HK1189386B - Designed repeat proteins binding to serum albumin - Google Patents

Description

Designed repeat proteins that bind to serum albumin

Technical Field

The present invention relates to designed repeat proteins having binding specificity for serum albumin, as well as nucleic acids encoding such serum albumin binding proteins, pharmaceutical compositions comprising such proteins, the use of such proteins for altering the pharmacokinetics of biologically active compounds, and the use of such proteins in the treatment of diseases.

Background Art

The pharmaceutical industry is eager to increase the effectiveness of bioactive compounds (such as protein therapeutics) by regulating or increasing their in vivo pharmacokinetic (PK) properties. This is especially true for bioactive compounds that are rapidly eliminated from the circulation by renal clearance. The kidneys typically filter out molecules with an apparent molecular weight lower than 60 kDa from the circulation. One strategy for improving the pharmacokinetic properties of such small bioactive compounds is to simply increase their apparent molecular size (i.e., increase their hydrodynamic radius), for example, by adding a non-proteinaceous polymer portion such as a polyethylene glycol polymer or a sugar residue, or adding a proteinaceous polymer portion such as a globular protein or an unstructured polypeptide, such as those described in WO 2007/103515 and WO 2008/155134.

Other strategies exploit the long circulatory half-life of serum proteins, such as immunoglobulins and serum albumin. Serum albumin, with a molecular weight of 67 kDa, is the most abundant protein in plasma, present at approximately 50 mg/ml (0.6 mM) and having a serum half-life of 19 days in humans. Serum albumin helps maintain plasma pH, contributes to colloidal blood pressure, acts as a carrier for many metabolites and fatty acids, and serves as the primary drug transporter in plasma. Several major small molecule binding sites have been described in albumin.

It has been demonstrated that non-covalent binding to serum albumin can extend the half-life of short-lived small molecules or polypeptides (WO 1991/001743). Polypeptides that specifically bind to serum albumin and can thereby extend the in vivo half-life of other molecules conjugated thereto include variants of bacterial albumin binding domains (e.g., WO 2005/097202 and WO 2009/016043), small peptides (e.g., Dennis, MS, et al., J. Biol. Chem. 277(3) , 35035-43, 2002 and WO 2001/045746) and immunoglobulin fragments (e.g., WO 2008/043822, WO 2004/003019, WO 2008/043821, WO 2006/040153, WO 2006/122787 and WO 2004/041865). WO 2008/043822 relates to binding proteins other than immunoglobulin fragments, such as molecules based on protein A domains, tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, and PDZ domains, which may be prepared to specifically bind to serum albumin. Nevertheless, WO 2008/043822 does not disclose the selection of designed ankyrin repeat domains with binding specificity for serum albumin (SA), nor does it disclose the specific repeat sequence motifs of the repeat domains that specifically bind to SA. Furthermore, it is claimed that the in vivo half-life of polypeptides can be extended by genetically fusing them to serum albumin (e.g., WO 1991/001743). Such alterations in the in vivo half-life of drugs may positively alter their pharmacokinetic (PK) and/or pharmacodynamic (PD) properties. This is a key issue in the development of new and effective therapeutic agents and disease treatment methods. Therefore, there is a need in the art for new approaches to alter the PK and/or PD of bioactive compounds.

In addition to antibodies, novel binding proteins or binding domains can also be used to specifically bind to target molecules (e.g., Binz, HK, Amstutz, P. and Plückthun, A., Nat. Biotechnol. 23 , 1257-1268, 2005). One such novel binding protein or binding domain is based on designed repeat proteins or designed repeat domains (WO 2002/020565; Binz, HK, Amstutz, P., Kohl, A., Stumpp, MT, Briand, C., Forrer, P., Grütter, MG, and Plückthun, A., Nat. Biotechnol. 22 , 575-582, 2004; Stumpp, MT, Binz, HK and Amstutz, P., Drug Discov. Today 13 , 695-701, 2008). WO 2002/020565 describes how to construct a large library of repeat proteins and their general use. Nevertheless, WO 2002/020565 neither discloses the selection of repeat domains with binding specificity to SA, nor discloses the specific repetitive sequence motif of the repeat domains specifically combined with SA. In addition, WO 2002/020565 does not prompt that the repeat domains with binding specificity to SA can be used to regulate the PK or PD of other molecules. The repeat domains of these designs utilize the modular properties of repeat proteins and have N-terminal and C-terminal capping modules to prevent the repeat domains of design from gathering by shielding the hydrophobic core of the domain (Forrer, P., Stumpp, MT, Binz, HK and Plückthun, A., FEBS letters 539 , 2-6, 2003). These capping modules are based on the capping repetitive sequences of natural guanine-adenine-binding proteins (GA-binding proteins). It has been shown that the thermal and thermodynamic stability of these designed ankyrin repeat domains can be further increased by modifying the C-terminal capping repeat sequence derived from GA-binding proteins (Interlandi, G., Wetzel, SK, Settanni, G., Plückthun, A., and Caflisch, A., J. Mol. Biol. 375 , 837-854, 2008; Kramer, MA, Wetzel, SK, Plückthun, A., Mittl, PRE, and Grütter, MG, J. Mol. Biol. 404 , 381-391, 2010). The authors introduced a total of eight mutations into the capping module and extended its C-terminal helix by adding three unique amino acids. Nevertheless, the introduction of these modifications into the C-terminal capping module resulted in an undesirable tendency for the designed repeat domains carrying this mutated C-terminal capping module to dimerize. Therefore, there is a need to prepare further optimized C-terminal capping modules or C-terminal capping repeat sequences of ankyrin repeat domains.

Targeting SA to modulate PK and/or PD using currently available approaches is not always effective. It is even becoming increasingly apparent that modulating the PK and/or PD of molecules by hijacking SA is complex and still not fully understood.

In summary, there is a need for improved binding proteins specific for SA that can improve the PK and/or PD of therapeutically relevant molecules or polypeptides for the treatment of cancer and other pathological conditions.

The technical problem addressed by the present invention is to identify novel binding proteins (such as repeat domains with binding specificity for SA) that are capable of altering the PK and/or PD of therapeutically relevant molecules for improved treatment of cancer and other pathological conditions. This technical problem is addressed by providing the embodiments characterized in the claims.

SUMMARY OF THE INVENTION

The present invention relates to a binding protein comprising at least one ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for mammalian serum albumin, and wherein the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51 and 52, and sequences wherein up to 9 amino acids in SEQ ID NOs: 49, 50, 51 and 52 are replaced by any amino acid.

In another embodiment, the invention relates to a binding protein comprising at least one ankyrin repeat domain, wherein the repeat domain has binding specificity for mammalian serum albumin, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least 70% amino acid sequence identity to an ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 17-31 and 43-48, wherein the G at position 1 and/or the S at position 2 of the ankyrin repeat domain is optionally deleted.

In particular, the present invention relates to a binding protein as defined above, wherein the ankyrin repeat domain competes for binding to mammalian serum albumin with an ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 17-31 and 43-48.

Furthermore, the present invention relates to such binding proteins comprising a biologically active compound, in particular binding proteins comprising a biologically active compound having a terminal plasma half-life in a mammal that is at least 2 times higher than the terminal plasma half-life of the unmodified biologically active compound.

The present invention further relates to nucleic acid molecules encoding the binding proteins of the present invention, and pharmaceutical compositions comprising one or more of the above-mentioned binding proteins or nucleic acid molecules.

The present invention further relates to methods of treating pathological conditions using the binding proteins of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Stability analysis of selected DARPins by SEC.

Elution profiles of size exclusion chromatography (SEC) runs of DARPins specific for xSA, analyzed using a Superdex 200 column 5/150 (Figure 1a or 1b) or Superdex 200 10/300GL (Figure 1c), before incubation (Figure 1a), after incubation at 40°C for 28 days at 30 mg/ml (approximately 2 mM) in PBS (Figure 1b), or after storage at -80°C for one month (Figure 1c). All samples were expressed and purified as described in Example 1. For SEC analysis, samples were diluted to a concentration of 500 µM. Molecular mass standards aprotinin (AP) 6.5 kDa, carbonic anhydrase (CA) 29 kDa, and conalbumin (CO) 75 kDa are indicated by arrows.

, mammalian serum albumin, A, absorbance at 280 nm; t, retention time in minutes;

DARPin #19 (SEQ ID NO: 19 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #20 (SEQ ID NO: 20 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #21 (SEQ ID NO: 21 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #22 (SEQ ID NO: 22 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #27 (SEQ ID NO: 27 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #28 (SEQ ID NO: 28 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #29 (SEQ ID NO: 29 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #30 (SEQ ID NO: 30 with a His-tag (SEQ ID NO: 15) fused at its N-terminus).

DARPin #43 (SEQ ID NO: 43 with a His-tag (SEQ ID NO: 15) fused at its N-terminus).

DARPin #44 (SEQ ID NO: 44 with a His-tag (SEQ ID NO: 15) fused at its N-terminus).

DARPin #45 (SEQ ID NO: 45 with a His-tag (SEQ ID NO: 15) fused at its N-terminus).

DARPin #46 (SEQ ID NO: 46 with a His-tag (SEQ ID NO: 15) fused at its N-terminus).

DARPin #47 (SEQ ID NO: 47 with a His-tag (SEQ ID NO: 15) fused at its N-terminus).

DARPin #48 (SEQ ID NO: 48 with a His-tag (SEQ ID NO: 15) fused at its N-terminus).

Figure 2. Thermal stability of selected DARPins.

Thermal denaturation traces (tracked by the increase in fluorescence intensity of the dye SYPRO Orange present in the buffer) of a DARPin specific for xSA in PBS at pH 7.4 ( FIG. 2 a ) and in MES buffer at pH 5.8 (250 mM (2-N-morpholino)-ethanesulfonic acid, pH 5.5), 150 mM NaCl mixed in a 1:4 (v/v) ratio with PBS at pH 7.4 and adjusted to pH 5.8) ( FIG. 2 b ).

, relative fluorescence units (RFU), excitation at 515-535 nm, detection at 560-580 nm; T, temperature in °C; see above for definition of DARPin.

Figure 3. Plasma clearance of selected DARPins in mice.

The plasma clearance of DARPins specific for MSA (mouse serum albumin) and a control DARPin was assessed in mice.

( FIG. 3 a ) DARPin comprising only one repeat domain with binding specificity for MSA, compared to DARPin #32 (see below), which has no binding specificity for MSA.

( FIG. 3 b ) A DARPin comprising two protein domains (one of which is a repeat domain with binding specificity for MSA) is shown in comparison to DARPin #32, which has no binding specificity for MSA.

DARPin was labeled with a ^99m Tc-carbonyl compound via a His-Tag and injected intravenously into mice. Radioactivity in the blood of the injected mice was measured at various time points after injection and displayed as the ratio of the injected dose (%ID) corrected for the radioactive decay of ^99m Tc. The fitted curve shows the results of a nonlinear regression of the radioactivity measured at various time points—a two-stage decay (Graphpad Prism). Each data point represents the mean of two mice per group.

, percentage of injected dose corrected for radioactive decay of ^99m Tc; t, time in hours;

DARPin #18 (SEQ ID NO: 18 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #23 (SEQ ID NO: 23 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #25 (SEQ ID NO: 25 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #32 (negative control DARPin with no binding specificity for xSA, SEQ ID NO: 32 having a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #33 (a DARPin comprising two repeat domains, one of which has binding specificity for xSA, SEQ ID NO: 33 having a His-tag (SEQ ID NO: 15) fused to its N-terminus);

DARPin #35 (a DARPin comprising two repeat domains, one of which has binding specificity for xSA, SEQ ID NO: 35 having a His-tag (SEQ ID NO: 15) fused to its N-terminus);

DARPin #36 (DARPin comprising two repeat domains, one of which has binding specificity for xSA, SEQ ID NO: 36 having a His-tag (SEQ ID NO: 15) fused to its N-terminus).

Figure 4. Plasma clearance of selected DARPins in cynomolgus monkeys.

The plasma clearance of DARPins specific for CSA (cynomolgus serum albumin) and a control DARPin was assessed in cynomolgus monkeys.

( FIG. 4 a ) DARPin #26 was compared with DARPin #32, which has no binding specificity for CSA.

(Figure 4b) DARPins #24, 34, and 17 were compared with DARPin #32, which has no binding specificity for CSA. At t = 0 hours, the following DARPins were intravenously injected into cynomolgus monkeys at a concentration of 0.5 mg/ml (DARPin #26, DARPin #24, DARPin #17, and DARPin #32) or 1 mg/ml (DARPin #34). At various time points after injection, DARPin concentrations in monkey plasma were measured by ELISA. The curves show the results of a nonlinear regression of the concentrations measured at different time points—a two-stage decay (Graphpad Prism). From the second stage, the terminal plasma half-life of the DARPin was determined. Each individual data point represents the average of two independent ELISA measurements from the same serum sample.

, DARPin concentration in nM; t, time in hours;

DARPin #17 (SEQ ID NO: 17 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #24 (SEQ ID NO: 24 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #26 (SEQ ID NO: 26 with a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #32 (negative control DARPin with no binding specificity for xSA, SEQ ID NO: 32 having a His-tag (SEQ ID NO: 15) fused at its N-terminus);

DARPin #34 (DARPin comprising two repeat domains, one of which has binding specificity for xSA, SEQ ID NO: 34 having a His-tag (SEQ ID NO: 15) fused to its N-terminus).

