Binding proteins for the enzyme acid alpha glucosidase (gaa) and uses thereof

Abstract

The present invention relates to the field of protein purification and, in particular, to a novel protein specifically binding to acid alpha-glucosidase (GAA). The present invention further relates to a fusion protein comprising the novel protein specifically binding to GAA. Furthermore, the present invention relates to an affinity matrix comprising the GAA binding protein of the present invention. The present invention also relates to the use of these GAA binding proteins or affinity matrices for affinity purification of GAA and to a method for affinity purification of GAA using the GAA binding proteins of the present invention. A further use relates to an analytical method for determining GAA in a liquid.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of protein purification, in particular to novel proteins specifically binding to acid alpha-glucosidase (GAA). The present invention further relates to fusion proteins comprising the novel proteins specifically binding to GAA. Furthermore, the present invention relates to affinity matrices comprising the GAA binding proteins of the present invention. The present invention also relates to the use of these GAA binding proteins or affinity matrices for the affinity purification of GAA and to methods for the affinity purification of GAA using the GAA binding proteins of the present invention. Further uses relate to analytical methods for the determination of GAA in a liquid. BACKGROUND

[0002] The lysosomal enzyme GAA (GAA) is essential in the process of glycogen degradation into glucose. Deficiency in GAA leads to the accumulation of glycogen in lysosomes and to the impairment of muscle and nerve cells, a condition known as glycogen storage disease II (Pompe's disease). The treatment of this metabolic disease is the replacement of GAA. It is therefore necessary to provide methods for the purification of the enzyme. There is a continuous need in the art for advanced tools allowing the efficient purification of GAA proteins.

[0003] The present invention meets this need by providing novel GAA binding polypeptides. These novel GAA binding polypeptides are particularly advantageous as affinity ligands for GAA as they allow the precise capture in affinity chromatography, thereby providing highly purified GAA for medical use.

[0004] The above summary of the invention does not necessarily describe all problems solved by the present invention. SUMMARY

[0005] The present disclosure provides the following items 1-15, but is not exclusively limited thereto:

[0006] 1. Acid alpha-glucosidase (GAA) binding protein comprising one or more GAA binding domains, wherein at least one domain comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18, or an amino acid sequence having at least 95% sequence identity thereto.

[0007] 2. The GAA binding protein according to item 1, wherein the protein has a binding affinity to GAA of less than 200 nM (as determined by surface plasmon resonance as described herein).

[0008] 3. The GAA binding protein according to item 1, wherein the GAA binding protein comprises 2, 3, 4, 5 or 6 domains connected to each other.

[0009] 4. The GAA binding protein according to clause 3, wherein the GAA binding protein is a homo-multimer.

[0010] 5. The GAA binding protein according to clause 3, wherein the GAA binding protein is a hetero-multimer.

[0011] 6. The GAA binding protein according to clause 3, wherein one or more of the domains are directly connected to each other or connected via one or more peptide linkers.

[0012] 7. A fusion protein comprising the protein according to any one of clauses 1-6.

[0013] 8. A polynucleotide encoding the protein according to any one of clauses 1-6 or the fusion protein according to clause 7.

[0014] 9. The GAA binding protein according to any one of clauses 1-6 or the fusion protein according to clause 7 for use in affinity purification of GAA.

[0015] 10. The GAA binding protein according to any one of clauses 1-6 or the fusion protein according to clause 7, further comprising one or more coupling sites for coupling to an affinity purification matrix, preferably wherein the GAA binding protein comprises one or more more cysteine residues coupled to an affinity purification matrix.

[0016] 11. An affinity purification matrix comprising the protein according to any one of clauses 1-6 or the fusion protein according to clause 7.

[0017] 12. Use of the GAA binding protein according to any one of clauses 1-6, 10 or the fusion protein according to clause 7 or the affinity purification matrix according to clause 11 for affinity purification of GAA.

[0018] 13. A method of affinity purifying GAA, the method comprising: (a) providing a liquid containing GAA; (b) providing an affinity purification matrix comprising at least one GAA binding protein according to any one of clauses 1-6, 10 or the fusion protein according to clause 7 coupled to the affinity purification matrix; (c) contacting the affinity purification matrix with the liquid under conditions that allow binding of at least one GAA binding protein according to any one of clauses 1-6, 10 to GAA; (d) eluting the GAA from the affinity purification matrix.

[0019] 14. Use of the GAA binding protein according to clauses 1-6 or the fusion protein according to clause 7 in a method of determining the presence of GAA.

[0020] 15. A method of analyzing a liquid sample for the presence of GAA, the method comprising the steps of:

[0021] (i) providing a liquid containing GAA,

[0022] (ii) providing a GAA binding protein according to any one of the preceding claims 1-6, or the fusion protein according to claim 7,

[0023] (iii) contacting the liquid of (i) with the GAA binding protein according to any one of the preceding claims 1-6 under conditions allowing binding of the at least one GAA binding protein according to claim 1 or the fusion protein according to claim 7 to GAA,

[0024] (iv) isolating the complex of GAA and the GAA binding protein according to any one of the preceding claims 1-6 or the fusion protein according to claim 7,

[0025] (v) determining the amount of the GAA binding protein according to any one of the preceding claims 1-6 in the liquid of (i).

[0026] The summary of the application is not limiting and other aspects and embodiments of the application will become apparent from the following description, examples, and drawings. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 : shows the amino acid sequence of the GAA binding protein.

[0028] Figure 2: shows a cycle study on Purolite Praesto 85 epoxy resin. Figure 2A shows DBC 10% of SEQ ID NO: 1 (with N-terminal extension) on Praesto 85 epoxy resin at 6 min residence time. The binding capacity was 19.5 mg / mL. Figure 2B shows DBC and base stability of GAA binding protein (SEQ ID NO: 1 with N-terminal extension) coupled to Praesto 85 resin and loaded with 2 mg / mL GAA. The remaining capacity after 50 cycles of loading, elution and 0.1 M NaOH regeneration was at least 90%. The DBC 10% was measured at the dotted line point indicated on the line. DETAILED DESCRIPTION

[0029] The present application provides novel polypeptides having binding affinity to GAA. The polypeptides of the present application represent advanced and powerful tools that fill a gap in the field of protein engineering and purification. In particular, the novel polypeptides provide advantageous effects in protein purification by virtue of said binding affinity to GAA. Thus, the novel polypeptides of the present application are particularly advantageous as they allow precise capture of GAA in affinity chromatography. The GAA binding proteins of the present application provide an efficient tool for purifying GAA which can then be used for medical purposes.

[0030] The binding affinity to GAA is provided by a polypeptide having SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18, or a polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18.

[0031] Before the application is described in detail below, it is to be understood that the application is not limited to the particular methodology, protocols and reagents described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects and embodiments only and is not intended to limit the scope of the present application which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the application. This includes technical and scientific terms and acronyms used in the field of protein engineering and purification, but also in the field of development of new GAA specific binding molecules for technical applications such as affinity chromatography as well as in the field of therapy and diagnostics.

[0032] The terms used herein are preferably defined as described in "A multilingual glossary of biotechnological terms: (lUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0033] Throughout this specification and the annexed claims, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term "comprising" or "including" is to be construed as meaning "consisting of or "consisting essentially of, if such a limitation is desired for any reason and to any extent.

