HK1213001B - Designed ankyrin repeat proteins binding to hepatocyte growth factor - Google Patents

Description

Designed ankyrin repeat proteins that bind to hepatocyte growth factor

Technical Field

The present invention relates to a designed ankyrin repeat protein with binding specificity to hepatocyte growth factor (HGF), a nucleic acid encoding the HGF binding protein, a pharmaceutical composition containing the protein, and use of the protein in treating diseases.

Background Art

The MET proto-oncogene encodes a receptor tyrosine kinase (MET or c-Met), also known as the hepatocyte growth factor receptor (HGFR; human HGFR has UniProtKB/SWISS-PROT No. P08581), which is essential for embryonic development and wound healing. Hepatocyte growth factor (HGF, also known as scatter factor; human HGF has UniProtKB/SWISS-PROT No. P14210) is the only known ligand for c-Met. This receptor is normally expressed by cells of epithelial origin, whereas HGF expression is restricted to cells of mesenchymal origin. When stimulated by HGF, c-Met induces multiple biological responses that collectively initiate a process known as invasive growth. Aberrant c-Met activation in cancer is associated with poor prognosis, with abnormally active c-Met triggering tumor growth, tumor cell survival, angiogenesis, and metastasis. c-Met is unregulated in many types of human malignancies, leading to kidney cancer, liver cancer, stomach cancer, breast cancer, and brain cancer. It is also a major factor in resistance to other targeted therapies (Bottaro, D.P., Rubin, J.S., Faletto, D.L., Chan, A.M., Kmiecik, T.E., Vande Woude, G.F. and Aaronson, S.A., Science 251(4995), 802-804, 1991; Comoglio, P.M, Giordano, S. and Trusolino, L., Nat. Rev. Drug Discov. 7(6), 504-516, 2008).

HGF is secreted as a single, inactive, single-chain precursor polypeptide (sc-HGF, sometimes also referred to as pro-HGF) and is cleaved by serine proteases into a 69 kDa α chain and a 34 kDa β chain. Active HGF is a disulfide-linked heterodimer consisting of the α and β chains. HGF has a high degree of homology to coagulation factors, as its α chain contains a plasminogen-like "kringle" structural motif, and the β chain contains a homology domain to serine proteases, but lacks enzymatic activity (Naldini, L., Weidner, K.M., Vigna, E., Gaudino, G., Bardelli, A., Ponzetto, C., Narsimhan, R.P., Hartmann, G., Zarnegar, R., Michalopoulos, G.K., et al., EMBO J. 10, 2867-2878, 1991).

HGF, through binding to its receptor, mediates several cellular responses, including the dispersion of various cell types, tubule and lumen formation, epithelial-mesenchymal transition, angiogenesis, liver regeneration, wound healing, and embryonic development. The HGF/c-Met signaling pathway is also known to be involved in tumor progression, invasion, and metastasis in various diseases, including many human solid tumors. In human cancers, paracrine and autocrine activation of c-Met by HGF, tumor expression of these molecules, and elevated circulating HGF concentrations are associated with poor prognosis and an increased risk of tumor metastasis (Comoglio et al., 2008, loc. cit.).

Because HGF plays an active role in the progression of cancer and other diseases, agents that block HGF activity could be beneficial in counteracting its effects. Various anti-HGF (aHGF) monoclonal antibodies (mAbs) are in clinical development, including AMG102 (rilotumumab, a humanized anti-human HGF IgG2 from Amgen), AV 299 from Schering/Aveo, and TAK-701 from Millennium.

In addition to antibodies, there are novel binding proteins or binding domains that can be used to specifically bind to target molecules, thereby acting as antagonists (e.g. Binz, H.K., Amstutz, P. and Plückthun, A., Nat. Biotechnol. 23, 1257-1268, 2005). Such novel binding proteins or binding domains without Fc are based on designed repeat proteins or designed repeat domains (WO 2002/020565; Binz, H.K., Amstutz, P., Kohl, A., Stumpp, M.T., Briand, C., Forrer, P., Grütter, M.G., and Plückthun, A., Nat. Biotechnol. 22, 575-582, 2004; Stumpp, M.T., Binz, H.K and Amstutz, P., Drug Discov. Today 13, 695-701, 2008). WO 2002/020565 describes how to construct large libraries of repeat proteins and their common applications. However, WO 2002/020565 does not disclose screening for repeat domains with HGF binding specificity, nor does it disclose specific repeat modules or repeat sequence motifs for repeat domains that specifically bind to HGF. Furthermore, WO 2002/020565 does not suggest that repeat domains with HGF binding specificity can be used to modulate HGF-mediated signaling pathways, thereby successfully treating diseases. These designed repeat domains exploit the modular nature of repeat proteins and can include N-terminal and C-terminal capping modules to prevent aggregation of the designed repeat domains by shielding the hydrophobic core of the domain (Forrer, P., Stumpp, M.T., Binz, H.K., and Plückthun, A., FEBS Letters 539, 2-6, 2003).

The present invention addresses the technical problem of identifying novel binding proteins with HGF binding specificity, such as ankyrin repeat proteins or domains, for use in modulating HGF-mediated signaling pathways to improve the treatment of certain cancers, vascular diseases, angiogenesis-related diseases, and other pathological conditions. The present invention addresses this technical problem by providing the embodiments recited in the claims.

Summary of the Invention

The present invention relates to a recombinant binding protein comprising at least one ankyrin repeat domain, wherein the ankyrin repeat domain binds to HGF in PBS (phosphate buffered saline) with a Kd lower than 10 ⁻⁷ M.

More specifically, the present invention relates to a recombinant binding protein comprising at least one ankyrin repeat domain that competes with an ankyrin repeat domain selected from SEQ ID NOs: 33 to 61 for binding to HGF.

The present invention also relates to a recombinant binding protein comprising at least one ankyrin repeat domain having binding specificity for HGF and comprising an ankyrin repeat module having an amino acid sequence selected from SEQ ID NOs: 12 to 25 and a sequence in which up to 9 amino acids in SEQ ID NOs: 12 to 25 are replaced by any amino acid.

In particular, the present invention relates to a recombinant binding protein comprising at least one ankyrin repeat domain, wherein the ankyrin repeat domain comprises at least one amino acid sequence having at least 90% amino acid sequence identity with any one ankyrin repeat domain selected from SEQ ID NOs: 33 to 61, wherein the G at position 1 and/or the S at position 2 of the ankyrin repeat domain are optionally deleted; and the L at the penultimate position and/or the N at the last position of the ankyrin repeat domain are optionally replaced by A.

In particular, the present invention relates to an HGF-binding recombinant protein comprising a peptide of any one of SEQ ID NOs: 1 to 25 and 33 to 62.

The present invention also relates to nucleic acids 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 also relates to a method of treating pathological conditions using the binding proteins of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Surface plasmon resonance analysis of selected DARPins.

Surface plasmon resonance (SPR) analysis of DARPin binding to HGF (using DARPin #51 as an example). DARPin at various concentrations (1.6, 3.1, 6.3, 12.5, and 25 nM) was added to a flow cell immobilized with human HGF for 120 seconds, followed by a buffer wash. The resulting traces were globally fitted to yield a Kd value of 51 pM for the interaction between DARPin #51 and human HGF. RU, resonance unit; s, time (seconds). See the definition of DARPin #51 below.

RU,Resonance Units;s,time in seconds;D1,DARPin#51;D2,DARPin#57;D3,DARPin#33.See below for the definition of DARPin#51,DARPin#57and DARPin#43.

Figure 2. Competitive surface plasmon resonance analysis.

SPR analysis of DARPin binding to human HGF (using DARPin #51, DARPin #57, and DARPin #43 as examples). 100 nM of DARPin #51 (i.e., D1) was added to a flow cell immobilized with human HGF for 120 seconds, followed by the injection of a second DARPin (DARPin #57 or DARPin #33; i.e., D2 or D3, respectively) at 100 nM for 60 seconds. Therefore, D1 and D3 compete for HGF binding, while D1 and D2 do not.

RU, resonance unit; s, time (seconds); D1, DARPin #51; D2, DARPin #57; D3, DARPin #33. See below for definitions of DARPin #51, DARPin #57, and DARPin #43.

Figure 3. Inhibition of HGF-stimulated c-Met phosphorylation

The figure shows the inhibitory effects of various concentrations of DARPins specific for human HGF (using DARPins #42 and #48 as examples) on HGF-stimulated c-Met phosphorylation in A549 cells, along with the corresponding fitted inhibition curves (using DARPins #42 and #48 as examples). The _IC50 values for DARPins #42 and #48 were determined to be 0.32 and 0.1 nM, respectively.

OD, optical density at 450-620 nm; C, DARPin concentration (nM); D1, DARPin #48; D2, DARPin #42; MA, maximum activation; MI, minimum activation. The X-axis is a logarithmic scale. See the definitions of DARPin #52 and DARPin #48 below.

Figure 4. Inhibition of U87 cell proliferation.

The figure shows the inhibition of U87 malignant glioma cell proliferation by DARPins with specific inhibitory effects on HGF at various concentrations and the corresponding fitted inhibition curves (using DARPin #51 and #43 as examples). The measured _IC50 values of DARPin #51 and #43 were 72 and 116 nM, respectively.

OD, optical density at 450-620 nm; C, DARPin concentration (nM); D1, DARPin #51; D2, DARPin #43. The X-axis is shown on a logarithmic scale. See below for definitions of DARPin #51 and DARPin #43.

Figure 5. c-Met competition experiment.

Shown are the inhibition of HGF-c-Met binding by various concentrations of human HGF-specific DARPins from a single experiment and the corresponding fitted inhibition curves. The calculated _IC50 values for DARPins #43 and #51 were approximately 1.36 and 0.62 nM, respectively. OD, optical density at 450-620 nm; C, DARPin concentration (nM); D1, DARPin #51; D2, DARPin #43. The X-axis is a logarithmic scale. See below for definitions of DARPins #51 and 43.

Figure 6. Efficacy of anti-HGF DARPin vs. vehicle in the U87 tumor xenograft model.

U87-MG tumor cells were injected subcutaneously into nude mice. Tumor-bearing animals were randomly divided into four groups and treated with different concentrations of PEGylated DARPin (intravenously three times per week, for a total of seven doses). PEGylated DARPin was generated as described in Example 6.

T, tumor volume (mm ³ ); d, time (day); V, vehicle (i.e., PBS); D1, PEGylated DARPin#62, the dose per injection was 0.11 mg/kg; D2, the dose per injection was 1.1 mg/kg; D3, the dose per injection was 11 mg/kg.

Figure 7. Multiple sequence alignment of SEQ ID NOs: 12 to 28. SEQ ID NOs: 12 to 28 were aligned based on residue position. The ankyrin repeat residue position numbers are indicated at the top. Residue positions 2, 5, 7-13, and 16-33 correspond to positions that typically contain framework residues. Residue positions 1, 3, 4, 6, 14, and 15 correspond to positions that may contain target interacting residues.

DETAILED DESCRIPTION

The recombinant binding protein or domain of the present invention is specific for mammalian HGF. Preferably, the recombinant binding protein of the present invention is specific for HGF of mouse, rat, dog, rabbit, monkey, or human origin. More preferably, the recombinant binding protein of the present invention is specific for HGF of human origin.

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

The term "recombinant" as used in recombinant proteins, recombinant protein domains, recombinant binding proteins, etc., 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., produced by gene synthesis) can be cloned into a bacterial expression plasmid (e.g., pQE30, Qiagen), a yeast expression plasmid, or a mammalian expression plasmid. For example, when the constructed recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., Escherichia coli), the bacterium can produce the polypeptide encoded by the recombinant DNA. The polypeptide produced is referred to as a recombinant polypeptide.