DETAILED DESCRIPTION

The binding domain according to the present invention is specific for mammalian serum albumin (xSA). Preferably, the binding domain according to the present invention is specific for serum albumin of mouse, rat, dog, rabbit, monkey or human origin. More preferably, the binding domain according to the present invention is specific for serum albumin of human origin (HSA).

The term "protein" refers to a polypeptide, wherein at least a portion of the polypeptide has or is capable of achieving a defined three-dimensional arrangement by forming secondary, tertiary, or quaternary structures within and/or between its polypeptide chains. If the protein comprises two or more polypeptides, each polypeptide chain may be non-covalently linked or covalently linked, for example, by disulfide bonds between the two polypeptides. Portions of a protein that each have or are capable of achieving a defined three-dimensional arrangement by forming secondary or tertiary structures are referred to as "protein domains." Such protein domains are well known to those skilled in the art.

The term "recombinant" as used in connection with recombinant proteins, recombinant protein domains, recombinant binding proteins, and the like, means that the polypeptide is produced using recombinant DNA techniques well known to those skilled in the relevant art. For example, a recombinant DNA molecule encoding a polypeptide (e.g., prepared by gene synthesis) can be cloned into a bacterial expression plasmid (e.g., pQE30, Qiagen), a yeast expression plasmid, or a mammalian expression plasmid. When, for example, the recombinant bacterial expression plasmid constructed in this manner is inserted into an appropriate bacterium (e.g., Escherichia coli ), the bacterium can produce the polypeptide encoded by the recombinant DNA. The resulting polypeptide is referred to as a recombinant polypeptide.

In the context of the present invention, the term "polypeptide" refers to a molecule consisting of one or more chains of multiple (ie two or more) amino acids linked by peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked by peptide bonds.

The term "polypeptide tag" refers to an amino acid sequence attached to a polypeptide/protein, wherein the amino acid sequence can be used to purify, detect or target the polypeptide/protein, or wherein the amino acid sequence improves the physicochemical behavior of the polypeptide/protein, or wherein the amino acid sequence has an effector function. Individual polypeptide tags, parts and/or domains of a binding protein may be linked to each other directly or via a polypeptide linker. Such polypeptide tags are well known in the art and are fully available to those skilled in the art. Examples of polypeptide tags are small polypeptide sequences that allow detection of the polypeptide/protein, such as His (e.g., the His-tag of SEQ ID NO: 15), myc, FLAG or Strep-tags, or parts such as enzymes (e.g., enzymes such as alkaline phosphatase), or that can be used for targeting (such as immunoglobulins or fragments thereof) and/or as part of an effector molecule.

The term "polypeptide linker" refers to an amino acid sequence that is capable of connecting, for example, two protein domains, a polypeptide tag and a protein domain, a protein domain and a non-polypeptide moiety such as polyethylene glycol, or two sequence tags. Such additional domains, tags, non-polypeptide moieties, and linkers are known to those skilled in the relevant art. A list of examples is provided in the specification of patent application WO 2002/020565. Specific examples of such linkers are glycine-serine linkers and proline-threonine linkers of various lengths; preferably, the linkers have a length of 2 to 24 amino acids; more preferably, the linkers have a length of 2 to 16 amino acids. An example of a glycine-serine linker is provided in SEQ ID NO: 16.

The term "polymer portion" refers to a proteinaceous polymer portion or a non-proteinaceous polymer portion. A "proteinaceous polymer portion" is preferably a polypeptide that does not form a stable tertiary structure when stored in phosphate-buffered saline (PBS) at a concentration of about 0.1 mM at room temperature (RT) for one month, and does not form more than 10%, preferably no more than 5%; further preferably no more than 2%; even more preferably no more than 1%; and most preferably no detectable amount of oligomers or aggregates as determined by size exclusion chromatography (SEC). When globulins are used as molecular weight standards for SEC, such proteinaceous polymer portions are run in SEC with an apparent molecular weight higher than their effective molecular weight. Preferably, the apparent molecular weight of the proteinaceous polymer portion as determined by SEC is 1.5x, 2x, or 2.5x its effective molecular weight calculated using its amino acid sequence. Also preferably, the apparent molecular weight of the non-proteinaceous polymer portion as determined by SEC is 2x, 4x, or 8x its effective molecular weight calculated using its molecular composition. Preferably, the proteinaceous polymer portion has more than 50%, 70%, or even 90% of its amino acids that do not form a stable secondary structure at about 0.1 mM concentration in PBS at RT, as determined by circular dichroism (CD). Most preferably, the proteinaceous polymer exhibits a typical near-UV CD spectrum of a random coil conformation. Such CD analysis is well known to those skilled in the art. Also preferred are proteinaceous polymer portions consisting of more than 50, preferably more than 100, 200, 300, 400, 500, 600, 700, or most preferably more than 800 amino acids. Examples of proteinaceous polymer portions are XTEN® (a registered trademark of Amunix; WO 2007/103515) polypeptides, or polypeptides comprising proline, alanine, and serine residues, as described in WO 2008/155134. Such proteinaceous polymer portions can be covalently linked to, for example, a binding domain of the present invention by preparing a gene fusion polypeptide using standard DNA cloning techniques, followed by standard expression and purification.

The polymer moiety of the present invention can vary widely in molecular weight (i.e., from about 1 kDa to about 150 kDa). Preferably, the polymer moiety has a molecular weight of at least 2, more preferably at least 5, 10, 20, 30, 50, 70, or most preferably at least 100 kDa. Preferably, the polymer moiety is linked to the binding domain via a polypeptide linker.

Examples of non-proteinaceous polymer moieties are hydroxyethyl starch (HES), polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes.The term "PEGylated" means that a PEG moiety is covalently attached, eg to a polypeptide of the invention.

In a specific embodiment, a PEG moiety or any other non-proteinaceous polymer can be coupled to a cysteine thiol group, for example via a maleimide linker, and the cysteine is coupled to the N- or C-terminus of a binding domain described herein via a peptide linker.

The term "binding protein" refers to a protein comprising one or more binding domains, one or more biologically active compounds and one or more polymer parts, as further explained below. Preferably, the binding protein comprises at most four binding domains. More preferably, the binding protein comprises at most two binding domains. Most preferably, the binding protein comprises only one binding domain. Moreover, any such binding protein may comprise additional protein domains other than the binding domain, a multimerization portion, a polypeptide tag, a polypeptide linker and/or a single Cys residue. Examples of multimerization portions are immunoglobulin heavy chain constant regions (which pair to provide functional immunoglobulin Fc domains), and leucine zippers or polypeptides comprising free sulfhydryl groups that form intermolecular disulfide bonds between two such polypeptides. A single Cys residue can be used to conjugate other moieties to the polypeptide, for example by using maleimide chemistry well known to those skilled in the art. Preferably, the binding protein is a recombinant binding protein. Also preferably, the binding domains of the binding protein have different target specificities.

The term "binding domain" means a protein domain that exhibits the same "fold" (three-dimensional arrangement) as a protein scaffold and has a predetermined property as defined below. Such binding domains can be obtained by techniques known in the art using reasonable, or most commonly combined, protein engineering techniques (Binz et al., 2005, in the above-mentioned citations). For example, a binding domain with a predetermined property can be obtained by a method comprising the following steps: (a) providing a set of different protein domains that exhibit the same fold as a protein scaffold as further defined below; and (b) screening the different sets and/or selecting from the different sets to obtain at least one protein domain with the predetermined property. The different sets of protein domains can be provided by several methods depending on the screening and/or selection system used, and can include the use of methods well known to those skilled in the art, such as phage display or ribosome display. Preferably, the binding domain is a recombinant binding domain.

The term "protein scaffold" refers to a protein having exposed surface regions that are highly tolerant to amino acid insertions, substitutions, or deletions. Examples of protein scaffolds that can be used to generate the binding domains of the present invention are antibodies or fragments thereof, such as single-chain Fv or Fab fragments, protein A from Staphylococcus aureus , meta-billin-binding protein from Pieris brassicae or other lipocalins, ankyrin repeat proteins or other repeat proteins, and human fibronectin. Protein scaffolds are known to those skilled in the art (Binz et al., 2005, loc. cit.; Binz et al., 2004, loc. cit.).

The term "predetermined property" refers to properties such as binding to a target, blocking a target, activating a target-mediated reaction, enzymatic activity, and other related properties. Depending on the type of property desired, one of ordinary skill will be able to identify the format and necessary steps for screening and/or selecting binding domains having the desired property. Preferably, the predetermined property is binding to a target.

The following definition of repeat proteins is based on patent application WO 2002/020565. Patent application WO 2002/020565 further contains a general description of repeat protein characteristics, technology and applications.

The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, each of the repeat proteins comprises at most four repeat domains. More preferably, each of the repeat proteins comprises at most two repeat domains. Most preferably, each repeat protein comprises only one repeat domain. Furthermore, the repeat protein may comprise additional non-repeat protein domains, polypeptide tags, and/or polypeptide linkers.

The term "repeat domain" refers to a protein domain comprising two or more consecutive repeating units (modules) as structural units, wherein the structural units have the same fold and are tightly stacked to produce, for example, a supercoiled structure with a joined hydrophobic core. Preferably, the repeat domain further comprises an N-terminal and/or C-terminal capping unit (or module). Even more preferably, the N-terminal and/or C-terminal capping unit (or module) is a capping repeat sequence.

The terms "designed repeat protein" and "designed repeat domain" refer to a repeat protein or repeat domain, respectively, obtained by the inventive procedure explained in patent application WO 2002/020565. Designed repeat proteins and designed repeat domains are synthetic and not naturally occurring. They are artificial proteins or domains, respectively, obtained by expressing corresponding designed nucleic acids. Preferably, expression is performed in eukaryotic or prokaryotic cells, such as bacterial cells, or by using a cell-free in vitro expression system. Thus, a designed ankyrin repeat protein (i.e., a DARPin) corresponds to a binding protein of the present invention comprising at least one ankyrin repeat domain.

The term "structural unit" refers to a locally ordered portion of a polypeptide formed by three-dimensional interactions between two or more secondary structure segments located adjacent to each other along the polypeptide chain. Such structural units exhibit a structural motif. The term "structural motif" refers to the three-dimensional arrangement of secondary structure elements present in at least one structural unit. Structural motifs are well known to those skilled in the art. Structural units alone cannot achieve a defined three-dimensional arrangement; however, their continuous arrangement, for example as a repeating module of a repeating domain, results in mutual stabilization of adjacent units, resulting in a supercoiled structure.

The term "repeat unit" refers to an amino acid sequence comprising a repeating sequence motif of one or more naturally occurring repeat proteins, wherein the "repeat unit" occurs in multiple copies and exhibits a defined folding topology common to all of the motifs that determine protein folding. Such repeat units correspond to the "repeating structural units (repeats)" of repeat proteins described by Forrer et al. (2003, loc. cit.) or the "continuous homologous structural units (repeats)" of repeat proteins described by Binz et al. (2004, loc. cit.). Such repeat units comprise framework residues and interacting residues. Examples of such repeat units are armadillo repeat units, leucine-rich repeat units, ankyrin repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units. Naturally occurring proteins containing two or more such repeat units are referred to as "naturally occurring repeat proteins." The amino acid sequences of individual repeat units of a repeat protein, when compared to one another, may have a significant number of mutations, substitutions, additions, and/or deletions while still substantially retaining the general pattern or motif of the repeat unit.

The term "ankyrin repeat unit" refers to a repeating unit which is an ankyrin repeat sequence as described, for example, in Forrer et al. (2003, loc. cit.) Ankyrin repeat sequences are well known to those skilled in the art.

The term "framework residue" refers to an amino acid residue of a repeating unit, or a corresponding amino acid residue of a repeating module, which participates in the folding topology, i.e., it participates in the folding of the repeating unit (or module) or participates in the interaction with adjacent units (or modules). Such participation can be an interaction with other residues in the repeating unit (or module), or an influence on the conformation of the polypeptide backbone present in an α-helix or β-sheet, or an amino acid stretch forming a linear polypeptide or a loop.

The term "target interaction residue" refers to an amino acid residue of a repeating unit, or a corresponding amino acid residue of a repeating module, that participates in an interaction with a target substance. Such participation can be a direct interaction with the target substance, or an influence on other directly interacting residues, such as by stabilizing the polypeptide conformation of the repeating unit (or module) to allow or enhance the interaction of the directly interacting residue with the target. Such framework and target interaction residues can be identified by analyzing structural data obtained by physicochemical methods (such as X-ray crystallography, NMR and/or CD spectroscopy), or by comparing with known and relevant structural information known to those skilled in the art of structural biology and/or bioinformatics.

Preferably, the repeat unit for inferring the tumor-necrosis factor glycoprotein motif is a homologous repeat unit, wherein the repeat unit comprises the identical structural motif, and wherein the framework residues exceeding 70% of the repeat unit are homologous to each other. Preferably, the framework residues exceeding 80% of the repeat unit are homologous. Most preferably, the framework residues exceeding 90% of the repeat unit are homologous. Computer programs (such as Fasta, Blast or Gap) for determining homology percentage between polypeptides are well known to those skilled in the art. Further preferably, the repeat unit for inferring the tumor-necrosis factor glycoprotein motif is a homologous repeat unit obtained from the repeat domain selected on the target (as described in Example 1) and having the same target specificity.