[0034] Throughout this specification, several documents (e.g., patents, patent applications, scientific publications, manufacturer's specifications, instruction manuals, GenBank Accession Number sequence submissions, etc.) can be cited. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosures by virtue of prior invention. Some of the documents cited herein can be characterized as "incorporated by reference." In the event of a conflict between the definitions or teachings of such incorporated references and the definitions or teachings within this specification, the text of this specification controls.

[0035] All sequences mentioned herein are disclosed in the attached sequence listing, which is incorporated by reference in its entirety and disclosure.

[0036] General definitions of important terms used in the application

[0037] The terms "GAA-binding protein" or "acid alpha-glucosidase binding polypeptide" or "acid alpha-glucosidase binding protein" or "GAA-binding polypeptide" are used interchangeably herein to describe a protein capable of binding acid alpha-glucosidase (GAA; Uniprot Identifier P10253). As described herein, "GAA-binding protein" refers to a protein having a detectable interaction with GAA, for example, as determined by SPR analysis or other appropriate techniques known to the skilled person.

[0038] The terms "binding affinity" and "binding activity" are used interchangeably herein and refer to the ability of a polypeptide of the application to bind to another protein, peptide or fragment or domain thereof. Binding affinity is typically measured by the equilibrium dissociation constant (KD) and reported, which constant is used to evaluate and rank the strength of a bimolecular interaction. Binding affinity and dissociation constant can be measured quantitatively. Methods for determining binding affinity are well known to the skilled person and can be selected from, for example, the following methods recognized in the art: surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), Kinetic Exclusion Assay (KinExA assay), Bio-Layer Interferometry (BLI), flow cytometry, fluorescence spectroscopy techniques, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) and enhanced chemiluminescence (ECL). Generally speaking, the dissociation constant KD is determined at a temperature in the range of 20-30°C. If not specifically stated otherwise, the K DValues are determined at 25°C by SPR. The most widely used SPR-based system is the BIAcore produced by BIAcore AB. In various embodiments of the application, the binding affinity for GAA can be determined by the BIAcore SPR system. In various embodiments, the concentration of the analyte is 1 mM. In various other embodiments, the concentration of the analyte is 10 mM. In various other embodiments of the application, the polypeptide of the application has a binding affinity for GAA as determined by SPR, wherein the concentration of the analyte in the SPR assay is 10 mM, preferably wherein the binding affinity is determined at 25°C.

[0039] The term "fusion protein" relates to a protein comprising at least a first protein genetically combined with at least a second protein. Fusion proteins are generated by joining two or more genes that originally coded for separate proteins. Thus, a fusion protein can comprise a multimer of the same or different proteins expressed as a single linear polypeptide.

[0040] The term "linker" as used herein refers in its broadest sense to a molecule that covalently links at least two other molecules.

[0041] The term "amino acid sequence identity" refers to the identity (or difference) of the amino acid sequences of two or more proteins over a specified amount. The "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. To determine sequence identity, the sequence of the query protein is aligned with the sequence of the reference protein or polypeptide, e.g., with the polypeptide of SEQ ID NO: 1. Methods of sequence alignment are well known in the art. For example, to determine the degree of amino acid sequence identity of an arbitrary polypeptide with respect to the amino acid sequence of, e.g., SEQ ID NO: 1, preferably the SIM Local similarity program (Xiaoquin Huang and Webb Miller (1991), Advances in Applied Mathematics, vol. 12:337-357) is used, which is freely available. For multiple alignment analysis, preferably ClustalW (Thompson et al. (1994) Nucleic Acids Res., 22(22):4673-4680) is used.

[0042] The terms "protein" and "polypeptide" refer to any chain of two or more amino acids linked by peptide bonds, and not to a specific length of the product. Thus, "peptide," "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" can be used interchangeably with any of these terms. The term "polypeptide" also means the product of a post-translational modification of a polypeptide, such as, for example, glycosylation, which is well known in the art.

[0043] The terms "alkali-stable" or "alkali stability" or "caustic-stable" or "caustic stability" refer to the ability of the GAA-binding protein of the present application to tolerate alkaline conditions without significantly losing the ability to bind to GAA. One of skill in the art can readily test for alkali stability by incubating the GAA-binding protein with a sodium hydroxide solution, for example, as described in the Examples, and subsequently determining the binding activity for GAA by routine experimentation known to one of skill in the art, for example, by chromatography.

[0044] The term "chromatography" refers to a separation technique that uses a mobile phase and a stationary phase to separate one molecule (e.g., GAA) from other molecules (e.g., contaminants) in a sample. The liquid mobile phase contains a mixture of molecules and transports these molecules through or across the stationary phase (such as a solid matrix). The molecules in the mobile phase can be separated because the different molecules in the mobile phase interact differently with the stationary phase.

[0045] The term "affinity chromatography" refers to a specific mode of chromatography in which a ligand coupled to the stationary phase interacts with a molecule (i.e., GAA) in the mobile phase (sample), i.e., the ligand has a specific binding affinity for the molecule to be purified. As understood in the context of the present application, affinity chromatography involves adding a sample containing GAA to a stationary phase comprising a chromatographic ligand (e.g., a GAA-binding protein of the present application).

[0046] For the stationary phase, the terms "solid support" or "solid matrix" can be used interchangeably.

[0047] The terms "affinity matrix" or "affinity purification matrix" or "affinity chromatography matrix," which are used interchangeably herein, refer to a matrix, e.g., a chromatography matrix, to which an affinity ligand, e.g., a GAA-binding protein of the present application, is attached. The ligand (e.g., GAA-binding protein) is capable of specifically binding to a target molecule (e.g., a VH3-containing protein) to be purified or removed from a mixture.

[0048] As used herein, the term "affinity purification" refers to a method for purifying GAA from a liquid by binding GAA to a GAA-binding protein immobilized on a matrix. As a result, components other than GAA in the mixture are removed. In a further step, the bound GAA can be eluted in a purified form.

[0049] Detailed description of embodiments of the present invention

[0050] The present invention will now be further described. Different aspects of the present invention are defined in more detail in the following paragraphs. Each aspect defined below, unless clearly indicated to the contrary, may be combined with any other aspect or aspects. Specifically, any feature indicated as preferred or advantageous may be combined with any other feature or features indicated as preferred or advantageous.

[0051] The novel polypeptides of the present invention (including SEQ ID NO: 1, 2, 16, 17, 18, 22, 23) exhibit binding affinity for GAA (determined herein by SPR). The novel binding protein of acid alpha glucosidase comprises an amino acid sequence having at least 95% identity to any one of the groups selected from SEQ ID NO: 1-9, 14-20, 22-23. The novel GAA binding protein comprises one or more GAA binding domains, wherein at least one GAA binding domain comprises, consists essentially of, or is composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or amino acids having at least 95% sequence identity thereto. The amino acid sequences of the GAA binding proteins SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 16 and SEQ ID NO: 17 and SEQ ID NO: 18 are as follows: Figure 1 Neutralize as shown here:

[0052] SEQ ID NO:1(192403)

[0053]

[0054] SEQ ID NO:2(192402)

[0055]

[0056] SEQ ID NO:16(192403del2N)

[0057]

[0058] SEQ ID NO:17(192402del2N)

[0059]

[0060] The novel GAA binding protein comprises one or more GAA binding domains, wherein at least one GAA binding domain comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid having at least 95% sequence identity thereto:

[0061]

[0062] X1may in some embodiments be any amino acid selected from alanine (A) or leucine (L). X1corresponds to position 46 in SEQ ID NO: 1 or SEQ ID NO: 2.

