RU2778346C2 - Recombinant binding proteins and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to the production of recombinant proteins binding a human epidermal growth factor receptor 2 (hereinafter – HER2) to ankyrin repeats; it can be used in medicine for the treatment of HER2-expressing cancer. Recombinant biparatopic binding protein is proposed, causing antagonism of transmission of ErbB signal, which contains two constructed domains with ankyrin repeats with binding specificity relatively to HER2, and two constructed domains with ankyrin repeats with binding specificity relatively to serum albumin.

EFFECT: invention provides for improved stability during storage of therapeutically active recombinant protein binding HER2 and serum albumin.

15 cl, 7 dwg, 3 tbl, 17 ex

Description

Link to related application

The present application claims the benefit and priority of European Patent Application EP 16190221 filed September 22, 2016 with the European Patent Office. The contents of European patent application EP 16190221 are incorporated herein by reference in their entirety for all purposes, including all tables, figures and claims, and including the inclusion of any element or part of the description, claims or drawings not contained in herein and referred to in PCT Rule 20.5(a), in accordance with PCT Rule 4.18.

The field of technology to which the present invention relates

New recombinant binding proteins are provided. The proteins contain two engineered ankyrin repeat domains with binding specificity for HER2 and two engineered ankyrin repeat domains with binding specificity for serum albumin linked by polypeptide linkers. Recombinant binding proteins are useful in the treatment of disease. In particular, recombinant binding proteins contain engineered ankyrin repeat domains with binding specificity for serum albumin, exhibiting high storage stability. Moreover, nucleic acids encoding said recombinant binding proteins, pharmaceutical compositions containing said recombinant binding proteins or nucleic acids, and the use of said recombinant binding proteins, nucleic acids or pharmaceutical compositions in the treatment of diseases are contemplated.

Background of the Invention

, M.G., and

, A., Nat. Biotechnol. 22, 575-582, 2004; Stumpp, M.T., Binz, H.K and Amstutz, P., Drug Discov. Today 13, 695-701, 2008) можно использовать для отбора целевых специфических сконструированных доменов с анкириновыми повторами. Такие целевые специфические сконструированные домены с анкириновыми повторами, в свою очередь, можно использовать в качестве ценных компонентов рекомбинантных связывающих белков для лечения заболеваний. Был описан отбор различных сконструированных доменов с анкириновыми повторами со специфичностью связывания в отношении рецептора 2 эпидермального фактора роста человека (HER2 или ErbB2; UniProt Р04626) (международная патентная публикация WO 2014/083208). Согласно настоящему изобретению раскрыты рекомбинантные связывающие белки, содержащие сконструированные домены с анкириновыми повторами со специфичностью связывания в отношении HER2.Libraries of engineered ankyrin repeat proteins (International Patent Publication WO 2002/020565; Binz, H.K., Amstutz, P., Kohl, A., Stumpp, MT, Briand, C., Forrer, P.,

, MG, and

, A., Nat. Biotechnol. 22, 575-582, 2004; Stumpp, MT, Binz, HK and Amstutz, P., Drug Discov. Today 13, 695-701, 2008) can be used to select target specific engineered ankyrin repeat domains. Such target specific engineered ankyrin repeat domains, in turn, can be used as valuable components of recombinant binding proteins for the treatment of diseases. A selection of different engineered ankyrin repeat domains with binding specificity for human epidermal growth factor receptor 2 (HER2 or ErbB2; UniProt P04626) has been described (International Patent Publication WO 2014/083208). The present invention discloses recombinant binding proteins containing engineered ankyrin repeat domains with binding specificity for HER2.

, D., Kontermann, R.E. Protein Eng. Des. Sel. 23, 827-834, 2010), авторы настоящего изобретения неожиданно обнаружили, что благодаря наличию двух сконструированных доменов с анкириновыми повторами со специфичностью связывания в отношении сывороточного альбумина вместо одного в рекомбинантном связывающем белке можно улучшить фармакокинетические свойства рекомбинантного связывающего белка.Unlike, for example, IgG antibodies, which exhibit long systemic half-lives mediated by FcRn recycling, proteins containing engineered ankyrin repeat domains typically exhibit rapid pharmacokinetic clearance and short terminal half-lives, unless the protein contains elements that improve pharmacokinetic properties, such as, for example, an engineered ankyrin repeat domain with serum albumin binding specificity described in international patent publication WO 2012/069654. The use of serum albumin binding to improve the pharmacokinetic properties of proteins is a technique well known in the art (see, for example, international patent publication WO 1991/001743; 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); and international patent publication WO 2012/069654). In order to use engineered ankyrin repeat domains with binding specificity for serum albumin in clinical drug candidates, it is desirable that the storage stability of known engineered ankyrin repeat domains with binding specificity for serum albumin be improved. Disclosed herein are recombinant binding proteins comprising engineered ankyrin repeat domains with binding specificity for serum albumin, said engineered ankyrin repeat domains with binding specificity for serum albumin exhibiting improved storage stability properties. Unlike previous publications (Kopr, J., Horning, N., Zettlitz, KA, Schwarz, A., Fuss, N.,

, D., Kontermann, RE Protein Eng. Des. Sel. 23, 827-834, 2010), the present inventors unexpectedly found that by having two engineered ankyrin repeat domains with binding specificity for serum albumin instead of one in the recombinant binding protein, the pharmacokinetic properties of the recombinant binding protein can be improved.

HER2 plays an important role in the pathogenesis and progression of some types of cancer. HER2 is a transmembrane receptor tyrosine kinase (RTK) belonging to the larger ErbB receptor family (Bublil, E. M. and Yarden, Y. Curr. Opin. Cell Biol. 19(2), 124-34, 2007). The ErbB receptor family is conserved across vertebrates and also includes ErbB1 or epidermal growth factor receptor (EGFR) or HER1 (UniProt P00533) and HER3 (ErbB3; UniProt P21860) and HER4 (ErbB4; UniProt Q15303) receptors. All ErbB receptors share extensive sequence and domain homologies and form functional homodimers (eg ErbB1-ErbB1, HER2-HER2 and HER4-HER4) and heterodimers in all combinations. Homo- and heterodimerization of receptors occurs upon ligand binding or receptor overexpression and in turn activates the intracellular domains of the receptor kinase by autophosphorylation. This then triggers the underlying intracellular signaling and biological responses. Well known ErbB signaling antagonists include the monoclonal antibodies trastuzumab (binding to HER2 extracellular domain IV and inhibiting HER2 homodimerization) and pertuzumab (binding to HER2 extracellular domain II and inhibiting HER2/HER3 heterodimerization). Importantly, trastuzumab has a predominantly antiproliferative effect, and tumors may avoid such treatment in advanced stages of the disease. Pertuzumab, which has an unexpectedly low therapeutic efficacy as a single agent, may complement the activity of trastuzumab by interfering with HER2/HER3 heterodimerization. Thus, the combination of trastuzumab with pertuzumab is attractive for the treatment of HER2-positive cancer (Capelan M., et al., Ann. Oncol., 24, 273-82, 2013).

The combination of trastuzumab and pertuzumab led to the concept that dual targeting of two domains in HER2 is required for superior antitumor efficacy. Mixtures of antibodies targeting HER2 domains II and IV, or simultaneously targeting domain I and another HER2 domain (US 20110033460; e.g. also domain IV) or domain I and domain IV have been reported (International Patent Publication WO 2014/060365; Jost, Ch., et al., Structure 27, 1-13, 2013; Tamaskovic, R., et al., Nat Commun 7, 11672, 2016), or domain II and domain IV (international patent publication WO 2014/083208), or simultaneously targeting a trastuzumab epitope to HER2 domain IV and a pertuzumab epitope to HER2 domain II (International Patent Publication WO 2009/068625). Some of the approaches have included biparatopic binding proteins. Interestingly, some of the biparatopic binding proteins tested in International Patent Publication WO 2009/068625 had an antagonistic effect, others an agonistic effect. Data in International Patent Publication WO 2014/083208 and Jost, Ch. et a.l (ibid.; international patent publication WO 2014/060365) indicate that the generation of antagonistic biparatope binding proteins is not direct. Instead, careful selection of individual domains (epitope, binder properties) as well as structural arrangement (orientation, spacing, linker length, linker composition) must be optimized for effective antagonism. In addition, the choice of fragment for pharmacokinetic engineering and its structural arrangement also needs to be optimized. Together, this represents a selection of at least 250,000 different options.

Disclosed herein is a recombinant binding protein comprising (i) a biparatopic binding protein that antagonizes ErbB signaling, consisting of two engineered ankyrin repeat domains with binding specificity for HER2 and (ii) two engineered ankyrin repeat domains with binding specificity for HER2. serum albumin ratio and with improved storage stability. Said recombinant binding protein, DARPin® drug candidate, has been shown to be a valuable drug candidate for the treatment of various diseases. DARPin® is a registered trademark of Molecular Partners AG, Switzerland.

Brief summary of the present invention

The present invention relates to recombinant binding proteins comprising four engineered ankyrin repeat domains, wherein two of the four engineered ankyrin repeat domains are engineered ankyrin repeat domains with binding specificity for HER2, and where two of the four engineered ankyrin repeat domains are ankyrin repeats are engineered ankyrin repeat domains with a binding specificity for serum albumin. In particular, the present invention relates to a recombinant binding protein containing an amino acid sequence that has at least 90% amino acid sequence identity with respect to SEQ ID NO: 21. The present invention also relates to such a recombinant binding protein, in which each of the two of engineered ankyrin repeat domains with binding specificity for serum albumin contains the amino acid sequence according to SEQ ID NO:14. of SEQ ID NO: 21. In one embodiment, each of said engineered ankyrin repeat domains with binding specificity for serum albumin, comprising SEQ ID NO: 14, exhibiting shows improved storage stability in PBS compared to an engineered ankyrin repeat domain with binding specificity for serum albumin containing the amino acid sequence of SEQ ID NO: 13. In one embodiment, the present invention provides a recombinant binding protein comprising the amino acid sequence of SEQ ID NO: 21. According to one embodiment, the present invention relates to a recombinant binding protein consisting of the amino acid sequence according to SEQ ID NO: 21. According to one embodiment, a recombinant binding protein containing the amino acid sequence according to SEQ ID NO: 21 exhibits higher stability storage than a recombinant binding protein comprising the amino acid sequence according to SEQ ID NO: 22. In one embodiment, a recombinant binding protein according to the present of the invention inhibits the proliferation of BT474 cells with an inhibition constant below 10 ^-7 M. In one embodiment, the recombinant binding protein of the present invention at a concentration of 100 nM exhibits a stronger downregulation of AKT-S473 phosphorylation in BT474 cells than trastuzumab at a concentration of component 100 nm. The present invention further relates to nucleic acids encoding the recombinant binding protein of the present invention. The present invention further relates to pharmaceutical compositions containing the recombinant binding protein and/or nucleic acid of the present invention and a pharmaceutically acceptable carrier and/or diluent. The present invention also relates to a pharmaceutical composition according to the present invention for use in the treatment of disease, neoplastic disease, cancer, breast cancer, ovarian cancer, gastric cancer, gastric cancer, uterine cancer, colon and rectal cancer, bladder cancer, overweight cancer. HER2 expression, HER2 expressing cancer, HER2 addictive cancer, HER2 partial addictive cancer, HER2 amplifying cancer, trastuzumab-resistant cancer, trastuzumab-responsive cancer, gastrointestinal cancer, or brain cancer. The present invention further relates to a kit containing a recombinant binding protein according to the present invention or a nucleic acid according to the present invention or a pharmaceutical composition according to the present invention. The present invention further relates to a method for producing a recombinant binding protein according to the present invention, the method comprising the steps of (i) expressing said recombinant binding protein in bacteria, and (ii) purifying said recombinant binding protein using chromatography.

Brief description of the figures

Figure 1. Graphical representation of recombinant binding proteins with binding specificity for HER2 containing engineered ankyrin repeat domains with binding specificity for serum albumin.

(a) Graphical representation of an engineered ankyrin repeat domain with binding specificity for serum albumin. Examples of such ankyrin repeat domains are engineered ankyrin repeat domains containing an amino acid sequence selected from the group consisting of SEQ ID NO: 12-15, in particular, an engineered ankyrin repeat domain containing the amino acid sequence of SEQ ID NO: 14. (b) Graphical representation of an engineered ankyrin repeat domain with binding specificity for HER2. Examples of such ankyrin repeat domains are engineered ankyrin repeat domains containing an amino acid sequence selected from the group consisting of SEQ ID NO: 16-20, in particular engineered ankyrin repeat domains containing amino acid sequences according to SEQ ID NO: 16 and 17. (c) Graphical representation of a polypeptide linker (eg, a polypeptide containing the amino acid sequence according to any of SEQ ID NO: 2-9, in particular, a polypeptide linker containing the amino acid sequence according to SEQ ID NO: 9). (d) Graphical representation of the N-terminal amino acid sequence. Examples of such N-terminal amino acid sequences are, for example, MGS or GS sequences (as, for example, represented at the N-terminus of any of SEQ ID NOs: 21-30 and 32 and 33), or polypeptide tags, as illustrated by the amino acid sequence, corresponding to SEQ ID NO: 1. (e) A graphical representation of the recombinant binding protein provided herein, containing two engineered ankyrin repeat domains with binding specificity for serum albumin and two engineered ankyrin repeat domains, each with binding specificity for relation to HER2 connected by polypeptide linkers and characterized by an N-terminal amino acid sequence. Two engineered ankyrin repeat domains with binding specificity for serum albumin are flanked by two other engineered ankyrin repeat domains. The recombinant binding protein containing the amino acids according to SEQ ID NO: 21 corresponds to this graphical representation.

Figure 2. Improved storage stability of the protein containing SEQ ID NO: 14

SDS analysis of 15% PAGE protein #13-His (#13) and protein #14-His (#14; see Example 1) stored at 10 mg/mL in PBS for 1 week at 4°C (1), 25°C (2), 40°C (3) and 60°C (4). The #14-His protein exhibits higher storage stability than the #13-His protein. M: Marker (lower band: 6.5 kDa; band at protein #14-His: 14.4 kDa; upper band for PAGE protein #14-His: 21.5 kDa).

Figure 3. Inhibition of BT474 cell proliferation by recombinant binding proteins with binding specificity for HER2.

Inhibition of proliferation of BT474 breast cancer cells was performed as described in Example 6. The recombinant binding protein containing SEQ ID NO: 21 exhibits a lower IC50 than the recombinant binding protein containing SEQ ID NO: 23, indicating that the presence SEQ ID NOs: 16 and 17 in the recombinant binding protein is more favorable than having SEQ ID NOs: 18 and 17. Solid circles and solid line: protein #21-His; filled squares and dashed line: protein #23-His; triangle: no inhibitor. OD (optical density): OD405-OD620; C: concentration in nM.