In the context of the present invention, the term "polypeptide" refers to a polypeptide consisting of one or more chains of multiple (i.e. two or more) amino acids linked by peptide bonds. Preferably, the polypeptide consists of eight or more 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 is used for purification, detection or targeting of the polypeptide/protein, or wherein the amino acid sequence enhances the physicochemical behavior of the polypeptide/protein, or wherein the amino acid sequence has an effector function. The various polypeptide tags, parts and/or domains of the binding protein can be linked to each other directly or via a polypeptide linker. These polypeptide tags are well known in the art and are fully accessible to those skilled in the art. Examples of polypeptide tags include small polypeptide sequences, such as histidine (His) (e.g., the His tag of SEQ ID NO: 9), proto-oncogenes (myc) and FLAG, or Streptococcus (Strep) tags or parts, such as enzymes (e.g., endonucleases like alkaline phosphatase), which allow the polypeptide/protein or part to be detected, can be used for targeting (e.g., immunoglobulins or fragments thereof) and/or act as effector molecules.

The term "polypeptide linker" refers to an amino acid sequence that can connect, 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 include glycine-serine linkers and proline-threonine linkers of variable length; preferably, the length of the linker is between 2 and 24 amino acids; more preferably, the length of the linker is between 2 and 16 amino acids. An example of a glycine-serine linker is shown in SEQ ID NO: 10, and an example of a proline-threonine linker is shown in SEQ ID NO: 11. Preferably, the proline-threonine linker shown in SEQ ID NO: 11 is preceded and/or followed by GS.

The term "polymer moiety" refers to a protein polymer moiety or a non-protein polymer moiety. A "protein polymer moiety" is preferably a polypeptide that does not form a stable tertiary structure. Examples of protein polymer moieties include (a registered trademark of Amunix; WO 2007/10351) polypeptides, or polypeptides comprising proline, alanine, and serine residues as described in WO 2008/155134. The protein polymer moiety can be covalently linked to, for example, a binding domain of the present invention by using standard DNA cloning techniques to produce a gene fusion polypeptide, followed by standard expression and purification. A "non-protein polymer moiety" is a polymer moiety that is not constructed from a polypeptide. Examples of non-protein polymer moieties include hydroxyethyl starch (HES), polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes. The term "pegylated" (PEGylated) refers to a PEG moiety covalently linked to, for example, a polypeptide of the present invention. The molecular weight of the polymer moiety of the present invention can vary widely. Preferably, the polymer moiety is linked to the binding domain by a polypeptide linker.

In a specific embodiment, a PEG moiety or any other non-protein polymer can be attached, for example, via a maleimide linker, to a cysteine thiol, wherein the cysteine is attached to the N-terminus 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 will be further explained below. Preferably, the binding protein comprises a maximum of four binding domains. More preferably, the binding protein comprises a maximum of two binding domains. Most preferably, the binding protein comprises only one binding domain. In addition, any of the binding proteins may include additional protein domains that are not binding domains, multimerization moieties, polypeptide tags, polypeptide linkers and/or one or more Cys residues. Examples of multimerization moieties include immunoglobulin heavy chain constant regions that pair to provide a functional immunoglobulin Fc domain, and leucine zippers or polypeptides that include free thiols that form intermolecular disulfide bonds between two of the polypeptides. Cys residues can be used to attach 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 targeting specificities.

The term "biologically active compound" refers to a compound that, when administered to a mammal suffering from the disease, is capable of disease modulation. The biologically active compound may have antagonistic or agonistic properties and may be a proteinaceous biologically active compound or a non-proteinaceous biologically active compound. The proteinaceous biologically active compound may, for example, be covalently linked to, for example, a binding domain of the present invention by generating a gene fusion polypeptide using standard DNA cloning techniques, followed by standard expression and purification. The non-proteinaceous biologically active compound may be covalently linked to, for example, a binding domain of the present invention by chemical means, for example, by linking to a cysteine thiol via a maleimide linker, wherein the cysteine is linked to the N-terminus or C-terminus of the binding domain described herein via a peptide linker, as described herein. Examples of proteinaceous biologically active compounds include domains with different targeting specificities (e.g., neutralizing growth factors by binding), cytokines (e.g., interleukins), growth factors (e.g., growth hormone), antibodies and fragments thereof, hormones (e.g., GLP-1), toxins (e.g., Pseudomonas aeruginosa exotoxin A), and any potential protein drug. Examples of non-protein bioactive compounds are toxins (eg DM1 from immunogens), small molecules targeting GPCRs, antibiotics and any possible non-protein drugs.

The term "binding domain" refers to a protein domain that exhibits the same "fold" (three-dimensional structure) as a protein backbone and has a predetermined property defined as follows. The binding domain can be obtained by deduction, or most commonly, by combined protein engineering techniques and skills known in the art (Binz et al., 2005, loc.cit.). For example, a binding domain with a predetermined property can be obtained by the following method, comprising the following steps: (a) providing a diversity set of protein domains that exhibit the same folding as the protein backbone as described below; and (b) screening and/or selecting from the diversity set to obtain at least one protein domain with the predetermined property. The diversity set of protein domains can be provided by several methods, according to the screening and/or selection system used, and can include utilizing methods well known to those skilled in the art, such as phage display or ribosome display methods. Preferably, the binding domain is a recombinant binding domain.

The term "protein scaffold" refers to a protein with exposed surface areas 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 include antibodies or fragments thereof, such as single-chain Fv or Fab fragments, protein A from Staphylococcus aureus, biletriene 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 "target" refers to an individual molecule, such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other natural 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 it can be any non-natural molecule or portion. Preferably, the target is a natural 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 HGF.

The term "predetermined property" refers to a property, such as binding to a target, blocking a target, activating a target-mediated reaction, enzymatic activity, and other related characteristics. Depending on the type of desired property, one of ordinary skill will be able to determine 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 definition of repeat proteins in the following text is based on the definition in patent application WO 2002/020565. Patent application WO 2002/020565 also 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 a maximum of four repeat domains. More preferably, each of the repeat proteins comprises a maximum of two repeat domains. Most preferably, each repeat protein comprises only one repeat domain. In addition, 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 form a supercoiled structure with a common hydrophobic core. Preferably, the repeat domain further comprises an N-terminal and/or C-terminal capping unit (or module). 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 according to the inventive method as described in patent application WO 2002/020565. Designed repeat proteins and designed repeat domains are synthetic proteins, not natural proteins. They are artificial proteins or domains, respectively, obtained by expressing corresponding designed nucleic acids. Preferably, the expression is carried out in eukaryotic or prokaryotic cells, such as bacterial cells, or by using a cell-free in vitro expression system. Therefore, a designed ankyrin repeat protein (i.e., DARPin) corresponds to a recombinant binding protein comprising at least one ankyrin repeat domain in the present invention.

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 structure of a secondary structure element 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 structure; however, their continuous organization, for example as a repeating module in a repeat domain, can stabilize adjacent units in a supercoiled structure.

The term "repeat unit" refers to an amino acid sequence comprising one or more repeating sequence motifs of a naturally occurring repeating protein, wherein the "repeat unit" exists in multiple copies and all of the motifs that define the folding of the protein have a common, defined folding topology. The repeat unit corresponds to a "repeat structural unit (repeat sequence)" of a repeating protein as described by Forrer et al., 2003, loc. cit., or to a "continuous homologous structural unit (repeat sequence)" of a repeating protein as described by Binz et al., 2004, loc. cit. The repeat unit comprises backbone residues and interacting residues. Examples of the repeat unit include armadillo repeat units, leucine-rich repeat units, ankyrin repeat units, tetratricopeptide repeat units, HEAT repeat units, and repeat units of leucine-rich variants. A naturally occurring protein containing two or more of these repeat units is referred to as a "naturally occurring repeat protein." When compared to one another, the amino acid sequences of each repeat unit of a repeating protein 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.

Thus, the term "ankyrin repeat unit" refers to a repeating unit, for example ankyrin repeat sequences according to Forrer et al., 2003, loc. cit. Ankyrin repeat sequences are well known to those skilled in the art. The term "ankyrin repeat domain" refers to a repeating domain comprising two or more consecutive ankyrin repeat units (modules) as structural units, and preferably comprising an N-terminal and/or C-terminal capping unit (or module).

The term "backbone residue" refers to an amino acid residue of a repeating unit or a corresponding amino acid residue of a repeating module that contributes to the folding topology, i.e., it contributes to the folding of the repeating unit (or module) or to the interaction with an adjacent unit (or module). The contribution can be an interaction with other residues in the repeating unit (or module), or an influence on the polypeptide backbone conformation, such as an alpha helix or a beta sheet, or an amino acid stretch that forms a linear polypeptide or a ring.

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 contributes to the interaction with the target substance. The contribution can be a direct interaction with the target substance, or an influence on other directly interacting residues, such as by stabilizing the conformation of the polypeptide of the repeating unit (or module) to allow or improve the interaction between the directly interacting residue and the target. The skeleton and target interaction residues can be analyzed and identified by structural data obtained by physicochemical methods (such as X-ray crystallography, NMR and/or CD spectral analysis), or by comparison with relevant structural information known to structural biology and/or bioinformatics technicians.

Preferably, the repeat units used to derive repeat sequence motifs are homologous repeat units, wherein the repeat units comprise the same structural motif and wherein more than 70% of the backbone residues in the repeat units are homologous to each other. Preferably, more than 80% of the backbone residues in the repeat units are homologous. Most preferably, more than 90% of the backbone residues in the repeat units are homologous. Computer programs such as Fasta, Blast or Gap for determining percent homology between polypeptides are well known to those skilled in the art. More preferably, the repeat units used to derive repeat sequence motifs are homologous repeat units of repeat domains selected from a determined target.

The term "repeat sequence motif" refers to an amino acid sequence derived from one or more repeating units or repeating modules. Preferably, the repeating unit or repeating module is a repeating domain with binding specificity for the same target. The repeating sequence motif includes backbone residue positions and target interaction residue positions. The backbone residue positions correspond to the positions of the backbone residues of the repeating unit (or module). Typically, positions 2, 5, 7-13 and 16-33 of the ankyrin repeat sequence contain backbone residues. The position numbering of the ankyrin repeat sequence is shown in Figure 7. Resti, the target interaction residue positions correspond to the positions of the target interaction residues of the repeating unit (or module). Typically, positions 1, 3, 4, 6, 14, and 15 of the ankyrin repeat sequence may contain target interaction residues. The repeating sequence motif includes fixed positions and random positions. The term "fixed position" refers to an amino acid position in the repeating sequence motif that is set to a specific amino acid. In most cases, the fixed position corresponds to the position of the backbone residue and/or target interaction residue with a specific target specificity. The term "random position" refers to an amino acid position in a repeat sequence motif that allows the presence of two or more amino acids. For example, any one of the twenty natural amino acids can generally be allowed, or most of the twenty natural amino acids can be allowed, such as amino acids other than cysteine, or other than glycine, cysteine, and proline. In most cases, the random position corresponds to the position of the target interaction residue. However, some positions of the framework residues can also be randomized.

The term "folding topology" refers to the tertiary structure of a repeating unit or module. The folding topology is determined by stretches of amino acids that form at least a partial α-helix or β-sheet, or stretches of amino acids that form a linear polypeptide or a circular structure, or any combination of α-helices, β-sheets, and/or linear polypeptide/circular structures. For example, an ankyrin repeat unit/module includes a β-turn, followed by two antiparallel α-helices, and a circular structure that connects the turn to the next repeating unit/module.