[0063] X2may in some embodiments be any amino acid selected from serine (S) or methionine (M). X2corresponds to position 54 in SEQ ID NO: 1 or SEQ ID NO: 2.

[0064] In some embodiments, the GAA-binding polypeptide has at least 95% sequence identity to the amino sequence of SEQ ID NO: 1. In some embodiments, the GAA-binding polypeptide has at least 95% sequence identity to the amino sequence of SEQ ID NO: 2. In some embodiments, the GAA-binding polypeptide has at least 95% sequence identity to the amino sequence of SEQ ID NO: 16. In some embodiments, the GAA-binding polypeptide has at least 95% sequence identity to the amino sequence of SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the GAA-binding polypeptide has at least 95% sequence identity to the amino sequence of SEQ ID NO: 22 or SEQ ID NO: 23. In other embodiments, the GAA-binding polypeptide has at least 95%, 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 22 or SEQ ID NO: 23. For example, "at least 95% identity to the amino acid of SEQ ID NO: 1" means 1 or 2 substitutions compared to the amino acid SEQ ID NO: 1; whereas "at least 98% identity to the amino acid of SEQ ID NO: 1" means 1 substitution compared to the amino acid SEQ ID NO: 1. For example, SEQ ID NO: 2 and SEQ ID NO: 1 differ in two amino acids, i.e., amino acid 46 and amino acid 54 are different, and are 96% identical. For example, SEQ ID NO: 16 and SEQ ID NO: 1 differ in two amino acids, i.e., amino acid 1 and amino acid 2 are missing in SEQ ID NO: 16. For example, SEQ ID NO: 17 and SEQ ID NO: 2 differ in two amino acids, i.e., amino acid 1 and amino acid 2 are missing in SEQ ID NO: 17.

[0065] One advantage of the disclosed GAA-binding domains and proteins comprising said domains is their functional property of specifically binding to GAA. This is a particular advantage for use as affinity ligands in GAA purification. The GAA-binding proteins of the present invention functionally have the characteristic of binding affinity to GAA of less than 200 nM. As shown in Example 3, the GAA-binding proteins of the present invention bind to GAA with a dissociation constant KD of less than 200 nM, preferably less than 100 nM, or more preferably less than 50 nM.

[0066] Multimer. In one embodiment of the application, the GAA-binding protein comprises 1, 2, 3, 4, 5, or 6 GAA-binding proteins linked to one another. In some embodiments, the GAA-binding protein can be, for example, a monomer, a dimer, a trimer, a tetramer, a pentamer, or a hexamer. Preferred GAA-binding proteins are monomers or dimers. Multimers of the proteins of the application are fusion proteins that are artificially produced, typically by recombinant DNA techniques, that are well known to the skilled artisan. In some embodiments, the multimer is a homo-multimer, e.g., the amino acid sequences of the GAA-binding proteins are identical, e.g., as set forth in SEQ ID NO: 7 (a dimer of SEQ ID NO: 1). In other embodiments, the multimer is a hetero-multimer, e.g., the amino acid sequences of the GAA-binding proteins are different.

[0067] Fusion protein. According to one embodiment, provided herein is a fusion protein comprising one or more, e.g., two, GAA-binding polypeptides as disclosed throughout this specification. More specifically, the fusion protein comprises one or more GAA-binding polypeptides as disclosed herein and a further polypeptide that is different from the disclosed polypeptides. In various embodiments, the further polypeptide that is different from the GAA-binding polypeptides disclosed herein can be a non-Ig binding protein, e.g., but not limited to, a protein that does not bind to the Fc portion of an immunoglobulin. In some embodiments, the non-Ig binding protein has at least 80% identity to SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 21. In some embodiments, the non-Ig binding protein has at least 89.5% identity to SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 21. Exemplary non-Ig binding proteins are set forth in the amino acid sequences of SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 21. Accordingly, some embodiments encompass fusion proteins comprising one or two GAA-binding polypeptides as disclosed herein and one or two non-Ig binding polypeptides.

[0068] In some embodiments, the fusion protein can comprise the following combinations (from N- to C-terminus):

[0069] (a) GAA-binding protein - non-Ig binding protein;

[0070] (b) non-Ig binding protein - GAA-binding protein;

[0071] (c) non-Ig binding protein - GAA-binding protein - non-Ig binding protein (see, e.g., SEQ ID NO: 9);

[0072] (d) GAA binding protein - non-Ig binding protein - non-Ig binding protein (see, e.g., SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20).

[0073] (e) GAA binding protein - GAA binding protein - non-Ig binding protein - non-Ig binding protein (see, e.g., SEQ ID NO: 15);

[0074] (f) non-Ig binding protein - non-Ig binding protein - GAA binding protein

[0075] Other combinations of non-Ig binding protein and GAA binding protein domains are also feasible to one of skill in the art.

[0076] In some embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 1, or a GAA binding protein having at least 95% amino acid identity thereto. In some embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 1, or a GAA binding protein having at least 95% amino acid identity thereto. In some embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 2, or a GAA binding protein having at least 95% amino acid identity thereto. In some embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 16, or a GAA binding protein having at least 95% amino acid identity thereto. In some embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 17, or a GAA binding protein having at least 95% amino acid identity thereto. In some embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 18, or a GAA binding protein having at least 95% amino acid identity thereto. In some embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 22 or SEQ ID NO: 23, or a GAA binding protein having at least 95% amino acid identity thereto. In some preferred embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 1. In other preferred embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 2. In other preferred embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 16. In other preferred embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 17. In other preferred embodiments, the fusion protein comprises the GAA binding protein of SEQ ID NO: 18.

[0077] The portions of the fusion protein can be connected directly to each other, end-to-end, or can be connected by a linker, which is preferably a peptide linker. In various embodiments, the peptide linker can be considered an amino acid sequence that spatially separates the two portions of the fusion protein. Typically, such a linker consists of 1-10 amino acids. The fusion protein can be characterized as a protein formed by genetic fusion or combination of a gene encoding a GAA-binding polypeptide of the application with a gene encoding a polypeptide other than the polypeptides described hereinabove. Thus, the fusion protein can be considered the product of the translation of two or more genes together (without a stop codon in between).

[0078] Molecules for purification or detection. In some embodiments, the GAA-binding protein or fusion protein comprising a GAA-binding protein can further comprise other amino acid residues at the N- and / or C-terminus, e.g., other sequences at the N- and / or C-terminus. The other sequences can include, for example, sequences introduced for purposes of purification or detection. Typical examples of such sequences include, but are not limited to, a Strep-tag (see, e.g., SEQ ID NO: 12), an oligo- octa-histidine tag, glutathione S-transferase, maltose-binding protein, an intein, an intein fragment, or an albumin-binding domain of protein G, etc. In one embodiment, the other amino acid sequence includes one or more peptide sequences that confer affinity for certain chromatography column materials. The GAA-binding protein or fusion protein comprising a GAA-binding protein can include a specific attachment site for attachment to a solid support, preferably at the C-terminus, such as a cysteine or lysine. Examples of GAA-binding proteins having a C-terminal cysteine are shown in SEQ ID NO: 3, 4, 5, 6, 8. Examples of GAA-binding proteins of the application having an N-terminal other amino acid are shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0079] Methods of producing GAA-binding proteins. The application further provides a method for producing a new GAA-binding polypeptide having binding affinity for GAA disclosed herein, the method comprising the steps of: (i) providing a population of polypeptides; (ii) contacting the population of polypeptides of (i) with GAA; (iii) identifying a complex comprising a GAA-binding polypeptide that binds to GAA; and (iv) obtaining the GAA-binding polypeptide that is capable of binding to GAA.

