Affinity ligand for a single chain antibody fragment specific for serum albumin

Abstract

The present invention relates to a novel affinity ligand that binds to a single chain antibody fragment (scFv) that is specific for serum albumin. The novel affinity ligands of the present invention are powerful tools because they allow efficient purification methods for fusion proteins comprising scFv via affinity chromatography.

Description

[0001] AFFINITY LIGAND FOR A SINGLE CHAIN ANTIBODY FRAGMENT SPECIFIC FOR SERUM

[0002] ALBUMIN

[0003] TECHNICAL FIELD

[0004] The present invention relates to a novel affinity ligand that binds to a single chain antibody fragment (scFv) that is specific for serum albumin. The novel affinity ligands of the present invention are powerful tools because they allow efficient purification methods for fusion proteins comprising scFv via affinity chromatography.

[0005] BACKGROUND OF THE INVENTION

[0006] Serum albumin binding domains such as single-chain antibody fragments (scFv) specific for human serum albumin are known to extend the half-lives of proteins that are used in therapeutic applications, such as interleukins, antibody fragments, or non-antibody proteins. Fusion proteins were developed that include scFv specific for albumin and therapeutically active proteins. Such fusion proteins that include such scFv exhibit longer serum half-lives and better in vivo pharmacokinetics than therapeutically active proteins without such scFv.

[0007] Fusion proteins that include scFv specific for human serum albumin may be purified for the use as therapeutic proteins in a variety of ways. Standard procedures include chromatographic techniques that use acidic pH ranges (for example, pH 3.7) for the elution of the proteins from the chromatographic columns. However, as consequence of the acidic elution condition, proteins may form aggregates.

[0008] Thus, there is a strong need in the art to provide methods for an efficient purification of therapeutically relevant proteins at mild isolation conditions.

[0009] The present invention meets this need by providing novel affinity ligands for single chain antibody fragment (scFv) specific for serum albumin. These affinity ligands are particularly advantageous because they allow a purification of therapeutically active proteins comprising scFv via affinity chromatography, allowing an isolation step at mild pH.

[0010] The above overview does not necessarily describe all problems solved by the present invention.

[0011] SUMMARY OF THE INVENTION

[0012] The present disclosure provides the following items 1 to 11 , without being specifically limited thereto:

[0013] 1. An affinity ligand for a single chain antibody fragment specific (scFv) specific for albumin, wherein the affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with at least 90 % sequence identity thereto. 2. The affinity ligand according to item 1 , wherein the affinity ligand has a binding affinity of less than 200 nM for the scFv.

[0014] 3. The affinity ligand according to any one of items 1-2, wherein the affinity ligand is a multimer.

[0015] 4. The affinity ligand according to any one of items 1-3, wherein the albumin is a serum albumin, preferably a human serum albumin.

[0016] 5. An affinity separation matrix comprising the affinity ligand according to any one of items 1-4.

[0017] 6. Use of the affinity ligand according to any one of items 1-4, or the affinity separation matrix according to item 5, for affinity purification.

[0018] 7. The use of item 6, which is for affinity purification of a fusion protein comprising a scFv specific for albumin.

[0019] 8. A method for purification of a fusion protein comprising a scFv specific for albumin, the method comprising:

[0020] (i) contacting a liquid containing a fusion protein comprising the scFv with affinity ligand according to any one of items 1-4, or the affinity matrix of item 5;

[0021] (ii) purifying the fusion protein comprising the scFv specific for albumin; and

[0022] (iii) obtaining the fusion protein.

[0023] 9. The method of purification of item 8, the method comprising:

[0024] (i) providing a liquid that contains a fusion protein comprising scFv specific for albumin;

[0025] (ii) providing an affinity separation matrix of item 5;

[0026] (iii) contacting said affinity separation matrix with the liquid;

[0027] (iv) purifying said fusion protein comprising scFv specific for albumin from the affinity separation matrix; and

[0028] (v) obtaining the fusion protein.

[0029] 10. The method of purification of items 8 or 9, the method comprising eluting the fusion protein comprising scFv specific for albumin from the affinity separation matrix at pH higher than 4.5. 11. A method for producing a fusion protein that comprises scFv specific for albumin, the method comprising:

[0030] (i) providing a liquid containing a fusion protein that comprises scFv specific for albumin;

[0031] (ii) contacting the liquid of (i) with affinity ligand according to any one of items 1-4, or the affinity separation matrix of item 5; and

[0032] (iii) purifying the fusion protein that comprises the scFv specific for albumin and thereby producing the fusion protein.

[0033] This summary of the invention is not limiting, and other aspects and embodiments of the invention will become evident from the following description, examples and drawings.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention provides novel proteins having specific binding affinity for scFv specific for albumin. The novel proteins of the present invention are particularly advantageous because as affinity ligands for fusion protein comprising scFv specific for albumin, they allow precise purification of fusion proteins, for example in affinity chromatography. In particular, purification methods with the affinity ligands of the invention can be performed at mild pH to avoid aggregation of fusion protein comprising scFv specific for albumin. Affinity ligands of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with at least 90 % identity to SEQ ID NO: 1 or SEQ ID NO: 3, respectively, bind to the single chain antibody fragment specific for human serum albumin (referred to as scFv or scFv HSA herein) with high affinity.

[0036] Before the present invention is described in more detail below it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may 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 invention, which is reflected by the appended items. Unless defined otherwise, 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 a skilled person working in the field of protein engineering and purification, but also including a skilled person working in the field of developing new specific binding molecules for scFv HSA for use as affinity ligands in the purification of fusion protein comprising scFv specific for albumin in affinity chromatography.

