature and based on polymers that expose a hydrophilic surface to the aqueous media used, i.e. expose hydroxy (-OH), carboxy (-COOH), carboxamido (CONH2, possibly in N- substituted forms), amino (-NH2, possibly in substituted form), oligo- or polyethylenoxy groups on their external surface and, if present, also on internal surfaces.

[0101] The IgA binding polypeptide or the IgA binding polypeptide multimer may be attached to the support via known coupling techniques utilizing e.g. thiol, amino and / or carboxy groups present in the IgA binding polypeptide or the IgA binding polypeptide multimer. Bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS) etc. are well-known coupling reagents. Between the support and the IgA binding polypeptide or the IgA binding polypeptide multimer, a spacer molecule can be introduced, which improves the availability of the first polypeptide moiety and / or facilitates the chemical coupling of the IgA binding polypeptide or the IgA binding polypeptide multimer to the support. Depending on the nature of the IgA binding polypeptide or the IgA binding polypeptide multimer and the coupling conditions, the coupling may be a random or multipoint coupling (e.g. via a plurality of lysines or histidines) or a single point coupling (e.g. via a single cysteine). In embodiments, to increase coupling control, it may be preferable that the IgA binding polypeptide or the IgA binding polypeptide multimer variant as such does not comprise any histidine residues. However also in such cases, the IgA binding polypeptide or the IgA binding polypeptide multimer as a whole may comprise a histidine tag, such as a His6 tag. Alternatively, the IgA binding polypeptide or the IgA binding polypeptide multimer may be attached to the support by non-covalent bonding, such as physical adsorption or biospecific adsorption.

[0102] The IgA binding polypeptide or the IgA binding polypeptide multimer may be coupled to the support via thioether bonds. Methods for performing such coupling are well-known in this field and easily performed by the skilled person in this field using standard techniques and equipment. Thioether bonds are flexible and stabile and generally suited for use in affinity chromatography. In particular when the thioether bond is via a terminal or near-terminal cysteine residue on the IgA binding polypeptide or the IgA binding polypeptide multimer, the mobility of the coupled IgA binding polypeptide or IgA binding polypeptide multimer is enhanced which provides improved binding capacity and binding kinetics. In some embodiments the IgA binding polypeptide or the IgA binding polypeptide multimer is coupled via a C- terminal cysteine provided on the IgA binding polypeptide or the IgA binding polypeptide multimer, as described above. This allows for efficient coupling of the cysteine thiol to electrophilic groups, e.g. epoxide groups, halohydrin groups etc. on a support, resulting in a thioether bridge coupling.

[0103] With regard to specific materials, the support may comprise a polymeric material. Polymeric materials include materials of natural or synthetic polymers, and combinations thereof. For example, the support may comprise a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides include e.g. dextran, starch, cellulose, pullulan, agar, agarose etc., including derivatives thereof. Polysaccharides are inherently hydrophilic with low degrees of nonspecific interactions, they provide a high content of reactive (activatable) hydroxyl groups and they are generally stable towards alkaline cleaning solutions used in bioprocessing. The support may comprise agar or agarose, such as crosslinked agarose. Such supports used in the present invention can easily be prepared according to standard methods, such as inverse suspension gelation (S Hjerten, Biochim Biophys Acta, 79(2), 393-398 (1964)). Alternatively, the base matrices are commercially available products, such as crosslinked agarose beads sold under the name of SEPHAROSE™ FF (Cytiva). In an embodiment, which is especially advantageous for large-scale separations, the support has been adapted to increase its rigidity using the methods described in US6602990 or US7396467, which are hereby incorporated by reference in their entirety, and hence renders the matrix more suitable for high flow rates.

[0104] Synthetic polymers useful as material for the support include polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides, etc. In case of hydrophobic polymers, such as matrices based on divinyl and monovinyl-substituted benzenes, the surface of the matrix can be hydrophilized to expose hydrophilic groups as defined above to a surrounding aqueous liquid. Such polymers are easily produced according to standard methods. As an alternative, a commercially available product, such as SOURCE™ (Cytiva) may be used.

[0105] Alternatively, the solid support according to the invention comprises a support of inorganic nature, e.g. silica, zirconium oxide, etc.

[0106] In embodiments, the IgA binding polypeptide or the IgA binding polypeptide multimer may be coupled to a support which is a convection-based chromatography matrix. Such convection-based chromatography matrix may comprise a porous polymer membrane, a filter, a fibrous matrix, or a porous monolith. Examples of a porous polymer membrane include MustangTM membranes (Cytiva) and SartobindTM membranes (Sartorius). A fibrous support may be based on electrospun polymeric fibers or cellulose fibers, optionally non-woven fibers. A fibrous matrix may thus be a non-woven fibrous matrix. The fibers may have a cross- sectional diameter of 10-1000 nm, such as 200-800 nm, 200-400 nm or 300-400 nm. Such a fibrous support can be found in a HiTrap Fibro™ unit (Cytiva). Alternative fibrous supports are disclosed in e.g. WO2019 / 137869 and W02018 / 011600. An adsorbent material or separation matrix as described herein may be used for the same purposes as mentioned above for the IgA binding polypeptide or the IgA binding polypeptide multimer. Thus, in one embodiment, the present disclosure provides a chromatography material comprising a separation matrix coupled to IgA binding polypeptides or multimers as disclosed herein, as well as chromatography columns or devices incorporating such separation matrix. In a particular embodiment, the solid support is thus a separation matrix. In one embodiment, the adsorbent material may be a sensor surface intended for use in a sensor or other detection or quantification devices.

[0107] In an eighth aspect of the present disclosure, there is provided a method of separating or isolating a target entity, such as IgA or a fragment thereof, wherein an adsorbent material, such as a separation matrix coupled to the IgA binding polypeptides and / or multimers thereof, as disclosed herein is used. The method comprises contacting a liquid sample comprising the target entity with a separation matrix as disclosed herein. Thus, a method of isolating IgA or a fragment thereof is provided, wherein said method comprises a) contacting a liquid sample comprising said IgA or said fragment thereof with the adsorbent material as defined in any one of the embodiments of the seventh aspect of the present disclosure. The contacting is performed under conditions under which the target entity can bind to the IgA binding polypeptide or the IgA binding polypeptide multimer moiety. The method may furthermore comprise washing said separation matrix with a washing liquid, eluting the target entity from the separation matrix with an elution liquid, and optionally cleaning the separation matrix with a cleaning liquid. The cleaning liquid can alternatively be called a cleaning-in-place (CIP) liquid. The cleaning liquid typically is an alkaline solution, such as comprising NaOH or KOH of at least 0.05 M, such as 0.05-1 M, such as 0.05-0.5 M, such as at least 0.1 M, such as 0.1-0.5 M, e.g. 0.3 M or 0.5 M. The contact (incubation) time may be at least 10 min. The binding, eluting and cleaning steps may advantageously be repeated at least 10 times, such as at least 20 times. The skilled person will understand that the liquid sample to be purified may be any sample comprising the target entity in an environment from which it is to be purified or separated. The sample may be obtained from a cell culture, such as a clarified cell culture harvest, and may have been subjected to one or more conventional steps of filtration, concentration, dilution and / or buffer exchange, and optionally one of more initial chromatography steps, in particular a step which is not based on affinity chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, multimodal chromatography, or size exclusion chromatography. For example, the sample may be a clarified and filtered cell culture harvest. In some cases the sample may have been subjected to one or more steps of filtration by tangential flow filtration (TFF).

[0108] Prior to contacting with the present separation matrix, liquid sample contains the target entity and at least one impurity, such as host cell protein (HCP) or host cell nucleic acids. The present separation matrix is useful for separating the target entity from such impurities, and after performing the above process, the eluate containing the target entity has a reduced level of at least one such impurity.

[0109] Optionally, after performing a separation based on affinity capture as described herein, the eluate containing purified target entity may be subjected to one or more steps of filtration, concentration, dilution or buffer exchange, or chromatography step(s) not based on affinity chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, multimodal chromatography, or size exclusion chromatography. A further chromatography step performed after the affinity separation of the present disclosure may be referred to as a polishing step.

[0110] While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment contemplated, but that the invention will include all embodiments falling within the scope of the appended claims.

[0111] Brief description of the drawings

[0112] Figure 1A and IB show representative sensorgrams of association and dissociation profiles of 16 IgA binding polypeptides according to the present disclosure (SEQ ID NO:106-121), as measured in Biacore using human polyclonal IgA as analyte.

[0113] Figure 2 shows representative sensorgrams of association and dissociation profiles of the IgA binding polypeptide, SEQ ID NO:121, wherein either human IgAl, human lgA2 or human polyclonal IgA are used as analyte.

[0114] Figure 3 shows representative sensorgrams of association and dissociation profiles of four IgA binding polypeptide multimers according to the present disclosure (SEQ ID NO:94-97), as measured in Biacore using human polyclonal IgA as analyte.

[0115] Figure 4 shows representative sensorgrams of association and dissociation profiles of three IgA binding polypeptides (SEQ ID NO:121-123) in comparison to two control constructs (SEQ ID NQ:104-105) for human polyclonal IgA (VH3 class) as analyte.