[0080] The method of producing a new GAA-binding polypeptide having binding affinity for GAA can include the further step of determining the binding affinity of the polypeptide for GAA. The binding affinity can be determined as described elsewhere herein.

[0081] Use of the novel GAA binding polypeptides in technical applications. Also provided herein is the use of any of the novel GAA binding polypeptides of the invention, including the fusion proteins, in technical applications, preferably as affinity ligands in affinity chromatography.

[0082] As described herein, affinity chromatography (also known as affinity purification) takes advantage of specific binding interactions between molecules. Methods for immobilizing proteins and methods for affinity chromatography are well known in the art of protein purification and can be readily performed by one of skill in the art using standard techniques and equipment.

[0083] In various embodiments, the affinity purification method can further comprise one or more washing steps performed under conditions sufficient to remove some or all of the molecules that are not specifically bound to the affinity purification matrix. Affinity purification matrices suitable for the disclosed uses and methods are known to one of skill in the art.

[0084] Binding to a solid support. In various aspects and / or embodiments of the invention, the novel polypeptides disclosed herein, including the novel polypeptides produced or obtained by any of the methods described above, are bound to a solid support. In some embodiments of the invention, the polypeptide comprises an attachment site for site-specific covalent coupling of the polypeptide to the solid support. Specific attachment sites include, but are not limited to, natural amino acids such as cysteine or lysine that are capable of undergoing specific chemical reactions with reactive groups of the solid phase or linkers between the solid phase and the protein.

[0085] Affinity purification matrix. In another embodiment, an affinity purification matrix comprising a GAA binding polypeptide is provided, including the polypeptides identified by any of the methods described above.

[0086] In preferred embodiments, the affinity purification matrix is a solid support. The affinity purification matrix comprises at least one GAA binding polypeptide provided by the invention. Thus, the novel GAA binding proteins disclosed herein encompass the use of the GAA purification by affinity matrices.

[0087] Solid support matrices for affinity chromatography are known in the art and include, for example, but are not limited to, agarose and stabilized derivatives of agarose, cellulose or cellulose derivatives, controlled pore glass, monoliths, silica, zirconia, titania or synthetic polymers, as well as hydrogels of various compositions and combinations of the foregoing.

[0088] The form of the solid support matrix can be any suitable well-known type. Such solid support matrices for coupling the novel proteins or polypeptides of the present application can include, for example, one of the following, but are not limited to: a column, a capillary, a particle, a membrane, a filter, a monolith, a fiber, a pad, a gel, a slide, a plate, a cartridge, or any other form commonly used in chromatography and known to one of skill in the art.

[0089] In one embodiment, the matrix is comprised of substantially spherical particles, also known as beads, such as Sepharose or Agarose beads. The particulate form of the matrix can be used in packed bed or suspended form, including an expanded bed. In other embodiments of the present application, the solid support matrix is a membrane, for example, a hydrogel membrane. In some embodiments, the affinity purification can involve a membrane as the matrix, to which the GAA binding protein of the present application is covalently bound. The solid support can also be in the form of a membrane in a cartridge.

[0090] In some embodiments, the affinity purification involves a chromatography column containing a solid support matrix, to which the novel protein of the present application is covalently bound. The novel protein or polypeptide of the present application can be attached to a suitable solid support matrix by conventional coupling techniques. Methods of immobilizing protein ligands to solid supports are well known in the field of protein engineering and purification, and can be readily performed by one of skill in the art using standard techniques and equipment.

[0091] GAA production methods. Further embodiments relate to a method of producing a GAA or a GAA-comprising protein, the method comprising at least one chromatography step using an affinity chromatography matrix having an affinity for specifically binding to a GAA protein, wherein a GAA affinity ligand (binding protein) as described above is coupled to said affinity chromatography matrix.

[0092] Methods of determining the presence of GAA. Furthermore, in some embodiments, the GAA binding protein described herein or the fusion protein described herein is used in a method of determining the presence of GAA. Some embodiments relate to a method of analyzing a liquid sample for the presence of GAA, the method comprising the steps of: (a) providing a liquid containing GAA, (b) providing a GAA binding protein (or fusion protein), (c) contacting the liquid containing GAA with the GAA binding protein (or fusion protein) described herein under conditions that allow the GAA to bind to the at least one GAA binding protein (or fusion protein), (d) isolating the complex of GAA and GAA binding protein (or fusion protein), and (e) determining the amount of GAA binding protein (or fusion protein) which is indicative of the amount of GAA in the liquid of (a).

[0093] Quantitative methods for GAA. A further embodiment relates to a method for quantifying GAA, the method comprising: (a) providing a liquid containing GAA; (b) providing a matrix to which a GAA binding protein (or fusion protein) as described herein has been covalently coupled; (c) contacting the affinity purification matrix with the liquid under conditions that allow GAA to bind to the at least one GAA binding protein (or fusion protein); (d) eluting the GAA; and optionally, (e) quantifying the amount of GAA eluted. Methods for determining the presence of GAA in a liquid sample can be quantitative or qualitative. Such methods are well known to the person skilled in the art and can be selected, for example, but not limited to, the following art-recognized methods: enzyme-linked immunosorbent assay (ELISA), enzymatic reaction, surface plasmon resonance (SPR) or chromatography.

[0094] Polynucleotides, vectors, host cells. One embodiment encompasses a (isolated) polynucleotide or nucleic acid molecule encoding a GAA binding polypeptide as disclosed herein. Another embodiment also encompasses a polypeptide encoded by a polynucleotide disclosed herein. Further provided is a vector, in particular an expression vector, comprising an isolated polynucleotide or nucleic acid molecule of the present application, as well as a host cell comprising said isolated polynucleotide or expression vector. For example, one or more polynucleotides encoding a polypeptide disclosed herein can be expressed in a suitable host, and the resulting protein can be isolated. A vector refers to any molecule or entity (e.g., a nucleic acid, a plasmid, a bacteriophage or a virus) that can be used to transfer protein encoding information into a host cell. Suitable vectors that can be used in the present application are known in the art.

[0095] Further, an isolated cell comprising a polynucleotide or nucleic acid or vector disclosed herein is provided. Suitable host cells include prokaryotic or eukaryotic organisms, for example, a bacterial host cell, a yeast host cell or a non-human host cell carrying a vector. Suitable bacterial expression host cells or systems are known in the art. Various mammalian or insect cell culture systems known in the art can also be used to express recombinant proteins.

[0096] Methods for producing a protein of the present application. In a further embodiment, a method for producing a GAA binding polypeptide as described is provided, the method comprising the steps of: (a) culturing a (suitable) host cell under conditions suitable for expressing a GAA binding polypeptide to obtain said GAA binding polypeptide; and (b) optionally isolating said GAA binding polypeptide. Suitable conditions for culturing prokaryotic or eukaryotic hosts are well known to the person skilled in the art.

[0097] GAA-binding polypeptides can be prepared by any of the conventional and well- known techniques, such as general organic synthesis strategies, solid phase assisted synthesis techniques, or by commercially available automated synthesizers. They can also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques.