[0037] 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 Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). Throughout this specification and the items, which follow, unless the context requires otherwise, the word “comprise”, and variants 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 “comprise(s)” or “comprising” may encompass a limitation to “consists of’ or “consisting of’, should such a limitation be necessary for any reason and to any extent.

[0038] Several documents (for example: patents, patent applications, scientific publications, manufacturer’s specifications, instructions, UniProt Accession Number, etc.) may be cited throughout the present specification. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein may be characterized as being “incorporated by reference". In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0039] All sequences referred to herein are disclosed in the attached sequence listing that, with its whole content and disclosure, forms part of the disclosure content of the present specification.

[0040] General Definitions of Important terms used in the Application

[0041] The term “scFv specific for albumin” or “scFv HSA” may be used interchangeably herein and describe a single-chain antibody fragment (scFv) that binds to albumin. In some embodiments, “scFv specific for albumin” or “scFv HSA” relates to a single-chain antibody fragment that binds to human serum albumin (HSA). The term “scFv specific for albumin” as understood herein refers to a scFv specific for albumin that comprises a variable heavy chain domain and a variable light chain domain. The scFv includes a variable heavy chain attached to a variable light chain domain by a linker. The scFv variable heavy chain includes a vhCDRI , a vhCDR2, and a vhCDR3, and the scFv variable light chain domain includes a vICDRI , a vlCRD2, and a vlCDR3. The term „scFv specific for albumin” or “scFv HSA” comprises all polypeptides which show an amino acid sequence identity of at least 70 %, 80 %, 85 %, 90 %, 95 %, 96 % or 97 % or more, or 100 % to SEQ I D NO: 5. In some embodiments, the term “scFv specific for albumin” refers to an amino acid sequence with at least 90 % to SEQ ID NO: 5. In some embodiments, the term “scFv specific for albumin” refers to an amino acid sequence of SEQ ID NO: 5.

[0042] The term “fusion protein” relates to a protein comprising at least a first protein joined genetically to at least a second protein. A fusion protein is created through joining of two or more genes that originally coded for separate proteins. Thus, a fusion protein may comprise a multimer of identical or different proteins which are expressed as a single, linear polypeptide.

[0043] The term “fusion protein comprising scFv specific for albumin” refers to a scFv specific for albumin as described above fused to at least one further protein. The further protein may be any protein wherein half-life extension is desired. The further protein may be, for example but not limited to, a cytokine (e.g. interleukins, interferons), growth factors, hormone (e.g. growth hormones, parathyroid hormone), an antibody fragment, or a non-antibody protein. For example, the fusion protein comprising scFv specific for albumin as understood herein includes the scFv specific for albumin as described above and one or two or more cytokines. Cytokines may be selected from the group of interleukins (e.g. IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 , IL-27, or variants thereof), GM-CSF, or IFNalpha. In some embodiments, the fusion protein comprises an interleukin and the scFv specific for albumin as described above. In some embodiments, the fusion protein comprising scFv specific for albumin refers to scFv specific for albumin fused to two cytokines. The cytokines can be the same or different. In some embodiments, the fusion protein comprising scFv specific for albumin as understood herein is having the formula, from N-terminus to C- terminus, selected from a) cytokine-scFv specific for albumin, or b) cytokine-scFv specific for albumin-cytokine, or c) scFv specific for albumin-cytokine. In some embodiment, the fusion protein comprising scFv specific for albumin as understood herein is having the formula, from N- terminus to C-terminus, selected from a) IL-12-scFv specific for albumin, or b) IL-12-scFv specific for albumin-IL-15, or c) IL-15-scFv specific for albumin-IL-12, c) IL-12-scFv specific for albumin- IL-18, or d) IL-18-scFv specific for albumin-IL-12. In some embodiments, the fusion protein comprising scFv specific for albumin refers to an scFv specific for albumin attached to the one or two or more cytokines by a linker.

[0044] The term “linker” refers to a peptide of 1 to 30 amino acids that connects two protein moieties, for example, heavy chain fragment and light chain fragment, or scFv specific for albumin and fusion partner. In some embodiments the peptide linker is based on glycine, serine, or alanine, including, for example, (GGGGS)n, (GSGGS)n, (GGGS)n, and (GS)nwherein n is an integer from 1-10. In some embodiments, the scFv specific for albumin as described above is connected to a fusion partner by (GGGGS)n, for example, (GGGGS)s linkers.

[0045] The term “serum albumin” refers to a member of a family of globular proteins and includes, but is not limited to, human serum albumin (Genbank accession number NP_000468) and mouse serum albumin (Genbank accession number NP_0033784).

[0046] The terms “affinity ligand” or ..binding protein" may be used interchangeably herein and describe a protein that is capable to bind to a single-chain antibody fragment (scFv) specific for albumin. As described herein, a binding protein refers to a protein with detectable interaction with scFv specific for albumin, as determined by suitable methods such as for example SPR analysis or BLI or other appropriate technology known to someone skilled in the art.

[0047] The term “binding affinity” refers to the ability of the proteins of the invention to bind to scFv specific for albumin.

[0048] The term “amino acid sequence identity” refers to a quantitative comparison of the identity (or differences) of the amino acid sequences of two or more proteins. “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 the sequence identity, the sequence of a query protein is aligned to the sequence of a reference protein or polypeptide, for example, to the polypeptide of SEQ ID NO: 1. Methods for sequence alignment are well known in the art. For example, for determining the extent of an amino acid sequence identity of an arbitrary polypeptide relative to the amino acid sequence of, for example, SEQ ID NO: 1 , the SIM Local similarity program is preferably employed (Xiaoquin Huang and Webb Miller (1991), Advances in Applied Mathematics, vol. 12: 337-357), that is freely available. For multiple alignment analysis, ClustalW is preferably used (Thompson et al. (1994) Nucleic Acids Res., 22(22): 4673-4680).