Figure 4. Experiments using xenograft tumor model in mice.

a.) Xenograft tumor model - BT474 breast cancer in mice. The model was run as described in Example 7. Triangles: PBS; open squares: trastuzumab; diamonds with a dashed line: trastuzumab and pertuzumab; solid circles: recombinant binding protein with binding specificity for HER2 (protein #21-His); V: tumor volume [mm ³ ]; t: time [d]. b.) BT474 breast cancer xenograft model in mice comparing protein #21-His (21), protein #23-His (23), protein #24-His (24) or protein #25-His ( 25) on the 18th day of treatment. The experiment was performed as described in Example 7. Protein #21-His is significantly more effective in inhibiting tumor growth than protein #24-His (p=0.0003), protein #23-His or protein #25. V: tumor volume [mm ³ ]; t: time [d]. c.) and d.) Tumor model using patient-derived gastric cancer xenograft GXA3039 in mice. The experiment was performed twice as described in Example 7. PBS (triangles), pertuzumab (open inverted triangles), trastuzumab (open squares), a mixture of trastuzumab and pertuzumab (dashed diamonds), and protein #21 were used in the indicated experiments, with .) Protein #21 shows strong tumor growth inhibition similar to the combination of trastuzumab and pertuzumab, while trastuzumab alone is less effective, d.) Protein #21 shows strong tumor growth inhibition similar to the combination of trastuzumab and pertuzumab. Trastuzumab alone and pertuzumab alone are significantly less effective. RTV: relative tumor volume relative to tumor volume at the start of treatment [%]; t: time [d]. e.) Tumor model using patient-derived gastric cancer xenograft GXF281 in mice. The experiment was performed as described in Example 7. PBS (triangles), lapatinib (open squares), and protein #21 (solid circles). Protein #21 shows strong tumor growth inhibition compared to lapatinib in this model. RTV: relative tumor volume relative to tumor volume at the start of treatment [%]; t: time [d].

Figure 5. Pharmacokinetic analyzes in mice.

a.) Mouse pharmacokinetic analysis comparing protein #21-His (solid circles) with protein #23-His (squares) according to Example 8. This experiment indicates a pharmacokinetic advantage of having SEQ ID NO: 16 in the recombinant binding protein compared to the presence of SEQ ID NO: 18. b.) Mouse pharmacokinetic analysis comparing protein #30-His (solid circles; contains two engineered ankyrin repeat domains with binding specificity for serum albumin) to protein #29-His (solid squares; contains one engineered ankyrin repeat domain with binding specificity for serum albumin) according to examples 8 and 11. The presence of two engineered ankyrin repeat domains with binding specificity for serum albumin improves the pharmacokinetic profile, going beyond the profile, achieved with a single engineered ankyrinovog domain o repeat with binding specificity for serum albumin. c.) Pharmacokinetic comparison of a recombinant binding protein containing GlySer polypeptide linkers (protein #26-His; solid squares) with a recombinant binding protein containing ProThr polypeptide linkers (protein #27-His; solid circles) as described in Examples 8 and 10. The presence of ProThr polypeptide linkers has a beneficial effect on pharmacokinetic properties in the mouse. %ID: percentage of dose administered by injection, normalized to the measurement value in 1 hour [%]; t: time [hours].

Figure 6. Pharmacokinetic analyzes in cynomolgus monkey.

a) Analysis of the pharmacokinetic properties of protein #21-His (solid circles) and protein #23-His (solid squares) at a dose of 1 mg/kg in cynomolgus monkey according to example 9. A longer terminal half-life is observed for protein #21 -His, indicating that the presence of SEQ ID NO: 16 in the recombinant binding protein is more favorable than the presence of SEQ ID NO: 18. b) Analysis of the pharmacokinetic properties of protein #21-His at a dose of 1 mg/kg ( solid circles), 5 mg/kg (solid squares), and 10 mg/kg (solid triangles) in the cynomolgus monkey according to Example 9. Protein #21-His shows a dose-dependent increase in terminal half-life from 47 hours at a dose of 1 mg /kg, up to 100 hours at a dose of 5 mg/kg, up to about 180 hours at a dose of 10 mg/kg. C: Concentration [nM]; t: time [hours].

Figure 7. Induction of apoptosis in BT474 cells.

Measurement of BT474 cell apoptosis induced by varying concentrations of protein #21 (solid circles), trastuzumab (open squares), pertuzumab (open inverted triangles) or a mixture of 100 nM trastuzumab and varying concentrations of pertuzumab (open diamonds; dashed line) according to Example 6. Results were plotted on the graph, including the fitting non-linear regression curves. Measurements for PBS (solid triangle) and 100 nM trastuzumab (solid inverted triangle) are shown as reference values at 50 pM. C: concentration in [nM]; RLU: relative light units.

Detailed disclosure of the present invention

The present invention relates to recombinant binding proteins comprising four engineered ankyrin repeat domains, wherein two of said four engineered ankyrin repeat domains are engineered ankyrin repeat domains with binding specificity for HER2, and where two of said four engineered ankyrin repeat domains are ankyrin repeats are engineered ankyrin repeat domains with a binding specificity for serum albumin. SEQ ID NO: 21 is an example of such a recombinant binding protein.

In one embodiment, each of said engineered ankyrin repeat domains with binding specificity for serum albumin is characterized by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with respect to SEQ ID NO: 14. In one embodiment, each of said engineered ankyrin repeat domains with binding specificity for serum albumin consists of an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 14, and/or (ii) sequences in which up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids according to SEQ ID NO: 14 are replaced by any amino acid. In one embodiment, each of said engineered ankyrin repeat domains with binding specificity for serum albumin consists of an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 14 and/or (ii) sequences wherein up to up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids according to SEQ ID NO: 14 are replaced by any amino acid, and each of these engineered ankyrin repeat domains with binding specificity for serum albumin is not is identical to the sequence of an engineered ankyrin repeat domain with binding specificity for serum albumin selected from the group consisting of SEQ ID NO: 12, 13 or 15 according to the present application, as well as SEQ ID NO: 17-25 and 43-48 according to International Patent publications WO 2012/069654.

In one embodiment, the penultimate amino acid of any of SEQ ID NOs: 10-20 is Ala or Leu, preferably Ala. In one embodiment, the C-terminal amino acid of any one of SEQ ID NOs: 10-20 is Ala or Asn, preferably Ala, or optionally absent. For example, SEQ ID NO: 29 contains, for example, SEQ ID NO: 15, in which the penultimate amino acid is Leu and there is no C-terminal amino acid. Preferably, both the penultimate amino acid and the C-terminal amino acid of any engineered ankyrin repeat domain (ie, any of SEQ ID NOs: 10-20) present in the recombinant binding protein of the present invention are Ala.

In one embodiment, Gly Ser amino acids at the N-terminus of any of SEQ ID NOs: 10-30 and 32-33 are optionally absent.

In one embodiment, said two engineered ankyrin repeat domains with binding specificity for serum albumin are 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% identical in terms of amino acid sequence. In one embodiment, said two engineered ankyrin repeat domains with binding specificity for serum albumin from said recombinant binding protein are identical in amino acid sequence.

In one embodiment, each of said two engineered ankyrin repeat domains with binding specificity for serum albumin of the recombinant binding protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 14. In one embodiment, each of said two engineered ankyrin repeat domains with binding specificity for serum albumin of the recombinant binding protein according to the present invention consists of SEQ ID NO: 14.

In one embodiment, two engineered ankyrin repeat domains with binding specificity for serum albumin from said recombinant binding protein are capable of simultaneously binding one serum albumin molecule each.

In one embodiment, the present invention provides recombinant binding proteins comprising four engineered ankyrin repeat domains, wherein two of said four engineered ankyrin repeat domains are engineered ankyrin repeat domains with binding specificity for HER2, and where two of said The four engineered ankyrin repeat domains are engineered ankyrin repeat domains with binding specificity for serum albumin, and wherein each of said two engineered ankyrin repeat domains with binding specificity for serum albumin consists of SEQ ID NO: 14.

In one embodiment, said engineered ankyrin repeat domain comprising SEQ ID NO: 14 exhibits improved storage stability in PBS compared to an engineered ankyrin repeat domain comprising SEQ ID NO: 13. In one embodiment, said engineered ankyrin repeat domain repeats containing SEQ ID NO: 14 exhibits improved storage stability in PBS compared to an engineered ankyrin repeat domain containing SEQ ID NO: 15. In one embodiment, said engineered ankyrin repeat domain consisting of SEQ ID NO: 14 , exhibits improved storage stability in PBS compared to the engineered ankyrin repeat domain consisting of SEQ ID NO: 13. In one embodiment, said engineered ankyrin repeat domain consisting of SEQ ID NO: 14 exhibits improved storage stability in PBS compared to the engineered ankyrin repeat domain consisting of SEQ ID NO: 15. The engineered ankyrin repeat domains consisting of SEQ ID NO: 12-15 are examples of engineered ankyrin repeat domains with binding specificity for serum albumin.

In one embodiment, said recombinant binding protein comprises two engineered ankyrin repeat domains with binding specificity for serum albumin, wherein said two engineered ankyrin repeat domains with binding specificity for serum albumin each comprise the amino acid sequence of SEQ ID NO: 14, and wherein each of said engineered ankyrin repeat domains with binding specificity for serum albumin exhibits improved storage stability in PBS, preferably reduced amounts of degradation products after storage at 40° C. for 1 month at 10 mg/mL in PBS, compared to the engineered ankyrin repeat domain containing the amino acid sequence of SEQ ID NO: 13.

In one embodiment, said recombinant binding protein comprising two engineered ankyrin repeat domains with binding specificity for serum albumin, wherein said two engineered ankyrin repeat domains with binding specificity for serum albumin each comprises an amino acid sequence according to SEQ ID NO: 14 , shows improved storage stability, preferably reduced amounts of degradation products after storage at 40° C. for 1 month at 10 mg/ml in PBS, compared to the corresponding recombinant binding protein, with each of these two engineered ankyrin repeat domains with binding specificity for serum albumin contains the amino acid sequence according to SEQ ID NO: 13. SEQ ID NO: 21 and 22 are examples of such recombinant binding proteins containing SEQ ID NO: 1 4 and 13, respectively.

In one embodiment, said recombinant binding protein comprising two engineered ankyrin repeat domains with binding specificity for serum albumin exhibits improved pharmacokinetic properties compared to a corresponding recombinant binding protein containing only one engineered ankyrin repeat domain with binding specificity for serum albumin. albumin. In one embodiment, said recombinant binding protein containing two engineered ankyrin repeat domains with binding specificity for serum albumin exhibits a longer terminal half-life in the cynomolgus monkey compared to the corresponding recombinant binding protein containing only one engineered ankyrin repeat domain with binding specificity for serum albumin. Example 11 (see Figure 5b) discloses recombinant binding proteins with improved pharmacokinetic properties.

In one embodiment, said two engineered ankyrin repeat domains with binding specificity for serum albumin are one N-terminal and one C-terminal of said two engineered ankyrin repeat domains with binding specificity for HER2.

In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 is characterized by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% amino acid sequence identity with respect to SEQ ID NO: 16. In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 comprises an amino acid sequence selected from the group consisting of (i) SEQ ID NO : 16, and/or (ii) sequences in which up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acids according to SEQ ID NO: 16 are replaced by any amino acid. In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 comprises SEQ ID NO: 16. In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 consists of SEQ ID NO: 16 .

In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 is characterized by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% amino acid sequence identity with respect to SEQ ID NO: 17. In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 comprises an amino acid sequence selected from the group consisting of (i) SEQ ID NO : 17, and/or (ii) sequences in which up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acids according to SEQ ID NO: 17 are replaced by any amino acid. In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 comprises SEQ ID NO: 17. In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 consists of SEQ ID NO: 17 .

In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 is characterized by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% amino acid sequence identity with respect to SEQ ID NO: 16, and one of these engineered ankyrin repeat domains with binding specificity for HER2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 17. In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 comprises the amino acid sequence selected from the group consisting of (i) SEQ ID NO: 16, and/or (ii) sequences in which up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids according to SEQ ID NO: 16 any amine is replaced acid, and one of these engineered ankyrin repeat domains with binding specificity for HER2 contains an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 17, and/or (ii) sequences in which up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acids according to SEQ ID NO: 17 are replaced by any amino acid. In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 comprises SEQ ID NO: 16, and one of said engineered ankyrin repeat domains with binding specificity for HER2 comprises SEQ ID NO: 17. In one embodiment, one of said engineered ankyrin repeat domains with binding specificity for HER2 consists of SEQ ID NO: 16, and one of said engineered ankyrin repeat domains with binding specificity for HER2 consists of SEQ ID NO: 17. In one embodiment, said recombinant binding protein contains SEQ ID NO: 16 at the N-terminus of SEQ ID NO: 17.

In one embodiment, said recombinant binding protein comprises an amino acid sequence that is characterized by 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity at in relation to SEQ ID NO: 32. According to one embodiment, said recombinant binding protein comprises an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 32 and/or (ii) sequences in which up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acids according to SEQ ID NO: 32 are replaced by any amino acid.

In one embodiment, the present invention provides a recombinant binding protein comprising four engineered ankyrin repeat domains, wherein two of said four engineered ankyrin repeat domains are engineered ankyrin repeat domains with binding specificity for HER2, consisting of SEQ ID NO: 16 and 17, wherein two of said four engineered ankyrin repeat domains are engineered ankyrin repeat domains with binding specificity for serum albumin, and wherein each of said two engineered ankyrin repeat domains with binding specificity for serum albumin are composed of SEQ ID NO: 14.

In one embodiment, the present invention provides a recombinant binding protein comprising SEQ ID NO: 32 and twice SEQ ID NO: 14. In one embodiment, the present invention relates to a recombinant binding protein comprising SEQ ID NO: 32 and twice SEQ ID NO: 14, wherein SEQ ID NO: 32 is flanked by one SEQ ID NO: 14 at the N-terminus and one SEQ ID NO: 14 at the C-terminus.