[0098] In one embodiment, a method of making a GAA-binding protein is provided, as described above, comprising the steps of: (a) providing a nucleic acid molecule encoding a GAA-binding polypeptide; (b) introducing the nucleic acid molecule into an expression vector; (c) introducing the expression vector into a host cell; (d) culturing the host cell in a culture medium; (e) subjecting the host cell to culture conditions suitable for expression of the GAA-binding polypeptide, thereby producing a GAA-binding polypeptide; optionally (f) isolating the protein or polypeptide produced in step (e); (g) optionally conjugating the protein or polypeptide to a solid matrix, as described above. In various embodiments of the application, production of the GAA-binding polypeptide is accomplished by cell-free in vitro transcription and translation.

[0099] The following examples are provided to further illustrate the application. The application is not limited in scope by the examples, which are merely illustrative of the application described above. The examples illustrate the utility of the application based on the above description.

[0100] Example

[0101] The following examples are provided to further illustrate the application. The application is not limited in scope by the examples, which are merely illustrative of the application described above. The examples illustrate the utility of the application based on the above description. For a complete disclosure of the application, reference is made to the documents cited in this application, which are incorporated by reference in their entirety.

[0102] Example 1. Selection of GAA-binding proteins of the application

[0103] Library construction and library cloning

[0104] Based on the library of stable non-immunoglobulin binding proteins having a protein A-like structure comprising random amino acid positions as set forth in SEQ ID NO: 10, internal synthesis by random oligonucleotides (ELLA Biotech) generated from synthetic trinucleotide phosphoramidites was performed to achieve a sufficiently balanced amino acid distribution while excluding cysteine and other amino acid residues at random positions. SEQ ID NO: 10 was randomized at least at amino acid positions 7, 8, 10, 11, 14, 15, 18, 20, 42, 43, 46, 47, 49, 50, 53, and 54.

[0105] The corresponding cDNA libraries were amplified by PCR and ligated to the pCD33-OmpA phagemid. Aliquots of the ligation mixture were used for electroporation of E. coli ER2738 (Lucigen). Established recombinant genetic methods were used unless otherwise stated.

[0106] Preliminary selection by TAT phage display. Using phage display as selection system, the naive library was enriched against biotinylated GAA (also known as Myozyme or Alglucosidase alpha; ). After transformation of competent bacterial ER2738 cells (Lucigene) with the phagemid pCD33-OmpA carrying the library, phage amplification and purification was performed using standard methods known to the skilled person. The solution- in- solution (SIS) method was used to allow binding between biotinylated GAA in solution and the phage library. The GAA concentration during phage cultivation was reduced from 200 nM (first round) to 100 nM (second round), to 50 nM (third round) and 25 nM (fourth round). In each round an empty magnetic bead preselection was performed. After each round of selection, GAA-binding phage were eluted by trypsin. To identify target-specific phage pools, eluted and reamplified phage of each round of selection were analyzed by phage pool ELISA. The wells of a microtiter plate (Greiner Bio-One) coated with streptavidin (10 pg / mL) were coated with biotinylated GAA (2.5 pg / mL). Bound phage were detected using an a-M13 HRP conjugated antibody (GE Healthcare).

[0107] Target-binding phage pool cloning into expression vector. The selected library showing specific binding to GAA in phage pool ELISA was amplified by PCR, cut with appropriate restriction nucleases and ligated into a derivative of the expression vector pET-28a (Merck, Germany) containing a 10 amino acid linker consisting of proline, serine and alanine and sfGFP according to methods known in the art.

[0108] Example 2. Expression and purification of GAA-binding proteins

[0109] The hits identified by selection as described in example 1 were characterized after screening and proteins were produced in p-scale (Phynexus).

[0110] The constructs were expressed in E. coli BL21(DE3) using a low copy plasmid system under the control of the T7 promoter. The proteins were produced in soluble form upon induction by lactose contained in the culture medium (auto-induction medium). BL21(DE3) competent cells were transformed with the expression plasmid, spread on selective agar plates (kanamycin) and incubated overnight at 37°C. A preculture was inoculated from a single colony into 3 mL 2xYT medium supplemented with 50 μg / mL kanamycin and incubated in a culture tube at 200 rpm at 37°C for 6 hours in a regular orbital shaker. The main culture was inoculated with 3 mL of the preculture in 300 mL ZYM-5052 (0.5% glycerol, 0.2% lactose, 0.05% glucose, 0.5% yeast extract, 1.0% casamino acids, 25 mM Na2HPO4, 25 mM KH2PO4, 5 mM Na2SO4, 2 mM MgSO4and trace elements) supplemented with 50 μg / mL kanamycin in a 1 L Erlenmeyer flask. The culture was transferred to an orbital shaker and incubated at 30°C and 200 rpm. Recombinant protein expression was induced by metabolizing the glucose and subsequently allowing lactose to enter the cell. The cells were grown overnight for about 17 hours to reach a final OD600 of about 2-4. Before harvesting, the OD600 was measured and samples adjusted to 0.6 / OD600 were taken out, pelleted and frozen at -20°C. To collect the biomass, the cells were centrifuged at 12000 x g for 15 minutes at 22°C. The pellets were weighed (wet weight). The cells were stored at -20°C before processing.

[0111] Proteins with affinity tags were purified by affinity chromatography and size exclusion. After affinity chromatography purification, the samples were analyzed by SDS-PAGE and the positive fractions were pooled and their protein concentration was measured. Further analysis included SDS-PAGE, SE-HPLC and RP-HPLC. Protein concentration was determined by absorbance measurement at 280 nm using the molar absorption coefficient. Reverse phase chromatography (RP-HPLC) was performed using a Dionex HPLC system and PLRP-S (5 μm, 100 A) chromatography column (Agilent). Size exclusion chromatography (SEHPLC or SEC) was performed using a Superdex TM 200 HiLoad 16 / 600 column (GE Healthcare). The volume of the SEC column was 120 mL and equilibrated with 2 CV. The samples were applied at a flow rate of 1 mL / min. Fractions were collected when the signal intensity reached 10 mAU. After SDS-PAGE analysis, the positive fractions were pooled and their protein concentration was measured. Further analysis included SDS-PAGE, SE-HPLC and RP-HPLC. Protein concentration was determined by absorbance measurement at 280 nm using the molar absorption coefficient. Reverse phase chromatography (RP-HPLC) was performed using a Dionex HPLC system and PLRP-S (5 μm, 100 A) chromatography column (Agilent).

[0112] ​Fusion proteins of GAA-binding protein and non-Ig-binding protein (SEQ ID NOs: 14, 15, 19, 20) were purified using the following strategy: Q-Sepharose FF 275 mL (pH 6) (Buffer A: 20 mM BisTris, 1 mM EDTA, pH 6; Buffer B: 20 ​​mM BisTris, 1 mM EDTA, 1 M NaCl, pH 6), Phenyl Sepharose HP 236 mL (Buffer A: 20 mM BisTris, 1 mM EDTA, 1 M (NH₄)₂SO₄, pH 6; Buffer B: 20 ​​mM BisTris, 1 mM EDTA, pH 6), and desalting 560 mL. For example, SEQ ID NO: 14 (CID204870) was successfully purified at a yield of 1 g protein per liter of cell culture volume. SEQ ID NO: 19 was successfully purified at a yield of 10 g protein per liter of cell culture volume.