The term "repeat sequence motif" refers to an amino acid sequence inferred from one or more repeating units or repeating modules. Preferably, the repeating units or repeating modules are derived from repeating domains with binding specificity to the same target. Such repeating sequence motifs include framework residue positions and target interaction residue positions. The framework residue positions correspond to the positions of the framework residues of the repeating unit (or module). Similarly, the target interaction residue positions correspond to the positions of the target interaction residues of the repeating unit (or module). Repeating sequence motifs include fixed positions and randomized positions. The term "fixed position" refers to an amino acid position in such a repeating sequence motif, wherein the position is set to a specific amino acid. Most commonly, such fixed positions correspond to the positions of framework residues and/or target interaction residues specific to a certain target. The term "randomized position" refers to an amino acid position in such a repeating sequence motif, wherein two or more amino acids are allowed at the amino acid position, for example, wherein any of the twenty common naturally occurring amino acids are allowed, or wherein most of the twenty naturally occurring amino acids are allowed, such as amino acids other than cysteine, or amino acids other than glycine, cysteine and proline. Most commonly, such randomized positions correspond to target-interacting residues, however, some framework residues can also be randomized.

The term "fold topology" refers to the tertiary structure of the repeating unit or repeating module. The fold topology is determined by the amino acid stretches that form at least part of an α-helix or β-sheet, or that form linear polypeptides or loops, or any combination of α-helices, β-sheets, and/or linear polypeptides/loops.

The term "continuously" refers to such arrangement, wherein repeat units or repeat modules are arranged in series. In the repeat protein of design, there are at least 2, generally about 2 to 6, particularly at least about 6, often 20 or more repeat units (or modules). In most cases, the repeat units (or modules) of the repeat domain will show a high degree of sequence identity (identical amino acid residues in corresponding positions) or sequence similarity (amino acid residues are different, but have similar physicochemical properties), and some amino acid residues may be very conservative key residues. However, between the different repeat units (or modules) in the repeat domain, the high degree of sequence variability due to amino acid insertion and/or disappearance and/or substitution will be possible, as long as the common folding topology of the repeat unit (or module) is maintained.

Methods for directly determining the folding topology of repeat proteins by physicochemical means such as X-ray crystallography, NMR or CD spectroscopy are well known to those skilled in the art. Methods for identifying and determining repeat units or repetitive sequence motifs or identifying families of related proteins comprising such repeat units or motifs are well established in the field of bioinformatics and are well known to those skilled in the art, such as homology searches (BLAST etc.). The step of refining the initial repetitive sequence motif may comprise an iterative process.

The term "repeat module" refers to the repeating amino acid sequence of a designed repeat domain that is originally derived from the repeating units of naturally occurring repeat proteins. Each repeat module contained in a repeat domain is derived from one or more repeating units of a naturally occurring repeat protein family or subfamily (e.g., the family of armadillo repeat proteins or ankyrin repeat proteins).

A "repeat module" may comprise positions with amino acid residues that are present in all copies of the corresponding repeat module ("fixed positions") and positions with different or "randomized" amino acid residues ("randomized positions").

The binding protein according to the present invention comprises at least one ankyrin repeat domain, wherein said repeat domain has binding specificity for mammalian serum albumin (xSA).

The terms "having binding specificity for a target,""specifically binds to a target," or "target specificity" and the like mean that the binding protein or binding domain binds to the target with a dissociation constant in PBS that is lower than the dissociation constant for binding to an unrelated protein, such as E. coli maltose binding protein (MBP). Preferably, the dissociation constant for the target in PBS is at least 10-fold, more preferably 10 ^- fold, even more preferably 10 ^- fold, or most preferably 10- ^fold lower than the corresponding dissociation constant for MBP.

The binding proteins of the present invention are not antibodies or fragments thereof, such as Fab or scFv fragments. Antibodies and fragments thereof are well known to those skilled in the art.

Furthermore, the binding domains of the present invention do not comprise an immunoglobulin fold (such as found in antibodies) and/or a type III fibronectin domain. The immunoglobulin fold is a common all-beta protein fold consisting of a two-layer sandwich of approximately seven antiparallel beta strands arranged between two beta sheets. Those skilled in the art are familiar with the immunoglobulin fold. For example, such binding domains comprising an immunoglobulin fold are described in WO 2007/080392 or WO 2008/097497.

In particular, the present invention relates to a binding protein comprising at least one ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for mammalian serum albumin, and wherein the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 50, 51 and 52, and sequences wherein up to 9 amino acids in SEQ ID NO: 49, 50, 51 and 52 are replaced by any amino acid.

Preferably, a maximum of 8 amino acids in SEQ ID NOs: 49, 50, 51 and 52 are replaced by other amino acids, more preferably a maximum of 7 amino acids, more preferably a maximum of 6 amino acids, more preferably a maximum of 5 amino acids, even more preferably a maximum of 4 amino acids, more preferably a maximum of 3 amino acids, more preferably a maximum of 2 amino acids, more preferably a maximum of 1 amino acid, and most preferably zero amino acids are replaced in SEQ ID NOs: 49, 50, 51 and 52.

Preferably, when amino acids in SEQ ID NOs: 49, 50, 51, and 52 are replaced, these amino acids are selected from the group consisting of A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; more preferably, they are selected from the group consisting of A, D, E, H, I, K, L, Q, R, S, T, V, and Y. Also preferably, the amino acids are replaced with homologous amino acids; that is, the amino acids are replaced with amino acids having side chains with similar biophysical properties. For example, the negatively charged amino acid D can be replaced with the negatively charged amino acid E, or a hydrophobic amino acid such as L can be replaced with A, I, or V. The replacement of amino acids with homologous amino acids is well known to those skilled in the art.

Preferred binding proteins comprise at least one ankyrin repeat domain, wherein the repeat domain binds to xSA in PBS with a dissociation constant (Kd) of less than 10 ⁻⁴ M. Preferably, the repeat domain binds to xSA in PBS with a Kd of less than 10 ⁻⁴ M, more preferably less than 10 ⁻⁵ M, 10 ⁻⁶ M, 10 ⁻⁷ M, or most preferably less than 10 ⁻⁸ M.

Methods for determining the dissociation constant of protein-protein interactions, such as techniques based on surface plasmon resonance (SPR) (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. If measured under different conditions (e.g., salt concentration, pH), the Kd value of a specific protein-protein interaction measured may vary. Thus, Kd values are preferably measured using standardized protein solutions and standardized buffers (such as PBS).

In the Examples, binding proteins comprising ankyrin repeat domains are shown that bind to xSA with a Kd below 10 ⁻⁴ M in PBS.

The ankyrin repeat domain of the binding protein of the present invention binds to xSA. Preferably, the binding protein comprises ankyrin repeat domain that binds to human serum albumin (HSA).

Further preferred are binding domains comprising 70-300 amino acids, in particular 100-200 amino acids.

Further preferred are binding proteins or binding domains that do not contain free Cys residues. "Free Cys residues" do not participate in the formation of disulfide bonds. Even more preferred are binding proteins or binding domains that do not contain any Cys residues.

The binding domain of the present invention is an ankyrin repeat domain or a designed ankyrin repeat domain (Binz et al., 2004, loc. cit.), preferably as described in WO 2002/020565. Examples of designed ankyrin repeat domains are shown in the Examples.

In another embodiment, the invention relates to a binding protein comprising at least one ankyrin repeat domain, wherein the repeat domain has binding specificity for mammalian serum albumin, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least 70% amino acid sequence identity to an ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 17-31 and 43-48, wherein the G at position 1 and/or the S at position 2 of the ankyrin repeat domain is optionally deleted.

Preferably, the ankyrin repeat domain in the binding protein of the present invention comprises an amino acid sequence having at least 70% amino acid sequence identity with an ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 21, 27 and 46; preferably 27 and 46. As defined above, the ankyrin repeat domain binds to xSA in PBS with a dissociation constant (Kd) of less than 10 ⁻⁴ M. Preferably, the repeat domain binds to xSA in PBS with a Kd of less than 10 ⁻⁴ M, more preferably less than 10 ⁻⁵ M, 10 ⁻⁶ M, 10 ⁻⁷ M, or most preferably less than 10 ⁻⁸ M.

Preferably, such ankyrin repeat domain in the binding protein of the present invention comprises an amino acid sequence having at least 70% amino acid sequence identity with a “randomized repeat unit” or a “randomized position” in an ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 17-31 and 43-48.

Preferably, instead of 70% amino acid sequence identity, such ankyrin repeat domains in the binding proteins of the present invention have an amino acid sequence with at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or most preferably at least 95% amino acid sequence identity.

In a specific embodiment, a binding protein with binding specificity for mammalian serum albumin (defined by replacing up to 9 amino acids in the ankyrin repeat module of SEQ ID NOs: 49, 50, 51, and 52, or by having at least 70% amino acid sequence identity with an ankyrin repeat domain selected from SEQ ID NOs: 17-31 and 43-48) has a terminal plasma half-life in mammals that is at least 5-fold greater than that of a corresponding binding protein that does not bind to mammalian serum albumin (e.g., the ankyrin repeat domain of SEQ ID NO: 32). In such preferred binding proteins, the minimum terminal plasma half-life in humans is at least 1 day, more preferably at least 3 days, and even more preferably at least 5 days.

In another embodiment, the present invention relates to a binding protein, wherein the ankyrin repeat domain comprises a repeat module having the following ankyrin repeat motif: X ₁ DX ₂ X ₃ X ₄ X ₅ TPLHLAAX ₆ X ₇ GHLX ₈ IVEVLLKX ₉ GADVNA (SEQ ID NO: 53)

wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , X ₈ and X ₉ independently represent an amino acid residue selected from the group consisting of A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y;

Preferably, wherein

_X1 represents an amino acid residue selected from the group consisting of A, D, M, F, S, I, T, N, Y and K; more preferably K and A;

_X2 represents an amino acid residue selected from the group consisting of E, K, D, F, M, N, I and Y; more preferably I, E and Y;

_X3 represents an amino acid residue selected from the group consisting of W, R, N, T, H, K, A and F; more preferably W, R and F;

_X4 represents an amino acid residue selected from the group consisting of G and S;

_X5 represents an amino acid residue selected from the group consisting of N, T and H;

X ₆ represents an amino acid residue selected from the group consisting of N, V and R;

_X7 represents an amino acid residue selected from the group consisting of E, Y, N, D, H, S, A, Q, T and G; more preferably E, Y and N;

X ₈ represents an amino acid residue selected from the group consisting of E and K;

_X9 represents an amino acid residue selected from the group consisting of S, A, Y, H and N; more preferably Y and H; and

wherein optionally up to 5 amino acids other than the positions indicated by X in SEQ ID NO: 53 are replaced by any amino acid.

In particular, the present invention relates to such binding proteins, wherein the ankyrin repeat domain comprises a repeat module having the following ankyrin repeat sequence motif: X ₁ DX ₂ X ₃ GX ₄ TPLHLAAX ₅ X ₆ GHLEIVEVLLKX ₇ GADVNA (SEQ ID NO: 10)

in

_X1 represents an amino acid residue selected from the group consisting of A, D, M, F, S, I, T, N, Y and K; preferably K and A;

_X2 represents an amino acid residue selected from the group consisting of E, K, D, F, M, N, I and Y; preferably I, E and Y;

_X3 represents an amino acid residue selected from the group consisting of W, R, N, T, H, K, A and F; preferably W, R and F;

_X4 represents an amino acid residue selected from the group consisting of N, T and H;

X ₅ represents an amino acid residue selected from the group consisting of N, V and R;

X ₆ represents an amino acid residue selected from the group consisting of E, Y, N, D, H, S, A, Q, T and G; preferably E, Y and N;

_X7 represents an amino acid residue selected from the group consisting of S, A, Y, H and N; preferably Y and H; and

wherein optionally up to 5 amino acids other than the positions indicated by X in SEQ ID NO: 10 are replaced by any amino acid.

In another embodiment, the present invention relates to a binding protein, wherein the ankyrin repeat domain comprises a repeat module having the following ankyrin repeat motif:

X ₁ DYFX ₂ HTPLHLAARX ₃ X ₄ HLX ₅ IVEVLLKX ₆ GADVNA (SEQ ID NO:11)

in

_X1 represents an amino acid residue selected from the group consisting of D, K and A; preferably K and A;

_X2 represents an amino acid residue selected from the group consisting of D, G and S; preferably G and S;

X ₃ represents an amino acid residue selected from the group consisting of E, N, D, H, S, A, Q, T and G; preferably Q, D and N; more preferably Q and N;

_X4 represents an amino acid residue selected from the group consisting of G and D;

_X5 represents an amino acid residue selected from the group consisting of E, K and G; preferably E and K;

X ₆ represents an amino acid residue selected from the group consisting of H, Y, A and N; preferably H, A and Y; more preferably A and Y; and

wherein optionally up to 5 amino acids other than the positions indicated by X in SEQ ID NO: 11 are replaced by any amino acid.

