Binding protein of enzyme acid α-glucosidase (GAA) and its use

Abstract

The present invention relates to the field of protein purification, and in particular to novel proteins that specifically bind to acid alpha-glucosidase (GAA). The present invention further relates to fusion proteins comprising the novel proteins that specifically bind 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. A further use relates to analytical methods for the determination of GAA in liquids.

Description

Technical Field

[0001] The present invention relates to the field of protein purification, and more particularly to novel proteins that specifically bind to acidic α-glucosidase (GAA). The present invention further relates to fusion proteins comprising a novel protein that specifically binds 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 the affinity purification of GAA and an affinity purification method of GAA using the GAA-binding protein of the present invention. A further use relates to an analytical method for the determination of GAA in a liquid.

Background Art

[0002] The lysosomal enzyme GAA (GAA) is essential for the breakdown of glycogen to glucose. Defects in GAA lead to the accumulation of glycogen in lysosomes, resulting in damage to muscle and nerve cells known as glycogenosis II (Pompe disease). Treatment of this metabolic disorder is the replacement of GAA. Therefore, it is essential to provide a method for purifying this enzyme. There is a continuing need in the art for advanced tools that enable efficient GAA protein purification.

[0003] The present invention meets this need by providing novel GAA-binding polypeptides. These novel GAA-binding polypeptides are particularly advantageous as GAA affinity ligands because they enable accurate capture in affinity chromatography to provide high-purity GAA for medical use.

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

Summary of the Invention

[0005] The present invention relates, without particularly being limited thereto, to the following items 1 to 15, 1. A GAA-binding protein comprising one or more acidic α-glucosidase (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 homology thereto, the GAA-binding protein. 2. The GAA-binding protein according to item 1, wherein this protein has a binding affinity for GAA of less than 200 nM (determined by the surface plasmon resonance method described herein). 3. The GAA-binding protein according to item 1, wherein this GAA-binding protein comprises 2, 3, 4, 5, or 6 domains bound to each other. 4. The GAA-binding protein according to item 3, wherein this GAA-binding protein is a homomultimer. 5. The GAA-binding protein according to item 3, wherein this GAA-binding protein is a heteromultimer. 6. The GAA-binding protein according to item 3, wherein one or more domains are bound to each other directly or by one or more peptide linkers. 7. A fusion protein comprising the protein according to any one of items 1 to 6. 8. A polynucleotide encoding the binding protein according to any one of items 1 to 6, or the fusion protein according to item 7. 9. The GAA-binding protein according to any one of items 1 to 6, or the fusion protein according to item 7, for use in affinity purification of GAA. 10. Further comprising one or more coupling sites for coupling to an affinity purification matrix, preferably this GAA-binding protein comprises one or more cysteine residues for coupling to an affinity purification matrix, the GAA-binding protein according to any one of items 1 to 6, or the fusion protein according to item 7. 11. An affinity purification matrix comprising the protein according to any one of items 1 to 6, or the fusion protein according to item 7. 12. Use of the GAA-binding protein according to any one of items 1 to 6, 10 for affinity purification of GAA, or the fusion protein according to item 7, or the affinity purification matrix according to item 11. 13. An affinity purification method of GAA, the method comprising: (a) providing a liquid containing GAA; (b) providing this affinity purification matrix containing at least one GAA-binding protein according to any one of items 1 to 6, 10 bound to the affinity purification matrix, or the fusion protein according to item 7; (c) contacting this affinity purification matrix with this liquid under conditions enabling binding of at least one GAA-binding protein according to any one of items 1 to 6, 10 to GAA; and (d) eluting this GAA from this affinity purification matrix. 14. Use of the GAA-binding protein according to items 1 to 6 or the fusion protein according to item 7 in a method for determining the presence of GAA. 15. A method for analyzing the presence of GAA in a liquid sample, the method comprising the following steps: (i) providing a liquid containing GAA; (ii) providing the GAA-binding protein according to items 1 to 6, or the fusion protein according to item 7; (iii) contacting the liquid of (i) with the GAA-binding protein according to items 1 to 6 under conditions enabling binding of at least one GAA-binding protein according to item 1 or the fusion protein according to item 7 to GAA; (iv) separating the complex of GAA and the GAA-binding protein according to items 1 to 6 or the fusion protein according to item 7; (v) quantifying the amount of the GAA-binding protein according to items 1 to 6 in the liquid of (i). Method. To provide.

[0006] This summary of the invention is not limiting, and other aspects and embodiments of the invention will become apparent from the following description, examples, and drawings.

Brief Description of the Drawings

[0007]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0008] The present invention provides a novel polypeptide having a binding affinity for GAA. The polypeptide of the present invention is an advanced and powerful tool that fills a gap in the fields of protein engineering and purification. In particular, the novel polypeptide brings an advantageous effect on protein purification due to its binding affinity for GAA. Therefore, the novel polypeptide of the present invention is particularly advantageous because it enables accurate capture of GAA in affinity chromatography. The GAA binding protein of the present invention then provides a very efficient tool for GAA purification that may be used for medical purposes next.