[0113] Example 3. Protein analysis by surface plasmon resonance (SPR)

[0114] 500-1500 RUGAA (ON-ligand) was immobilized on a CM-5 sensor chip (GE Healthcare); the chip was equilibrated with SPR running buffer. GAA was biotinylated using sulfo-NHS-biotin standard reagent and purified by size exclusion chromatography (Superdex 200). The target protein was immobilized by injecting the biotinylated target onto a streptavidin-coated sensor chip. Once the ligand binds, the protein analyte accumulates on the surface, increasing the refractive index. This change in refractive index is measured in real time and plotted as response or resonance units versus time. The analytes were applied to the chip in serial dilutions at a flow rate of 30 μL / min. Association was performed for 120 seconds and dissociation for 360 seconds. After each run, the chip surface was regenerated with 30 μL regeneration buffer (10 mM glycine) and equilibrated with running buffer. Binding studies were performed using BIAcore 3000 (GE Healthcare); data were evaluated using the Langmuir 1:1 model (RI = 0) using the manufacturer's BIAevaluation 3.0 software. The estimated dissociation constant (K D) against GAA standardization and indication. Shown is the real-time measured refractive index change and plotted as a function of response or resonance units [RU] versus time [sec]. Results: The GAA binding proteins as disclosed herein show strong specific binding to immobilized GAA with affinities of less than 100 nM (see Table 1). The GAA binding proteins of the present invention do not bind hlgG. The fusion protein of SEQ ID NO: 19 does not bind hlgG; this affinity is specific for GAA with affinities of less than 100 nM.

[0115] Table 1. Specific affinity of GAA affinity ligands against GAA

[0116]

[0117] Example 4. GAA binding proteins of the present invention as affinity ligands for purification of GAA

[0118] Coupling efficiency: According to the manufacturer's instructions, each milliliter of Praesto TM Epoxy 85 was coupled with 20 mg of purified GAA binding protein (with C-terminal cysteine) (coupling buffer: 50 mM Na2HP04, 150 mM NaCl, 5 mM TCEP, 2.05 M Na2S04(or 175 mg Na2S04 / mL resin), pH 9.5, coupling conditions: 3 hours at 35 °C). The results are shown in Table 2.

[0119] Table 2. Coupling efficiency of GAA affinity ligands

[0120] GAA affinity ligand Coupling efficiency SEQ ID NO: 1 17.0 mg / mL resin SEQ ID NO: 1 (N-terminal cap) 17.4 mg / mL resin SEQ ID NO: 1 (dimer) 19.0 mg / mL resin SEQ ID NO: 2 17.4 mg / mL resin SEQ ID NO: 2 (N-terminal cap) 16.5 mg / mL resin

[0121] DBC 10%: Running buffer: 20 mM citrate, 150 mM NaCl, 1 mM EDTA, pH 6,2. First elution buffer: 100 mM citrate buffer, 20% (v / v) hexylene glycol, pH 3.5; second elution buffer: 0.1 M citrate, pH 2.0 (determination of elution ratio). Dynamic binding capacity (DBC) was determined from the mass of GAA injected at 10% breakthrough at a 6 minute residence time. Results: The coupled resin showed good dynamic binding capacity (DBC 10%) for the affinity ligand (with c-terminal Cys):

[0122] • SEQ ID NO: 1, DBC 10% at least 27 mg / mL

[0123] • SEQ ID NO: 1 dimer, DBC 10% at least 24 mg / mL

[0124] • SEQ ID NO: 1 fusion protein (SEQ ID NO: 19) at least 22.7 mg / mL

[0125] • SEQ ID NO: 2, DBC 10% at least 16 mg / mL.

[0126] Base stability (cycle study). The affinity ligand (coupled via C- terminal Cys) showed good base stability in the cycle study: TM Epoxy 85 coupled sample (coupling conditions: 20 mg / mL, 3 hours, 35 °C, 2.05 M Na2SO4pH 8.5) treated with 0.1 M NaOH for at least 10 hours (610 min) had a total elution at pH 3.5 of 100%.

[0127] Results: The affinity ligand (coupled via C-terminal Cys) showed good base stability in the cycle study:

[0128] • SEQ ID NO: 1 (N-terminal extension) with 95% remaining capacity

[0129] • SEQ ID NO: 1 dimer

[0130] o 94.4% remaining capacity after 610 minutes (about 10 hours)

[0131] o 84.4% remaining capacity after 1515 minutes (25 hours) (equivalent to 101 cycles).

[0132] Figure 2 shows the cycle study on Praesto TM Epoxy 85 resin. Figure 2A shows the DBC 10% of SEQ ID NO: 1 (with N-terminal extension) at 6 minute residence time on Praesto TM Epoxy 85. The binding capacity was 19.5 mg / mL. Figure 2B shows the DBC 10% and base stability of GAA binding protein (SEQ ID NO: 1 with N-terminal extension) coupled to Praesto TM Epoxy 85 resin and loaded with 2 mg / mL GAA. The remaining capacity was at least 90% after 50x loading, elution and 0.1 M NaOH regeneration cycles.

[0133] Table 3 shows the results of immobilized affinity ligands in NaOH incubation in packed column format.

[0134] Table 3. Base stability

[0135]