In one embodiment, the polypeptide linkers connecting engineered domains to ankyrin repeats present in the recombinant binding protein of the present invention comprise amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NO: 2-9, more preferably SEQ ID NO: 3 -9, more preferably SEQ ID NO: 4-9, more preferably SEQ ID NO: 6 or 9, more preferably SEQ ID NO: 9, in which up to 4, 3, 2, 1.0 amino acids from the specified SEQ ID NO : 2-9, more preferably SEQ ID NO: 3-9, more preferably SEQ ID NO: 4-9, more preferably SEQ ID NO: 6 or 9, more preferably SEQ ID NO: 9 is replaced by any amino acid. In one embodiment, the flanking N-terminal Gly Ser of any of SEQ ID NOs: 7-9 and/or the flanking C-terminal Gly Ser of any of SEQ ID NOs: 2-6 are optionally absent. According to one embodiment, any of SEQ ID NO: 2-9 optionally additionally contains Arg Ser at the C-terminus (as, for example, shown in SEQ ID NO: 29). In one embodiment, said polypeptide linkers comprise an amino acid sequence selected from any of the amino acid sequences of SEQ ID NO: 2-9, more preferably SEQ ID NO: 3-9, more preferably SEQ ID NO: 4-9, more preferably SEQ ID NO: : 6 or 9, more preferably SEQ ID NO: 9. In one embodiment, the polypeptide linkers connecting the engineered domains to the ankyrin repeats present in the recombinant binding protein of the present invention consist of an amino acid sequence selected from any of the amino acid sequences of SEQ ID NO : 2-9, more preferably SEQ ID NO: 3-9, more preferably SEQ ID NO: 4-9, more preferably SEQ ID NO: 6 or 9, more preferably SEQ ID NO: 9. In one embodiment, each of the polypeptide linkers connecting the engineered domains to ankyrin repeats present in the recombinant binding protein of the present invention consists of SEQ ID NO: 9. In one embodiment, said polypeptide linkers present in the recombinant binding protein of the present invention are 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% identical, preferably identical. In one embodiment, each of said two engineered ankyrin repeat domains with binding specificity for serum albumin consists of the amino acid sequence according to SEQ ID NO: 14, and said four engineered ankyrin repeat domains are linked via polypeptide linkers, each consisting of amino acid sequence according to SEQ ID NO: 9.

According to one embodiment, the present invention provides a recombinant binding protein comprising, in N-terminal to C-terminal direction: SEQ ID NO: 14 - SEQ ID NO: 16 - SEQ ID NO: 17 - SEQ ID NO: 14, connected using polypeptide linkers. According to one embodiment, the present invention relates to a recombinant binding protein comprising in N-terminal to C-terminal direction: SEQ ID NO: 14 - SEQ ID NO: 9 - SEQ ID NO: 16 - SEQ ID NO: 9 - SEQ ID NO: 17 - SEQ ID NO: 9 - SEQ ID NO: 14.

In one embodiment, the present invention provides a recombinant binding protein comprising an amino acid sequence that is characterized by at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% amino acid sequence identity with respect to a recombinant binding protein consisting of SEQ ID NO: 21. According to one embodiment, the present invention relates to a recombinant binding protein containing an amino acid sequence selected from the group consisting of ( i) SEQ ID NO: 21 and/or (ii) amino acid sequences up to 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 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 according to SEQ ID NO: 21 are replaced by others mi amino acids. In one embodiment, the present invention provides a recombinant binding protein consisting of an amino acid sequence that is characterized by at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100% amino acid sequence identity with respect to a recombinant binding protein consisting of SEQ ID NO: 21. According to one embodiment, the present invention relates to a recombinant binding protein consisting of an amino acid sequence selected from the group consisting of from (i) SEQ ID NO: 21 and (ii) amino acid sequences up to 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 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 according to SEQ ID NO: 21 are replaced by others mi amino acids. Preferably, said amino acid substitutions in SEQ ID NO: 21 are located at positions 127-444 according to SEQ ID NO: 21.

In one embodiment, the present invention provides a recombinant binding protein comprising an amino acid sequence that is characterized by at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% amino acid sequence identity with respect to a recombinant binding protein consisting of SEQ ID NO: 21, wherein said recombinant binding protein contains two engineered ankyrin repeat domains with binding specificity for serum albumin, and each of these two engineered ankyrin repeat domains with binding specificity for serum albumin contains the amino acid sequence of SEQ ID NO: 14. In one embodiment, the present invention provides a recombinant binding protein comprising an amino acid sequence that is characterized by at least 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to a recombinant binding protein consisting of SEQ ID NO: 21, wherein said recombinant binding protein contains two engineered domains with ankyrin repeats with binding specificity for serum albumin, and wherein each of said two engineered ankyrin repeat domains with binding specificity for serum albumin consists of the amino acid sequence of SEQ ID NO: 14. In one embodiment, the present invention relates to a recombinant binding protein , containing an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 21 and/or (ii) amino acid sequences, in which up to 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 3 1, 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 according to SEQ ID NO: 21 are replaced by other amino acids, wherein said recombinant binding protein contains two engineered ankyrin repeat domains with binding specificity for serum albumin, and each of said two of engineered ankyrin repeat domains with binding specificity for serum albumin comprises the amino acid sequence of SEQ ID NO: 14. In one embodiment, the present invention provides a recombinant binding protein comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 21 and/or (ii) amino acid sequences in which up to 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 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 according to SEQ ID NO: 21 are replaced by other amino acids, wherein said recombinant binding protein contains two engineered ankyrin repeat domains with binding specificity for serum albumin, and wherein each of said two engineered ankyrin repeat domains with binding specificity for serum albumin consists of the amino acid sequence of SEQ ID NO: 14.

In one embodiment, the present invention relates to a recombinant binding protein comprising the amino acid sequence consisting of SEQ ID NO: 21. In one embodiment, the present invention relates to a recombinant binding protein comprising the amino acid sequence according to SEQ ID NO: 21. Preferred is a recombinant binding protein. a protein containing the amino acid sequence according to SEQ ID NO: 21. Preferred is SEQ ID NO: 21. Preferred is a recombinant binding protein, wherein the amino acid sequence is SEQ ID NO: 21. Preferred is a protein in which the amino acid sequence is SEQ ID NO: : 21. Preferred is a recombinant binding protein consisting of SEQ ID NO: 21. Preferred is a recombinant binding protein consisting of the amino acid sequence according to SEQ ID NO: 21.

Many features make the protein encoded by SEQ ID NO: 21 the preferred recombinant binding protein of the present invention. It contains two engineered ankyrin repeat domains with binding specificity for HER2 and two engineered ankyrin repeat domains with binding specificity for serum albumin, each of said two engineered ankyrin repeat domains with binding specificity for serum albumin consisting of SEQ ID NO: 14. Two engineered ankyrin repeat domains with binding specificity for HER2 bind to HER2 on two different epitopes. It is the first recombinant binding protein to combine serum albumin binding and biparatopic binding to HER2. The engineered ankyrin repeat domain according to SEQ ID NO: 14 shows improved storage stability properties (see Example 2 and 3; Figure 2) compared to known engineered ankyrin repeat domains with binding specificity for serum albumin. These two engineered ankyrin repeat domains with binding specificity for serum albumin surprisingly result in improved pharmacokinetic properties of the recombinant binding protein compared to the corresponding recombinant binding protein having only one engineered ankyrin repeat domain with binding specificity for serum albumin (Example 11, figures 5). When two engineered ankyrin repeat domains with binding specificity for serum albumin flank other engineered ankyrin repeat domains with binding specificity for HER2, better pharmacokinetic properties are observed (Example 11). The choice of engineered ankyrin repeat domains with binding specificity for HER2 as well as their structural arrangement results in maximum compound activity (examples 5, 6 and 7). The engineered ankyrin repeat domains are coupled using a PT-enriched linker, which unexpectedly results in improved pharmacokinetic properties (examples 10 and 11). The molecules combine the functionality of at least two drugs (eg, trastuzumab and pertuzumab) in one molecule, and additionally can induce apoptosis in HER2 expressing cells (example 6, figure 7).

In one embodiment, said recombinant binding protein of SEQ ID NO: 21 exhibits improved storage stability compared to the corresponding recombinant binding protein of SEQ ID NO: 22. In one embodiment, said recombinant binding protein of SEQ ID NO: 21, shows reduced amounts of degradation products after storage at 40°C for 1 month at a concentration of 10 mg/ml in PBS compared to the corresponding recombinant binding protein consisting of SEQ ID NO: 22.

In one embodiment, said recombinant binding protein of SEQ ID NO: 21 exhibits improved pharmacokinetic properties compared to a recombinant binding protein of SEQ ID NO: 21 wherein a C-terminal engineered ankyrin repeat domain with binding specificity in serum albumin was removed. In one embodiment, said recombinant binding protein of SEQ ID NO: 21 exhibits improved pharmacokinetic properties compared to a recombinant binding protein of amino acids 1-422 of SEQ ID NO: 21. In one embodiment, said recombinant binding protein, consisting of SEQ ID NO: 21 exhibits improved pharmacokinetic properties compared to the recombinant binding protein consisting of SEQ ID NO: 33.

In one embodiment, each of said engineered ankyrin repeat domains with binding specificity for serum albumin binds mouse, rat, dog, cynomolgus or human serum albumin, more preferably mouse, cynomolgus or human serum albumin, more preferably cynomolgus monkey serum albumin. or human, more preferably human serum albumin, in PBS with a dissociation constant (Kd) below 10 ^-5 M; preferably below 10 ^-6 M; or more preferably below 10 ^-7 M. In one embodiment, each of said engineered ankyrin repeat domains with binding specificity for serum albumin consists of SEQ ID NO: 14 and binds human serum albumin in PBS with a dissociation constant (Kd) below 10 ^-5 M; preferably below 10 ^-6 M; or more preferably below 10 ^-7 M. Examples of the determination of the dissociation constant using surface plasmon resonance are given in example 5 and in international patent publication WO 2014/083208.

In one embodiment, each of said engineered ankyrin repeat domains with binding specificity for HER2 binds to human HER2 in PBS with a dissociation constant (Kd) below 10 ^-6 M; preferably below 10 ^-7 M; more preferably below 10 ^-8 M or more preferably below 10 ^-9 M. In one embodiment, said engineered ankyrin repeat domain with binding specificity for HER2, consisting of SEQ ID NO: 16, binds to human HER2 in PBS with a dissociation constant (Kd) below 10 ^-6 M; preferably below 10 ^-7 M; more preferably below 10 ^-8 M, more preferably below 10 ^-9 M, more preferably below 10 ^-10 M, or more preferably below 10 ^-11 M. In one embodiment, said engineered ankyrin repeat domain with binding specificity for HER2, consisting from SEQ ID NO: 17, binds to human HER2 in PBS with a dissociation constant (Kd) below 10 ^-6 M; preferably below 10 ^-7 M; more preferably below 10 ^-8 M or more preferably below 10 ^-9 M.

In one embodiment, said recombinant binding protein binds to human HER2 in PBS with a dissociation constant (Kd) below 10 ^-7 M; preferably below 10 ^-8 M; more preferably below 10 ^-9 M or more preferably below 10 ^-10 M. In one embodiment, said recombinant binding protein with binding specificity for HER2 comprising SEQ ID NO: 32 is bound to human serum albumin in PBS with a dissociation constant (Kd ) below 10 ^-8 M; preferably below 10 ^-9 M or more preferably below 10 ^-10 M. In one embodiment, said recombinant binding protein with binding specificity for HER2, comprising SEQ ID NO: 21, binds to human HER2 in PBS with a dissociation constant (Kd) below 10 ^-7 M; preferably below 10 ^-8 M; more preferably below 10 ^-9 M or more preferably below 10 ^-10 M. In one embodiment, said recombinant binding protein with binding specificity for HER2, consisting of SEQ ID NO: 21, binds to human HER2 in PBS with a dissociation constant (Kd ) below 10 ^-7 M; preferably below 10 ^-8 M; more preferably below 10 ^-9 M or more preferably below 10 ^-10 M. In one embodiment, said recombinant binding protein binds to human serum albumin with a dissociation constant (Kd) below 10 ^-5 M, preferably below 10 ^-6 M or more preferably below 10 ^-7 M. In one embodiment, said recombinant binding protein with binding specificity for HER2, comprising SEQ ID NO: 21, binds to human serum albumin in PBS with a dissociation constant (Kd) below 10 ^-5 M; preferably below 10 ^-6 M or more preferably below 10 ^-7 M. In one embodiment, said recombinant binding protein with binding specificity for HER2, consisting of SEQ ID NO: 21, binds to human serum albumin in PBS with a dissociation constant (Kd ) below 10 ^-5 M; preferably below 10 ^-6 M or more preferably below 10 ^-7 M.

In one embodiment, said recombinant binding protein inhibits cell proliferation of HER2-expressing cells with an inhibition constant (IC ₅₀ ) below 10 ^-6 M; preferably below 10 ^-7 M or more preferably below 10 ^-8 M. Examples of such cells include BT474, SKBR-3, SKOV-3, NCI-N87, ZR-75-30, HCC1419 or MDA-MB175 cells. In one embodiment, said recombinant binding protein inhibits proliferation of BT474 cells with an inhibition constant (IC ₅₀ ) below 10 ^-6 M; preferably below 10 ^-7 M or more preferably below 10 ^-8 M. In one embodiment, the recombinant binding protein comprising SEQ ID NO: 32 inhibits proliferation of BT474 cells with an inhibition constant (IC ₅₀ ) below 10 ^-6 M; preferably below 10 ^-7 M or more preferably below 10 ^-8 M. In one embodiment, the recombinant binding protein comprising SEQ ID NO: 21 inhibits proliferation of BT474 cells with an inhibition constant (IC ₅₀ ) below 10 ^-6 M; preferably below 10 ^-7 M or more preferably below 10 ^-8 M. According to one embodiment, the recombinant binding protein consisting of SEQ ID NO: 21 inhibits the proliferation of BT474 cells with an inhibition constant (IC ₅₀ ) below 10 ^-6 M; preferably below 10 ^-7 M or more preferably below 10 ^-8 M. Cell inhibition assays are well known in the art. In one embodiment, said recombinant binding protein of SEQ ID NO: 21 inhibits BT474 cell proliferation more than a recombinant binding protein having only one engineered ankyrin repeat domain with binding specificity for HER2. In one embodiment, said recombinant binding protein of SEQ ID NO: 21 inhibits proliferation of BT474 cells without the aid of antibody-dependent cell-mediated cytotoxicity (ADCC; well known to those skilled in the art). In one embodiment, said recombinant binding protein of SEQ ID NO: 21 inhibits proliferation of BT474 cells without the aid of an IgG1-Fc fragment.

In one embodiment, said recombinant binding protein comprising, preferably consisting of SEQ ID NO: 21, induces apoptosis in BT474 cells. In one embodiment, said recombinant binding protein comprising, preferably consisting of SEQ ID NO: 21, induces apoptosis in BT474 cells with a half maximum effective concentration (EC ₅₀ ) value of less than 100 nM. Preferably, said recombinant binding protein induces apoptosis in BT474 cells with an EC ₅₀ value of less than 90, 80, 70, 60, 50, 40, 30, 20, or 10 nM. Preferably, said recombinant binding protein at 100 nM induces apoptosis in BT474 to a greater extent than 100 nM trastuzumab, 100 nM pertuzumab, or a mixture of 100 nM trastuzumab and 100 nM pertuzumab. In one embodiment, said recombinant binding protein comprising, preferably consisting of SEQ ID NO: 21, induces apoptosis in HER2 expressing cells.

