Designed ankyrin repeat domains having binding specificity for serum albumin

Abstract

This invention describes a novelly designed ankyrin repeat domain with binding specificity to serum albumin, a recombinant binding protein comprising at least two designed ankyrin repeat domains with binding specificity to serum albumin, a recombinant binding protein comprising at least one designed ankyrin repeat domain with binding specificity to hepatocyte growth factor (HGF), at least one designed ankyrin repeat domain with binding specificity to vascular endothelial growth factor (VEGF-A), and at least two designed ankyrin repeat domains with binding specificity to serum albumin, a nucleic acid encoding such designed ankyrin repeat domains and recombinant binding proteins, a pharmaceutical composition comprising such designed ankyrin repeat domains, recombinant binding proteins, or nucleic acids, and the use of such designed ankyrin repeat domains, recombinant binding proteins, nucleic acids, or pharmaceutical compositions in the treatment of diseases.

Description

[0001] Cross-referencing of related patent applications

[0002] This application claims the benefit and priority of European patent application EP15162502, filed with the European Patent Office on 2 April 2015. This application also claims the benefit and priority of European patent application EP15162511, filed with the European Patent Office on 2 April 2015. The contents of European patent applications EP15162502 and EP 15162511 are incorporated herein by reference in their entirety for all purposes, including all tables, drawings and claims, and, pursuant to Article 4.18 of the PCT, any element or portion of the description, claims or drawings not contained herein but referred to in Article 20.5(a) of the PCT. Technical Field

[0003] A novel ankyrin repeat domain with binding specificity to serum albumin is provided, exhibiting improved storage stability. A recombinant binding protein comprising at least two ankyrin repeat domains with binding specificity to serum albumin is also provided, exhibiting improved pharmacokinetic characteristics compared to a recombinant binding protein comprising only one ankyrin repeat domain with binding specificity to serum albumin. Specifically, a recombinant binding protein is provided comprising at least one ankyrin repeat domain with binding specificity to hepatocyte growth factor (HGF), at least one ankyrin repeat domain with binding specificity to vascular endothelial growth factor A (VEGF-A), and at least two ankyrin repeat domains with binding specificity to serum albumin. Furthermore, nucleic acids encoding these ankyrin repeat domains and / or recombinant binding proteins, pharmaceutical compositions comprising these ankyrin repeat domains, recombinant binding proteins, or nucleic acids are provided, along with the use of these ankyrin repeat domains, recombinant binding proteins, nucleic acids, or pharmaceutical compositions in the treatment of diseases. Background Technology

[0004] The following discussion of the background art is provided merely to help the reader understand the present invention, and is not intended to acknowledge the prior art that describes or constitutes the present invention.

[0005] In addition to antibodies, novel binding proteins or binding domains exist that can be used to specifically bind target molecules (e.g., Binz, HK, Amstutz, P., Plückthun, A., Nat. Biotechnol. 23, 1257-1268, 2005). One such novel binding protein or binding domain without an Fc is based on a designed repeating protein or a designed repeating domain, such as a designed ankyrin repeating protein or a designed ankyrin repeating domain (WO 2002 / 020565; Binz, HK, Amstutz, P., Kohl, A., Stumpp, MT, Briand, C., Forrer, P., Grütter, MG, Plückthun, A., Nat. Biotechnol. 22, 575-582, 2004). WO 2002 / 020565 describes methods for constructing large libraries of repeating proteins (such as ankyrin repeating proteins) and their general applications. WO 2012 / 069654 describes a recombinant binding protein containing a designed ankyrin repeat domain with binding specificity to serum albumin. WO 2010 / 060748 describes a recombinant binding protein containing a designed ankyrin repeat domain with binding specificity to VEGF-A, and WO 2011 / 135067 describes a modified form of this recombinant binding protein that specifically binds to VEGF-A. WO 2014 / 191574 describes a recombinant binding protein containing a designed ankyrin repeat domain with binding specificity to HGF. None of these patent applications disclose recombinant binding proteins containing designed ankyrin repeat domains with binding specificity to both VEGF-A and HGF.

[0006] Unlike IgG antibodies, which exhibit, for example, a longer systemic half-life mediated by FcRn recycling, proteins containing engineered ankyrin repeat domains typically exhibit rapid pharmacokinetic clearance and a shorter terminal half-life, unless the protein contains elements that improve pharmacokinetic characteristics, such as engineered ankyrin repeat domains with binding specificity to serum albumin, as described, for example, in WO 2012 / 069654. Using serum albumin binding to improve the pharmacokinetic profile of proteins is a well-known method in the art (see, for example, WO 9101743; Frejd FY, 2012 (in Kontermann, R (Ed.) “Therapeutic proteins: strategies to modulate their plasma half-lives”, Wiley-VCH Verlag GmbH, 2012, ISBN 978-3-527-32849-9); Nguyen, A., Reyes, AIE II., Zhang, M., McDonald, P., Wong, WL, Damico, LA, Dennis, MS Protein Eng. Des. Sel. 19, 291-297, 2006; WO 2008 / 096158; WO 2006 / 122787; WO 2011 / 095545 and WO 2012 / 069654). To enable the use of designed ankyrin repeat domains with serum albumin binding specificity in clinical drug candidates, the storage stability of designed ankyrin repeat domains known to have serum albumin binding specificity must be improved. This article discloses designed ankyrin repeat domains with improved serum albumin binding specificity.

[0007] The effect of the valence of a designed ankyrin repeat domain with specificity for binding to serum albumin on the pharmacokinetic characteristics of recombinant albumin-binding proteins has not been investigated. Based on studies of albumin-binding domains (Hopp, J., Horning, N., Zettlitz, KA, Schwarz, A., Fuss, N., Müller, D., Kontermann, REProtein Eng. Des. Sel. 23, 827-834, 2010), those skilled in the art predicted that recombinant albumin-binding proteins containing two albumin-binding protein domains (such as a designed ankyrin repeat domain with specificity for binding to serum albumin) would not have improved pharmacokinetic characteristics compared to recombinant albumin-binding proteins containing only one designed ankyrin repeat domain with specificity for binding to serum albumin. Surprisingly, we found that this was not the case. Therefore, this paper discloses recombinant binding proteins containing at least two designed ankyrin repeat domains with binding specificity to serum albumin, which exhibit improved pharmacokinetic characteristics (i.e., prolonged terminal half-life, increased exposure, decreased clearance, and / or increased percentage of injected dose) compared to recombinant binding proteins containing only one designed ankyrin repeat domain with binding specificity to serum albumin.

[0008] It is well known that angiogenesis plays a crucial role in tumorigenesis and maintenance (Ferrera, N., and Kerbel, RS, Nature 438, 967-974, 2005). Therefore, inhibiting angiogenesis, particularly targeting vascular endothelial growth factor (VEGF) and its receptor, has become a cornerstone of modern clinical oncology (Hurwitz, H., Clin. Colorectal Cancer, Suppl. 2, 62-68, 2004; Escudier, B., Clin. Adv. Hematol. Oncol. 5, 530-531, 2007). The mammalian VEGF family consists of five glycoproteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D (also known as FIGF), and placental growth factor (PlGF, also known as PGF). VEGF-A has been shown to be an effective target for anti-angiogenic therapy (Weis, SM, and Cheresh, DA, Nat. Med. 17, 1359-1370, 2011). VEGF-A ligands bind to and activate three structurally similar type III receptor tyrosine kinases named VEGFR-1 (also known as FLT1), VEGFR-2 (also known as KDR), and VEGFR-3 (also known as FLT4). To date, several angiogenesis inhibitors have been approved by regulatory authorities, and these angiogenesis inhibitors, when used in combination with chemotherapy, have shown effects in prolonging progression-free survival (PFS) and / or overall survival across various cancer types. Unfortunately, the use of VEGF / VEGFR inhibitors (such as the VEGF-A inhibitor bevacizumab) has been limited. During treatment, resistance inevitably occurs, suggesting that achieving excellent clinical outcomes may necessarily involve the inhibition of other targets and resistance pathways (Kerbel, RS, N. Engl. J. Med. 358, 2039-2049, 2008; Hurwitz, 2004 (cited above); Escudier, 2007 (cited above)).

[0009] cMet tyrosine kinase is a cell surface receptor for hepatocyte growth factor (HGF, also known as dispersing factor, SF) that is mainly expressed on epithelial cells (Comoglio, PM, Giordano, S., and Trusolino, L., Nat. Rev. Drug Discov. 7, 504-516, 2008). Although cMet and HGF are expressed at low levels in normal adult tissues, their expression is often upregulated in various human tumors, which is associated with tumor cell survival, growth, angiogenesis, invasion, and metastasis in preclinical models (Rong, S., Segal, S., Anver, M., Resau, JH, Vande Woude, GF, Proc. Natl. Acad. Sci. USA 91, 4731-4735, 1994; Michieli, P., Mazzone, M., Basilico, C., Cavassa, S., Sotile, A., Naldini, L., Comaglio, PM, Cancer Cell 6, 61-73, 2004). Upregulation of HGF and / or cMet expression and signaling has been found to be associated with poor prognosis and drug resistance in many clinical tumor types (Fasolo, A., Sessa, C., Gianni, L., Broggini, M., Ann. Oncol. 24, 14-20, 2013). In summary, these studies suggest that the HGF-cMet axis is an important target for therapeutic interventions (Comoglio, 2008 (cited above); Fasolo et al., 2013 (cited above)). HGF mediates numerous cellular responses by binding to its receptor, including 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 has also been shown to play a role in various diseases, including many human solid tumors, where it is involved in tumorigenesis, invasion, and metastasis. Currently, HGF / cMet pathway inhibitors used in Phase II or III clinical development include monoclonal antibodies (mAbs) targeting the extracellular domain of cMet (i.e., MetMab from Genentech-Roche) or small molecule inhibitors targeting the intracellular kinase domain. Small molecule inhibitors (such as tivantinib) and cabozantinib While highly effective, HGF / cMet inhibitors are less specific than mAbs and may have higher toxicity. Anti-HGF / cMet biologics include rilotumumab (AMG102) (a humanized anti-HGF mAb) and ficlatuzumab (AV-299) (a humanized anti-HGF IgG1). Combining HGF / cMet inhibitors with other targeted agents is a hot research area, aiming to simultaneously inhibit various signaling pathways with redundant or synergistic tumor functions. HGF / cMet triggers potent angiogenesis signals that synergistically induce new tumor blood vessels with VEGF and can induce anti-angiogenic therapies (such as those used in glioblastoma and renal cell carcinoma, respectively). and Resistance to sunitinib (Jahangiri, A., De Lay, M., Miller, LM, Carbonell, WS, Hu, YL, Lu, K., Tom, MW, Paquette, J., Tokuyasu, TA, Tsao, S., Marshall, R., Perry, A., Bjorgan, KM, Chaumeil, MM, Ronen, SM, Bergers, G., Aghi, MK, Clin. Cancer Res. 19, 1773-1783, 2013).

[0010] Currently, various anti-HGF / cMet compounds are being investigated in combination with other targeted agents (such as anti-VEGF receptor inhibitors), and these targeted agents have demonstrated good safety profiles across various tumor types (Sharma, PS, Sharma, R., Tyagi, T. Curr. Cancer Drug Targets. 11, 624-653, 2011). However, this combination therapy means that patients must receive two separate treatments, each with varying safety profiles, which could lead to unintended increases in toxicity and potentially limit treatment options. Furthermore, the administration regimens for the different treatments may differ, which could make drug administration more cumbersome for patients. Last but not least, concurrent administration of various agents can significantly increase the costs associated with treatment and patient care.

[0011] One commercially available drug with dual cMet and VEGF inhibitory activity is cabozantinib. (; a small molecule drug) is an oral multispecific tyrosine kinase inhibitor that targets cMet and VEGFR1-3 (in addition to RET, KIT, AXL and FLT3). Clinical methods have been validated for the single-agent formulation of cabozantinib to simultaneously inhibit HGF and VEGF in tumors (Yakes, FM, Chen, J., Tan, J., Yamaguchi, K., Shi, Y., Yu, P., Qian, F., Chu, F., Bentzien, F., Cancilla, B., Orf, J., You, A., Laird, AD, Engst, S., Lee, L., Lesch, J., Chou, YC, Joly, AH, Mol. Cancer Ther. 10, 2298-2308, 2011; Castellone, MD, Carlomagno, F., Salvatore, G., Santoro, M., Best Pract. Res. Clin. Endocrinol. Metab. 22, 1023-1038, 2008). For example, in castration-resistant prostate cancer, studies have shown that anti-HGF mAbrilotumumab failed to demonstrate efficacy as a single agent in a phase II study, while cabozantinib showed antitumor activity in a higher proportion of patients (Smith, DC, Smith, MR, Sweeney, C., Elfiky, AA, Logothetis, C., Corn, PG, Vogelzang, NJ, Small, EJ, Harzstark, AL, Gordon, MS, Vaishampayan, UN, Haas, NB, Spira, AI, Lara, PNJr., Lin, CC, Srinivas, S., Sella, A., P., Scheffold, C., Weitzman, AL, Hussain, M., J. Clin. Oncol. 31, 412-419, 2013). However, this activity is accompanied by a high incidence of adverse events, resulting in dose reductions in 62% of patients, raising concerns about the safety and tolerability of this pleiotropic modality.

[0012] Simultaneous targeting of VEGF-A and HGF / cMet can advantageously disrupt angiogenesis and tumor progression. As mentioned above, current treatments that simultaneously act on the VEGF-A / VEGFR-2 and HGF / cMet pathways are based on single-agent therapy that is nonspecific and leads to safety consequences, or involve several specific agents that must be combined, resulting in the need for co-administration or multiple administrations. Furthermore, some current drugs exhibit short systemic half-lives. Therefore, there is a need for modified drugs that block the VEGF-A / VEGFR-2 and HGF / cMet pathways. This is technically difficult to achieve using antibody drugs, as antibody drugs further require costly production in mammalian cells. This article provides recombinant binding proteins that address these issues. In some embodiments, the recombinant binding proteins provided herein comprise at least one engineered ankyrin repeat domain with binding specificity to VEGF-A, at least one engineered ankyrin repeat domain with binding specificity to HGF, and at least two engineered ankyrin repeat domains with binding specificity to serum albumin (for improving pharmacokinetic characteristics). Summary of the Invention

[0013] This invention relates to a novel ankyrin repeat domain with binding specificity to serum albumin, the domain comprising the amino acid sequence of SEQ ID NO:50, and exhibiting improved storage stability compared to known ankyrin repeat domains with binding specificity to serum albumin. In one embodiment, this invention relates to a recombinant binding protein comprising at least two ankyrin repeat domains with binding specificity to serum albumin, each of which comprises SEQ ID NO:50. In one embodiment, this invention relates to a recombinant binding protein comprising a first ankyrin repeat domain, a second ankyrin repeat domain, a third ankyrin repeat domain, and a fourth ankyrin repeat domain, wherein the first ankyrin repeat domain is specific for binding to VEGF-A, and the second ankyrin repeat domain is specific for binding to HGF, and the third and fourth ankyrin repeat domains are each specific for binding to serum albumin and comprise the amino acid sequence of SEQ ID NO:50. In one embodiment, the first designed ankyrin repeating domain, the second designed ankyrin repeating domain, the third designed ankyrin repeating domain, and the fourth designed ankyrin repeating domain of the recombinant binding protein are arranged in the order of third-second-first-fourth from the N-terminus to the C-terminus. In one embodiment, the first designed ankyrin repeat domain of the recombinant binding protein comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:12 to 21, and an amino acid sequence in which up to 10 amino acids from SEQ ID NO:12 to 21 are arbitrarily interchanged; the second designed ankyrin repeat domain of the recombinant binding protein comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:23 to 37, and an amino acid sequence in which up to 10 amino acids from SEQ ID NO:23 to 37 are arbitrarily interchanged; the third and fourth designed ankyrin repeat domains of the recombinant binding protein each comprise an amino acid sequence SEQ ID NO:50; and the designed ankyrin repeat domains are linked by a polypeptide linker comprising an amino acid sequence selected from amino acid sequences SEQ ID NO:2 to 9, and an amino acid sequence in which up to 4 amino acids from SEQ ID NO:2 to 9 are arbitrarily interchanged.In one embodiment, the first designed ankyrin repeat domain of the recombinant binding protein comprises the amino acid sequence SEQ ID NO:18, the second designed ankyrin repeat domain of the recombinant binding protein comprises the amino acid sequence SEQ ID NO:26, and the third and fourth designed ankyrin repeat domains of the recombinant binding protein each comprise the amino acid sequence SEQ ID NO:50, and the designed ankyrin repeat domains are linked by a polypeptide linker consisting of the amino acid sequence SEQ ID NO:9. In one embodiment, the present invention relates to a recombinant binding protein comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO:134. In one embodiment, the present invention relates to a recombinant binding protein comprising an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:134. In a preferred embodiment, the present invention relates to a recombinant binding protein consisting of the amino acid sequence of SEQ ID NO:134.

[0014] The present invention also relates to nucleic acids encoding amino acid sequences of the designed ankyrin repeating domains or recombinant binding proteins of the present invention.

[0015] The present invention also relates to pharmaceutical compositions comprising the recombinant binding protein of the present invention and / or a designed ankyrin repeating domain or nucleic acid, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0016] This invention also relates to the use of the pharmaceutical compositions of the invention for treating diseases. In one embodiment, the invention further relates to the use of the pharmaceutical compositions of the invention for treating cancer, gastric cancer, or kidney cancer. Attached Figure Description

[0017] Figure 1 : Recombinant structures containing engineered ankyrin repeating domains with binding specificity to serum albumin Illustration of synthesized protein .

[0018] (a) Illustration of a designed ankyrin repeating domain having binding specificity to serum albumin. An example of such ankyrin repeating domain is a designed ankyrin repeating domain having an amino acid sequence selected from SEQ ID NO:40 to 56, particularly a designed ankyrin repeating domain having the amino acid sequence of SEQ ID NO:50. (b) Illustration of a designed ankyrin repeating domain having binding specificity to a target other than serum albumin. An example of such an ankyrin repeating domain is a designed ankyrin repeating domain having an amino acid sequence selected from SEQ ID NO:12 to 39. (c) Illustration of a peptide linker (e.g., a peptide having an amino acid sequence corresponding to any one of SEQ ID NO:2 to 9). (d) Illustration of an N-terminal amino acid sequence. An example of such an N-terminal amino acid sequence is, for example, the sequence MGS or GS, or a peptide tag with an amino acid sequence corresponding to SEQ ID NO:1. (e) Illustration of a bioactive compound. Such portions can be, for example, proteins or protein domains having competitive activity (e.g., hormones or enzymes), antagonistic activity (e.g., receptor domains or antibody fragments), or toxic activity (e.g., toxins). Such portions can also be, for example, small molecule compounds exhibiting, for example, competitive, antagonistic, or toxic activity. (f) An illustration of a recombinant binding protein provided herein comprising two engineered ankyrin repeat domains having binding specificity to serum albumin, and one engineered ankyrin repeat domain having binding specificity to another target besides serum albumin, these domains being linked by a polypeptide linker and having an N-terminal amino acid sequence. For example, a recombinant binding protein having an amino acid sequence corresponding to any one of SEQ ID NO:73 to 81 consists of three engineered ankyrin repeat domains such that SEQ ID NO:73, 75, 78, and 80 have two engineered ankyrin repeat domains having binding specificity to serum albumin, flanking a corresponding third engineered anchoring repeat domain, as illustrated in the illustration. (g) An illustration of the recombinant binding protein provided herein, comprising two designed ankyrin repeat domains specific for binding to serum albumin, and two designed ankyrin repeat domains specific for binding to targets other than serum albumin. These domains are linked by a polypeptide linker and have an N-terminal amino acid sequence. The two designed ankyrin repeat domains specific for binding to serum albumin are located flanking the other two designed ankyrin repeat domains.For example, a recombinant binding protein having an amino acid sequence corresponding to any one of SEQ ID NO: 95 to 107, 110, 116, 122, 129 to 131, 134 to 144, 149 to 172, and 175 to 179, particularly SEQ ID NO: 134, corresponds to this illustration. (h) An illustration of a recombinant binding protein provided herein, comprising two designed ankyrin repeat domains with binding specificity to serum albumin, and two designed ankyrin repeat domains with binding specificity to targets other than serum albumin, these domains being linked by a polypeptide linker and having an N-terminal amino acid sequence. The two designed ankyrin repeat domains with binding specificity to serum albumin are located at the N-terminus of the other two designed ankyrin repeat domains. For example, a recombinant binding protein having an amino acid sequence corresponding to any one of SEQ ID NO: 112, 119, 124, 128, 132, and 133 corresponds to this illustration. (i) An illustration of a pharmaceutical compound comprising two engineered ankyrin repeating domains with binding specificity to serum albumin and a bioactive compound. The bioactive compound may be covalently linked to the two engineered ankyrin repeating domains with binding specificity to serum albumin via chemical coupling or protein fusion (in the case of a peptide).

[0019] Figure 2 Improved storage stability of the recombinant binding protein containing SEQ ID NO:50 The results of SDS 15% PAGE analysis of proteins #49 and #50 (corresponding to SEQ ID NO:49 and 50, respectively, and having SEQ ID NO:1 at the N-terminus, prepared according to Example 4) after being stored in PBS at 10 mg / mL for 1 week at 4℃ (1), 25℃ (2), 40℃ (3), and 60℃ (4) were obtained. M: Label (lower band: 6.5 kDa; band at protein #50 level: 14.4 kDa; upper band in protein #50 PAGE case: 21.5 kDa).