[0136] SEQUENCE LISTING <110> Navigo Proteins GmbH <120> BINDING PROTEINS FOR THE ENZYME ACID ALPHA-GLUCOSIDASE AND USES THEREOF <130> NP43 WO <160> 23 <170> PatentIn version 3.5 <210> 1 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein (monomer) <400> 1 Ile Ala Ala Lys Phe Asp Met Lys Gin Ala Trp Ala Asp His Phe Ile 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin 20 25 30 Ser Leu Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Leu Gin Ala 35 40 45 Gln Lys Leu Asn Gin Met Gin Ala Pro Lys 50 55 <210> 2 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein <400> 2 Ile Ala Ala Lys Phe Asp Met Lys Gin Ala Trp Ala Asp His Phe Ile 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin 20 25 30 Ser Leu Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Ala Gin Ala 35 40 45 Gln Lys Leu Asn Gin Ser Gin Ala Pro Lys 50 55 <210> 3 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein (with C-terminal Cys) <400> 3 Ile Ala Ala Lys Phe Asp Met Lys Gin Ala Trp Ala Asp His Phe Ile 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin 20 25 30 Ser Leu Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Leu Gin Ala 35 40 45 Gln Lys Leu Asn Gin Met Gin Ala Pro Lys Cys 50 55 <210> 4 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein (with C-terminal Cys) <400> 4 Val Asp Ala Lys Phe Asp Ile Ala Ala Lys Phe Asp Met Lys Gln Ala 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln 20 25 30 Ser Leu Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Ala Gln Ala 35 40 45 Gln Lys Leu Asn Gln Ser Gln Ala Pro Lys Cys 50 55 <210> 5 <211> 65 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein (N-cap and C-terminal Cys) <400> 5 Val Asp Ala Lys Phe Asp Ile Ala Ala Lys Phe Asp Met Lys Gln Ala 1 5 10 15 Trp Ala Asp His Phe Ile Leu Glu Leu Pro Asn Leu Thr Glu Glu Gln 20 25 30 Arg Asn Ala Phe Arg Gln Ser Leu Ser Asp Asp Pro Ser Val Ser Asp 35 40 45 Leu Val Leu Leu Gln Ala Gln Lys Leu Asn Gln Met Gln Ala Pro Lys 50 55 60 Cys 65 <210> 6 <211> 65 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein (N cap and C-terminal Cys) <400> 6 Val Asp Ala Lys Phe Asp lie Ala Ala Lys Phe Asp Met Lys Gin Ala 1 5 10 15 Trp Ala Asp His Phe lie Leu Glu Leu Pro Asn Leu Thr Glu Glu Gin 20 25 30 Arg Asn Ala Phe Arg Gin Ser Leu Ser Asp Asp Pro Ser Val Ser Asp 35 40 45 Leu Val Leu Ala Gin Ala Gin Lys Leu Asn Gin Ser Gin Ala Pro Lys 50 55 60 Cys 65 <210> 7 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein (two bodies) <400> 7 Ile Ala Ala Lys Phe Asp Met Lys Gin Ala Trp Ala Asp His Phe lie 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin 20 25 30 Ser Leu Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Leu Gin Ala 35 40 45 Gln Lys Leu Asn Gln Met Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp 50 55 60 Met Lys Gln Ala Trp Ala Asp His Phe Ile Leu Glu Leu Pro Asn Leu 65 70 75 80 Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln Ser Leu Ser Asp Asp Pro 85 90 95 Ser Val Ser Asp Leu Val Leu Leu Gln Ala Gln Lys Leu Asn Gln Met 100 105 110 Gln Ala Pro Lys 115 <210> 8 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein (two-body, with C-terminal Cys) <400> 8 Ile Ala Ala Lys Phe Asp Met Lys Gln Ala Trp Ala Asp His Phe Ile 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln 20 25 30 Ser Leu Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Leu Gln Ala 35 40 45 Gln Lys Leu Asn Gln Met Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp 50 55 60 Met Lys Gln Ala Trp Ala Asp His Phe Ile Leu Glu Leu Pro Asn Leu 65 70 75 80 Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln Ser Leu Ser Asp Asp Pro 85 90 95 Ser Val Ser Asp Leu Val Leu Leu Gln Ala Gln Lys Leu Asn Gln Met 100 105 110 Gln Ala Pro Lys Cys 115 <210> 9 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein GAA binding protein <400> 9 Ile Ala Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Asp Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln 20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala 35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp 50 55 60 Met Lys Gln Ala Trp Ala Asp His Phe Ile Leu Glu Leu Pro Asn Leu 65 70 75 80 Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln Ser Leu Ser Asp Asp Pro 85 90 95 Ser Val Ser Asp Leu Val Leu Leu Gln Ala Gln Lys Leu Asn Gln Met 100 105 110 Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala 115 120 125 Asp Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn 130 135 140 Ala Phe Arg Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val 145 150 155 160 Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys 165 170 <210> 10 <211> 58 <212> PRT <213> artificial sequence <220> <223> Non-Fc binding protein; scaffold protein <400> 10 Ile Ala Ala Lys Phe Asp Glu Ala Gln Ser Ala Ala Asp Ser Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln 20 25 30 Ser Leu Ser Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala 35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 11 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Non-Fc binding protein <400> 11 Ile Ala Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Asp Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln 20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala 35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Strep tag <400> 12 Ser Ala Trp Ser His Pro Gln Phe Glu Lys 1 5 10 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> flag tag (flag-tag) <400> 13 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 14 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> 204870 cor1-PAdelFc2-PAdelFc2 <400> 14 Ile Ala Ala Lys Phe Asp Met Lys Gln Ala Trp Ala Asp His Phe Ile 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln 20 25 30 Ser Leu Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Leu Gln Ala 35 40 45 Gln Lys Leu Asn Gln Met Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp 50 55 60 Glu Ala Gln Gln Ala Ala Asp Tyr Glu Ile Leu His Leu Pro Asn Leu 65 70 75 80 Thr Glu Glu Gln Arg Asn Ala Phe Arg Gln Ser Leu Arg Asp Asp Pro 85 90 95 Ser Val Ser Leu Glu Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser 100 105 110 Gln Ala Pro Lys lie Ala Ala Lys Phe Asp Glu Ala Gin Gin Ala Ala 115 120 125 Asp Tyr Glu lie Leu His Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn 130 135 140 Ala Phe Arg Gin Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val 145 150 155 160 Leu Gly Glu Ala Gin Lys Leu Asn Asp Ser Gin Ala Pro Lys 165 170 <210> 15 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> corel duplex - PAdelFc2 duplex <400> 15 lie Ala Ala Lys Phe Asp Met Lys Gin Ala Trp Ala Asp His Phe lie 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin 20 25 30 Ser Leu Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Leu Gin Ala 35 40 45 Gln Lys Leu Asn Gin Met Gin Ala Pro Lys lie Ala Ala Lys Phe Asp 50 55 60 Met Lys Gln Ala Trp Ala Asp His Phe lie Leu Glu Leu Pro Asn Leu 65 70 75 80 Thr Glu Glu Gln Arg Asn Ala Phe Arg Gin Ser Leu Ser Asp Asp Pro 85 90 95 Ser Val Ser Asp Leu Val Leu Leu Gin Ala Gin Lys Leu Asn Gin Met 100 105 110 Gln Ala Pro Lys lie Ala Ala Lys Phe Asp Glu Ala Gin Gin Ala Ala 115 120 125 Asp Tyr Glu lie Leu His Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn 130 135 140 Ala Phe Arg Gin Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val 145 150 155 160 Leu Gly Glu Ala Gin Lys Leu Asn Asp Ser Gin Ala Pro Lys lie Ala 165 170 175 Ala Lys Phe Asp Glu Ala Gin Gin Ala Ala Asp Tyr Glu lie Leu His 180 185 190 Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin Ser Leu 195 200 205 Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala Gin Lys 210 215 220 Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin Ser Leu 225 230 <210> 16 <211> 56 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein <400> 16 Ala Lys Phe Asp Met Lys Gin Ala Trp Ala Asp His Phe He Leu Glu 1 5 10 15 Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin Ser Leu 20 25 30 Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Leu Gin Ala Gin Lys 35 40 45 Leu Asn Gin Met Gin Ala Pro Lys 50 55 <210> 17 <211> 56 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein <400> 17 Ala Lys Phe Asp Met Lys Gin Ala Trp Ala Asp His Phe He Leu Glu 1 5 10 15 Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin Ser Leu 20 25 30 Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Ala Gin Ala Gin Lys 35 40 45 Leu Asn Gln Ser Gln Ala Pro Lys 50 55 <210> 18 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein <220> <221> Xaa <222> (38)..(38) <223> Xaa can be selected from any amino acid <220> <221> Xaa <222> (46)..(46) <223> Xaa can be selected from any amino acid <400> 18 Gln Ala Trp Ala Asp His Phe Ile Leu Glu Leu Pro Asn Leu Thr Glu 1 5 10 15 Glu Gln Arg Asn Ala Phe Arg Gln Ser Leu Ser Asp Asp Pro Ser Val 20 25 30 Ser Asp Leu Val Leu Xaa Gln Ala Gln Lys Leu Asn Gln Xaa 35 40 45 <210> 19 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein comprising a GAA binding protein <400> 19 Ala Lys Phe Asp Met Lys Gln Ala Trp Ala Asp His Phe Ile Leu Glu 1 5 10 15 Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin Ser Leu 20 25 30 Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Leu Gin Ala Gin Lys 35 40 45 Leu Asn Gin Met Gin Ala Pro Lys Ala Gin His Asp Lys Asp Gin Gin 50 55 60 Ala Ala Asp Lys Glu lie Leu His Leu Pro Asn Leu Thr Glu Glu Gin 65 70 75 80 Arg Asn Lys Phe Arg Gin Ser Leu Arg Asp Asp Pro Ser Val Ser Ala 85 90 95 Glu lie Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gin Ala Pro Lys 100 105 110 lie Ala Ala Gin His Asp Lys Asp Gin Gin Ala Ala Asp Lys Glu lie 115 120 125 Leu His Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Lys Phe Arg Gin 130 135 140 Ser Leu Arg Asp Asp Pro Ser Val Ser Ala Glu lie Leu Ala Glu Ala 145 150 155 160 Lys Lys Leu Asn Asp Ala Gin Ala Pro Lys Cys 165 170 <210> 20 <211> 171 <212> PRT <213> Artificial sequence <220> <223> Fusion protein comprising a GAA binding protein <400> 20 Ala Lys Phe Asp Met Lys Gin Ala Trp Ala Asp His Phe He Leu Glu 1 5 10 15 Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Ala Phe Arg Gin Ser Leu 20 25 30 Ser Asp Asp Pro Ser Val Ser Asp Leu Val Leu Ala Gin Ala Gin Lys 35 40 45 Leu Asn Gin Ser Gin Ala Pro Lys Ala Gin His Asp Lys Asp Gin Gin 50 55 60 Ala Ala Asp Lys Glu He Leu His Leu Pro Asn Leu Thr Glu Glu Gin 65 70 75 80 Arg Asn Lys Phe Arg Gin Ser Leu Arg Asp Asp Pro Ser Val Ser Ala 85 90 95 Glu He Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gin Ala Pro Lys 100 105 110 He Ala Ala Gin His Asp Lys Asp Gin Gin Ala Ala Asp Lys Glu He Leu 115 120 125 Leu His Leu Pro Asn Leu Thr Glu Glu Gin Arg Asn Lys Phe Arg Gin 130 135 140 Ser Leu Arg Asp Asp Pro Ser Val Ser Ala Glu Ile Leu Ala Glu Ala 145 150 155 160 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Cys 165 170 <210> 21 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Non-Ig binding protein <400> 21 Ile Ala Ala Gln His Asp Lys Ile Gln Gln Ala Ala Asp Lys Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Lys Phe Arg Gln 20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Ala Glu Ile Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 22 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein <400> 22 Gln Ala Trp Ala Asp His Phe Ile Leu Glu Leu Pro Asn Leu Thr Glu 1 5 10 15 Glu Gin Arg Asn Ala Phe Arg Gin Ser Leu Ser Asp Asp Pro Ser Val 20 25 30 Ser Asp Leu Val Leu Leu Gin Ala Gin Lys Leu Asn Gin Met 35 40 45 <210> 23 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> GAA binding protein <400> 23 Gln Ala Trp Ala Asp His Phe He Leu Glu Leu Pro Asn Leu Thr Glu 1 5 10 15 Glu Gin Arg Asn Ala Phe Arg Gin Ser Leu Ser Asp Asp Pro Ser Val 20 25 30 Ser Asp Leu Val Leu Ala Gin Ala Gin Lys Leu Asn Gin Ser 35 40 45