[0116] Figure 5A-5E shows representative sensorgrams of association and dissociation profiles of three IgA binding polypeptides (SEQ ID NO:121-123) in comparison to two control constructs (SEQ ID NQ:104-105) for human polyclonal IgA (VH3 class), Ixekizumab (IgGl (VHl class)) and Denosumab (lgG2 (VH3 class)) as analytes.

[0117] Figure 6 shows representative sensorgrams of association and dissociation profiles of three IgA binding polypeptides (SEQ ID NO:121-123) in comparison to two control constructs (SEQ ID NQ:104-105) for 20 pM Denosumab (lgG2 (VH3 class)) as analyte.

[0118] Figure 7 is a representative SDS-PAGE gel image of protein preparations according to Examples 4 and 5: human polyclonal IgA, human polyclonal IgA Fc and human polyclonal IgA Fab (VH3 class) fragments.

[0119] Figure 8 shows representative sensorgrams of association and dissociation profiles of three IgA binding polypeptides (SEQ ID NO:121-123) in comparison to two control constructs (SEQ ID NQ:104-105) for human polyclonal IgA Fc fragment as analyte. Figure 9 shows representative sensorgrams of association and dissociation profiles of an IgA binding polypeptide (SEQ ID NO:123) in comparison to two control constructs (SEQ ID NQ:104-105) for human polyclonal IgA Fab fragment (VH3 class) as analyte.

[0120] Figure 10 shows a representative chromatogram for frontal analysis using 1 mg / mL human polyclonal IgA on a separation matrix coupled to the IgA binding polypeptide multimer SEQ ID NQ:100 (absorbance at 280 nm (mAU), conductivity and pH are shown, "equ..." corresponds to "equilibration" and "col..." corresponds to "column cleaning").

[0121] Figure 11A-D is a listing of amino acid sequences of examples of IgA binding polypeptides and polypeptide multimers according to the present disclosure as well as amino acid sequences of domain A, B, C, D and E of SpA and derivatives thereof. In IgA binding polypeptides of the present disclosure, the deduced Sequence A, Sequence B and Sequence C extend from residue 9 to residue 35, from residue 9 to residue 51 and from residue 5 to residue 35, respectively, in each sequence.

[0122] EXAMPLES

[0123] Summary of Examples

[0124] This project aimed for providing IgA binding polypeptides derived from SpA or any domain thereof, that have an improved binding profile, such as a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NO:9 for the same IgA polypeptide. Moreover, it was aimed to provide IgA binding polypeptides that selectively bind human IgA among other human immunoglobulin isotypes.

[0125] Thirteen amino acid positions (positions X9, X10, Xu, X13, X14, X17, Xis, X24, X25, X27, X28, X32 and X35) were identified with the highest impact on IgA binding, and the present inventors have found certain amino acid variants surprisingly beneficial in these key positions. Accordingly, IgA binding polypeptides were constructed and further characterised for specificity, pH sensitivity and alkaline stability, as described in Example 1. Specificity for human IgAl and lgA2 isotypes was also evaluated. A subset of the IgA binding polypeptides was chosen for further studies and modifications, as described in Examples 2-7. These included multimerization of the IgA binding polypeptides to tetramers and / or hexamers and expression of said tetramers in E. coli. Using some of these IgA binding polypeptide multimers, a primary characterization was performed, wherein the multimers were evaluated for static binding capacity (SBC), dynamic binding capacity (DBC), pH elution and alkaline stability. To further improve selectivity for human IgA among other human immunoglobulin isotypes, amino acid substitutions were introduced for reduced or abolished ability to bind the VH3 region of immunoglobulins, as described in WO2023 / 046886. Furthermore, amino acid substitutions were tested for improved solubility and increased extinction coefficient of the IgA binding polypeptides and multimers thereof. Examples 4-5 also describe comparative data on binding characteristics of the IgA binding polypeptides of the present disclosure in comparison to known constructs.

[0126] Example 1

[0127] Biacore evaluation of 16 IgA binding polypeptides

[0128] The present inventors have found that certain amino acid variants in key positions for IgA binding are surprisingly advantageous. Based on these amino acid variants, 16 different IgA binding polypeptides (SEQ ID NO:106-121) - comprising SEQ ID NO:14-29, respectively, were constructed and characterised as described below. These IgA binding polypeptides show the following amino acid diversity in the key amino acid positions (Table 1):

[0129] Table 1: Amino acid variants in key positions of 16 IgA binding polypeptides

[0130] The 16 IgA binding polypeptides were analysed in Biacore using polyclonal human

[0131] IgA, human IgAl and human lgA2 as analytes. Binding response, relative binding quality to polyclonal human IgA, human IgAl and human lgA2 and NaOH-stability over 60 cycles with 0.5 M NaOH were evaluated. In addition, the pH at which binding is lost (elution pH) was also assessed.

[0132] Material and Methods

[0133] Generation of 16 IgA binding polypeptides with ABD-His-tag

[0134] Nucleic acid sequences encoding SEQ ID NO:14-29 were cloned into pAM183 expression vectors for production of 16 different IgA binding polypeptides as ABD- His-tagged constructs (SEQ ID NO:106-121). Each ABD-His-tagged construct comprised a leader sequence (SEQ ID NQ:101) at the N-terminal of the amino acid sequence selected from SEQ ID NO:14-29, and SEQ ID NQ:102 (comprising a linker- ABD-tag-linker-His-tag motif) at the C terminal thereof. For the avoidance of doubt, each construct consisted of SEQ ID NQ:101-SEQ ID NO:X-SEQ ID NQ:102, wherein SEQ ID NO:X corresponds to a sequence selected from SEQ ID NO:14-29. For protein production, materials and equipment used were as follows: glycerol stocks for the IgA binding polypeptides; 2YT culture medium, Carbenicillin, IPTG, 100 ml baffled glass shake flasks, Infors HT Shaking incubator, His-GraviTrap and kit (Cytiva), PD10 GraviTraps (Cytiva).

[0135] Protein expression culture medium (2YT medium supplemented with 200 pg / ml Carbenicillin and 1 mM IPTG) was prepared and added to filled baffled shake flasks (20 ml per flask, 22 flasks in total). Each flask was inoculated with 5 pl from the corresponding glycerol stock. The flasks were incubated in Infors HT shaking incubator for 24 hours at 27°C and 190 rpm shaking, whereafter the cultures were pelleted and re-suspended in 5 ml PBS in Falcon tubes and frozen. To generate crude IgA binding polypeptide variant lysates the frozen Falcon tubes were incubated in 80°C water bath for 2 hours followed by pelleting of cell debris by centrifugation at 12000xg for 30 min. Imidazole was added to the lysates to achieve the concentration recommended by the manufacturer and the samples were purified using His GraviTraps kit according to kit instructions. Buffer exchange into PBS+P (27 mM KCI, 1.37 M NaCI and 05% v / v Surfactant P20 - running buffer used in Biacore) was performed using PD10 Gravitraps and kit instructions. Concentration of buffer exchanged samples was measured using NanoDrop (Thermo Scientific) according to manufacturer's instructions. Following purification, samples were kept in refrigerator until all measurements performed and were then transferred to Eppendorf tubes and kept in freezer (-20 C).

[0136] Evaluation of binding to polyclonal human IgA, IgAl and lgA2 using Biacore Materials and equipment used were as follows: CM5 sensor chips (Cytiva), Biacore NHS coupling kit (Cytiva), IgA binding polypeptide variants from above, Biacore 8K+ (Cytiva); polyclonal human IgA (Sigma, #14036), human IgAl (Calbiochem, #400109) and human lgA2 (Sigma, 400110) as analytes, diluted in PBS+P (P=0.05% Tween 20). Immobilization was performed using a standard method in Biacore software with coupling of the IgA binding polypeptide variants in FC2 and activation / inactivation in FC1 on a CM5 sensor chip. The IgA binding polypeptide variants were diluted in 10 mM acetate buffer (pH 5) at approximately 30 pg / ml. The immobilization levels did vary somewhat between different IgA binding polypeptide variants (1947 to 2089 RU).

[0137] Channel 1 to channel 8 were immobilized with the IgA binding polypeptides. Two chips were used until all polypeptides were tested, and the same two chips were used for binding analyses, elution pH testing and alkali stability studies. Injections for each channel (cycles) were as follows: buffer, 0.5 pM polyclonal human IgA, 0.5 pM human IgAl and 0.5 pM human lgA2.

[0138] Biacore method:

[0139] Running buffer: PBS+P

[0140] Flow rate: 5 pl / min

[0141] Sample injection: 300s over both Flow Cells (FC1 and FC2) Dissociation time: 300s

[0142] Regeneration: 10 mM Glycine-HCI pH 1.5, 30 pl / min, 2x30s

[0143] Evaluation of elution pH using Biacore For testing at which pH IgA binding is lost, the method below was repeated 7 times using different buffers (Phosphate buffer (PBS) pH 7.2, 50 mM Citrate buffer pH 5.0, 4.0, 3.5, 3.0, 2.5, and 2.0).

[0144] Materials and equipment used were as follows: the same CM5 sensor chips with IgA binding polypeptide variants from above, Biacore 8K+ (Cytiva); polyclonal human IgA as above, 0.5 M NaOH.