In one embodiment, said recombinant binding protein comprising SEQ ID NO: 21 inhibits the RAS pathway. In one embodiment, HER2 in BT474 is less phosphorylated when treated with said recombinant binding protein comprising SEQ ID NO: 21 than when treated with trastuzumab.

In one embodiment, said recombinant binding protein is capable of binding to HER2 and human serum albumin simultaneously. In one embodiment, said recombinant binding protein is capable of binding to two molecules of human serum albumin simultaneously. Such simultaneous binding can be demonstrated using surface plasmon resonance experiments (example 5) or size exclusion chromatography combined with static light scattering techniques (example 14), well known to the person skilled in the art.

In one embodiment, said recombinant binding protein binds to HER2 in a biparatopic fashion. In one embodiment, said recombinant binding protein binds to HER2 at two different epitopes.

In one embodiment, the present invention provides a recombinant binding protein comprising first, second, third, and fourth engineered ankyrin repeat domains, wherein said first and second engineered ankyrin repeat domains each have a binding specificity for HER2, and each of said third and fourth engineered ankyrin repeat domains are characterized by binding specificity for serum albumin. Preferably, said recombinant binding protein consists of a single polypeptide chain. More preferably, said first, second, third and fourth engineered ankyrin repeat domains are linked via polypeptide linkers. SEQ ID NO: 21 is an example of such a recombinant binding protein.

In one embodiment, said first engineered ankyrin repeat domain with binding specificity for HER2 binds to domain II of HER2. In one embodiment, said second engineered ankyrin repeat domain with binding specificity for HER2 binds to domain IV of HER2. In one embodiment, said first engineered ankyrin repeat domain with binding specificity for HER2 binds to HER2 domain II, and said second engineered ankyrin repeat domain with binding specificity for HER2 binds to domain IV of HER2. In one embodiment, said first engineered ankyrin repeat domain with binding specificity for HER2 comprises SEQ ID NO: 16. In one embodiment, said second engineered ankyrin repeat domain with binding specificity for HER2 comprises SEQ ID NO: 17. In one embodiment, in an embodiment, said first engineered ankyrin repeat domain with binding specificity for HER2 comprises SEQ ID NO: 16, and said second engineered ankyrin repeat domain with binding specificity for HER2 comprises SEQ ID NO: 17.

In one embodiment, said first engineered ankyrin repeat domain with binding specificity for HER2, comprising SEQ ID NO: 16, binds to human HER2 in PBS with a dissociation constant (Kd) below 10 ^-6 M; preferably below 10 ^-7 M; more preferably below 10 ^-8 M, more preferably below 10 ^-9 M, more preferably below 10 ^-10 M, or more preferably below 10 ^-11 M. In one embodiment, said second engineered ankyrin repeat domain with binding specificity for HER2, containing SEQ ID NO: 17, binds to human HER2 in PBS with a dissociation constant (Kd) below 10 ^-6 M; preferably below 10 ^-7 M; more preferably below 10 ^-8 M or more preferably below 10 ^-9 M.

In one embodiment, each of said third and fourth engineered ankyrin repeat domains with binding specificity for serum albumin binds to human serum albumin in PBS with a dissociation constant (Kd) below 10 ^-5 M, preferably below 10 ^-6 M, or more preferably below 10 ^-7 M. In one embodiment, each of said third and fourth engineered ankyrin repeat domains with binding specificity for serum albumin, comprising SEQ ID NO: 14, binds to human serum albumin in PBS with a dissociation constant (Kd) below 10 ^-5 M; preferably below 10 ^-6 M or more preferably below 10 ^-7 M.

The terms "first", "second", "third", and optionally "fourth" as used in the phrases "first engineered ankyrin repeat domain", "second engineered ankyrin repeat domain", "third engineered ankyrin repeat domain", and "fourth engineered ankyrin repeat domain" ankyrin repeat domain" do not indicate or imply any positional arrangement of said engineered ankyrin repeat domains within the recombinant binding protein. In one embodiment, said four engineered ankyrin repeat domains are N-terminal to C-terminal: first - second - fourth.

In one embodiment, the recombinant binding protein inhibits proliferation of BT474 cells in a mouse xenograft tumor model. In one embodiment, the recombinant binding protein inhibits tumor growth in a patient-derived xenograft expressing HER2 cancer model in mice. In one embodiment, the recombinant binding protein inhibits tumor growth in a patient-derived HER2-expressing xenograft model of gastric cancer in mice. The term "patient-derived HER2-expressing xenograft" means a xenograft of patient-derived cancer tissue, wherein at least one cell in said tissue expresses HER2 above background.

According to any embodiment of the present invention relating to an engineered ankyrin repeat domain or recombinant binding protein comprising an amino acid sequence that is characterized by at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or 100% amino acid sequence identity with respect to a given amino acid sequence, non-identical amino acids can be located at any position of the engineered ankyrin repeat domain or recombinant binding protein.

Similarly, according to any embodiment of the present invention relating to an engineered ankyrin repeat domain or recombinant binding protein, wherein up to 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 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 replaced by any amino acid, the replaced amino acids can be located at any position of the engineered ankyrin repeat domain. Techniques for modifying, for example by point mutation, a recombinant binding protein of the present invention are well known to those skilled in the art. Starting with SEQ ID NO: 21, one skilled in the art knows how and where to replace amino acids (e.g., using the knowledge disclosed in International Patent Publication WO 2002/020565) to generate high probability sequence variants according to SEQ ID NO: 21 unchanged functional activity.

In one embodiment, said recombinant binding protein exhibits an increase in terminal half-life, preferably an increase in terminal half-life of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%, compared to the corresponding recombinant binding protein lacking said fourth engineered ankyrin repeat domain with binding specificity for serum albumin. In one embodiment, said recombinant binding protein exhibits an increase in terminal half-life, preferably an increase in terminal half-life of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% by compared with the corresponding recombinant binding protein containing only one engineered ankyrin repeat domain with binding specificity for serum albumin. Examples of such an increase in the terminal half-life are presented in example 11.

In one embodiment, said recombinant binding protein, comprising, preferably consisting of, SEQ ID NO: 21, exhibits a terminal half-life of approximately 47 hours at 1 mg/kg in cynomolgus monkey. In one embodiment, said recombinant binding protein comprising, preferably consisting of, SEQ ID NO: 21 exhibits a terminal half-life of approximately 100 hours at 5 mg/kg in cynomolgus monkey. In one embodiment, said recombinant binding protein comprising, preferably consisting of, SEQ ID NO: 21 exhibits a terminal half-life of greater than 100 hours at 10 mg/kg in cynomolgus monkey. In one embodiment, the recombinant binding protein comprising, preferably consisting of, SEQ ID NO: 21 exhibits a terminal half-life of greater than 100 hours at 100 mg/kg in cynomolgus monkey.

In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of an engineered ankyrin repeat domain or a recombinant binding protein of the present invention, preferably a recombinant binding protein of the present invention. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of a recombinant binding protein of the present invention. According to one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence consisting of SEQ ID NO: 21. According to one embodiment, the present invention relates to a nucleic acid encoding said recombinant binding protein. According to one embodiment, the present invention relates to a nucleic acid encoding a recombinant binding protein consisting of SEQ ID NO: 21. Moreover, the present invention relates to vectors containing any nucleic acid according to the present invention. Nucleic acids are well known to the skilled person. In the examples, nucleic acids were used to generate engineered ankyrin repeat domains or recombinant binding proteins of the present invention in E. coli.

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

According to one embodiment, the present invention provides 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.

Pharmaceutically acceptable carriers and/or diluents are known to those skilled in the art and are explained in more detail below. In addition, a diagnostic composition is provided containing one or more of the recombinant binding proteins and/or engineered ankyrin repeat domains mentioned above and/or nucleic acids, in particular recombinant binding proteins and/or nucleic acids.

The pharmaceutical composition contains a recombinant binding protein and/or an engineered ankyrin repeat domain and/or a nucleic acid, preferably a recombinant binding protein and/or nucleic acid as described herein, and a pharmaceutically acceptable carrier, excipient or stabilizer, for example, as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980. Suitable carriers, excipients or stabilizers known to those skilled in the art are sodium chloride aqueous solution, Ringer's solution, dextrose solution, Hank's solution , fixed oils, ethyl oleate, 5% dextrose in aqueous sodium chloride solution, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not by itself induce the production of antibodies harmful to the individual receiving the composition, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition may also be a combination formulation containing an additional active agent, such as an anticancer agent or an antiangiogenic agent, or an additional biologically active compound.

One embodiment of the present invention relates to the use of a recombinant binding protein of the present invention comprising two engineered ankyrin repeat domains with binding specificity for serum albumin for the manufacture of a pharmaceutical composition, said recombinant binding protein exhibiting an increased terminal half-life, preferably a terminal half-life , increased by at least 5%, preferably by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or 250% compared to the corresponding recombinant binding protein containing only one engineered ankyrin repeat domain with binding specificity for serum albumin.

In one embodiment, the pharmaceutical composition comprises at least one recombinant binding protein as described herein and a detergent such as a non-ionic detergent, a buffer such as phosphate, and a sugar such as sucrose. In one embodiment, such a composition contains recombinant binding proteins as described above and PBS.

According to one embodiment, the present invention relates to the use of a pharmaceutical composition or a recombinant binding protein of the present invention for the treatment of a disease. To this end, the pharmaceutical composition or recombinant binding protein of the present invention is administered to a patient in need thereof in a therapeutically effective amount. Administration may include topical administration, oral administration, and parenteral administration. A typical route of administration is parenteral administration. When administered parenterally, the pharmaceutical composition of the present invention will be formulated into a unit dosage injectable form such as a solution, suspension or emulsion, together with pharmaceutically acceptable excipients as defined above. The dosage and route of administration will depend on the individual being treated and the particular disease.

In addition, any of the aforementioned pharmaceutical composition or recombinant binding protein is provided for the treatment of a disorder.

In one embodiment, said recombinant binding protein or such other pharmaceutical composition as described herein is administered intravenously. For parenteral use, the recombinant binding protein or said pharmaceutical composition may be administered by injection as a bolus injection or by slow infusion in a therapeutically effective amount.

In one embodiment, the present invention relates to a method for treating a medical condition, the method comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of a recombinant binding protein of the present invention. In one embodiment, the present invention relates to a method for treating a medical condition, the method comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition of the present invention. Example 7 (Figure 4) illustrates the applicability of using a recombinant binding protein containing SEQ ID NO: 21 for the treatment of cancer. According to one embodiment, the present invention relates to the use of a pharmaceutical composition according to the present invention for the treatment of a disease. According to one embodiment, the present invention relates to a pharmaceutical composition for use in the treatment of a disease. According to one embodiment, the present invention relates to a pharmaceutical composition for use in the treatment of a medical condition.

According to one embodiment, the present invention relates to a recombinant binding protein consisting of SEQ ID NO: 21 for use as a drug. According to one embodiment, the present invention relates to a recombinant binding protein consisting of SEQ ID NO: 21 for use in the treatment of a disease. In one embodiment, said pharmaceutical composition, recombinant binding protein or nucleic acid molecule is provided for use in the treatment of a disease. In one embodiment, the present invention relates to a medicament containing said pharmaceutical composition, recombinant binding protein or nucleic acid molecule. According to one embodiment, the present invention relates to the use of a recombinant binding protein consisting of SEQ ID NO: 21 in a pharmaceutical composition for the treatment of a disease. According to one embodiment, the present invention relates to said pharmaceutical composition for use in the treatment of a disease. In one embodiment, the present invention relates to said recombinant binding protein for use in the treatment of a disease. According to one embodiment, the present invention relates to said nucleic acid for use in the treatment of a disease. According to one embodiment, the present invention relates to the use of said pharmaceutical composition, recombinant binding protein or nucleic acid molecule as a drug for the treatment of a disease. According to one embodiment, the present invention relates to the use of said pharmaceutical composition, recombinant binding protein or nucleic acid molecule for the treatment of a disease. According to one embodiment, the present invention relates to the use of said pharmaceutical composition, recombinant binding protein or nucleic acid molecule for the manufacture of a medicament. According to one embodiment, the present invention relates to the use of said pharmaceutical composition, recombinant binding protein or nucleic acid molecule for the manufacture of a medicament for the treatment of a disease. According to one embodiment, the present invention relates to a method for the manufacture of a medicament for the treatment of a disease, wherein said pharmaceutical composition, recombinant binding protein, or nucleic acid molecule is the active ingredient of the medicament. According to one embodiment, the present invention relates to a method for treating a disease using said pharmaceutical composition, recombinant binding protein, or nucleic acid molecule.

et al., 2012. Modern Pathology 25, 637-650; Wolff et al., 2013. J. Clin. Oncol. 31 (31), 3997-4014).The term "medical condition" (or disorder or disease) includes autoimmune disorders, inflammatory disorders, retinopathies (especially proliferative retinopathies), neurodegenerative disorders, infections, metabolic diseases, and neoplastic diseases. Any of the recombinant binding proteins described herein can be used to produce a medicament for the treatment of such a disorder, in particular a disorder selected from the group consisting of the following: autoimmune disorder, inflammatory disorder, retinopathy, and neoplastic disease. A "medical condition" may be a condition that is characterized by inappropriate cell proliferation. The medical condition may be a hyperproliferative condition. In particular, the present invention relates to a method for treating a medical condition, the method comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of a recombinant binding protein or said pharmaceutical composition according to the present invention. In a preferred embodiment, said medical condition is a neoplastic disease. As used herein, the term "neoplastic disease" refers to a pathological condition or condition of cells or tissue characterized by the growth of rapidly proliferating cells or neoplasm. In one embodiment, said medical condition is a malignant neoplastic disease. In one embodiment, said medical condition is cancer. In one embodiment, said medical condition refers to the following: HER2 expressing cancer, HER2 addictive cancer, HER2 partial addictive cancer, HER2 overexpressing cancer, HER2 amplifying cancer, trastuzumab-resistant cancer, trastuzumab-responsive cancer. In one embodiment, said medical condition is breast cancer and/or gastrointestinal cancer and/or brain cancer. In one embodiment, said medical condition is breast cancer, ovarian cancer, gastric cancer, gastric cancer, uterine cancer, colon and rectal cancer, bladder cancer, and/or HER2 overexpressing cancer. The term "brain cancer" may refer to brain metastasis of HER2 overexpressing cancer, brain metastasis of HER2 amplifying cancer, HER2 overexpressing brain cancer, or HER2 amplifying brain cancer. The term "cancer of the gastrointestinal tract" may refer to stomach cancer, esophageal cancer, colon and rectal cancer, bile duct cancer, bladder cancer, or pancreatic adenocarcinoma. The term "therapeutically effective amount" means an amount that is sufficient to produce the desired effect on the patient. In one embodiment, said medical condition is cancer, wherein said cancer cells exhibit levels of HER2 expression above baseline as determined by IHC. In one embodiment, said medical condition is cancer, wherein said cancer cells exhibit levels of HER2 expression above levels of HER2 expression of healthy cells in their environment, as determined by IHC. Such levels of HER2 expression above background are well known to those skilled in the art, for example from assays such as HercepTest® (Dako). Preferably, HER2 expression levels above baseline refer to a HercepTest® score of 1+, 2+ or 3+, more preferably 2+ or 3+. Overexpression of HER2 is well known to those skilled in the art (

et al., 2012. Modern Pathology 25, 637-650; Wolff et al., 2013. J. Clin. oncol. 31(31), 3997-4014).