[0009] The binding affinity for 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 an amino acid sequence having at least 95% homology 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.

[0010] Before the present invention is described in more detail below, it should be understood that since the methods, protocols and reagents described herein may vary, the present invention is not limited to the specific methods, protocols and reagents described herein. The terms used herein are for the purpose of describing particular aspects and embodiments only and are not intended to limit the scope of the present invention, which is also to be understood as being represented by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. This includes those skilled in the art working in the fields of protein engineering and purification, but also those skilled in the art working in the technical applications such as affinity chromatography, and in the field of developing novel GAA-specific binding molecules for use in therapy and diagnosis.

[0011] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms:(IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Koelbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0012] Throughout this specification and the claims, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" and variations thereof are to be understood to mean that they include the stated integer or step or group of integers or steps but do not exclude any other integer or step or group of integers or steps. The term "comprise(s)" or "comprising" can include a limitation to "consists of" or "consisting of" if such a limitation is necessary for any reason or to any extent.

[0013] Throughout this specification, several documents may be cited (e.g., patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank accession number sequences, etc.). Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of a prior invention. Some of the documents cited in this specification may be characterized as being "incorporated by reference". In the event of a conflict between the definition or teaching of such incorporated reference and the definitions or teachings recited in this specification, the language of this specification shall control.

[0014] All sequences referred to in this specification are disclosed in the accompanying sequence listing, which forms part of the disclosure of this specification together with all of the content and disclosure of the accompanying sequence listing.

[0015] General definitions of important terms used in this application The terms "GAA-binding protein" or "acid alpha-glucosidase-binding polypeptide" or "acid alpha-glucosidase-binding protein" or "GAA-binding polypeptide" can be used interchangeably herein to describe a protein capable of binding to acid alpha-glucosidase (GAA; Uniprot identifier P10253). As described herein, "GAA-binding protein" refers to a protein that undergoes a detectable interaction with GAA, as measured, for example, by SPR analysis or other suitable techniques known to those skilled in the art.

[0016] The terms "binding affinity" and "binding activity" may be used interchangeably herein and refer to the ability of a polypeptide of the invention to bind to another protein, peptide, or fragment or domain thereof. Binding affinity is typically used to assess and rank the strength of a bimolecular interaction and is measured and reported by the equilibrium dissociation constant (K D ). This binding affinity and dissociation constant can be measured quantitatively. Methods for measuring binding affinity are well known to those skilled in the art and can be selected, for example, from the following established methods in the art: surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), binding equilibrium exclusion analysis (KinExA assay), biolayer interferometry (BLI), flow cytometry, fluorescence spectroscopy techniques, isothermal titration calorimetry (ITC), analytical ultracentrifugation systems, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Typically, the dissociation constant K D is measured at a temperature in the range of 20°C to 30°C. Unless otherwise indicated, the K DThe value is measured by SPR at 25°C. The most widely used SPR-based system is BIAcore manufactured by BIAcore AB. In various embodiments of the present invention, the binding affinity for GAA may be measured by a BIAcore SPR system. In various embodiments, the concentration of the analyte is 1 μM. In various other embodiments, the concentration of the analyte is 10 μM. In various other embodiments of the present invention, the polypeptide of the present invention has a binding affinity for GAA measured by SPR, the concentration of the analyte in the SPR assay is 10 μM, and preferably, the binding affinity is measured at 25°C.

[0017] The term "fusion protein" refers to a protein comprising at least a first protein genetically linked to at least a second protein. A fusion protein is created by joining two or more genes originally encoded for separate proteins. Thus, a fusion protein may contain the same protein, or a multimer of different proteins expressed as a single linear polypeptide.

[0018] As used herein, the term "linker" refers, in the broadest sense, to a molecule that covalently binds at least two different molecules.

[0019] The term "amino acid sequence homology" refers to a quantitative comparison of the homology (or difference) of the amino acid sequences of two or more proteins. The "percent (%) amino acid sequence homology" with respect to a reference polypeptide sequence is defined, if necessary, after aligning the sequences to achieve the maximum percent sequence homology and introducing gaps, as the percent of amino acid residues in the sequence that are identical to the amino acid residues in the reference polypeptide sequence. To determine sequence homology, the sequence of the query protein is aligned with the sequence of a reference protein or polypeptide, such as the polypeptide of SEQ ID NO:1. Sequence alignment methods are well known in the art. For example, to determine the degree of amino acid sequence homology of any polypeptide with respect to the amino acid sequence of SEQ ID NO:1, preferably the freely available SIM Local similarity program (Xiaoquin Huang and Webb Miller (1991), Advances in Applied Mathematics, vol.12:337-357) is utilized. For multiple alignment analysis, preferably ClustalW is used (Thompson et al. (1994) Nucleic Acids Res., 22(22):4673-4680).

[0020] The terms "protein" and "polypeptide" refer to any chain of two or more amino acids joined by peptide bonds and do not refer to products of a specific length. Thus, the terms "peptide", "protein", "amino acid chain", or any other term used to refer to a chain of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" may be used in place of any of these terms or interchangeably with these terms. The term "polypeptide" is also intended to refer to products of post-translational modification of polypeptides, such as glycosylation, which are well known in the art.