[0145] Biacore method:

[0146] Running buffer: PBS+P

[0147] Flow rate: 10 pl / min

[0148] Sample injection 1 (polyclonal human IgA, 0.5 pM): 300s over both Flow Cells Dissociation time 1: 60s

[0149] Sample injection 2 (50 mM Citrate buffer, different pH each round): 300s over both Flow Cells

[0150] Dissociation time 2: 60s

[0151] Regeneration: 10 mM Glycine-HCI pH 1.5, 30 pl / min, 2x30

[0152] Alkali stability (0.5 M NaOH) evaluation using Biacore

[0153] After binding analyses and elution pH testings were done, on the same CM5 sensor chips with immobilized IgA-binding polypeptide variants from above, alkali stability tests were performed.

[0154] Materials and equipment used were as follows: the same CM5 sensor chips with IgA binding polypeptide variants from above, Biacore 8K+(Cytiva); polyclonal human IgA as above, 0.5 M NaOH.

[0155] Biacore method (cycle):

[0156] Running buffer: PBS+P

[0157] Flow rate: 5 pl / min

[0158] Sample injection 1 (polyclonal human IgA, 0.5 pM): 300s over both Flow Cells Dissociation time 1: 60s

[0159] Sample injection 2 (0.5 M NaOH): 300s over both Flow Cells

[0160] Dissociation time 2: 60s Regeneration: 10 mM Glycine-HCI pH 1.5, 30 pl / min, 2x30s

[0161] This cycle was repeated 60 times to follow stability of polyclonal human IgA response values. Results and Conclusions

[0162] Results of the evaluation of the 16 IgA binding polypeptides are summarized in Table

[0163] 2 below. Elution pH was between 3.5-3.0 for each polypeptide.

[0164] Table 2: Evaluation of 16 IgA binding polypeptides in Biacore. *50% binding capacity is retained after the indicated number of cycles using 0.5 M NaOH. (SEQ ID NOs terminal seguences described above.)

[0165] Representative sensorgrams of association and dissociation profiles of the 16 IgA binding polypeptides (SEQ ID NO:106-121) using polyclonal human IgA as analyte are shown in Fig. 1A and IB. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before injection and signal just before end of injection. High, medium and low association and dissociation rates are relative and were determined in comparison of the response levels to each other. The response levels of polyclonal human IgA interaction of the tested IgA binding polypeptides show that each variant exhibited high or medium association rates and low or medium dissociation rates. These association-dissociation profiles indicate that the tested IgA binding polypeptide variants are particularly advantageous for IgA binding with optimal characteristics. As shown in Table 2, the response levels for human IgAl and human lgA2 interactions relative to that for polyclonal human IgA demonstrate that all isomers of IgA are bound successfully and with desired characteristics to the tested polypeptides. This is further demonstrated in Fig. 2 for the IgA binding polypeptide comprising SEQ ID NO:29 (SEQ ID NO:121). Surprisingly, the IgA binding polypeptide comprising SEQ ID NO:21 (SEQ ID NO:113) exhibited preferred binding to human IgAl, while the IgA binding polypeptide comprising SEQ ID NO:25 (SEQ ID NO:117) exhibited preferred binding to human lgA2. These IgA binding polypeptides may be particularly advantageous for certain applications, for example wherein IgA isoforms are required to be separately purified.

[0166] IgA selectivity of the 16 IgA binding polypeptides was also assessed in Biacore, using human IgM, human IgD, human IgE, adalimumab, belimumab or trastuzumab as analyte. No binding or, in the single case of trastuzumab, weak binding was observed. Accordingly, the 16 IgA binding polypeptides showed selective binding to IgA over IgM, IgD and IgE, moreover, selective or preferred binding to IgA in comparison to IgG. Further evaluation of IgA vs IgG binding is presented in Example 4 below.

[0167] As demonstrated in Table 2, 50% of binding capacity was maintained for each tested polypeptide after 13 or more washing cycles using 0.5 M NaOH.

[0168] The inventors have found based on the above evaluation that some of the tested IgA binding polypeptides (SEQ ID NO:118-121, comprising SEQ ID NO:26-29, respectively) exhibit surprisingly beneficial characteristics with respect to their binding profile to human IgA and alkaline stability. These variants were selected for multimerization and further studies. Example 2

[0169] Biacore evaluation of four IgA binding polypeptide multimers

[0170] Four of the above tested IgA binding polypeptides (SEQ ID NO:118-121) were synthesized as homomeric tetramers (SEQ ID NO:94-97). All constructs comprised a leader sequence (SEQ ID NQ:103) and were made with a C-terminal His6-tag and a cysteine to facilitate purification with IMAC and coupling through the cysteine . Binding profile of the tetramers was assessed using Biacore and polyclonal human IgA as analyte.

[0171] Materials and Methods

[0172] Preparation of IgA binding polypeptide multimers

[0173] Sequences were ordered as DNA-synthesized plasmids from ATUM (Newark, CA, USA) in pJ401 plasmid backbone. The plasmids were transformed into chemically competent E. coli K12-017 cells and research cell-banks were made by inoculating 10 mL LB-broth with a single colony and grow for 5 h in 37°C. 1 L fermentation was performed with the multimers, and the products of the fermentations were purified using Ni Sepharose 6FF (Cytiva) for capture, followed by a polishing step using Source 30Q (Cytiva). The purified protein was concentrated to approx. 50 g / L and stored frozen.

[0174] Biacore analysis of IgA binding polypeptide multimers (SEQ ID NO:94-97)for polyclonal human IgA binding

[0175] Materials and equipment used were as follows: CM5 sensor chips (Cytiva), Biacore Thiol Coupling kit (Cytiva), IgA binding polypeptide multimer variants according to SEQ ID NO:94-97 and a hexameric construct of SEQ ID NO:1 with a C-terminal cysteine tag and an N-terminal leader sequence, Biacore 8K+ (Cytiva); polyclonal human IgA (Sigma, #14036), human IgAl (Calbiochem, #400109) and human lgA2 (Sigma, 400110) as analytes.

[0176] Immobilization was performed using a standard method in Biacore software with thiol coupling of the IgA binding polypeptide multimer variants in FC2 and activation / i nactivation in FC1. The IgA binding polypeptide multimer variants were diluted in acetate buffer (pH 5) at approximately 30 pg / ml. The immobilization levels did vary somewhat between different IgA binding polypeptide multimer variants (116-153 RU).

[0177] Biacore method:

[0178] Running buffer: PBS+P

[0179] Flow rate: 10 pl / min

[0180] Sample injection: 300s / 2 min over both Flow Cells (FC1 and FC2)

[0181] Dissociation time: 300s

[0182] Regeneration: 10 mM Glycin-HCI pH 1.5, 30 pl / min, 2x30s

[0183] Five channels were immobilized with the IgA binding polypeptide multimers variants (SEQ ID NO:94-97) and the hexameric construct of SEQ ID NO:1, respectively. Analytes were IgA (polyclonal, IgAl and lgA2) and trastuzumab, all at a concentration of 0.5 pM.

[0184] Results and Conclusions

[0185] Binding characteristics of the tested multimers (SEQ ID NO:94-97) for polyclonal human IgA in Biacore are shown in Fig. 3. Each multimer bound polyclonal human IgA with a high association rate. SEQ ID NO:94 and 97 exhibited medium, while SEQ ID 95 and 96 showed low dissociation rates. The data demonstrate beneficial association and dissociation profiles of the IgA polypeptide multimers according to the present disclosure.

[0186] Example 3

[0187] Evaluation of static binding capacity and introduction of further amino acid substitutions

[0188] Two IgA binding polypeptides from Example 1 (SEQ ID NO:118 and 121, comprising SEQ ID NO:26 and 29, respectively) were selected for testing further amino acid substitutions, namely F5Y for increasing the extinction coefficient of the polypeptides and A43E for improving solubility. SEQ ID NO:26 and 29 were prepared as homomeric tetramers (SEQ ID NO:98 and 99) with a C-terminal His6-tag and an N- terminal leader sequence (SEQ ID NO:103). Static binding capacity (SBC) of these multimers was assessed in plate studies.

[0189] Materials and Methods

[0190] Static binding capacity measurement

[0191] PreDictor plate studies were performed on SEQ ID NO:98 and 99 to evaluate static binding capacity (SBC). Hexamers of SEQ ID NO:1 with a C-terminal cysteine tag and an N-terminal leader sequence were produced and used as control.

[0192] 96-well predictor plate with resin was prepared as follows: the resin was settled for at least 1 hour and the concentration was adjusted to 50% slurry by withdrawing or adding 20% ethanol. 1 ml cube was used. The PIAB pump was set to working flow 550 mbar. A 5 pm polypropylene filter was placed on the top of the bottom part of the cube. The resin was added until the cone was full, then the vacuum was put on. A timer was started when the resin looked dry from the top and after ca 55 sec the cube was disassembled, and the cubed resin was removed from the vacuum at 60 sec. Afterwards, each resin was put in a 50 ml falcon tube and 20% ethanol was added until the resin slurry volume was 20 ml, obtaining a 5% resin slurry. The resin slurry was shaken on a vortex to obtain a homogeneous slurry then, 8 ml were transferred to a glass vial and put on the Gilson liquid handler. The stirring apparatus was put in the resin slurry and kept going during transfer of the resin to the wells. 120 pl resin slurry (6 pl resin) were transferred into each well.