In particular, the present invention relates to the treatment of a medical condition using the pharmaceutical composition of the present invention, said medical condition being cancer.

The use of the recombinant binding protein of the present invention or said pharmaceutical compositions for the treatment of cancer may also be carried out in combination with one or more other therapies known in the art. Used in this document, the term "use in combination with" should refer to the joint introduction, which is carried out according to this scheme. This includes the simultaneous administration of different compounds, as well as the administration of different compounds with a time shift (for example, compound A is administered once and compound B is administered several times thereafter, or vice versa, or both compounds are administered simultaneously and one of the two is also administered on later stages). Examples of compounds that can, for example, be co-administered include the following: taxanes, anthracyclines, platinum-based chemotherapy drugs, 5-FU, PI3K inhibitors (such as, for example, apitolisib, taselisib or alpelisib), MEK inhibitors (such as, for example, , trametinib or cobimetinib), RAS inhibitors (such as, for example, salirazib), RAF inhibitors, mTOR inhibitors (including, for example, apitolisib and everolimus), EGFR inhibitors of any form, microtubule inhibitors (including eribulin), targeted therapies, including cell cycle kinase inhibitors (such as, for example, palbociclib), HER2 inhibitors, trastuzumab, trastuzumab-DM1, pertuzumab, cetuximab, panitumumab, nimotuzumab, bevacizumab, ranibizumab, MP0250, ipilimumab, pembrolizumab, nivolumab, urelumab, utolimumab, or atezolizumab . Preferably, these compounds are used at the recommended dosages. In one embodiment, said recombinant binding protein comprising SEQ ID NO: 21 potentiates the action of PI3K inhibitors, mTOR inhibitors, eribulin, trastuzumab, or lapatinib. In one embodiment, said recombinant binding protein comprising SEQ ID NO: 21 will allow lower doses of PI3K inhibitors, mTOR inhibitors, eribulin, trastuzumab, or lapatinib to be used to treat a disease. Examples of such combinations are presented in example 17.

In a further embodiment, the present invention relates to the use of a recombinant binding protein of the present invention for the manufacture of a medicament that is used to treat a medical condition, preferably a neoplastic disease, more preferably cancer.

According to one embodiment, the present invention relates to the use of a pharmaceutical composition according to the present invention for the manufacture of a medicament that is used to treat a medical condition, which may be a neoplastic disease, in particular cancer.

Formulations to be used for in vivo administration must be aseptic or sterile. This is easily accomplished by filtration through sterile filter membranes.

According to one embodiment, the present invention relates to a recombinant binding protein containing any of the aforementioned repeat domains.

According to one embodiment, the present invention provides a kit containing said recombinant binding protein. In one embodiment, the present invention relates to a kit containing a nucleic acid encoding said recombinant binding protein. According to one embodiment, the present invention relates to a kit containing the specified pharmaceutical composition. According to one embodiment, the present invention provides a kit containing said recombinant binding protein and/or a nucleic acid encoding said recombinant binding protein and/or said pharmaceutical composition. According to one embodiment, the present invention provides a kit comprising a recombinant binding protein comprising, preferably consisting of SEQ ID NO: 21 and/or a nucleic acid encoding a recombinant binding protein comprising, preferably consisting of SEQ ID NO: 21, and/ or a pharmaceutical composition containing a recombinant binding protein containing, preferably consisting of SEQ ID NO: 21, and/or a nucleic acid encoding a recombinant binding protein containing, preferably consisting of SEQ ID NO: 21.

According to one embodiment, the present invention relates to a method for producing a recombinant binding protein according to the present invention. In one embodiment, the present invention relates to a method for producing a recombinant binding protein comprising, preferably consisting of, SEQ ID NO: 21, the method comprising the steps of (i) expressing said recombinant binding protein in bacteria, and (ii) purifying said recombinant binding protein using chromatography. Said method may include additional steps. Such a method for obtaining a recombinant binding protein according to the present invention is presented in example 1.

The present invention is not limited to the specific embodiments described in the examples. Other sources may be used and processed following the general principles described below.

A number of documents are cited in the present description of the invention. The disclosure of the contents of these documents is incorporated herein by reference.

The present disclosure relates to a number of amino acid sequences from the amino acid sequence listing of the present disclosure titled "P015_Sequence_Listing.txt", and the amino acid sequences from the sequence protocol are incorporated herein by reference.

DEFINITIONS

In the context of the present invention, the term "protein" refers to a polypeptide, wherein at least a portion of the polypeptide has or is capable of acquiring a specific three-dimensional arrangement by forming secondary, tertiary or quaternary structures within a single polypeptide chain and/or between several polypeptide chains. If the protein contains two or more polypeptide chains, the individual polypeptide chains may be linked non-covalently or covalently, for example, by a disulfide bond between the two polypeptides. The portion of a protein that individually has or is capable of acquiring a particular three-dimensional arrangement by forming a secondary or tertiary structure is referred to as a "protein domain". Such protein domains are well known to those skilled in the art.

The term "recombinant" as used in a recombinant protein, recombinant protein domain, recombinant binding protein, and the like, means that said polypeptides are produced using recombinant DNA techniques well known to those skilled in the art. For example, a recombinant DNA molecule (e.g., derived from gene synthesis) encoding a polypeptide 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 a DNA that allows expression in vitro. If, for example, such a recombinant bacterial expression plasmid is inserted into suitable bacteria (eg, Escherichia coli), these bacteria can produce the polypeptide encoded by the recombinant DNA. Accordingly, the polypeptide produced is referred to as a recombinant polypeptide or recombinant protein.

In the context of the present invention, the term "binding protein" refers to a protein containing two or more, preferably three or more, more preferably four or more binding domains. Preferably said binding protein is a recombinant binding protein. Preferably, said binding protein contains four repeat domains. More preferably, said binding protein contains four engineered ankyrin repeat domains. Preferably, each of the two of said binding domains of said binding protein has a target specificity for serum albumin. Also preferably, each of the two binding domains of the specified binding protein is characterized by target specificity for HER2.

Moreover, any such binding protein may contain additional polypeptides (such as, for example, polypeptide tags, polypeptide linkers, fusion with other protein binding domains, cytokines, hormones, or antagonists) or chemical modifications (such as combination with polyethylene glycol, toxins (for example, DM1 from Immunogen), small molecules, antibiotics, and the like) are well known to those skilled in the art.

, A., 2005. Nat. Biotech. 23, 1257-1268). Например, связывающий домен, характеризующийся предварительно определенным свойством, можно получить с помощью способа, предусматривающего следующие стадии: (а) обеспечение разнообразной коллекции белковых доменов, проявляющих такую же укладку, что и белковый каркас, как определено дополнительно ниже; и (b) скрининг указанной разнообразной коллекции и/или отбор из указанной разнообразной коллекции для получения по меньшей мере одного белкового домена, обладающего указанным предварительно определенным свойством. Разнообразную коллекцию белковых доменов можно получить несколькими способами в соответствии с используемой системой скрининга и/или отбора, и это может включать в себя использование способов, хорошо известных специалисту в настоящей области техники, таких как фаговый дисплей или рибосомный дисплей. Предпочтительно указанный связывающий домен представляет собой рекомбинантный связывающий домен.The term "binding domain" means a protein domain exhibiting the same "fold" (ie, secondary, tertiary and/or quaternary structure) as the protein backbone and having a predetermined property as defined below. Such a binding domain can be obtained using rational or, most commonly, combinatorial protein engineering techniques that are known to those skilled in the art (Binz, H.K., Amstutz, P.,

, A., 2005. Nat. Biotech. 23, 1257-1268). For example, a binding domain having a predetermined property can be obtained using a method comprising the following steps: (a) providing a diverse collection of protein domains exhibiting the same fold as the protein backbone, as defined further below; and (b) screening said diverse collection and/or selecting from said diverse collection to obtain at least one protein domain having said predetermined property. A diverse collection of protein domains can be obtained in a number of ways, according to the screening and/or selection system used, and this may include using methods well known to one of skill in the art, such as phage display or ribosome display. Preferably said binding domain is a recombinant binding domain.

The term "protein backbone" means a protein with exposed surface regions in which amino acid insertions, substitutions or deletions are highly tolerated. Examples of protein scaffolds that can be used to create binding domains of the present invention are antibodies or fragments thereof, such as single chain Fv or Fab fragments, protein A from Staphylococcus aureus, bilin binding protein from Pieris brassicae or other lipocalins, ankyrin repeat proteins, or proteins with other repeats and human fibronectin. Protein scaffolds are known to the person skilled in the art (Binz et al., 2005, ibid.; Binz et al., 2004, ibid.).

The term "target" refers to a single molecule, such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other naturally occurring molecule, including any portion of such a single molecule or complexes of two or more such molecules. The target may be a whole cell or tissue sample, or it may be any non-natural compound. Preferably, the target is a naturally occurring or non-natural polypeptide or a polypeptide containing chemical modifications, for example modified by natural or non-natural phosphorylation, acetylation or methylation. In a specific application according to the present invention, the targets are serum albumin and HER2.

The term "predetermined property" refers to a property such as target binding, target blocking, activation of a target-mediated reaction, enzymatic activity, and additional properties associated therewith. Depending on the type of property desired, one skilled in the art will be able to identify the format and necessary steps to perform screening and/or selection of a binding domain with the desired property. Preferably, said predetermined property is target specific binding.

In the context of the present invention, the term "polypeptide" refers to a molecule consisting of a chain consisting of many, i. two or more amino acids linked by peptide bonds. Preferably, the polypeptide is composed of more than eight amino acids linked by peptide bonds. The term "polypeptide" also includes a plurality of amino acid chains linked together by S-S cysteine bridges. The polypeptides are well known to the person skilled in the art.

The term "polypeptide tag" refers to an amino acid sequence attached to a polypeptide/protein, wherein said amino acid sequence is useful for purifying, detecting, or "targeting" (i.e., localizing to a target site) said polypeptide a/protein, or wherein said the amino acid sequence improves the physicochemical behavior of the polypeptide a/protein, or wherein said amino acid sequence has an effector function. Individual polypeptide tags of the binding protein can be connected to other parts of the binding protein directly or through polypeptide linkers. All of these polypeptide tags are well known in the art and are fully available to a person skilled in the art. Examples of polypeptide tags are small polypeptide sequences, such as His tags (for example, a histidine tag according to SEQ ID NO: 1), myc, FLAG, or Strep, or polypeptides such as enzymes (for example, alkaline phosphatase), which allow the detection of said polypeptide a /protein or polypeptides that can be used for targeting (such as immunoglobulins or fragments thereof) and/or as effector molecules.

The term "polypeptide linker" refers to an amino acid sequence that is capable of linking, for example, two protein domains, a polypeptide 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, labels, non-proteinaceous compounds or polymers and linkers are known to the person skilled in the art. A list of examples is given in the description of patent application WO 2002/020565. Specific examples of such linkers are glycine-serine linkers and proline-threonine linkers of variable length. Examples of glycine-serine linkers are GS and the amino acid sequences shown in SEQ ID NOs: 2-6, and examples of proline-threonine linkers are shown in the amino acid sequences of SEQ ID NOs: 7-9.

, А., 2003. FEBS Letters 539, 2-6) содержат общее описание признаков белков с повторами и признаков доменов с повторами, техник и применений. Термин "белок с повторами" относится к белку, содержащему один или несколько доменов с повторами. Предпочтительно белок с повторами содержит вплоть до шести доменов с повторами. Более предпочтительно белок с повторами содержит вплоть до пяти доменов с повторами. Более предпочтительно белок с повторами содержит вплоть до четырех доменов с повторами. Более того, указанный белок с повторами может содержать дополнительные не содержащие повтор белковые домены, полипептидные метки и/или полипептидные линкеры. Домены с повторами могут представлять собой связывающие домены, как описано в настоящем документе ранее.International Patent Publication WO 2002/020565 and Forrer et al., 2003 (Forrer, P., Stumpp, M.T., Binz, H.K.,

, A., 2003. FEBS Letters 539, 2-6) provide a general description of repeat protein features and repeat domain features, techniques, and applications. The term "repeated protein" refers to a protein containing one or more repeat domains. Preferably, the repeat protein contains up to six repeat domains. More preferably, the repeat protein contains up to five repeat domains. More preferably, the repeat protein contains up to four repeat domains. Moreover, said repeat protein may contain additional non-repeat protein domains, polypeptide tags and/or polypeptide linkers. Repeat domains may be binding domains, as previously described herein.

The term "domain with repeats" refers to a protein domain containing two or more successive repeat modules as structural units, and these structural units are characterized by the same folding and closely stacked on top of each other, forming a supercoiled structure characterized by the presence of a joint hydrophobic core. Along with structural homology, such repeat modules are additionally characterized by sequence homology. Preferably, the repeat domain further comprises an N-terminal and/or C-terminal capping repeat. For clarity, a capping repeat may be a repeat module. Such repeat domains, repeat modules and capping repeats, sequence motifs, and structural and sequence homology are well known to the person skilled in the art from examples of ankyrin repeat domains (International Patent Publication WO 2002/020565), leucine rich repeat domains (international patent publication WO 2002/020565), tetratricopeptide repeat domains (Main, E.R., Xiong, Y., Cosso, M.J., D'Andrea, L., Regan, L., Structure 11(5), 497-508, 2003) and armadillo repeat domains (international patent publication WO 2009/040338). In addition, one skilled in the art is well aware that such repeat domains differ from proteins containing repeat amino acid sequences, where each repeat amino acid sequence is capable of forming a separate domain (e.g., the FN3 domains of fibronectin) or where the repeat amino acid sequences are not structural units. , i.e. these repeating amino acid sequences do not stack tightly on top of each other to form a supercoiled structure characterized by the presence of a common hydrophobic core. Methods for identifying and determining repeat modules or repeat sequence motifs or identifying families of related proteins containing such repeat units or motifs, such as homology search (BLAST®, etc.), are well known in the field of bioinformatics and are well known to a person skilled in the art. .