[0021] The terms "alkali-stable" or "alkali stability" or "corrosion-stable" or "corrosion stability" refer to the ability of the GAA-binding protein of the present invention to withstand alkaline conditions without significantly losing its binding ability to GAA. A person skilled in the art can easily test the alkali stability of a GAA-binding protein by incubating it with sodium hydroxide as described in the examples and subsequently testing its binding ability to GAA by routine experiments known to those skilled in the art, such as chromatographic techniques.

[0022] The term "chromatography" refers to a separation technique that uses a mobile phase and a stationary phase to separate one type of molecule (e.g., GAA) from other molecules (e.g., contaminants) in a sample. The liquid mobile phase contains a mixture of multiple molecules, which are transported across or through a stationary phase (such as a solid matrix). The molecules in the mobile phase can be separated due to differences in their interactions with the stationary phase.

[0023] The term "affinity chromatography" refers to a specific type of chromatography in which a ligand bound 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 invention, affinity chromatography involves adding a sample containing GAA to a stationary phase containing a chromatography ligand, such as the GAA-binding protein of the present invention.

[0024] The terms "solid support" or "solid matrix" are used interchangeably with respect to the stationary phase.

[0025] As used interchangeably herein, the terms "affinity matrix", "affinity purification matrix", or "affinity chromatography matrix" refer to a matrix, such as a chromatography matrix, to which an affinity ligand, such as the GAA-binding protein of the present invention, is attached. The ligand (e.g., GAA-binding protein) can specifically bind to a molecule of interest (e.g., VH3-containing protein) to be removed from a purification or mixture.

[0026] As used herein, the term "affinity purification" refers to a method of purifying liquid GAA by binding it to a GAA-binding protein immobilized on a matrix. Thereby, other components of the mixture excluding GAA are removed. In a further step, the bound GAA can be eluted in a purified form.

[0027] Detailed Description of Embodiments of the Present Invention The present invention will be further described. In the following section, other aspects of the present invention are defined in more detail. Each aspect defined below may be combined with any one or more other aspects, unless a clear indication to the contrary is given. Specifically, any feature shown to be preferred or advantageous may be combined with any one or more other features shown to be preferred or advantageous.

[0028] The novel polypeptides of the present invention (including SEQ ID NO: 1, 2, 16, 17, 18, 22, 23) exhibit binding affinity for GAA (measured herein by SPR). The novel binding proteins for acidic α-glucosidase comprise an amino acid sequence having at least 95% homology to any one selected from the group of SEQ ID NO: 1-9, 14-20, 22-23. The novel GAA binding protein comprises one or more GAA binding domains, and at least one GAA binding domain consists essentially of or consists 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 an amino acid having at least 95% sequence homology 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 shown in Figure 1 and herein. SEQ ID NO:1(192403) IAAKFDMKQAWADHFILELPNLTEEQRNAFRQSLSDDPSVSDLVLLQAQKLNQMQAPK SEQ ID NO:2(192402) IAAKFDMKQAWADHFILELPNLTEEQRNAFRQSLSDDPSVSDLVLAQAQKLNQSQAPK SEQ ID NO:16(192403 del2N) AKFDMKQAWADHFILELPNLTEEQRNAFRQSLSDDPSVSDLVLLQAQKLNQMQAPK SEQ ID NO:17(192402 del2N) AKFDMKQAWADHFILELPNLTEEQRNAFRQSLSDDPSVSDLVLAQAQKLNQSQAPK.

[0029] The novel GAA-binding protein contains one or more GAA-binding domains, and at least one GAA-binding domain contains the amino acid sequence of SEQ ID NO:18 or an amino acid having at least 95% sequence homology thereto. QAWADHFILELPNLTEEQRNAFRQSLSDDPSVSDLVLX 1 QAQKLNQX 2

[0030] X 1 may be any amino acid and, in some embodiments, may be an amino acid selected from alanine (A) or leucine (L). X 1 corresponds to position 46 in SEQ ID NO:1 or SEQ ID NO:2.

[0031] X 2 may be any amino acid and, in some embodiments, may be an amino acid selected from serine (S) or methionine (M). X 2 corresponds to position 54 in SEQ ID NO:1 or SEQ ID NO:2.

[0032] In some embodiments, the GAA-binding polypeptide has at least 95% sequence homology to the amino acid sequence of SEQ ID NO:1. In some embodiments, the GAA-binding polypeptide has at least 95% sequence homology to the amino acid sequence of SEQ ID NO:2. In some embodiments, the GAA-binding polypeptide has at least 95% sequence homology to the amino acid sequence of SEQ ID NO:16. In some embodiments, the GAA-binding polypeptide has at least 95% sequence homology to the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:18. In some embodiments, the GAA-binding polypeptide has at least 95% sequence homology to the amino acid 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 homology 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% homology to the amino acids of SEQ ID NO:1" refers to one or two substitutions compared to the amino acids of SEQ ID NO:1, and "at least 98% homology to the amino acids of SEQ ID NO:1" refers to one substitution compared to the amino acids of SEQ ID NO:1. For example, SEQ ID NO:2 and SEQ ID NO:1 differ by two amino acids, namely amino acid 46 and amino acid 54, and are 96% identical. For example, SEQ ID NO:16 and SEQ ID NO:1 lack two amino acids, namely amino acid 1 and amino acid 2 of SEQ ID NO:16. For example, SEQ ID NO:17 and SEQ ID NO:2 lack two amino acids, namely amino acid 1 and amino acid 2 of SEQ ID NO:17.