[0020] Figure 3 Mouse pharmacokinetic studies showed that the recombinant binding protein contained two molecules that bind to serum albumin. Benefits of specific, designed ankyrin repeat domains As described in Example 5, using m99Pharmacokinetic studies of Tc-tagged proteins were conducted in mice. The figure shows the percentage of the injected dose (%ID) over time (t; hours) (relative to earlier measurement time points (a: 4 hours; bd: 1 hour)). Unless otherwise stated, the proteins used contain an N-terminal His tag (SEQ ID NO:1) in addition to the indicated sequence. (a) Comparison of pharmacokinetic characteristics of protein #57 (a single engineered ankyrin repeat domain with binding specificity to serum albumin; SEQ ID NO:57, which contains SEQ ID NO:51; solid circle) with proteins #62 and #63 (proteins containing two engineered ankyrin repeat domains (twice SEQ ID NO:51) with binding specificity to serum albumin, wherein the two domains are linked by a GS-rich (SEQ ID NO:63; solid rhombus) or PT-rich (SEQ ID NO:62; solid square) polypeptide linker). The inclusion of two designed ankyrin repeat domains with binding specificity to serum albumin resulted in higher %ID at, for example, 24 hours (+57% GS; +59% PT), 48 hours (+76% GS; +82% PT), or 72 hours (+79% GS; +94% PT) after injection, and improved terminal half-life (+38% GS; +48% PT) compared to proteins containing only a single designed ankyrin repeat domain with binding specificity to serum albumin. (b) Pharmacokinetic characteristics of protein #64 (solid circle) containing SEQ ID NO:22 (designed ankyrin repeat domain with binding specificity to another target besides serum albumin) and SEQ ID NO:51 (designed ankyrin repeat domain with binding specificity to serum albumin) compared to those of protein #73 (solid square) and protein #74 (solid rhombus) containing SEQ ID NO:22 and two SEQ ID NO:51, respectively. Protein #73 has SEQ ID NO:51 flanking SEQ ID NO:22, and protein #74 has twice the number of SEQ ID NO:51 flanking SEQ ID NO:22. The inclusion of two engineered ankyrin repeat domains with binding specificity to serum albumin results in higher %ID, for example, at 24 hours post-injection (+62% N-terminus; +89% flanking) or 48 hours (+136% N-terminus; +175% flanking), and improves the terminal half-life (+>63% for both N-terminus and flanking) compared to proteins containing only a single engineered ankyrin repeat domain with binding specificity to serum albumin.(c) Pharmacokinetic characteristics of protein #82 (solid circle) containing SEQ ID NO:11 (twice; designed ankyrin repeat domain without known binding specificity) and SEQ ID NO:51 (ankyrin repeat domain with binding specificity to serum albumin) compared to protein #109 (solid square) containing SEQ ID NO:11 (twice; N-terminus). The presence of two designed ankyrin repeat domains with binding specificity to serum albumin resulted in higher %ID, for example, at 24 hours (+12%) or 48 hours (+35%) post-injection, and improved terminal half-life (+71%) compared to proteins containing only a single designed ankyrin repeat domain with binding specificity to serum albumin. (d) Pharmacokinetic characteristics of protein #83 (solid circle) containing SEQ ID NO:38 and 39 (designed ankyrin repeat domains, respectively, with binding specificity to a target other than serum albumin) and SEQ ID NO:50 (designed ankyrin repeat domain with binding specificity to serum albumin) compared to protein #110 (solid square) containing SEQ ID NO:38, 39, and 50 (twice; flanking SEQ ID NO:38 and 39). The presence of two designed ankyrin repeat domains with binding specificity to serum albumin resulted in higher %IDs, for example, at 24 hours (+198%), 48 hours (+198%), or 72 hours (+228%) post-injection, and improved terminal half-life (+19%) compared to proteins containing only a single designed ankyrin repeat domain with binding specificity to serum albumin. Note that the measurements for protein #83 are close to the lower limit of quantitation.

[0021] Figure 4 Pharmacokinetic studies in cynomolgus monkeys showed that the recombinant binding protein contained two molecules that interact with serum albumin. Combining the benefits of specific, designed ankyrin repeat domainsPharmacokinetic studies were performed on cynomolgus monkeys as described in Example 6. The concentrations of the corresponding proteins over time (expressed in days (a) or hours (b)) are shown, where (a) is shown in nM and (b) is shown as relative values ​​at measurement points 10 minutes after injection. Unless otherwise stated, the proteins used contain an N-terminal His tag (SEQ ID NO:1) in addition to the indicated sequence. (a) Comparison of pharmacokinetic characteristics of protein #57 (0.5 mg / Kg; 27.7 nmol / kg; a single designed ankyrin repeat domain with binding specificity to serum albumin; SEQ ID NO:57, which contains SEQ ID NO:51; solid circle) with protein #62 (1.04 mg / Kg; 34.5 nmol / kg; a protein containing two designed ankyrin repeat domains with binding specificity to serum albumin (twice SEQ ID NO:51), wherein the two domains are linked by a PT-rich polypeptide linker). The inclusion of two engineered ankyrin repeat domains with binding specificity to serum albumin resulted in higher exposure (2138 d*nmol / L vs. 4676 d*nmol / L, i.e., +119% calculated up to day 7), lower clearance (0.0108 L / (d*kg) vs. 0.0031 L / (d*kg); i.e., -71%), and improved terminal half-life (4.57 days vs. 9.00 days, i.e., +97% calculated from day 1 to day 7) compared to proteins containing only a single engineered ankyrin repeat domain with binding specificity to serum albumin. (b) Pharmacokinetic characteristics of protein #97 (recombinant binding protein consisting of the amino acid sequence of SEQ ID NO:97; solid square) and protein #134 (recombinant binding protein consisting of the amino acid sequence of SEQ ID NO:134, without additional sequence tag; solid circle) administered intravenously at 1 mg / kg to cynomolgus monkeys are shown. Compared with protein #97, protein #134 has improved pharmacokinetic characteristics.

[0022] Figure 5 : Contains two designed ankyrin repeat domains that are specific for binding to serum albumin Size exclusion chromatography coupled with static light scattering analysis of group-binding protein (protein #134) As described in Example 7, experiments were conducted using protein #134 (a recombinant binding protein consisting of SEQ ID NO: 134; solid line), human serum albumin (dashed line), and a mixture of the two (dash line). The experiments showed that protein #134, containing two engineered ankyrin repeating domains (twice the size of SEQ ID NO: 50) with binding specificity to serum albumin, can bind to two human serum albumin molecules simultaneously.

[0023] Figure 6: ELISA analysis of recombinant binding proteinsAs outlined in Example 10, protein #134 (a recombinant binding protein consisting of the amino acid sequence of SEQ ID NO: 134) was analyzed in various binding ELISAs. Protein #134 contains a designed ankyrin repeat domain specific to VEGF-A, a designed ankyrin repeat domain specific to HGF, and two designed ankyrin repeat domains specific to serum albumin, and correspondingly, interactions between protein #134 and these target proteins were predicted. (a) VEGF-A ELISA. Binding signals of protein #134 at various concentrations with fixed VEGF-A (solid circle), rat (solid square), and mouse (solid rhombus) as well as human VEGF-C (hollow inverted triangle) and human PDGF-AB (hollow circle) are shown, along with corresponding fitted inhibition curves. Protein #134 binds VEGF-A with high affinity to these species but not to VEGF-C and PDGF-AB. (b) HGF ELISA. The binding signals of protein #134 with immobilized HGF in humans (solid circles), cynomolgus monkeys (solid triangles), and mice (solid rhombuses) at various concentrations are shown, along with the corresponding fitted inhibition curves. Protein #134 binds to HGF in these species with high affinity. (c) Serum albumin ELISA. The binding signals of protein #134 with immobilized serum albumin in humans (solid circles), cynomolgus monkeys (hollow inverted triangles), rats (hollow triangles), mice (hollow squares), and dogs (hollow rhombuses) at various concentrations are shown, along with the corresponding fitted inhibition curves. Protein #134 binds to serum albumin in these species with high affinity. In the logarithmic scale, OD (optical density) is the OD at 450 nm minus the OD at 620 nm; c[pM] is the concentration of the recombinant binding protein (in pM).

[0024] Figure 7: VEGF-A / VEGF-R2 and HGF / cMet receptor competition assay As described in Example 11, protein #134 (a recombinant binding protein consisting of the amino acid sequence of SEQ ID NO: 134) was analyzed in various competitive assays. (a) VEGF-A / VEGFR-2 HTRF binding competitive assay. Protein #134 inhibits the VEGF-A / VEGFR-2 interaction. The dashed line represents the baseline, and the circle symbol represents no competitive signal. R: the ratio of the 665 nm signal to the 620 nm signal, c: the concentration of protein #134 (in nM). (b) HGF / cMet competitive binding assay. Protein #134 inhibits the HGF / cMet interaction. OD: OD at 450 nm minus OD at 620 nm, c: the concentration of protein #134 (in nM). (c) VEGF-A competitive binding ELISA. Protein #134 binds at an IC50 concentration better than 10 pM. 50Combined with VEGF-A. OD: OD at 450nm minus OD at 620nm, c: concentration of protein #134 (in pM).

[0025] Figure 8 SPR analysis of recombinant binding proteins As described in Example 12, the binding of protein #134 (a recombinant binding protein consisting of the amino acid sequence of SEQ ID NO: 134) to VEGF-A, HGF, and HSA was analyzed using a ProteOn instrument. Human HGF was immobilized on a biosensor chip, and protein #134, human VEGF-A, or human serum albumin was injected according to the following injection protocols: (1) protein #134–hVEGF-A–HSA, (2) protein #134–hVEGF-A–PBST, (3; dashed line) protein #134–PBST–HSA, (4) PBST–PBST–PBST, (5) PBST–hVEGF-A–PBST, (6) PBST–PBST–HSA. Curves 1 and 2 show that protein #134 can bind to both human HGF and human VEGF-A simultaneously. Furthermore, since VEGF-A binding reaches saturation in curve 1, curve 1 indicates that protein #134 can bind to human HGF, human VEGF-A, and human serum albumin simultaneously. Control injections showed no nonspecific interactions. RU: Resonance unit; t: Time (in seconds).

[0026] Figure 9: Effects of recombinant binding proteins on cell proliferation and migration. As described in Example 13, the effects of protein #134 (a recombinant binding protein consisting of the amino acid sequence of SEQ ID NO: 134) were evaluated in different cell assays. (a) Inhibition of HUVEC proliferation by protein #134. HUVECs (3 × 10⁻⁶ cells) were stimulated with 8 ng / mL human VEGF-A. 3 Cells / well). The proliferation status of HUVECs was analyzed in the absence (hollow circles) or with increasing concentrations of protein #134 (solid circles). Inhibition was quantified by adding BrdU during the last 24 hours of incubation after 3 days of culture at 37°C and 5% CO2. Protein #134 exhibited an IC50 value in the range of 100 pM to 150 pM. 50Error bars reflect the standard deviation of independent replicates. OD: OD at 450 nm minus OD at 620 nm, c: concentration of protein #134 (ng / mL). X-axis is displayed on a logarithmic scale. (b) Effect of protein #134 in Oris cell migration assay using A549 cells. Protein #134 significantly inhibited HGF-induced cell migration. Cells were seeded 24 hours before stimulation with HGF (500 pM; H and D) or PBS (N) in the presence (D) and absence (H, N) of protein #134 (5 μM). After removing obstructions, migration was detected and quantified 48 hours after staining cells with calcein. Images were taken for accurate quantification of uncovered areas in cell culture plates. U: uncovered area of ​​8 independent wells (μm). 2 H: HGF, without protein #134, N: without HGF, without protein #134, D: HGF and protein #134. (c) Inhibition of cMet phosphorylation in A549 cells by protein #134. A549 cells were starved overnight and stimulated for 10 min with 1 nM human HGF (negative control without HGF) in the presence of PBS or at progressively increasing concentrations of protein #134. OD450-620 was measured by ELISA, and P-cMet was detected in cell lysates. Relative signal (% phosphorylation) was calculated using the maximum signal (HGF, without protein #134) and minimum signal (without HGF, without protein #134). Protein #134 showed an IC50 greater than 1 nM. 50 Inhibit cMet phosphorylation. %P: percentage of phosphorylation, c: concentration of protein #134 (in nM).

[0027] Figure 10: Effects of recombinant binding proteins on tumor growth in vivo. As described in Example 14, protein #134 (a recombinant binding protein consisting of the amino acid sequence of SEQ ID NO: 134; comprising (i) a designed ankyrin repeat domain specific for binding to VEGF-A, (ii) a designed ankyrin repeat domain specific for binding to HGF, and (iii) two designed ankyrin repeat domains specific for binding to serum albumin, see [link to relevant documentation]) was evaluated in a mouse model of tumor xenograft. Figure 1(a) The efficacy of other recombinant binding proteins. Quantitative analysis of proliferating cells and mean vascular area in tumor tissues of a U87M mouse model treated with protein #134, protein #60 (consisting of the amino acid sequence of SEQ ID NO:60 and additionally having SEQ ID NO:1 at the N-terminus; comprising a designed ankyrin repeat domain (identical to the domain in protein #134) with binding specificity to HGF and a designed ankyrin repeat domain (identical to the domain in protein #134) with binding specificity to serum albumin) or protein #61 (consisting of the amino acid sequence of SEQ ID NO:61 and additionally having SEQ ID NO:1 at the N-terminus; comprising a designed ankyrin repeat domain (identical to the domain in protein #134) with binding specificity to VEGF-A and a designed ankyrin repeat domain (identical to the domain in protein #134) with binding specificity to serum albumin) as described in Example 14. Regarding the inhibition of U87M tumor xenograft cell proliferation (P; measured as percentage of proliferating cells %pc), protein #60 showed slight inhibition, similar to protein #61, while protein #134 had a significantly stronger effect. Similarly, regarding the inhibition of angiogenesis (A; measured as percentage of mean vascular area %mva), protein #60 showed slight inhibition, protein #61 showed moderate inhibition, while protein #134 showed the strongest effect. PBS (white column), protein #60 (horizontal stripe column), protein #61 (vertical stripe column), protein #134 (black column). (b) Effect of protein #134 on tumor growth in a mouse model of human kidney tumor xenograft compared to sorafenib and PBS. Details of the model are described in Example 14. As expected, sorafenib inhibited tumor growth. Interestingly, protein #134 inhibited tumor growth to a greater extent than sorafenib, controlling tumor volume at its initial level. V: Tumor volume (mm) 3 ), d: treatment days, hollow circle: medium, solid circle: protein #134 (4 mg / kg), hollow square: sorafenib (200 mg / kg). (c) Effect of protein #134 on tumor growth in a mouse model of human kidney tumor xenograft compared to paclitaxel and the combination of protein #134 and sorafenib. Details of the model are described in Example 14. Paclitaxel and protein #134 inhibited tumor growth to approximately the same level. The combination of paclitaxel and protein #134 inhibited tumor growth even better, controlling tumor volume at its initial level. V: tumor volume (in mm) 3), d: number of days after treatment, hollow circle: medium, solid circle: protein #134 (4 mg / kg intravenous injection), hollow square: paclitaxel (15 mg / kg intravenous injection), hollow triangle: protein #134 (4 mg / kg) and paclitaxel (15 mg / kg). Detailed Implementation

[0028] In the context of this invention, the term "protein" refers to a polypeptide, wherein at least a portion of the polypeptide has or is capable of acquiring a defined three-dimensional arrangement by forming secondary, tertiary, or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. If the protein comprises two or more polypeptide chains, the individual polypeptide chains may be non-covalently or covalently linked, for example, by disulfide bonds between the two polypeptides. Protein portions that individually have or are capable of acquiring a defined three-dimensional arrangement by forming secondary or tertiary structures are referred to as "protein domains." Such protein domains are well known to those skilled in the art.

[0029] The term "recombinant," as used in recombinant proteins, recombinant protein domains, recombinant binding proteins, etc., refers to the production of the polypeptide using recombinant DNA techniques well known to those skilled in the art. For example, a recombinant DNA molecule encoding a polypeptide (e.g., produced through gene synthesis) can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), a yeast expression plasmid, a mammalian expression plasmid, or a plant expression plasmid, or into DNA capable of in vitro expression. If, for example, such a recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., *Escherichia coli*), the bacterium can produce a polypeptide encoded by the recombinant DNA. The resulting polypeptide is referred to as a recombinant polypeptide or recombinant protein.

[0030] In the context of this invention, the term "binding protein" refers to a protein comprising two or more, preferably three or more, more preferably four or more binding domains. Preferably, the binding protein is a recombinant binding protein. Preferably, the binding protein comprises two or more repeating domains. More preferably, the binding protein comprises three repeating domains. More preferably, the binding protein comprises four repeating domains. Also preferably, the binding protein comprises three or more designed ankyrin repeating domains. Further preferably, the binding protein comprises four or more designed ankyrin repeating domains. More preferably, the binding protein comprises four designed ankyrin repeating domains. Optionally, the binding protein comprises one or more bioactive compounds. Each of the binding domains of the binding protein has target specificity. Preferably, two or more of the binding domains of the binding protein each have target specificity for serum albumin. Preferably, the binding protein comprises at least three binding domains that bind to at least two different targets. More preferably, the binding protein comprises at least four binding domains that bind to at least three different targets.

[0031] In addition, any such binding protein may contain other polypeptides known to those skilled in the art, such as, for example, polypeptide tags or polypeptide linkers.

[0032] The term "bioactive compound" refers to a compound that, when applied to a mammal suffering from a disease, alleviates said disease. Bioactive compounds may have antagonistic or competitive properties and may be protein-based or non-protein-based bioactive compounds. Such protein-based bioactive compounds can be covalently linked to binding domains, such as those of the present invention, by generating a genetic fusion polypeptide using standard DNA cloning techniques, followed by standardized expression and purification. Non-protein-based bioactive compounds can be covalently linked to binding domains, such as those of the present invention, chemically (e.g., by coupling to a cysteine ​​thiol via a maleimide linker, wherein the cysteine ​​is coupled to the N or C terminus of the binding domain as described above via a polypeptide linker). Examples of protein-based bioactive compounds are binding domains with different target specificities (e.g., by binding to a growth factor to neutralize it), cytokines (e.g., interleukins), growth factors (e.g., human growth hormone), antibodies and fragments thereof, hormones (e.g., GLP-1), or protein-based drugs. Examples of non-protein-based bioactive compounds are toxins (e.g., DM1 from an immunogen), small molecules targeting GPCRs, antibiotics, or non-protein-based drugs.

[0033] The term "binding domain" refers to a protein domain that exhibits the same "folding" (i.e., secondary, tertiary, and / or quaternary structure) as a protein scaffold and has predetermined properties as defined below. Such binding domains can be obtained through reasonable or most common combinatorial protein engineering techniques known in the art (Binz et al., 2005 (cited above)). For example, a binding domain with predetermined properties can be obtained by a method comprising the steps of: (a) providing different sets of protein domains having the same folding as a protein scaffold as further defined below; and (b) screening the different sets and / or selecting from the different sets to obtain at least one protein domain having the predetermined properties. Different sets of protein domains can be provided by several methods conforming to the screening and / or selection systems used, and may include methods well known to those skilled in the art, such as phage display or ribosome display. Preferably, the binding domain is a recombinant binding domain.

[0034] The term "protein scaffold" refers to a protein having a highly resistant surface area to amino acid insertions, substitutions, or deletions. Examples of protein scaffolds that can be used to generate the binding domains of the present invention are antibodies or fragments thereof (such as single-chain Fv or Fab fragments), protein A from Staphylococcus aureus, choletriene-binding proteins or other apolipoproteins from Pieris brassicae, ankyrin repeats or other repeating proteins, and human fibronectin. Protein scaffolds are known to those skilled in the art (Binz et al., 2005 (cited above); Binz et al., 2004 (cited above)).

[0035] The term "target" refers to a single molecule, such as a nucleic acid molecule, polypeptide or protein, carbohydrate, or any other naturally occurring molecule, including any part of such a single molecule, or a complex of two or more such molecules. A target can be a whole cell or tissue sample, or it can be any non-natural compound. Preferably, the target is a naturally occurring or non-natural polypeptide or a polypeptide containing chemical modifications (e.g., by natural or non-natural phosphorylation, acetylation, or methylation). In a specific application of the invention, the target is serum albumin, HGF, and VEGF-A.

[0036] The term "predetermined property" refers to properties such as target binding, target blocking, target activation of target-mediated reactions, enzyme activity, and other related properties. Depending on the type of property desired, those skilled in the art will be able to identify the manner and necessary steps for performing screening and / or selecting binding domains with the desired property. Preferably, the predetermined property refers to specific binding to a target.

[0037] In the context of this invention, the term "peptide" refers to a molecule consisting of chains of multiple (i.e., two or more) amino acids linked together by peptide bonds. Preferably, a polypeptide consists of eight or more amino acids linked together by peptide bonds. The term "peptide" also includes multiple chains of amino acids linked together by SS bridges of cysteine ​​residues. Polypeptides are well known to those skilled in the art.

[0038] The term "peptide tag" refers to an amino acid sequence attached to a peptide / protein, wherein the amino acid sequence can be used to purify, detect, or "target" (i.e., locate to a target site) the peptide / protein, or wherein the amino acid sequence improves the physicochemical behavior of the peptide / protein, or wherein the amino acid sequence has an effector function. A single peptide tag binding to a protein can be directly or via a peptide linker to other parts of the binding protein. These peptide tags are well known in the art and are readily available to those skilled in the art. Examples of peptide tags are smaller peptide sequences, such as His (e.g., the His tag of SEQ ID NO:1), myc, FLAG, or Strep tags; or peptides, such as enzymes (e.g., alkaline phosphatases) that allow detection of the peptide / protein, or peptides that can be targeted (e.g., immunoglobulins or fragments thereof) and / or peptides that can be used as effector molecules.

[0039] The term "peptide linker" refers to an amino acid sequence capable of linking, for example, two protein domains, a peptide tag and a protein domain, a protein domain and a non-protein compound or polymer (such as polyethylene glycol), or two sequence tags. Such additional domains, tags, non-protein compounds or polymers, and linkers are known to those skilled in the art. A list of examples is provided in the specification of patent application WO 2002 / 020565. Specific examples of such linkers are variable-length glycine-serine linkers and proline-threonine linkers; preferably, the linkers have a length between 2 and 30 amino acids; more preferably, the linkers have a length between 2 and 24 amino acids. Examples of glycine-serine linkers are the amino acid sequences provided in GS and SEQ ID NO:2 to 6, and examples of proline-threonine linkers are provided in amino acid sequences SEQ ID NO:7 to 9.

[0040] The aforementioned citations to patent application WO 2002 / 020565 and Forrer et al. (2003) contain a general description of the characteristics, techniques, and applications of repeating proteins and repeating domains. The term "repeating protein" refers to a protein containing one or more repeating domains. Preferably, a repeating protein contains up to six repeating domains. More preferably, a repeating protein contains up to five repeating domains. Even more preferably, a repeating protein contains up to four repeating domains. Furthermore, the repeating protein may contain additional non-repeating protein domains, peptide tags, and / or peptide linkers. Repeating domains may be binding domains as described above.

[0041] The term "repetitive domain" refers to a protein domain comprising two or more consecutive repeating modules as structural units, wherein these structural units have identical folds and are tightly stacked to produce a supercoiled structure with a nodal hydrophobic core. In addition to structural homology, such repeating modules also exhibit sequence homology. Preferably, the repeating domain further comprises N-terminal and / or C-terminal capped repeats. For clarity, capped repeats can be repeating modules. Such repetitive domains, repetitive modules and end-capped repeats, sequence motifs, and structural and sequence homology are well known to those skilled in the art based on examples of: designed ankyrin repetitive domains (WO2002 / 020565), leucine-rich repetitive domains (WO 2002 / 020565), 34-peptide repetitive domains (Main, ER, Xiong, Y., Cocco, MJ, D'Andrea, L., Regan, L., Structure 11(5), 497-508, 2003), and armadillo repetitive domains (WO 2009 / 040338). It is further well known to those skilled in the art that these repetitive domains differ from proteins containing repetitive amino acid sequences, in which each repetitive amino acid sequence is capable of forming a single domain (e.g., the FN3 domain of fibronectin), or where the repetitive amino acid sequences are not structural units, i.e., the repetitive amino acid sequences are not tightly stacked to create a supercoiled structure with a nodal hydrophobic core. Methods for identifying and determining repetitive modules or repetitive sequence motifs, or for identifying related protein families containing such repetitive units or motifs (such as homology search methods (BLAST, etc.)), are well-established in the field of bioinformatics and are well known to those skilled in the art.