[0049] The terms “protein” and “polypeptide” refer to any chain of two or more amino acids linked by peptide bonds and does not refer to a specific length of the product. Thus, “peptides”, “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 instead of, or interchangeably with, any of these terms. The term “polypeptide” is also intended to refer to the products of post-translational modifications of the polypeptide like, e.g., glycosylation, which are well known in the art.

[0050] The term “chromatography” refers to separation technologies which employ a mobile phase and a stationary phase to separate one type of molecules (e.g., fusion protein comprising scFv specific for albumin) from other molecules (e.g., contaminants) in the sample. The liquid mobile phase contains a mixture of molecules and transports these across or through a stationary phase (such as a solid matrix). Due to the differential interaction of the different molecules in the mobile phase with the stationary phase, molecules in the mobile phase can be separated.

[0051] The term “affinity chromatography” refers to a specific mode of chromatography in which a ligand (e.g., affinity ligand of the invention) coupled to a stationary phase (e.g. solid support) interacts with a molecule (i.e. scFv specific for albumin) in the mobile phase (the liquid sample), i.e. the ligand has a specific binding affinity for the molecule to be captured. As understood in the context of the invention, affinity chromatography involves the addition of a (liquid) sample containing fusion protein comprising scFv specific for albumin to a stationary phase which comprises a chromatography ligand, such as the affinity ligand of the invention. The terms “solid support” or “solid matrix” are used interchangeably for the stationary phase.

[0052] The terms "affinity matrix" or "affinity separation matrix", as used interchangeably herein, refer to a matrix, e.g., a chromatographic matrix, onto which an affinity ligand of the invention is attached. The attached affinity ligand is capable of specific binding to a protein of interest (e.g., fusion protein comprising scFv specific for albumin) which is to be purified from a liquid. The term “affinity purification” as used herein refers to a method of purifying fusion protein comprising scFv specific for albumin from a liquid by binding fusion protein comprising scFv specific for albumin to a ligand of the invention that is immobilized to a matrix. Thereby, fusion protein comprising scFv specific for albumin is removed from the liquid and thereby purified.

[0053] Detailed description of the embodiments of the invention

[0054] The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect defined below may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0055] The novel affinity ligand exhibits a binding affinity for an scFv specific for albumin. The invention provides an affinity ligand for an scFv specific for albumin, wherein the affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with at least 90 % sequence identity thereto.

[0056] Structural characteristics of the affinity ligand. In some embodiments, the affinity ligand for scFv specific for albumin comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the affinity ligand for scFv specific for albumin comprises or an amino acid with at least 90 % sequence identity to SEQ ID NO: 1 .

[0057] In some embodiments, the affinity ligand for scFv specific for albumin comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the affinity ligand for scFv specific for albumin comprises or an amino acid with at least 90 % sequence identity to SEQ ID NO: 3.

[0058] One embodiment that refers to the affinity ligand for scFv specific for albumin comprises an amino acid sequence with at least 90 % sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.

[0059] In some embodiments, the affinity ligand for scFv specific for albumin has at least 90 %, 91 %, 92 %, 93 %, 94 %, 95%, 96 %, 97 %, 98 %, 99 %, or 100 % sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.

[0060] In some embodiments, amino acids corresponding to positions 1-22, 24-40, 42-47, 49-54, 62-73, 75-78, 80-100, 102-107, 109-117 of SEQ ID NO: 1 or SEQ ID NO: 3 are identical in affinity ligands for scFv specific for albumin as described above. In some embodiments, the affinity ligand for scFv specific for albumin with at least 90 % sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3 may have substitutions in one or more positions corresponding to positions 23, 41 , 48, 55, 56, 57, 58, 59, 60, 61 , 74, 79, 101 , 108 of SEQ ID NO: 1 or SEQ ID NO: 3, respectively.

[0061] Functional characteristics of the affinity ligand. One advantage of the herein disclosed affinity ligand is the binding to scFv specific for albumin. In specific embodiments, the scFv specific for albumin has the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence of at least 90 % identity thereto. Some embodiments refer to an affinity ligand for an scFv specific for albumin, wherein the affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with at least 90 % sequence identity thereto. Some embodiments refer to an affinity ligand for a scFv specific for albumin, wherein the affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with at least 90 % sequence identity thereto, and wherein the scFv comprises the amino acid sequence of SEQ ID NO: 5.

[0062] The affinity ligand has a binding affinity of less than 200 nM for scFv specific for albumin as described herein. In some embodiments, the affinity ligand has a binding affinity of less than 100 nM for scFv specific for albumin as described herein. In some embodiments, the affinity ligand has a binding affinity of less than 50 nM for scFv specific for albumin as described herein. In preferred embodiments, the affinity ligand has a binding affinity of less than 10 nM for scFv specific for albumin as described herein. Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD) which is used to evaluate and rank the strength of bimolecular interactions. The binding affinity and dissociation constants can be measured quantitatively. Methods for determining binding affinities are well known to the skilled person and can be selected, for instance, from the following methods that are well established in the art: surface plasmon resonance (SPR), Bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), kinetic exclusion analysis (KinExA assay), flow cytometry, fluorescence spectroscopy techniques, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Typically, the dissociation constant KD is determined at temperatures in the range of 20°C and 30°C. If not specifically indicated otherwise, KD values recited herein are determined at 25°C by SPR. In various embodiments of the present invention, the binding affinity for scFv specific for albumin may be determined by the Sierra SPR-32 system (Bruker).