[0033] One advantage of the disclosed GAA binding domains and proteins containing these domains is their functional characteristic of specifically binding to GAA. This is particularly advantageous for use as an affinity ligand in the purification of GAA. The GAA binding proteins of the present invention are functionally characterized by a binding affinity for GAA of less than 200 nM. The GAA binding proteins of the present invention bind to GAA with a dissociation constant K D of less than or equal to 200 nM, preferably less than or equal to 100 nM, or more preferably less than or equal to 50 nM, as shown in Example 3.

[0034] Multimer. In one embodiment of the present invention, the GAA binding protein comprises 1, 2, 3, 4, 5, or 6 GAA binding proteins bound to each other. In some embodiments, the GAA binding protein can be, for example, a monomer, dimer, trimer, tetramer, pentamer, or hexamer. Preferred GAA binding proteins are monomers or dimers. The multimers of the proteins of the present invention are generally artificially generated fusion proteins by recombinant DNA techniques well known to those skilled in the art. In some embodiments, the multimer is a homomultimer, for example, the amino acid sequences of the GAA binding proteins are identical as shown in SEQ ID NO:7 (dimer of SEQ ID NO:1). In other embodiments, the multimer is a heteromultimer, for example, the amino acid sequences of the GAA binding proteins are different.

[0035] Fusion protein. According to one embodiment, provided herein is a fusion protein comprising one or more, for example two, GAA-binding polypeptides disclosed throughout this specification. More specifically, the fusion protein comprises one or more GAA-binding polypeptides disclosed herein and an additional polypeptide different from the disclosed polypeptides. In various embodiments, the additional polypeptide different from the GAA-binding polypeptides disclosed herein may be, for example, but not limited to, a non-Ig-binding protein such as 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% homology 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% homology to SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:21. Exemplary non-Ig-binding proteins are shown in the amino acid sequences of SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:21. Thus, some embodiments include a fusion protein comprising one or two GAA-binding polypeptides and one or two non-Ig-binding polypeptides disclosed herein.

[0036] In some embodiments, the fusion protein is one of the following (a) GAA-binding protein - non-Ig-binding protein; (b) non-Ig-binding protein - GAA-binding protein; (c) non-Ig-binding protein - GAA-binding protein - non-Ig-binding protein (see, for example, SEQ ID NO:9); (d) GAA-binding protein - non-Ig-binding protein - non-Ig-binding protein (see, for example, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20). (e) GAA-binding protein - GAA-binding protein - non-Ig-binding protein - non-Ig-binding protein (see, for example, SEQ ID NO:15); (f) A combination of non-Ig binding protein - non-Ig binding protein - GAA binding protein (from N-terminus to C-terminus) may be included.

[0037] Other combinations of non-Ig binding proteins and GAA binding protein domains are also feasible for those skilled in the art.

[0038] 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 homology 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 homology 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 homology 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 homology 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 homology 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 homology 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 homology 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.

[0039] The portions of the fusion protein may be directly linked to each other in a head-to-tail manner or may be linked by a linker, which is preferably a peptide linker. In various embodiments, the peptide linker can be regarded as an amino acid sequence that sterically separates two portions of the fusion protein. Typically, such a linker consists of 1 to 10 amino acids.

[0040] The fusion protein can be characterized as a protein formed by genetically fusing or ligating a gene encoding the GAA-binding polypeptide of the present invention with a gene encoding a polypeptide different from the polypeptides described hereinabove. Thus, the fusion protein can be regarded as the product of two or more genes translated simultaneously (without a stop codon in between).

[0041] Molecules for purification or detection. In some embodiments, the GAA-binding protein or a fusion protein containing the GAA-binding protein may also contain additional amino acid residues at the N-terminus and / or C-terminus, such as additional sequences at the N-terminus and / or C-terminus. The additional sequences may include, for example, sequences introduced for purposes such as purification or detection. Typical examples of such sequences include, but are not limited to, Strep-tags (see SEQ ID NO: 12, etc.), oligohistidine tags, glutathione S-transferase, maltose-binding protein, intein, intein fragments, or the albumin-binding domain of protein G or others. In one embodiment, the additional amino acid sequence contains one or more peptide sequences that confer affinity for a specific chromatographic column material. The GAA-binding protein or a fusion protein containing the GAA-binding protein may preferably contain a specific attachment site for attachment to a solid support, such as cysteine or lysine, at the C-terminus. Examples of GAA-binding proteins having a C-terminal cysteine are shown in SEQ ID NO: 3, 4, 5, 6, 8. Examples of the GAA-binding proteins of the present invention having an N-terminal additional amino acid are shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0042] Method for producing a GAA-binding protein. The present invention further provides a method for producing a novel GAA-binding polypeptide disclosed herein that has a binding affinity for GAA, the method comprising the following steps: (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 bound to GAA, and (iv) obtaining a GAA-binding polypeptide capable of binding to GAA.