[0042] The terms "designed repeat protein" and "designed repeat domain" refer to repeat proteins or repeat domains obtained as a result of an innovative procedure (e.g., as described in patent application WO 2002 / 020565). The term "designed" implies that such repeat proteins and repeat domains are artificial, synthetic, and not derived from nature. The designed repeat proteins or designed repeat domains in WO 2002 / 020565 include designed ankyrin repeat proteins or designed ankyrin repeat domains, respectively. Therefore, the designed ankyrin repeat protein as used herein corresponds to the protein of the present invention comprising at least one designed ankyrin repeat domain. Furthermore, the term "not derived from nature" means that the sequence of the binding protein or the binding domain is not present as a non-artificial sequence entry in sequence databases (e.g., GenBank, EMBL-Bank, or Swiss-Prot). These databases and other similar sequence databases are well known to those skilled in the art. The recombinant binding protein or designed ankyrin repeat domain of the present invention is not naturally occurring.

[0043] The terms “repetitive module,” “repetitive unit,” “terminal repeat,” “terminal module,” and other terms related to repetitive proteins and repetitive domains are defined in WO 2002 / 020565, and these definitions are incorporated herein by reference.

[0044] The terms "target-specific binding," "specific binding to the target," "highly specific binding to the target," "target-specific," or "target-specific" refer to the binding protein or binding domain in PBS binding to the target with a lower dissociation constant (i.e., binding with higher affinity) than it binds to unrelated proteins (such as E. coli maltose-binding protein (MBP)). Preferably, the dissociation constant ("Kd") of the target in PBS is at least 1 / 10 of the corresponding dissociation constant of MBP. 2 More preferably at least 1 / 10 3 More preferably at least 1 / 10 4 or more preferably at least 1 / 10 5 .

[0045] Methods for determining the dissociation constant of protein-protein interactions, such as surface plasmon resonance (SPR)-based techniques (e.g., SPR isostatic analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. The measured Kd value for a specific protein-protein interaction can vary if measurements are performed under different conditions (e.g., salt concentration, pH). Therefore, it is preferable to use standardized protein solutions and standardized buffers (such as PBS) for Kd value measurements.

[0046] The term "PBS" refers to an aqueous phosphate buffer solution containing 137 mM NaCl, 10 mM phosphate and 2.7 mM KCl with a pH of 7.4.

[0047] In the context of the binding domains of this invention, the term "inhibitory binding" refers to the ability of the binding domain to prevent its target from binding to another protein (typically a natural ligand of the target or another antagonist). The strength of inhibition is typically assessed by evaluating the half-maximum inhibitory concentration (IC50). 50 The measurement is performed using [the specific technology / method]. The terms "inhibition" and IC [are also relevant]. 50 The evaluation of values ​​is well-established in the field. For example, the designed ankyrin repeat domain of SEQ ID NO:18 inhibits VEGF-A binding to its natural ligand VEGFR-2.

[0048] This invention relates to a designed ankyrin repeat domain that is specific for binding to serum albumin, and to a recombinant binding protein comprising at least two designed ankyrin repeat domains that are specific for binding to serum albumin. It also relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain is specific for binding to VEGF-A, and wherein the second designed ankyrin repeat domain is specific for binding to HGF, and wherein the third and fourth designed ankyrin repeat domains are each specific for binding to serum albumin.

[0049] In one embodiment, the present invention relates to a designed ankyrin repeating domain having binding specificity to serum albumin. Examples of designed ankyrin repeating domains having binding specificity to serum albumin are given in SEQ ID NO:40 to 56 (see also the examples), and other examples are described in WO 2012 / 069654. Specifically, the present invention relates to a designed ankyrin repeating domain having binding specificity to serum albumin selected from SEQ ID NO:48 to 50, more preferably SEQ ID NO:49 and 50, more preferably SEQ ID NO:50, wherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acids are exchanged by any amino acid. In one embodiment, the present invention relates to a designed ankyrin repeat domain having binding specificity to serum albumin, wherein the designed ankyrin repeat domain has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a designed ankyrin repeat domain selected from SEQ ID NO:48 to 50, more preferably SEQ ID NO:49 and 50, more preferably SEQ ID NO:50. In one embodiment, the present invention relates to a designed ankyrin repeat domain having binding specificity to serum, wherein the designed ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NO:48 to 50, more preferably SEQ ID NO:49 and 50, more preferably SEQ ID NO:50. In one embodiment, the present invention relates to a designed ankyrin repeat domain having binding specificity to serum, wherein the designed ankyrin repeat domain is composed of an amino acid sequence selected from SEQ ID NO:48 to 50, more preferably SEQ ID NO:49 and 50, more preferably SEQ ID NO:50. In one embodiment, the present invention relates to a designed ankyrin repeat domain having binding specificity to serum albumin: the designed ankyrin repeat domain comprising the amino acid sequence of SEQ ID NO:50. A preferred designed ankyrin repeat domain having binding specificity to serum albumin is SEQ ID NO:50. Preferably, the designed ankyrin repeat domain having binding specificity to serum albumin has a content of less than 10 -5 M, preferably below 10 -6 M or more preferably less than 10 -7The dissociation constant (Kd) of M binds to serum albumin from mice, rats, dogs, cynomolgus monkeys, or humans in PBS, more preferably to serum albumin from mice, cynomolgus monkeys, or humans, even more preferably to serum albumin from cynomolgus monkeys or humans, and even more preferably to human serum albumin. The term "mouse serum albumin" refers to UniProt accession number P07724, the term "cynomolgus monkey serum albumin" (i.e., macacafascicularis) refers to UniProt accession number A2V9Z4, and the term "human serum albumin" refers to UniProt accession number P02768. In one embodiment, the invention relates to a designed ankyrin repeating domain having binding specificity to serum albumin: the designed ankyrin repeating domain comprises an amino acid sequence selected from SEQ ID NO:48 to 50, more preferably SEQ ID NO:49 and 50, even more preferably SEQ ID NO:50 (which exhibits improved storage stability compared to SEQ ID NO:51), more preferably consisting of said amino acid sequence. In the context of this invention, "improved storage stability" refers to an increase of 0.5°C, 1°C, 1.5°C, 2°C, 2.5°C, 3°C, 3.5°C, or 4°C in the midpoint of denaturation temperature (i.e., the midpoint of co-unfolding after temperature increase), and / or the amount of degradation bands detected by Coomassie-stained SDS-PAGE after storage at 40°C for 1 month in PBS at 10 mg / mL, preferably a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in the amount of degradation products. Methods for assessing storage stability by SDS-PAGE and for determining the midpoint of denaturation using fluorescence assays or circular dichroism are well known to those skilled in the art. In one embodiment, the present invention relates to a designed ankyrin repeating domain having binding specificity to serum albumin: the designed ankyrin repeating domain comprises, more preferably consists of, the amino acid sequence SEQ ID NO:50, which exhibits improved storage stability compared to SEQ ID NO:49, and preferably exhibits a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in the amount of degradation products detected by SDS-PAGE after storage at 10 mg / mL in PBS at 40°C for 1 month compared to SEQ ID NO:49. Examples of designed ankyrin repeating domains with improved storage stability and recombinant binding proteins are given in Example 9.

[0050] In one embodiment, the invention relates to a designed ankyrin repeat domain having binding specificity to serum albumin: the designed ankyrin repeat domain is selected from the amino acid sequences SEQ ID NO:44 to 49, 51 and 52, more preferably SEQ ID NO:48, 49, 51 and 52, more preferably SEQ ID NO:48 and 49, and more preferably SEQ ID NO:49 (containing glutamic acid at position 78). In one embodiment, the invention relates to SEQ ID NO:49 in which the aspartic acid at position 78 has been exchanged for glutamic acid, corresponding to SEQ ID NO:50. SEQ ID NO:49 contains a large number of potential degradation sites. Among other potential degradation sites, degradation may occur, for example, near any one of the five asparagine (including asparagine-glycine dipeptide), 13 aspartic, or 10 glycine in SEQ ID NO:49. SEQ ID NO:49 also contains a large number of potential oxidation sites. Surprisingly, the major effect on storage stability can be achieved by mutating only position 78 of SEQ ID NO:49. Furthermore, by mutating position 78 of SEQ ID NO:49 from aspartic acid to glutamine, the function of the designed ankyrin repeat with binding specificity to serum albumin is preserved. The designed ankyrin repeat domain consisting of SEQ ID NO:49 containing glutamic acid at position 78 exhibits higher storage stability compared to the designed ankyrin repeat domain containing aspartic acid at position 78.

[0051] In one embodiment, the present invention relates to recombinant binding proteins comprising at least two, preferably two, designed ankyrin repeating domains that are specific for binding to serum albumin. Preferred recombinant binding proteins of the present invention comprise two designed ankyrin repeating domains that are specific for binding to serum albumin. Examples of such recombinant binding proteins are given in the amino acid sequences SEQ ID NO: 62, 63, 73 to 81 and 95 to 179.

[0052] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two, more preferably two, designed ankyrin repeat domains with binding specificity to serum albumin, wherein the recombinant binding protein exhibits improved pharmacokinetic characteristics compared to a recombinant binding protein comprising only one designed ankyrin repeat domain with binding specificity to serum albumin. Examples of the present invention disclose such a recombinant binding protein.

[0053] The statement "a recombinant binding protein containing only one designed ankyrin repeat domain with binding specificity to serum albumin" refers to a recombinant binding protein having the composition of the recombinant binding protein of the present invention, wherein the number of designed ankyrin repeat domains with binding specificity to serum albumin is reduced to one by removing all (except one) designed ankyrin repeat domains with binding specificity to serum albumin and their corresponding polypeptide linkers. Preferably, the remaining designed ankyrin repeat domain with binding specificity to serum albumin is located at a position in the recombinant binding protein corresponding to the position of the designed ankyrin repeat domain with binding specificity to serum albumin in the recombinant binding protein of the present invention, and the remaining designed ankyrin repeat domain with binding specificity to serum albumin is the same as the designed ankyrin repeat domain with binding specificity to serum albumin located at the corresponding position in the recombinant binding protein of the present invention. For example, the recombinant binding protein composed of SEQ ID NO:85 is a recombinant binding protein composed of SEQ ID NO:95 in which the designed ankyrin repeat domain (SEQ ID NO:50 in this case) with specific binding to serum albumin at the C-terminus and the adjacent polypeptide linker (SEQ ID NO:9 in this case) have been removed. Importantly, the remaining designed ankyrin repeat domain (SEQ ID NO:50) with specific binding to serum albumin in SEQ ID NO:85 is N-terminal, and SEQ ID NO:95 contains the same SEQ ID NO:50 at the same position. Similarly, the recombinant binding protein composed of SEQ ID NO:83 is a recombinant binding protein composed of SEQ ID NO:110 in which the designed ankyrin repeat domain (SEQ ID NO:50 in this case) with specific binding to serum albumin at the C-terminus and the adjacent polypeptide linker (SEQ ID NO:9 in this case) have been removed.

[0054] In this invention, the expressions "exhibiting improved pharmacokinetic characteristics," "improved pharmacokinetic characteristics," or "improved pharmacokinetic characteristics" have the following meanings: the pharmacokinetic parameters of the recombinant binding protein are improved compared to the corresponding pharmacokinetic parameters of the compared protein. Corresponding examples are provided in Examples 5 and 6, and... Figure 3 and Figure 4As shown in the figure. For example, when protein #110 (a protein consisting of SEQ ID NO:110 and an additional SEQ ID NO:1 at the N-terminus) was compared with protein #83 (a protein consisting of SEQ ID NO:83 and an additional SEQ ID NO:1 at the N-terminus) in a cynomolgus monkey pharmacokinetics study, protein #110 had a higher exposure rate (+32%), a lower clearance rate (-47%), and a longer terminal half-life (+168%, calculated based on day 1 to day 6) compared to protein #83. For example, when protein #62 (a protein consisting of SEQ ID NO:62 and an additional SEQ ID NO:1 at the N-terminus) was compared with protein #57 (a protein consisting of SEQ ID NO:57 and an additional SEQ ID NO:1 at the N-terminus) in a cynomolgus monkey pharmacokinetics study, protein #62 had a higher exposure rate (+119%), a lower clearance rate (-71%), and a longer terminal half-life (i.e., +97% calculated from day 1 to day 7) compared to protein #57. Alternatively, when protein #109 (composed of SEQ ID NO: 109 and an additional SEQ ID NO: 1 at the N-terminus) was compared with protein #82 (composed of SEQ ID NO: 82 and an additional SEQ ID NO: 1 at the N-terminus) in a cynomolgus monkey pharmacokinetics study, protein #109 showed a higher exposure rate (+19%), a lower clearance rate (-37%), and a longer terminal half-life (i.e., +55% calculated from day 1 to day 7) compared to protein #82. As another example, when protein #97 (composed of SEQ ID NO: 97 and an additional SEQ ID NO: 1 at the N-terminus) was compared with protein #68 (composed of SEQ ID NO: 68 and an additional SEQ ID NO: 1 at the N-terminus) in a cynomolgus monkey pharmacokinetics study, protein #97 showed a longer terminal half-life (i.e., +264% calculated from day 1 to day 7) compared to protein #68. Further examples are given in Examples 5 and 6, and... Figure 3 and Figure 4As shown in the figure. Preferably, the improved pharmacokinetic characteristics are reduced clearance and / or increased exposure, and / or prolonged terminal half-life. More preferably, the improved pharmacokinetic properties are a prolonged terminal half-life. In one embodiment, compared to recombinant binding proteins containing only one designed ankyrin repeat domain with binding specificity to serum albumin, the recombinant binding proteins of the present invention containing at least two, more preferably two, designed ankyrin repeat domains with binding specificity to serum albumin exhibit a prolonged terminal half-life and / or reduced clearance and / or increased exposure by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 250%. In one embodiment, compared with recombinant binding proteins containing only one designed ankyrin repeat domain with binding specificity to serum albumin, the recombinant binding protein of the present invention containing at least two, more preferably two designed ankyrin repeat domains with binding specificity to serum albumin, exhibits an extended terminal half-life, preferably an extended terminal half-life of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 250%.

[0055] Preferably, clearance rate and / or exposure rate and / or terminal half-life are evaluated in mammals, more preferably mice and / or cynomolgus monkeys, more preferably cynomolgus monkeys. Preferably, when measuring clearance rate and / or exposure rate and / or terminal half-life in mice, data from up to 48 hours post-injection are considered for evaluation. More preferably, when evaluating terminal half-life in mice, data from 24 to 48 hours are used for calculation. Preferably, when measuring clearance rate and / or exposure rate and / or terminal half-life in cynomolgus monkeys, data from up to day 7 post-injection are considered for evaluation. More preferably, when evaluating terminal half-life in cynomolgus monkeys, data from day 1 to day 7 are used for calculation. The term "terminal half-life" for drugs (such as the recombinant binding protein of the present invention) refers to the time required for the plasma concentration of the drug to reach half the concentration applied to a mammal after pseudo-equilibrium (e.g., calculated using data from 24 to 48 hours in mice, or from day 1 to day 7 in cynomolgus monkeys). Terminal half-life is not defined as the time required for half the dose of drug applied to a mammal to be eliminated. The term "terminal half-life" is well known to those skilled in the art. Preferably, pharmacokinetic comparisons are performed at any dose, more preferably at an equivalent dose (i.e., the same mg / kg dose) or an equimolar dose (i.e., the same mol / kg dose), more preferably at an equimolar dose (i.e., the same mol / kg dose). Those skilled in the art will understand that experimental dose variations in animals with equivalent and / or equimolar administration are at least 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Preferably, the dose used for pharmacokinetic measurements is selected from 0.001 mg / kg to 1000 mg / kg, more preferably from 0.01 mg / kg to 100 mg / kg, more preferably from 0.1 mg / kg to 50 mg / kg, and even more preferably from 0.5 mg / kg to 10 mg / kg.

[0056] In one embodiment, compared to recombinant binding proteins containing only one designed ankyrin repeat domain with binding specificity to serum albumin, the recombinant binding protein of the present invention, containing at least two, more preferably two designed ankyrin repeat domains with binding specificity to serum albumin, exhibits a higher percentage of the injected dose in mice at 24 hours and / or 48 hours and / or 72 hours post-injection, preferably 24 hours, preferably 48 hours, more preferably 72 hours, more preferably 72 hours and 48 hours, and more preferably 24 hours, 48 ​​hours and 72 hours post-injection. Preferably, the percentage of the injected dose in mice is calculated by comparing it with a concentration measurement taken at 1 hour or 4 hours, preferably 1 hour, post-injection. In one embodiment, compared to recombinant binding proteins containing only one designed ankyrin repeat domain with binding specificity to serum albumin, the recombinant binding protein of the present invention, containing at least two, more preferably two designed ankyrin repeat domains with binding specificity to serum albumin, exhibits a higher percentage of the injected dose in cynomolgus monkeys at 4 and / or 5 and / or 6 days post-injection, preferably 4 days, preferably 5 days, more preferably 6 days, more preferably 5 and 6 days post-injection, and more preferably 4, 5 and 6 days post-injection. Preferably, the percentage of the injected dose in cynomolgus monkeys is calculated by comparing it with a concentration measurement taken at 10 minutes or 1 hour post-injection, preferably 10 minutes. A higher percentage of the injected dose refers to an increase in the dose percentage of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 250%.

[0057] In one embodiment, the recombinant binding protein of the present invention comprises at least three engineered ankyrin repeat domains, wherein at least two of the engineered ankyrin repeat domains are engineered ankyrin repeat domains that are specific for binding to serum albumin. Examples of such recombinant binding proteins are given in the amino acid sequences SEQ ID NO: 73 to 81 and 95 to 179.

[0058] In one embodiment, the recombinant binding protein comprises at least four designed ankyrin repeat domains, wherein at least two of the designed ankyrin repeat domains are designed ankyrin repeat domains that are specific for binding to serum albumin. Examples of such recombinant binding proteins are given in the amino acid sequences SEQ ID NO: 95 to 179.

[0059] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeat domains having binding specificity to serum albumin, wherein each of the designed ankyrin repeat domains having binding specificity to serum albumin in PBS has binding specificity to mammalian serum albumin, more preferably mouse, rat, dog, cynomolgus monkey or human serum albumin, more preferably mouse, cynomolgus monkey or human serum albumin, more preferably cynomolgus monkey or human serum albumin, more preferably human serum albumin.

[0060] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two engineered ankyrin repeat domains having binding specificity to serum albumin, wherein each of the engineered ankyrin repeat domains having binding specificity to serum albumin has a binding frequency of less than 10. -5 M, preferably below 10 -6 M, preferably less than 10 -7 The dissociation constant (Kd) of M binds to serum albumin in PBS, more preferably mammalian serum albumin, more preferably mouse, rat, dog, cynomolgus monkey, or human serum albumin, more preferably mouse, cynomolgus monkey, or human serum albumin, more preferably cynomolgus monkey, or human serum albumin, more preferably human serum albumin. Examples of such designed ankyrin repeating domains with binding specificity to serum albumin are given in Example 2 and SEQ ID NO:40 to 56.

[0061] In one embodiment, the present invention relates to a recombinant binding protein comprising two designed ankyrin repeat domains specific to binding serum albumin, wherein the two designed ankyrin repeat domains specific to binding serum albumin are located at any position compared to any other protein domain (preferably included in any other designed ankyrin repeat domain in the recombinant binding protein), preferably wherein the two designed ankyrin repeat domains specific to binding serum albumin are both N-termini of any other protein domain (preferably included in any other designed ankyrin repeat domain in the recombinant binding protein), or wherein the two designed ankyrin repeat domains specific to binding serum albumin are one N-terminus and one C-terminus of any other protein domain (preferably included in any other designed ankyrin repeat domain in the recombinant binding protein), or more preferably, wherein the two designed ankyrin repeat domains specific to binding serum albumin are one N-terminus and one C-terminus of any other protein domain (preferably included in any other designed ankyrin repeat domain in the recombinant binding protein). Preferably, the two designed ankyrin repeat domains with binding specificity to serum albumin are not both C-termini of any other protein domain (preferably included in any other designed ankyrin repeat domain in the recombinant binding protein). Examples of different arrangements of the designed ankyrin repeat domains within the recombinant binding protein are given in SEQ ID NO: 95 to 179 and described in the embodiments. SEQ ID NO: 134 shows a preferred arrangement of the two designed ankyrin repeat domains with binding specificity to serum albumin in the recombinant binding protein of the present invention.

[0062] In one embodiment, the present invention relates to a recombinant binding protein comprising at least three, preferably at least four, more preferably four designed ankyrin repeat domains, wherein two of the at least three, preferably at least four, more preferably four designed ankyrin repeat domains are each specific for binding to serum albumin, and / or preferably, wherein the at least three, preferably at least four, more preferably four designed ankyrin repeat domains are linked by a polypeptide linker.

[0063] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeating domains having binding specificity to serum albumin, wherein the designed ankyrin repeating domains having binding specificity to serum albumin have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any amino acid sequence selected from SEQ ID NO:44 to 52, preferably SEQ ID NO:48 to 50, more preferably SEQ ID NO:49 and 50, and more preferably SEQ ID NO:50.

[0064] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeat domains having binding specificity to serum albumin, wherein the designed ankyrin repeat domains having binding specificity to serum albumin are selected from any amino acid sequence selected from SEQ ID NO:44 to 52, preferably SEQ ID NO:48 to 50, more preferably SEQ ID NO:49 and 50, more preferably SEQ ID NO:50, and wherein in each of the two designed ankyrin repeat domains having binding specificity to serum albumin, at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acids are exchanged by any amino acid.

[0065] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeat domains having binding specificity to serum albumin, wherein the designed ankyrin repeat domains having binding specificity to serum albumin are selected from any amino acid sequence, wherein the any amino acid sequence is selected from SEQ ID NO:44 to 52, preferably SEQ ID NO:48 to 50, more preferably SEQ ID NO:49 and 50, and even more preferably SEQ ID NO:50. In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeat domains having binding specificity to serum albumin, wherein each of the designed ankyrin repeat domains having binding specificity to serum albumin comprises the amino acid sequence of SEQ ID NO:50.

[0066] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two engineered ankyrin repeating domains specific for binding to serum albumin, wherein 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% of the amino acid sequence of the engineered ankyrin repeating domains specific for binding to serum albumin are identical. In one embodiment, the amino acid sequence of the engineered ankyrin repeating domains specific for binding to serum albumin in the recombinant binding protein is identical. For example, the two designed ankyrin repeat domains with binding specificity to serum albumin contained in SEQ ID NO:130 are at least 95% identical (6 residues different out of 124 amino acids). Similarly, the two designed ankyrin repeat domains with binding specificity to serum albumin contained in SEQ ID NO:129 are at least 80% identical (24 residues different out of 124 amino acids).

[0067] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two engineered ankyrin repeat domains with binding specificity to serum albumin, wherein each engineered ankyrin repeat domain with binding specificity to serum albumin is capable of simultaneously binding one serum albumin molecule. Preferably, the serum albumin is human. An example of the recombinant binding protein of the present invention comprising two engineered ankyrin repeat domains with binding specificity to serum albumin simultaneously binding two serum albumin molecules is shown in Example 7.

[0068] In one embodiment, the present invention relates to a recombinant binding protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10 designed ankyrin repeating domains that are specific for binding to serum albumin.