[0063] As described elsewhere herein, purification methods with the affinity ligands of the invention can be performed at mild pH, in particular mild acidic to neutral pH, to avoid aggregation of fusion protein comprising scFv specific for albumin. As disclosed elsewhere herein, mild pH in the context of the present invention, i.e., in the context of affinity purification I chromatography, specifically means a pH (at or) higher than 4.5, for example, at pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, or pH 6.7. Accordingly, affinity ligands of the present invention, comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with at least 90 % identity to SEQ ID NO: 1 or SEQ ID NO: 3, respectively, allow (or enable) the elution of an scFv specific for albumin, or a fusion protein comprising an scFv specific for albumin, at mild pH, in particular mild acidic to neutral pH, which means a pH (at or) higher than 4.5, more specifically (at or) higher than pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, or pH 6.7. In particular, the elution means an elution in affinity purification or affinity chromatography of an scFv specific for albumin or a fusion protein comprising an scFv specific for albumin using an affinity separation matrix as described elsewhere herein, more specifically an elution in affinity purification or affinity chromatography using an affinity separation matrix as described elsewhere herein, or an elution in affinity purification using a chromatography matrix as described elsewhere herein. As described herein, the terms “allow the elution” or “enable the elution” or “is capable of eluting” may be used interchangeably herein.

[0064] Multimers. Some embodiments refer to an affinity ligand for an scFv specific for albumin, wherein the affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with at least 90 % sequence identity thereto, wherein the affinity ligand is a multimer. In one embodiment of the invention, the affinity ligand for scFv specific for albumin comprises one or more affinity ligands of SEQ ID NO: 1 or SEQ ID NO: 3 linked to each other. In one embodiment of the invention, the affinity ligand for scFv specific for albumin comprises one affinity ligand or two affinity ligands linked to each other. In some embodiments, the affinity ligand comprises two affinity ligands of SEQ ID NO: 1 directly linked (for example, the dimer of SEQ ID NO: 1 is shown in SEQ ID NO: 2). In some embodiments, the affinity ligand comprises two affinity ligands of SEQ ID NO: 3 directly linked (for example, the dimer of SEQ ID NO: 1 is shown in SEQ ID NO: 4). Multimers, e.g. dimers, of the affinity ligand are generated artificially, generally by recombinant DNA technology well-known to a skilled person.

[0065] Molecules for purification or detection. In some embodiments, the affinity ligand for scFv specific for albumin may also comprise additional amino acid residues at the N- and / or C-terminal end, such as for example an additional sequence at the N- and / or C-terminal end. Additional sequences may include for example sequences introduced e.g. for purification or detection. Typical examples for such sequences include, without being limiting, Strep-tags, oligohistidine- tags, glutathione S-transferase, maltose-binding protein, inteins, intein fragments, or the albuminbinding domain of protein G, or others. In one embodiment, additional amino acid sequences include one or more peptide sequences that confer an affinity to certain chromatography column materials. The affinity ligand for scFv specific for albumin may include specific attachment sites for the attachment to solid supports, preferably at the C-terminal end, such as cysteine or lysine. Affinity separation matrix. One embodiment of the invention refers to an affinity separation matrix comprising the affinity ligand for the scFv specific for albumin, wherein the affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with at least 90 % sequence identity thereto.

[0066] In preferred embodiments, the affinity separation matrix is a solid support. The affinity separation matrix comprises at least one affinity ligand for scFv specific for albumin as described above.

[0067] Solid support matrices for affinity chromatography are known in the art and include, e.g., without being limited thereto, agarose and stabilized derivatives of agarose, cellulose or derivatives of cellulose, controlled pore glass, monolith, silica, zirconium oxide, titanium oxide, or synthetic polymers, and hydrogels of various compositions and combinations of the above.

[0068] The formats for solid support matrices can be of any suitable well-known kind. Such solid support matrix for coupling a novel affinity ligand of the present invention might comprise, e.g., one of the following, without being limited thereto: columns, capillaries, particles, membranes, filters, monoliths, fibers, pads, gels, slides, plates, cassettes, or any other format commonly used in chromatography and known to someone skilled in the art.

[0069] In one embodiment, the matrix is comprised of substantially spherical beads, for example Sepharose or Agarose beads. Matrices in particle form can be used as a packed bed or in a suspended form including expanded beds. In other embodiments of the invention, the solid support matrix is a membrane, for example a hydrogel membrane. In some embodiments, the affinity purification may involve a membrane as a matrix to which the affinity ligand for scFv specific for albumin as described herein is covalently bound. The solid support can also be in the form of a membrane in a cartridge.

[0070] Use in affinity purification. Some embodiments relate to the use of the novel affinity ligand for scFv specific for albumin as disclosed herein, for the affinity purification of fusion proteins comprising scFv specific for albumin. Accordingly, the novel affinity ligand as disclosed herein is encompassed for use in the affinity purification of a fusion protein comprising scFv specific for albumin.

[0071] Some embodiments relate to the use of the affinity separation matrix comprising the novel affinity ligand for scFv specific for albumin as disclosed herein, for the affinity purification of fusion protein comprising scFv specific for albumins. Accordingly, the affinity separation matrix comprising affinity ligand as disclosed herein is encompassed for use in the affinity purification of a fusion protein comprising scFv specific for albumin.