[0043] The method for producing a novel GAA-binding polypeptide having a binding affinity for GAA may include an additional step of determining the binding affinity of the polypeptide for GAA. The binding affinity may be determined as described elsewhere herein.

[0044] Use of a novel GAA-binding polypeptide in a technical application. Also provided herein is the use of any novel GAA-binding polypeptide of the present invention for use in a technical application, preferably as an affinity ligand in affinity chromatography, comprising a fusion protein.

[0045] As described herein, affinity chromatography (also referred to as affinity purification) uses specific binding interactions between molecules. Methods for immobilizing proteins and affinity chromatography methods are well known in the field of protein purification and can be readily performed by those skilled in the art using standard techniques and equipment.

[0046] In various embodiments, the affinity purification method may further include one or more washing steps performed under conditions sufficient to remove some or all of the molecules that do not specifically bind thereto from the affinity purification matrix. Affinity purification matrices suitable for the disclosed uses or methods are known to those skilled in the art.

[0047] Binding to a solid support. In various aspects and / or embodiments of the present invention, the novel polypeptides disclosed herein, including the novel polypeptides generated or obtained by any of the methods described above, bind to a solid support. In some embodiments of the present invention, the polypeptide includes an attachment site for site-specific covalent binding 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 can undergo a specific chemical reaction with a reactive group of the solid phase, or a linker between the solid phase and the protein.

[0048] Affinity purification matrix. In another embodiment, there is provided an affinity purification matrix containing a GAA-binding polypeptide, including the polypeptide identified by any of the methods described above.

[0049] In a preferred embodiment, the affinity purification matrix is a solid support. The affinity purification matrix includes at least one GAA-binding polypeptide provided by the present invention. Thus, the novel GAA-binding proteins disclosed herein also include use for purifying GAA by the affinity matrix.

[0050] Solid supports for affinity chromatography are known in the art and include, but are not limited to, agarose and stabilized derivatives of agarose, cellulose or derivatives of cellulose, porous glass, monoliths, silica, zirconium oxide, titanium oxide, or synthetic polymers, and hydrogels of various compositions, as well as combinations of the above.

[0051] The form of the solid support matrix can be of any suitable well-known type. Such solid support matrices for binding the novel proteins or polypeptides of the present invention can be, for example, but not limited to, the following: columns, capillaries, particles, membranes, filters, monoliths, fibers, pads, gels, glass slides, plates, cassettes, or any other form commonly used in chromatography and known to those skilled in the art.

[0052] In one embodiment, the matrix consists of substantially spherical particles, also known as beads, such as, for example, Sepharose or agarose beads. The particulate matrix can be used as a packed bed or in a suspension form including an expanded bed. In other embodiments of the present invention, the solid support matrix is a membrane, such as, for example, a hydrogel membrane. In some embodiments, affinity purification may include a membrane as a matrix to which the GAA-binding protein of the present invention is covalently bound. The solid support can also be in the form of a membrane in a cartridge.

[0053] In some embodiments, affinity purification includes a chromatography column containing a solid support matrix to which the novel protein of the present invention is covalently bound. The novel proteins or polypeptides of the present invention may be attached to a suitable solid support matrix by conventional binding techniques. Methods for immobilizing protein ligands to solid supports are well known in the fields of protein engineering and purification and can be readily performed by those skilled in the art using standard techniques and equipment.

[0054] A method for producing GAA. Further embodiments relate to these processes for producing GAA or proteins containing GAA, which include at least one chromatography step using an affinity chromatography matrix having an affinity for specifically binding the GAA protein, wherein the affinity ligand (binding protein) for GAA described above binds to this affinity chromatography matrix.

[0055] Method for determining the presence of GAA. Further, in some embodiments, the GAA-binding protein or the fusion protein described herein is used in a method for determining the presence of GAA. Some embodiments relate to a method for analyzing the presence of GAA in a liquid sample, the method comprising the following steps: (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 described herein under conditions that allow binding of at least one GAA-binding protein (or fusion protein) to GAA; (d) separating the complex of GAA and the GAA-binding protein (or fusion protein); and (e) determining the amount of the GAA-binding protein (or fusion protein) that indicates the amount of GAA in the liquid of (a).

[0056] Method for quantifying GAA. Further embodiments relate to a method for quantifying GAA, the method comprising: (a) providing a liquid containing GAA; (b) providing a matrix to which the GAA-binding protein (or fusion protein) described herein is covalently bound; (c) contacting this affinity purification matrix with the liquid under conditions that allow binding of at least one GAA-binding protein (or fusion protein) to GAA; (d) eluting this GAA; and optionally (e) quantifying the amount of the eluted GAA. The method for determining the presence of GAA in a liquid sample may be quantitative or qualitative. Such methods are well known to those skilled in the art and can be selected from established methods in the art, such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme reaction, surface plasmon resonance (SPR), or chromatography.