The terms "engineered repeat protein" and "engineered repeat domain" refer to a repeat protein or repeat domain, respectively, resulting from the procedure of the present invention, for example as explained in International Patent Publication WO 2002/020565. The term "engineered" refers to the property that such repeat proteins and repeat domains, respectively, are artificial, synthetic, and non-natural. The engineered repeat proteins or engineered repeat domains of International Patent Publication WO 2002/020565 include engineered ankyrin repeat proteins or engineered ankyrin repeat domains, respectively. Accordingly, an engineered ankyrin repeat protein herein corresponds to a protein of the present invention comprising at least one engineered ankyrin repeat domain. In addition, the term "non-natural" means that the sequence of said binding protein or said binding domain is not present as a non-artificial sequence value in a sequence database such as GenBank, EMBL-Bank or Swiss-Prot. These databases and other similar sequence databases are well known to the person skilled in the art. The recombinant binding proteins or engineered ankyrin repeat domains of the present invention do not occur naturally.

The terms "repeat module", "repeat unit", "capping repeat", "capping module" and additional terms relating to repeat proteins and repeat domains are defined in International Patent Publication WO 2002/020565 and the definitions are incorporated by reference.

The term "characterized by target specificity", "specifically binds to a target", "binds to a target with high specificity", "target specificity" or "target specificity" and the like means that the binding protein or binding domain binds to PBS targeting with a lower dissociation constant (ie, it binds with higher affinity) than it binds to an unrelated protein such as E. coli maltose binding protein (MBP). Preferably the dissociation constant ("Kd") in PBS for the target is at least 10 ² ; more preferably at least 10 ³ ; more preferably at least 10 ⁴ ; or more preferably at least 10 ⁵ times lower than the corresponding dissociation constant for MBP. Methods for determining dissociation constants of protein-protein interactions, such as surface plasmon resonance (SPR) techniques (eg, SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. The measured Kd values of a particular protein-protein interaction may vary if measured under different conditions (eg, salt concentration, pH). Thus, measurements of Kd values are preferably carried out using standard protein solutions and a standardized buffer such as PBS.

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

The term "inhibit cell proliferation" and the like in the context of the present invention refers to the ability of said recombinant binding protein to inhibit cell proliferation. The strength of inhibition is usually measured by assessing the concentration of half-maximal inhibition (IC ₅₀ ). The term "inhibition" and the evaluation of IC ₅₀ values are well known in the art.

The term "mouse serum albumin" refers to the UniProt identification number P07724, the term "cynomolgus monkey serum albumin" (i.e. Macaca fascicularis) refers to the UniProt identification number A2V9Z4, and the term "human serum albumin" refers to the UniProt identification number P02768.

As used herein, the term "HER2" refers to human epidermal growth factor receptor 2, also known as Neu, ErbB-2, CD340 (differentiation cluster 340) or p185. HER2 is a member of the epidermal growth factor receptor (EGFR/ErbB) family. HER2 in humans is encoded by ERBB2, a known proto-oncogene located on the long arm of human chromosome 17 (17q12). HER2 is numbered P04626 according to UniProtKB/Swiss-Prot. Human HER2 is composed of 1255 amino acids with a 21 amino acid signal sequence, a 631 amino acid extracellular region (eg, an ectodomain containing domains I to IV), a 23 amino acid transmembrane region, and a 580 amino acid cytoplasmic domain.

"Improved storage stability" in the context of the present invention means a reduction in the number of degradation bands, preferably a reduction in the number of degradation products, as detected by Coomassie-stained SDS-PAGE, occurring after storage at 40°C for 1 month at a concentration of 10 mg/ml in PBS, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or 50%. Methods for evaluating storage stability using SDS-PAGE are well known to those skilled in the art. Examples of engineered ankyrin repeat domains and recombinant binding proteins with improved storage stability properties are shown in Examples 2 and 3.

The expression "recombinant binding protein containing only one engineered ankyrin repeat domain with binding specificity for serum albumin" means recombinant binding, which is characterized by the composition of the recombinant binding protein according to the present invention, in which the number of engineered ankyrin repeat domains with binding specificity for serum albumin albumin is reduced to one by removing all but one engineered ankyrin repeat domains with binding specificity for serum albumin and the corresponding polypeptide linkers.

The term "biparatopic binding protein" means a binding protein directed against two different epitopes located on the same target protein. For example, a biparatopic binding protein that targets HER2 contains at least a first binding domain that targets a first epitope on HER2 and a second binding domain that targets a different second epitope on HER2. The protein of SEQ ID NO: 32 is a biparatopic binding protein containing two engineered ankyrin repeat domains with binding specificity for HER2 targeting different epitopes on HER2. The recombinant binding protein consisting of SEQ ID NO: 21 contains SEQ ID NO: 32.

The expression "shows improved pharmacokinetic properties", "improved pharmacokinetic properties" or "improved pharmacokinetic properties" according to the present invention means that the pharmacokinetic parameter of the recombinant binding protein is improved compared to the corresponding pharmacokinetic parameter of the protein with which it is compared. Relevant examples are shown in examples 8, 9, 10 and 11 (see figures 5 and 6). Preferably, the improved pharmacokinetic property is reduced clearance and/or increased exposure and/or increased terminal half-life. More preferably, the improved pharmacokinetic property is an increased terminal half-life. In one embodiment, a recombinant binding protein of the present invention comprising at least two, more preferably two engineered ankyrin repeat domains with binding specificity for serum albumin exhibits an increased terminal half-life and/or reduced clearance and/or increased impact of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250% or 250% compared to the corresponding recombinant binding protein containing only one engineered ankyrin repeat domain with binding specificity for serum albumin. In one embodiment, a recombinant binding protein of the present invention comprising at least two, more preferably two, engineered ankyrin repeat domains with binding specificity for serum albumin exhibits an increased terminal half-life, preferably an increased terminal half-life of at least 5 %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250% or 250% compared to the corresponding recombinant binding protein containing only one engineered ankyrin repeat domain with binding specificity for serum albumin.

Preferably clearance and/or exposure and/or terminal half-life is measured in a mammal, more preferably a mouse and/or cynomolgus monkey, more preferably a cynomolgus monkey. Preferably, when measuring clearance and/or exposure and/or final half-life in the mouse, the assessment was made up to 48 hours after injection. More preferably, the estimated terminal half-life in the mouse is calculated from 24 to 48 hours. Preferably, when measuring clearance and/or exposure and/or terminal half-life in the cynomolgus monkey, the assessment was made up to 7 days post-injection. More preferably, the terminal half-life of the cynomolgus monkey is estimated from 1 day to 5 days. In addition, effects such as target-mediated clearance can be identified by one skilled in the art and taken into account when calculating the terminal half-life. The term "final half-life" of a drug, such as a recombinant binding protein of the present invention, refers to the period of time required to reach half of the plasma drug concentration applied to a mammal after pseudo-equilibrium has been achieved (e.g., calculated from 24 hours to 48 hours in a mouse or calculated from day 1 to day 5 in a cynomolgus macaque). The terminal half-life is not defined as the time required to eliminate half of the dose of drug administered to a mammal. The term "final half-life" is well known to those skilled in the art. Preferably, the pharmacokinetic comparison is made at any dose, more preferably at an equivalent dose (i.e. the same dose in mg/kg) or an equimolar dose (i.e. the same dose in mol/kg), more preferably an equimolar dose (i.e. e. the same dose in mol/kg). One skilled in the art will recognize that administration of equivalent and/or equimolar doses to animals is subject to experimental dose changes of at least 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90 % or 100%. Preferably the dose used for the pharmacokinetic measurement is selected from 0.001 to 1000 mg/kg, more preferably from 0.01 to 100 mg/kg, more preferably from 0.1 to 50 mg/kg, more preferably from 0.5 to 10 mg /kg.

EXAMPLES

General materials and methods. 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. Cell lines were purchased from LGC/ATCC (France/USA). Cell culture media were from Invitrogen/Lubio (Switzerland). Unless otherwise indicated, the methods are performed according to established protocols described (e.g., Sambrook J., Fritsch E.F. and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, New York) well known to those skilled in the art. technology. Some of the methods used have previously been described in international patent publications WO 2012/069654 and WO 2014/083208.

Engineered ankyrin repeat domains: Methods for producing engineered ankyrin repeat domains with binding specificity for serum albumin are described in International Patent Publication WO 2012/069654. Methods for producing engineered ankyrin repeat domains with binding specificity for HER2 and examples of such engineered ankyrin repeat domains and methods for producing biparatope binding proteins are described in International Patent Publication WO 2014/083208. The engineered ankyrin repeat domains are generally described in detail in International Patent Publication WO 2002/020565.

Example 1: Recombinant DNA, Protein Expression and Protein Purification

DNA encoding engineered ankyrin repeat domains or recombinant binding proteins was generated using genetic methods well known to those skilled in the art. Recombinant binding proteins selected from the group of amino acid sequences according to SEQ ID NO: 21-33, optionally additionally containing SEQ ID NO: 1 at the N-terminus, or engineered ankyrin repeat domains selected from the group of amino acid sequences of SEQ ID NO: 10-20 , optionally additionally containing SEQ ID NO: 1 at the N-terminus, were expressed in the cytoplasm of Escherichia coli using standard techniques using, for example, the pQE expression system from Qiagen (Germany). In the case where the GS amino acids were at the N-terminus, the Met residue additionally encoded by the expression vector was successfully cleaved in the cytoplasm of E. coli from the expressed polypeptide, since the initial Met was followed by a small Gly residue (i.e., the amino acid at position 1, for example , SEQ ID NO: 21). Cells were lysed using a French press and proteins were purified to near homogeneity from the crude cell extract using standard chromatographic techniques well known to one of skill in the art.

The following list defines the proteins used in the present invention:

Protein #10-His: SEQ ID NO: 10 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #11-His: SEQ ID NO: 11 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #12-His: SEQ ID NO: 12 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #13-His: SEQ ID NO: 13 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #14-His: SEQ ID NO: 14 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #15-His: SEQ ID NO: 15 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #16-His: SEQ ID NO: 16 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #17-His: SEQ ID NO: 17 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #18-His: SEQ ID NO: 18 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #19-His: SEQ ID NO: 19 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #20-His: SEQ ID NO: 20 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #21: SEQ ID NO: 21

Protein #21-His: SEQ ID NO: 21 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #22: SEQ ID NO: 22

Protein #22-His: SEQ ID NO: 22 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #23: SEQ ID NO: 23

Protein #23-His: SEQ ID NO: 23 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #24: SEQ ID NO: 24

Protein #24-His: SEQ ID NO: 24 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #25: SEQ ID NO: 25

Protein #25-His: SEQ ID NO: 25 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #26: SEQ ID NO: 26

Protein #26-His: SEQ ID NO: 26 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #27: SEQ ID NO: 27

Protein #27-His: SEQ ID NO: 27 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #28: SEQ ID NO: 28

Protein #28-His: SEQ ID NO: 28 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #29: SEQ ID NO: 29

Protein #29-His: SEQ ID NO: 29 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #30: SEQ ID NO: 30

Protein #30-His: SEQ ID NO: 30 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #31-His: SEQ ID NO: 31 (includes a histidine tag at its N-terminus)

Protein #32: SEQ ID NO: 32

Protein #32-His: SEQ ID NO: 32 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Protein #33: SEQ ID NO: 33

Protein #33-His: SEQ ID NO: 33 with histidine tag (SEQ ID NO: 1) fused to its N-terminus

Example 2 Construction of an improved stability engineered ankyrin repeat domain with binding specificity for serum albumin (SEQ ID NO: 14).

Contrary to conventional knowledge about ankyrin repeats, the present inventors have found that protein #12-His, protein #13-His and protein #15-His (International Patent Publication WO 2012/069654; see Example 1) are degraded upon prolonged incubation in PBS at 10 mg/kg at 40° C., and thus are not ideal for use as components in drug candidates. Analysis of the amino acid sequence according to SEQ ID NO: 13 reveals a large number of potential degradation sites. Degradation can, for example, occur near any of the 5 asparagine residues (including asparagine-glycine dipeptides), 13 aspartate residues, or 10 glycine residues according to SEQ ID NO: 13, among additional potential degradation sites. SEQ ID NO: 13 further contains a number of potential oxidation sites. Thus, it is necessary to analyze a large number of mutants and combination mutants in order to isolate a variant that exhibits improved storage stability properties. Surprisingly, the present inventors were able to create a functional variant that has a significant effect on storage stability by mutating only position 80 according to SEQ ID NO: 13. By mutating aspartate at position 80 according to SEQ ID NO: 13 on glutamate, a functional engineered domain with ankyrin repeats was created with binding specificity for serum albumin, corresponding to SEQ ID NO: 14, which shows a marked improvement in storage stability (example 3; figure 2). Protein #14-His, like protein #13-His, showed low nanomolar affinity (dissociation constant (Kd) below 100 nM) for human, mouse, and cynomolgus serum albumin at pH 7.4 in PBS (a measurement of surface plasmon ProteOn resonance according to the manufacturer (BioRad)). Surprisingly, the #14-His protein further showed an improved thermal denaturation midpoint compared to the #13-His protein as determined by thermal unfolding using well-known techniques (Niesen, F.H., Berglund, H., Vedadi, M., Nature Protocols 2, 2212-2221, 2007; protein #13-His: Tm=86°C; protein #14-His, Tm=89.5°C; pH 6.0).

Example 3 Improved protein storage stability using SEQ ID NO: 14.

#13-His protein, #14-His protein and #15-His protein were prepared as described in Example 1 and the samples were concentrated to 10 mg/mL in PBS. Protein #14-His and protein #15-His were then stored for 1 month at -80° C. or at 40° C. in glass vials followed by SDS 15% PAGE analysis. While Protein #14-His and Protein #15-His showed equivalent storage stability at -80°C, Protein #14-His showed significantly reduced amounts of degradation products with >50% reduction compared to Protein #15 -His on SDS 15% PAGE after 1 month storage at 40°C. Similarly, when stored at 4°C, 25°C, 40°C and 60°C for one week at a concentration of 10 mg/ml in PBS, protein #14-His showed significantly reduced amounts of degradation products compared to protein #13- His. In particular, protein #14-His showed a >50% reduction in degradation products compared to protein #13-His on SDS 15% PAGE, either when stored at 40°C or 60°C, respectively (Figure 2). These data illustrate that #14-His protein has improved storage stability compared to #13-His proteins and #15-His protein. Similarly, when comparing the storage stability of protein #12-His, protein #13-His, protein #14-His and protein #15-His (see example 1) by incubating proteins at 10 mg/ml in PBS in glass vials within 1 month at 40°C protein #12-His, protein #13-His and protein #14-His show a decrease in degradation products of >30% compared to protein #15-His.