The term "continuous" refers to a structure in which the repeating units or repeating units are arranged in series. In the design of repeat proteins, there are at least 2, usually about 2 to 6, in particular at least about 6, and often 20 or more repeating units (or modules). In most cases, the repeating units (or modules) of the repeat domain show a high degree of sequence identity (the amino acid residues in the corresponding positions are the same) or a high degree of sequence similarity (the amino acid residues are different, but the physicochemical properties are similar), and some of the amino acid residues may be highly conserved key residues. However, as long as the repeating units (or modules) also maintain a common folding topology, it is possible that a high degree of sequence variability may result from amino acid insertions and/or deletions between different repeating units (or modules) of the repeat domain.

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

The term "repeat module" refers to the repetitive amino acid sequence of a designed repeat domain, which is originally derived from the repeat unit of a natural repeat protein. Each repeat module contained in the repeat domain is derived from one or more repeat units of a natural repeat protein family or subfamily, for example, the armadillo repeat protein or ankyrin repeat protein family. More preferably, each repeat module contained in the repeat domain comprises a repeat sequence motif derived from homologous repeat units obtained by screening for the same target (e.g., HGF), as described in Example 1.

Thus, the term "ankyrin repeat module" refers to a repeat module originally derived from the repeating unit of the natural ankyrin repeat protein. Ankyrin repeat proteins are well known to those skilled in the art.

A "repeat unit" can include amino acid residue positions that are present in all copies of the corresponding repeat module ("fixed positions"), as well as positions that have different or "randomized" amino acid residues ("random positions").

The term "capping module" refers to a polypeptide fused to a repeating module at the N or C terminus of a repeating domain, wherein the capping module forms a tight tertiary interaction (i.e., an interaction of the tertiary structure) with the repeating module, thereby providing a cap, and the cap shields the hydrophobic core of the repeating module from the side that is not in contact with the consecutive repeating modules, so that it is not in contact with the solvent. The N and/or C terminal capping module can be, or can be derived from, a capping unit or other structural unit present in a natural repeating protein adjacent to the repeating unit. The term "capping unit" refers to a naturally folded polypeptide, wherein the polypeptide defines a specific structural unit fused to a repeating unit at the N or C terminus, wherein the polypeptide form forms a tight tertiary interaction with the repeating unit, thereby providing a cap, and the cap shields the hydrophobic core of the repeating unit from the side that is not in contact with the consecutive repeating modules, so that it is not in contact with the solvent. Preferably, the capping module or capping unit is a capping repeat sequence. The term "capping repeat" refers to a capping module or capping unit that has a similar or identical fold to the adjacent repeat unit (or module) and/or has sequence similarity to the adjacent repeat unit (or module). Capping modules and capping repeat sequences can be found in WO 2002/020565, WO 2012/069655 and Interlandi et al., 2008 (loc. cit.). Examples of N-terminal ankyrin capping modules (i.e., N-terminal capping repeat sequences) include SEQ ID NOs: 1 to 3, and examples of ankyrin C-terminal capping modules (i.e., C-terminal capping repeat sequences) include SEQ ID NOs: 4 to 8.

For example, the N-terminal ankyrin capping module of SEQ ID NO: 33 is encoded by amino acids at positions 1 to 32, and the C-terminal capping module of SEQ ID NO: 33 is encoded by amino acids at positions 132 to 159.

The recombinant binding protein of the present invention comprises at least one ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for mammalian HGF.

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

Recombinant binding proteins comprising an ankyrin repeat domain with HGF binding specificity are shown in the Examples.

In particular, the present invention relates to a recombinant binding protein as defined herein comprising at least one ankyrin repeat domain, wherein said ankyrin repeat domain specifically binds to HGF (Hepatocyte Growth Factor).

Specific binding refers to the selective affinity of the ankyrin repeat domains described herein for HGF. All ankyrin repeat domains or epitopes thereof that bind to HGF are considered to specifically bind to that substance. Thus, all ankyrin repeat domains that bind to HGF have specific binding to HGF.

Preferably, the ankyrin repeat domain binds to HGF in PBS with a dissociation constant (Kd) lower than 10 ⁻⁷ M; more preferably, lower than 10 ⁻⁸ M, 10 ⁻⁹ M or 10 ⁻¹⁰ M, or most preferably lower than 10 ⁻¹¹ M.

The method for the dissociation constant of the protein-protein interaction determined, such as surface plasmon resonance (SPR)-based technology (such as SPR equilibrium or kinetic analysis) or isothermal titration calorimetry (ITC), is well known to those skilled in the art. The Kd value measured for the specific protein-protein interaction measured under different conditions (such as, salt concentration, pH value) may vary. Therefore, the measurement of the Kd value preferably uses protein standard solution and standard buffer, such as PBS.

Example 2 shows a possible method for determining the IC ₅₀ value of the ankyrin repeat sequence for inhibiting the binding of HGF to c-Met, and a recombinant binding protein comprising ankyrin repeat domain that binds to HGF with a Kd value of less than 10 ⁻⁷ M in PBS.

Preferred are recombinant binding proteins comprising an ankyrin repeat domain having binding specificity for human HGF.

More preferred are recombinant binding proteins comprising an ankyrin repeat domain comprising 70 to 300 amino acids, particularly 90-200 amino acids.

The binding domain of the present invention is an ankyrin repeat domain or a designed ankyrin repeat domain, preferably as described in WO 2002/020565. Examples of ankyrin repeat domains with HGF binding specificity are described in the Examples.

In yet another embodiment, the present invention relates to an ankyrin repeat domain comprising at least one mammalian HGF binding specific ankyrin repeat domain, wherein the ankyrin repeat domain inhibits the binding of HGF to c-Met in PBS with an IC ₅₀ value of less than 10 ⁻⁷ M. Preferably, the ankyrin repeat domain inhibits the binding of HGF to c-Met in PBS with an IC ₅₀ value of less than 10 ⁻⁸ M, more preferably less than 10 ⁻⁹ M, 10 ⁻¹⁰ M, or most preferably less than 10 ⁻¹¹ M.

The half maximal inhibitory concentration ( _IC50 ) is a measure of the effectiveness of a compound (e.g., a binding domain of the present invention) in inhibiting a biological, biochemical, or biophysical function. Methods for determining the _IC50 for inhibition of protein-protein interactions, such as competitive ELISA, are well known to those skilled in the art. The _IC50 values of specific inhibitors of protein-protein interactions measured under different conditions (e.g., salt concentration, pH) may vary. Therefore, the _IC50 values are preferably measured using a protein standard solution and a standard buffer, such as PBS.

Example 5 shows a possible method for determining the IC ₅₀ value of the ankyrin repeat sequence for inhibiting the binding of HGF to c-Met, and can identify and recombinant binding proteins, and recombinant binding proteins comprising ankyrin repeat domains that bind to HGF with a Kd value of less than 10 ^-7 M in PBS.

In yet another embodiment, the present invention relates to recombinant binding proteins comprising at least one HGF-binding ankyrin repeat domain that inhibits c-Met phosphorylation in HGF-stimulated A549 cells (ATCC, Product No. CCL-185). A549 cells were starved for 24 hours and stimulated with HGF in the presence of a titration of an ankyrin repeat domain specific for HGF. The ability of the compounds of the present invention to inhibit cMet phosphorylation in HGF-stimulated A549 cells was measured using standards known to those skilled in the art. Example 4 illustrates a method for determining the _IC50 for inhibition of c-Met phosphorylation by the ankyrin repeat domain or protein, as well as recombinant binding proteins comprising ankyrin repeat domains that exhibit an _IC50 value of less than 10^{^-7} M for inhibition of c-Met phosphorylation in HGF-stimulated A549 cells.

Preferably, the repeat domain has an IC ₅₀ value for inhibition of HGF- ^{stimulated c-Met phosphorylation in A549 cells of less than 10 −8 M} , more preferably less than 10 ⁻⁹ M, 10 ⁻¹⁰ M, or most preferably less than 10 ⁻¹¹ M.

In another embodiment, the present invention relates to a recombinant binding protein comprising at least one ankyrin repeat domain having binding specificity for HGF, which inhibits HGF-stimulated U87 cell proliferation (ATCC, Product No.: HTB-14) with an IC ₅₀ value of less than 10 ⁻⁶ M. Preferably, the repeat domain inhibits HGF-stimulated U87 cell proliferation with an IC ₅₀ value of less than 10 ⁻⁷ M, more preferably less than 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, or most preferably less than 10 ⁻¹¹ M.

U87 cells can grow in response to HGF and can therefore be used to measure the functional inhibitory ability of the compounds of the present invention. U87 cells were cultured in culture medium and seeded in DMEM containing 1% FBS (experimental medium). After 24 hours of cell culture, HGF was added and titrated with anti-HGF DARPin. The ability of the compounds of the present invention to inhibit HGF was assessed by measuring the proliferation capacity of U87 cells using standard measurement methods known to those skilled in the art. Example 4 shows a method for determining the _IC50 value of the ankyrin repeat protein for inhibiting U87 cell proliferation, as well as a recombinant binding protein containing ankyrin repeat domain with an _IC50 value of less than ^10-6 M for inhibiting U87 cell proliferation.

The present invention relates to a recombinant binding protein comprising at least one ankyrin repeat domain with HGF binding specificity, wherein the binding protein and/or the ankyrin repeat domain has a midpoint denaturation temperature (Tm) of heat-induced unfolding in PBS higher than 40°C and forms less than 5% (w/w) insoluble aggregates after incubation in PBS at 37°C for 1 day at a concentration of up to 10 g/L.

The term "PBS" refers to a phosphate-buffered saline solution containing 137 mM NaCl, 10 mM phosphoric acid, and 2.7 mM KCl at a pH of 7.4.

Preferably, the midpoint denaturation temperature (Tm) of the recombinant binding protein and/or binding domain during heat-induced unfolding in PBS at pH 7.4 is higher than 45°C, more preferably higher than 50°C, more preferably higher than 55°C, and most preferably higher than 60°C. The binding protein or binding domain of the present invention has a defined secondary and tertiary structure under physiological conditions. The heat-induced unfolding of the polypeptide causes it to lose its tertiary and secondary structure, which can be subsequently measured, for example, by circular dichroism (CD). The midpoint denaturation temperature of the binding protein or binding domain during heat-induced unfolding corresponds to the cooperative transition midpoint temperature when the protein or domain undergoes heat denaturation in a physiological buffer at a temperature slowly increased from 10°C to about 100°C. The determination of the midpoint denaturation temperature of heat-induced unfolding is well known to those skilled in the art. The midpoint denaturation temperature of the binding protein or binding domain during heat-induced unfolding is indicative of the thermal stability of the polypeptide.

It is also preferred that the recombinant binding protein and/or ankyrin repeat domain form less than 5% (w/w) insoluble aggregates after incubation 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, more preferably up to 80 g/L, and most preferably up to 100 g/L. The formation of insoluble aggregates can be detected by visible precipitation, which increases significantly when insoluble aggregates form, gel filtration, or dynamic light scattering. Insoluble aggregates can be removed from the protein sample by centrifugation at 10,000 x g for 10 minutes. Preferably, the recombinant binding protein and/or ankyrin repeat 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 after incubation in PBS at 37°C under the above-mentioned culture conditions. The proportion 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 using standard quantitative methods.

It is also preferred that the recombinant binding protein and/or ankyrin repeat domain does not lose its original three-dimensional structure after being incubated 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 recombinant binding protein comprising an ankyrin repeat domain having HGF binding specificity and the above-specified or preferred midpoint denaturation temperature and non-aggregation properties.