In another embodiment, the present invention relates to a binding protein, wherein the ankyrin repeat domain comprises a repeat module having the following ankyrin repeat motif:

X ₁ DFX ₂ GX ₃ TPLHLAAX ₄ X ₅ GHLEIVEVLLKX ₆ GADVNA (SEQ ID NO:54)

in

_X1 represents an amino acid residue selected from the group consisting of F, S, L and K; preferably S and K;

_X2 represents an amino acid residue selected from the group consisting of: V and A;

X ₃ represents an amino acid residue selected from the group consisting of R and K;

_X4 represents an amino acid residue selected from the group consisting of S and N;

X ₅ represents an amino acid residue selected from the group consisting of N, D, Q, S, A, T and E; preferably D and Q;

X ₆ represents an amino acid residue selected from the group consisting of A, H, Y, S and N; preferably A and H; and

wherein optionally up to 5 amino acids other than the positions indicated by X in SEQ ID NO: 54 are replaced by any amino acid.

In particular, the present invention relates to binding proteins, wherein the ankyrin repeat domain comprises a repeat module having the following ankyrin repeat motif:

X ₁ DFX ₂ GX ₃ TPLHLAAX ₄ DGHLEIVEVLLKX ₅ GADVNA (SEQ ID NO:12)

in

_X1 represents an amino acid residue selected from the group consisting of F, S, L and K; preferably S and K;

_X2 represents an amino acid residue selected from the group consisting of: V and A;

X ₃ represents an amino acid residue selected from the group consisting of R and K;

_X4 represents an amino acid residue selected from the group consisting of S and N;

_X5 represents an amino acid residue selected from the group consisting of A, H, Y, S and N; preferably A and H; and

wherein optionally up to 5 amino acids other than the positions indicated by X in SEQ ID NO: 12 are replaced by any amino acid.

Preferred is a binding protein, wherein the ankyrin repeat domain comprises a repeat module having ankyrin repeat motif of SEQ ID NO:12, which is preceded by a repeat module having ankyrin repeat motif of SEQ ID NO:11.

In another embodiment, the present invention relates to a binding protein, wherein the ankyrin repeat domain comprises a repeat module having the following ankyrin repeat motif:

X ₁ DX ₂ X ₃ GTTPLHLAAVYGHLEX ₄ VEVLLKX ₅ GADVNA (SEQ ID NO:13)

in

_X1 represents an amino acid residue selected from the group consisting of K, A, D, M, F, S, I, T, N and Y; preferably K and A;

_X2 represents an amino acid residue selected from the group consisting of E, K, D, F, M, N and Y; preferably E, D and Y;

_X3 represents an amino acid residue selected from the group consisting of R, N, T, H, K, A and F; preferably R and F;

_X4 represents an amino acid residue selected from the group consisting of I and M;

_X5 represents an amino acid residue selected from the group consisting of H, Y, K, A and N; preferably K and A; and

wherein optionally up to 5 amino acids other than the positions indicated by X in SEQ ID NO: 13 are replaced by any amino acid.

In another embodiment, the present invention relates to a binding protein, wherein the ankyrin repeat domain comprises a repeat module having the following ankyrin repeat motif:

X ₁ NETGYTPLHLADSSGHX ₂ EIVEVLLKX ₃ X ₄ X ₅ DX ₆ NA (SEQ ID NO:14)

in

_X1 represents an amino acid residue selected from the group consisting of Q and K;

_X2 represents an amino acid residue selected from the group consisting of L and P;

_X3 represents an amino acid residue selected from the group consisting of H, N, Y and A; preferably H and A;

_X4 represents an amino acid residue selected from the group consisting of G and S;

X ₅ represents an amino acid residue selected from the group consisting of A, V, T and S; preferably S and A;

X ₆ represents an amino acid residue selected from the group consisting of V and F; and wherein optionally up to 5 amino acids other than the positions indicated by X in SEQ ID NO: 14 are replaced by any amino acid.

Preferred is a binding protein, wherein the ankyrin repeat domain comprises a repeat module having ankyrin repeat motif of SEQ ID NO:14, which is preceded by a repeat module having ankyrin repeat motif of SEQ ID NO:13.

The term "capping module" refers to a polypeptide fused to the N-terminal or C-terminal repeat module of a repeat domain, wherein the capping module forms a tight tertiary interaction (i.e., a tertiary structural interaction) with the repeat module, thereby providing a cap that separates the hydrophobic core of the repeat module from the solvent on the side that is not in contact with the consecutive repeat module. The N-terminal and/or C-terminal capping module can be, or can be derived from, a capping unit or other structural unit present in a naturally occurring repeat protein adjacent to the repeat unit. The term "capping unit" refers to a naturally occurring folded polypeptide, wherein the polypeptide is defined as a specific structural unit fused to a repeat unit at the N-terminus or C-terminus, wherein the polypeptide forms a tight tertiary structural interaction with the repeat unit, thereby providing a cap that separates the hydrophobic core of the repeat unit from the solvent on one side. Preferably, the capping module or capping unit is a capping repeat sequence. The term "capping repeat sequence" refers to a capping module or capping unit that has a similar or identical fold to the adjacent repeat unit (or module) and/or sequence similarity to the adjacent repeat unit (or module). Capping modules and capping repeats are described in WO 2002/020565 and Interlandi et al., 2008 (cited above). For example, WO 2002/020565 describes an N-terminal capping module (i.e., a capping repeat) having the following amino acid sequence:

GSDLGKKLLEAARAGQDDEVRILMANGADVNA (SEQ ID NO:1), and

A C-terminal capping module (i.e., capping repeat sequence) having the following amino acid sequence:

QDKFGKTAFDISIDNGNEDLAEILQKLN (SEQ ID NO:2).

et al., 2008 (loc. cit.) described C-terminal capping modules having the following amino acid sequences: QDKFGKTPFDLAIREGHEDIAEVLQKAA (SEQ ID NO: 3) and QDKFGKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 4).

For example, the N-terminal capping module of SEQ ID NO: 17 is encoded by amino acids 1-32, and the C-terminal capping module of SEQ ID NO: 17 is encoded by amino acids 99-126.

A preferred N-terminal capping module comprises the following sequence motif

X ₁ LX ₂ KKLLEAARAGQDDEVRILX ₃ AX ₄ GADVNA (SEQ ID NO:5)

wherein X ₁ represents an amino acid residue G, A or D;

Wherein X ₂ represents amino acid residue G or D;

wherein X ₃ represents an amino acid residue L, V, I, A or M; preferably, L or M; and

wherein X ₄ represents an amino acid residue A, H, Y, K, R or N; preferably, A or N.

Further preferred is any such N-terminal capping module comprising an N-terminal capping repeat sequence, wherein one or more amino acid residues in the capping repeat sequence are replaced by specific amino acid residues that are present at corresponding positions when aligning the corresponding capping units or repeat units.

A preferred C-terminal capping module comprises the following sequence motif

X ₁ DKX ₂ GKTX ₃ X ₄ DX ₅ X ₆ X ₇ DX ₈ GX ₉ EDX ₁₀ AEX ₁₁ LQKAA (SEQ ID NO:6)

Wherein X ₁ represents amino acid residue Q or K;

wherein X ₂ represents an amino acid residue A, S or F; preferably, S or F;

wherein X ₃ represents an amino acid residue A or P;

wherein X ₄ represents amino acid residue A or F;

wherein X ₅ represents amino acid residue I or L;

wherein X ₆ represents an amino acid residue S or A;

wherein X ₇ represents amino acid residue I or A;

wherein X ₈ represents an amino acid residue A, E or N; preferably, A or N;

wherein X ₉ represents an amino acid residue N or H;

wherein X ₁₀ represents amino acid residue L or I;

wherein X ₁₁ represents amino acid residue I or V; and

If _X4 represents F, _X7 represents I, and _X8 represents N or E, then _X2 does not represent F.

Another preferred C-terminal capping module comprises the following sequence motif

X ₁ DKX ₂ GKTX ₃ ADX ₄ X ₅ X ₆ DX ₇ GX ₈ EDX ₉ AEX ₁₀ LQKAA (SEQ ID NO:7)

Wherein X ₁ represents amino acid residue Q or K;

wherein X ₂ represents an amino acid residue A, S or F; preferably, S or F;

wherein X ₃ represents an amino acid residue A or P;

wherein X ₄ represents amino acid residue I or L;

wherein X ₅ represents an amino acid residue S or A;

wherein X ₆ represents amino acid residue I or A;

wherein X ₇ represents an amino acid residue A, E or N; preferably, A or N;

wherein X ₈ represents an amino acid residue N or H;

wherein X ₉ represents an amino acid residue L or I; and

wherein _X10 represents amino acid residue I or V.

Another preferred C-terminal capping module comprises the following sequence motif

X ₁ DKX ₂ GKTX ₃ ADX ₄ X ₅ ADX ₆ GX ₇ EDX ₈ AEX ₉ LQKAA (SEQ ID NO:8)

Wherein X ₁ represents amino acid residue Q or K;

wherein X ₂ represents an amino acid residue A, S or F; preferably, S or F;

wherein X ₃ represents an amino acid residue A or P;

wherein X ₄ represents amino acid residue I or L;

wherein X ₅ represents an amino acid residue S or A;

wherein X ₆ represents an amino acid residue A, E or N; preferably, A or N;

wherein X ₇ represents an amino acid residue N or H;

wherein X ₈ represents an amino acid residue L or I; and

wherein X ₉ represents amino acid residue I or V.

Preferably, such a C-terminal capping module comprising the sequence motif of SEQ ID NO: 6, 7 or 8 has an amino acid residue A, I or K at the position corresponding to position 3 of said sequence motif; preferably, I or K.

Also preferably, such a C-terminal capping module comprising the sequence motif of SEQ ID NO: 6, 7 or 8 has an amino acid residue R or D at the position corresponding to position 14 of said sequence motif.

A preferred C-terminal capping module is a C-terminal capping module having the following amino acid sequence: QDKSGKTPADLAADAGHEDIAEVLQKAA (SEQ ID NO: 9).

Further preferred is a C-terminal capping module having the amino acid sequence of SEQ ID NO: 9, wherein

The amino acid residue at position 1 is Q or K;

The amino acid residue at position 4 is S or F;

The amino acid residue at position 9 is A or F;

The amino acid residue at position 13 is A or I;

The amino acid residue at position 15 is A, E, or N; and

wherein the C-terminal capping module does not have the amino acid sequence of SEQ ID NO: 2, 3 or 4.

Further preferred is a C-terminal capping module having an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90% or most preferably at least 95% amino acid sequence identity when aligned with SEQ ID NO: 9 or 2. Preferably, the amino acid residue at the position of the C-terminal capping module corresponding to position 4 of SEQ ID: 9 when aligned is S, the amino acid residue at the position of the C-terminal capping module corresponding to position 9 of SEQ ID: 9 when aligned is A, the amino acid residue at the position of the C-terminal capping module corresponding to position 13 of SEQ ID: 9 when aligned is A, and/or the amino acid residue at the position of the C-terminal capping module corresponding to position 15 of SEQ ID: 9 when aligned is A. Further preferably, the amino acid residue at the position of the C-terminal capping module corresponding to position 9 of SEQ ID: 9 when aligned is A, and/or the amino acid residue at the position of the C-terminal capping module corresponding to position 13 of SEQ ID: 9 when aligned is A. Also preferably, the C-terminal capping module comprises 28 amino acids.

Further preferred is a C-terminal capping module having an amino acid sequence of SEQ ID NO: 2 or 9, wherein

One or more amino acid residues of the C-terminal capping module are replaced by specific amino acids that are present at corresponding positions when aligning corresponding C-terminal capping repeat sequences or capping units, and wherein

The amino acid residue at position 4 is S;

The amino acid residue at position 9 is A;

The amino acid residue at position 13 is A; and/or

The amino acid residue at position 15 is A.

Preferably, at most 30% of the amino acid residues of the C-terminal capping module are replaced, more preferably at most 20% and even more preferably at most 10% of the amino acid residues are replaced. Also preferably, such a C-terminal capping module is a naturally occurring C-terminal capping repeat sequence.

Also preferred is a C-terminal capping module comprising amino acids 1-25 or 1-26 of any of the above C-terminal capping modules based on SEQ ID NO: 9.

Further preferred is a C-terminal capping module having an amino acid sequence which does not comprise the amino acid N followed by G.

Also preferred are C-terminal capping modules that have at least 70%, preferably at least 75%, 80%, 85%, 90%, or most preferably at least 95% amino acid sequence identity with any of the above C-terminal capping modules based on SEQ ID NO: 9 or with SEQ ID NO: 9 itself.

Further preferred is a C-terminal capping module having at least 70%, preferably at least 75%, 80%, 85%, 90% or most preferably at least 95% amino acid sequence identity to SEQ ID NO: 2 or 9, and wherein the C-terminal capping module has amino acid A at position 9; preferably, the C-terminal capping module has amino acid A at positions 9 and 13; more preferably, the C-terminal capping module has amino acid A at positions 9, 13 and 15; most preferably, the C-terminal capping module has amino acid A at positions 9, 13 and 15 and amino acid S at position 4.

Further preferred is a C-terminal capping module which does not have the amino acid R at position 14 and/or does not have the amino acid E at position 15.

Also preferred are C-terminal capping modules that do not have the same amino acid sequence as SEQ ID NO: 2, 3 or 4.