[0057] Polynucleotides, vectors, host cells. One embodiment includes a isolated polynucleotide or nucleic acid molecule encoding a GAA binding polypeptide disclosed herein. Further embodiments also include a polypeptide encoded by a polynucleotide disclosed herein. Further, vectors, particularly expression vectors comprising the isolated polynucleotide or nucleic acid molecule of the invention, and host cells comprising the isolated polynucleotide or expression vector are provided. For example, one or more polynucleotides encoding a polypeptide disclosed herein may be expressed in a suitable host and the resulting protein can be isolated. A vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) that can be used to introduce information encoding a protein into a host cell. Suitable vectors applicable to the present invention are known in the art.

[0058] Further provided are isolated cells comprising a polynucleotide or nucleic acid or vector disclosed herein. Suitable host cells include prokaryotes or eukaryotes such as, for example, a bacterial host cell, a yeast host cell, or a non-human host cell carrying the vector. Suitable bacterial expression host cells or systems are known in the art. A variety of mammalian or insect cell culture systems known in the art can also be used to express recombinant proteins.

[0059] Methods for producing the proteins of the invention. In further embodiments, provided are methods for producing the described GAA binding polypeptide, the method comprising the steps of: (a) culturing a (suitable) host cell under conditions suitable for the expression of the GAA binding polypeptide so as to obtain the GAA binding polypeptide; and (b) optionally isolating the GAA binding polypeptide. Suitable conditions for culturing prokaryotic or eukaryotic hosts are well known to those skilled in the art.

[0060] The GAA-binding polypeptide may be prepared by any conventional well-known technique such as simple organic synthesis strategies, solid-phase assisted synthesis techniques, or a commercially available automated synthesizer. These may also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques.

[0061] In one embodiment, as detailed above, a method for preparing a GAA-binding protein is provided, the method comprising: (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 medium; (e) subjecting the host cell to culture conditions suitable for the expression of the GAA-binding polypeptide, thereby producing the GAA-binding polypeptide; optionally (f) separating the protein or polypeptide produced in step (e); and (g) optionally binding the protein or polypeptide to the solid matrix described above. In various embodiments of the invention, the production of the GAA-binding polypeptide is carried out by cell-free in vitro transcription and translation.

[0062] The following examples provide further illustration of the invention. However, the invention is not limited thereto, and the following examples merely demonstrate the practicability of the invention based on the above description.

Examples

[0063] The following examples are provided for further illustration of the invention. However, the invention is not limited thereto, and the following examples merely demonstrate the practicability of the invention based on the above description. For a complete disclosure of the invention, reference is also made to the documents cited in this application, which are hereby incorporated by reference in their entirety.

[0064] Example 1. Selection of the GAA-binding Protein of the Invention Library construction and library cloning. A library based on a stable non-immunoglobulin binding protein having a protein A-like structure shown in SEQ ID NO:10 containing randomized amino acid positions was synthesized in-house with randomized oligonucleotides generated by synthetic trinucleotide phosphoramidites (ELLA Biotech) to achieve a balanced amino acid distribution that simultaneously excluded cysteine and other amino acid residues at the randomized positions. At least amino acid positions 7, 8, 10, 11, 14, 15, 18, 20, 42, 43, 46, 47, 49, 50, 53 and 54 of SEQ ID NO:10 were randomized.

[0065] The corresponding cDNA library was amplified by PCR and ligated with pCD33-OmpA phagemid. An aliquot of the ligation mixture was used for electroporation of E. coli ER2738 (Lucigen). Unless otherwise indicated, established recombinant gene methods were used.

[0066] Primary selection by the TAT phage display method. A naive library was enriched against biotinylated GAA (Lumizyme™, also known as myozyme or alglucosidase alpha) using the phage display method as the selection system. Competent bacteria ER2738 cells (Lucigene) were transformed with phagemid pCD33-OmpA carrying the library, and then phage amplification and purification were performed using standard methods known to those skilled in the art. To enable binding between biotinylated GAA in solution and the phage library, solution-based selection (SIS) was utilized. The GAA-phage complex was captured by streptavidin / neutravidin magnetic beads. The GAA concentration during phage incubation was diluted from 200 nM (first round) to 100 nM (second round), to 50 nM (third round), and then to 25 nM (fourth round). Pre-selection with empty magnetic beads was performed in each round. After each selection round, phage bound to GAA was eluted with trypsin. To identify the target-specific phage pool, phage from each eluted and re-amplified selection round was analyzed by phage pool ELISA. Wells of a medium-binding microtiter plate (Greiner Bio-One) were pre-coated with streptavidin (10 μg / ml) and then coated with biotinylated GAA (2.5 μg / ml). Bound phage was detected using an α-M13 HRP-conjugated antibody (GE Healthcare).

[0067] Cloning of the target-binding phage pool into an expression vector. The selection pool that showed specific binding to GAA in phage pool ELISA was amplified by PCR according to methods known in the art, digested with appropriate restriction enzymes, 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.

[0068] Example 2. Expression and purification of GAA-binding proteins After screening the hits identified by the selection described in Example 1, the protein was produced in μ units (Phynexus).