In another embodiment, the present invention relates to a recombinant binding protein comprising at least one ankyrin repeat domain that binds specifically to mammalian HGF, wherein the ankyrin repeat domain competes with an ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 33 to 61 (preferably SEQ ID NOs: 33, 37, 41, 42, 43, 48, 51, 52, 57, 58, 59, 60 and 61, more preferably 37, 42, 43, 51, 52 and 60, more preferably 36, 43, 51 and 60, especially 51 and 60) for binding to mammalian HGF.

Also preferably, the ankyrin repeat domain competes for binding to mammalian HGF with a binding protein selected from DARPins #33 to 61. Preferably, the ankyrin repeat domain competes for binding to mammalian HGF with a binding protein selected from DARPins #24, 45, and 50. More preferably, the ankyrin repeat domain competes for binding to mammalian HGF with binding proteins DARPin #24 or 50.

The term "competitive binding" means that although two different binding domains of the present invention can bind to the same target individually, they cannot bind to the same target at the same time. Therefore, the two binding domains compete to bind to the target. Preferably, the two competing binding domains bind to overlapping or identical binding epitopes on the target. Methods for determining whether two binding domains compete to bind to the target, such as competitive enzyme-linked immunosorbent assay (ELISA) or competitive SPR assay (e.g., using a Proteon instrument from BioRad), are well known in the art. An example of the competitive SPR measurement is shown in Example 2.

In another embodiment, the present invention relates to a recombinant binding protein comprising at least one mammalian HGF binding-specific ankyrin repeat domain, wherein the ankyrin repeat domain comprises an ankyrin repeat domain having at least 70% amino acid sequence with an ankyrin repeat domain selected from SEQ ID NOs: 33 to 61, wherein the G at position 1 and/or the S at position 2 of the ankyrin repeat domain are optionally deleted, and the L at the second to last position and/or the N at the first to last position of the ankyrin repeat domain are optionally replaced by A.

Preferably, the ankyrin repeat domain in the recombinant binding protein of the present invention comprises an ankyrin repeat domain having at least 70% amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 37, 41, 42, 43, 48, 51, 52, 57, 58, 59, 60 and 61; more preferably, 37, 42, 43, 51, 52 and 60; more preferably, 37, 43, 51 and 60, in particular SEQ ID NOs: 51 and 60.

Preferably, the ankyrin repeat domain in the recombinant binding protein of the present invention comprises an amino acid sequence having at least 70% amino acid sequence identity with one, two or three ankyrin repeat modules between the N-terminal cap module and the C-terminal cap module of the ankyrin repeat domain selected from SEQ ID NOs: 33 to 61.

Preferably, the ankyrin repeat domain or the one, two or three repeat modules comprises an amino acid sequence having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% amino acid sequence identity.

Preferably, up to 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid in the repeat domain of SEQ ID NOs: 33 to 61 is substituted by another amino acid.

Preferably, when amino acids are replaced in the capping modules of the repeat modules of SEQ ID NOs: 1 to 8, SEQ ID NOs: 12 to 25, or the repeat domains of SEQ ID NOs: 33 to 61, 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. Techniques for replacing one amino acid in a polypeptide with another are well known to those skilled in the art.

In another embodiment, the present invention relates to a recombinant binding protein comprising at least one ankyrin repeat domain having mammalian HGF binding specificity, wherein the ankyrin repeat sequence domain is selected from SEQ ID NOs: 33 to 61, wherein the G at position 1 and/or the S at position 2 of the ankyrin repeat domain are optionally deleted; and the L at the penultimate position and/or the N at the last position of the ankyrin repeat domain are optionally replaced by A.

Preferably, the ankyrin repeat domain is selected from SEQ ID NOs: 33, 37, 41, 42, 43, 48, 51, 52, 57, 58, 59, 60 and 61; more preferably, 37, 42, 43, 51, 52 and 60; more preferably, 37, 43, 51 and 60, in particular SEQ ID NOs: 51 and 60.

In another embodiment, the present invention relates to a recombinant binding protein comprising at least one mammalian HGF binding-specific ankyrin repeat domain, wherein the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from SEQ ID NOs: 12 to 25; and a sequence in which up to 9 amino acids in SEQ ID NOs: 12 to 25 are replaced by any amino acid.

Preferably, the recombinant binding protein comprises at least one ankyrin repeat module having an amino acid sequence selected from SEQ ID NOs: 12 to 25; and a sequence in which up to 9 amino acids in SEQ ID NOs: 12 to 25 are replaced by any amino acid; wherein 6, preferably 5, more preferably 4, more preferably 3, more preferably 2, and most preferably 1 of the 9 replaced amino acids are located at positions 1, 3, 4, 6, 14 and/or 15 of SEQ ID NOs: 12 to 25.

Preferably, the ankyrin repeat module of the ankyrin repeat domain is selected from SEQ ID NOs: 12, 15, 19, 21 and 24; more preferably, 19 and 21.

More preferably, the ankyrin repeat module of the ankyrin repeat domain is selected from SEQ ID NO: 12, 13, 14, 15, 18, 19, 20, 21, and 22; more preferably, 12, 15, 18, 19, 20, 21 and 24; more preferably, 18, 19, 20 and 21.

Preferably, at most 8 amino acids, more preferably at most 7 amino acids, more preferably at most 6 amino acids, more preferably at most 5 amino acids, more preferably at most 4 amino acids, more preferably at most 3 amino acids, more preferably at most 2 amino acids, and most preferably 1 amino acid in the repeating modules of SEQ ID NOs: 12 to 25 are replaced by another amino acid.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

KDRYGDTPLHLAADIGHLEIVEVLLKAGADVNA (SEQ ID NO: 18) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 18 are substituted with any amino acid, wherein

K at position 1 is optionally substituted by Q, H, N, I, T, Y, V, F, D, R, L or S; preferably, it is optionally substituted by Q, H, N, I, T, Y, V or F; further preferably, it is optionally substituted by Q, H, I, T, Y, V; most preferably, it is optionally substituted by Q or H;

R at position 3 is optionally substituted by A, T, K, H, N, Y, F, S or D; most preferably, it is optionally substituted by A, T, K, H or N; most preferably, it is optionally substituted by A;

Y at position 4 is optionally substituted by F, W, R, L, S or T; preferably, it is optionally substituted by F or W;

The D at position 6 is optionally substituted by L, A, N, E, S or T; most preferably, it is optionally substituted by L or A;

The D at position 14 is optionally substituted by S, N, M, T, or F; preferably by S or N;

The I at position 15 is optionally substituted by Y, A, N, T, R, V, K, D, L, or S; most preferably, it is optionally substituted by Y, A, N, T, R, or V; and most preferably, it is optionally substituted by Y or A; and

The E at position 19 is optionally replaced by a K.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

KDRYGDTPLHLAADIGHLEIVEVLLKAGADVNA (SEQ ID NO: 18) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 18 are replaced by any amino acids, wherein

The K in position 1 can be optionally replaced by Q, H, I, or V;

R at position 3 is optionally substituted by A, T or N, preferably A;

Y at position 4 is optionally replaced by F or W;

D in position 6 is optionally replaced by N;

D at position 14 is optionally replaced by S;

The I at position 15 is optionally substituted by Y or A; and

The E at position 19 is optionally replaced by a K.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

EDYFGNTPLHLAASYGHLEIVEVLLKAGADVNA (SEQ ID NO: 19) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 19 are substituted with any amino acid, wherein

E at position 1 is optionally substituted by D, H, N, F, A, L, or Q; preferably, D, H, or N; and most preferably, D or H.

Y at position 3 is optionally substituted by W, S, H, V, F, A or D; preferably, it is optionally substituted by W, S, H, V, A or F; most preferably, it is optionally substituted by W or S;

F at position 4 is optionally substituted by Y, A or W; most preferably, it is optionally substituted by Y or A;

N at position 6 is optionally substituted by D, H, I or M; most preferably, it is optionally substituted by D or H; most preferably, it is optionally substituted by D;

The S at position 14 is optionally substituted by N or H; and

Y at position 15 is optionally substituted by M, W, L, T, V, S, A, I or N; most preferably, it is optionally substituted by M, W, L, T, A or V; further preferably, it is optionally substituted by M, A or W; and most preferably, it is optionally substituted by A.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

EDYFGNTPLHLAASYGHLEIVEVLLKAGADVNA (SEQ ID NO: 19) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 19 are replaced by any amino acids, wherein

The E at position 1 is optionally substituted by D, F, A or L; most preferably by D or A;

Y in position 3 can be optionally replaced by W, S or V;

F at position 4 is optionally replaced by Y;

N in position 6 is optionally replaced by D;

The Y at position 15 is optionally substituted by M, L, S, or A; most preferably, it is optionally substituted by M, A, or L; most preferably, it is optionally substituted by A or L.

Preferred is a recombinant binding protein, wherein the ankyrin repeat domain comprises the ankyrin repeat module of SEQ ID NO: 18 and the ankyrin repeat module of SEQ ID NO: 19. Preferably, in the ankyrin repeat domain, the ankyrin repeat module of SEQ ID NO: 19 directly follows the ankyrin repeat module of SEQ ID NO: 18. For example, in the ankyrin repeat domain of DARPin #51, the ankyrin repeat module of SEQ ID NO: 19 directly follows the ankyrin repeat module of SEQ ID NO: 18.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

KDDYGNTPLHLAANTGHLEIVEVLLKAGADVNA (SEQ ID NO: 20) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 20 are replaced by any amino acids, wherein

K at position 1 is optionally substituted by M, I, D, L, Q, V, R, T or H; most preferably, it is optionally substituted by M, I or D; most preferably, it is optionally substituted by I or D;

R at position 3 is optionally substituted by H, Y, T, W or F; preferably, it is optionally substituted by H or Y;

Y at position 4 is optionally substituted by S, N, A, G, F or R; most preferably, it is optionally substituted by S, N, A or G; most preferably, it is optionally substituted by S;

The N at position 6 is optionally substituted by S, T, L or Y; most preferably by S;

The D at position 14 is substituted by L, optionally by F, M or I; most preferably, it is optionally substituted by L or I;

The T at position 15 is optionally substituted by S, W, V, E, F or A; most preferably by S;

H at position 17 is optionally replaced by R; and

The E at position 19 is optionally replaced by a K.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

KDDYGNTPLHLAANTGHLEIVEVLLKAGADVNA (SEQ ID NO: 20) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 20 are replaced by any amino acids, wherein

The K in position 1 can be optionally replaced by M, D, L, Q or V;

The R at position 3 is optionally replaced by Y or T;

Y at position 4 is optionally replaced by A or N;

T at position 15 is optionally replaced by S;

H at position 17 is optionally replaced by R; and

The E at position 19 is optionally replaced by a K.

Also preferred is a recombinant binding protein, wherein the ankyrin repeat domain comprises the ankyrin repeat module of SEQ ID NO: 19 and the ankyrin repeat module of SEQ ID NO: 20. Preferably, in the ankyrin repeat domain, the ankyrin repeat module of SEQ ID NO: 20 directly follows the ankyrin repeat module of SEQ ID NO: 19. For example, in the ankyrin repeat domain of DARPin #51, the ankyrin repeat module of SEQ ID NO: 20 directly follows the ankyrin repeat module of SEQ ID NO: 19.