[0193] Polyclonal human IgA sample was purified using an IgA polypeptide multimer according to the present disclosure (SEQ ID NO:99) and was diluted to 1.8 g / l concentration in the two different equilibration buffers: 20 mM phosphate, 150 mM NaCI, pH 7.2 and 20 mM phosphate, 500 mM NaCI, pH 7.2. Standard dilutions were made in respective phosphate buffer.

[0194] The plate experiment was performed as follows: for equilibration, 200 pl of equilibration buffer was added to each well, followed by incubation for 1 min on a shaker at 1100 rpm. The buffer was then removed by vacuum. This was repeated three times. The last equilibration buffer was collected on a UV plate by centrifugation at 500 g for 1 min and kept as blank. Phosphate buffer with 150 mM NaCI or 500 mM NaCI was added to the wells. For sample loading, 200 pl of 1.8 g / l polyclonal human IgA sample was loaded into each well, followed by incubation for 1 hour on a saker at 1100 rpm. Then the flowthrough was collected on a UV plate by centrifugation at 500 g for 1 min. A standard curve was constructed on a UV plate from the prepared polyclonal human IgA sample.

[0195] All standards and samples were measured at three wavelengths 250, 280 and 300 nm on a plate reader. A calibration curve was calculated from the standards and used to estimate the concentration in the flowthrough. Then capacity calculations were made based on the concentration in the flowthrough according to the following equation where q is the binding capacity of the medium under given conditions at termination of incubation, Vmedium is the volume of medium in the well, Vnq is the volume of liquid in the well, co is the concentration of target protein at the start of the experiment (t=0), and Cunbound is the concentration of target protein found in the flowthrough (liquid phase) at termination of incubation.

[0196] Results and Conclusions

[0197] The inventors have selected two IgA binding polypeptides (SEQ ID NO:118 and 121, comprising SEQ ID NO:26 and 29, respectively) for further amino acid substitutions, namely F5Y for increasing the extinction coefficient of the polypeptides and A43E for improving solubility. Firstly, A43E mutation was made to lower the pl from 6.75 to 6.23 for these constructs, thereby improving solubility at neutral pH (pH 7).

[0198] Moreover, introducing the mutation Y in position Xs (F5Y) was found to increase the extinction coefficient from 0.106 to 0.309 of these constructs, thereby improving their applicability for chromatography and detection by UV. Tetramers of these IgA binding polypeptides (SEQ ID NO:98 and 99) were selected for SBC studies. As presented in Table 3, both SEQ ID NO:98 and 99 showed increased IgA binding capacity in SBC measurements in comparison to the control (hexamer of SEQ ID NO:1).

[0199] An increase in salt concentration had a minor effect on SBC with maintaining optimal IgA binding capacity. Data are presented with baseline subtraction of signal for empty wells.

[0200] Table 3: SBC results with different salt concentrations in the absorption buffers.

[0201] The SBC measurements confirmed improved and preferred IgA binding properties of IgA binding polypeptide multimers according to the present disclosure (SEQ ID NO:98 and 99) in comparison to the control (hexamer of SEQ ID NO:1). SBC results for the control also revealed that a minimal residual binding affinity to immunoglobulins other than IgA may be retained by the tested multimers. This was tested further, as described in Example 4.

[0202] Example 4

[0203] Comparative evaluation of IgA binding profiles and assessment of specificity

[0204] The IgA binding polypeptide comprising SEQ ID NO:29 (SEQ ID NO:121) was selected for further evaluation of IgA binding specificity. The inventors in this Example surprisingly demonstrate that the polypeptide has a binding affinity for VH3 class IgG, which may be diminished by additional amino acid substitutions (namely, V40G and L51V) thereby leading to improved binding selectivity for human polyclonal IgA (Fig. 5 and 6). Based on the sequence of SEQ ID NO:29, an IgA binding polypeptide comprising the mutations V40G and L51V was produced (SEQ ID NO:122), wherein the monomeric construct thus comprised SEQ ID NO:45 coupled to an N-terminal leader sequence (SEQ ID NQ:101) and a C-terminal ABD-His-tag (SEQ ID NQ:102), as described in Example 1. A monomeric construct comprising the mutations of V40G and L51V together with the above described substitutions (A43E and F5Y) was also generated (SEQ ID NO:123), wherein the polypeptide comprised SEQ ID NO:77 coupled to an N-terminal leader sequence (SEQ ID NO:101) and a C-terminal ABD- His-tag (SEQ ID NQ:102). This Example presents binding characteristics of these IgA binding polypeptides for polyclonal IgA (VH3 class), IgGl (VHl class) and lgG2 (VH3 class) in comparison to two polypeptides designed based on prior art sequences known for IgA binding. These two monomeric control constructs (SEQ ID NQ:104 and 105) comprised SEQ ID NO:9 and SEQ ID NQ:10, respectively, and each was coupled to the N-terminal leader sequence (SEQ ID NQ:101) and the C-terminal ABD-His-tag (SEQ ID NQ:102).

[0205] Materials and Methods

[0206] Preparation of IgA binding polypeptides

[0207] Essentially as described in Example 1, nucleic acid sequences encoding SEQ ID NO:9, 10, 29, 45 and 77 were cloned into pAM183 expression vector for production as ABD-His-tagged constructs (SEQ ID NQ:104, 105 and 121-123). Protein expression was induced in 20 ml 2YT medium supplemented with 200 pg / ml Carbenicillin with addition of 1 mM IPTG at QD600 at ~0.7. Thereafter, the flasks were incubated in Infors HT shaking incubator for 24 hours at 27°C and at 190 rpm shaking. The cells were harvested by centrifugation, re-suspended in 5 ml PBS and thereafter incubated in 80°C water bath for 2 hours. Cell debris was pelleted by centrifugation at 12000xg for 30 min. For Biacore experiments below, the samples were diluted 100 times in PBS+P (P=0.05% Tween 20).

[0208] Biacore evaluation of binding characteristics for polyclonal IgA (VH3 class), IgGl (VHl class) and lgG2 (VH3 class)

[0209] Materials and equipment used for Biacore experiments were as follows: Biacore 8K+ (Cytiva), CM5 sensor chips (Cytiva), Biacore NHS coupling kit (Cytiva), Rat Serum Albumin (RSA, Sigma), IgA binding polypeptide variants from above, Denosumab (lgG2 VH3, Absolute Antibodies), Ixekizumab (IgGl VHl, Absolute Antibodies), polyclonal human IgA (VH3 class, as described below). Analytes (Denosumab, Ixekizumab, polyclonal human IgA) were diluted to 2 pM in PBS+P and Denosumab was also prepared in a 20 pM concentration. Immobilization of a CM5 sensor chip was performed using a standard method in Biacore software with coupling of the RSA (50 pg / ml, 10 mM acetate buffer, pH 5) in FC2 and activation / inactivation in FC1. The immobilization levels resulted in around 1999 RU for all surfaces.

[0210] Biacore method:

[0211] Running buffer: PBS+P Flow rate: 10 pl / min Sample injection for capture of affinity ligand: 600s over Flow Cell FC2 Wait 30 seconds

[0212] Analyte injection: 600s over both Flow Cells (FC1 and FC2) Dissociation time: 600s

[0213] Regeneration: 10 mM Glycine-HCI pH 1.5, 30 pl / min, 2x30s

[0214] Purification of human polyclonal IgA (VH3 class)

[0215] Human plasma was diluted 1:1 in phosphate buffered saline (PBS), pH 7.4 and filtered using ULTA Capsule HC 0.2 pm sterile filter (Cytiva) prior to chromatography. IgA was purified from human plasma by IgA affinity chromatography resin (SEQ ID NO:100) packed into HiScale 26 / 110 chromatography column (Cytiva) using AKTA Pure 25 FLPC system (Cytiva). Plasma sample was applied onto the column equilibrated in 20 mM sodium phosphate, 150 mM NaCI, pH 7.2 maintaining residence time (RT) of 6 minutes. Column was washed with 20 mM sodium phosphate, 500 mM NaCI, pH 7.2 maintaining RT of 6 minutes, followed by wash with 20 mM sodium citrate pH 6.0. IgA was eluted with 25 mM sodium citrate, pH 2.5. The estimated content of IgA monomers was approximately 70%.

[0216] Eluate from the previous purification step was neutralized to pH 7.5, filtered using 0.2 pm syringe filter (Sarstedt) and purified further by affinity to MabSelect™ VH3 (Cytiva) packed into Tricorn 5 / 100 chromatography column (Cytiva) using AKTA Pure 25 FPLC system (Cytiva). Sample was applied onto the column pre-equilibrated in PBS, pH 7.4 (Medicago) and washed using the same buffer, RT of 4 minutes. During the second wash step, column was washed with 50 mM sodium acetate, pH 6.0, with a RT of 4 minutes. Protein was eluted using 50 mM sodium acetate pH 3.5. Elution peak fractions were pooled and buffer-exchanged into PBS, pH 7.4 (Medicago) using PD MidiTrap columns prepacked with Sephadex G-25 resin (Cytiva).

[0217] To isolate IgA monomers, protein was purified by size exclusion chromatography using HiLoad Superdex 200 26 / 60 pg column (Cytiva), coupled to AKTA Pure P150 FPLC system (Cytiva). PBS, pH 7.4 (Medicago) was used as a mobile phase. Final protein sample contained >95% of monomeric poly IgA (VH3 class, Fig. 7).