[0069] The construct was expressed in E. coli BL21(DE3) using a low-copy plasmid system under the control of the T7 promoter. After induction with lactose contained in the medium (auto-induction medium), the protein was produced in a soluble form. 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 in 3 ml of 2xYT medium supplemented with 50 μg / ml kanamycin and cultured in a culture test tube at 37°C for 6 hours at 200 rpm in a conventional orbital shaker. The main culture was inoculated with 3 ml of the preculture in 300 mL of ZYM-5052 (0.5% glycerol, 0.2% lactose, 0.05% glucose, 0.5% yeast extract, 1.0% casamino acids, 25 mM Na 2 HPO 4 、25 mM KH 2 PO 4 、5 mM Na 2 SO 4 、2 mM MgSO 4 and 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. The expression of the recombinant protein was induced by metabolizing glucose and then introducing lactose into the cells. The cells were grown overnight for about 17 hours to achieve a final OD600 of about 2 - 4. Before harvesting, the OD600 was measured, and a sample adjusted to 0.6 / OD600 was taken out, pelleted, and frozen at -20°C. To collect the biomass, the cells were centrifuged at 12,000 xg for 15 minutes at 22°C. The pellet was weighed (wet weight). The cells were stored at -20°C before processing.

[0070] Proteins with affinity tags were purified by affinity chromatography and size exclusion. After affinity chromatography purification, size exclusion chromatography (SE HPLC or SEC) was performed using an Äkta system and a Superdex™ 200 HiLoad 16 / 600 column (GE Healthcare). The SEC column had a volume of 120 ml and was equilibrated with 2 CV. The sample was applied at a flow rate of 1 ml / min. Fraction collection was started when the signal intensity reached 10 mAU. After SDS-PAGE analysis, positive fractions were pooled and the protein concentration was measured. Further analysis included SDS-PAGE, SE-HPLC, and RP-HPLC. The protein concentration was determined by measuring the absorbance at 280 nm using the molar extinction coefficient. Reverse phase chromatography (RP-HPLC) was performed using a Dionex HPLC system and a PLRP-S (5 μm, 300 Å) column (Agilent).

[0071] Fusion proteins of GAA-binding protein and non-Ig-binding protein (SEQ ID NO:14, 15, 19, 20) were purified by the following methods: 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 (NH 4 ) 2 SO 4 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 of protein per liter of cell culture volume. SEQ ID NO:19 was successfully purified at a yield of 10 g of protein per liter of cell culture volume.

[0072] Example 3. Analysis of Proteins by Surface Plasmon Resonance (SPR) The GAA (ON - ligand) of 500 - 1500 RU was immobilized on a CM - 5 sensor chip (GE Healthcare), and this chip was equilibrated with SPR running buffer. The GAA was biotinylated with 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 sensor chip coated with streptavidin. When the ligand bound, the protein analyte accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real - time and plotted as response or resonance units versus time. The analyte was applied to the chip at a flow rate of 30 μl / min with serial dilutions. Association was carried out for 120 seconds and dissociation for 360 seconds. After each run, the chip surface was regenerated with 30 μl of regeneration buffer (10 mM glycine) and equilibrated with the running buffer. The binding assay was performed by using BIAcore 3000 (GE Healthcare), and the data evaluation was carried out by using the Langmuir 1:1 model (RI = 0) with BIAevaluation 3.0 software provided by the manufacturer. The evaluated dissociation constant (K D ) was normalized with respect to GAA and displayed. The change in refractive index measured in real - time and plotted as response or resonance units [RU] versus time [seconds] is shown. Results. The GAA - binding proteins disclosed herein showed strong specific binding to the immobilized GAA with an affinity of less than 100 nM (see Table 1). The GAA - binding proteins of the present invention do not bind to hIgG. The fusion protein of SEQ ID NO:19 does not bind to hIgG and is specific for GAA with an affinity of less than 100 nM.

[0073] TIFF0007689746000001.tif56170

[0074] Example 4. The GAA - binding proteins of the present invention as affinity ligands for GAA purification Coupling efficiency: 20 mg of purified GAA-binding protein (having a C-terminal cysteine) was immobilized per mL of Praesto™ Epoxy 85 according to the manufacturer's instructions (coupling buffer: 50 mM Na 2 HPO 4 4, 150 mM NaCl, 5 mM TCEP, 2.05 M Na 2 2 4 (or 175 mg of Na 2 2 4 per mL of resin), pH 9.5, coupling conditions: 3 h at 35 °C). The results are shown in Table 2.

[0075] TIFF0007689746000002.tif54170

[0076] DBC10%: 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 (for determination of elution ratio). The dynamic binding capacity (DBC) was determined by the mass of GAA injected at a residence time of 6 min and 10% breakthrough. Results. The bound resin showed an excellent dynamic binding capacity (DBC10%) for the affinity ligand (having a C-terminal Cys). ● Having a DBC10% of at least 27 mg / ml with SEQ ID NO:1 ● Having a DBC10% of at least 24 mg / ml with the SEQ ID NO:1 dimer ● At least 22.7 mg / ml with the SEQ ID NO:1 fusion protein (SEQ ID NO:19) ● Having a DBC10% of at least 16 mg / ml with SEQ ID NO:2

[0077] Corrosion stability (cycle test). Samples coupled to Praesto™ Epoxy 85 (coupling conditions: 20 mg / ml, 3 h, 35 o °C, 2.05 M Na 2 2 4It was treated with 0.1 M NaOH at room temperature (pH 8.5) for at least 10 hours (610 minutes).