Further preferred is a C-terminal capping module having an amino acid sequence based on SEQ ID NO: 9, wherein the C-terminal capping module has amino acids at positions 26, 27 and 28 selected from the group consisting of: A, L, R, M, K and N; more preferably A, L, R and K; and most preferably K, A and L.

By combining techniques known to those skilled in the art (such as amino acid sequence alignment, mutagenesis, and gene synthesis), the capping module of the repeat domain can be replaced with the capping module of the present invention. For example, the C-terminal capping repeat sequence of SEQ ID NO: 9 can be replaced with the C-terminal capping repeat sequence of SEQ ID NO: 17 as follows: (i) determining the C-terminal capping repeat sequence of SEQ ID NO: 17 (i.e., sequence positions 99-126) by sequence alignment with SEQ ID NO: 9, (ii) replacing the determined sequence of the C-terminal capping repeat sequence of SEQ ID NO: 17 with the sequence of SEQ ID NO: 9, (iii) preparing a gene encoding a repeat domain encoding the replaced C-terminal capping module, (iv) expressing the modified repeat domain in the cytoplasm of Escherichia coli, and (v) purifying the modified repeat domain by standard means.

In addition, the repeat domain of the present invention can be constructed genetically as follows: by gene synthesis, an N-terminal capping module (i.e., the N-terminal capping repeat sequence of SEQ ID NO: 1) is assembled, followed by one or more repeat modules (i.e., a repeat module comprising amino acid residues 33-98 of SEQ ID NO: 17) and a C-terminal capping module (i.e., the C-terminal capping repeat sequence of SEQ ID NO: 9). The genetically assembled repeat domain gene can then be expressed in E. coli as described above.

Also preferred are binding proteins, wherein the ankyrin repeat domain or designed ankyrin repeat domain comprises a C-terminal capping module having a sequence motif of SEQ ID NO: 6, 7 or 8, wherein the capping module has amino acid 1 at position 3, and wherein the repeat module is preceded by a repeat module having ankyrin repeat sequence motif of SEQ ID NO: 12.

Further preferred are binding proteins, repeat domains, N-terminal capping modules or C-terminal capping modules having an amino acid sequence that does not contain amino acids C, M or N.

Further preferred are binding proteins, repeat domains, N-terminal capping modules or C-terminal capping modules having an amino acid sequence that does not contain the amino acid N followed by G.

Further preferred is any such C-terminal capping module comprising a C-terminal capping repeat sequence, wherein one or more amino acid residues in the capping repeat sequence are replaced by specific amino acid residues that are present at corresponding positions when aligning the corresponding capping units or repeat units.

Further preferred are binding proteins comprising any such N-terminal or C-terminal capping modules.

Examples of amino acid sequences of such C-terminal capping modules are the amino acid sequences at positions 99-126 in SEQ ID NOs: 19, 21, 27, 28, 38, 40, and 42. Example 6 demonstrates that the thermal stability of the repeat domains can be increased by replacing their C-terminal capping modules with the capping modules of the present invention.

The term "target" refers to an individual molecule such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other naturally occurring molecule, including any portion of such an individual molecule, or a complex of two or more such molecules. The target can be a whole cell or tissue sample, or can be any non-natural molecule or portion. Preferably, the target is a naturally occurring or non-natural polypeptide or a polypeptide containing a chemical modification, such as modification by natural or non-natural phosphorylation, acetylation, or methylation. In a specific application of the present invention, the target is xSA.

The term "xSA" refers to mammalian serum albumin, such as serum albumin from mouse, rat, rabbit, dog, pig, monkey, or human. The term "MSA" refers to mouse serum albumin (UniProtKB/Swiss-Prot Basic Accession No. P07724), the term "CSA" refers to cynomolgus monkey ( i.e. , macaca fascicularis ) serum albumin (UniProtKB/Swiss-Prot Basic Accession No. A2V9Z4), and the term "HSA" refers to human serum albumin (UniProtKB/Swiss-Prot Basic Accession No. P02768).

The term "consensus sequence" refers to an amino acid sequence, wherein the consensus sequence is obtained by structural and/or sequence alignment of a plurality of repeating units. Using two or more repeating units that have been structurally and/or sequence aligned and allowing for gaps in the alignment, the most common amino acid residues at each position can be determined. A consensus sequence is a sequence comprising the most commonly occurring amino acids at each position. In cases where two or more amino acids occur at a single position in excess of the average, the consensus sequence may comprise a subset of those amino acids. The two or more repeating units may be taken from repeating units contained in a single repeating protein or from two or more different repeating proteins.

Those skilled in the art are familiar with consensus sequences and methods for determining them.

A "consensus amino acid residue" is an amino acid that is found at a particular position in a consensus sequence. If two or more, such as three, four or five, amino acid residues are found in two or more repeating units with similar probability, the consensus amino acid may be one of the most common amino acids or a combination of the two or more amino acid residues.

Further preferred are non-naturally occurring capping modules, repeat modules, binding proteins or binding domains.

The term "non-naturally occurring" means synthetic or not from nature, more specifically, the term means made by human hands. The term "non-naturally occurring binding protein" or "non-naturally occurring binding domain" means that the binding protein or the binding domain is synthetic (i.e., prepared by chemical synthesis with amino acids) or recombinant and not from nature. "Non-naturally occurring binding protein" or "non-naturally occurring binding domain" are artificial proteins or domains obtained by expressing nucleic acids of corresponding design, respectively. Preferably, expression is carried out in eukaryotic or bacterial cells, or by using a cell-free in vitro expression system. Moreover, the term means that the sequence of the binding protein or the binding domain does not exist as a non-artificial sequence entry in a sequence database, such as GenBank, EMBL-Bank or Swiss-Prot. Those skilled in the art are familiar with these databases and other similar sequence databases.

The present invention relates to a binding protein comprising a binding domain, wherein the binding domain is an ankyrin repeat domain and specifically binds to xSA, wherein the binding protein and/or binding domain has a midpoint denaturation temperature (Tm) above 40°C after thermal unfolding in PBS and forms less than 5% (w/w) insoluble aggregates when incubated in PBS at 37°C for 1 day at a concentration of up to 10 g/L.

The term "PBS" means an aqueous phosphate-buffered saline solution containing 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl and having a pH of 7.4.

Preferably, the binding protein and/or binding domain has a midpoint denaturation temperature (Tm) of greater than 45°C, more preferably greater than 50°C, more preferably greater than 55°C, and most preferably greater than 60°C after thermal unfolding in PBS at pH 7.4 or in MES buffer at pH 5.8. The binding protein or binding domain of the present invention has a defined secondary and tertiary structure under physiological conditions. Thermal unfolding of such a polypeptide will result in the loss of its tertiary and secondary structure, which can be tracked by, for example, circular dichroism (CD) measurements. The midpoint denaturation temperature of the binding protein or binding domain after thermal unfolding corresponds to the temperature at the midpoint of the cooperative transition in a physiological buffer after thermal denaturation of the protein or domain by slowly increasing the temperature from 10°C to about 100°C. The determination of the midpoint denaturation temperature after thermal unfolding is well known to those skilled in the art. This midpoint denaturation temperature of the binding protein or binding domain after thermal unfolding will indicate the thermal stability of the polypeptide.

Also preferred are binding proteins and/or binding domains that form less than 5% (w/w) insoluble aggregates when incubated in PBS at 37°C for more than 5 days, preferably more than 10 days, more preferably more than 20 days, more preferably more than 40 days, and most preferably more than 100 days at a concentration of up to 20 g/L, preferably up to 40 g/L, more preferably up to 60 g/L, even more preferably up to 80 g/L, and most preferably up to 100 g/L. The formation of insoluble aggregates can be detected by the appearance of a visible precipitate, gel filtration, or dynamic light scattering, which increases strongly after the formation of insoluble aggregates. Insoluble aggregates can be removed from protein samples by centrifugation at 10'000 x g for 10 minutes. Preferably, the binding protein and/or binding domain forms less than 2%, more preferably less than 1%, 0.5%, 0.2%, 0.1%, or most preferably less than 0.05% (w/w) insoluble aggregates in PBS under the described incubation conditions at 37°C. The percentage of insoluble aggregates can be determined by separating the insoluble aggregates from the soluble protein and then determining the amount of protein in the soluble and insoluble fractions by standard quantitative methods.

Also preferred are binding proteins and/or binding domains that do not lose their native three-dimensional structure upon incubation in PBS containing 100 mM dithiothreitol (DTT) at 37°C for 1 or 10 hours.

In a specific embodiment, the present invention relates to a binding protein comprising a binding domain which is an ankyrin repeat domain which specifically binds to xSA and has the indicated or preferred midpoint denaturation temperature and non-aggregation properties as defined above, wherein the terminal plasma half-life of the binding protein in a mammal is at least 5-fold higher than a binding domain which does not bind to a serum protein such as xSA.

Preferably, the terminal plasma half-life of the binding domain in a mammal is at least 10-fold, more preferably at least 20-fold, 40-fold, 100-fold, 300-fold or most preferably at least 10 ³ -fold greater than a binding domain that does not bind to a serum protein such as xSA.

Also preferably, the binding domain does not bind xSA, as evidenced by binding to xSA with a Kd higher than 10 ⁻⁴ M, more preferably higher than 10 ⁻³ M or most preferably higher than 10 ⁻² M. An example of a binding domain that does not bind xSA is the repeat domain of SEQ ID NO: 32.

Further preferably, the binding domain is a repeat domain and has a terminal plasma half-life in a mammal that is at least 5-fold higher (i.e., longer) than the repeat domain of SEQ ID NO: 32, or compared to DARPin #32, DARPin #41, or DARPin #42, more preferably at least 10-fold, 20-fold, 40-fold, 100-fold, 300-fold, or most preferably at least 10 ³ -fold higher.

A preferred binding protein comprises a binding domain with binding specificity for HSA that has a terminal plasma half-life in humans of greater than 1 day, more preferably greater than 3 days, 5 days, 7 days, 10 days, 15 days, or most preferably greater than 20 days.

The terminal plasma half-life of the binding domain can be determined by assays well known to those skilled in the art (Toutain, PL, and Bousquet-Mélou, A., J. Vet. Pharmacol. Ther. 27(5), 427-439, 2004). Examples of determining the terminal plasma half-life are given in the Examples.

The term "terminal plasma half-life" of a drug (such as a binding protein or binding domain of the invention) refers to the time required for the drug administered to a mammal to reach half its plasma concentration after reaching pseudo-equilibrium. This half-life is not defined as the time required to eliminate half of the dose of the drug administered to the mammal.

In a specific embodiment, the present invention relates to a binding protein comprising a binding domain that is an ankyrin repeat domain that specifically binds to xSA, and the binding protein comprises a biologically active compound.

The term "biologically active compound" refers to a compound that, when administered to a mammal having a disease, alters the disease. A biologically active compound may have antagonistic or agonistic properties and may be a proteinaceous or non-proteinaceous biologically active compound.

Such proteinaceous biologically active compounds can be covalently linked to, for example, a binding domain of the present invention by preparing a gene fusion polypeptide using standard DNA cloning techniques, followed by standard expression and purification. For example, DARPin #36 comprises a repeat domain with binding specificity for human growth factor (i.e., the biologically active compound) followed by a repeat domain with binding specificity for HSA.

Such non-proteinaceous biologically active compounds can be covalently linked to, for example, a binding domain of the invention by chemical means, for example, coupling via a maleimide linker to a cysteine thiol group, wherein the cysteine is coupled to the N- or C-terminus of a binding domain described herein via a peptide linker.

Examples of proteinaceous bioactive compounds are binding domains with unique target specificity (e.g., by binding to a growth factor and thereby neutralizing it), cytokines (e.g., interleukins), growth factors (e.g., human growth hormone), antibodies and fragments thereof, hormones (e.g., GLP-1), and any possible proteinaceous drug.

Examples of non-proteinaceous bioactive compounds are toxins (eg DM1 from ImmunoGen), small molecules targeting GPCRs, antibiotics and any possible non-proteinaceous drugs.

In a specific embodiment, the present invention is directed to a binding protein comprising an ankyrin repeat domain that specifically binds xSA, and said binding protein further comprises a biologically active compound, wherein the terminal half-life of said binding protein in a mammal is at least 2-fold higher than the terminal half-life of said unmodified biologically active compound, wherein said higher terminal half-life is conferred to said binding protein by said repeat domain.

Preferably, the terminal plasma half-life of the binding protein in a mammal is at least 5-fold, more preferably at least 10-fold, 20-fold, 40-fold, 100-fold, 300-fold or most preferably at least 10 ³ -fold greater than that of the unmodified biologically active compound.

Another preferred embodiment is a recombinant binding protein comprising a binding domain that specifically binds to xSA, and wherein the binding domain is an ankyrin repeat domain or a designed ankyrin repeat domain. Such ankyrin repeat domains may comprise 1, 2, 3 or more internal repeat modules that will participate in binding to xSA. Preferably, such ankyrin repeat domains comprise an N-terminal capping module, 2-4 internal repeat modules and a C-terminal capping module. Preferably, the binding domain is an ankyrin repeat domain or a designed ankyrin repeat domain. Also preferably, the capping module is a capping repeat sequence.