[0072] Method for affinity purification of fusion protein comprising scFv. As described herein, affinity chromatography (also called affinity purification) makes use of specific binding interactions between molecules. Methods for immobilization of protein and methods for affinity chromatography are well-known in the field of protein purification and can be easily performed by a skilled person in this field using standard techniques and equipment.

[0073] Some embodiments refer to a method for purification of a fusion protein comprising scFv specific for albumin, the method comprising: (i) contacting a liquid containing a fusion protein comprising scFv specific for albumin with the affinity ligand as described herein, or the affinity separation matrix as described above; (ii) purifying the fusion protein comprising scFv specific for albumin; and (iii) obtaining the fusion protein.

[0074] Some embodiments refer to a method for purification of fusion protein comprising scFv specific for albumin, the method comprising: (i) providing a liquid that contains an fusion protein comprising scFv specific for albumin; (ii) providing an affinity separation matrix comprising at least one affinity ligand as described above coupled to said affinity separation matrix; (iii) contacting said affinity separation matrix of (ii) with the liquid of (i); (iv) purifying said fusion protein comprising scFv specific for albumin from said affinity separation matrix, and (v) obtaining the fusion protein.

[0075] Some embodiments refer to a method of purification as described above, the method comprising eluting the fusion protein comprising scFv specific for albumin from the affinity separation matrix at pH higher than 4.5.

[0076] Various embodiments refer to a method of purification as described above, the method comprising eluting the fusion protein comprising scFv specific for albumin from the affinity separation matrix at pH higher than 4.5, for example, at pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, or pH 6.7. By eluting the fusion protein at mild acidic to neutral pH, aggregation of the fusion protein is prevented.

[0077] In various embodiments, the method of affinity purification may further comprise one or more washing steps.

[0078] In various embodiments, the method of affinity purification may further comprise one or more steps for isolating the purified fusion protein comprising scFv specific for albumin via methods that are known to a person skilled in the art.

[0079] In some embodiments, the method of purification involves a chromatography column containing a solid support (i.e. affinity separation matrix) to which the affinity ligand as described herein is covalently bound. The affinity ligand as described above may be attached to a suitable solid support via conventional coupling techniques. Methods for immobilization of protein ligands to solid supports are well-known in the field of protein engineering and purification and can easily be performed by a skilled person in this field using standard techniques and equipment.

[0080] In various embodiments of the present invention, the affinity ligands disclosed herein generated or obtained by any of the methods as described above are conjugated to a solid support. In some embodiments, the affinity ligand comprises an attachment site for site-specific covalent coupling of affinity ligand to a solid support. Specific attachment sites comprise without being limited thereto, natural amino acids, such as cysteine or lysine, which enable specific chemical reactions with a reactive group of the solid phase, or a linker between the solid phase and the protein.

[0081] Method for producing a fusion protein. Some embodiments refer to a method for producing a fusion protein comprising scFv specific for albumin, the method comprising: (i) providing a liquid containing a fusion protein comprising scFv specific for albumin; (ii) contacting the liquid of (i) with affinity ligand as described above, or contacting the liquid of (i) with the affinity separation matrix as described above; and (iii) purifying the fusion protein comprising scFv specific for albumin and thereby producing the fusion protein.

[0082] Various embodiments refer to a method for producing a fusion protein as described above, the method comprising eluting the fusion protein comprising scFv specific for albumin from the affinity separation matrix at pH higher than 4.5, for example, at pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, or pH 6.7. By eluting the fusion protein at mild acidic to neutral pH, aggregation of the fusion protein is prevented.

[0083] In various embodiments, the method of producing a fusion protein may further comprise one or more washing steps.

[0084] In one embodiment, a method of producing a fusion protein comprising scFv specific for albumin, the method comprising: (i) providing a nucleic acid molecule encoding the fusion protein; (ii) introducing said nucleic acid molecule into an expression vector; (iii) introducing said expression vector into a host cell; (iv) culturing the host cell in a culture medium; (v) subjecting the host cell to culturing conditions suitable for expression thereby producing the fusion protein; (vi) contacting the fusion protein with affinity ligand as described above, or contacting the fusion protein with the affinity separation matrix as described above; and (vii) purifying the fusion protein, optionally by eluting the fusion from the affinity separation matrix as described above under conditions of pH higher than 4.5; and thereby producing the fusion protein

[0085] Methods to determine the presence of scFv. In some embodiments refer to a use of the affinity ligand for scFv specific for albumin as described above or the affinity matrix as described above in methods to determine the presence of scFv specific for albumin.

[0086] Some embodiments, the affinity ligand for scFv specific for albumin as described herein is used in methods to determine the presence of a fusion protein comprising scFv specific for albumin. Some embodiments relate to a method of analyzing the presence of fusion protein comprising scFv specific for albumin in liquid samples, the method comprising the following steps: (i) providing a liquid that contains fusion protein comprising scFv specific for albumin, (ii) providing the affinity ligand, (iii) contacting the liquid that contains fusion protein comprising scFv specific for albumin with the affinity ligand as described herein under conditions that permit binding, (iv) isolating (eluting) the fusion protein comprising scFv specific for albumin, preferably at pH higher than 4.5, and (v) determining the amount of fusion protein comprising scFv specific for albumin.