Also preferred is a recombinant binding protein, wherein the ankyrin repeat domain comprises the ankyrin repeat module of SEQ ID NO: 18, the ankyrin repeat module of SEQ ID NO: 19, and the ankyrin repeat module of SEQ ID NO: 20. Preferably, in the ankyrin repeat domain, the ankyrin repeat module of SEQ ID NO: 19 directly follows the ankyrin repeat module of SEQ ID NO: 18, and the ankyrin repeat module of SEQ ID NO: 20 directly follows the ankyrin repeat module of SEQ ID NO: 19. For example, in the ankyrin repeat domain of DARPin #51, the ankyrin repeat module of SEQ ID NO: 19 directly follows the ankyrin repeat module of SEQ ID NO: 18, and the ankyrin repeat module of SEQ ID NO: 20 directly follows the ankyrin repeat module of SEQ ID NO: 19.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

HDYSGFTPLHLAAYYGHLEIVEVLLKHGADVNA (SEQ ID NO: 12) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 12 are replaced by any amino acids, wherein

H at position 1 is optionally substituted by T, S, N, Q, I, K, F, Y or V; most preferably, it is optionally substituted by T, S, N, Q or I; most preferably, it is optionally substituted by T or S;

Y at position 3 is optionally substituted by R, N, D, M, W, E, A, Q or L; most preferably, it is optionally substituted by R, N or D; most preferably, it is optionally substituted by R or N;

The S at position 4 is optionally substituted by N, T or W; most preferably by N or T;

F in position 6 is optionally replaced by I;

Y at position 14 is optionally replaced by F;

Y at position 15 is optionally substituted by W or H; and

The I at position 20 is optionally replaced by V or L.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

FDDWGHTPLHLAARYGHLEIVEVLLKYGADVNA (SEQ ID NO: 13) C-terminal capping module, and the following sequence: in which up to 9 amino acids in SEQ ID NO: 13 are replaced by any amino acids, wherein

F at position 1 is optionally substituted by T, H, L, D, Y, K or E; preferably, it is optionally substituted by T, H, L or D; most preferably, it is optionally substituted by T or H;

D at position 3 is optionally substituted by R, K, A, N, Y, S, L, T or F; preferably, it is optionally substituted by R, K, A or N; most preferably, it is optionally substituted by A, R or K;

W at position 4 is optionally substituted by F, Y or R; most preferably by F;

H at position 6 is replaced by L, optionally by I, M, N or K; most preferably, it is optionally replaced by L or I;

R at position 14 is optionally substituted by H, Y, S, F, A, N or I; preferably optionally substituted by H, Y or S; most preferably optionally substituted by H; and

Y at position 15 is optionally substituted by F, L, T, K or R; preferably, it is optionally substituted by F, L or T; and most preferably, it is optionally substituted by F.

Preferably, the recombinant binding protein comprises an ankyrin repeat module of SEQ ID NO: 12 and an ankyrin repeat module of SEQ ID NO: 13. Preferably, in the ankyrin repeat module of SEQ ID NO: 13, the ankyrin repeat module of SEQ ID NO: 12 follows the ankyrin repeat module of SEQ ID NO: 13. For example, in the ankyrin repeat domain of DARPin #13, the ankyrin repeat module of SEQ ID NO: 13 follows the ankyrin repeat module of SEQ ID NO: 12.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

KYEDGLTPLHLAAFYGHLEIVEVLLRHGADVNA (SEQ ID NO: 15) C-terminal capping module, and the following sequence: in which up to 9 amino acids in SEQ ID NO: 15 are replaced by any amino acids, wherein

The A at position 13 is optionally replaced by V; and

The R at position 26 may optionally be replaced by K.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

TDAWGHTPLHLAAYLGHLEIVEVLLKYGADVNA (SEQ ID NO: 16) C-terminal capping module, and the following sequence: in which up to 9 amino acids in SEQ ID NO: 16 are replaced by any amino acids, wherein

The P at position 8 is optionally replaced by T;

The A at position 12 is optionally replaced by T;

Y at position 14 is optionally substituted by S, H or A; preferably, it is optionally substituted by S or H;

L at position 15 is optionally substituted by Y, N or S; most preferably, it is optionally substituted by Y or N; and

The A at position 33 may optionally be replaced by T.

Preferred is a recombinant binding protein, wherein the ankyrin repeat domain comprises the ankyrin repeat module of SEQ ID NO: 15 and the ankyrin repeat module of SEQ ID NO: 16. Preferably, in the ankyrin repeat domain, the ankyrin repeat module of SEQ ID NO: 16 directly follows the ankyrin repeat module of SEQ ID NO: 15. For example, in the ankyrin repeat domain of DARPin #41, the ankyrin repeat module of SEQ ID NO: 16 directly follows the ankyrin repeat module of SEQ ID NO: 15.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

HDTWGLTPLHLAAFHGHQEIVEVLLKHGADVNA (SEQ ID NO: 21) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 21 are substituted with any amino acids.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

QDFYGKTPLHLAALRGHLEIVEVLLKYGADVNA (SEQ ID NO: 22) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 22 are replaced by any amino acids.

Preferred is a recombinant binding protein, wherein the ankyrin repeat domain comprises the ankyrin repeat module of SEQ ID NO: 21 and the ankyrin repeat module of SEQ ID NO: 22. Preferably, in the ankyrin repeat domain, the ankyrin repeat module of SEQ ID NO: 22 directly follows the ankyrin repeat module of SEQ ID NO: 21. For example, in the ankyrin repeat domain of DARPin #60, the ankyrin repeat module of SEQ ID NO: 22 directly follows the ankyrin repeat module of SEQ ID NO: 21.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

HDYLGLTPLHLAASDGHLEIVEVLLKHGADVNA (SEQ ID NO: 23) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 23 are replaced by any amino acids, wherein

H at position 1 is optionally substituted by N, L, K, R, F, Q or D; preferably, it is optionally substituted by N, L or K; most preferably, it is optionally substituted by N;

Y at position 3 is optionally substituted by F, Q, T, R, N, S or D; most preferably, it is optionally substituted by F, Q or T; most preferably, it is optionally substituted by F or Q;

L at position 4 is optionally substituted by V, T, Q, Y, D or F; preferably, it is optionally substituted by V or T;

The L in position 6 is optionally replaced by D;

The S at position 14 is optionally substituted by A, N or F; preferably by A or N; and

D at position 15 is optionally substituted by I, T, S, R, A, Y or M; preferably, it is optionally substituted by I, T, S or R; most preferably, it is optionally substituted by I or T.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

YDYNGLTPLHLAANNGHLEIVEVLLKYGADVNA (SEQ ID NO: 24) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 24 are replaced by any amino acids, wherein

Y at position 1 is optionally substituted by S, I, T, Q, E, M, K, D or V; preferably, it is optionally substituted by S, I or T; most preferably, it is optionally substituted by S or I;

Y at position 3 is optionally substituted by A, F, V, M, W, T, R or Q; most preferably, it is optionally substituted by A, F, V or M; most preferably, it is optionally substituted by A or V;

N at position 4 is optionally substituted by Y, F, T or W; most preferably by Y;

L at position 6 is optionally substituted by F, H, Y, N or W; preferably, it is optionally substituted by F, H, Y or N; most preferably, it is optionally substituted by F or H;

H at position 10 is optionally replaced by Y;

The A at position 12 is optionally replaced by T or V;

N at position 14 is optionally replaced by S;

N at position 15 is optionally substituted by V, M or T; most preferably by V; and

The I at position 20 may be optionally replaced by V or L.

In another embodiment, the present invention relates to a recombinant binding protein, wherein the ankyrin repeat domain with HGF binding specificity comprises a repeat module having:

FDVAGYTPLHLAAYFGHLEIVEVLLKYGADVNA (SEQ ID NO: 25) ankyrin repeat sequence, and sequences in which up to 9 amino acids in SEQ ID NO: 25 are replaced by any amino acids, wherein

F at position 1 is optionally substituted by I, M, T, D, Y, Q, E, H, A or S; most preferably, it is optionally substituted by I, M, T, D, Y or Q; most preferably, it is optionally substituted by I, T, D or Y; most preferably, it is optionally substituted by I or Y;

V at position 3 is optionally substituted by I, H, S, A, D or W; most preferably, it is optionally substituted by I, H or S; most preferably, it is optionally substituted by I or S;

The A at position 4 is optionally substituted by F, Y, V, M, N, L, T, H or I; preferably, it is optionally substituted by F, Y, V or M; most preferably, it is optionally substituted by F or Y;

Y at position 6 is optionally substituted by F, H, T, W, M, N, Q or S; preferably, it is optionally substituted by F, H, T or W; most preferably, it is optionally substituted by F or T;

Y at position 14 is optionally substituted by H, M, L, N, I, R, W or T; preferably, it is optionally substituted by H, L, N, I, R or T; most preferably, it is optionally substituted by H or L; and

F at position 15 is optionally substituted by Y, H, M, T, V, L, N or I; most preferably by Y, H, T or V; most preferably by T or V.

Preferred is a recombinant binding protein, wherein the ankyrin repeat domain comprises the ankyrin repeat module of SEQ ID NO: 23 and the ankyrin repeat module of SEQ ID NO: 24. Preferably, in the ankyrin repeat domain, the ankyrin repeat module of SEQ ID NO: 24 directly follows the ankyrin repeat module of SEQ ID NO: 23. For example, in the ankyrin repeat domain of DARPin #57, the ankyrin repeat module of SEQ ID NO: 24 directly follows the ankyrin repeat module of SEQ ID NO: 23.

Also preferred is a recombinant binding protein, wherein the ankyrin repeat domain comprises the ankyrin repeat module of SEQ ID NO: 24 and the ankyrin repeat module of SEQ ID NO: 25. Preferably, in the ankyrin repeat domain, the ankyrin repeat module of SEQ ID NO: 25 directly follows the ankyrin repeat module of SEQ ID NO: 24. For example, in the ankyrin repeat domain of DARPin #57, the ankyrin repeat module of SEQ ID NO: 24 directly follows the ankyrin repeat module of SEQ ID NO: 23.

Also preferred is a recombinant binding protein, wherein the ankyrin repeat domain comprises the ankyrin repeat module of SEQ ID NO: 23, the ankyrin repeat module of SEQ ID NO: 24, and the ankyrin repeat module of SEQ ID NO: 25. Preferably, in the ankyrin repeat domain, the ankyrin repeat module of SEQ ID NO: 24 directly follows the ankyrin repeat module of SEQ ID NO: 23, and the ankyrin repeat module of SEQ ID NO: 25 directly follows the ankyrin repeat module of SEQ ID NO: 24. For example, in the ankyrin repeat domain of DARPin #57, the ankyrin repeat module of SEQ ID NO: 24 directly follows the ankyrin repeat module of SEQ ID NO: 23, and the ankyrin repeat module of SEQ ID NO: 24 directly follows the ankyrin repeat module of SEQ ID NO: 24.

Further preferably, an N-terminal or C-terminal ankyrin capping module comprises an N-terminal or C-terminal ankyrin capping repeat sequence, respectively, wherein one or more amino acid residues in the capping repeat sequence are replaced by the amino acid residues at the corresponding positions when aligned with the corresponding ankyrin capping unit or ankyrin repeat unit.

Amino acid substitutions can be any of the 20 common natural amino acids, preferably selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; more preferably, selected from A, D, E, H, I, K, L, Q, R, S, T, V, and Y. Also preferably, amino acid substitutions are made with homologous amino acids; that is, an amino acid is replaced with an amino acid having a side chain 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. Replacing an amino acid with a homologous amino acid is a technique well known to those skilled in the art.

Also preferred is a C-terminal ankyrin capping module comprising amino acids A at positions 27 and 28 of the above C-terminal capping modules based on SEQ ID NOs: 28 to 48.

Also preferred is a C-terminal capping module comprising amino acids A at positions 27 and 28 of the above C-terminal capping modules based on SEQ ID NOs: 4 to 8.