[0069] In one embodiment, the present invention relates to a recombinant binding protein comprising at least three, preferably at least four, and more preferably four designed ankyrin repeat domains, wherein at least two, more preferably two, of the designed ankyrin repeat domains are designed ankyrin repeat domains that are specific for binding to serum albumin, more preferably human serum albumin, and wherein the at least two, more preferably two, designed ankyrin repeat domains that are specific for binding to serum albumin are at least, preferably, an N-terminus and a C-terminus of any other designed ankyrin repeat domain (preferably two other designed ankyrin repeat domains), and wherein the at least two, more preferably two, designed ankyrin repeat domains that are specific for binding to serum albumin are at least 10 - 5 M, preferably below 10 -6 Or more preferably less than 10 -7 The dissociation constant (Kd) of M binds to serum albumin, preferably human serum albumin, in PBS, and wherein the recombinant binding protein exhibits an extended terminal half-life, preferably at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, compared to a recombinant binding protein containing only one designed ankyrin repeat domain with binding specificity to serum albumin, and wherein the at least three, preferably at least four, more preferably four designed ankyrin repeat domains are linked by a polypeptide linker. In one embodiment, 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% of the at least two, more preferably two, designed ankyrin repeating domains that are specific to binding to serum albumin are identical, and more preferably the designed ankyrin repeating domains are identical.

[0070] In one embodiment, the protein domain or designed ankyrin repeat domain present in the recombinant binding protein of the present invention is linked by a polypeptide linker composed of any amino acid sequence components. In one embodiment, the polypeptide linker linking the protein or designed ankyrin repeat domain present in the recombinant binding protein of the present invention comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:2 to 9, more preferably SEQ ID NO:3 to 9, more preferably SEQ ID NO:4 to 9, more preferably SEQ ID NO:6 or 9, more preferably SEQ ID NO:9, wherein up to 4, 3, 2, 1, or 0 amino acids are exchanged by any amino acid. In one embodiment, the polypeptide linker comprises an amino acid sequence selected from any one of amino acid sequences SEQ ID NO:2 to 9, more preferably SEQ ID NO:3 to 9, more preferably SEQ ID NO:4 to 9, more preferably SEQ ID NO:6 or 9, more preferably SEQ ID NO:9. In one embodiment, the flanking N-terminal Gly Ser of SEQ ID NO:7 to 9 and / or the flanking C-terminal Gly Ser of SEQ ID NO:2 to 9 are optionally omitted. In one embodiment, SEQ ID NO:7 to 9 further comprises Arg Ser at the C-terminus (as is present, for example, in SEQ ID NO:68 and 109). In one embodiment, the second to C-terminal amino acids glycine of the polypeptide linker of SEQ ID NO:2 to 6 may be exchanged for arginine (as is present, for example, in SEQ ID NO:70 and 88). In one embodiment, the polypeptide linker connecting the designed ankyrin repeating domain present in the recombinant binding protein of the present invention consists of an amino acid sequence selected from any of the amino acid sequences SEQ ID NO:2 to 9, more preferably SEQ ID NO:3 to 9, more preferably SEQ ID NO:4 to 9, more preferably SEQ ID NO:6 or 9, more preferably SEQ ID NO:9. In one embodiment, 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% of the polypeptide linkers present in the recombinant binding protein of the present invention are identical, preferably the polypeptide linkers are identical.

[0071] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeating domains having binding specificity to serum albumin, wherein the polypeptide linker comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:2 to 9, more preferably SEQ ID NO:3 to 9, more preferably SEQ ID NO:4 to 9, more preferably SEQ ID NO:6 or 9, more preferably SEQ ID NO:9, wherein up to 4, 3, 2, 1, or 0 amino acids are exchanged by any amino acid, and wherein the designed ankyrin repeating domain having binding specificity to serum albumin has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any amino acid sequence selected from SEQ ID NO:44 to 52, preferably SEQ ID NO:48 to 50, more preferably SEQ ID NO:49 and 50, more preferably SEQ ID NO:50.

[0072] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two engineered ankyrin repeating domains having binding specificity to serum albumin, wherein the polypeptide linker consists of an amino acid sequence selected from the amino acid sequence SEQ ID NO: 6 or 9, wherein up to 4, 3, 2, 1, or 0 amino acids are arbitrarily interchanged, and wherein each engineered ankyrin repeating domain having binding specificity to serum albumin is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any amino acid sequence selected from SEQ ID NO: 48 to 50.

[0073] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeat domains having binding specificity to serum albumin, wherein each of the at least two designed ankyrin repeat domains having binding specificity to serum albumin comprises the amino acid sequence of SEQ ID NO:50.

[0074] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeat domains specifically binding to serum albumin, wherein each of the at least two designed ankyrin repeat domains specifically binding to serum albumin comprises the amino acid sequence of SEQ ID NO: 50, and wherein, compared to a recombinant binding protein wherein each of the at least two designed ankyrin repeat domains specifically binding to serum albumin comprises the amino acid sequence of SEQ ID NO: 49, and / or compared to a recombinant binding protein wherein each of the at least two designed ankyrin repeat domains specifically binding to serum albumin comprises the amino acid sequence of SEQ ID NO: 51, preferably compared to a recombinant binding protein wherein each of the at least two designed ankyrin repeat domains specifically binding to serum albumin comprises the amino acid sequence of SEQ ID NO: 49, the recombinant binding protein exhibits improved storage stability and preferably reduced degradation products after being stored at 10 mg / mL in PBS at 40°C for 1 month.

[0075] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeat domains having binding specificity to serum albumin, wherein each of the at least two designed ankyrin repeat domains having binding specificity to serum albumin comprises the amino acid sequence of SEQ ID NO:50, and wherein the designed ankyrin repeat domains are linked by peptide linkers each comprising the amino acid sequence of SEQ ID NO:9.

[0076] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeat domains having binding specificity to serum albumin, wherein each of the at least two designed ankyrin repeat domains having binding specificity to serum albumin is composed of the amino acid sequence of SEQ ID NO:50.

[0077] In one embodiment, the present invention relates to a recombinant binding protein comprising at least two designed ankyrin repeat domains having binding specificity to serum albumin, wherein each of the at least two designed ankyrin repeat domains having binding specificity to serum albumin is composed of the amino acid sequence of SEQ ID NO:50, and wherein the designed ankyrin repeat domains are linked by a polypeptide linker composed of the amino acid sequence of SEQ ID NO:9.

[0078] In one embodiment, the present invention relates to a recombinant binding protein comprising four designed ankyrin repeat domains, wherein two of the designed ankyrin repeat domains are designed ankyrin repeat domains that are specific for binding to serum albumin, wherein each of the two designed ankyrin repeat domains that are specific for binding to serum albumin comprises the amino acid sequence of SEQ ID NO:50, and wherein the designed ankyrin repeat domains are linked by a polypeptide linker comprising the amino acid sequence of SEQ ID NO:9, and wherein the designed ankyrin repeat domains are arranged (from the N-terminal side to the C-terminal side) as follows: SEQ ID NO:50–SEQ ID NO:9–XXX–SEQ ID NO:9–YYY–SEQ ID NO:9–SEQ ID NO:50, wherein XXX and YYY each represent a designed ankyrin repeat domain that is specific for binding to another target besides serum albumin.

[0079] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain is specific for binding to VEGF-A, and wherein the second designed ankyrin repeat domain is specific for binding to HGF, and wherein the third designed ankyrin repeat domain and the fourth designed ankyrin repeat domain are each specific for binding to serum albumin. Preferably, the recombinant binding protein consists of a single polypeptide chain. More preferably, the first designed ankyrin repeat domain, the second designed ankyrin repeat domain, the third designed ankyrin repeat domain, and the fourth designed ankyrin repeat domain are linked by a polypeptide linker. In one embodiment, the present invention relates to a recombinant binding protein comprising a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain is specific for binding to VEGF-A, wherein the second designed ankyrin repeat domain is specific for binding to HGF, and wherein the third designed ankyrin repeat domain and the fourth designed ankyrin repeat domain are each specific for binding to serum albumin. Examples of such recombinant binding proteins are given in the amino acid sequences SEQ ID NO: 95 to 108 and 116 to 179.

[0080] Preferably, the designed ankyrin repeat domain with binding specificity to VEGF-A binds to VEGF-A from mice, rats, dogs, rabbits, cynomolgus monkeys, or humans; more preferably, it binds to VEGF-A from mice, cynomolgus monkeys, or humans; even more preferably, it binds to VEGF-A from cynomolgus monkeys or humans; even more preferably, it binds to VEGF-A from humans. Preferably, the VEGF-A is human VEGF-A165. Examples of designed ankyrin repeat domains with binding specificity to VEGF-A are given herein (SEQ ID NO: 12 to 21; see Examples), and other examples are described in WO 2010 / 060748 and WO 2011 / 135067.

[0081] Preferably, the designed ankyrin repeat domain with binding specificity to HGF binds to mouse, rat, dog, rabbit, cynomolgus monkey, or human HGF, more preferably mouse, cynomolgus monkey, or human HGF, even more preferably cynomolgus monkey or human HGF, and even more preferably human HGF. Examples of designed ankyrin repeat domains with binding specificity to HGF are given herein (SEQ ID NO: 23 to 37; see Examples), and other examples are described in WO 2014 / 191574.

[0082] In one embodiment, the recombinant binding protein or the designed ankyrin repeating domain does not contain free Cys residues. "Free Cys residues" do not participate in disulfide bond formation. In one embodiment, the present invention relates to a binding protein or binding domain that does not contain any Cys residues. In one embodiment, the designed ankyrin repeating domain and / or the recombinant binding protein does not contain any disulfide bonds. It is known to those skilled in the art that, for example, disulfide bonds in antibody fragments hinder the simple preparation of antibody fragments in bacteria.

[0083] The techniques for modifying the recombinant binding protein of this invention are well known to those skilled in the art.

[0084] Specifically, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeating domain, a second designed ankyrin repeating domain, a third designed ankyrin repeating domain, and a fourth designed ankyrin repeating domain, wherein the first designed ankyrin repeating domain is at a concentration of less than 10 -7 M, preferably below 10 -8 M, preferably less than 10 -9 M or more preferably less than 10 -10 The dissociation constant (Kd) of M binds to VEGF-A in PBS; and wherein the second designed ankyrin repeating domain binds to VEGF-A at a concentration of less than 10. -7 M, preferably below 10 -8 M, preferably less than 10 -9 M or more preferably less than 10- 10 M's Kd binds to HGF in PBS; and wherein the third and fourth designed ankyrin repeat domains each bind at less than 10 -5 M, preferably below 10 -6 M or more preferably less than 10 -7 M's Kd binds to serum albumin in PBS. Examples of designed ankyrin repeat domains with binding specificity to VEGF-A, HGF, and serum albumin are given herein (SEQ ID NO: 12 to 56; see Examples).

[0085] Furthermore, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeating domain, a second designed ankyrin repeating domain, a third designed ankyrin repeating domain, and a fourth designed ankyrin repeating domain, wherein the first designed ankyrin repeating domain is at a concentration of less than 10 -7 M, Preferred 10 -8 M, more preferably 10 -9 M's IC 50 The value inhibits human VEGF-A binding to human VEGFR-2 in PBS, and wherein the second engineered ankyrin repeat domain is at a value of less than 10. -7 M, Preferred 10 -8 M, more preferably 10 -9 M's IC 50 The value inhibits the binding of human HGF to human cMet in PBS. Different examples of designed ankyrin repeating domains selected from SEQ ID NO:12 to 37 are given in the examples.

[0086] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeating domain, a second designed ankyrin repeating domain, a third designed ankyrin repeating domain, and a fourth designed ankyrin repeating domain, wherein the first designed ankyrin repeating domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with an amino acid repeating domain selected from amino acid sequences SEQ ID NO:12 to 21, more preferably SEQ ID NO:17 to 21, more preferably SEQ ID NO:18 to 20, and more preferably SEQ ID NO:18. In one embodiment, the first designed ankyrin repeating domain comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:12 to 21, more preferably SEQ ID NO:17 to 21, more preferably SEQ ID NO:18 to 20, more preferably SEQ ID NO:18, and wherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids of SEQ ID NO:12 to 21, more preferably SEQ ID NO:17 to 21, more preferably SEQ ID NO:18 to 20, more preferably SEQ ID NO:18 are arbitrarily interchanged. In one embodiment, the first designed ankyrin repeating domain comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:14 to 21, wherein a single amino acid is substituted by an arbitrary amino acid, the substitution occurring at the same position aligned with the amino acid sequences of SEQ ID NO:14 to 21. In one embodiment, the first designed ankyrin repeating domain comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:12 to 21, more preferably SEQ ID NO:17 to 21, more preferably SEQ ID NO:18 to 20, and more preferably SEQ ID NO:18. In another embodiment, the first designed ankyrin repeating domain is composed of an amino acid sequence selected from amino acid sequences SEQ ID NO:12 to 21, more preferably SEQ ID NO:17 to 21, more preferably SEQ ID NO:18 to 20, and more preferably SEQ ID NO:18.Furthermore, the second designed ankyrin repeating domain of the recombinant binding protein preferably comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with a designed ankyrin repeating domain selected from amino acid sequences SEQ ID NO:23 to 37, more preferably SEQ ID NO:23 to 27, more preferably SEQ ID NO:25 to 27, and more preferably SEQ ID NO:26. In one embodiment, the second designed ankyrin repeating domain comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:23 to 37, more preferably SEQ ID NO:23 to 27, more preferably SEQ ID NO:25 to 27, more preferably SEQ ID NO:26, and an amino acid sequence in which up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids of SEQ ID NO:23 to 37, more preferably SEQ ID NO:23 to 27, more preferably SEQ ID NO:25 to 27, more preferably SEQ ID NO:26 are arbitrarily interchanged. In another embodiment, the second designed ankyrin repeating domain comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:23 to 27, wherein a single amino acid is substituted by an arbitrary amino acid, the substitution occurring at the same position aligned with the amino acid sequences of SEQ ID NO:23 to 27. In one embodiment, the second designed ankyrin repeating domain comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:23 to 37, more preferably SEQ ID NO:23 to 27, more preferably SEQ ID NO:25 to 27, and more preferably SEQ ID NO:26. In another embodiment, the second designed ankyrin repeating domain is composed of an amino acid sequence selected from amino acid sequences SEQ ID NO:23 to 37, more preferably SEQ ID NO:23 to 27, more preferably SEQ ID NO:25 to 27, and more preferably SEQ ID NO:26. In one embodiment, the third and fourth designed ankyrin repeat domains of the recombinant binding protein each comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with an ankyrin repeat domain selected from amino acid sequences SEQ ID NO:40 to 56, preferably SEQ ID NO:48 to 52, more preferably SEQ ID NO:48 to 50, and even more preferably SEQ ID NO:50.In one embodiment, the third and fourth designed ankyrin repeating domains each comprise an amino acid sequence selected from amino acid sequences SEQ ID NO:40 to 56, preferably SEQ ID NO:48 to 52, more preferably SEQ ID NO:48 to 50, more preferably SEQ ID NO:50, and an amino acid sequence in which up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids from SEQ ID NO:40 to 56, preferably SEQ ID NO:48 to 52, more preferably SEQ ID NO:48 to 50, more preferably SEQ ID NO:50 are exchanged for any other amino acid. In one embodiment, the third and fourth designed ankyrin repeating domains each comprise an amino acid sequence selected from amino acid sequences SEQ ID NO:42 to 51, wherein a single amino acid is replaced by any amino acid, the replacement occurring at the same position aligned with the amino acid sequences of SEQ ID NO:42 to 51. In one embodiment, the third and fourth designed ankyrin repeating domains each comprise an amino acid sequence selected from amino acid sequences SEQ ID NO:40 to 56, preferably SEQ ID NO:48 to 52, more preferably SEQ ID NO:48 to 50, and even more preferably SEQ ID NO:50. In another embodiment, the third and fourth designed ankyrin repeating domains each consist of an amino acid sequence selected from amino acid sequences SEQ ID NO:40 to 56, preferably SEQ ID NO:48 to 52, more preferably SEQ ID NO:48 to 50, and even more preferably SEQ ID NO:50. In another embodiment, the third and fourth designed ankyrin repeating domains are identical. Furthermore, in this embodiment, the designed ankyrin repeating domain is connected by a polypeptide linker selected from amino acid sequences SEQ ID NO:2 to 9, more preferably SEQ ID NO:3 to 9, more preferably SEQ ID NO:4 to 9, more preferably SEQ ID NO:6 or 9, more preferably SEQ ID NO:9, and amino acid sequences in which up to 4, 3, 2, 1, or 0 amino acids of SEQ ID NO:2 to 9, more preferably SEQ ID NO:3 to 9, more preferably SEQ ID NO:4 to 9, more preferably SEQ ID NO:6 or 9, more preferably SEQ ID NO:9 are exchanged for any amino acid.In one embodiment, the polypeptide linker comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:2 to 9, more preferably SEQ ID NO:3 to 9, more preferably SEQ ID NO:4 to 9, more preferably SEQ ID NO:6 or 9, more preferably SEQ ID NO:9. In one embodiment, the flanking N-terminal Gly Ser of SEQ ID NO:7 to 9 and / or the flanking C-terminal Gly Ser of SEQ ID NO:2 to 9 are optionally omitted. In one embodiment, SEQ ID NO:7 to 9 further comprises Arg Ser at the C-terminus (as present, for example, in SEQ ID NO:97 and 98). In one embodiment, the second to C-terminal amino acids glycine of the polypeptide linker of SEQ ID NO:2 to 6 may be exchanged for arginine (as present, for example, in SEQ ID NO:99 and 100). In one embodiment, the polypeptide linker connecting the designed ankyrin repeating domain present in the recombinant binding protein of the present invention comprises an amino acid sequence selected from any of the amino acid sequences SEQ ID NO:2 to 9, more preferably SEQ ID NO:3 to 9, more preferably SEQ ID NO:4 to 9, more preferably SEQ ID NO:6 or 9, more preferably SEQ ID NO:9. In one embodiment, the polypeptide linker present in the recombinant binding protein of the present invention is identical. Examples of such polypeptide linkers, variations thereof, and the use of such polypeptide linkers in recombinant binding proteins are given in the embodiments.

[0087] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeating domain, a second designed ankyrin repeating domain, a third designed ankyrin repeating domain, and a fourth designed ankyrin repeating domain, wherein the first designed ankyrin repeating domain comprises an amino acid sequence selected from amino acid sequences SEQ ID NO: 12 to 21, preferably SEQ ID NO: 17 to 21, more preferably SEQ ID NO: 18 to 20, and more preferably SEQ ID NO: 18; wherein the second designed ankyrin repeating domain comprises an amino acid sequence selected from amino acid sequences SEQ ID NO: 23 to 37, more preferably SEQ ID NO: 23 to 27, more preferably SEQ ID NO: 25 to 27, and more preferably SEQ ID NO: 26; and wherein the third designed ankyrin repeating domain and the fourth designed ankyrin repeating domain each comprise an amino acid sequence selected from amino acid sequences SEQ ID NO: 40 to 56, preferably SEQ ID NO: 48 to 52, more preferably SEQ ID NO: 48 to 50, and more preferably SEQ ID NO: 18. The amino acid sequence of NO:50, wherein the designed ankyrin repeating domain is connected by a polypeptide linker selected from amino acid sequences SEQ ID NO:2 to 9, more preferably SEQ ID NO:3 to 9, more preferably SEQ ID NO:4 to 9, more preferably SEQ ID NO:6 or 9, more preferably SEQ ID NO:9.

[0088] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain is specific for binding VEGF-A, and wherein the second designed ankyrin repeat domain is specific for binding HGF, and wherein the third and fourth designed ankyrin repeat domains are each specific for binding serum albumin, wherein the designed ankyrin repeat domains are linked by a peptide linker, and wherein the recombinant binding protein can simultaneously bind VEGF-A and HGF, more preferably simultaneously bind VEGF-A, HGF, and serum albumin, and even more preferably simultaneously bind human VEGF-A, human HGF, and human serum albumin. In one embodiment, the recombinant binding protein can simultaneously bind two serum albumin molecules, more preferably simultaneously bind two serum albumin molecules.

[0089] The terms “first,” “second,” “third,” and optionally “fourth” used in “first designed ankyrin repeating domain,” “second designed ankyrin repeating domain,” “third designed ankyrin repeating domain,” and “fourth designed ankyrin repeating domain” do not indicate or imply any arrangement of the designed ankyrin repeating domains within the recombinant-binding protein.

[0090] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain linked by a peptide linker. In one embodiment, the first designed ankyrin repeat domain is the N-terminus of a C-terminal designed ankyrin repeat domain and the C-terminus of two other designed ankyrin repeat domains. In one embodiment, the second designed ankyrin repeat domain, which is specific for binding to HGF, is the C-terminus of an N-terminal designed ankyrin repeat domain and the N-terminus of two other designed ankyrin repeat domains. In one embodiment, the third and fourth designed ankyrin repeat domains (each specific for binding to serum albumin) are an N-terminus and a C-terminus of two other designed ankyrin repeat domains, or they are the N-terminus of two other designed ankyrin repeat domains; more preferably, the third and fourth designed ankyrin repeat domains are an N-terminus and a C-terminus of two other designed ankyrin repeat domains. In one embodiment, the third designed ankyrin repeating domain is the N-terminus of three other designed ankyrin repeating domains, the fourth designed ankyrin repeating domain is the C-terminus of three other designed ankyrin repeating domains, the second designed ankyrin repeating domain is the C-terminus of the third designed ankyrin repeating domain and the N-terminus of the first designed ankyrin repeating domain, and the first designed ankyrin repeating domain is the C-terminus of the second designed ankyrin repeating domain and the N-terminus of the fourth designed ankyrin repeating domain.

[0091] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain is specific for binding to VEGF-A, and wherein the second designed ankyrin repeat domain is specific for binding to HGF, and wherein the third designed ankyrin repeat domain and the fourth designed ankyrin repeat domain are each specific for binding to serum albumin, and wherein the designed ankyrin repeat domains are linked by a peptide linker. In one embodiment, the order (from N-terminus to C-terminus) of the first designed ankyrin repeat domain, the second designed ankyrin repeat domain, the third designed ankyrin repeat domain, and the fourth designed ankyrin repeat domain (from N-terminus to C-terminus) is third-second-first-four, third-four-second-first, fourth-second-first-third, or fourth-third-second-first, even more preferably third-second-first-four, or fourth-second-first-third, even more preferably third-second-first-four.

[0092] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain is specific for binding to VEGF-A, and wherein the second designed ankyrin repeat domain is specific for binding to HGF, and wherein the third designed ankyrin repeat domain and the fourth designed ankyrin repeat domain are each specific for binding to serum albumin, wherein the recombinant binding protein binds to EC. 50 Less than 10 -7 M, preferably less than 10 -8 M, more preferably less than 10 -9 M, more preferably less than 10 -10 M's VEGF-A, preferably human VEGF-A.

[0093] In one embodiment, the present invention relates to a recombinant-binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the third designed ankyrin repeat domain and the fourth designed ankyrin repeat domain each comprise the amino acid sequence of SEQ ID NO:50. In another embodiment, the present invention relates to a recombinant-binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO:18, and the second designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO:26, and the third designed ankyrin repeat domain and the fourth designed ankyrin repeat domain each comprise the amino acid sequence of SEQ ID NO:50, and wherein the designed ankyrin repeat domains are linked by peptide linkers, each peptide linker comprising the amino acid sequence of SEQ ID NO:9.