[0087] Method of quantification of a fusion protein comprising scFv specific for albumin. Further embodiments relate to a method of quantification of an fusion protein comprising scFv specific for albumin, the method comprising: (i) providing a liquid that contains fusion protein comprising scFv specific for albumin; (ii) providing a matrix to which the affinity ligand as described herein has been covalently coupled; (iii) contacting said affinity separation matrix with the liquid under conditions that permit binding; (iv) eluting said fusion protein comprising scFv specific for albumin, preferably at pH higher than 4.5; and (v) quantitating the amount of eluted fusion protein comprising scFv specific for albumin. Methods to determine the presence of fusion protein comprising scFv specific for albumin in liquid samples might be quantitative or qualitative. Such methods are well known to the skilled person and can be selected, for instance but limited to, from the following methods that are well established in the art: enzyme-linked immunosorbent assay (ELISA), enzymatic reactions, surface plasmon resonance (SPR), or chromatography. Polynucleotides, vectors, host cells. One embodiment covers an isolated polynucleotide or nucleic acid molecule encoding an affinity ligand for scFv specific for albumin as described herein. A further embodiment also encompasses proteins encoded by polynucleotides.

[0088] Further provided is a vector, in particular an expression vector, comprising the isolated polynucleotide or nucleic acid molecule for the affinity ligand as described herein, as well as a host cell comprising the isolated polynucleotide or the expression vector. For example, one or more polynucleotides, which encode the affinity ligand as disclosed herein may be expressed in a suitable host and the produced protein can be isolated. A vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used for transfer of proteinencoding information into a host cell. Suitable vectors that may be applied are known in the art.

[0089] Suitable host cells include prokaryotes or eukaryotes, for example a bacterial host cell, a yeast host cell or a non-human host cell carrying a vector. A suitable host may be a microbial expression host that is capable to express the protein of the invention. Suitable bacterial expression host cells or systems are known in the art. Various mammalian or insect cell culture systems as known in the art can also be employed to express recombinant proteins.

[0090] Furthermore, an isolated cell comprising a polynucleotide or nucleic acid, or a vector as disclosed herein is provided.

[0091] Method of producing the affinity ligand of the invention. In a further embodiment, a method for the production of the affinity ligand as described is provided, the method comprising the step(s): (i) culturing a (suitable) host cell under conditions suitable for the expression of the affinity ligand so as to obtain said affinity ligand; and (ii) optionally isolating said affinity ligand. Suitable conditions for culturing a prokaryotic or eukaryotic host are well known to a person skilled in the art.

[0092] The affinity ligand as described above may be prepared by any conventional and well-known techniques such as plain organic synthetic strategies, solid phase-assisted synthesis techniques, or by commercially available automated synthesizers. They may also be prepared by conventional recombinant techniques, alone or in combination with conventional synthetic techniques.

[0093] In one embodiment, a method for the preparation of the affinity ligand for scFv specific for albumin is provided, as detailed above, said method comprising the steps: (i) providing a nucleic acid molecule encoding the affinity ligand; (ii) introducing said nucleic acid molecule into an expression vector; (iii) introducing said expression vector into a host cell; (iv) culturing the host cell in a culture medium; (v) subjecting the host cell to culturing conditions suitable for expression thereby producing the affinity ligand; optionally (vi) isolating the polypeptide produced in step (v); and (vii) optionally conjugating the affinity ligand to a solid matrix as described above. In various embodiments of the present invention the production of the affinity ligand for scFv specific for albumin is performed by cell-free in vitro transcription and translation. EXAMPLES

[0094] The following Examples are provided for further illustration of the invention. The invention, however, is not limited thereto, and the following Examples merely show the practicability of the invention on the basis of the above description. For a complete disclosure of the invention reference is made also to the literature cited in the application which is incorporated completely into the application by reference.

[0095] EXAMPLE 1. Expression and purification of affinity ligands

[0096] The untagged proteins were purified by using an AKTA avant system (Cytiva). The initial capturing step was performed by using a Q Sepharose anion exchange chromatography column (Cytiva; binding buffer: 20 mM BisTris, 1 mM EDTA pH 6.5; elution buffer: 20 mM BisTris, 1 mM EDTA, 1 M NaCI pH 6.5). Afterwards the pooled fractions were incubated with a final concentration of 1 M (NH4)2SO4 followed by a hydrophobic interaction chromatography (Phenyl Sepharose HP, Cytiva; binding buffer: 20 mM BisTris, 1 mM EDTA, 1 M (NH4)2SO4 pH 6.5; elution buffer: 20 mM BisTris,

[0097] I mM EDTA pH 6.5). For polishing of the eluted fractions a size exclusion chromatography was performed (Sephacryl S-200HR, Cytiva) in 20 mM citric acid, 150 mM NaCI, 1 mM EDTA, 5 mM TCEP pH 6.0. Protein concentrations were determined by absorbance measurement at 280 nm using the molar absorbent coefficient. Further analysis included RP-HPLC and SE-HPLC. Reversed phase chromatography (RP-HPLC) has been performed using an Agilent 1290 Infinity

[0098] II System and a PLRP-S (5 pm, 300 A) column (Agilent). The resulting purity of all proteins was >95 %. Analytic size exclusion chromatography (SE-HPLC) has been performed using an Agilent 1290 Infinity II System and a Superdex75 increase 5 / 150 GL (Cytiva). No aggregation or oligomers were obtained.