The amino acid G at position 1 and/or the S at position 2 of SEQ ID NOs: 1 to 3 can be removed from the N-terminal ankyrin capping module without any significant effect on performance. These two amino acids act as linkers to connect the ankyrin repeat domain to other amino acids and proteins. The present invention also includes the ankyrin repeat domain comprising an N-terminal ankyrin capping module, wherein the amino acid G at position 1 and/or the S at position 2 are removed. It should be understood that the amino acid positions (e.g., "position 33") in the ankyrin repeat domain described herein are adjusted accordingly, resulting in some shifts, for example, if one amino acid is deleted, "position 33" will become "position 32", or if two amino acids are deleted, "position 33" will become "position 31".

The ankyrin capping module of the ankyrin repeat domain of the present invention can be replaced with a capping module by ankyrin binding techniques known to those skilled in the art, such as amino acid sequence alignment, induced mutagenesis, and gene synthesis. For example, the C-terminal capping repeat sequence of SEQ ID NO: 33 can be replaced with the C-terminal capping repeat sequence of SEQ ID NO: 8 by the following method: (i) determining the C-terminal capping repeat sequence of SEQ ID NO: 33 (i.e., sequence positions 132 to 159) by sequence alignment with SEQ ID NO: 8, (ii) replacing the determined C-terminal capping repeat sequence of SEQ ID NO: 33 with the sequence of SEQ ID NO: 8, (iii) generating a gene encoding a repeat domain, wherein the repeat domain encodes the C-terminal capping module for replacement, (iv) expressing the modified repeat domain in the cytoplasm of Escherichia coli, and (v) purifying the modified repeat domain by standard methods. As another example, the N-terminal capping repeat sequence of SEQ ID NO:33 can be replaced with the N-terminal capping repeat sequence of SEQ ID NO:2, wherein the following method is used: (i) determining the N-terminal capping repeat sequence of SEQ ID NO:33 (i.e., sequence positions 1 to 32) by alignment with the sequence of SEQ ID NO:2, (ii) replacing the determined N-terminal capping repeat sequence of SEQ ID NO:33 with the sequence of SEQ ID NO:2, (iii) generating a gene encoding a repeat domain, wherein the repeat domain encodes the N-terminal capping module for replacement, (iv) expressing the modified repeat domain in the cytoplasm of Escherichia coli, and (v) purifying the modified repeat domain by standard methods.

In addition, the ankyrin repeat domain of the present invention can be constructed by assembling an N-terminal ankyrin capping module (e.g., the N-terminal capping repeat sequence of SEQ ID NO: 2) followed by one or more repeat modules (e.g., three ankyrin repeat modules comprising ankyrin repeat sequences from amino acid residues 33 to 131 of SEQ ID NO: 33) and a C-terminal capping module (e.g., the C-terminal capping module of SEQ ID NO: 8) by gene synthesis. The genetically assembled repeat domain gene can be expressed in Escherichia coli as described herein.

Further preferred are recombinant binding proteins, repeat domains, repeat modules, N-terminal capping modules or C-terminal capping modules having an amino acid sequence lacking amino acids C, M or N.

Further preferred are recombinant binding proteins, repeat domains, repeat modules, N-terminal capping modules or C-terminal capping modules lacking amino acid D, E or N and followed by G.

Further preferred are recombinant binding proteins or repeat domains comprising any of said N-terminal or C-terminal capping modules.

In another preferred embodiment of the recombinant binding protein comprising the ankyrin repeat domain of the present invention, one or more amino acid residues of the N-terminal capping module of the repeat domain are replaced with amino acid residues at corresponding positions in 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, the N-terminal capping unit is a native N-terminal capping unit.

In another preferred embodiment of the recombinant binding protein comprising the ankyrin repeat domain of the present invention, one or more amino acid residues of the C-terminal capping module of the repeat domain are replaced with amino acid residues at corresponding positions in 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, the C-terminal capping unit is a native C-terminal capping unit.

In yet another specific embodiment, up to 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% of the amino acid residues are replaced with an amino acid that is not present at the corresponding position in the repeat unit, N-terminal capping unit or C-terminal capping unit.

The term "consensus sequence" refers to an amino acid sequence, wherein the consensus sequence is obtained by aligning the structure and/or sequence of multiple repeating units. Using two or more repeating units that have been aligned in structure and/or sequence, and allowing for gaps in the alignment, the most frequently occurring amino acid residues at each position can be determined. A consensus sequence is a sequence that contains the amino acids that occur most frequently at each position. When two or more amino acids are present in excess at a single position, the consensus sequence may include a subset of the amino acids. The two or more repeating units may be obtained from a single repeating protein or from repeating units contained in two or more repeating proteins.

Consensus sequences and methods for determining them are well known to those skilled in the art.

A "consensus amino acid residue" is an amino acid at a specific position in a consensus sequence. If two or more (e.g., three, four, or five) amino acid residues have similar occurrence rates in two or more repeating units, the consensus amino acid can be one of the most common amino acids or a combination of two or more amino acid residues.

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

The term "non-natural" refers to synthetic or non-natural, more specifically, the term refers to artificially made. The term "non-natural binding protein" or "non-natural binding domain" means that the binding protein or the binding domain are synthesized (i.e., obtained by amino acid chemical synthesis) or recombinantly obtained, and are not naturally present. "Non-natural binding protein" or "non-natural binding domain" are artificial proteins or domains obtained by the expression of corresponding designed nucleic acids, respectively. Preferably, the expression is in eukaryotic or bacterial cells, or a cell-free in vitro expression system is used. In addition, the term means that the sequence of the binding protein or the binding domain does not exist as a non-artificial sequence entry in sequence databases such as GenBank, EMBL-bank or SWISS-PROT. These databases and other similar sequence databases are well known to those skilled in the art.

In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain that specifically binds HGF and further comprising an ankyrin repeat domain that specifically binds vascular endothelial growth factor A (VEGF-A). Examples of ankyrin repeat domains specific for HGF are provided herein, and examples of ankyrin repeat domains specific for VEGF-A are described in WO 2010/060748 or WO 2011/135067. Also preferably, the recombinant binding protein further comprises one or more, preferably one or two, ankyrin repeat domains specific for human serum albumin. Examples of ankyrin repeat domains specific for human serum albumin are described in WO 2012/069654. Any of the repeat domains specific for serum albumin, VEGF-A, or HGF can be linked by genetic means using a polypeptide linker (e.g., SEQ ID NO: 10 or 11) using techniques known to those skilled in the art.

Another preferred embodiment provides a recombinant binding protein comprising an ankyrin repeat domain having HGF binding specificity and containing one, two, three, or more internal repeat modules that can participate in HGF binding. Preferably, the ankyrin repeat domain comprises an N-terminal capping module, two to four internal repeat modules, and a C-terminal capping module. Preferably, the capping module is a capping repeat sequence. Also preferably, the capping module can participate in HGF binding.

Further preferred is a recombinant binding protein comprising two or more of the ankyrin repeat domains having HGF binding specificity. Preferably, the binding protein comprises 2 or 3 of the repeat domains. The two or more repeat domains have the same or different amino acid sequences.

Another preferred embodiment is a recombinant binding protein comprising an ankyrin repeat domain according to the present invention, wherein one or more amino acid residues of the repeat module of the ankyrin repeat domain are replaced with amino acid residues at corresponding positions when the repeat unit is aligned. Preferably, no more than 30% of the amino acid residues are replaced, more preferably no more than 20%, and even more preferably no more than 10% of the amino acid residues are replaced. Most preferably, the repeat unit is a natural repeat unit.

In yet another embodiment, at most 30%, more preferably at most 20%, even more preferably at most 10% of the amino acid residues are substituted with amino acids that are not present at the corresponding positions in the repeat unit.

In yet another embodiment, any of the recombinant HGF binding proteins or domains described herein can be covalently linked to one or more other moieties, including, for example, moieties that bind to other targets to create bispecific binding agents, biologically active compounds, labeling moieties (e.g., fluorescent labels (e.g., fluorescein) or radioactive tracers), moieties that facilitate protein purification (e.g., small peptide tags, such as His- or Strep-tags), moieties that provide effector function to enhance therapeutic efficacy (e.g., antibody Fc portions for providing antibody-dependent cell-mediated cytotoxicity), toxic protein moieties (e.g., Pseudomonas aeruginosa exotoxin A (ETA) or small molecule toxic agents such as maytansine or DNA alkylating agents), or moieties that improve pharmacokinetics. Improved pharmacokinetics can be determined based on sensory perception. Therapeutic needs are assessed. It is often necessary to increase bioavailability and/or increase the time between doses, which may be achieved by increasing the time the available protein remains in the serum after administration. In some examples, it is necessary to increase the time duration of the protein serum concentration (e.g., reducing the difference between the concentration in a short period after administration and the concentration in a short period before the next administration). Moieties that tend to slow the blood clearance of proteins include hydroxyethyl starch (HES), polyethylene glycol (PEG), sugars (e.g., sialic acid), well-tolerated protein moieties (e.g., Fc fragments or serum albumin), and binding domains or peptides that are specific and clear for a large number of serum proteins, such as antibody Fc fragments or serum albumin. Examples of binding domains with affinity for serum proteins are described in WO 2012/069654. The recombinant binding proteins of the present invention can be attached to a portion that reduces the clearance rate of mammalian (e.g., mouse, rat, or human) polypeptides to more than three times that of unmodified polypeptides.

In yet another embodiment, the present invention relates to a nucleic acid molecule encoding the specific recombinant binding protein, the specific ankyrin repeat domain, the specific ankyrin repeat module and the specific capping module. In addition, a vector comprising the nucleic acid molecule is also contemplated.

Furthermore, a pharmaceutical composition comprising one or more of the above-described recombinant binding proteins, particularly comprising repeat domains or nucleic acid molecules encoding specific binding proteins, and optionally a pharmaceutically acceptable carrier and/or diluent is contemplated. Pharmaceutically acceptable carriers and/or diluents are known to those skilled in the art and are explained in more detail below. Even further, a diagnostic composition comprising one or more of the above-described recombinant binding proteins, particularly binding proteins comprising repeat domains, is contemplated.

A pharmaceutical composition comprises a recombinant binding protein as described above and a pharmaceutically acceptable carrier, excipient, or stabilizer, such as those described in Remington's Pharmaceutical Sciences ^16th edition, Osol, A. Ed. [1980]. Suitable carriers, excipients, or stabilizers known to those skilled in the art include saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies 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 may also be a combination preparation that includes other active agents, such as anticancer drugs or anti-angiogenic agents.

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

The pharmaceutical composition can be administered by any suitable method within the knowledge of those skilled in the art.

Furthermore, any of the pharmaceutical compositions described above are contemplated for use in treating a disease.

The present invention further provides a method of treatment, which comprises administering a therapeutically effective dose of the recombinant binding protein of the present invention to a patient in need thereof.

In addition, a method for treating pathological symptoms in mammals, including humans, is also contemplated, which comprises administering a therapeutically effective dose of the above-mentioned pharmaceutical composition to a patient in need thereof.

Examples of such pathological conditions include atherosclerosis, restenosis, pulmonary hypertension, inflammatory joint diseases, eye and retinal diseases, and fibrotic diseases, including pulmonary fibrosis, cirrhosis and other liver diseases, scleroderma, glomerulosclerosis, and cardiac fibrosis. In addition, anti-HGF therapy is useful for tumor pathological conditions such as solid tumors and hematological tumors. For example, gliomas, sarcomas, osteogenic sarcomas, multiple myeloma, leukemias, lymphomas, and epithelial cancers, including bone metastases.