[0094] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO:18, and wherein the second designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO:26, and wherein the third designed ankyrin repeat domain and the fourth designed ankyrin repeat domain each comprise the amino acid sequence of SEQ ID NO:50, and wherein the designed ankyrin repeat domains are linked by peptide linkers, each peptide linker comprising the amino acid sequence of SEQ ID NO:9, and wherein the designed ankyrin repeat domains are arranged (from the N-terminal side to the C-terminal side) as follows: SEQ ID NO:50–SEQ ID NO:9–SEQ ID NO:26–SEQ ID NO:9–SEQ ID NO:18–SEQ ID NO:9–SEQ ID NO:50.

[0095] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain is specific for binding to VEGF-A, and wherein the second designed ankyrin repeat domain is specific for binding to HGF, and wherein the third and fourth designed ankyrin repeat domains are each specific for binding to serum albumin, and wherein the designed ankyrin repeat domains are linked by peptide linkers, each peptide linker comprising the amino acid sequence of SEQ ID NO:9, and wherein the recombinant binding protein binds to EC compared to the recombinant binding protein. 50 Lower (i.e., better) VEGF-A and / or HGF, preferably VEGF-A, wherein the designed ankyrin repeat domains are linked via peptide linkers, each peptide linker comprising the amino acid sequence SEQ ID NO:6. Linker to EC 50 Examples of the effects are given in Example 8, and the term "lower EC" is used. 50 "This is well known to those skilled in the art. Preferably, the term "lower EC" is used. 50 "This indicates an improvement in EC by a coefficient of 1.1, more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0." 50 value.

[0096] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain is specific for binding to VEGF-A, and wherein the second designed ankyrin repeat domain is specific for binding to HGF, and wherein the third designed ankyrin repeat domain and the fourth designed ankyrin repeat domain are each specific for binding to serum albumin, wherein the recombinant binding protein binds EC compared to a recombinant binding protein comprising only one designed ankyrin repeat domain specific for binding to serum albumin. 50 Lower (i.e., better) VEGF-A and / or HGF, preferably VEGF-A. An example is given in Example 8.

[0097] In one embodiment, the present invention relates to a recombinant binding protein comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with an amino acid sequence selected from amino acid sequences SEQ ID NO: 134 to 179, preferably SEQ ID NO: 134 to 158, more preferably SEQ ID NO: 134 to 149, more preferably SEQ ID NO: 134 to 140, and more preferably SEQ ID NO: 134.

[0098] In one embodiment, the present invention relates to a recombinant binding protein comprising an amino acid sequence selected from amino acid sequences SEQ ID NO:134 to 179, preferably SEQ ID NO:134 to 158, more preferably SEQ ID NO:134 to 149, more preferably SEQ ID NO:134 to 140, and more preferably SEQ ID NO:134, wherein up to 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 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, 1, or 0 amino acids are exchanged by any amino acid.

[0099] In any embodiment of the present invention relating to a designed ankyrin repeating domain or recombinant binding protein (which comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a given amino acid sequence), dissimilar amino acids may be located at any position in the designed ankyrin repeating domain or the recombinant binding protein.

[0100] Similarly, in any embodiment of the present invention relating to a designed ankyrin repeating domain or recombinant binding protein (in which up to 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 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, 1, or 0 amino acids are exchanged by any amino acid), the exchanged amino acids may be located at any position within the designed ankyrin repeating domain.

[0101] In one embodiment, the present invention relates to a recombinant binding protein comprising an amino acid sequence selected from amino acid sequences SEQ ID NO:134 to 179, preferably SEQ ID NO:134 to 158, more preferably SEQ ID NO:134 to 149, more preferably SEQ ID NO:134 to 140, and even more preferably SEQ ID NO:134.

[0102] The present invention particularly relates to recombinant binding proteins comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 134.

[0103] In one embodiment, the present invention relates to a recombinant binding protein comprising the amino acid sequence SEQ ID NO:134, wherein up to 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 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, 1, or 0 amino acids are exchanged by any amino acid.

[0104] The present invention also particularly relates to recombinant binding proteins comprising an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:134. In one embodiment, the present invention relates to recombinant binding proteins comprising the amino acid sequence of SEQ ID NO:134.

[0105] In one embodiment, the present invention relates to a recombinant binding protein composed of an amino acid sequence selected from amino acid sequences SEQ ID NO:134 to 179, preferably SEQ ID NO:134 to 158, more preferably SEQ ID NO:134 to 149, more preferably SEQ ID NO:134 to 140, and even more preferably SEQ ID NO:134.

[0106] Preferably, it is SEQ ID NO:134. Preferably, it is a recombinant binding protein, wherein the amino acid sequence is SEQ ID NO:134. Preferably, it is a protein, wherein the amino acid sequence is SEQ ID NO:134. Preferably, it is a recombinant binding protein composed of the amino acid sequence of SEQ ID NO:134.

[0107] Several features make SEQ ID NO:134 a preferred recombinant binding protein of the present invention. It comprises two designed ankyrin repeat domains, each separate from SEQ ID NO:50 and specific for binding to serum albumin. Compared to known designed ankyrin repeat domains specific for binding to serum albumin, the designed ankyrin repeat domains exhibit improved storage stability (see Example 9). Figure 2 It contains two engineered ankyrin repeat domains with binding specificity to serum albumin, which surprisingly improve pharmacokinetic characteristics (Examples 5 and 6). Figure 3 and Figure 4 The two designed ankyrin repeat domains with binding specificity to serum albumin are located flanking other designed ankyrin repeat domains, resulting in the observed optimal pharmacokinetic characteristics (Example 6). The designed ankyrin repeat domains with binding specificity to VEGF-A and HGF, and their structural arrangement, were selected to maximize the activity of the compound (Example 8). The designed ankyrin repeat domains are linked using PT-rich linkers, which surprisingly improves the activity of each designed ankyrin repeat domain (Example 8) and surprisingly improves the pharmacokinetic characteristics (Example 5).

[0108] In one embodiment, the present invention relates to a recombinant binding protein comprising at least a first designed ankyrin repeat domain, a second designed ankyrin repeat domain, a third designed ankyrin repeat domain, and a fourth designed ankyrin repeat domain, wherein the first designed ankyrin repeat domain is specific for binding to VEGF-A, and wherein the second designed ankyrin repeat domain is specific for binding to HGF, and wherein the third designed ankyrin repeat domain and the fourth designed ankyrin repeat domain are each specific for binding to serum albumin, wherein the first designed ankyrin repeat domain, the second designed ankyrin repeat domain, the third designed ankyrin repeat domain, and the fourth designed ankyrin repeat domain are linked by a peptide linker, and wherein, compared to a recombinant binding protein lacking the fourth designed ankyrin repeat domain specific for binding to serum albumin, the recombinant binding protein exhibits a prolonged terminal half-life, preferably a prolonged terminal half-life of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%. Examples 5 and 6 and Figure 3 and Figure 4 An example of this extended terminal half-life is given.

[0109] In one embodiment, the present invention relates to nucleic acids encoding the amino acid sequence of the designed ankyrin repeat domain or recombinant binding protein of the present invention, more preferably the recombinant binding protein of the present invention. In one embodiment, the present invention relates to nucleic acids encoding the amino acid sequence of any recombinant binding protein of the present invention, said recombinant binding protein comprising at least two, more preferably two designed ankyrin repeat domains having binding specificity to serum albumin. In one embodiment, the present invention relates to nucleic acids encoding the amino acid sequence of the recombinant binding protein of the present invention. Furthermore, the present invention relates to vectors comprising any nucleic acid of the present invention. Nucleic acids are well known to those skilled in the art. In embodiments, nucleic acids are used to generate the designed ankyrin repeat domain or recombinant binding protein of the present invention in *E. coli*.

[0110] In one embodiment, the present invention relates to a pharmaceutical composition comprising the recombinant binding protein and / or a designed ankyrin repeat domain of the present invention, or a nucleic acid encoding the recombinant binding protein and / or a designed ankyrin repeat domain of the present invention, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0111] In one embodiment, the present invention relates to a pharmaceutical composition comprising a recombinant binding protein or a nucleic acid encoding a recombinant binding protein, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0112] Pharmaceutically acceptable carriers and / or diluents are known to those skilled in the art and will be described in more detail below. Furthermore, diagnostic compositions comprising one or more of the above-described recombinant binding proteins and / or designed ankyrin repeat domains and / or nucleic acids (especially recombinant binding proteins) are considered.

[0113] The pharmaceutical composition comprises the recombinant binding protein described herein and / or a designed ankyrin repeating domain and / or nucleic acid, as well as a pharmaceutically acceptable carrier, excipient, or stabilizer, such as Remington's PharmaceuticalSciences 16 th As described in edition, Osol, A.Ed., 1980. Suitable carriers, excipients, or stabilizers known to those skilled in the art are saline, Ringer's solution, dextran solution, Hank's solution, fixed oil, ethyl oleate, 5% dextran 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 formulation containing additional active agents, such as anticancer agents or antiangiogenic agents, or other bioactive compounds.

[0114] One embodiment of the present invention relates to the use of the recombinant binding protein of the present invention in the manufacture of a pharmaceutical composition, said recombinant binding protein comprising at least two, preferably two, designed ankyrin repeating domains having binding specificity to serum albumin, wherein, compared to a recombinant binding protein comprising only one designed ankyrin repeating domain having binding specificity to serum albumin, said recombinant binding protein exhibits an extended terminal half-life, preferably an extended terminal half-life of at least 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 250%.

[0115] In one embodiment, the pharmaceutical composition comprises at least one recombinant binding protein as described herein and a detergent such as a nonionic detergent, a buffer such as a phosphate, and a sugar such as sucrose. In one embodiment, such a composition comprises the recombinant binding protein as described above and PBS.

[0116] In one embodiment, the present invention relates to the use of a pharmaceutical composition or recombinant binding protein according to the invention for the treatment of a disease. For this purpose, the pharmaceutical composition or recombinant binding protein according to the invention is administered to a patient in need at a therapeutically effective amount. Administration may include topical administration, oral administration, and parenteral administration. A typical route of administration is parenteral administration. In parenteral administration, the pharmaceutical composition of the invention is formulated in combination with the aforementioned pharmaceutically acceptable excipients into a unit-dose injectable form, such as a solution, suspension, or emulsion. The dosage and mode of administration will depend on the individual to be treated and the specific disease.

[0117] In addition, consideration is being given to using any of the above-mentioned pharmaceutical compositions or recombinant binding proteins for the treatment of the disorder.

[0118] The pharmaceutical compositions of the present invention can be administered, for example, via parenteral administration. In parenteral administration, the pharmaceutical composition of the present invention will be formulated in combination with the pharmaceutically acceptable excipients described above into a unit-dose injectable form, such as a solution, suspension, or emulsion. The dosage and mode of administration will depend on the individual to be treated and the specific disease. In one embodiment, the recombinant binding protein or other such pharmaceutical compositions described herein are administered intravenously. For parenteral application, the recombinant binding protein or the pharmaceutical composition may be administered by rapid bolus injection or by slow infusion in a therapeutically effective amount.

[0119] In one embodiment, the present invention relates to a method of treating a medical condition, the method comprising the step of administering a therapeutically effective amount of the recombinant binding protein of the present invention to a patient requiring such treatment. In one embodiment, the present invention relates to a method of treating a medical condition, the method comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a patient requiring such treatment. Example 14 (Figure 10) illustrates the application of using the recombinant binding protein composed of SEQ ID NO:134 to treat cancer. In one embodiment, the present invention relates to the use of the pharmaceutical composition of the present invention for treating a disease. In one embodiment, the present invention relates to a pharmaceutical composition for treating a disease.

[0120] A “medical condition” (or condition) can be a condition characterized by improper angiogenesis. A medical condition may be a condition of excessive proliferation. Examples of medical conditions suitable for treatment include autoimmune disorders, inflammatory disorders, retinopathy (particularly proliferative retinopathy), neurodegenerative disorders, infections, and neoplastic diseases. Any of the recombinant binding proteins described herein can be used to prepare a medicament for treating such disorders, particularly those selected from autoimmune disorders, inflammatory disorders, retinopathy, and neoplastic diseases. The present invention specifically relates to a method of treating a medical condition, comprising the step of administering a therapeutically effective amount of the recombinant binding protein of the present invention or the pharmaceutical composition thereof to a patient in need of such treatment. In some embodiments, the medical condition is a neoplastic disease. As used herein, the term “neoplastic disease” refers to an abnormal state or condition of cells or tissues characterized by cell growth or rapid proliferation of tumors. In a more specific sense, the term refers to cancer. In a more specific sense, the term may refer to renal cell carcinoma and / or gastric cancer and / or multiple myeloma. The term “therapeutically effective amount” refers to an amount sufficient to produce the desired effect on a patient.

[0121] Specifically, the present invention relates to the treatment of a medical condition using the pharmaceutical composition of the present invention, wherein the medical condition is cancer.

[0122] The use of the recombinant binding protein of the present invention or the pharmaceutical composition thereof for the treatment of cancer can also be combined with any other therapies known in the art. As used herein, the term "in combination with" means simultaneous administration according to a given method of administration. This includes simultaneous administration of different compounds as well as staggered administration of different compounds (e.g., compound A is administered once, followed by compound B administered multiple times, or vice versa, or two compounds are administered simultaneously and one of the compounds is also administered in a subsequent phase).

[0123] The use of recombinant proteins for the treatment of diseases including pathological angiogenesis is also being considered. The term "pathological angiogenesis" refers to the formation and growth of blood vessels during the maintenance and development of several disease states.

[0124] In another embodiment, the present invention relates to the use of the recombinant binding protein of the present invention in the manufacture of a medicament for treating medical conditions, preferably neoplastic diseases, more preferably cancer.

[0125] In one embodiment, the present invention relates to the use of the pharmaceutical composition of the invention in the manufacture of a medicament for treating a medical condition that may be neoplastic, particularly cancer.

[0126] Preparations intended for internal administration must be sterile or sterilized. This can be easily achieved through filtration using sterile filter membranes.

[0127] The term “selected from” in conjunction with a single choice in this invention has the meaning of that particular choice. For example, in one embodiment, “This invention relates to recombinant binding proteins comprising an amino acid sequence selected from the amino acid sequence SEQ ID NO:134” has the meaning of “This invention relates to recombinant binding proteins comprising an amino acid sequence SEQ ID NO:134”.

[0128] In one embodiment, the present invention relates to a recombinant binding protein comprising any of the aforementioned repeating domains. In one embodiment, the present invention relates to a recombinant binding protein comprising any of the aforementioned SEQ ID NO: 134 to 179.

[0129] This invention is not limited to the specific embodiments described in the examples. Other sources may use and process it in accordance with the summary below.

[0130] Numerous references have been cited throughout this specification. The contents of these references are incorporated herein by reference.

[0131] This specification relates to multiple amino acid sequences of the amino acid sequence listing named "MD41211_Sequence_Listing.txt" in this specification, and the amino acid sequences of that listing are incorporated herein by reference.

[0132] Example

[0133] All standard materials and reagents disclosed herein are known to those skilled in the art and are commercially available or can be prepared using well-known techniques.

[0134] Material

[0135] Chemicals were purchased from Sigma-Aldrich (Switzerland). Oligonucleotides were obtained from Microsynth (Switzerland). Unless otherwise specified, DNA polymerase, restriction enzymes, and buffers were obtained from New England Biolabs (USA) or Thermo Fisher Scientific Fermentas (Lithuania). Cloning and protein production strains were *E. coli* XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). Recombinant VEGF-A (human, mouse, rat), VEGF-C, PDGF-AB, and HGF (human, cynomolgus monkey, mouse) were obtained from R&D Systems (Biotechne; Minneapolis, USA), Peprotech (Rocky Hill, USA), Sino Biological (Beijing, China), ReliaTech (Wolfenbüttel, Germany), or were prepared in Chinese hamster ovary cells or *Pichia pastoris* and purified according to standard protocols. Serum albumins from different species were obtained from Sigma-Aldrich, Innovative Research (Novi, USA), CSL Behring (Switzerland), or directly from animals using standard methods. Biotinylated VEGF-A or HGF was chemically obtained by coupling the biotin moiety to the primary amine of the protein using standard biotinylation reagents and methods (Thermo Fisher Scientific Inc., USA). Antibodies were obtained from Thermo Fisher Scientific or QIAgen (Germany), or generated in mice or rabbits using standard immunization and hybridoma protocols well-known to those skilled in the art. Cell culture reagents were obtained from Lonza (Switzerland), Roche (Switzerland), Thermo Fisher Scientific, and Promocell (Germany).

[0136] Molecular biology

[0137] Unless otherwise stated, the procedure shall be performed in accordance with the protocol described (Sambrook J., Fritsch EF and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, New York).

[0138] Designed ankyrin repeating domains, libraries, and selection

[0139] Methods for generating a designed ankyrin repeat protein library, examples of designed ankyrin repeat protein libraries, and methods for selecting designed ankyrin repeat proteins from a designed ankyrin repeat protein library are described (WO 2002 / 020565; WO 2010 / 060748; WO 2012 / 069654; WO 2012 / 069655; WO 2014 / 001442; Binz et al., 2004 (cited above)).

[0140] Example 1: Designed ankyrin repeats with binding specificity to VEGF-A, HGF, or serum albumin Domain selection, expression, purification and analysis

[0141] Using ribosome display (Binz et al., 2004 (cited above)), designed ankyrin repeat domains with binding specificity to VEGF-A were selected from combinatorial libraries using methods described in WO2010 / 060748 for generating designed ankyrin repeat domains with binding specificity to HGF, WO2014 / 191574 for generating designed ankyrin repeat domains with binding specificity to serum albumin, and WO2012 / 069654 for generating designed ankyrin repeat domains with binding specificity to serum albumin. The binding of the selected clones to specific (VEGF-A, HGF, or serum albumin, respectively) and non-specific (e.g., MBP, E. coli, maltose-binding protein) targets was evaluated by crude extract ELISA, demonstrating that hundreds of designed ankyrin repeat domains with binding specificity to VEGF-A, HGF, or serum albumin were successfully selected for each target. For example, the designed ankyrin repeat domains of SEQ ID NO:12 to 22 constitute an amino acid sequence of an ankyrin repeat domain with binding specificity to VEGF-A, the designed ankyrin repeat domains of SEQ ID NO:23 to 37 constitute an amino acid sequence of an ankyrin repeat domain with binding specificity to HGF, and the designed ankyrin repeat domains of SEQ ID NO:40 to 56 constitute an amino acid sequence of an ankyrin repeat domain with binding specificity to serum albumin.

[0142] These designed ankyrin repeat domains, which are specific for binding to VEGF-A, HGF, or serum albumin, and a negative control designed ankyrin repeat domain (i.e., proteins #10 and #11) without known binding specificity, were cloned into a pQE (QIAgen, Germany)-based expression vector that provides an N-terminal His tag for easy protein purification. The proteins were prepared and purified using methods known to those skilled in the art, such as those described, for example, in WO2010 / 060748.

[0143] Example 2: Characterization of designed ankyrin repeating domains using surface plasmon resonance

[0144] SPR was measured using a ProteOn instrument (BioRad), and the measurements were performed according to standard procedures known to those skilled in the art. The Kd values ​​for the selected proteins are listed in Tables 1 through 3.

[0145] Table 1: Examples of dissociation constants of designed ankyrin repeating domains binding to human VEGF-A

[0146]

[0147] * Proteins #12, #13, and #16 in this table represent designed ankyrin repeating domains consisting of the corresponding amino acid sequences of SEQ ID NO:12, 13, and 16, and also including an N-terminal His tag (SEQ ID NO:1).

[0148] Proteins #14, #15, and #17 through #22 obtained similar VEGF-A dissociation constant values.

[0149] Table 2: Examples of dissociation constants of designed ankyrin repeating domains binding to human HGF

[0150]

[0151] * Proteins #23 to #29 in this table represent designed ankyrin repeating domains consisting of the corresponding amino acid sequences of SEQ ID NO:23 to 29, and also including an N-terminal His tag (SEQ ID NO:1).

[0152] Proteins #30 to #37 obtained similar HGF dissociation constant values.

[0153] Table 3: Examples of dissociation constants of designed ankyrin repeating domains binding to human HSA

[0154]

[0155] * Proteins #44 to #55 in this table represent designed ankyrin repeating domains consisting of the corresponding amino acid sequences of SEQ ID NO:44, 45, 46, 47, 48, 49, 50, 51, 52, 54, and 55, and also containing an N-terminal His tag (SEQ ID NO:1).

[0156] Proteins #40 to #43, #53, #56, and #57 yielded similar human serum albumin dissociation constants.

[0157] Example 3: Competitive binding assay and receptor competitive binding assay

[0158] Designed ankyrin repeat domains with binding specificity to VEGF-A, HGF, or serum albumin were characterized by competitive assays. These assays are well known to those skilled in the art. For designed ankyrin repeat domains with binding specificity to VEGF-A, a quantitative sandwich enzyme immunoassay (VEGF-A Quantikine Kit DVE00, R&D Systems) was used according to the manufacturer's instructions. A VEGF-A-specific monoclonal antibody was pre-coated onto a microplate. VEGF-A standards and mixtures of different concentrations of VEGF-A (20 pM) and proteins #18, #19, or #20 were applied to the wells, and any free VEGF-A present (i.e., not bound to the designed ankyrin repeat domain) was bound by the immobilized antibody. After washing away any unbound material, an enzyme-linked polyclonal antibody specific to VEGF-A was added to the wells. After washing to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells, and color development was performed proportionally to the amount of VEGF-A bound in the initial step. The color development was stopped, and the color intensity was measured. In this assay, the designed ankylosing spicule tested showed high VEGF-A inhibitory potency. IC was calculated from this titration curve obtained as described above using Graph Pad Prism software and standard procedures known to those skilled in the art. 50 Values. For the designed ankyrin repeat domains with binding specificity to HGF, a cMet receptor competitive assay was performed. For this purpose, 5 nM human cMet receptor in PBS solution was fixed overnight at 4 °C on Maxisorp plates. After washing with 0.05% TWEEN 20 PBS, the plates were blocked for 2 hours at room temperature with PBS containing 0.05% TWEEN 20 and 0.25% casein, accompanied by shaking at 300 rpm. Constant concentrations of 5 nM human HGF were pre-incubated on diluted plates in PBS 0.05% TWEEN 20 for 30 minutes at room temperature with 1000 nM–1 pM protein #23, #26, #28, and #29 (each a 1:4 dilution series). After washing the ELISA plates with 0.05% TWEEN PBS, the pre-incubated samples were transferred to the ELISA plates and incubated at room temperature for 2 hours with shaking at 300 rpm. After washing with 0.25% TWEEN PBS, 200 ng / mL anti-human HGF antibody was added at room temperature for 1 hour with shaking at 300 rpm. After washing with 0.05% TWEEN PBS, 100 ng / mL HRP-conjugated polyclonal anti-HGF species antibody was added at room temperature for 30 minutes with shaking at 300 rpm. Detection was performed using 1:3 diluted BM blue POD (Roche). After 15 minutes, the colorimetric reaction was stopped by adding 1M H2SO4. Readouts were performed at A450 wavelength using A620 as the reference wavelength.