An improvement in storage stability is also observed when studying the storage stability of protein #21 and protein #22 (recombinant binding proteins consisting of amino acid sequences corresponding to SEQ ID NOs: 21 and 22, respectively). Protein #21 and Protein #22 were prepared as described in Example 1, samples were concentrated to 10 mg/ml in PBS and stored for 1 month at -80°C in or at 40°C in glass vials, followed by analysis using a standard size exclusion chromatography. While Protein #21 and Protein #22 show equivalent elution profiles when stored at -80°C, Protein #21 shows significantly more monomeric forms compared to Protein #22 when stored at 40°C. This indicates that the presence of SEQ ID NO: 21 in the recombinant binding protein is more favorable in terms of storage stability than the presence of SEQ ID NO: 22. Similarly, protein #21 exhibits lower amounts of degradation products than protein #22 when analyzed by SDS-PAGE after storage for 1 month at 40°C in glass vials in PBS at a concentration of 10 mg/ml, which confirms the higher storage stability of the recombinant binding protein containing SEQ ID NO: 21, compared with a recombinant binding protein containing SEQ ID NO: 22. In turn, this indicates that the presence of engineered ankyrin repeat domains with binding specificity for serum albumin, consisting of SEQ ID NO: 14, in a recombinant binding protein is more favorable in terms of storage stability than having engineered ankyrin repeat domains with binding specificity for serum albumin, consisting of SEQ ID NO: 13.

Example 4: Creation of a drug candidate

, A., J. Mol. Biol. 382, 1211-1227, 2008; Zahnd, C, Pecorari, F., Straumann, N., Wyler, E. and

, A., J. Biol. Chem. 281(46), 35167-35175, 2006). В международных патентных публикациях WO 2014/083208 и WO 2014/060365 сконструированные домены с анкириновыми повторами со специфичностью связывания в отношении HER2, связывающиеся с различными доменами, объединяли с получением бипаратопным связывающих белков, которые показывают усиленное ингибирование роста опухолевых клеток.Examples of engineered ankyrin repeat domains with binding specificity for HER2 have been previously disclosed (International Patent Publications WO 2014/083208; WO 2014/060365; Steiner, D., Forrer, P. and

, A., J. Mol. Biol. 382, 1211-1227, 2008; Zahnd, C, Pecorari, F., Straumann, N., Wyler, E. and

, A., J. Biol. Chem. 281(46), 35167-35175, 2006). In International Patent Publications WO 2014/083208 and WO 2014/060365, engineered ankyrin repeat domains with binding specificity for HER2 binding to different domains were combined to produce biparatopic binding proteins that show enhanced inhibition of tumor cell growth.

The recombinant binding protein of SEQ ID NO: 21 is a candidate drug containing two engineered ankyrin repeat domains with binding specificity for HER2 (SEQ ID NO: 16 and 17, respectively) and two flanking engineered domains with ankyrin repeats with binding specificity for serum albumin (each SEQ ID NO: 14) linked by polypeptide linkers (each SEQ ID NO: 9) (see figure 1). This recombinant binding protein was identified through a complex procedure including, among others, protein engineering and optimization steps. In a first step, hundreds of combinations of engineered ankyrin repeat domains with binding specificity for HER2 were generated as biparatope binding proteins by cloning, expression and purification (see Example 1). The biparatope binding proteins were then screened for affinity for HER2 as well as for cellular activity and inhibition of cell proliferation as described in International Patent Publication WO 2014/083208 and as described below. Representative results of the biparatopic binding proteins resulting from these actions are shown in Examples 5 and 6. It is important that the molecule be biparatopic since having only one engineered ankyrin repeat domain with binding specificity for HER2 results in molecules with no cellular inhibitory activity. Recombinant binding proteins were further engineered. This step involved (among other things) pharmacokinetic engineering by adding engineered ankyrin repeat domains with binding specificity for serum albumin in various formats (i.e. adding different amounts of engineered ankyrin repeat domains with binding specificity for serum albumin in different formats). positions in the molecule) to biparatopic binding proteins, and by testing different polypeptide linkers. These proteins were tested for in vivo potency (Representative Example 7), pharmacokinetics in mice (Representative Example 8) and pharmacokinetics in cynomolgus monkey (Representative Example 9), as well as cellular activity. Optimization of the polypeptide linker included testing the effect of the linker on the pharmacokinetic profile (representative example 10). The optimization of the pharmacokinetic engineering attempt was assessed using pharmacokinetic measurements (representative example 11) and activity measurements (representative example 12). Moreover, a variety of drug candidates were tested for their recombinant expression levels in E. coli (Example 13). The recombinant binding protein of SEQ ID NO: 21 is the protein resulting from these efforts. It is a drug candidate for use in humans.

Protein #31, a recombinant binding protein of SEQ ID NO: 31, corresponds to a protein of protein #21 and amino acids according to SEQ ID NO: 1 at the N-terminus.

Example 5: High affinity binding of HER2 with a recombinant binding protein containing SEQ ID NO: 21.

The #21-His protein was expressed and purified as described in Example 1. Its affinity for HER2 was assessed by surface plasmon resonance as described in International Patent Publication WO 2014/083208. The dissociation constant of 27 pM was determined by global fitting of resonant unit values obtained for various concentrations, a method well known to those skilled in the art. In comparison, the #23-His protein (see Example 1) containing a different combination of engineered ankyrin repeat domains with binding specificity for HER2 exhibited a dissociation constant of 64 pM. Similarly, the combination of engineered ankyrin repeat domains corresponding to SEQ ID NOs: 16 and 17 exhibited a better dissociation constant than most combinations based on engineered ankyrin repeat domains with binding specificity for HER2 known from International Patent Publication WO 2014/ 083208.

Surface plasmon resonance measurements further showed that the recombinant binding protein consisting of SEQ ID NO: 21 does not interact with the extracellular domains of HER3 or EGFR.

Surface plasmon resonance measurements further showed that engineered ankyrin repeat domains consisting of SEQ ID NO: 16 or 17 (each additionally containing SEQ ID NO: 1 at the N-terminus) exhibited dissociation constants of 9 pM and 152 pM, respectively, in relation to binding to HER2.

Experiments using surface plasmon resonance further showed that protein #21 can bind to human HER2 and human serum albumin simultaneously.

Experiments using surface plasmon resonance further showed that protein #21 at a concentration of 100 nM binds to human serum albumin with a dissociation constant (Kd) of 21 nM.

Example 6: High cellular activity and induction of apoptosis of a recombinant binding protein containing SEQ ID NO: 21.

#21-His protein and #23-His protein were expressed and purified as described in Example 1. The effects of recombinant binding proteins on BT474 cell proliferation were determined by measuring DNA synthesis using BrdU (bromodeoxyuridine) labeling (BrdU, Cell Proliferation ELISA, Roche) . Briefly, 10,000 BT474 cells were seeded per well in a 96-well plate in 100 μl of complete medium and incubated for 24 hours. Recombinant binding proteins or controls were added for an additional 72 hours. BrdU for cell labeling was added for the last 24 hours. Labeled (proliferating) cells were detected according to the manufacturer's protocol. Data was analyzed using GraphPad prism software plotting log [c] on the x-axis and OD450-602 nm on the y-axis. Data were fitted using a non-linear regression fit (logarithm (antagonist) vs. response curve - variable slope model (four parameters)) to give IC ₅₀ values. The results are shown in Figure 3. #21-His protein inhibits BT474 cell proliferation with an observed IC ₅₀ value of 1.5 nM. A 60% higher IC ₅₀ value (i.e. worse) of 2.4 nM is observed for protein #23-His indicating that the presence of SEQ ID NOs: 16 and 17 in the recombinant binding protein is more favorable than the presence of SEQ ID NOs: 18 and 17. Similarly, the combination of engineered ankyrin repeat domains corresponding to SEQ ID NOs: 16 and 17 inhibited BT474 cells with a lower observed IC ₅₀ value than that observed for most combinations, based on engineered ankyrin repeat domains with binding specificity for HER2 known from international patent publication WO 2014/083208.

The recombinant binding protein consisting of SEQ ID NO: 21 (protein #21) further exhibited potent inhibition of proliferation in a variety of cancer cell lines, including SKBR-3, SKOV-3, NCI-N87, ZR-75-30 cell lines. , HCC1419 or MDA-MB175.

Apoptosis induction by protein #21 was determined by measuring caspase 3/7 activation using Caspase 3/7-Glo systems (Promega, Switzerland). Briefly, 10,000 BT474 cells were seeded per well in a 96-well plate in 100 μl of complete medium and incubated for 24 hours. Protein #21 and reference standards were added for an additional 24 hours. The Caspase Glo reagent was added according to the manufacturers protocol for 1 hour. Caspase 3/7 activation was monitored by measuring luciferase activity. Alternatively, apoptosis induction was determined using the ELISAPLUS cell death detection system (Roche, Switzerland). The assay was performed according to the manufacturers protocol. The cell count and incubation time were similar to the Caspase Glo reading. Data was analyzed using GraphPad prism software plotting concentration on the x-axis and OD405/490 nm or relative light units (RLU; measured on a Tecan M-1000 reader) on the y-axis. Data were fitted using a non-linear regression fit (logarithm (agonist) versus response curve - variable slope model (four parameters)). The results are shown in Figure 7. For protein #21, an EC ₅₀ value of 2.4 nM was observed. Protein #21 exhibits similarly high activity in inducing apoptosis for SKBR-3, SKOV-3, NCI-N87, ZR-75-30, HCC1419 or MDA-MB175 cell lines. In contrast, the antibodies trastuzumab, pertuzumab, or a mixture of trastuzumab and pertuzumab did not induce apoptosis in BT474 cells (Figure 7).

It is important to note that these analyzes show that protein #21, in which SEQ ID NO: 32 is flanked by SEQ ID NO: 14, exhibits high cellular activity, similar to the situation using SEQ ID NO: 32 alone.

Example 7 High Efficacy of Recombinant Binding Protein Containing SEQ ID NO: 21 in Mouse Xenograft Tumor Models.

#21-His protein, #23-His protein, #24-His protein, #25-His protein were expressed and purified as described in Example 1. In one study, #21-His protein was compared with trastuzumab and PBS. The results are shown in figure 4a. Mouse BT474 breast cancer xenograft tumor models were essentially performed as follows using procedures well known to one of skill in the art: BT474 human breast carcinoma cells were cultured, resuspended and 2×10 ⁷ cells in 200 μl of RPMI 1640 medium containing 50 :50 Matrigel (BD Biosciences) was injected into the right flank of female Balb/c nude mice per mouse. At tumor volumes of approximately 200-300 mm ³ , mice were randomized into groups of 8 animals and tumor treatment was initiated with iv administration of PBS, trastuzumab (10 mg/kg), trastuzumab (10 mg/kg), and pertuzumab (10 mg/kg) or protein #21-His (35 mg/kg) 7 doses at 3-day intervals (Q3Dx7). Tumor volumes were measured over 32 days. Protein #21-His is more effective in inhibiting tumor growth than trastuzumab.

In a similar study using BT474 cells (identical setup and procedure; all doses 35 mg/kg), #21-His protein was compared to #23-His protein, #24-His protein, and #25-His protein. The results on the 18th day after treatment are shown in Figure 4b. Importantly, protein #21-His is more effective in suppressing tumors than protein #23-His, protein #24-His or protein #25-His.

In addition, protein #21 was expressed and purified as described in Example 1. PBS, protein #21 (60 mg/kg), trastuzumab (10 mg/kg), pertuzumab (10 mg/kg), and a combination of trastuzumab and pertuzumab (each at a dose of 10 mg/kg) was used in a patient-derived mouse gastric cancer xenograft tumor model well known to those skilled in the art. All groups were dosed every three days 6 times. Briefly, tumor fragments were obtained from xenografts in serial passage in nude mice. After removal from donor mice, tumors were cut into fragments (4-5 mm in diameter) for subcutaneous implantation. Mice were randomized into groups when the tumors reached a volume of approximately 100-120 mm ³ . The day of randomization and initiation of treatment was designated as day 0 in each experiment. Tumor growth was monitored by 2D caliper measurement on the day of randomization and twice a week thereafter. Relative volumes of individual tumors (individual RTVs) for day x were calculated by dividing the volume of an individual tumor on day x (Tx) by the individual volume of the same tumor on day 0 (T0) multiplied by 100%. Tumor inhibition for a given day (T/C in %) was calculated from the ratio of median RTVs in study and control groups, multiplied by 100%. Figure 4c shows the efficacy of protein #21 in the PDX model GXA3039 of gastric cancer compared to trastuzumab and the combination of trastuzumab and pertuzumab. Protein #21 exhibits strong tumor growth inhibition similar to the combination of trastuzumab and pertuzumab, while trastuzumab alone is less effective. Figure 4d shows a second experiment in the same model comparing protein #21 with trastuzumab, pertuzumab, and a combination of trastuzumab and pertuzumab. Protein #21 exhibits strong tumor growth inhibition similar to the combination of trastuzumab and pertuzumab. Trastuzumab alone and pertuzumab alone are significantly less effective. This indicates that the tumor model may have acquired resistance to trastuzumab over time. This, in turn, suggests that protein #21 is effective even in trastuzumab-resistant cancers.

In a similar experiment, protein #21 was further analyzed in the PDX mouse gastric cancer model GXA281, well known to those skilled in the art, compared to standard therapy, lapatinib (Figure 4e). Briefly, tumor implantation and tumor growth monitoring were performed as described above. Lapatinib was administered at a dose of 100 mg/kg/day for 21 days daily i.v. and protein #21 at a dose of 40 mg/kg every three days 6 times i.v.

These experiments of this example illustrate the applicability of proteins containing SEQ ID NO: 21 for use as a drug in the treatment of disease.

Example 8: Favorable pharmacokinetic properties of the recombinant binding protein containing SEQ ID NO: 21 in mouse.

, A., Cancer Res. 70, 1595-1605, 2010). Результаты показаны на фигуре 5. Белок #21-His проявляет конечный период полужизни, составляющий 30,4 часов с 19,5% ID (введенной с помощью инъекции дозы), оставшейся через 48 часов. Для сравнения, белок #23-His проявляет конечный период полужизни, составляющий 24,7 часов с 6,5% ID, оставшейся через 48 часов. Таким образом, наличие SEQ ID NO: 16 в рекомбинантном связывающем белке оказывается более благоприятным, чем наличие SEQ ID NO: 18.The measurements shown in this example are the result of an attempt to engineer the half-life as described in example 11. Protein #21-His and protein #23-His were expressed and purified as described in example 1. The pharmacokinetic properties of His-tagged proteins were evaluated in mouse, as described elsewhere (Zahnd, C., Kawe, M., Stumpp, M.T., de Pasquale, C., Tamaskovic, R., Nagy-Davidescu, G., Dreier, B., Schibli, R. , Binz, H.K., Waibel, R.,

A., Cancer Res. 70, 1595-1605, 2010). The results are shown in Figure 5. The #21-His protein exhibits a terminal half-life of 30.4 hours with 19.5% ID (injected dose) remaining at 48 hours. In comparison, #23-His protein exhibits a terminal half-life of 24.7 hours with 6.5% ID remaining at 48 hours. Thus, the presence of SEQ ID NO: 16 in the recombinant binding protein is more favorable than the presence of SEQ ID NO: 18.