The recombinant binding proteins or ankyrin repeat domains of the present invention can be obtained and/or improved 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 intracellular expression and selection/screening, for example, by protein complementation (WO 1998/341120). Such methods are well known to those skilled in the art.

Ankyrin repeat protein libraries for selection/screening of recombinant binding proteins or ankyrin repeat domains of the present invention can be obtained according to techniques known to those skilled in the art (WO 2002/020565, Binz, H.K., et al., J. Mol. Biol., 332, 489-503, 2003, and Binz et al., 2004, loc. cit). The use of such libraries for screening ankyrin repeat domains specific for HGF is shown in Example 1. In addition, ankyrin repeat domains of the present invention can be assembled using the ankyrin repeat modules of the present invention and appropriate capping modules or capping repeat sequences (Forrer, P., et al., FEBS Letters 539, 2-6, 2003) using standard recombinant DNA techniques (e.g., WO 2002/020565, Binz et al., 2003, loc. cit. and Binz et al., 2004, loc. cit).

The present invention is not limited to the specific embodiments described in the Examples. Other raw materials may be used and processed as outlined below.

Example

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

Experimental Materials

Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were purchased from Microsynth (Switzerland). Unless otherwise stated, DNA polymerase, restriction endonucleases, and buffers were purchased from New England Biolabs (USA) or Fermentas (Lithuania). Cloning and protein production strains were Escherichia coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). Recombinant human HGF was purchased from Peprotech (USA, product number 100-39), and recombinant mouse HGF was purchased from RND Systems (USA; product number 2207-HG/CF). Biotinylated HGF was obtained by chemically linking the biotin moiety to the protein using standard biotinylation reagents and methods (Pierce, USA).

Cell lines were purchased from ATCC (France/USA; Cat. Nos. A549-CCL-185, U87MG–HTB-14). Cell culture medium was purchased from Invitrogen/Lubio (Switzerland). Fetal bovine serum was purchased from Promocell (Germany; C-37350). Cell proliferation assays, including cell proliferation ELISA and BrdU (colorimetric) (Cat. No. 1164722900), were purchased from Roche (Switzerland). Assays for P-c-Met were purchased from RnD Systems (Human Phospho-HGF R/c-METDuoSet IC; Cat. No. DYC2480-5; Sample Diluent Concentrate 2; DXC002). Human HGF for cell-based assays was purchased from Peprotech (Cat. No. 100-39) and reconstituted in cell culture medium containing 10% FBS.

Molecular Biology

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

Design of ankyrin repeat protein libraries

Methods for generating libraries of designed ankyrin repeat proteins have been described (WO 2002/020565; Binz et al. 2003, loc. cit.; Binz et al. 2004, loc. cit.). Using these methods, libraries of ankyrin repeat proteins having randomized ankyrin repeat modules and/or randomized capping modules can be constructed. For example, the libraries can be constructed based on a fixed N-terminal capping module (e.g., the N-terminal capping module of SEQ ID NO: 2) or a randomized N-terminal capping module as shown in SEQ ID NO: 29, one or more randomized repeat units within the sequence motifs of SEQ ID NOs: 26, 27, or 28, and a fixed C-terminal capping module (e.g., the C-terminal capping module of SEQ ID NO: 8) or a randomized C-terminal capping module as shown in SEQ ID NO: 30. Preferably, such libraries are constructed such that the randomized positions of the repeat or capping modules do not contain amino acids C, G, M, N (before the G residue), or P. In addition, the random repeating unit based on the sequence motif of SEQ ID NO: 26, 27 or 28 can be further randomized at positions 10 and/or 17; the random N-terminal capping module based on the sequence motif of SEQ ID NO: 29 can be further randomized at positions 7 and/or 9; and the randomized C-terminal capping module based on the sequence motif of SEQ ID NO: 30 can be further randomized at positions 10, 11 and/or 17.

In addition, the randomization module in the library can include additional polypeptide loops inserted at random amino acid positions. Examples of polypeptide loop insertions include complement determining region (CDR) loop libraries of antibodies or peptide libraries synthesized from scratch. For example, the insertion of the loop can be guided by the structure of the N-terminal ankyrin repeat domain of human ribonuclease L (Tanaka, N., Nakanishi, M, Kusakabe, Y, Goto, Y., Kitade, Y, Nakamura, K.T., EMBO J. 23 (30), 3929-3938, 2004). Similar to the ankyrin repeat domain with 10 amino acids inserted in the β-turn near the boundary of two ankyrin repeat sequences, the ankyrin repeat protein library can contain variable length (e.g., 1 to 20 amino acids) random loops (fixed and randomized positions) inserted into one or more β-turns of the ankyrin repeat domain.

Any of the N-terminal capping modules of the ankyrin repeat protein library preferably has a RELLKA or RILKAA motif instead of a RILLAA motif (e.g., present at positions 21 to 26 of SEQ ID NO: 29), and any of the C-terminal capping modules of the ankyrin repeat protein library preferably has a KAA or KLA motif instead of a KLN motif (e.g., the last three amino acids of SEQ ID NO: 30).

The design of the ankyrin repeat protein library can be carried out under the guidance of known ankyrin repeat domain structures that interact with the target. The protein data bank (Protein Data Bank, PDB) unique query codes or identification codes (PDB-ID) of examples of the structures are 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1N11, 1S70 and 2ZGD.

Examples of designed ankyrin repeat protein libraries, such as N2C and N3C, have been 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 random repeat modules between the N-terminal and C-terminal capping modules.

The nomenclature used to define positions within repeat units and modules is based on Binz et al. 2004, loc. cit., with the improvement that the boundaries of the ankyrin repeat modules and 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, and 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 verified by mass spectrometry analysis.

Example 1: Selection of proteins containing ankyrin repeat domains with HGF binding specificity

Using ribosome display technology (Hanes, J. and Plückthun, A., PNAS 94, 4937-42, 1997), a number of designed HGF-binding-specific ankyrin repeat proteins (DARPins) were selected from the DARPins library described by Binz et al. 2004 (loc. cit.). The selected clones were evaluated for binding to both specific targets (HGF) and nonspecific targets (MBP, Escherichia coli maltose binding protein) using crude extract ELISA, demonstrating the successful selection of hundreds of HGF-binding proteins. For example, the ankyrin repeat domains of SEQ ID NOs: 33 to 61 constitute the amino acid sequences of selected binding proteins containing HGF-binding-specific ankyrin repeat domains. Individual ankyrin repeat modules derived from these HGF-binding-specific ankyrin repeat domains are provided in SEQ ID NOs: 12 to 25.

Screening of HGF-specific ankyrin repeat proteins by ribosome display technology

Screening for HGF-specific ankyrin repeat proteins was performed using ribosome display technology (Hanes and Plückthun, loc. cit.) using human and/or mouse HGF as the target protein and the designed ankyrin repeat protein library described above and an established protocol (Zahnd, C., Amstutz, P. and Plückthun, A., Nat. Methods 4, 69-79, 2007). After each selection cycle, the number of reverse transcription (RT)-PCR cycles was successively reduced from 45 to 25, based on the enrichment of binding proteins and the yield. The first four selection cycles used standard ribosome display screening, with decreasing target concentrations and increasing wash stringency to increase the selection pressure from rounds 1 to 4 (Binz et al., 2004, loc. cit.). To enrich for high-affinity anti-HGF DARPins, the output from round 4 of the standard ribosome display screening (described above) was subjected to one or two rounds of off-rate selection with increased stringency (Zahnd, 2007, loc. cit.). Following each off-rate selection, a final round of standard selection was performed to amplify and recover binding proteins selected in the off-rate selection. To enhance the cross-reactivity of selected binding proteins with human and mouse HGF, an additional round of selection with mouse HGF was performed after the off-rate selection with human HGF.

Specific binding of selected clones to HGF was demonstrated by crude extract receptor competitive ELISA

Individual selected HGF-binding specific DARPins were identified using a receptor competition enzyme-linked immunosorbent assay (ELISA) using crude E. coli extracts from standard DARPin-expressing cells. DARPins selected using ribosome display technology were cloned into the pQE30 (Qiagen) expression vector, transformed into E. coli XL1-Blue (Stratagene), and grown overnight at 37°C in 96-deep-well plates (each containing a single clone) containing 1.2 ml of growth medium (TB containing 1% glucose and 50 μg/ml ampicillin). In a new 96-deep-well plate, 50 μl of the overnight culture was inoculated into 0.9 mL of fresh LB medium containing 50 μg/ml ampicillin. After incubation at 37°C for 60-90 minutes, expression was induced with IPTG (0.5 mM final concentration) for another 3-4 hours. Cells were harvested, resuspended in 50 μl of B-PERII (Pierce), and incubated with shaking at room temperature for 15 minutes. Then, 950 μl of PBS solution was added, and cell debris was removed by centrifugation. A 1:50 dilution of extract from each lysed clone in PBSTC (PBS supplemented with 0.1% Tween and 0.25% (w/v) casein, pH 7.4) was added to a protein G-coated Maxisorp plate containing human or mouse c-Met-Fc fusions, along with 1 nM biotinylated human or mouse HGF, and incubated at room temperature for 10 minutes. The plate was then washed extensively with PBS-T (PBS supplemented with 0.1% Tween, pH 7.4), incubated using a standard ELISA procedure with streptavidin-HRP conjugate (11089153001, Roche), and binding was detected using POD substrate (Roche). The assay was colorimetric at 405 nm. Screening of hundreds of clones using crude cell extract ELISA revealed over a hundred different DARPins specific for HGF. Examples of selected HGF binding-specific ankyrin repeat domain amino acid sequences are provided in SEQ ID NOs: 33-61.

The HGF binding specific ankyrin repeat domain and negative control DARPins without HGF binding specificity (e.g., DARPins #28 and 29) were cloned into an expression vector based on pQE (QIAgen, Germany) that provides an N-terminal tag for easy protein purification (as described below). For example, expression vectors encoding the following DARPins were constructed:

DARPin #33 (SEQ ID NO: 33 with a histidine tag (SEQ ID NO: 9) fused to the N-terminus);

DARPin #34 (SEQ ID NO: 34 with a histidine tag (SEQ ID NO: 9) fused to the N-terminus);

DARPin #35 (SEQ ID NO: 35 with a histidine tag (SEQ ID NO: 9) fused to the N-terminus);

DARPin #36 (SEQ ID NO: 36 with a histidine tag (SEQ ID NO: 9) fused to the N-terminus);

DARPin #37 (SEQ ID NO: 37 with a histidine tag (SEQ ID NO: 9) fused to the N-terminus);

DARPin #38 (SEQ ID NO: 38 with a histidine tag (SEQ ID NO: 9) fused to the N-terminus);

DARPin #39 (SEQ ID NO: 39 with a histidine tag fused to the N-terminus);

DARPin #40 (SEQ ID NO: 40 with a histidine tag fused to the N-terminus);

DARPin #41 (SEQ ID NO: 41 with a histidine tag fused to the N-terminus);

DARPin #42 (SEQ ID NO: 42 with a histidine tag fused to the N-terminus);

DARPin #43 (SEQ ID NO: 43 with a histidine tag fused to the N-terminus);

DARPin #44 (SEQ ID NO: 44 with a histidine tag fused to the N-terminus);

DARPin #45 (SEQ ID NO: 45 with a histidine tag fused to the N-terminus);

DARPin #46 (SEQ ID NO: 46 with a histidine tag fused to the N-terminus);

DARPin #47 (SEQ ID NO: 47 with a histidine tag fused to the N-terminus);

DARPin #48 (SEQ ID NO: 48 with a histidine tag fused to the N-terminus);

DARPin #49 (SEQ ID NO: 49 with a histidine tag fused to the N-terminus);

DARPin #50 (SEQ ID NO: 50 with a histidine tag fused to the N-terminus);

DARPin #51 (SEQ ID NO: 51 with a histidine tag fused to the N-terminus);

DARPin #52 (SEQ ID NO: 52 with a histidine tag fused to the N-terminus);

DARPin #53 (SEQ ID NO: 53 with a histidine tag fused to the N-terminus);

DARPin #54 (SEQ ID NO: 54 with a histidine tag fused to the N-terminus);

DARPin #55 (SEQ ID NO: 55 with a histidine tag fused to the N-terminus);

DARPin #56 (SEQ ID NO: 56 with a histidine tag fused to the N-terminus);

DARPin #57 (SEQ ID NO: 57 with a histidine tag fused to the N-terminus);

DARPin #58 (SEQ ID NO: 58 with a histidine tag fused to the N-terminus);

DARPin #59 (SEQ ID NO: 59 with a histidine tag fused to the N-terminus);

DARPin #60 (SEQ ID NO: 60 with a histidine tag fused to the N-terminus);

DARPin #61 (SEQ ID NO: 61 with a histidine tag fused to the N-terminus); and

DARPin #62 (SEQ ID NO: 62 with a histidine tag fused to the N-terminus).