[0159] Example ICs obtained through these measurements 50 The values ​​are given in Tables 4 and 5. Similar VEGF-A IC50 values ​​were obtained with proteins #12 to #17 and #21. 50 Similar HGF IC values ​​were obtained using proteins #24, #25, #27, and #30 to #37. 50 value.

[0160] Table 4: Inhibition of antibody VEGF-A binding by designed ankyrin repeat domains (mean IC50) 50 value)

[0161]

[0162] * Proteins #18 to #20 in this table represent designed ankyrin repeating domains consisting of the corresponding amino acid sequences of SEQ ID NO:18 to 20, and also including an N-terminal His tag (SEQ ID NO:1).

[0163] Table 5: Inhibition of HGF-cMET binding by designed ankyrin repeating domains (mean IC50) 50 value)

[0164]

[0165] * Proteins #23, #26, #28, and #29 in this table represent designed ankyrin repeating domains consisting of the corresponding amino acid sequences of SEQ ID NO:23, 26, 28, and 29, and also containing an N-terminal His tag (SEQ ID NO:1).

[0166] Example 4: Generation of recombinant binding proteins (especially those containing two, three, or four designed ankyrin repeats) Recombinant proteins with domains and other repeating proteins

[0167] DNA encoding a designed ankyrin repeat domain or recombinant binding protein was generated using genetic methods well known to those skilled in the art. The recombinant binding protein, selected from amino acid sequences SEQ ID NO:58 to 133 and additionally having an amino acid GS at SEQ ID NO:1 or an N-terminus, or a recombinant binding protein selected from amino acid sequences SEQ ID NO:134 to 179, or a designed ankyrin repeat domain selected from amino acid sequences SEQ ID NO:10 to 57 and additionally having an amino acid GS at SEQ ID NO:1 or an N-terminus, was expressed in the cytoplasm of *E. coli* using standard techniques employing the pQE expression system from Qiagen (Germany). In the case where the amino acid GS is at the N-terminus, because the initial Met is followed by a small Gly residue (i.e., the amino acid at position 1 of SEQ ID NO:134 to 179), the Met residue additionally encoded by the expression vector is effectively cleaved from the expressed polypeptide in the *E. coli* cytoplasm. Cells were lysed using a French press, and the protein was purified from the crude cell extract to near homogeneity using standard chromatographic techniques well known to those skilled in the art.

[0168] Example 5: Pharmacokinetic characteristics with the binding of serum albumin contained in the recombinant binding protein Improved pharmacokinetic effects in mice from a specific increase in the number of designed ankyrin repeat domains. .

[0169] For mouse pharmacokinetic studies, proteins #57, #62, #63, #64, #68, #73, #74, #82, #83, #97, #109, and #110 (corresponding to SEQ ID NO: 57, 62, 63, 64, 68, 73, 74, 82, 83, 97, 109, and 110, and additionally having SEQ ID NO: 1 at the N-terminus) prepared as described in Example 4 were radiolabeled according to the instructions described (Zahnd, C., Kawe, M., Stumpp, MT, de Pasquale, C., Tamaskovic, R., Nagy-Davidescu, G., Dreier, B., Schibli, R., Binz, HK., Waibel, R., Plückthun, A., Cancer Res. 70, 1595-1605, 2010), and administered a single intravenous bolus of 10 μg / 100 μL to the tail vein of female BALB / c mice. Serum samples were collected from each mouse at each time point, and cumulative radioactivity was measured using a gamma scintillation counter.

[0170] In these experiments, proteins containing two designed ankyrin repeat domains specific to serum albumin consistently exhibited improved pharmacokinetic characteristics compared to similar constructs containing only one designed ankyrin repeat domain with binding specificity to serum albumin. Figure 3 For example, a comparison of protein #57 (which contains a single designed ankyrin repeat domain specific to serum albumin binding) (SEQ ID NO:57, containing SEQ ID NO:51 plus a C-terminal polypeptide) with proteins #62 and #63 (which contain two designed ankyrin repeat domains specific to serum albumin binding) (twice the size of SEQ ID NO:51, linked by a GS-rich (SEQ ID NO:63) or PT-rich (SEQ ID NO:62) polypeptide linker) showed that: the inclusion of two designed ankyrin repeat domains specific to serum albumin binding resulted in higher %ID, for example, at 24 hours post-injection (+57% GS; +59% PT), 48 hours (+76% GS; +82% PT), or 72 hours (+79% GS; +94% PT), and resulted in an improved terminal half-life (+38% GS; +48% PT) compared to proteins containing only a single designed ankyrin repeat domain specific to serum albumin binding. Figure 3 a). Specifically, the use of PT-rich linkers, particularly SEQ ID NO:9, improves pharmacokinetic characteristics. Figure 3a). The following three examples demonstrate that the method of improving pharmacokinetic characteristics by having two (instead of one) engineered ankyrin repeat domains with binding specificity to serum albumin in a protein can be transferred to different proteins containing different engineered ankyrin repeat domains. For example, protein #64, containing SEQ ID NO:22 (engineered ankyrin repeat domain with binding specificity to another target besides serum albumin) and 51 (engineered ankyrin repeat domain with binding specificity to serum albumin), is compared with protein #74, which contains SEQ ID NO:22 and twice the number of SEQ ID NO:51 (protein #73 has SEQ ID NO:51 flanking SEQ ID NO:22, and protein #74 has SEQ ID NO:51 located at the N-terminus of SEQ ID NO:22). A comparison of the pharmacokinetic characteristics of proteins #73 and #74 (NO:51) showed that the inclusion of two engineered ankyrin repeat domains with binding specificity to serum albumin resulted in higher %IDs, for example, at 24 hours post-injection (+62% N-terminus; +89% flanking) or 48 hours (+136% N-terminus; +175% flanking), and improved terminal half-life (both N-terminus and flanking) compared to proteins containing only a single engineered ankyrin repeat domain with binding specificity to serum albumin (+>63%). Figure 3 b). Similarly, a pharmacokinetic comparison of protein #82, containing twice the amount of SEQ ID NO:11 (a designed ankyrin repeat domain without known binding specificity) and a single SEQ ID NO:51 (a designed ankyrin repeat domain with binding specificity to serum albumin), with protein #109, containing twice the amount of SEQ ID NO:11 and twice the amount of SEQ ID NO:51 (N-terminus), showed that the inclusion of two designed ankyrin repeat domains with binding specificity to serum albumin resulted in higher %ID, for example, at 24 hours (+12%) or 48 hours (+35%) after injection, and improved terminal half-life (+71%) compared to proteins containing only a single designed ankyrin repeat domain with binding specificity to serum albumin. Figure 3c). Furthermore, a pharmacokinetic comparison of protein #83, containing SEQ ID NO:38 and 39 (each a designed ankyrin repeat domain specifically binding to another target besides serum albumin) and 50 (a designed ankyrin repeat domain specifically binding to serum albumin), with protein #110, containing SEQ ID NO:38 and 39 and twice as many SEQ ID NO:50 (flanking SEQ ID NO:38 and 39), showed that the inclusion of two designed ankyrin repeat domains specifically binding to serum albumin resulted in higher %ID, for example, at 24 hours (+198%), 48 hours (+198%), or 72 hours (+228%) post-injection, and improved terminal half-life (+19%) compared to proteins containing only a single designed ankyrin repeat domain specifically binding to serum albumin. Figure 3 d). Furthermore, protein #97 exhibited significantly improved pharmacokinetic characteristics compared to protein #68, for example, terminal half-lives of 21 hours and 16 hours, respectively, indicating that the inclusion of two designed ankyrin repeat domains with binding specificity to serum albumin is more beneficial than the inclusion of only one such domain.

[0171] These results surprisingly demonstrate that, as further discussed in Example 6, the use of two, rather than one, designed ankyrin repeat domains with binding specificity to serum albumin in recombinant binding proteins improves pharmacokinetic characteristics.

[0172] Example 6: Pharmacokinetic characteristics with the binding of serum albumin contained in the recombinant binding protein Improved pharmacokinetics in cynomolgus monkeys by increasing the number of specifically designed ankyrin repeating domains. .

[0173] For the pharmacokinetic studies in cynomolgus monkeys, proteins #57, #62, and #97 (corresponding to proteins SEQ ID NO: 57, 62, and 97, and additionally having SEQ ID NO: 1 at the N-terminus) and protein #134 (corresponding to protein SEQ ID NO: 134), prepared as described in Example 4, were administered to cynomolgus monkeys via intravenous infusion over 30 minutes at target dose levels between 0.5 and 100 mg / kg. Blood samples were collected prior to administration and again at selected time points, such as 5 minutes, 10 minutes, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, and 168 hours after the infusion (i.e., post-injection). The blood samples were allowed to stand at room temperature and centrifuged to produce serum, which was then stored at -80°C for analysis. Pharmacokinetic parameters were determined using procedures well known to those skilled in the art. A rabbit monoclonal antibody designed with ankyrin repeat domains was used as the capture reagent, and a mouse monoclonal antibody designed with ankyrin repeat domains was used as the detection reagent. Serum concentrations of proteins #57, #62, #97, and #134 were determined by sandwich ELISA using a standard curve. Pharmacokinetic parameters were determined using standard software such as Phoenix WinNonLin (Certara, Princeton, USA) or GraphPadPrism (GraphPad Software, La Jolla, USA) and standard analyses such as non-compartmental analysis. The obtained pharmacokinetic characteristics were... Figure 4As shown in the figure. Proteins containing two designed ankyrin repeat domains with specific binding to serum albumin consistently exhibit improved pharmacokinetic characteristics compared to similar constructs containing only one designed ankyrin repeat domain with specific binding to serum albumin. For example, comparing protein #57 (0.5 mg / kg; 27.7 nmol / kg) containing a single designed ankyrin repeat domain with specific binding to serum albumin (SEQ ID NO: 51) with a design ankyrin repeat domain with specific binding to serum albumin (twice the size of SEQ ID NO: 51)... Protein #62 (1.04 mg / kg; 34.5 nmol / kg) linked via a PT-rich peptide linker (NO:51) showed that the presence of two designed ankyrin repeat domains with specific binding to serum albumin in the protein resulted in higher exposure (2138 d*nmol / L vs. 4676 d*nmol / L, i.e., +119% calculated up to day 7), lower clearance (0.0108 L / (d*kg) vs. 0.0031 L / (d*kg); i.e. -71%), and improved terminal half-life (4.57 days vs. 9.00 days, i.e., +97% calculated from day 1 to day 7) compared to proteins containing only a single designed ankyrin repeat domain with specific binding to serum albumin. Figure 4 a). Furthermore, protein #57 was compared with protein #62 on day 4 (23.39% vs. 57.72%; +148%), day 5 (19.00% vs. 48.41%; +155%), and day 6 (18.5% vs. 51.94%; +175%), the percentage increase in concentration measured 10 minutes after injection being normalized to the injected dose. As another example of cynomolgus monkeys, protein #134 (corresponding to the protein in SEQ ID NO: 134), prepared as described in Example 4, was tested at different doses in 10 animals (5 males and 5 females for each dose), and the terminal half-life was assessed using WinNonLin, considering concentration values ​​up to day 7. When administered to cynomolgus monkeys at 1 mg / kg, protein #134 exhibited a mean terminal half-life of 4.0 days (95 hours) (+46% compared to protein #97, which showed a terminal half-life of 2.7 days (65 hours) at 1 mg / kg), 5.3 days (127 hours) at 10 mg / kg, and 5.8 days (139 hours) at 100 mg / kg. A comparison of the pharmacokinetic characteristics of protein #134 and protein #97 in cynomolgus monkeys was conducted in... Figure 4Shown in

[0174] In the absence of any albumin-binding activity, the recombinant binding protein has a terminal half-life in the range of minutes in both mice and cynomolgus monkeys (see WO 2012 / 069654). Proteins containing at least one engineered ankyrin repeat domain with binding specificity for serum albumin showed a much longer terminal half-life compared to the case where there is no engineered ankyrin repeat domain with binding specificity for serum albumin. The pharmacokinetic characteristics of a protein containing one engineered ankyrin repeat domain with binding specificity for serum albumin are shown in Figure 3 a and Figure 4 a.

[0175] This technique includes a study in which the valence effect of another serum albumin-binding protein domain (the albumin-binding domain (ABD) from streptococcal protein G) was investigated (Hopp et al., 2010; cited above). Another study used a C-terminal fusion peptide of a non-protein domain (WO 2011 / 095545). The ABD is a helical protein domain with binding specificity for serum albumin. Importantly, Hopp et al., 2010 (cited above) showed that compared to a recombinant binding protein containing only one ABD (C-terminal), having two such ABDs (one N-terminal and one C-terminal) in a recombinant binding protein did not result in a significant improvement in the terminal half-life in mice (37.9 ± 1.1 hours vs. 36.4 ± 4.8 hours). Specifically, at 24 hours and 72 hours after injection, the recombinant binding protein containing one ABD showed the same percentage of the injected dose as the recombinant binding protein containing two ABDs, indicating comparable pharmacokinetic characteristics of the two recombinant binding proteins. Based on these findings regarding ABDs, those skilled in the art predicted that compared to a recombinant binding protein containing only one engineered ankyrin repeat domain with binding specificity for serum albumin, a recombinant binding protein containing two albumin-binding protein domains (such as an engineered ankyrin repeat domain with binding specificity for serum albumin) would not have improved pharmacokinetic characteristics. Surprisingly, we found that this is not the case. Contrary to Hopp et al. (cited above), a recombinant binding protein containing two engineered ankyrin repeat domains with binding specificity for serum albumin surprisingly showed a significantly extended terminal half-life compared to a recombinant binding protein containing only one engineered ankyrin repeat domain with binding specificity for serum albumin.

[0176] These examples illustrate several other findings. For instance, the pharmacokinetic profile of protein #134 was superior to that of protein #97, highlighting the importance of selecting a single, designed ankyrin repeat domain. SEQ ID NO 134 was chosen to consist of components that produce the maximum activity and optimal pharmacokinetic profile. Furthermore, the arrangement of designed ankyrin repeat domains within protein #134 was selected to produce the optimal pharmacokinetic profile. When recombinant binding proteins containing four designed ankyrin repeat domains (including two designed ankyrin repeat domains with binding specificity to serum albumin, which is relevant to the pharmacokinetics of mice and cynomolgus monkeys) were analyzed, recombinant binding proteins containing two designed ankyrin repeat domains with binding specificity to serum albumin (flanking the other two designed ankyrin repeat domains) were observed to have the most favorable pharmacokinetic profile.

[0177] Since the examples in this embodiment include different combinations of designed ankyrin repeat domains that are specific to binding to another target besides serum albumin, the method of using at least two designed ankyrin repeat domains that are specific to binding to serum albumin to improve pharmacokinetic characteristics appears to be generally applicable to proteins that contain several designed ankyrin repeat domains.

[0178] Example 7: Simultaneous binding of two human serum albumin molecules via protein #134.

[0179] Protein #134 (a recombinant binding protein consisting of SEQ ID NO: 134, with an additional GS at the N-terminus) was prepared as described in Example 4. Protein #134, purified human serum albumin (HSA), and a protein #134 / HSA mixture (1:2 stoichiometry) were analyzed by size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS). SEC-MALS was performed on an Agilent 1200 system (Life Technologies, USA) connected to a Wyatt (USA) MALS and refractive index detector using the proteins listed in Table 6 at concentrations of 30 μM (protein #134) or 60 μM (HSA) (flow rate: 0.6 mL / min; injection volume: 100 μL; column: GE Healthcare (USA) Superdex 20010 / 300GL). The protein #134 / HSA mixture was pre-incubated at 20 °C for 3 hours prior to injection. Chromatograms are shown below. Figure 5As shown in Table 6, the molecular weight of the eluent was determined and compared with the theoretical molecular weight. For this experiment, 100 mg HSA (CSL Behring, 20% solution) was purified using a Superdex 200_26 60 column on an AEKTA prime system (GE Healthcare; 2.0 mL / min, PBS, isocratic flow, injection volume: 10 mL 1:20 PBS diluted HSA, 4 mL fraction collected). SEC-MALS experiments were performed using the peak fraction of the main peak.

[0180] Protein #134 at 30 μM is monodisperse, and the elution fraction contains the protein of the expected molecular weight. Figure 5 Similarly, the 60 μM purified HSA was monodisperse, and the elution fraction contained proteins of the expected molecular weight (Table 6). A mixture of 30 μM protein #134 and 60 μM HSA produced two peaks in the SEC. One peak contained a protein complex with a molecular weight corresponding to the 1:2 (protein #134 / HSA) complex, indicating that both designed ankyrin repeat domains with binding specificity to serum albumin were functioning simultaneously. Furthermore, at the tail of this peak, a protein complex with a molecular weight corresponding to the 1:1 (protein #134 / HSA) complex was detected. Additionally, free HSA was detected. Due to the small amount, the case where the main peak was the 2:1 (protein #134 / HSA) complex can be disregarded (which would theoretically be consistent with the observed weight), but a larger fraction corresponding to 75% free HSA is still expected. Free protein #134 / HSA was not detected. No peaks with molecular weights greater than one of the 1:2 (protein #134 / HSA) complexes were detected. SEC-MALS measurements and the variations observed in SEC-MALS measurements are well known to those skilled in the art.

[0181] Table 6: Size exclusion chromatography and static light scattering chromatography of protein #134 and HSA, and the complex protein #134 / HSA Using the analysis results .

[0182] peak Theoretical stoichiometry and MW Measured MW HSA 69366.6Da 63350Da Protein #134 62397.0Da 58700Da Protein #134 / HSA tail 1:1, 131763.6 Da 132500Da Protein #134 / HSA Middle For 1:1 and 1:2 mixtures, MW depends on the ratio. 173700Da Protein #134 / HSA front 1:2, 201130.2Da 197500Da

[0183] When proteins #97, #102, #109, and #110 were analyzed using size exclusion chromatography coupled with static light scattering, a similar recombinant binding protein was observed to bind to two human serum albumin molecules simultaneously.

[0184] Example 8: By selecting the composition of the adapter and by selecting the adapter with binding specificity to serum albumin. The number of ankyrin repeating domains is calculated to maximize target binding activity.

[0185] Peptide linkers connecting protein domains are well known to those skilled in the art. Gly-Ser-rich linkers are well known for their use in linking two Fv polypeptide chains within single-chain Fv antibody fragments. Various other peptide linkers exist, including, for example, antibody hinge regions, or unstructured peptides (such as sequences primarily containing the amino acids Ala, Glu, Lys, Pro, Ser, Thr (WO2007 / 103515) or Ala, Pro, and Ser (WO 2008 / 155134)). Furthermore, Pro-Thr-rich linkers have been disclosed (WO 2014 / 191574). It is necessary to evaluate the effect of each linker / domain combination on the properties of the protein domains linked by that linker. In addition to the properties of the peptide linkers, we surprisingly found that the number of serum albumin-binding domains can also affect protein function. To maximize the target-binding activity of the recombinant binding proteins of the present invention, recombinant binding proteins containing Gly-Ser-rich and Pro-Thr-rich polypeptide linkers were compared with recombinant binding proteins containing one or two designed ankyrin repeat domains specific for binding to serum albumin. For this purpose, the binding of proteins #69, #71, and #107, each additionally having SEQ ID NO:1 at the N-terminus and prepared according to the method described in Example 4, to VEGF-A and HGF was analyzed by ELISA (method see Example 4). The results are shown in Table 7. EC50 of protein #69 and protein #71 50 Comparison of values ​​showed that, compared with recombinant binding proteins with Gly-Ser-rich linkers, recombinant binding proteins with Pro-Thr-rich linkers were more effective in binding VEGF-A (coefficient 2) and HGF (coefficient 1.3), respectively. EC50 values ​​for protein #69 and protein #107... 50 Comparison of values ​​showed that the recombinant binding protein containing two designed ankyrin repeat domains specific to serum albumin was more effective at binding VEGF-A (coefficient 1.4) and HGF (coefficient 1.1), respectively, compared to a recombinant binding protein containing only one designed ankyrin repeat domain specific to serum albumin. The presence of two albumin-binding domains in the construct had a negative impact on the molecule's functionality in previous results, so these results are surprising given previous findings (Hopp et al., 2010). This result suggests that the recombinant binding protein preferentially contains a Pro-Thr-rich linker and two designed ankyrin repeat domains specific to serum albumin, rather than a Gly-Ser-rich linker and one designed ankyrin repeat domain specific to serum albumin.

[0186] Table 7: Designed anchor proteins with different linkers and different numbers exhibiting binding specificity to serum albuminELISA analysis of recombinant binding proteins with white repeat domains

[0187]

[0188] * Proteins #69, #71, and #107 in this table represent designed ankyrin repeating domains consisting of the corresponding amino acid sequences of SEQ ID NO:69, 71, and 107, and also containing an N-terminal His tag (SEQ ID NO:1).

[0189] The number of designed ankyrin repeat domains with binding specificity to serum albumin

[0190] Example 9: Improved protein stability when using SEQ ID NO:50.

[0191] Proteins #48, #49, and #51 were further characterized for the midpoint of denaturation temperature (i.e., the midpoint of co-unfolding after temperature elevation) by mixing proteins (25 μL; 100 μM PBS solution) with a fluorescent dye (25 μL Sypro orange diluted 1 / 2500 in PBS (Life Technologies, USA)) and measuring melting curves using a thermal cycler including a fluorescence reader (CFX96 Real-Time PCR Detection System; BioRad; holding time 25 seconds at 0.5 °C, then reading fluorescence). This is essentially as described by Niesen et al., 2007 (Niesen, FH, Berglund, H., Vedadi, M., Nature Protocols). As described in 2,2212-2221, 2007. In PBS, protein #48 showed a denaturation midpoint of 83.5°C, protein #49 showed a denaturation midpoint of 84.5°C, and protein #51 showed a denaturation midpoint of 79.5°C.

[0192] To identify the designed ankyrin repeating domain with optimal storage stability and binding specificity to serum albumin, proteins #49, #50, and #51 (corresponding to SEQ ID NO:49, 50, and 51, respectively, and additionally having SEQ ID NO:1 at the N-terminus) were prepared as described in Example 4, and the samples were concentrated to 10 mg / mL in PBS. Proteins #50 and #51 were then stored in glass vials at -80°C or 40°C for one month, followed by analysis on SDS 15% PAGE. While proteins #50 and #51 showed equivalent stability at -80°C, after one month of storage at 40°C, protein #50 showed a significantly reduced amount of degradation products on SDS 15% PAGE compared to protein #51. Similarly, after one week of storage in 10 mg / mL PBS solution at 4°C, 25°C, 40°C, and 60°C, protein #50 showed a significantly reduced amount of degradation products compared to protein #49. Specifically, when stored at 40°C or 60°C, protein #50 showed a >50% reduction in degradation products compared to protein #49 on SDS 15% PAGE. Figure 2 These findings indicate that protein #50 exhibits improved storage stability compared to proteins #49 and #51. Similarly, when comparing the storage stability of proteins #48 to #51 (corresponding to SEQ ID NO: 48 to 51, and additionally having SEQ ID NO: 1 at the N-terminus; prepared as described in Example 4), by incubating the proteins at a concentration of 10 mg / mL in PBS at 40°C for 1 month in glass vials, proteins #48 to #50 showed a >30% reduction in degradation products compared to protein #51.