[0099] EXAMPLE 2. Binding analysis of affinity ligands by SPR

[0100] The purified proteins were immobilized on a High Capacity Amine sensor chip (Bruker) using PDEA after NHS / EDC activation with Sierra SPR-32 system (Bruker). The chip was equilibrated with SPR running buffer (PBS 0.05 % Tween pH 7.3). Upon binding, target analyte was accumulated on the surface increasing the refractive index. This change in the refractive index was measured in real time and plotted as response or resonance units versus time. The analyte (scFv specific for albumin) was applied to the chip in serial dilutions with a flow rate of 30 pl / min. The association was performed for 180 seconds and the dissociation for 240 seconds. After each run, the chip surface was regenerated with 30 pl regeneration buffer (10 mM Glycin pH 2.0) and equilibrated with running buffer. Binding studies were carried out using Sierra SPR-32 system (Bruker); data evaluation was operated via Sierra Analyser software, provided by the manufacturer, using the Langmuir 1 :1 model (RMin=0). Evaluated dissociation constants (KD) were standardized against the immobilized protein and indicated. Shown is the change in refractive index measured in real time and plotted as response or resonance unit [RU] versus time [sec]. Ligands bind to scFv specific for human serum albumin (scFv HSA; SEQ ID NO: 5) with a KD below 10 nM. As control, ligands with similar scaffold but different target specificity (for example, immunoglobulin or SARS Cov-2) do not bind to scFv HSA.

[0101] Table 1. Binding affinity of ligands to scFv specific for serum albumin

[0102] EXAMPLE 3. Purification of fusion protein comprising scFv HSA via affinity ligands

[0103] High coupling density to resin. Affinity ligands were purified to homogeneity and immobilized at 20 mg per mL activated Praesto Epoxy Jetted 50 (Purolite) according to the manufacturer’s instructions, coupling conditions: 35 °C for 3 h, pH 9.5 per mL resin. All ligands were successfully coupled to epoxy activated Praesto Jetted 50 resin. The coupling density was about 15-18 mg / ml for coupled ligands.

[0104] Elution of fusion protein comprising scFv specific for albumin at high pH above 4.5.

[0105] Resin with coupled 227873 or 227875 was loaded with 0.2 mg / ml target (HSA scFv) in solution (liquid) that contained scFv HSA or a fusion protein comprising scFv HSA and an interleukin (IL12- scFv HSA or IL12-HSA scFv-IL15) with contact time of 6 minutes. A wash step with 1xPBS, 0.02% Tween at pH 7.3 removed unbound sample. The protein of interest was eluted from the resin with different elution steps:

[0106] (1) 100 mM citrate, 0.02% Tween 20, 1 mM EDTA pH 6.0;

[0107] (2) 100 mM citrate, 0.02% Tween 20, 1 mM EDTA pH 5.5;

[0108] (3) 100 mM citrate, 0.02% Tween 20, 1 mM EDTA pH 5.0;

[0109] (4) 100 mM citrate, 0.02% Tween 20, 1 mM EDTA pH 4.5.

[0110] The elution of scFv HSA or fusion protein comprising scFv HSA and interleukin started at pH 5.0. The scFv HSA or fusion protein comprising scFv HSA and interleukin was eluted completely at pH 4.5.

[0111] In a different experiment, resin with coupled affinity ligand 227873 or 227875 was loaded with 3 mL of a solution with a fusion protein comprising scFv HSA and interleukin (IL12-scFv HSA, IL12- scFv HSA-IL15, or IL18-scFv HSA-IL12) with contact time of 6 minutes. A wash step with 100 mM citrate, 0.02% Tween 20, 1 mM EDTA pH 6.7 was included to remove unbound sample. The bound sample was eluted with 100 mM Citrate, 0.02% Tween 20, 1 M L-Arginine, 1 mM EDTA at pH 6.7 followed by a gradient elution with 100 mM Citrate, 0.02% Tween 20, 1 M L-Arginine, 1 mM EDTA at pH 6.7 to 5.0. The fusion protein comprising scFv HSA could be purified with all affinity ligands at pH 6.7. Eluted fusion protein comprising scFv HSA showed high purity on SDS- PAGE. A strong enrichment of fusion protein comprising scFv HSA compared to the original load was observed.

[0112] DBC10%. Coupled resin was packed into super compact 5 / 50 column (Gdtec GmbH). Fusion protein comprising scFv HSA was used as target (cone. 1.5 mg / ml; in 1x PBS, 0.02% Tween20, pH 7.3). Target sample was applied to the matrix comprising immobilized affinity ligand until 10 % target breakthrough at 6 min residence time. Unbound sample was washed with 1xPBS, pH 7.3. Loaded target was quantified and calculated as dynamic binding capacity DBC10 %. Results: The binding capacity (DBC10) for affinity ligand 227873 was 15.7 mg / ml and for 227875 11.6 mg / ml. Caustic stability reflects the remaining binding capacity (in %) compared to the binding capacity at 0 h.

[0113] Caustic stability. Columns were incubated with 0.1 M NaOH for 12.5 h at room temperature (22 °C + / - 3 °C); 2 sets were measured. DBC10 was measured after incubation with 0.1 M NaOH and compared to DBC10 values without NaOH incubation. Results: After 12.5 h in 0.1 M NaOH, 227873 showed 90.7% and 227875 96.6% remaining target binding capacity (DBC10 in %).

[0114] EXAMPLE 4. Validation of purified fusion protein comprising HSA scFv

[0115] Purified fusion protein comprising scFv HSA and cytokine (IL-12, IL-15, and / or IL-18) was analyzed for homogeneity using size exclusion chromatography (SE-HPLC). Analysis has been performed on a Agilent 1290 Infinity II HPLC system and a Superdex200 increase 5 / 150 GL (Cytiva). No aggregation of eluted fusion protein comprising scFv HSA and cytokine IL-12, IL-15, and / or IL- 18 was detected.