In particular, the present invention relates to a binding protein as defined above, wherein the ankyrin repeat domain competes for binding to mammalian serum albumin with ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 17-31 and 43-48; preferably SEQ ID NOs: 17-31; more preferably SEQ ID NOs: 19, 21, 27 and 28, in particular SEQ ID NOs: 19 and 27.

Most preferred is a binding protein wherein the ankyrin repeat domain is selected from the group consisting of: SEQ ID NOs: 17-31 and 43-48, wherein the G at position 1 and/or the S at position 2 of the ankyrin repeat domain is optionally deleted.

Also preferably, the repeat domain competes for xSA binding with a binding protein selected from DARPins #17-31 and 43-48. Preferably, the repeat domain competes for xSA binding with a binding protein selected from DARPins #19, 21, 27, 28, 45, 46, 47, and 48. More preferably, the repeat domain competes for xSA binding with binding proteins DARPin #19, 45, 46, 48, or 27; even more preferably, the repeat domain competes for xSA binding with binding proteins DARPin #46 or 27.

The term "competitive binding" means that two different binding domains of the present invention cannot bind to the same target at the same time, but each of them can bind to the same target. Therefore, such two binding domains compete for binding to the target. Preferably, the two competitive binding domains bind to overlapping or identical binding epitopes on the target. Methods for determining whether two binding domains compete for binding to a target are well known to those skilled in the art, such as competitive enzyme-linked immunosorbent assays (ELISAs) or competitive SPR measurements (e.g., by using a BioRad Proteon instrument).

Another preferred embodiment is a binding protein comprising a repeat domain with binding specificity for xSA selected from the group consisting of the repeat domains of SEQ ID NOs: 17-31. Preferably, the repeat domain is a repeat domain of SEQ ID NOs: 19, 21, 27, or 28. More preferably, the repeat domain is a repeat domain of SEQ ID NO: 19. Even more preferably, the repeat domain is a repeat domain of SEQ ID NO: 21. Even more preferably, the repeat domain is a repeat domain of SEQ ID NO: 27. Even more preferably, the repeat domain is a repeat domain of SEQ ID NO: 28.

Further preferred are binding proteins wherein the repeat domain with binding specificity for xSA comprises an amino acid sequence that has at least 70% amino acid sequence identity with a repeat domain of the set of repeat domains. Preferably, the amino acid sequence identity is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

Further preferred are binding proteins wherein the repeat domain with binding specificity for xSA comprises a repeat module that has at least 70% amino acid sequence identity with a repeat module of a repeat domain of the set of repeat domains. Preferably, the amino acid sequence identity is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

Further preferred is a binding protein comprising two or more repeat domains having binding specificity for xSA. Preferably, the binding protein comprises two or three repeat domains. The two or more repeat domains have the same or different amino acid sequences.

In another preferred embodiment of the binding protein comprising a repeat domain according to the present invention, one or more amino acid residues of the repeat module of the repeat domain are replaced by amino acid residues found at the corresponding position when aligning the repeat units. Preferably, at most 30% of the amino acid residues are replaced, more preferably at most 20%, and even more preferably at most 10% of the amino acid residues are replaced. Most preferably, such repeat units are naturally occurring repeat units.

In another preferred embodiment of the binding protein comprising a repeat domain according to the present invention, one or more amino acid residues of the N-terminal capping module of the repeat domain are replaced by amino acid residues found at corresponding positions when aligning the N-terminal capping unit. Preferably, at most 30% of the amino acid residues are replaced, more preferably at most 20%, and even more preferably at most 10% of the amino acid residues are replaced. Most preferably, such N-terminal capping unit is a naturally occurring N-terminal capping unit.

In another preferred embodiment of the binding protein comprising a repeat domain according to the present invention, one or more amino acid residues of the C-terminal capping module of the repeat domain are replaced by amino acid residues found at corresponding positions when aligning the C-terminal capping unit. Preferably, at most 30% of the amino acid residues are replaced, more preferably at most 20%, and even more preferably at most 10% of the amino acid residues are replaced. Most preferably, such C-terminal capping unit is a naturally occurring C-terminal capping unit.

In another specific embodiment, up to 30% of the amino acid residues, more preferably, up to 20%, and even more preferably, up to 10% of the amino acid residues are replaced with an amino acid not found at the corresponding position in the repeat unit, the N-terminal capping unit or the C-terminal capping unit.

In other embodiments, any of the xSA binding proteins or domains described herein can be covalently bound to one or more additional moieties, including, for example, moieties that bind to different targets to create bispecific binding agents, biologically active compounds, labeling moieties (e.g., fluorescent labels such as fluorescein, or radioactive tracers), moieties that facilitate protein purification (e.g., small peptide tags such as His- or strep-tags), moieties that provide effector functions that provide improved therapeutic efficacy (e.g., antibody Fc portions that provide antibody-dependent cell-mediated cytotoxicity, toxic protein moieties such as Pseudomonas aeruginosa exotoxin A (ETA) or small molecule toxic agents such as maytansinoids or DNA alkylating agents), or moieties that provide improved pharmacokinetics. Improved pharmacokinetics can be assessed based on perceived therapeutic need. It is often desirable to increase bioavailability and/or increase the time between doses, which may be achieved by increasing the time that a protein remains available in the serum following administration. In some cases, it is desirable to improve the continuity of protein serum concentration over time (e.g., to reduce the difference in protein serum concentration between the concentration shortly after administration and the concentration shortly before the next administration).

In another embodiment, the present invention relates to a nucleic acid molecule encoding a specific binding protein, a specific N-terminal capping module or a specific C-terminal capping module. In addition, it also relates to a vector comprising the nucleic acid molecule.

In addition, the present invention relates to a pharmaceutical composition comprising one or more of the above-mentioned binding proteins (especially binding proteins comprising repeat domains), or nucleic acid molecules encoding specific binding proteins, and optionally a pharmaceutically acceptable carrier and/or diluent. Pharmaceutically acceptable carriers and/or diluents are known to those skilled in the art and will be explained in more detail below. Further, the present invention relates to a diagnostic composition comprising one or more of the above-mentioned binding proteins (especially binding proteins comprising repeat domains).

The pharmaceutical composition comprises a binding protein as described above and a pharmaceutically acceptable carrier, excipient or stabilizer, for example as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. [1980]. Suitable carriers, excipients or stabilizers known to those skilled in the art are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose saline, substances that improve isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself cause the production of antibodies that are harmful to the individual receiving the composition, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. The pharmaceutical composition can also be a combination formulation that includes another active agent, such as an anticancer agent or an anti-angiogenic agent.

Preparations to be used for in vivo administration must be sterile or aseptic. This is readily accomplished by filtration through sterile filtration membranes.

The pharmaceutical composition can be administered by any suitable method within the knowledge of the skilled person. The preferred route of administration is parenteral. In parenteral administration, the drug of the present invention is formulated into a unit dose injectable form, such as a solution, suspension or emulsion, in combination with a pharmaceutically acceptable excipient as defined above. The dosage and mode of administration will depend on the individual and specific disease to be treated. In general, the pharmaceutical composition is administered so that the binding protein of the present invention is administered at a dose of 1 μg/kg to 20 mg/kg, more preferably 10 μg/kg to 5 mg/kg, and most preferably 0.1 to 2 mg/kg. It is preferably administered in a bolus dose. Continuous infusion can also be used, including continuous subcutaneous delivery via an osmotic minipump. If so, the pharmaceutical composition can be infused at a dose of 5 to 20 μg/kg/minute, more preferably 7 to 15 μg/kg/minute.

Furthermore, it is contemplated that any of the above-described pharmaceutical compositions may be used in the treatment of a disorder.

The present invention further provides a method of treatment comprising administering a therapeutically effective amount of a binding protein of the present invention to a patient in need thereof.

Furthermore, methods of treating a pathological condition in a mammal, including a human, are contemplated, comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition described above.

Binding proteins according to the present invention can be obtained and/or further evolved by several methods, such as display on the surface of phage (WO 1990/002809, WO 2007/006665) or bacterial cells (WO 1993/010214), ribosome display (WO 1998/048008), plasmid display (WO 1993/008278), or by using covalent RNA-repeat protein hybrid constructs (WO 2000/032823), or for example by intracellular expression and selection/screening by protein complementation assays (WO 1998/341120). Such methods are known to those skilled in the art.

Ankyrin repeat protein libraries for selecting/screening binding proteins according to the present invention can be obtained according to protocols known to those skilled in the art (WO 2002/020565, Binz, HK, et al., J. Mol. Biol., 332 , 489-503, 2003, and Binz et al., 2004, loc. cit.). The use of such libraries for selecting xSA-specific DARPins is described in Example 1. Similarly, the ankyrin repeat sequence motifs described above can be used to create ankyrin repeat protein libraries that can be used to select or screen xSA-specific DARPins. Furthermore, the repeat domains of the invention can be assembled modularly from the repeat modules of the invention and appropriate capping modules or capping repeat sequences using standard recombinant DNA techniques (e.g., WO 2002/020565, Binz et al., 2003, loc. cit. and Binz et al., 2004, loc. cit.) (Forrer, P., et al., FEBS letters 539 , 2-6, 2003).

The present invention is not limited to the specific embodiments described in the examples. Other sources can be used and processed according to the outline described below.

Example

All starting materials and reagents disclosed below are known to those skilled in the art and are either commercially available or can be prepared using well-known techniques.

Material

Chemicals were purchased from Fluka (Switzerland). Oligonucleotides were from Microsynth (Switzerland). Unless otherwise stated, DNA polymerases, restriction enzymes, and buffers were from New England Biolabs (USA) or Fermentas (Lithuania). Cloning and protein preparation strains were Escherichia coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). Purified serum albumin and serum from various species were purchased (e.g., from Sigma-Aldrich, Switzerland or Innovative Research, USA). Biotinylated serum albumin from various species was chemically obtained by coupling the biotin moiety to primary amines of purified serum albumin using standard biotinylation reagents and methods (Pierce, USA).

Molecular Biology

Unless otherwise stated, methods were performed according to the described protocols (Sambrook J., Fritsch E.F. and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, New York).

Designed ankyrin repeat protein library

Ankyrin repeat protein libraries based on N2C and N3C designs are described (WO 2002/020565; Binz et al. 2003, loc. cit.; Binz et al. 2004, loc. cit.). The numbers in N2C and N3C describe the number of randomized repeat modules present between the N-terminal and C-terminal capping modules. The nomenclature used to define repeat units and positions within the modules is based on that of Binz et al. 2004, loc. cit., with the modification that the ankyrin repeat modules and the edges of the ankyrin repeat units are shifted by one amino acid position. For example, position 1 of the ankyrin repeat module of Binz et al. 2004 (loc. cit.) corresponds to position 2 of the ankyrin repeat module of the present disclosure. Thus, position 33 of the ankyrin repeat module of Binz et al. 2004 (loc. cit.) corresponds to position 1 of the next ankyrin repeat module of the present disclosure.

All DNA sequences were confirmed by sequencing, and the calculated molecular weights of all proteins were confirmed by mass spectrometry.

Example 1: Selection of Binding Proteins Comprising Repeated Domains with Binding Specificity for xSA

Using ribosome display (Hanes, J. and Plückthun, A., PNAS 94 , 4937-42, 1997), a number of designed ankyrin repeat proteins (DARPins) with binding specificity for xSA were selected from the N2C or N3C DARPin library described by Binz et al. 2004 (loc. cit.). Selected clones were evaluated for binding to specific (xSA; i.e., MSA, HSA, or CSA) and nonspecific (MBP, E. coli maltose binding protein) targets by crude extract ELISA, indicating successful selection of xSA binding proteins. The repeat domains of SEQ ID NOs: 17-31 constitute the amino acid sequences of the selected binding proteins, which contain repeat domains with binding specificity for xSA. Sequence analysis of the selected binders revealed specific ankyrin repeat sequence motifs inherent to some of the selected binder families. Such ankyrin repeat motifs present in the repeat domains having binding specificity for xSA are provided in SEQ ID NOs: 11-14.

Selection of serum albumin-specific ankyrin repeat proteins by ribosome display

Selection of serum albumin-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Plückthun, loc. cit.) using HSA, CSA, or MSA as target proteins using ankyrin repeat protein libraries designed as described (WO 2002/020565, Binz et al., 2003, loc. cit. and Binz et al., 2004, loc. cit.) and established protocols (Zahnd, C., Amstutz, P. and Plückthun, A., Nat. Methods 4 , 69-79, 2007). Ribosome display selection cycles were performed on HSA, CSA, or MSA (including biotinylated variants of HSA or MSA immobilized with Neutravidin or streptavidin) using both N2C and N3CDARPin libraries using established protocols (Binz et al., 2004, loc. cit.). The number of reverse transcription (RT)-PCR cycles after each selection cycle was constantly reduced from 40 to 30, adjusted for the yield of enriched binders. Four selection cycles for HSA, CSA, or MSA yielded a library of DARPins with micromolar to nanomolar affinities, as revealed by ELISA and SPR measurements of individual clones. The affinity of certain DARPins was further improved using affinity maturation by methods well known to those skilled in the art (e.g., diversification of DARPin clones by error-prone PCR and selection and screening of improved binders as described above).