[0193] These findings were confirmed by testing the storage stability of proteins #102 and #103 (recombinant binding proteins consisting of amino acid sequences corresponding to SEQ ID NO: 102 and 103, and both having SEQ ID NO: 1 at the N-terminus). Proteins #102 and #103 were prepared as described in Example 4, the samples were concentrated to 10 mg / mL in PBS, and stored in glass vials at -80°C or 40°C for one month, followed by analysis by standard size exclusion chromatography. At -80°C, proteins #102 and #103 showed equivalent elution profiles, while at 40°C, protein #102 showed 98.72% monomeric material, and protein #103 showed 100% monomeric material. This indicates that the presence of SEQ ID NO: 50 in the recombinant binding protein is more desirable in terms of storage stability than the presence of SEQ ID NO: 49. Similarly, when protein #103 was stored in PBS at a concentration of 10 mg / mL in glass vials at 40°C for one month, SDS-PAGE analysis showed that protein #103 exhibited a lower amount of degradation products than protein #102, confirming that the recombinant binding protein containing SEQ ID NO:50 has higher storage stability compared to the recombinant binding protein containing SEQ ID NO:49.

[0194] Similar results were obtained when comparing protein #134, prepared as described in Example 4, with protein #143 or protein #150 (recombinant binding proteins composed of amino acid sequences corresponding to SEQ ID NO: 134, 143, and 150, respectively). After one month of storage in glass vials at a concentration of 10 mg / mL in PBS at 40°C, analysis on SDS 15% PAGE showed that protein #134 exhibited a >50% reduction in the amount of degradation products compared to proteins #143 and #150. This indicates that the presence of SEQ ID NO: 50 in the recombinant binding protein is more desirable in terms of storage stability than the presence of SEQ ID NO: 49 or SEQ ID NO: 51.

[0195] Example 10: Characterization of recombinant binding proteins using ELISA

[0196] The purified recombinant binding protein consisting of the amino acid sequence SEQ ID NO:134, prepared according to the method described in Example 4, was analyzed by ELISA. 100 μL or 50 μL of 20 nM target (VEGF-A, HGF, or serum albumin) PBS solution per well was fixed overnight in Maxisorp plates (Nunc, Denmark) at 4°C. After washing five times with 300 μL PBST (supplemented with 0.1% Tween 20), the wells were blocked for 2 hours at room temperature with 300 μL PBST-C (supplemented with 0.25% casein PBST), while shaking at 450 rpm on a Titramax 1000 shaker (Heidolph, Germany). After washing five times as described above, add 100 μL / well or 50 μL / well of PBST-C solution containing protein #134 (concentration range 100 nM to 0.01 pM), and incubate at room temperature for 1 to 2 hours with shaking at 450 rpm. After washing five times as described above, detect the binding of protein #134 in PBST-C at room temperature using 100 μL / well or 50 μL / well of rabbit anti-designed ankyrin repeat domain monoclonal antibody with shaking at 450 rpm for 1 hour. After washing five times as described above, detect the bound anti-designed ankyrin repeat domain antibody in PBST-C at room temperature using 100 μL / well or 50 μL / well of goat anti-rabbit IgG-HRP conjugate with shaking at 450 rpm for 1 hour. After washing five times as described above, perform an ELISA using 100 μL of soluble BM blue POD substrate (Roche, Switzerland) diluted 1:4 in water. After 5 minutes, the reaction was stopped with 100 μL of 1M H2SO4. Then the OD was recorded (OD450nm-OD620nm).

[0197] ELISA results showed that protein #134 bound VEGF-A in humans, cynomolgus monkeys, rats, and mice with equivalent potency (Table 8 and 134). Figure 6a The cynomolgus monkey's VEGF-A is identical to that of humans, therefore it was not tested separately. No binding of protein #134 to VEGF-C and PDGF-AB was detected (Table 9 and...). Figure 6a In humans, cynomolgus monkeys, and mice, HGF was bound by protein #134 with equivalent potency (EC). 50 Values ​​are in the range of 20-50 pM; Table 8 and Figure 6b In addition, protein #134 binds with equivalent potency to serum albumin (EC50) in humans, cynomolgus monkeys, rats, dogs, and mice. 50 Values ​​are in the range of 10-20 pM; Table 8 and Figure 6cThe comparison of protein #134 (i.e., the protein consisting of SEQ ID NO: 134) with protein #60 or protein #61 (i.e., the protein consisting of SEQ ID NO: 60 or 61, additionally having SEQ ID NO: 1 at the N-terminus, prepared according to the method described in Example 4) showed that the observed protein #134 binds to the EC of human serum albumin. 50 Significantly superior to the EC observed for proteins #60 or #61 (225 pM and 322 pM, respectively). 50 .

[0198] Table 8: Epigenetic EC50 of protein #134 binding to VEGF-A, HGF, and serum albumin in different species 50 value

[0199]

[0200] * CI confidence interval. With 100% sequence identity to human VEGF-A, the values ​​for human VEGF-A are listed; na was not analyzed.

[0201] Table 9: Epigenetic EC of protein #134 binding to VEGF and PDGF in different individuals 50 value

[0202]

[0203] * CI confidence interval

[0204] Example 11: Characterization of recombinant binding proteins using a competition assay

[0205] The purified recombinant protein consisting of the amino acid sequence SEQ ID NO:134, prepared according to the method described in Example 4, was subjected to competitive ELISA and FRET analyses. Such competitive FRET and ELISA assays are well known to those skilled in the art. Protein #134 was measured in a VEGF-A / VEGFR-2 competitive FRET assay. For this purpose, protein #134 and biotinylated VEGF-A165 (Reliatech, #300-076Bi-L) were prepared and dissolved in PBS containing 0.2% BSA and 0.01% Tween (PBST-BSA) as an eight-fold stock solution. A competitive mixture of 5 μL of eight-fold protein #134 and 5 μL of eight-fold biotinylated VEGF-A165 was preheated at room temperature for 1 hour (competitive mixture). Simultaneously, 5 μL of streptomycin-Tb (streptomycin-Lumi4-terbium cavitation compound donor, Cisbio #610SATLB) and 5 μL of PAb anti-hIgG-de (D2-conjugated goat anti-human IgG, Cisbio #61HFCDAA) were added to 500 μL of PBST-BSA buffer and incubated for 20 min (2× reagent). 10 μL / well of 2× reagent was dispensed into 384-well HTRF white plates (Thermo Fisher Scientific Inc.), and 5 μL / well of a four-fold concentration of hVEGF-R2-Fc fusion (Reliatech, #SFC-008) was added. Then, 5 μL of the pre-incubated competitive mixture was added to each well. The complete reaction mixture was incubated in the dark at room temperature for 1 hour, and then fluorescence was read using a fluorescence reader. The final mixture contained 10 nM soluble VEGF-R2-Fc fusion, 10 nM biotinylated VEGF-A, and different concentrations of protein #134. Readouts were performed at A665 nm and A595 nm wavelengths (excitation wavelength 340 nm). The results are shown below. Figure 7a In this assay, protein #134 was detected at an IC50 concentration of 0.6 nM. 50 The value inhibits VEGF-A / VEGFR-2 interaction. Protein #134 was further measured in an HGF / cMet competitive ELISA assay as described in Example 3. The results are shown in... Figure 7b In this assay, protein #134 was detected at an IC50 value of 0.92 nM. 50 The value inhibits HGF / cMet interaction. Protein #134 was also measured in a VEGF-A competitive ELISA assay, as described in Example 3. Results are shown in... Figure 7c In this assay, protein #134 was detected with an IC50 value in the single-digit pM range. 50 Value (IC) 50 (4.5pM) inhibits VEGF binding.

[0206] Example 12: Characterization of recombinant binding proteins and target binding using surface plasmon resonance

[0207] SPR was measured using the following setup, similar to that described in Example 2. 2700 RU of human HGF was immobilized on the sensor chip. Then, 100 nM protein #134 or PBST was injected for 180 seconds, followed by a 360-second PBST wash. Subsequently, 100 nM human VEGF-A or PBST was injected for 180 seconds (to reach saturation), followed by a 360-second PBST wash. Finally, 100 nM human serum albumin or PBST was injected for 180 seconds, followed by a 600-second PBST wash. The resulting signal was... Figure 8 The results show that protein #134 can bind to HGF, VEGF-A, and serum albumin. Furthermore, the results indicate that protein #134 can bind to both HGF and VEGF-A, as well as HGF, VEGF-A, and serum albumin simultaneously.

[0208] Example 13: Characterization of recombinant binding proteins in cell cultures

[0209] Further cellular assays were performed on the purified recombinant binding protein consisting of the amino acid sequence SEQ ID NO:134, prepared in accordance with the method described in Example 4. These assays included the HUVEC proliferation assay for assessing VEGF-A inhibition, the A549 cell migration assay for assessing HGF inhibition, and the cMet phosphorylation assay, all of which are well known to those skilled in the art.

[0210] Inhibition of VEGF-A-induced HUVEC proliferation was determined by titrating progressively increasing concentrations of protein #134 in a HUVEC proliferation assay. Human VEGF-A was used at a concentration of 8 ng / mL (corresponding to EC80 as determined in the proliferation assay). Protein #134 was titrated from 200 ng / mL to 0.195 ng / mL. Cells were seeded in 50 μL of analytical medium. Protein dilutions (in analytical medium) were prepared by serial dilutions at a 1:2 ratio in dilution plates; the concentration was four times the final concentration. The protein #134 dilutions were mixed 1:1 with four times the concentration of concentrated VEGF-A (32 ng / mL; final 8 ng / mL). 50 μL of the mixture was added to cells for 72 hours. Cell proliferation was assessed by BrdU incorporation into replicated DNA or by monitoring metabolic activity using WST-1. Results were presented in... Figure 9a The results show that protein #134 exhibits an IC50 of 5.7 ng / mL (91.35 pM). 50 .

[0211] Inhibition of HGF / cMet interaction was measured using Protein #134 in the Oris cell migration assay (Platypus Technologies, USA). The assay was performed according to the manufacturer's protocol. Briefly, 100,000 A549 cells were seeded in serum-free DMEM. After 24 hours, cell adhesion occurred, and the medium was replaced with the analytical medium; i.e., DMEM with and without 0.5 nM HGF, containing and without 5 μM protein. HGF and the neutralizing protein were pre-incubated at room temperature for 1 hour before adding the cells. The Oris assay was then removed. TM The cells were then incubated for 48 hours to allow cell migration. Cells were stained with calcein (2.5 ng / mL) for 40 minutes and images were taken. The migration zone was measured using an inverted Olympus microscope and its CellSensDimension software. The migration zone was calculated as the coverage zone by subtracting the uncovered area of ​​the pre-migration well from the uncovered area of ​​the corresponding sample well. The uncovered area was the cell-free region and was measured using the diameter function in the software's processing folder. Results are in... Figure 9b The results show that protein #134 can inhibit HGF-induced cell migration in A549 cells.

[0212] The inhibition of cMet phosphorylation by protein #134 was measured using A549 cells and a DuoSet P-cMet-ELISA (R&D Systems). Cells were seeded in 96-well plates at 200,000 cells per well in complete medium. After 24 hours, the medium was replaced with serum-free medium. Cells were incubated for another 24 hours in both the presence and absence of protein #134 and stimulated with 1 nM human HGF (or PBS for the negative control). HGF and protein #134 were pre-incubated at room temperature for at least 30 minutes before adding cells. Cells were stimulated at room temperature for 10 minutes. Stimulation was terminated by removing the cell supernatant (by flicking) and adding cell lysis buffer according to the protocol. Cell lysates were stored at -20°C until the ELISA assay. Results were presented in […]. Figure 9c The diagram shows that protein #134 can achieve an IC50 concentration of 184 pM. 50 Inhibit HGF-mediated cMet phosphorylation.

[0213] Example 14: Effects of recombinant binding proteins on tumor growth in vivo

[0214] The U87MG xenograft mouse model was used to evaluate the benefits of a combination containing both a designed ankyrin repeat domain that is specific to VEGF-A binding and a designed ankyrin repeat domain that is specific to HGF binding, compared to containing either domain alone. Protein #134, composed of SEQ ID NO:134 (containing two designed ankyrin repeat domains, each composed of amino acids from SEQ ID NO:50, specifically binding to serum albumin, a designed ankyrin repeat domain, composed of SEQ ID NO:18, specifically binding to VEGF-A, and a designed ankyrin repeat domain, composed of amino acids from SEQ ID NO:26, specifically binding to HGF), was prepared according to the method described in Example 4. Protein #61, composed of SEQ ID NO:61 (containing a designed ankyrin repeat domain, composed of amino acids from SEQ ID NO:50, specifically binding to serum albumin, and a designed ankyrin repeat domain, composed of amino acids from SEQ ID NO:18, specifically binding to VEGF-A), and further having SEQ ID NO:1 at its N-terminus, was also prepared. Protein #61, composed of SEQ ID NO:60 (containing a designed ankyrin repeat domain, composed of amino acids from SEQ ID NO:50, specifically binding to serum albumin, and further having SEQ ID NO:1 at its N-terminus), was also prepared. The amino acid composition of NO:26 is a designed ankyrin repeat domain that binds specifically to HGF, and additionally contains protein #60 of SEQ ID NO:1 at the N-terminus. For in vivo analysis, 2 × 10⁻⁶ mice were used. 6 U87MG cells were subcutaneously transplanted into the right rib of female NMRI nu / nu mice (Harlan), and the mice were divided into groups with equal tumor volume. Mice were treated intravenously with PBS or 4 mg / kg protein on days 29 and 32. Tumors were harvested and frozen on day 35. Tumor volume was measured each day of treatment using the following formula: Volume = (width) 2 × length / 2. Weight measurements showed no significant differences among the four treatment groups. Tumor cross sections were then stained with an antibody against Ki67 (ab66155; Abcam, UK) to quantify proliferation, or stained with an antibody against CD-31 (BD550274; BD Biosciences, USA) to quantify angiogenesis using standard IHC methods. The percentage of proliferating cells and the percentage of mean vascular area were measured using ImageJ software. Results are shown in... Figure 10aIn contrast to PBS, proteins #60 and #61 inhibited proliferation, and proteins #60 (mildly) and #61 inhibited angiogenesis, as expected. However, the combination of both (resulting in protein #134) improved the inhibition of both proliferation and angiogenesis. This suggests that the combination of anti-VEGF-A and anti-HGF activities is key to good therapeutic efficacy.

[0215] Protein #134 was further characterized in two patient-derived xenograft mouse models, a gastric cancer model, and a renal cancer model. Patient-derived tumor xenograft mouse models are well known to those skilled in the art. Protein #134 was prepared according to the method described in Example 4.

[0216] For xenograft mouse models derived from patients with renal cell carcinoma, renal cell carcinoma specimens were subcutaneously implanted into NMRI nu / nu mice from surgical specimens and passaged three to five times until a stable growth pattern was established. After removal from donor mice, tumor fragments with a diameter of 4-5 mm were cut and subcutaneously implanted into NMRI nu / nu mice. After significant solid tumor growth, mice were randomly assigned to three groups, and the test products were administered to one group each as described below: PBS 10 mL / kg intravenously three times a week; Protein #134 4 mg / kg intravenously three times a week; and Sorafenib 200 mg / kg orally daily for 21 days. Tumor volume was assessed as described above on the day of treatment initiation and on days 3, 7, 10, 14, 18, and 21. Results are shown in... Figure 10b In this model, protein #134 was more effective than sorafenib, which is currently the standard of care for renal cell carcinoma.