Claims

1. An acid alpha-glucosidase (GAA) binding protein consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 17, or a homodimer of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

2. The GAA binding protein of claim 1, wherein the GAA binding protein has a binding affinity for GAA of less than 200 nM.

3. The GAA binding protein of claim 1, wherein the GAA binding protein is a homodimer of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

4. A fusion protein comprising the GAA binding protein of any one of claims 1-3, consisting of the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO:

20.

5. A polynucleotide encoding the GAA binding protein of any one of claims 1-3 or the fusion protein of claim 4.

6. The GAA binding protein of any one of claims 1-3, for use in affinity purification of GAA.

7. The fusion protein of claim 4, for use in affinity purification of GAA.

8. The GAA binding protein of any one of claims 1-3, further comprising one or more coupling sites for coupling to an affinity purification matrix.

9. The GAA binding protein of claim 8, wherein the GAA binding protein comprises one or more cysteine residues for coupling to an affinity purification matrix.

10. The fusion protein of claim 4, further comprising one or more coupling sites for coupling to an affinity purification matrix.

11. The fusion protein of claim 10, wherein the GAA binding protein comprises one or more cysteine residues for coupling to an affinity purification matrix.

12. An affinity purification matrix comprising the protein of any one of claims 1-3 or the fusion protein of claim 4.

13. Use of the GAA binding protein of any one of claims 1-3, 8-9 or the fusion protein of claim 4 or the affinity purification matrix of claim 12 for affinity purification of GAA.

14. A method of affinity purifying GAA, the method comprising: (a) providing a liquid containing GAA; (b) providing an affinity purification matrix comprising at least one GAA binding protein of any one of claims 1-3, 8-9 or the fusion protein of claim 4 coupled to the affinity purification matrix; (c) contacting the affinity purification matrix with the liquid under conditions that allow binding of the at least one GAA binding protein of any one of claims 1-3, 8-9 to GAA; and (d) eluting the GAA from the affinity purification matrix.

15. Use of a GAA binding protein according to claims 1 to 3 or a fusion protein according to claim 4 for non-diagnostic purposes in a method for determining the presence of GAA.

16. A method for non-diagnostic purposes for analyzing the presence of GAA in a liquid sample, the method comprising the steps of: (i) providing a liquid containing GAA, (ii) providing a GAA binding protein according to claims 1 to 3 or a fusion protein according to claim 4, (iii) contacting the liquid of (i) with the GAA binding protein according to claims 1 to 3 or the fusion protein according to claim 4 under conditions that allow binding of at least one GAA binding protein according to claim 1 or the fusion protein according to claim 4 to GAA, (iv) isolating the complex of GAA and the GAA binding protein according to claims 1 to 3 or the fusion protein according to claim 4, and (v) determining the amount of the GAA binding protein according to claims 1 to 3 or the fusion protein according to claim 4 in the liquid of (i).

17. A method for non-diagnostic purposes for analyzing the presence of GAA in a liquid sample, the method comprising the steps of: (i) providing a liquid containing GAA, (ii) providing a GAA binding protein according to claims 1 to 3 or a fusion protein according to claim 4, (iii) contacting the liquid of (i) with the GAA binding protein according to claims 1 to 3 or the fusion protein according to claim 4 under conditions that allow binding of at least one GAA binding protein according to claim 1 or the fusion protein according to claim 4 to GAA, (iv) isolating the complex of GAA and the GAA binding protein according to claims 1 to 3 or the fusion protein according to claim 4, and (v) determining the amount of the GAA binding protein according to claims 1 to 3 or the fusion protein according to claim 4 in the liquid of (i).

18. A method for non-diagnostic purposes for analyzing the presence of GAA in a liquid sample, the method comprising the steps of: (i) providing a liquid containing GAA, (ii) providing a GAA binding protein according to claims 1 to 3 or a fusion protein according to claim 4, (iii) contacting the liquid of (i) with the GAA binding protein according to claims 1 to 3 or the fusion protein according to claim 4 under conditions that allow binding of at least one GAA binding protein according to claim 1 or the fusion protein according to claim 4 to GAA, (iv

Images