[0218] Results and Conclusions

[0219] Fig. 4 shows representative sensorgrams of association and dissociation profiles for human polyclonal IgA (VH3 class) as analyte, of the three IgA binding polypeptides (SEQ ID NO:121-123 comprising SEQ ID NO:29, 45 and 77, respectively) in comparison to the two control constructs (SEQ ID NQ:104-105 comprising SEQ ID NO:9 and 10, respectively). The data demonstrate that all tested IgA binding polypeptides according to the present disclosure show improved binding characteristics to human polyclonal IgA (VH3 class) when compared to the control polypeptides. These improved binding characteristics include higher maximum binding capacity (in RU), higher binding affinity as well as more favourable association and dissociation profiles, as measured in Biacore.

[0220] Fig. 5A-E presents Biacore results for each tested analyte: human polyclonal IgA (VH3 class), Ixekizumab (IgGl (VHl class)) and Denosumab (lgG2 (VH3 class)). The tested polypeptides are shown in separate figure panels as indicated. Results for human polyclonal IgA correspond to the data shown in Fig. 4, i.e. each polypeptide binds this analyte, but IgA binding polypeptides of the present disclosure show improved binding characteristics. Fig. 5 also reveals that none of the tested polypeptides binds VHl class IgG (Ixekizumab). The inventors also demonstrate that the IgA binding polypeptide comprising SEQ ID NO:29 (SEQ ID NO:121) and the control construct comprising SEQ ID NO:10 (SEQ ID NO:105) exhibit binding affinity for VH3 class IgG (Denosumab). This binding affinity cannot be observed in cases of SEQ ID NO:122 and 123, comprising SEQ ID NO:45 and 77, respectively. Accordingly, the present inventors have found that IgA binding selectivity of the herein disclosed polypeptides may be improved by introducing amino acid substitutions for reduced or abolished ability to bind the VH3 region of immunoglobulins. This improvement may particularly be relevant for IgA binding polypeptides which derive from certain SpA domains, such as SEQ ID NO:4-8. As shown in panels Fig. 5D and 5E, amino acid substitutions V40G and L51V in the tested constructs in fact lead to improved binding selectivity for human polyclonal IgA by diminished binding to VH3 class IgG. Using Denosumab at a higher (20 pM) concentration, VH3 class IgG binding ability of SEQ ID NO:121 and SEQ ID NQ:105 becomes more pronounced (Fig. 6). Moreover, it also appears that the control construct comprising SEQ ID NO:9 (SEQ ID NQ:104) exhibits a minimal binding affinity for VH3 class IgG. This affinity is lower than the affinity observed for SEQ ID NO:121 and SEQ ID NQ:105. The IgA binding polypeptides comprising either SEQ ID NO:45 or SEQ ID NO:77 show essentially no binding affinity for VH3 class IgG. Thus, introducing amino acid substitutions for reduced or abolished ability to bind the VH3 region of immunoglobulins may be advantageous for the IgA binding polypeptides.

[0221] Example 5

[0222] Evaluation the IgA binding polypeptide binding site

[0223] In this Example, the IgA binding polypeptides described in Example 4 (SEQ ID NO:121-123, comprising SEQ ID NO:29, 45 and 77, respectively) were tested for binding to intact human polyclonal IgA Fc and / or Fab (VH3 class) fragments. Similarly as above, two monomeric prior art constructs (SEQ ID NQ:104-105, comprising SEQ ID NO:9 and SEQ ID NQ:10, respectively) were used as controls for comparative evaluation of binding affinities and capacities for the IgA fragments.

[0224] Materials and methods Generation of human polyclonal IgA (VH3 class) Fc and Fab regions

[0225] Digestion of monomeric human polyclonal IgA (VH3 class, produced as described in Example 4) was done with IgASAP Subl+2 (WI0-IA3-010, Genovis) which digests human IgA at once specific site above the hinge, generating intact Fab and Fc fragments. The digestion was performed, with 360 pg IgA material from above (Example 4), according to the manufacturer's protocol. The separation of IgA Fc and VH3 IgA Fab fragments was done with MabSelect™ VH3 protein A affinity resin using standard procedure, as described above in Example 4. The purity was over 95% for both Fc and Fab fragments (Fig. 7).

[0226] The preparation of the IgA binding polypeptides and the Biacore evaluation were performed as described above. IgA Fc and IgA Fab (VH3 class) fragments were used as analyte at a concentration of 2 pM, diluted in PBS+P.

[0227] Results and Conclusions

[0228] Fig. 8 shows representative sensorgrams of association and dissociation profiles for human polyclonal IgA Fc fragment as analyte, of the three IgA binding polypeptides (SEQ ID NO:121-123 comprising SEQ ID NO:29, 45 and 77, respectively) in comparison to the two control constructs (SEQ ID NQ:104-105 comprising SEQ ID NO:9 and 10, respectively). The data are similar to that presented in Fig. 4, and demonstrate that all tested IgA binding polypeptides according to the present disclosure show improved binding characteristics to human polyclonal IgA Fc fragment when compared to the control polypeptides. These improved binding characteristics include higher maximum binding capacity (in RU), higher binding affinity as well as more favourable association and dissociation profiles, as measured in Biacore.

[0229] As shown in Fig. 9, neither the IgA binding polypeptide comprising SEQ ID NO:77 (SEQ ID NO:123) nor any of the control polypeptides (SEQ ID NQ:104 and 105) exhibited apparent binding affinity for human polyclonal IgA Fab fragment (VH3 class). In conclusion, IgA binding polypeptides of the present disclosure are shown to have binding affinity for the Fc region of human polyclonal IgA. Moreover, IgA binding polypeptides according to the present disclosure show improved binding characteristics to human polyclonal IgA Fc fragment when compared to known control polypeptides.

[0230] Example 6

[0231] Evaluation of dynamic binding capacity

[0232] Prototypes of IgA binding polypeptide multimer (hexamer and tetramer) ligands of different ligand density were produced. Frontal analysis of the tetrameric ligands is presented herein to determine dynamic binding capacity (DBG) for polyclonal human IgA.

[0233] Materials and Methods

[0234] Frontal analyses with polyclonal human IgA were performed on IgA binding polypeptide tetramers of SEQ ID NO:77 (SEQ ID NO: 100), which were produced with an N-terminal leader sequence (SEQ ID NO:103) and His6-tag, and were packed in Tricorn 5 / 50 columns (Cytiva).

[0235] The polyclonal human IgA test material was purified from human plasma using the prototype of SEQ ID NQ:100 packed in HiScale 26 / 110 column (Cytiva). The IgA sample was diluted in adsorption buffer to 1 mg / mL and filtered using a Sterivex 0.22 pm filter. The final sample concentration was determined by online 100% absorbance signal using the extinction coefficient 1.32.

[0236] For frontal analysis, the following buffers were used: adsorption / equilibration buffer: phosphate buffer 20 mM + 0.15 M NaCI, pH 7.2 elution buffer: citrate buffer 25 mM, pH 2.5

[0237] - CIP: 0.1 M NaOH

[0238] For the determination of 100% absorbance signal, PBS buffer was run through the bypass position until a stable baseline was reached. Auto zero was executed and the IgA solution was applied through bypass to obtain a 100% signal at a flow rate of 0.167 mL / min. Afterwards, PBS buffer was run through the bypass position until a stable baseline was reached.

[0239] The IgA-sample was loaded to the columns via the sample-pump at flow rate 0.167 mL / min (6 min RT) until the UV signal of approx. 20%-of-maximum was reached. The column was then washed with adsorption buffer at flow rate 0.167 mL / min. The protein was eluted with elution buffer at flow rate 0.167 mL / min. The column was cleaned with 0.1 M NaOH at a flow rate of 0.2 mL / min during 3 CV followed by reequilibration with adsorption buffer.

[0240] The breakthrough capacity was calculated using Extensions-DBC Calculations- Analyze tool in UNICORN 7.7 (Cytiva) according to manufacturer's instructions. Accordingly, breakthrough capacity at 10% was calculated, which equals to the amount of IgA that is loaded onto the column until the concentration of IgA in the column effluent is 10% of the IgA concentration in the feed.

[0241] Results and Conclusions

[0242] Frontal analysis results for separation matrices coupled to the tetrameric construct SEQ ID NO:100 with different ligand densities are shown in Table 4. Moreover, a DBC run with 1 mg / mL IgA until 20% breakthrough is shown in Fig. 10 on the separation matrix coupled to SEQ ID NQ:100 at low ligand density, wherein offset absorbance was observed to approximately 40 mAU during sample application and wherein IgA breakthrough occurred at about 35 mL elution volume.

[0243] Table 4: DBC results for SEQ ID NQ:100 prototypes at varying ligand densities.

[0244] The herein presented frontal analysis results demonstrate optimal coupling yield and optimal binding capacity for IgA binding polypeptide multimers, such as SEQ ID NQ:100, according to the present disclosure. Optimal binding capacity may be achieved with low ligand density, which is considered particularly advantageous for cost-efficient production of IgA binding polypeptide ligands for chromatography applications.

[0245] Example 7

[0246] Evaluation of alkaline stability on column

[0247] An accelerated alkaline stability study was performed on a prototype of the tetrameric construct SEQ ID NQ:100, with high ligand density (about 16 mg / mL).