High-level soluble expression of DARPins

Selected clones that demonstrated HGF binding specificity in crude cell extract ELISA were expressed in E. coli BL21 or XL1-Blue cells and purified using their histidine tags using standard protocols for further analysis. 25 ml of a stationary overnight culture (TB, 1% glucose, 100 mg/l ampicillin; 37°C) was inoculated into a 500 ml culture (same medium). When the absorbance at 600 nm reached 1.0, the culture was induced with 0.5 mM IPTG and incubated at 37°C for 4-5 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. Protein purification was performed using a Ni-nitrilotriacetic acid column (2.5 ml column volume) according to the manufacturer's instructions (QIAgen, Germany). Alternatively, DARPins lacking the 6xHis tag or selected repeat domains were 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 HGF binding specificity were purified from 1 L of E. coli culture with a purity of >95% (estimated from SDS-15% PAGE). The purified DARPins were used for further characterization.

Example 2: Specific surface plasmon resonance analysis of DARPins binding to hepatocyte growth factor Sign

Human HGF molecules were directly immobilized on the flow cell and their interactions with various selected DARPins were analyzed.

Kd determination by surface plasmon resonance (SPR) analysis

SPR measurements were performed using a ProteOn instrument (BioRad) according to standard procedures known to those skilled in the art. Human HGF was covalently immobilized on a GLC chip (BioRad) using PBS, pH 7.4, containing 0.005% Tween in running buffer to a level of approximately 5,000 resonance units (RU). To determine the interaction between DARPins and HGF, 200 μL of serial dilutions of DARPins at concentrations of 25, 12.5, 6.26, 3.13, and 1.67 nM (based on rate measurements) in running buffer (PBS containing 0.005% Tween) were injected, followed by a 25-minute flow of running buffer at a constant rate of 100 μL/min (not based on rate measurements). The interval signal (i.e., resonance units (RU)) and the baseline injection (i.e., injection of running buffer alone) were subtracted from the RU trace obtained after DARPin injection (double baseline). The corresponding association and dissociation rates were obtained from the SPR traces obtained from the association and dissociation rate measurements of the DARPin interaction with HGF. For example, Figure 1 shows the SPR trace of the resulting DARPin #51. Dissociation constants (Kd values) can be calculated from estimated association and dissociation rates using standard methods known to those skilled in the art. Kd values for selected DARPins were determined to be between 10 pM and 20 nM. Table 1 summarizes Kd values for selected DARPins as examples.

Table 1: Kd values for the interaction between DARPin and human HGF

Competitive SPR analysis

To determine whether the binding proteins of the present invention compete with the ankyrin repeat domain for HGF binding, a competition SPR assay was performed by allowing a first DARPin to bind to human HGF to saturation, followed by the injection of a second DARPin. If no signal increase was observed upon injection of the second DARPin, competition between the first and second DARPins for HGF binding was considered; if a signal increase was observed, no competition between the first and second DARPins for HGF binding was considered. Figure 2 provides an example analysis of two DARPins that competed for HGF binding and two DARPins that did not compete for HGF binding.

Example 3: Inhibition of Hepatocyte Growth Factor-Induced c-Met Phosphorylation by DARPins with HGF Binding Properties opposite sex

A549 cells were seeded at 50,000 cells per well in a 96-well plate containing complete medium (DMEM; 10% FBS). After 24 hours, the medium was replaced with serum-free medium. Cells were cultured for an additional 24 hours and stimulated with 1 nM human HGF in the presence or absence of DARPin. HGF and DARPin were preincubated at room temperature for at least 30 minutes before addition to the cells. Cells were stimulated for 10 minutes under cell culture conditions. Stimulation was terminated by removing the cell supernatant (flicking) and adding cell lysis buffer (RIPA; Sample Diluent Concentrate 2; RnD Systems). Phospho-c-Met in cell lysates was measured using the Human Phospho-HGF R/c-MET DuoSet IC Kit (RnD Systems) according to the manufacturer's instructions. Data were analyzed using GraphPad Prism software (GraphPad Software Inc., CA, USA). Percent inhibition of phosphorylation was calculated by setting the OD value obtained in the unstimulated control to 100% inhibition and the OD value obtained in the uninhibited control to 0% inhibition. For inhibition experiments, data were fit to the log(inhibitor) vs. response/variable slope.

The results of the examples are summarized in Table 3. _IC50 values were calculated from the titration curves obtained above using standard procedures known to those skilled in the art. Percent inhibition was calculated as described above. Example titration curves for DARPins #51 and #43 are provided in FIG3 .

Table 3: Inhibitory potential of DARPins on HGF-stimulated c-Met phosphorylation

Example 4: Inhibitory effect of HGF-binding specific DARPins on U87 cell proliferation

U87 cells were cultured in DMEM containing 10% FBS (Promocell-Cytokine-Low) at 37°C and 5% CO₂. Cells were split twice weekly at a 1:3 ratio. 2500 cells were seeded per 96-well plate in 100 μl of DMEM containing 1% FBS and cultured overnight. The next day, DARPins (10 μl, 10× final concentration) were added and cultured for an additional 5 days. On day 4, 10 μl of BrdU labeling solution (10 μl, 1:1000 dilution) was added per well. On day 5, assays were performed according to the manufacturer's instructions. DARPins were titrated between 1,000 and 2 nM. Experiments were analyzed using GraphPad Prism software. _IC₅₀ was calculated from nonlinear fits of the resulting titration curves.

The results of the examples are summarized in Table 4. _IC50 values were calculated based on the above titration curves obtained by standard procedures known to those skilled in the art. Example titration curves for DARPins #51 and #43 are provided in FIG5.

Table 4: Inhibition of U87 proliferation by DARPins combined with HGF

Example 5: Characterization of HGF-binding specific DARPins by receptor competition assay

The potency of anti-HGF DARPins in inhibiting human HGF binding to its receptor was determined by a receptor competition ELISA. c-Met (Fc chimera; RnD Systems, USA, 358-MT-100/CF) was pre-coated on a microplate. DARPins and HGF were pre-mixed in PBSTC and incubated at room temperature for 0.5 hours. The pre-incubation mixture was then transferred to the c-Met-precoated wells, and any HGF not blocked by the DARPins was bound to the immobilized receptor. After washing away any unbound material, an HGF-specific polyclonal antibody (RnD Systems, USA, AF-294-NA) was added to the wells. After washing away any unbound material, a horseradish peroxidase-conjugated antibody (Novus Biologicals, USA, NB7357) conjugated to the HGF-specific polyclonal antibody was added to each well. After washing to remove any unbound antibody-enzyme reagent, substrate solution (Roche, Switzerland, 11484281001) was added to the wells, and color development was proportional to the amount of HGF bound. Color development was terminated, and color intensity was measured at 405 nm. In this assay, the DARPins tested demonstrated high HGF inhibitory potency. The results of the examples are summarized in Table 5. Figure 5 provides an example titration curve for a panel of DARPins. Based on the titration curves generated as described above, _IC50 values were calculated using standard methods known to those skilled in the art.

Table 5: Inhibition of the interaction between HGF and its receptor c-Met by DARPin

Example 6: Inhibitory effect of HGF-binding specific DARPins on U87 tumor growth

¹⁰⁷ U87-MG cells were injected subcutaneously into the right flank of Swiss nude mice. After 25 days, when tumors reached an average size of 126 ± 51 ^mm3 , mice were randomly assigned to treatment groups of 8 mice per group, and treatment began. Mice were intravenously injected with 0.1 ml of vehicle solution/PBS or various DARPins in PBS (doses of 0.11 mg/kg, 1.1 mg/kg, and 11 mg/kg), with a 3-day interval between doses (Q3D x 7). Body weight and tumor volume were monitored twice weekly. The experiment was terminated 21 days after the start of treatment. As an example, Figure 6 shows a plot of tumor volume versus treatment time (number of days after treatment initiation) for PEGylated DARPin #62. The DARPins used in this tumor growth inhibition experiment contain an N-terminal histidine tag (SEQ ID NO: 9), an HGF-binding-specific ankyrin repeat domain described herein, a GS-linker peptide (SEQ ID NO: 10), and a C-terminal Cys residue. The protein portion of the DARPins was purified using standard methods by utilizing its histidine tag and conjugated to 40 kDa polyethylene glycol using standard maleimide chemistry. Other known methods for extending the terminal half-life of the ankyrin repeat domains of the invention (e.g., PEGylation, see above) can also be used in the tumor model to test the in vivo efficacy of the domains.

Claims (10)

1. A recombinant protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain specifically binds to human hepatocyte growth factor, wherein the ankyrin repeat domain consists of any amino acid sequence selected from SEQ ID NO: 33, 37, 38, 41 to 43, 48, 51, 56 to 61, wherein the G at position 1 and/or the S at position 2 of the ankyrin repeat domain are optionally deleted; and the L at the penultimate position and/or the N at the last position of the ankyrin repeat domain are optionally replaced with A. 2. A recombinant protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain binds to hepatocyte growth factor in phosphate buffer, and wherein the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO:51, wherein the G at position 1 and/or the S at position 2 of the ankyrin repeat domain are optionally deleted; and the L at the penultimate position and/or the N at the last position of the ankyrin repeat domain are optionally replaced with A. 3. The recombinant protein of claim 1 or 2, wherein the ankyrin repeat domain binds to hepatocyte growth factor in phosphate buffer with a dissociation constant ( _KD ) of less than ^10⁻⁷ M. 4. The recombinant protein of claim 1 or 2, wherein the ankyrin repeat domain inhibits the binding of hepatocyte growth factor to c-Met in phosphate buffer at an _IC50 value of less than ^10⁻⁷ M. 5. The recombinant protein of claim 3, wherein the ankyrin repeat domain inhibits the binding of hepatocyte growth factor to c-Met in phosphate buffer at an _IC50 value of less than ^10⁻⁷ M. 6. A nucleic acid encoding a recombinant protein according to any one of claims 1-5. 7. A pharmaceutical composition comprising a recombinant protein according to any one of claims 1-5 or a nucleic acid according to claim 6, and optionally comprising a pharmaceutically acceptable carrier. 8. Use of the recombinant protein according to any one of claims 1-5 in the preparation of a medicament, wherein the medicament is used to treat a pathological condition characterized by the expression of c-Met. 9. The use according to claim 8, wherein the pathological condition is cancer characterized by the expression of c-Met. 10. The use according to claim 9, wherein the cancer is glioma.