[0078] Result: The affinity ligand (coupled via the C-terminal Cys) showed excellent corrosion stability in the cycle test. ● Having a residual capacity of 95% with SEQ ID NO:1 (N-terminal extension) ● SEQ ID NO:1 dimer 〇 Having a residual capacity of 94.4% after 610 minutes (about 10 hours) 〇 Having a residual capacity of 84.4% after 1515 minutes (25 hours) (equivalent to 101 cycles)

[0079] Figure 2 shows the cycle test on Praesto (trademark) Epoxy 85 resin. Figure 2A shows the DBC 10% for SED ID NO:1 (with N-terminal extension) on Praesto (trademark) Epoxy 85 at a residence time of 6 minutes. The binding capacity was 19.5 mg / ml. Figure 2B shows the DBC 10% and corrosion stability of the GAA-binding protein (SEQ ID NO:1 with N-terminal extension) coupled to Praesto (trademark) Epoxy 85 resin and loaded with 2 mg / ml GAA. The residual capacity after 50 loading, elution, and 0.1 M NaOH regeneration cycles was at least 90%.

[0080] Table 3 shows the results for the immobilized affinity ligand incubated in NaOH in a packed column format.

[0081] TIFF0007689746000003.tif79170

Claims

**Claim 1** A GAA-binding protein comprising one or more acidic α-glucosidase (GAA) binding domains, wherein at least one domain comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 95% sequence homology to SEQ ID NO: 1 or SEQ ID NO: 2, a GAA-binding protein. **Claim 2** The GAA-binding protein according to claim 1, wherein the GAA-binding protein has a binding affinity for GAA of less than 200 nM. **Claim 3** The GAA-binding protein according to claim 1, wherein the GAA-binding protein comprises 2, 3, 4, 5, or 6 domains bound to each other. **Claim 4** The GAA-binding protein according to claim 3, wherein the GAA-binding protein is a homomultimer. **Claim 5** The GAA-binding protein according to claim 3, wherein the GAA-binding protein is a heteromultimer. **Claim 6** The GAA-binding protein according to claim 3, wherein one or more domains are bound to each other directly or by one or more peptide linkers. **Claim 7** A fusion protein comprising the GAA-binding protein according to any one of claims 1 to 6. **Claim 8** A polynucleotide encoding the GAA-binding protein according to any one of claims 1 to 6, or the fusion protein according to claim 7. **Claim 9** The GAA-binding protein according to any one of claims 1 to 6, or the fusion protein according to claim 7, for use in affinity purification of GAA. **Claim 10** The GAA-binding protein according to any one of claims 1 to 6, or the fusion protein according to claim 7, further comprising one or more coupling sites for coupling to an affinity purification matrix. **Claim 11** The GAA-binding protein or fusion protein according to claim 10, wherein the GAA-binding protein comprises one or more cysteine residues for coupling to an affinity purification matrix. **Claim 12** An affinity purification matrix comprising the GAA-binding protein according to any one of claims 1 to 6, or the fusion protein according to claim 7. **Claim 13** A method for affinity purification of GAA, the method comprising: (a) providing a liquid containing GAA; (b) providing the affinity purification matrix containing at least one GAA-binding protein according to any one of claims 1 to 6, 10, or the fusion protein according to claim 7, which is bound 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 claims 1 to 6, 10 to GAA; and (d) eluting the GAA from the affinity purification matrix.

14. Use of a GAA-binding protein according to any one of claims 1 to 6 or a fusion protein according to claim 7 in a method for determining the presence of GAA.

15. A method for analyzing the presence of GAA in a liquid sample, the method comprising the following: (i) providing a liquid containing GAA; (ii) providing a GAA-binding protein according to any one of claims 1 to 6 or a fusion protein according to claim 7; (iii) contacting the liquid of (i) with a GAA-binding protein according to any one of claims 1 to 6 under conditions that allow binding of at least one GAA-binding protein according to claim 1 or a fusion protein according to claim 7 to GAA; (iv) separating a complex of GAA and a GAA-binding protein according to any one of claims 1 to 6 or a fusion protein according to claim 7; and (v) quantifying the amount of a GAA-binding protein according to any one of claims 1 to 6 or a fusion protein according to claim 7 in the liquid of (i). A method comprising the steps.

16. A method for preparing a GAA-binding protein, the method comprising the following: (a) providing a nucleic acid molecule encoding a GAA-binding protein according to any one of claims 1 to 6; (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 medium; and (e) subjecting the host cell to culture conditions suitable for the expression of the GAA-binding protein, thereby producing the GAA-binding protein. A method comprising the steps.

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