Selected clones specifically bound serum albumin as shown by crude extract ELISA

Using standard protocols, crude E. coli extracts from DARPin-expressing cells were used to identify each selected DARPin that specifically binds to xSA by enzyme-linked immunosorbent assay (ELISA). Selected clones were cloned into the pQE30 (Qiagen) expression vector by ribosome display, transformed into E. coli XL1-Blue (Stratagene), and cultured overnight at 37°C in a 96-deep-well plate (each clone in a single well) containing 1 ml of growth medium (2YT containing 1% glucose and 100 µg/ml ampicillin). In a new 96-deep-well plate, 100 µl of the overnight culture was inoculated with 1 ml of fresh 2YT containing 50 µg/ml ampicillin. After incubation at 37°C for 2 hours, expression was induced with IPTG (1 mM final concentration) for another 3 hours. Cells were harvested, resuspended in 100 µl of B-PERII (Pierce), and incubated with shaking at room temperature for 15 minutes. 900µl of PBS-TC (PBS supplemented with 0.25% casein hydrolysate, 0.1% Tween 20®, pH 7.4) was then added, and cell debris was removed by centrifugation. 100µl of each lysed clone was applied to the wells of a NeutrAvidin-coated MaxiSorp plate containing xSA or an irrelevant MBP immobilized via its biotin moiety and incubated at room temperature for 1 hour. After extensive washing with PBS-T (PBS supplemented with 0.1% Tween 20®, pH 7.4), the plate was developed using a standard ELISA procedure using a monoclonal anti-RGS(His) ₄ antibody (34650, Qiagen) as the primary antibody and a polyclonal goat anti-mouse antibody conjugated to alkaline phosphatase (A3562, Sigma) as the secondary reagent. Binding was then detected using disodium 4-nitrophenyl phosphate (4NPP, Fluka) as the substrate for alkaline phosphatase. Color development was measured at 405 nm. Screening of several hundred clones by such crude cell extract ELISA revealed over a hundred different DARPins specific for xSA. These binding proteins were selected for further analysis. Examples of amino acid sequences of selected repeat domains that specifically bind to xSA are provided in SEQ ID NOs: 17-31, 37-40, and 43-48.

Inference of repeat sequence motifs from selected repeat domains with binding specificity for xSA

The amino acid sequences of the selected repeat domains with binding specificity for xSA were further analyzed using sequence analysis tools known to those skilled in the art (WO 2002/020565; Forrer et al., 2003, loc. cit.; Forrer, P., Binz, HK, Stumpp, MT and Plückthun, A., ChemBioChem, 5(2) , 183-189, 2004). However, in contrast to WO 2002/020565, in which naturally occurring repeat motifs were used to infer repeat sequence motifs, repeat sequence motifs were inferred from the repeat units of the selected repeat domains with binding specificity for xSA. This resulted in the identification of a family of selected repeat domains containing a common repeat sequence motif. Such repeat sequence motifs present in the repeat domains with binding specificity for xSA are provided in SEQ ID NOs: 11-14.

High-level and soluble expression of DARPins

For further analysis, selected clones that demonstrated specific xSA binding in the crude cell extract ELISA as described above were expressed in Escherichia coli BL21 or XL1-Blue cells and purified using their His-tag using standard protocols. 1 liter of culture (same medium) was inoculated with 25 ml of a stationary overnight culture (LB, 1% glucose, 100 mg/l ampicillin; 37°C). The culture was induced with 0.5 mM IPTG at an absorbance of 0.7 at 600 nm and incubated at 37°C for 4 hours. The culture was centrifuged, and the resulting pellet was resuspended in 40 ml of TBS500 (50 mM Tris–HCl, 500 mM NaCl, pH 8) and sonicated. The lysate was centrifuged again, and glycerol (10% (v/v) final concentration) and imidazole (20 mM final concentration) were added to the resulting supernatant. The protein was purified using a Ni-nitrilotriacetic acid column (2.5 ml column volume) according to the manufacturer's instructions (QIAgen, Germany). Alternatively, DARPins or selected repeat domains without a 6xHis tag can be purified by anion exchange chromatography followed by size exclusion chromatography using standard resins and protocols known to those skilled in the art. Up to 200 mg of highly soluble DARPins with binding specificity for serum albumin can be purified from 1 liter of E. coli culture with a purity estimated at >95% by SDS-15% PAGE. The purified DARPins were used for further characterization.

Example 2: Stability Analysis and Size Exclusion Chromatography of DARPins with Binding Specificity for xSA

DARPins #19-22 and DARPin #27-30, which have binding specificity for xSA, were purified to near homogeneity using their His-tags as described above and stored at 30 mg/ml (approximately 2 mM) in PBS at 40°C for 28 days (stability study). On day 0 (Figure 1a) and day 28 (Figure 1b), samples were removed, diluted to 500 µM, and analyzed by size exclusion chromatography (SEC) to assess their apparent molecular weight and stability over time (i.e., their tendency to aggregate, multimerize, or degrade).

In another experiment (Figure 1c), DARPins #19 and 43-48 with binding specificity for xSA were purified to near homogeneity using their His-tags as described above and stored at -80°C in PBS at approximately 100 mg/ml for 28 days, diluted to 500 µM, and analyzed by size exclusion chromatography (SEC) for characterization (i.e., their tendency to aggregate, multimerize, or degrade). Of note, a larger column was used compared to the first analytical series (see below).

Size Exclusion Chromatography (SEC)

Analytical SEC is carried out at 20 ℃ using an HPLC system (Agilent 1200 series) using a Superdex 200 5/150 column (Fig. 1a and Fig. 1b) or a Superdex 200 10/300GL column (Fig. 1c) (GE Healthcare). The Superdex 200 5/150 column has a bed volume of 3.0 ml and an external water volume of 1.08 ml (determined experimentally using blue dextran). The Superdex 200 10/300GL column has a bed volume of 24 ml and an external water volume of approximately 8 ml. Measured according to standard procedures known to those skilled in the art. At a flow rate of 0.2 ml/min and a maximum pressure of 15 bar (Superdex 200 5/150), or at a flow rate of 0.6 ml/min and a maximum pressure of 18 bar (Superdex 200 10/300GL), run in PBS. Protein samples were diluted to approximately 20-500 µM in PBS, filtered (0.22 µm), and 20-100 µl of the diluted sample was injected onto the column for separation. The elution profile of the protein sample was recorded by reading the absorbance at 280 nm. A calibration curve was generated using aprotinin (AP) with a molecular weight of 6.5 kDa, carbonic anhydrase (CA) with a molecular weight of 29 kDa, and conalbumin (CO) with a molecular weight of 75 kDa as standard proteins. The apparent molecular weight of the sample protein was determined from this calibration curve.

The results are shown in Figure 1. DARPins #19-22 and 27-30 showed indistinguishable SEC chromatograms (i.e., indistinguishable elution profiles) on days 0 and 28 of the stability study. It was concluded that all DARPins eluted as monomers under the assay conditions, and that DARPins #19-23 and 27-30 were stable in PBS at 40°C for at least 1 month (i.e., their elution profiles did not reveal any tendency toward aggregation, multimerization, or degradation).

Example 3: Thermal stability of DARPins with binding specificity for xSA

The thermal stability of DARPins specific for xSA was analyzed using a fluorescence-based thermal stability assay (Niesen, FH, Nature Protocols 2(9) : 2212-2221, 2007). The temperature at which a protein (i.e., such a DARPin) unfolds is measured by the increase in fluorescence of a dye (e.g., SYPRO Orange; Invitrogen, catalog number S6650) that has affinity for the hydrophobic portion of the protein (which is exposed as the protein unfolds). The temperature at the midpoint of the resulting fluorescence transition (from lower fluorescence intensity to higher fluorescence intensity) corresponds to the midpoint denaturation temperature (Tm) of the analyzed protein.

Fluorescence-based thermal stability assay

Thermal denaturation of DARPins was measured using a real-time PCR instrument (i.e., a C1000 Thermal Cycler (BioRad) coupled with a CFX96 optical system (BioRad)) using SYPRO Orange as the fluorescent dye. DARPins were prepared at an 80 µM concentration in either PBS (pH 7.4) or MES buffer (pH 5.8) containing 1x SYPRO Orange (diluted from a 5'000x SYPRO Orange stock solution, Invitrogen), and 50 µl of this protein solution or buffer alone was added to a white 96-well PCR plate (Bio-Rad). The plate was sealed with Microseal 'B' Adhesive Seals (Bio-Rad) and heated in a real-time PCR instrument from 20°C to 95°C in 0.5°C increments, including a 25-second incubation step after each temperature increment. Following thermal denaturation of the DARPins, the relative fluorescence units of the samples at each temperature increment were measured. Using channel 2 of the real-time PCR instrument (i.e., excitation at 515-535 nm and detection at 560-580 nm), the relative fluorescence units in the wells were measured and the corresponding values obtained with buffer alone were subtracted. From the resulting thermal denaturation transition midpoint, the Tm value of the analyzed DARPin can be determined.

The results of thermal denaturation of DARPins in PBS at pH 7.4 or MES buffer at pH 5.8, which was followed by an increase in the fluorescence intensity of SYPRO Orange, are shown in Figures 2 and 3. The measured thermal denaturation transitions confirmed that all analyzed DARPins with binding specificity for xSA had Tm values well above 40°C (at both pH 7.4 and pH 5.8).

Example 4: Characterization of DARPins with Binding Specificity for xSA by Surface Plasmon Resonance Analysis

DARPins with binding specificity for xSA were immobilized via their His-tags to coated α-RGS-His antibodies (Qiagen, Cat. No. 34650) in a flow cell, and the interaction of human, cynomolgus monkey (cyno), mouse, rat, rabbit, and dog serum albumin with the immobilized DARPins was analyzed.

Surface Plasmon Resonance (SPR) Analysis

SPR measurements were performed using a ProteOn instrument (BioRad) according to standard protocols known to those skilled in the art. The running buffer was PBS, pH 7.4, containing 0.01% Tween 20®. Anti-RGS-His antibodies were covalently immobilized on a GLC chip (BioRad) to a level of approximately 2000 resonance units (RU). DARPin immobilization on the antibody-coated chip was then performed by injecting 150 µl of a 1 µM DARPin solution over 300 s (flow rate = 30 µl/min). Interactions with serum albumin from different species were then measured as follows: a 100 µl volume of running buffer (PBS containing 0.01% Tween) containing serum albumin at concentrations of 400 nM, 200 nM, 100 nM, and 50 nM was injected over 60 seconds (on-rate measurement), followed by a 10-30 minute flow of running buffer (flow rate = 100 µl/min) (off-rate measurement). The signals (i.e., resonance unit (RU) values) of an uncoated reference cell and a reference injection (i.e., running buffer alone) were subtracted from the RU traces obtained after serum albumin injection (double referencing). The association and dissociation rates of the corresponding DARPin-serum albumin interactions can be determined from the SRP traces obtained from the association and dissociation rate measurements.

The results are summarized in Tables 1 and 2. The dissociation constant (Kd) was calculated from the estimated association and dissociation rates using standard procedures known to those skilled in the art and was found to be in the range of about 3 nM to about 300 nM. Although all DARPins analyzed bound to human and cynomolgus monkey serum albumin, rabbit, mouse, rat, and dog serum albumin were bound by only a subset of these DARPins.

Example 5: Terminal plasma half-life of DARPins with binding specificity for xSA

The terminal plasma half-life of DARPins in mice and cynomolgus monkeys (also abbreviated as "cyno") is determined according to standard procedures known to those skilled in the art (Toutain et al., loc. cit.). A predetermined amount of DARPin is injected intravenously into the mammals, and the clearance of the DARPin from the plasma is tracked over time by tracking the DARPin plasma concentration. The DARPin concentration initially decreases until a pseudo-equilibrium is reached (the α-phase), followed by an exponential further decrease in the DARPin plasma concentration (the β-phase). The terminal plasma half-life of the DARPin can then be calculated from this β-phase.

Determination of plasma clearance of DARPins in mice

To evaluate the plasma clearance of DARPins with binding specificity for xSA, the test proteins were radiolabeled and injected into the tail vein of naive Balb/c mice. The following DARPins were injected: DARPin #19, DARPin #21, DARPin #23, DARPin #25, DARPin #18, DARPin #32, DARPin #35, DARPin #36, DARPin #33, DARPin #34, DARPin #37, DARPin #38, DARPin #43, DARPin #44, DARPin #45, DARPin #46, DARPin #47, and DARPin #48. DARPins were radiolabeled with ^99m Tc-carbonyl complexes as previously described (Waibel, R., et al., Nature Biotechnol. 17(9) , 897-901, 1999). DARPin (40 µg) was incubated with ^99m Tc-carbonyl (0.8-1.6 m Ci) for 1 hour and then diluted to 400 µl in PBS (pH 7.4). Each mouse was injected intravenously with 100 µl of the resulting labeled DARPin solution (equivalent to 10 µg protein and 0.2-0.4 m Ci). Blood samples were collected from the mice 1, 4, 24, and 48 hours after the initial injection, and the radioactivity in the samples was measured. The radioactivity level measured at a specific time point is a direct measure of the amount of DARPin still present in the plasma at that time point. The % injected dose is the percentage of the total radioactivity measured in the mouse whole blood (1.6 ml for an 18 g mouse) at a specific time point relative to the total radioactivity in the injected sample corrected for radioactive decay of ^99m Tc.