[0248] Material and Methods

[0249] The method included frontal analysis, as described above, followed by incubation of the column in 0.1 M NaOH for 4 hours corresponding to 16 CIP cycles (15 min contact time). Sample application was performed until 85% of the 100% absorbance signal was reached. Equilibration of the column and frontal analysis were performed between incubations.

[0250] Results and Conclusions

[0251] The results of the accelerated alkaline stability study are listed in Table 5 and show that the tested prototype withstood 80 CIP cycles with 0.1 M NaOH with >80% residual capacity (QBIO and QBSO). NO reduction in QBSO capacity was observed for the first 32 cycles. Thereafter, the DBC gradually decreased. After 112 cycles (28 hours contact time) the remaining capacity was approximately 70% of the DBC at start

[0252] (QBIO and BSO).

[0253] Table 5: Results from accelerated alkaline study on a prototype SEO ID NO: 100 with high ligand density

[0254] These data demonstrate that the herein tested IgA binding polypeptide multimers are particularly beneficial for chromatography applications with retained binding capacity over several runs, wherein the column may be cleaned in alkaline conditions.

[0255] ITEMIZED LIST OF EMBODIMENTS

[0256] 1. An IgA binding polypeptide derived from Staphylococcus Protein A (SpA) or any domain thereof, wherein said polypeptide has a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NO:9 for the same IgA polypeptide.

[0257] 2. The IgA binding polypeptide according to item 1, wherein said polypeptide has binding affinity to an Fc region of an immunoglobulin, and has lower binding affinity for a VH3 region of trastuzumab compared to the binding affinity of SEQ ID NO:3 for the same VH3 region.

[0258] 3. The IgA binding polypeptide according to item 1 or 3, wherein said polypeptide comprises an amino acid sequence that has at least 70% identity to a sequence selected from the group consisting of SEQ ID NO:l-8.

[0259] 4. The IgA binding polypeptide according to item 3, wherein said amino acid sequence has at least 70% identity to SEQ ID NO:1 and / or SEQ ID NO:2, such as SEQ ID NO:2.

[0260] 5. An IgA binding polypeptide comprising an amino acid sequence comprising Sequence A, which Sequence A consists of an amino acid sequence selected from i), ii) and iii), wherein i), ii) and iii) are defined as follows: i) X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35 (SEQ ID NO:11), wherein X33 is selected from T, S, G, Q, A, E, H, R, P, D, K and N; ii) an amino acid sequence which has at least 81% identity to a sequence defined by i); iii) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 9-35 in SEQ ID NO:1, residues 9-35 in SEQ ID NO:2, residues 9-35 in SEQ ID NO:3, residues 9- 35 in SEQ ID NO:4, residues 9-35 in SEQ ID NO:5, residues 9-35 in SEQ ID NO:6, residues 12-38 in SEQ ID NO:7 and residues 2-28 in SEQ ID NO:8; wherein additionally, in each of i), ii) and iii), independently from each other,

[0261] X9 is selected from T and S,

[0262] X10 is I,

[0263] Xu is selected from Q, E, V and L,

[0264] X13 is S,

[0265] X14 is selected from Q and R,

[0266] X17 is R;

[0267] Xis is selected from L, Q, E and V,

[0268] X24 is selected from R and H,

[0269] X25 is selected from R and Q,

[0270] X27is K,

[0271] X28 is L,

[0272] X32 is H, and

[0273] X35 is L.

[0274] 6. The IgA binding polypeptide according to item 5, wherein X9 is S.

[0275] 7. The IgA binding polypeptide according to item 5 or 6, wherein Xu is selected from E and V.

[0276] 8. The IgA binding polypeptide according to any one of items 5-7, wherein Xu is E.

[0277] 9. The IgA binding polypeptide according to any one of items 5-8, wherein X14 is R.

[0278] 10. The IgA binding polypeptide according to any one of items 5-9, wherein Xis is selected from L and Q.

[0279] 11. The IgA binding polypeptide according to any one of items 5-10, wherein Xis is Q. 12. The IgA binding polypeptide according to any one of items 5-11, wherein X24 is H.

[0280] 13. The IgA binding polypeptide according to any one of items 5-12, wherein X25 is Q.

[0281] 14. The IgA binding polypeptide according to any one of items 5-13, wherein X33 in sequence i), ii) and / or iii) is selected from T, S, G, Q, A, E, H, R, P, D, K and N, such as from S, G, A and E.

[0282] 15. The IgA binding polypeptide according to any one of items 5-14, wherein X33 in sequence i), ii) and / or iii) is selected from A and S.

[0283] 16. The IgA binding polypeptide according to any one of items 5-15, wherein X33 in sequence i), ii) and / or iii) is S.

[0284] 17. The IgA binding polypeptide according to any one of items 5-16, wherein sequence i) corresponds to the sequence from position X9 to position X35 in a sequence selected from the group consisting of SEQ ID NO:14-29, such as the group consisting of SEQ ID NO:26-29.

[0285] 18. The IgA binding polypeptide according to any one of items 5-17, wherein sequence i) corresponds to the sequence from position X9 to position X35 in SEQ ID NO:29.

[0286] 19. The IgA binding polypeptide according to any one of items 5-18, wherein said polypeptide has a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NO:9 for the same IgA polypeptide.

[0287] 20. The IgA binding polypeptide according to any one of items 5-19, wherein said polypeptide has binding affinity to an Fc region of an immunoglobulin, and has lower binding affinity for a VH3 region of trastuzumab compared to the binding affinity of SEQ ID NO:3 for the same VH3 region.

[0288] 21. The IgA binding polypeptide according to any one of items 5-20, wherein the amino acid sequence of said polypeptide has at least 70% identity to a sequence selected from the group consisting of SEQ ID NO:l-8.

[0289] 22. The IgA binding polypeptide according to item 21, wherein said amino acid sequence has at least 70% identity to SEQ ID NO:1 and / or SEQ ID NO:2, such as SEQ ID NO:2.

[0290] 23. The IgA binding polypeptide according to item 3 or 4, where the amino acid sequence of said polypeptide comprises Sequence A as defined in any one of items 5-18.

[0291] 24. The IgA binding polypeptide according to any one of items 5-23, wherein Sequence A as defined in any one of items 5-18 is comprised in the amino acid sequence of said polypeptide in an amino acid region that corresponds to positions X9to X35in SEQ ID NO:3.

[0292] 25. The IgA binding polypeptide according to any one of items 3-24, wherein said polypeptide comprises an amino acid selected from T, S, G, Q, A, E, H, R, P, D, K and N, such as from S, G, A and E, such as from A and S, in a position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; an amino acid selected from E, G, R, D, K, Q, N, H and S, such as from G, R, E and D, such as from R and G, in a position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; and an amino acid selected from L, V, S, I, R and G, such as from V, L, I and R, such as from I, V and R, in a position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3.

[0293] 26. The IgA binding polypeptide according to item 25, wherein when the amino acid in the position corresponding to position X51 in SEQ ID NO:3 is L then the amino acids in positions that correspond to positions X33 and X40 in SEQ ID NO:3 are selected from AD, HK, EG, ER, GR, AK, AR, PK, RR and KK, respectively, and when the amino acid in the position corresponding to position X51 in SEQ ID NO:3 is G then the amino acids in positions that correspond to positions X33 and X40 in SEQ ID NO:3 are TK, respectively.

[0294] 27. The IgA binding polypeptide according to item 25 or 26, wherein said polypeptide comprises a S in the position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3.

[0295] 28. The IgA binding polypeptide according to any one of items 25-27, wherein said polypeptide comprises a G in the position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3.

[0296] 29. The IgA binding polypeptide according to any one of items 25-28, wherein said polypeptide comprises a V in the position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3.

[0297] 30. The IgA binding polypeptide according to any one of items 25-29, wherein said polypeptide comprises an E in a position in the amino acid sequence thereof that corresponds to position X43 in SEQ ID NO:3. 31. The IgA binding polypeptide according to any one of items 25-30, wherein said polypeptide comprises a Y in a position in the amino acid sequence thereof that corresponds to position Xs in SEQ ID NO:3.

[0298] 32. The IgA binding polypeptide according to any one of items 5-25, wherein the amino acid sequence of said polypeptide comprises Sequence B, which Sequence B consists of an amino acid sequence selected from iv), v) and vi), wherein iv), v) and vi) are defined as follows: iv) X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35DDPSX40SX42X43X4 4LX46X47AX49KX51 (SEQ ID NO:12); v) an amino acid sequence which has at least 81% identity to a sequence defined by iv); vi) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 9-51 in SEQ ID NO:1, residues 9-51 in SEQ ID NO:2, residues 9-51 in SEQ ID NO:3, residues 9- 51 in SEQ ID NO:4, residues 9-51 in SEQ ID NO:5, residues 9-51 in SEQ ID NO:6, residues 12-54 in SEQ ID NO:7 and residues 2-44 in SEQ ID NO:8; wherein the amino acid sequence from position X9 to position X35 is Sequence A is as defined in any one of items 5-18; and wherein additionally, in each of iv), v) and vi), independently from each other,

[0299] X40 is selected from V, E, G, R, D, K, Q, N, H and S, such as from E, G, R, D, K, Q, N, H and S, such as from G, R, E and D, such as from R and G, X42 is selected from K, A and T,

[0300] X43 is selected from A, E and N,

[0301] X44 is selected from I, L and V,

[0302] X46 is selected from A, S and G,

[0303] X47 is selected from E and K,

[0304] X49 is selected from K and Q, and Xsi is selected from L, V, S, I, R and G, such as from V, L, I and R, such as from I, V and R.