[0217] For xenograft mouse models derived from gastric cancer patients, gastric cancer specimens were implanted from surgical specimens into NMRI nu / nu mice and passaged three to five times until a stable growth pattern was established. After being removed from the donor mice, the tumors were cut into segments with a diameter of 4-5 mm and subcutaneously implanted into NMRI nu / nu mice. After significant solid tumor growth, mice were randomly assigned to groups of eight, and the test products were administered to one group each as described below: PBS was administered intravenously at 10 mL / kg on days 0, 3, 6, 9, 12, 15, and 18; Protein #134 was administered intravenously at 4 mL / kg on days 0, 3, 6, 9, 12, 15, and 18; Paclitaxel was administered intravenously at 15 mg / kg on days 0, 7, and 14; or Protein #134 plus Paclitaxel was administered intravenously at 4 mg / kg on days 0, 3, 6, 9, 12, 15, and 18, and at 15 mg / kg on days 0, 7, and 14. Tumor volume was assessed as described above on the day of treatment initiation and on days 2, 6, 13, 16, and 20. Results are shown in... Figure 10c In this model, protein #134 is at least as effective as paclitaxel, and the combination of the two is significantly more effective than either component alone. sequence list <110> Molecular Combination Company <120> Designed ankyrin repeat domains with binding specificity to serum albumin <130> MD41211 <150> EP 15 162 502 <151> 2015-04-02 <150> EP 15 162 511 <151> 2015-04-02 <160> 181 <170> BiSSAP 1.3 <210> 1 <211> 12 <212> PRT <213> Artificial sequence <220> <223> His tags <400> 1 Met Arg Gly Ser His His His His His Gly Ser 1 5 10 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> GS-Connector <400> 2 Gly Gly Gly Gly Ser 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial sequence <220> <223> GS-Connector <400> 3 Gly Gly Gly Gly Ser Gly Ser 1 5 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <220> <223> peptide linkers <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser 1 5 10 <210> 5 <211> 17 <212> PRT <213> Artificial sequence <220> <223> GS-Connector <400> 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Ser <210> 6 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> GS-Connector <400> 6 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Ser 20 <210> 7 <211> 10 <212> PRT <213> Artificial sequence <220> <223> PT connector <400> 7 Gly Ser Pro Thr Pro Thr Pro Thr Gly Ser 1 5 10 <210> 8 <211> 17 <212> PRT <213> Artificial sequence <220> <223> PT connector <400> 8 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly 1 5 10 15 Ser <210> 9 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> PT connector <400> 9 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 1 5 10 15 Pro Thr Pro Thr Pro Thr Gly Ser 20 <210> 10 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 10 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Thr Asp 20 25 30 Asn Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Ser Asn Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ser 50 55 60 Asp Leu Thr Gly Ile Thr Pro Leu His Leu Ala Ala Ala Thr Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Tyr Asp Asn Asp Gly His Thr Pro Leu His Leu Ala Ala Lys Tyr Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 11 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 11 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 12 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 12 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Phe Asp 20 25 30 Trp Met Gly Trp Thr Pro Leu His Leu Ala Ala His Glu Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Thr 50 55 60 Asp Val Ser Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Thr 85 90 95 Lys Asp Asn Thr Gly Trp Thr Pro Leu His Leu Ser Ala Asp Leu Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 13 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 13 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Leu Gly Trp Thr Pro Leu His Leu Ala Ala His Glu Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Val Ser Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Lys Asp Asn Thr Gly Trp Thr Pro Leu His Leu Ser Ala Asp Leu Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 14 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 14 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Thr Ala Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Pro 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala His 50 55 60 Asp Tyr Gln Gly Trp Thr Pro Leu His Leu Ala Ala Thr Leu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 15 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 15 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Thr Ala Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Val Pro Trp Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Ile Gly His 65 70 75 80 Gln Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 16 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 16 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Ser Thr Gly Tyr Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Tyr Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 17 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 17 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Arg Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Pro 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ala 50 55 60 Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 18 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 18 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Pro 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Ala Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 19 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 19 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Pro 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Ala Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 20 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 20 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Pro 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> twenty one <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> twenty one Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Pro 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Tyr Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> twenty two <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> twenty two Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> twenty three <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> twenty three Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Arg Phe Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ile Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala Glu 50 55 60 Asp Trp Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser Met Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Met Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 24 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> Engineered ankyrin repeat domain <400> 24 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Arg Phe Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ile Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Trp Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser Phe Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 25 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 25 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ile Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Glu 50 55 60 Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser Tyr Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala 130 135 140 Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 145 150 155 <210> 26 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 26 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ile Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Glu 50 55 60 Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser Tyr Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala 130 135 140 Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 145 150 155 <210> 27 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 27 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ala Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Glu 50 55 60 Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser Ala Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Lys Asp Asp Ala Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala 130 135 140 Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 145 150 155 <210> 28 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 28 Asp Leu Gly Met Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala His Asp 20 25 30 Thr Trp Gly Leu Thr Pro Leu His Leu Ala Ala Phe His Gly His Gln 35 40 45 Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala Gln 50 55 60 Asp Phe Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Leu Arg Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Gln Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 29 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 29 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Tyr 20 25 30 Glu Asp Gly Leu Thr Pro Leu His Leu Ala Ala Phe Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala Thr 50 55 60 Asp Ala Trp Gly His Thr Pro Leu His Leu Ala Ala Tyr Tyr Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Thr 85 90 95 His Asp Lys Glu Gly Met Thr Ala Leu His Leu Ala Ala Leu Thr Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 30 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 30 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Thr His Asp 20 25 30 Asn Phe Gly Asp Thr Pro Leu His Leu Ala Ala Ser Ile Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala Phe 50 55 60 Asp Ser Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Ser Ser Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Leu Asp Tyr Asn Gly Asn Thr Pro Leu His Leu Ala Ala Asn Ser Gly 100 105 110 Arg Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 31 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 31 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Val Asp 20 25 30 Ala Trp Gly Asp Thr Pro Leu His Leu Ala Ala Ser Ile Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ala 50 55 60 Asp Tyr Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Ser Ala Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Asp Asn Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 Arg Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 32 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 32 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Ile Asp 20 25 30 Thr Trp Gly Asn Thr Pro Leu His Leu Ala Ala Asp Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Leu 50 55 60 Asp Trp Phe Gly Asp Thr Pro Leu His Leu Ala Ala Ser Leu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala 85 90 95 Val Asp Thr Tyr Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 Arg Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 33 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 33 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Gln Asp 20 25 30 Arg Trp Gly Asp Thr Pro Leu His Leu Ala Ala Ser Ala Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Asp 50 55 60 Asp Val Phe Gly Asp Thr Pro Leu His Leu Ala Ala Ser Leu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Asp Asp Tyr Ala Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 34 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 34 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Gln Asp 20 25 30 Arg Trp Gly Asp Thr Pro Leu His Leu Ala Ala Ser Ala Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Asp 50 55 60 Asp Val Phe Gly Asp Thr Pro Leu His Leu Ala Ala Ser Leu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Asp Asp Tyr Ala Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 35 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 35 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Asn Asp 20 25 30 Phe Leu Gly Leu Thr Pro Leu His Leu Ala Ala Ser Thr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ile 50 55 60 Asp Ala Tyr Gly His Thr Pro Leu His Leu Ala Ala Asn Asn Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Ile Asp His Phe Gly Tyr Thr Pro Leu His Leu Ala Ala Met Phe Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 36 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 36 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Asn Asp 20 25 30 Ser Ser Gly Leu Thr Pro Leu His Leu Ala Ala Phe Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Asp 50 55 60 Asp Asp Trp Gly His Thr Pro Leu His Leu Ala Ala His Tyr Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Ile Asp Thr Arg Gly Leu Thr Pro Leu His Leu Ala Ala Ile Ala Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 37 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 37 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Ser Asp 20 25 30 Asp Thr Gly Leu Thr Pro Leu His Leu Ala Ala Asn Arg Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala Asn 50 55 60 Asp Phe Ala Gly Met Thr Pro Leu His Leu Ala Ala Asn Val Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 His Asp Asp Tyr Gly Leu Thr Pro Leu His Leu Ala Ala Asn Trp Arg 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 38 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 38 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Lys Ser Gly Asn Thr Pro Leu His Leu Ala Ala Arg Phe Gly His Leu 35 40 45 Glu Ile Val Glu Phe Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Glu Thr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Trp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Lys Asp Leu Tyr Gly Arg Thr Pro Leu His Leu Ala Ala Lys Leu Arg 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 145 150 155 <210> 39 <211> 157 <212> PRT <213> artificial sequence <220> <223> Engineered ankyrin repeat domain <400> 39 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Leu Asp 20 25 30 Gln Ile Gly Trp Thr Pro Leu His Leu Ala Ala Asn Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Leu Trp Gly Gln Thr Pro Leu His Leu Ala Ala Trp Lys Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Lys Asp Thr Asp Gly Leu Thr Pro Leu His Leu Ala Ala Ile Arg Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala 130 135 140 Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 145 150 155 <210> 40 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 40 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Ser Gly Ala Asp Val Asn Ala Ala Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asp Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asp Ala Ser 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Asp Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 41 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 41 Asp Leu Gly Lys Glu Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Ala Asp 20 25 30 Tyr Phe Gly His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ser 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Ala Phe Glu Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 42 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 42 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Ala Asp 20 25 30 Tyr Phe Gly His Thr Pro Leu His Leu Ala Ala Arg Asp Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ser 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 43 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 43 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Ala Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asp Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ser 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 44 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 44 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 45 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 45 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 46 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 46 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 47 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 47 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 48 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 48 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 49 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 49 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 50 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 50 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 51 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 51 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 52 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 52 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 53 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 53 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Phe Leu Met Ala Asn Gly Ala Asp Val Asn Ala Ala Asp 20 25 30 Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Gln 50 55 60 Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 <210> 54 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 54 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 55 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 55 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 56 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeating domain <400> 56 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Ser Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120 <210> 57 <211> 159 <212> PRT <213> Artificial sequence <220> <223> Designed ankyrin repeats with C-terminal peptide linkers domain <400> 57 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Asp Ile Thr Gly Leu Lys Leu Asn 145 150 155 <210> 58 <211> 302 <212> PRT <213> Artificial sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 58 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 130 135 140 Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp 145 150 155 160 Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys 165 170 175 Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ile Gly His 180 185 190 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 195 200 205 Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser Tyr Gly 210 215 220 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 225 230 235 240 Ala Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr 245 250 255 Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 260 265 270 Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp 275 280 285 Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn 290 295 300 <210> 59 <211> 269 <212> PRT <213> Artificial sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 59 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 130 135 140 Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp 145 150 155 160 Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 165 170 175 Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His 180 185 190 Pro Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 195 200 205 Lys Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly 210 215 220 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 225 230 235 240 Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala 245 250 255 Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn 260 265 <210> 60 <211> 305 <212> PRT <213> Artificial sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 60 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp 180 185 190 Ile Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala 210 215 220 Ser Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala 245 250 255 Ala Asn Thr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 260 265 270 Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu 275 280 285 Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala 290 295 300 Ala 305 <210> 61 <211> 272 <212> PRT <213> Artificial sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 61 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro 180 185 190 Trp Gly His Pro Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala 210 215 220 Ala Ala Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 245 250 255 Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 260 265 270 <210> 62 <211> 273 <212> PRT <213> Artificial sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 62 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala 180 185 190 Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala 210 215 220 Ala Asn Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly 225 230 235 240 Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile 245 250 255 Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu 260 265 270 Asn <210> 63 <211> 269 <212> PRT <213> Artificial sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 63 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 130 135 140 Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp 145 150 155 160 Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys 165 170 175 Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His 180 185 190 Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 195 200 205 Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly 210 215 220 His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn 225 230 235 240 Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala 245 250 255 Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn 260 265 <210> 64 <211> 273 <212> PRT <213> Artificial sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 64 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala 180 185 190 Pro Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala 210 215 220 Ala Ala Val Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 225 230 235 240 Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile 245 250 255 Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu 260 265 270 Asn <210> 65 <211> 273 <212> PRT <213> Artificial sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 65 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala 180 185 190 Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala 210 215 220 Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 225 230 235 240 Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile 245 250 255 Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu 260 265 270 Asn <210> 66 <211> 272 <212> PRT <213> Artificial sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 66 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro 180 185 190 Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala 210 215 220 Ala Val Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser 245 250 255 Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 260 265 270 <210> 67 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Proteins containing two engineered ankyrin repeat domains <400> 67 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg 180 185 190 Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala 210 215 220 Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala 245 250 255 Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 260 265 270 <210> 68 <211> 488 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 68 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala 180 185 190 Asp Ala Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala 210 215 220 Ala Ser Ala Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 225 230 235 240 Ala Asp Val Asn Ala Lys Asp Asp Ala Gly Asn Thr Pro Leu His Leu 245 250 255 Ala Ala Asn Thr Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala 260 265 270 Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp 275 280 285 Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 290 295 300 Leu Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr 305 310 315 320 Thr Pro Thr Pro Thr Pro Thr Gly Ser Arg Ser Asp Leu Asp Lys Lys 325 330 335 Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu 340 345 350 Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Leu Gly Trp Thr 355 360 365 Pro Leu His Leu Ala Ala His Glu Gly His Leu Glu Ile Val Glu Val 370 375 380 Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val Ser Gly Tyr 385 390 395 400 Thr Pro Leu His Leu Ala Ala Ala Asp Gly His Leu Glu Ile Val Glu 405 410 415 Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asn Thr Gly 420 425 430 Trp Thr Pro Leu His Leu Ser Ala Asp Leu Gly His Leu Glu Ile Val 435 440 445 Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe 450 455 460 Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu 465 470 475 480 Ala Glu Ile Leu Gln Lys Leu Asn 485 <210> 69 <211> 488 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 69 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Tyr Leu Gly Trp Thr Pro Leu His Leu Ala Ala 180 185 190 His Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Lys Asp Val Ser Gly Tyr Thr Pro Leu His Leu Ala 210 215 220 Ala Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 225 230 235 240 Ala Asp Val Asn Ala Lys Asp Asn Thr Gly Trp Thr Pro Leu His Leu 245 250 255 Ser Ala Asp Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala 260 265 270 Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp 275 280 285 Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys 290 295 300 Leu Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr 305 310 315 320 Thr Pro Thr Pro Thr Pro Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys 325 330 335 Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu 340 345 350 Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Tyr Gly Asp Thr 355 360 365 Pro Leu His Leu Ala Ala Asp Ala Gly His Leu Lys Ile Val Glu Val 370 375 380 Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Glu Asp Tyr Phe Gly Asn 385 390 395 400 Thr Pro Leu His Leu Ala Ala Ser Ala Gly His Leu Glu Ile Val Glu 405 410 415 Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Ala Gly 420 425 430 Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly His Leu Lys Ile Val 435 440 445 Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser 450 455 460 Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile 465 470 475 480 Ala Glu Val Leu Gln Lys Leu Asn 485 <210> 70 <211> 480 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 70 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 130 135 140 Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp 145 150 155 160 Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 165 170 175 Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ala Gly His 180 185 190 Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 195 200 205 Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser Ala Gly 210 215 220 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 225 230 235 240 Ala Lys Asp Asp Ala Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr 245 250 255 Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 260 265 270 Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp 275 280 285 Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Gly Gly 290 295 300 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 305 310 315 320 Ser Arg Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly 325 330 335 Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn 340 345 350 Ala Lys Asp Tyr Leu Gly Trp Thr Pro Leu His Leu Ala Ala His Glu 355 360 365 Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 370 375 380 Asn Ala Lys Asp Val Ser Gly Tyr Thr Pro Leu His Leu Ala Ala Ala 385 390 395 400 Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 405 410 415 Val Asn Ala Lys Asp Asn Thr Gly Trp Thr Pro Leu His Leu Ser Ala 420 425 430 Asp Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 435 440 445 Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser 450 455 460 Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn 465 470 475 480 <210> 71 <211> 480 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 71 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 130 135 140 Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp 145 150 155 160 Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 165 170 175 Asp Tyr Leu Gly Trp Thr Pro Leu His Leu Ala Ala His Glu Gly His 180 185 190 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 195 200 205 Lys Asp Val Ser Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Asp Gly 210 215 220 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 225 230 235 240 Ala Lys Asp Asn Thr Gly Trp Thr Pro Leu His Leu Ser Ala Asp Leu 245 250 255 Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 260 265 270 Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp 275 280 285 Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Gly Gly Gly 290 295 300 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 305 310 315 320 Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly 325 330 335 Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn 340 345 350 Ala Lys Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ala 355 360 365 Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 370 375 380 Asn Ala Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser 385 390 395 400 Ala Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 405 410 415 Val Asn Ala Lys Asp Asp Ala Gly Asn Thr Pro Leu His Leu Ala Ala 420 425 430 Asn Thr Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala 435 440 445 Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 450 455 460 Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn 465 470 475 480 <210> 72 <211> 455 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 72 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala 180 185 190 Asp Ala Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala 210 215 220 Ala Ser Ala Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 225 230 235 240 Ala Asp Val Asn Ala Lys Asp Asp Ala Gly Asn Thr Pro Leu His Leu 245 250 255 Ala Ala Asn Thr Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala 260 265 270 Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp 275 280 285 Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 290 295 300 Leu Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr 305 310 315 320 Thr Pro Thr Pro Thr Pro Thr Gly Ser Arg Ser Asp Leu Asp Lys Lys 325 330 335 Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu 340 345 350 Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Ser Thr Gly Tyr Thr 355 360 365 Pro Leu His Leu Ala Ala Pro Trp Gly His Leu Glu Ile Val Glu Val 370 375 380 Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Gln Gly Trp 385 390 395 400 Thr Pro Leu His Leu Ala Ala Ala Val Gly His Leu Glu Ile Val Glu 405 410 415 Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430 Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala 435 440 445 Glu Val Leu Gln Lys Leu Asn 450 455 <210> 73 <211> 422 <212> PRT <213> Synthetic Sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 73 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala 180 185 190 Pro Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala 210 215 220 Ala Ala Val Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 225 230 235 240 Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile 245 250 255 Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu 290 295 300 Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met 305 310 315 320 Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro 325 330 335 Leu His Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu 340 345 350 Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr 355 360 365 Pro Leu His Leu Ala Ala Asn Asp Gly His Leu Glu Ile Val Glu Val 370 375 380 Leu Leu Lys His Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys 385 390 395 400 Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu 405 410 415 Val Leu Gln Lys Leu Asn 420 <210> 74 <211> 422 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 74 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala 180 185 190 Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala 210 215 220 Ala Asn Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly 225 230 235 240 Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile 245 250 255 Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Arg Ser Asp Leu Asp Lys Lys Leu 290 295 300 Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met 305 310 315 320 Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro 325 330 335 Leu His Leu Ala Ala Pro Trp Gly His Leu Glu Ile Val Glu Val Leu 340 345 350 Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Gln Gly Trp Thr 355 360 365 Pro Leu His Leu Ala Ala Ala Val Gly His Leu Glu Ile Val Glu Val 370 375 380 Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys 385 390 395 400 Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu 405 410 415 Ile Leu Gln Lys Leu Asn 420 <210> 75 <211> 422 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 75 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala 180 185 190 Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala 210 215 220 Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 225 230 235 240 Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile 245 250 255 Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu 290 295 300 Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met 305 310 315 320 Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro 325 330 335 Leu His Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu 340 345 350 Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr 355 360 365 Pro Leu His Leu Ala Ala Asn Asp Gly His Leu Glu Ile Val Glu Val 370 375 380 Leu Leu Lys His Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys 385 390 395 400 Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu 405 410 415 Val Leu Gln Lys Leu Asn 420 <210> 76 <211> 422 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 76 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala 180 185 190 Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala 210 215 220 Ala Asn Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly 225 230 235 240 Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile 245 250 255 Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu 290 295 300 Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met 305 310 315 320 Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro 325 330 335 Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu 340 345 350 Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr 355 360 365 Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val 370 375 380 Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys 385 390 395 400 Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu 405 410 415 Ile Leu Gln Lys Leu Asn 420 <210> 77 <211> 420 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 77 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val 180 185 190 Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp 210 215 220 Ser Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala 225 230 235 240 Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 245 250 255 Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu 290 295 300 Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn 305 310 315 320 Gly Ala Asp Val Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His 325 330 335 Leu Ala Ala Pro Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys 340 345 350 Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu 355 360 365 His Leu Ala Ala Ala Val Gly His Leu Glu Ile Val Glu Val Leu Leu 370 375 380 Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala 385 390 395 400 Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu 405 410 415 Gln Lys Ala Ala 420 <210> 78 <211> 420 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 78 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Glu Arg Gly Thr Thr Pro Leu His Leu Ala Ala Val Tyr Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asn Glu Thr Gly Tyr Thr Pro Leu His Leu Ala Asp Ser Ser Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Ser Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro 180 185 190 Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala 210 215 220 Ala Val Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser 245 250 255 Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu 290 295 300 Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn 305 310 315 320 Gly Ala Asp Val Asn Ala Lys Asp Glu Arg Gly Thr Thr Pro Leu His 325 330 335 Leu Ala Ala Val Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys 340 345 350 Ala Gly Ala Asp Val Asn Ala Lys Asn Glu Thr Gly Tyr Thr Pro Leu 355 360 365 His Leu Ala Asp Ser Ser Gly His Leu Glu Ile Val Glu Val Leu Leu 370 375 380 Lys His Ser Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro 385 390 395 400 Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu 405 410 415 Gln Lys Ala Ala 420 <210> 79 <211> 420 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 79 Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 100 105 110 Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg 180 185 190 Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala 210 215 220 Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala 245 250 255 Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu 290 295 300 Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala 305 310 315 320 Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His 325 330 335 Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys 340 345 350 Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu 355 360 365 His Leu Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu 370 375 380 Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro 385 390 395 400 Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu 405 410 415 Gln Lys Ala Ala 420 <210> 80 <211> 420 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 80 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala Pro 180 185 190 Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala Ala 210 215 220 Ala Val Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser 245 250 255 Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu 290 295 300 Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala 305 310 315 320 Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His 325 330 335 Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys 340 345 350 Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu 355 360 365 His Leu Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu 370 375 380 Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro 385 390 395 400 Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu 405 410 415 Gln Lys Ala Ala 420 <210> 81 <211> 420 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 81 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg 180 185 190 Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala 210 215 220 Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala 245 250 255 Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu 290 295 300 Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn 305 310 315 320 Gly Ala Asp Val Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His 325 330 335 Leu Ala Ala Pro Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys 340 345 350 Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu 355 360 365 His Leu Ala Ala Ala Val Gly His Leu Glu Ile Val Glu Val Leu Leu 370 375 380 Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala 385 390 395 400 Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu 405 410 415 Gln Lys Ala Ala 420 <210> 82 <211> 422 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 82 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Ser Pro Thr Pro 115 120 125 Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 130 135 140 Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 145 150 155 160 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 165 170 175 Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala 180 185 190 Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 195 200 205 Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala 210 215 220 Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 225 230 235 240 Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile 245 250 255 Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu 260 265 270 Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr 275 280 285 Pro Thr Pro Thr Pro Thr Gly Ser Arg Ser Asp Leu Gly Lys Lys Leu 290 295 300 Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met 305 310 315 320 Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro 325 330 335 Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu 340 345 350 Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr 355 360 365 Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val 370 375 380 Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys 385 390 395 400 Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu 405 410 415 Ile Leu Gln Lys Leu Asn 420 <210> 83 <211> 486 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 83 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Lys Ser Gly Asn Thr Pro Leu His Leu Ala Ala Arg 180 185 190 Phe Gly His Leu Glu Ile Val Glu Phe Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asp Glu Thr Gly Lys Thr Pro Leu His Leu Ala Ala 210 215 220 Ile Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Lys Asp Leu Tyr Gly Arg Thr Pro Leu His Leu Ala 245 250 255 Ala Lys Leu Arg His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 260 265 270 Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile 275 280 285 Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala 290 295 300 Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr 305 310 315 320 Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu 325 330 335 Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys 340 345 350 Ala Gly Ala Asp Val Asn Ala Leu Asp Gln Ile Gly Trp Thr Pro Leu 355 360 365 His Leu Ala Ala Asn Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu 370 375 380 Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Leu Trp Gly Gln Thr Pro 385 390 395 400 Leu His Leu Ala Ala Trp Lys Gly His Leu Glu Ile Val Glu Val Leu 405 410 415 Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Thr Asp Gly Leu Thr 420 425 430 Pro Leu His Leu Ala Ala Ile Arg Gly His Leu Glu Ile Val Glu Val 435 440 445 Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys 450 455 460 Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu 465 470 475 480 Val Leu Gln Lys Ala Ala 485 <210> 84 <211> 486 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 84 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Lys Ser Gly Asn Thr Pro Leu His Leu Ala Ala Arg Phe Gly His Leu 35 40 45 Glu Ile Val Glu Phe Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Glu Thr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Trp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Lys Asp Leu Tyr Gly Arg Thr Pro Leu His Leu Ala Ala Lys Leu Arg 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro 145 150 155 160 Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro 165 170 175 Thr Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg 180 185 190 Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Leu Asp Gln Ile Gly Trp Thr Pro Leu His Leu Ala Ala 210 215 220 Asn Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Lys Asp Leu Trp Gly Gln Thr Pro Leu His Leu Ala 245 250 255 Ala Trp Lys Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 260 265 270 Ala Asp Val Asn Ala Lys Asp Thr Asp Gly Leu Thr Pro Leu His Leu 275 280 285 Ala Ala Ile Arg Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala 290 295 300 Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp 305 310 315 320 Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 325 330 335 Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 340 345 350 Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 355 360 365 Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu 370 375 380 Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro 385 390 395 400 Leu His Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu 405 410 415 Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr 420 425 430 Pro Leu His Leu Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val 435 440 445 Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys 450 455 460 Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu 465 470 475 480 Val Leu Gln Lys Ala Ala 485 <210> 85 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 85 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp 180 185 190 Ile Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala 210 215 220 Ser Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala 245 250 255 Ala Asn Thr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 260 265 270 Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu 275 280 285 Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala 290 295 300 Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr 305 310 315 320 Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu 325 330 335 Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys 340 345 350 Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu 355 360 365 His Leu Ala Ala Pro Trp Gly His Pro Glu Ile Val Glu Val Leu Leu 370 375 380 Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro 385 390 395 400 Leu His Leu Ala Ala Ala Ala Gly His Leu Glu Ile Val Glu Val Leu 405 410 415 Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr 420 425 430 Pro Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val 435 440 445 Leu Gln Lys Ala Ala 450 <210> 86 <211> 449 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 86 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln 145 150 155 160 Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 165 170 175 Lys Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ile Gly 180 185 190 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 195 200 205 Ala Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser Tyr 210 215 220 Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 225 230 235 240 Asn Ala Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala Ala Asn 245 250 255 Thr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 260 265 270 Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala 275 280 285 Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly 290 295 300 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 305 310 315 320 Gly Gly Ser Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg 325 330 335 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp 340 345 350 Val Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala 355 360 365 Pro Trp Gly His Pro Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 370 375 380 Asp Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala 385 390 395 400 Ala Ala Ala Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 405 410 415 Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu 420 425 430 Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala 435 440 445 Ala <210> 87 <211> 453 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 87 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ile Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Glu 50 55 60 Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser Tyr Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala Ala Asn Thr Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala 130 135 140 Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro 145 150 155 160 Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro 165 170 175 Thr Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg 180 185 190 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu His Leu Ala Ala 210 215 220 Pro Trp Gly His Pro Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro Leu His Leu Ala 245 250 255 Ala Ala Ala Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 260 265 270 Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu 275 280 285 Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala 290 295 300 Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr 305 310 315 320 Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu 325 330 335 Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys 340 345 350 Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu 355 360 365 His Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu 370 375 380 Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro 385 390 395 400 Leu His Leu Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu 405 410 415 Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr 420 425 430 Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val 435 440 445 Leu Gln Lys Ala Ala 450 <210> 88 <211> 480 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 88 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 130 135 140 Ser Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp 145 150 155 160 Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 165 170 175 Asp Tyr Leu Gly Trp Thr Pro Leu His Leu Ala Ala His Glu Gly His 180 185 190 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 195 200 205 Lys Asp Val Ser Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Asp Gly 210 215 220 His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 225 230 235 240 Ala Lys Asp Asn Thr Gly Trp Thr Pro Leu His Leu Ser Ala Asp Leu 245 250 255 Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 260 265 270 Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp 275 280 285 Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Gly Gly Gly 290 295 300 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 305 310 315 320 Ser Arg Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly 325 330 335 Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn 340 345 350 Ala Lys Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp Ala 355 360 365 Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 370 375 380 Asn Ala Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala Ser 385 390 395 400 Ala Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 405 410 415 Val Asn Ala Lys Asp Asp Ala Gly Asn Thr Pro Leu His Leu Ala Ala 420 425 430 Asn Thr Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala 435 440 445 Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 450 455 460 Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn 465 470 475 480 <210> 89 <211> 453 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 89 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Asp Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp 180 185 190 Ile Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala 210 215 220 Ser Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala 245 250 255 Ala Asn Thr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 260 265 270 Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu 275 280 285 Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala 290 295 300 Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr 305 310 315 320 Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Asp Lys Lys Leu Leu 325 330 335 Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys 340 345 350 Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Thr Gly Trp Thr Pro Leu 355 360 365 His Leu Ala Ala Pro Trp Gly His Pro Glu Ile Val Glu Val Leu Leu 370 375 380 Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Gln Gly Trp Thr Pro 385 390 395 400 Leu His Leu Ala Ala Ala Val Gly His Leu Glu Ile Val Glu Val Leu 405 410 415 Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr 420 425 430 Pro Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val 435 440 445 Leu Gln Lys Ala Ala 450 <210> 90 <211> 453 <212> PRT <213> Artificial sequence <220> <223> Proteins containing three engineered ankyrin repeat domains <400> 90 Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 20 25 30 Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu 35 40 45 Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 50 55 60 Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 85 90 95 Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly 100 105 110 His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr 115 120 125 Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 130 135 140 Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala 145 150 155 160 Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val 165 170 175 Asn Ala Lys Asp Arg Tyr Gly Asp Thr Pro Leu His Leu Ala Ala Asp 180 185 190 Ile Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 195 200 205 Val Asn Ala Glu Asp Tyr Phe Gly Asn Thr Pro Leu His Leu Ala Ala 210 215 220 Ser Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 225 230 235 240 Asp Val Asn Ala Lys Asp Asp Tyr Gly Asn Thr Pro Leu His Leu Ala 245 250 255 Ala Asn Thr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 260 265 270 A...

Claims

1. A recombinant binding protein comprising the amino acid sequence of SEQ ID NO: 134, wherein the binding protein has binding specificity for HGF, VEGF-A and serum albumin.

2. A nucleic acid encoding the amino acid sequence of the binding protein of claim 1.

3. A pharmaceutical composition comprising the binding protein of claim 1, and a pharmaceutically acceptable carrier and / or diluent.

4. A pharmaceutical composition comprising the nucleic acid of claim 2, and a pharmaceutically acceptable carrier and / or diluent.

5. Use of the binding protein of claim 1 in the preparation of a medicament, wherein the medicament is for treating a medical condition, and wherein the medical condition is lung cancer, glioblastoma, kidney cancer, or gastric cancer.

6. The use of claim 5, wherein the condition is lung cancer.

7. The use of claim 5, wherein the condition is glioblastoma.

8. The use of claim 5, wherein the condition is kidney cancer.

9. The use of claim 5, wherein the condition is gastric cancer.

Images