Compared to DARPin #32, which has no binding specificity for xSA, DARPins with binding specificity for MSA had significantly increased terminal plasma half-lives in mice (Figure 4). DARPin #19, DARPin #21, DARPin #23, DARPin #33, DARPin #37, DARPin #43, DARPin #44, DARPin #45, DARPin #46, DARPin #47, and DARPin #48 had terminal plasma half-lives of approximately 2–2.5 days in mice.

Determination of plasma clearance of DARPin in cynomolgus monkeys

DARPins diluted in PBS were injected as a bolus into the cephalic vein of cynomolgus monkeys. The following DARPins were injected: DARPin #26 (0.5 mg/kg), DARPin #24 (0.5 mg/kg), DARPin #17 (0.5 mg/kg), DARPin #34 (1 mg/kg), and DARPin #32 (0.5 mg/kg). At various time points after injection, plasma was prepared from blood drawn from the femoral vein of the animals. DARPin concentrations in the plasma samples were then determined by sandwich ELISA using standard protocols known to those skilled in the art and appropriate DARPin standard curves with known DARPin concentrations.

Cynomolgus monkey plasma samples were serially diluted in PBS-C (PBS containing 0.25% casein, pH 7.4) on MaxiSorp ELISA plates coated with anti-DARPin-specific rabbit monoclonal antibodies. After extensive washing with PBS-T (PBS supplemented with 0.1% Tween 20®, pH 7.4), the plates were developed with a horseradish peroxidase (HRP)-conjugated monoclonal anti-RGS(His)4 antibody (Qiagen). Binding was detected using 100 µl of BM-Blue POD substrate (Roche Diagnostics). The reaction was stopped by adding 50 µl _of 1 M _H₂SO₄ , and the absorbance at 450 nm was measured (and the absorbance at 620 nm was subtracted). The DARPin concentration in the plasma samples was calculated by single exponential regression (GraphPad Prism) against a standard curve of DARPin diluted in monkey serum. The terminal plasma half-life of DARPins was calculated by nonlinear regression (two-phase decay) of concentrations measured up to 240 h after injection. The second (β) phase half-life corresponds to the terminal plasma half-life.

Compared to DARPin #32, which has no binding specificity for xSA, DARPins with binding specificity for xSA had increased terminal plasma half-lives in cynomolgus monkeys ( Figure 4 , Table 3 ). DARPin #19, DARPin #21, DARPin #43, DARPin #44, DARPin #45, DARPin #46, DARPin #47, and DARPin #48 had terminal plasma half-lives of approximately 10-15 days in cynomolgus monkeys.

Example 6: Higher thermal stability of DARPins with improved C-terminal capping modules

As described in Example 3, the thermal stability of DARPins was analyzed using a fluorescence-based thermal stability assay. Alternatively, the thermal stability of a DARPin was analyzed by CD spectrometry; that is, its thermal denaturation was measured by tracking its circular dichroism (CD) signal at 222 nm using techniques well known to those skilled in the art. The sample's CD signal was recorded at 222 nm in a Jasco J-715 instrument (Jasco, Japan) while slowly heating a 0.02 mM concentration of the protein (in PBS, pH 7.4) from 20°C to 95°C using a temperature increment of 1°C/min. This is an effective way to track the denaturation of DARPins, as DARPins are primarily composed of α-helices, which exhibit a strong change in their CD signal at 222 nm upon unfolding. The midpoint of the observed transition of the DARPin's CD signal trace measured in this manner corresponds to its Tm value.

The thermal stability of DARPin #37 (SEQ ID NO: 37 with a His-tag (SEQ ID NO: 15) fused to its N-terminus) was compared to that of DARPin #38 (SEQ ID NO: 38 with a His-tag (SEQ ID NO: 15) fused to its N-terminus) using a fluorescence-based thermal stability assay. These two DARPins have identical amino acid sequences, except for the C-terminal capping module of their repeat domains. The repeat domain of DARPin #38 contains the improved C-capping module described herein, while DARPin #37 does not. The Tm values of DARPin #37 and DARPin #38 were measured in PBS at pH 7.4 at approximately 63°C and approximately 73°C, respectively. The Tm values of DARPin #37 and DARPin #38 were measured in MES buffer at pH 5.8 at approximately 54.5°C and approximately 66°C, respectively.

Using a fluorescence-based thermostability assay, the thermostability of DARPin #39 (SEQ ID NO: 39 with a His-tag (SEQ ID NO: 15) fused to its N-terminus) was compared to that of DARPin #40 (SEQ ID NO: 40 with a His-tag (SEQ ID NO: 15) fused to its N-terminus). These two DARPins have identical amino acid sequences, except for the C-terminal capping module of their repeat domains. The repeat domain of DARPin #40 contains the improved C-capping module described herein, while DARPin #39 does not. The T values of DARPin #39 and DARPin #40 were measured to be approximately 51°C and approximately 55°C, respectively, in MES buffer at pH 5.8.

The thermal stability of DARPin #41 (SEQ ID NO:41) was compared with that of DARPin #42 (SEQ ID NO:42) using CD spectroscopy. These two DARPins share the same amino acid sequence, except for the C-terminal capping module of their repeat domains. The repeat domain of DARPin #42 contains the improved C-capping module described herein, while DARPin #41 does not. The Tm values of DARPin #41 and DARPin #42 in PBS at pH 7.4 were measured to be approximately 59.5°C and approximately 73°C, respectively.

Sequence Listing

<110> Molecular Partners AG

Steiner, Daniel

Binz, Hans Kaspar

Gulotti-Georgieva, Maya

Merz, Frieder W.

Phillips, Douglas

Sonderegger, Ivo

<120> Designed repeat proteins that bind to serum albumin

<130> P393A

<150> EP10192711.9

<151> 2010-11-26

<160> 54

<170> PatentIn version 3.5

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65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Asp Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp

100 105 110

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

115 120 125

<210> 25

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 25

Gly Ser Asp Leu Gly Lys Glu Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Ala Asp Tyr Phe Gly His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn

50 55 60

Ala Ser Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Ala Phe Glu Ile Ser Ile Asp

100 105 110

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

115 120 125

<210> 26

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 26

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Phe Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Ala Asp Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly

35 40 45

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

50 55 60

Ala Gln Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp

100 105 110

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

115 120 125

<210> 27

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 27

Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly

35 40 45

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 28

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 28

Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly

35 40 45

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 29

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 29

Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly

35 40 45

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 30

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 30

Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly

35 40 45

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Ser Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 31

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 31

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Val Asp Ile Trp Gly Asn Thr Pro Leu His Leu Ala Ala Asn Glu Gly

35 40 45

His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn

50 55 60

Ala Leu Asp His Trp Gly Asp Thr Pro Leu His Leu Ala Ala Met Trp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

85 90 95

Asn Ala Leu Asp Asn Asn Gly Phe Thr Pro Leu His Leu Gly Tyr Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

130 135 140

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

145 150 155

<210> 32

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 32

Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Arg Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly

35 40 45

His Pro Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

50 55 60

Ala Ala Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp

100 105 110

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

115 120 125

<210> 33

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 33

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly

145 150 155 160

Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn

165 170 175

Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu

180 185 190

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

195 200 205

Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg

210 215 220

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

225 230 235 240

Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile

245 250 255

Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

260 265 270

<210> 34

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 34

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Ala Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn

50 55 60

Ala Ser Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp

100 105 110

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly

145 150 155 160

Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn

165 170 175

Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu

180 185 190

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

195 200 205

Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg

210 215 220

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

225 230 235 240

Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile

245 250 255

Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

260 265 270

<210> 35

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 35

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly

35 40 45

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp

100 105 110

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly

145 150 155 160

Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn

165 170 175

Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn

180 185 190

Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

195 200 205

Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn

210 215 220

Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp

225 230 235 240

Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala

245 250 255

Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

260 265 270

<210> 36

<211> 302

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 36

Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Arg Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly

35 40 45

His Pro Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

50 55 60

Ala Ala Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp

100 105 110

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly

145 150 155 160

Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn

165 170 175

Ala Val Asp Ile Trp Gly Asn Thr Pro Leu His Leu Ala Ala Asn Glu

180 185 190

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

195 200 205

Asn Ala Leu Asp His Trp Gly Asp Thr Pro Leu His Leu Ala Ala Met

210 215 220

Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp

225 230 235 240

Val Asn Ala Leu Asp Asn Asn Gly Phe Thr Pro Leu His Leu Gly Tyr

245 250 255

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

260 265 270

Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp

275 280 285

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

290 295 300

<210> 37

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 37

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Ala Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn

50 55 60

Ala Ser Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp

100 105 110

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

115 120 125

<210> 38

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 38

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala

20 25 30

Ala Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn

50 55 60

Ala Ser Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp

100 105 110

Asn Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 39

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 39

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala

20 25 30

Ala Asp Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly

35 40 45

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

50 55 60

Ala Gln Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp

100 105 110

Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn

115 120 125

<210> 40

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 40

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala

20 25 30

Ala Asp Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly

35 40 45

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

50 55 60

Ala Gln Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val

85 90 95

Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp

100 105 110

Asn Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 41

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 41

Met Arg Gly Ser His His His His His Gly Ser Asp Leu Gly Lys

1 5 10 15

Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile

20 25 30

Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr

35 40 45

Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu

50 55 60

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly

65 70 75 80

Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala

85 90 95

Glu Ile Leu Gln Lys Leu Asn

100

<210> 42

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 42

Met Arg Gly Ser His His His His His Gly Ser Asp Leu Gly Lys

1 5 10 15

Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile

20 25 30

Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr

35 40 45

Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu

50 55 60

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly

65 70 75 80

Lys Thr Pro Ala Asp Leu Ala Ala Asp Asn Gly His Glu Asp Ile Ala

85 90 95

Glu Val Leu Gln Lys Ala Ala

100

<210> 43

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 43

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 44

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 44

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 45

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 45

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 46

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 46

Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 47

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 47

Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 48

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 48

Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln

1 5 10 15

Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

35 40 45

His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

50 55 60

Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp

65 70 75 80

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

85 90 95

Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp

100 105 110

Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120 125

<210> 49

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 49

Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly

1 5 10 15

His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 50

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 50

Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 51

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 51

Lys Asp Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 52

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 52

Lys Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val Asn

20 25 30

Ala

<210> 53

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (3)..(6)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (14)..(15)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (19)..(19)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (27)..(27)

<223> Xaa can be any naturally occurring amino acid

<400> 53

Xaa Asp Xaa Xaa Xaa Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly

1 5 10 15

His Leu Xaa Ile Val Glu Val Leu Leu Lys Xaa Gly Ala Asp Val Asn

20 25 30

Ala

<210> 54

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (14)..(15)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (27)..(27)

<223> Xaa can be any naturally occurring amino acid

<400> 54

Xaa Asp Phe Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Xaa Gly Ala Asp Val Asn

20 25 30

Ala

Claims (13)

1. A binding protein comprising at least one ankyrin repeat domain, wherein the ankyrin repeat domain is specific for binding to mammalian serum albumin, and wherein the ankyrin repeat domain comprises an ankyrin repeat module consisting of an amino acid sequence selected from the following: SEQ ID NO: 49, 50, 51 and 52. 2. A binding protein comprising at least one ankyrin repeating domain, wherein the repeating domain is specific for binding to mammalian serum albumin, and wherein the ankyrin repeating domain is selected from SEQ ID NO: 17-31 and 43-48. 3. The binding protein of claim 1 or 2, wherein the ankyrin repeat domain has a terminal plasma half-life in mammals that is at least 5 times longer than that of the ankyrin repeat domain of SEQ ID NO:32. 4. The binding protein of claim 1 or 2, wherein the ankyrin repeating domain has a terminal plasma half-life of at least 1 day in humans. 5. The binding protein of claim 1 or 2, wherein the binding protein further comprises a bioactive compound, and wherein the terminal plasma half-life of the binding protein in mammals is at least twice as long as the terminal plasma half-life of the bioactive compound alone, wherein the at least twice as long terminal plasma half-life is conferred by the ankyrin repeating domain of the binding protein. 6. The binding protein of claim 1 or 2, wherein the ankyrin repeat domain competes with the ankyrin repeat domain selected from SEQ ID NO: 17-31 and 43-48 for binding to mammalian serum albumin. 7. The binding protein of claim 1, wherein the ankyrin repeat module comprises the amino acid sequence of SEQ ID NO:50. 8. The binding protein of claim 1, wherein the ankyrin repeat module consists of the amino acid sequence of SEQ ID NO:49. 9. The binding protein of claim 8, wherein the ankyrin repeating domain further comprises ankyrin repeating modules consisting of an amino acid sequence selected from SEQ ID NO:50, and such sequences wherein up to two amino acids in SEQ ID NO:50 are replaced by other amino acids. 10. The binding protein of claim 2, wherein the ankyrin repeat domain is the ankyrin repeat domain of SEQ ID NO: 46. 11. The binding protein of any one of claims 1, 2 and 7-10, wherein the ankyrin repeating domain has a dissociation constant of less than ^10⁻⁷ M with human serum albumin in PBS. 12. A nucleic acid encoding the binding protein as described in any one of claims 1-11. 13. A pharmaceutical composition comprising a binding protein as described in any one of claims 1-11 or a nucleic acid as described in claim 12, and optionally a pharmaceutically acceptable carrier and/or diluent.