[0305] 33. The IgA binding polypeptide according to item 32, wherein when the amino acid in position X51 is L then the amino acids in positions X33 and X40 are selected from SV, SQ, AD, HK, EG, ER, GR, AK, AR, PK, RR and KK, such as from AD, HK, EG, ER, GR, AK, AR, PK, RR and KK, respectively, and when the amino acid in position X51 is G then the amino acids in positions X33 and X40 is TK, respectively.

[0306] 34. The IgA binding polypeptide according to item 32 or 33, wherein X40 is G.

[0307] 35. The IgA binding polypeptide according to any one of items 32-34, wherein X42 is K.

[0308] 36. The IgA binding polypeptide according to any one of items 32-35, wherein X43 is selected from A and E.

[0309] 37. The IgA binding polypeptide according to any one of items 32-36, wherein X43 is E.

[0310] 38. The IgA binding polypeptide according to any one of items 32-37, wherein X44 is I.

[0311] 39. The IgA binding polypeptide according to any one of items 32-38, wherein X46 is A.

[0312] 40. The IgA binding polypeptide according to any one of items 32-39, wherein X47 is E. 41. The IgA binding polypeptide according to any one of items 32-40, wherein X49 is

[0313] K.

[0314] 42. The IgA binding polypeptide according to any one of items 32-41, wherein X51 is V.

[0315] 43. The IgA binding polypeptide according to any one of items 32-42, wherein sequence iv) corresponds to the sequence from position X9 to position X51 in a sequence selected from the group consisting of SEQ ID NO:14-61, such as the group consisting of SEQ ID NO:30-61, such as the group consisting of SEQ ID NO:46-61.

[0316] 44. The IgA binding polypeptide according to any one of items 32-43, wherein sequence iv) corresponds to the sequence from position X9 to position X51 in a sequence selected from the group consisting of SEQ ID NO:26-29, SEQ ID NO:42-45 and SEQ ID NO:58-61; such as the group consisting of SEQ ID NO:42-45 and SEQ ID NO:58-61; such as the group consisting of SEQ ID NO:58-61.

[0317] 45. The IgA binding polypeptide according to any one of items 32-44, wherein sequence iv) corresponds to the sequence from position X9 to position X51 in a sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:45 and SEQ ID NO:61; such as the group consisting of SEQ ID NO:45 and SEQ ID NO:61; such as the sequence from position X9 to position X51 in SEQ ID NO:61.

[0318] 46. The IgA binding polypeptide according to any one of items 32-45, wherein the amino acid sequence of said polypeptide comprises Sequence C, which Sequence C consists of an amino acid sequence selected from vii), viii) and ix), wherein vii), viii) and ix) are defined as follows: vii) X5X6KX8X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35DDPSX40S X42X43X44LX46X47AX49KX51 (SEQ ID NO:13); viii) an amino acid sequence which has at least 81% identity to a sequence defined by vii); ix) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 5-51 in SEQ ID NO:1, residues 5-51 in SEQ ID NO:2, residues 5-51 in SEQ ID NO:3, residues 5- 51 in SEQ ID NO:4, residues 5-51 in SEQ ID NO:5, residues 5-51 in SEQ ID NO:6, residues 8-54 in SEQ ID NO:7; wherein the amino acid sequence from position X9 to position X51 is Sequence B is as defined in any one of items 32-45; and wherein additionally, in each of vii), viii) and ix), independently from each other,

[0319] X5 is selected from F and Y,

[0320] Xe is selected from D and N, and

[0321] Xs is selected from E, D and A.

[0322] 47. The IgA binding polypeptide according to item 46, wherein X5 is Y.

[0323] 48. The IgA binding polypeptide according to item 46 or 47, wherein Xe is D.

[0324] 49. The IgA binding polypeptide according to any one of items 46-48, wherein Xs is E.

[0325] 50. The IgA binding polypeptide according to item 46-49, wherein sequence vii) corresponds to the sequence from position X5 to position X51 in a sequence selected from the group consisting of SEQ ID NO:14-77, such as the group consisting of SEQ ID NQ:30-77, such as the group consisting of SEQ ID NO:46-77, such as the group consisting of SEQ ID NO:62-77.

[0326] 51. The IgA binding polypeptide according to item 46-50, wherein sequence vii) corresponds to the sequence from position X5 to position X51 in a sequence selected from the group consisting of SEQ ID NO:26-29, SEQ ID NO:42-45, SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:42-45, SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:74-77.

[0327] 52. The IgA binding polypeptide according to item 46-51, wherein sequence vii) corresponds to the sequence from position Xs to position X51 in a sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:45, SEQ ID NO:61 and SEQ ID NO:77; such as the group consisting of SEQ ID NO:45, SEQ ID NO:61 and SEQ ID NO:77; such as the group consisting SEQ ID NO:61 and SEQ ID NO:77; such as the sequence from position Xs to position X51 in SEQ ID NO:77.

[0328] 53. The IgA binding polypeptide according to any one of items 3-52, wherein the amino acid sequence comprised in said polypeptide is a sequence selected from the group consisting of SEQ ID NO:14-77, such as the group consisting of SEQ ID NQ:30- 77, such as the group consisting of SEQ ID NO:46-77, such as the group consisting of SEQ ID NO:62-77.

[0329] 54. The IgA binding polypeptide according to any one of items 3-53, wherein the amino acid sequence comprised in said polypeptide is a sequence selected from the group consisting of SEQ ID NO:26-29, SEQ ID NO:42-45, SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:42-45, SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:58-61 and SEQ ID NO:74-77; such as the group consisting of SEQ ID NO:74-77.

[0330] 55. The IgA binding polypeptide according to any one of items 3-54, wherein the amino acid sequence comprised in said polypeptide is a sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:45, SEQ ID NO:61 and SEQ ID NO:77; such as the group consisting of SEQ ID NO:45, SEQ ID NO:61 and SEQ ID NO:77; such as the group consisting of SEQ ID NO:61 and SEQ ID NO:77. 56. The IgA binding polypeptide according to any one of items 3-55, wherein the amino acid sequence comprised in said polypeptide is SEQ ID NO:77.

[0331] 57. The IgA binding polypeptide according to any one of items 1-56, wherein said polypeptide derived from SpA or any domain thereof, said Sequence A, said Sequence B or said Sequence C forms part of a three-helix bundle protein domain.

[0332] 58. The IgA binding polypeptide according to item 57, wherein said polypeptide derived from SpA or any domain thereof, said Sequence A, said Sequence B or said Sequence C forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.

[0333] 59. The IgA binding polypeptide according to item 57 or 58, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.

[0334] 60. The IgA binding polypeptide according to item 59, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.

[0335] 61. The IgA binding polypeptide according to any one of items 1-60, wherein the IgA binding polypeptide is capable of binding to said IgA polypeptide with a binding affinity that is more than 100%, such as more than 110%, such as more than 120%, of the corresponding binding affinity of SEQ ID NO:9 and / or SEQ ID NQ:10 to the same IgA polypeptide.

[0336] 62. The IgA binding polypeptide according to any one of items 1-61, wherein the IgA binding polypeptide has minimal or essentially no binding affinity for an immunoglobulin selected from the group consisting of IgM, IgD, IgE and IgG, such as IgG. 63. An IgA binding polypeptide multimer, wherein each monomer of the multimer comprises an IgA binding polypeptide which is independently selected from any IgA binding polypeptide defined in any one of items 1-62.

[0337] 64. The IgA binding polypeptide multimer according to item 63, wherein said multimer is selected from the group consisting of dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer and decamer.

[0338] 65. The IgA binding polypeptide multimer according to item 63 or 64, wherein said multimer is selected from the group consisting of tetramer and hexamer.

[0339] 66. The IgA binding polypeptide multimer according to any one of items 63-65, wherein said multimer is a tetramer.

[0340] 67. The IgA binding polypeptide multimer according to any one of items 63-66, wherein said multimer is a homomer.

[0341] 68. The IgA binding polypeptide multimer according to any one of items 63-67, wherein said multimer comprises an amino acid sequence selected from the group consisting of SEQ ID NO:78-93, such as the group consisting of SEQ ID NO:82-93, such as the group consisting of SEQ ID NO:86-93, such as the group consisting of SEQ ID NQ:90-93.

[0342] 69. The IgA binding polypeptide multimer according to any one of items 63-68, wherein said multimer comprises an amino acid sequence selected from the group consisting of SEQ ID NO:81, SEQ ID NO:85, SEQ ID NO:89 and SEQ ID NO:93; such as the group consisting of SEQ ID NO:85, SEQ ID NO:89 and SEQ ID NO:93; such as the group consisting of SEQ ID NO:89 and SEQ ID NO:93. 70. The IgA binding polypeptide multimer according to any one of items 63-69, wherein said multimer comprises SEQ ID NO:93.