[0116] EXAMPLE 5. Ligand detection in Protein A ELISA (leaching assay)

[0117] To determine low levels of leached variants in affinity chromatography is important for obtaining reliable results. Protein A ELISA Kits for the detection of native and recombinant Protein A (Repligen, Cat. No. 9000-1) were used for leaching assays according to manufacturer’s instructions, except using 0.1 % PBST as dilution buffer. Samples: eluted fraction of Praesto Jetted Epoxy50_227873 (SEQ ID NO: 2 immobilized to Praesto Jetted Epoxy50). The leaching of the affinity ligand was 4.5 ng per mg fusion protein comprising scFv HSA (IL12-scFv HSA-IL15) and showed good detectability in PBST buffer, comparable to rProtein A standard. SEQUENCES

[0118] SEQ ID NO: 1: Affinity ligand for scFv specific for albumin (227873)

[0119] AKFDEAQQAAFYEILHLPNLTEEQRNQFIQWLRDDPSVSLEVLGHAQYLNDSQARGEVNYLEK

[0120] FDEAQQEAFYRILHLYNLTEEQRNAFIQSLRDDPSVSREVLGEALKLNASQAPK

[0121] SEQ ID NO: 2: Affinity ligand for scFv specific for albumin (dimer of SEQ ID NO: 1 , 227873)

[0122] AKFDEAQQAAFYEILHLPNLTEEQRNQFIQWLRDDPSVSLEVLGHAQYLNDSQARGEVNYLEK

[0123] FDEAQQEAFYRILHLYNLTEEQRNAFIQSLRDDPSVSREVLGEALKLNASQAPKAKFDEAQQAA

[0124] FYEILHLPNLTEEQRNQFIQWLRDDPSVSLEVLGHAQYLNDSQARGEVNYLEKFDEAQQEAFY

[0125] RILHLYNLTEEQRNAFIQSLRDDPSVSREVLGEALKLNASQAPK

[0126] SEQ ID NO: 3: Affinity ligand for scFv specific for albumin (227875)

[0127] AKFDEAQQAAFYEILHLPNLTEQQRNQFIQWLRDDPSVSLQVLGHAQHLNDSQATKSLSTIEKF

[0128] DEAQQEAFYVILHLINLTEEQRNAFIQSLRDDPSVSWEVLGEAKKLNASQAPK

[0129] SEQ ID NO: 4: Affinity ligand for scFv specific for albumin (dimer of SEQ ID NO: 3, 227875)

[0130] AKFDEAQQAAFYEILHLPNLTEQQRNQFIQWLRDDPSVSLQVLGHAQHLNDSQATKSLSTIEKF

[0131] DEAQQEAFYVILHLINLTEEQRNAFIQSLRDDPSVSWEVLGEAKKLNASQAPKAKFDEAQQAAF

[0132] YEILHLPNLTEQQRNQFIQWLRDDPSVSLQVLGHAQHLNDSQATKSLSTIEKFDEAQQEAFYVIL

[0133] HLINLTEEQRNAFIQSLRDDPSVSWEVLGEAKKLNASQAPK

[0134] SEQ ID NO: 5: scFv specific for albumin

[0135] EVQLVESGGGLIQPGRSLRLSCAASGITFDDAVMHWVRQAPGKGLEWVAGISSNSGYIGYADS

[0136] VKGRFTISRDNAKNSLYLQMNRLRAEDTAVYYCVKGLYSNPRGGAFDIWGQGTMVTVSSAST

[0137] GGGGSGGGGSGGGGSVHSSYVLTQPPSVSVAPGQTATITCGGNNIGTKSVHWYQQKPGQAP

[0138] VLVVYADSDRPSGIPERVSGSNSGNTATLTISRVEAGDEADYYCQVWDSRSDHLWVFGGGTK

[0139] LTVLGGLNDIFEAQKIEWHE

Claims

Claims1. An affinity ligand for a single chain antibody fragment (scFv) specific for albumin, wherein the affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with at least 90 % sequence identity thereto, wherein the affinity ligand has a binding affinity of less than 200 nM for the scFv.

2. The affinity ligand according to claim 1 , wherein the affinity ligand is a multimer.

3. The affinity ligand according to claim 1 or 2, wherein the albumin is a human serum albumin.

4. An affinity separation matrix comprising the affinity ligand according to any one of claims 1-3.

5. Use of the affinity ligand according to any one of claims 1-3, or the affinity separation matrix according to claim 4, for affinity purification.

6. The use of claim 5, which is for affinity purification of a fusion protein comprising a scFv specific for albumin.

7. A method of purification of a fusion protein comprising a scFv specific for albumin, the method comprising:(i) contacting a liquid containing a fusion protein comprising the scFv specific for albumin with affinity ligand according to any one of claims 1-3, or the affinity matrix of claim 4;(ii) purifying the fusion protein comprising the scFv specific for albumin; and(iii) obtaining the fusion protein.

8. The method of purification of claim 7, the method comprising:(i) providing a liquid that contains a fusion protein comprising scFv specific for albumin;(ii) providing an affinity separation matrix of claim 4;(iii) contacting said affinity separation matrix with the liquid;(iv) purifying said fusion protein comprising scFv specific for albumin from the affinity separation matrix; and(v) obtaining the fusion protein.

9. The method of purification of claim 7 or 8, the method comprising eluting the fusion protein comprising scFv specific for albumin from the affinity separation matrix at pH higher than 4.5.

10. A method for producing a fusion protein that comprises scFv specific for albumin, the method comprising:(i) providing a liquid containing a fusion protein that comprises scFv specific for albumin;(ii) contacting the liquid of (i) with affinity ligand according to any one of claims 1-3, or the affinity matrix of claim 4; and(iii) purifying the fusion protein that comprises scFv specific for albumin and thereby producing the fusion protein.

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