[0343] 71. The IgA binding polypeptide multimer according to any one of items 63-67, further comprising at least one linker.

[0344] 72. The IgA binding polypeptide multimer according to item 71, wherein said linker comprises up to 15 amino acid residues.

[0345] 73. The IgA binding polypeptide according to any one of items 1-62 or the IgA binding polypeptide multimer according to any one of items 63-72, which comprises additional amino acids at the C-terminal and / or N-terminal end thereof.

[0346] 74. The IgA binding polypeptide or the IgA binding polypeptide multimer according to item 73, wherein said additional amino acid(s) improve(s) production, purification, stabilization and / or in vitro coupling of the polypeptide or the polypeptide multimer.

[0347] 75. The IgA binding polypeptide or the IgA binding polypeptide multimer according to item 74, wherein said additional amino acid(s) improve(s) coupling of the polypeptide or the polypeptide multimer and are selected from the group consisting of one or more cysteine residues, a plurality of lysine residues and a plurality of histidine residues.

[0348] 76. The IgA binding polypeptide multimer according to any one of items 73-75, wherein said multimer comprises SEQ ID NQ:100.

[0349] 77. The IgA binding polypeptide according to any one of items 1-62 and 73-75, or the IgA binding polypeptide multimer according to in any one of items 63-76, wherein the IgA binding polypeptide or the IgA binding polypeptide multimer exhibits at least

[0350] 80% residual binding capacity after about 80 cleaning cycles with 0.1 M NaOH.

[0351] 78. A polynucleotide encoding the IgA binding polypeptide as defined in any one of items 1-62, 73-75 and 77, or the IgA binding polypeptide multimer as defined in any one of items 63-77.

[0352] 79. An expression vector comprising the polynucleotide as defined in item 78.

[0353] 80. A host cell comprising the expression vector as defined in item 79.

[0354] 81. An adsorbent material comprising the IgA binding polypeptide as defined in any one of items 1-62, 73-75 and 77, or the IgA binding polypeptide multimer as defined in any one of items 63-77, coupled to a solid support.

[0355] 82. The adsorbent material according to item 81, wherein the solid support is selected from a particle, a bead, a fiber, a fibrous membrane, a filter, a sheet, a porous monolith, a chip, a plate, and a well.

[0356] 83. The adsorbent material according to item 81 or 82, wherein the solid support is a separation matrix.

[0357] 84. A method of isolating IgA or a fragment thereof comprising a) contacting a liquid sample comprising said IgA or said fragment thereof with the adsorbent material as defined in any one of items 81-83.

Claims

74CLAIMS1. An IgA binding polypeptide derived from Staphylococcus Protein A (SpA) or any domain thereof, wherein said polypeptide has a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NO:9 for the same IgA polypeptide.

2. The IgA binding polypeptide according to claim 1, wherein said polypeptide has binding affinity to an Fc region of an immunoglobulin, and has lower binding affinity for a VH3 region of trastuzumab compared to the binding affinity of SEQ ID NO:3 for the same VH3 region.

3. An IgA binding polypeptide comprising an amino acid sequence comprising Sequence A, which Sequence A consists of an amino acid sequence selected from i), ii) and iii), wherein i), ii) and iii) are defined as follows: i) X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35 (SEQ ID NO:11), wherein X33 is selected from T, S, G, Q, A, E, H, R, P, D, K and N; ii) an amino acid sequence which has at least 81% identity to a sequence defined by i); iii) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 9-35 in SEQ ID NO:1, residues 9-35 in SEQ ID NO:2, residues 9-35 in SEQ ID NO:3, residues 9- 35 in SEQ ID NO:4, residues 9-35 in SEQ ID NO:5, residues 9-35 in SEQ ID NO:6, residues 12-38 in SEQ ID NO:7 and residues 2-28 in SEQ ID NO:8; wherein additionally, in each of i), ii) and iii), independently from each other, X9 is selected from T and S,X10 is I,Xu is selected from Q, E, V and L,X13 is S,X14 is selected from Q and R,X17 is R;75Xis is selected from L, Q, E and V,X24 is selected from R and H,X25 is selected from R and Q,X27is K,X28 is L,X32 is H, andX35 is L.

4. The IgA binding polypeptide according to claim 3, wherein sequence i) corresponds to the sequence from position X9 to position X35 in a sequence selected from the group consisting of SEQ ID NO:14-29, such as the group consisting of SEQ ID NO:26-29.

5. The IgA binding polypeptide according to claim 3 or 4, wherein said polypeptide has a higher binding affinity for an IgA polypeptide compared to the binding affinity of SEQ ID NO:9 for the same IgA polypeptide.

6. The IgA binding polypeptide according to any one of claims 3-5, wherein said polypeptide has binding affinity to an Fc region of an immunoglobulin, and has lower binding affinity for a VH3 region of trastuzumab compared to the binding affinity of SEQ ID NO:3 for the same VH3 region.

7. The IgA binding polypeptide according to any one of claims 1-6, wherein said polypeptide comprises an amino acid sequence that has at least 70% identity to a sequence selected from the group consisting of SEQ ID NO:1-8.

8. The IgA binding polypeptide according to any one of claims 3-7, wherein said polypeptide comprises76 an amino acid selected from T, S, G, Q, A, E, H, R, P, D, K and N, such as from S, G, A and E, such as from A and S, in a position in the amino acid sequence thereof that corresponds to position X33 in SEQ ID NO:3; an amino acid selected from E, G, R, D, K, Q, N, H and S, such as from G, R, E and D, such as from R and G, in a position in the amino acid sequence thereof that corresponds to position X40 in SEQ ID NO:3; and an amino acid selected from L, V, S, I, R and G, such as from V, L, I and R, such as from I, V and R, in a position in the amino acid sequence thereof that corresponds to position X51 in SEQ ID NO:3.

9. The IgA binding polypeptide according to any one of claims 3-8, wherein said polypeptide comprises an E in a position in the amino acid sequence thereof that corresponds to position X43 in SEQ ID NO:3.

10. The IgA binding polypeptide according to any one of claims 3-9, wherein said polypeptide comprises a Y in a position in the amino acid sequence thereof that corresponds to position X5 in SEQ ID NO:3.

11. The IgA binding polypeptide according to any one of claims 3-8, wherein the amino acid sequence of said polypeptide comprises Sequence B, which Sequence B consists of an amino acid sequence selected from iv), v) and vi), wherein iv), v) and vi) are defined as follows: iv) X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35DDPSX40SX42X43X4 4LX46X47AX49KX51 (SEQ ID NO:12); v) an amino acid sequence which has at least 81% identity to a sequence defined by iv); vi) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 9-51 in SEQ ID NO:1, residues 9-51 in SEQ ID NO:2, residues 9-51 in SEQ ID NO:3, residues 9-7751 in SEQ ID NO:4, residues 9-51 in SEQ ID NO:5, residues 9-51 in SEQ ID NO:6, residues 12-54 in SEQ ID NO:7 and residues 2-44 in SEQ ID NO:8; wherein the amino acid sequence from position Xg to position X35 is Sequence A is as defined in in claim 3 or 4; and wherein additionally, in each of iv), v) and vi), independently from each other,X40 is selected from V, E, G, R, D, K, Q, N, H and S, such as from E, G, R, D, K, Q, N, H and S, such as from G, R, E and D, such as from R and G, X42 is selected from K, A and T,X43 is selected from A, E and N,X44 is selected from I, L and V,X46 is selected from A, S and G,X47 is selected from E and K,X49 is selected from K and Q, andX51 is selected from L, V, S, I, R and G, such as from V, L, I and R, such as from I, V and R.

12. The IgA binding polypeptide according to claim 11, wherein the amino acid sequence of said polypeptide comprises Sequence C, which Sequence C consists of an amino acid sequence selected from vii), viii) and ix), wherein vii), viii) and ix) are defined as follows: vii) X5X6KX8X9X10X11AX13X14EIX17X18LPNLTX24X25QX27X28AFIX32X33LX35DDPSX40S X42X43X44LX46X47AX49KX51 (SEQ ID NO:13); viii) an amino acid sequence which has at least 81% identity to a sequence defined by vii); ix) an amino acid sequence which has at least 70% identity to any sequence selected from the group consisting of: residues 5-51 in SEQ ID NO:1, residues 5-51 in SEQ ID NO:2, residues 5-51 in SEQ ID NO:3, residues 5- 51 in SEQ ID NO:4, residues 5-51 in SEQ ID NO:5, residues 5-51 in SEQ ID NO:6, residues 8-54 in SEQ ID NO:7;78 wherein the amino acid sequence from position Xg to position X51 is Sequence B is as defined in claim 11; and wherein additionally, in each of vii), viii) and ix), independently from each other,X5 is selected from F and Y,Xe is selected from D and N, andXs is selected from E, D and A.

13. An IgA binding polypeptide multimer, wherein each monomer of the multimer comprises an IgA binding polypeptide which is independently selected from any IgA binding polypeptide defined in any one of claims 1-12.

14. An adsorbent material comprising the IgA binding polypeptide as defined in any one of claims 1-12, or the IgA binding polypeptide multimer as defined in claim 13, coupled to a solid support.

15. A method of isolating IgA or a fragment thereof comprising a) contacting a liquid sample comprising said IgA or said fragment thereof with the adsorbent material as defined in claim 14.

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