Example 9: Favorable pharmacokinetic properties of the recombinant binding protein of SEQ ID NO: 21 in cynomolgus monkey.

The measurements shown in this example are the result of an attempt to engineer the half-life as described in example 11. Protein #21-His, protein #23-His and protein #24-His were expressed and purified as described in example 1. Pharmacokinetic properties of proteins evaluated in the Javanese macaque. Proteins were administered to cynomolgus monkeys by intravenous infusion over 30 minutes at a target dose level of 1 mg/kg. Blood samples were collected before dosing and again at selected time points, e.g. 5 min, 10 min, 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours and 240 hours after the end of the infusion (i.e. after the injection). Blood samples were left to stand at room temperature and centrifuged to obtain serum, followed by storage at -80°C pending analysis. Pharmacokinetic parameters were determined using procedures well known to those skilled in the art. Serum protein concentrations were determined by sandwich ELISA using rabbit anti-ankyrin repeat domain engineered monoclonal antibody as capture reagent and mouse anti ankyrin repeat domain engineered monoclonal antibody as detection reagent and 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 assays such as non-compartmental assays. The results for Protein #21-His and Protein #23-His are shown in Figure 6. Protein #21-His exhibited a terminal half-life of 47 hours while Protein #23-His exhibited a terminal half-life of 35 hours , and the #24-His protein exhibited a terminal half-life of 45 hours. Proteins also exhibit significant target-mediated clearance once the concentration falls below 100 nM. Target-mediated clearance is well known to those skilled in the art and is known, for example, from antibodies targeting HER2.

Similarly, #21-His protein was expressed and purified as described in Example 1. Protein pharmacokinetic properties were evaluated in cynomolgus monkey as described above at 1 mg/kg, 5 mg/kg and 10 mg/kg doses. Pharmacokinetic parameters were evaluated as described above and the results are shown in Figure 6. A dose-dependent increase in terminal half-life was observed from 47 hours at the 1 mg/kg dose to 100 hours at the 5 mg/kg dose to more than 100 hours at the 10 mg dose. /kg. Target-mediated clearance is observed as soon as the concentration falls below 100 nM.

Similar results were observed for protein #21 (expressed and purified as described in Example 1). Protein pharmacokinetic properties were evaluated in cynomolgus monkey as described above at 1 mg/kg, 10 mg/kg and 100 mg/kg. Pharmacokinetic parameters are evaluated as described above. A dose-dependent increase in terminal half-life is observed. For protein #21, a terminal half-life of 109.5 hours or 130.6 hours was observed at a dose of 100 mg/kg in the cynomolgus monkey.

Example 10: Improvement of drug properties through the choice of polypeptide linkers.

Polypeptide linkers that connect protein domains are well known to those skilled in the art. Gly-Ser rich linkers are well known from Fv single chain antibody fragments, where they have proven to be most suitable for joining two Fv polypeptide chains. Surprisingly, the present inventors have found that Pro-Thr rich linkers have a positive effect on the pharmacokinetic properties of a recombinant binding protein containing two engineered ankyrin repeat domains with binding specificity for HER2 and two engineered ankyrin repeat domains with binding specificity for serum albumin compared to a rich Gly-Ser linker. Protein #26-His and protein #27-His were expressed and purified as described in Example 1. Pharmacokinetic analyzes in mice were performed as described in Example 8. Protein #27-His had 7% of the injected dose remaining after 48 hours after intravenous injection, while protein #26-His was characterized by only 5.3% of the remaining dose. Similarly, the #27-His protein exhibited a longer terminal half-life than the #26-His protein.

Example 11: Engineering of pharmacokinetic properties

Many pharmacokinetic engineering options are known in the art, including but not limited to pegylation, polypeptide extension, Fc fusion, serum albumin fusion, and serum albumin binding (Kontermann, R (Ed.) "Therapeutic proteins: strategies to modulate their plasma half-lives", Wiley-VCH Verlag GmbH, 2012, ISBN 978-3-527-32849-9). Starting, for example, with a combination of engineered ankyrin repeat domains with binding specificity for HER2 containing SEQ ID NOs: 16 and 17, there are hundreds of potential drug candidate options (selection of pharmacokinetic modification technique, selection of polypeptide linker (e.g., SEQ ID NO : 2-9 among many others), among other aspects). The present inventors found that using two engineered ankyrin repeat domains with binding specificity for serum albumin, consisting of SEQ ID NO: 14 (see Examples 2 and 3), and using Pro-Thr rich polypeptide linkers (SEQ ID NO: 9), resulting in SEQ ID NO: 21, favorable pharmacokinetic properties are achieved and this format has clear advantages over other formats tested. The results of the polypeptide linker selection effect are shown, for example, in Example 10. Comparison of the pharmacokinetic profiles of protein #29-His and protein #30-His in mice unexpectedly revealed the advantage of having two engineered ankyrin repeat domains with binding specificity for serum albumin compared to having only one engineered ankyrin repeat protein with binding specificity for serum albumin (see Figure 5), as the literature on the albumin-binding domain has shown that having two albumin-binding domains present in the molecule does not result in any advantage in pharmacokinetic properties compared to having only one albumin-binding domain (Hopp et al., ibid.). Similarly, when comparing protein #21 (recombinant binding proteins consisting of SEQ ID NO: 21) with a variant of protein #21 in which the C-terminal engineered domain with ankyrin repeats with binding specificity for serum albumin is absent (amino acids 1-422 according to SEQ ID NO: 21, resulting in protein #33), protein #21 exhibits markedly improved pharmacokinetic properties, in particular a longer terminal half-life. Comparison of the pharmacokinetic profile of cynomolgus protein #21 with pegylated protein #28 (which contains the same engineered ankyrin repeat domains with binding specificity for HER2 (SEQ ID NOs: 16 and 17), but protein #28 contains a 40 kD PEG fragment instead of two engineered ankyrin repeat domains with binding specificity for serum albumin) at doses of 5 mg/kg indicates that a protein containing two engineered ankyrin repeat domains with binding specificity for serum albumin exhibits markedly improved pharmacokinetic properties. . As described in Example 9, a comparison of the pharmacokinetic profiles of protein #21-His and protein #24-His revealed that it was more favorable to have two engineered ankyrin repeat domains with binding specificity for serum albumin flanking SEQ ID NOs: 16 and 17, instead of having both of them at the N-terminus (see figure 6).

For studies in this example, protein #21-His, protein #24-His, protein #28-His, protein #29-His and protein #30-His were prepared according to example 1. Protein #28-His was additionally pegylated using 40 kDa PEG moiety (NOF Sunbright GL2-400MA) by attaching the PEG moiety to the free C-terminal cysteine residue of protein #28-His with a maleimide reagent, followed by purification to homogeneity using standard purification methods, procedures well known to the person skilled in the art. Pharmacokinetic assays in mice or cynomolgus monkeys were performed as described in Examples 8 and 9.

Example 12: Recombinant binding protein containing SEQ ID NO: 21 shows better in vivo activity compared to recombinant binding protein containing SEQ ID NO: 24

#21-His protein and #24-His protein were expressed and purified as described in Example 1. Xenograft experiments using mice were performed as described in Example 7. The results are shown in Figure 4b. After 32 days of treatment, #21-His protein exhibited significantly better tumor suppression than #24-His protein. This indicates that having engineered ankyrin repeat domains with binding specificity for serum albumin flanking engineered ankyrin repeat domains with binding specificity for HER2 (as found in SEQ ID NO: 21) is more favorable than having both engineered ankyrin repeat domains with binding specificity for serum albumin at the N-terminus of engineered ankyrin repeat domains with binding specificity for HER2 (as found in SEQ ID NO: 24).

Example 13: High yield of recombinant expression in E. coli.

Protein #21, protein #23 and protein #25 coding sequences were cloned into a standard vector with a T7 promoter and the proteins were expressed in E. coli HMS 174 (DE3). Protein #21 achieved titers of 4.4 g/L while Protein #23 showed a titer of 1.9 g/L and Protein #25 showed a titer of 2.1 g/L indicating best expression squirrel #21. Surprisingly, the arrangement of engineered ankyrin repeat domains used in protein #21 thus results in higher recombinant expression than, for example, observed for another arrangement used in protein #25, despite the same components used (engineered ankyrin repeat domains and polypeptide linkers). Interestingly, the combination of engineered ankyrin repeat domains corresponding to SEQ ID NOs: 16 and 17 exhibited better expression of the recombinant protein than most combinations based on engineered ankyrin repeat domains with binding specificity for HER2 known from International Patent Publication WO 2014/083208.

Example 14: Simultaneous binding of two molecules of serum albumin protein #21.

Protein #21 was expressed and purified as described in Example 1. SEC combined with static light scattering (Superdex 200 10/300GL (GE Healthcare), Agilent 1200 (Life Technologies), Wyatt Optilab Trex (RI) and MiniDawnTreos (MALS)) was performed using 30 μM protein #21, 60 μM human serum albumin (20% CSL Behring solution was used to purify the monomeric fraction of human serum albumin by size exclusion chromatography on a Superdex 200 26/60 column (GE Healthcare)) or a 1:2 mixture of the same and another on PBS.

Protein #21 eluted as a monomeric peak and a molecular weight of 65.2 kDa was determined by MALS (multi-angle laser light scattering) which is within 1.2 kDa of its theoretical molecular weight (66437 Da). Serum albumin eluted as a monomeric peak of 55.1 kDa (theoretical molecular weight 58917 Da). A 1:2 mixture (molar ratio) of the two molecules resulted in a main peak with an average molecular weight of 169.8 kDa (range 186.2 kDa - 136.6 kDa) containing molecules with a molecular weight corresponding to the 1:2 complex ( theoretical molecular weight: 191.8 kDa), as well as the 1:1 complex (132.8 kDa). In addition, a small peak in free serum albumin was detected, corresponding to 6.5% serum albumin used in the experiment. No peak was found containing a molecular weight corresponding to free protein #21. These results indicate that protein #21 can bind to two serum albumin molecules at the same time.

Example 15: High thermal stability of protein #21.

Protein #21 was expressed and purified as described in Example 1. Thermal stability was assessed using circular dichroism using a Jasco J-815 CD spectrometer under nitrogen (3 L/min). Protein #21 was measured at a concentration of 0.4 μM in PBS in 117.100F-QS cuvettes (Hellma, d=1 cm) using temperature control sensors in solution by heating from 20°C to 95°C (below 45°C: + 3°C/min, above 45°C: +1°C/min, recording signal at 222 nm, with 1 min delay at 95°C and subsequent cooling phase (reverse program compared to heating phase) The CD signal was normalized to mean residual ellipticity (MRE) in units of degrees×cm ² /dmol In addition, the far ultraviolet CD spectrum was recorded.

Protein #21 exhibits a CD spectrum typical of alpha helical proteins with characteristic minima at 208 nm and 222 nm. Protein #21 exhibits high thermal stability after a stable baseline (no change in slope), signal loss at 222 nm is detected above 78°C.

Example 16: Effect of Protein #21 on the Phosphoproteome.

, C, Jung, S.,

, G., Schmid, P., Prefot, P., Hoehle, C, Koncarevic, S., Gee, J., Nicholson, R., Ward, M., Castellano, L., Stebbing, J., Zucht, H.D., Sarker, D., Heaton, N., and Pike, I., 2014. PLOS One 9 (3), e90948). Анализ выявил, что обработка клеток с помощью белка #21 дифференциально регулирует многочисленные фосфорилированные пептиды по сравнению с трастузумабом, пертузумабом и их комбинацией. Результаты показаны в таблице 1. Важно, что белок #21 проявляет некоторые заметные отличия от трастузумаба, пертузумаба и смеси трастузумаба и пертузумаба в том, что он сильнее ингибирует фосфорилирование HER2 на сайтах 1073/1078/1083, 1139/1151, 1172/1174, 1174 и 1240. В других сайтах фосфорилирования фосфорилирование увеличивается (772). Находящиеся ниже HER2 специфические представители сигнального каскада mTOR, как было установлено, дифференциально регулируются после обработки белком #21; например, фосфопептид для киназы S6 (S441/T444/S447) подвергался отрицательной регуляции в 0,63 раза по отношению к РВ S по сравнению с другими группами, где отрицательная регуляция составляла только в 0,88 раз. Более того, фосфорилированные пептиды, которые относятся к MAPK1 и MAPK3, подверглись значительной отрицательной регуляции.Protein #21 was expressed and purified as described in Example 1. BT474 or NCI-N87 cells were treated for 18 hours with any of protein #21 (100 nM), trastuzumab (100 nM), pertuzumab (100 nM), trastuzumab mix (100 nM) and pertuzumab (100 nM) or PBS. The cells were then subjected to mass spectrometric analysis of the phosphoproteome as described by Britton D. et al. (Britton, D., Zen, Y., Quaglia, A., Selzer, S., Mitra, Vl.,

, C, Jung, S.,

, G., Schmid, P., Prefot, P., Hoehle, C, Koncarevic, S., Gee, J., Nicholson, R., Ward, M., Castellano, L., Stebbing, J., Zucht, HD, Sarker, D., Heaton, N., and Pike, I., 2014. PLOS One 9(3), e90948). The analysis revealed that treatment of cells with protein #21 differentially regulates numerous phosphorylated peptides compared to trastuzumab, pertuzumab, and the combination thereof. The results are shown in Table 1. Importantly, protein #21 shows some notable differences from trastuzumab, pertuzumab and the trastuzumab/pertuzumab mixture in that it more strongly inhibits HER2 phosphorylation at sites 1073/1078/1083, 1139/1151, 1172/1174, 1174 and 1240. At other phosphorylation sites, phosphorylation is increased (772). Downstream of HER2, specific members of the mTOR signaling cascade have been shown to be differentially regulated after protein #21 treatment; for example, the phosphopeptide for S6 kinase (S441/T444/S447) was down-regulated 0.63-fold relative to PB S compared to the other groups, where it was only 0.88-fold downregulated. Moreover, the phosphorylated peptides that belong to MAPK1 and MAPK3 were significantly downregulated.

Analysis of the same samples by AKT-S473 phosphorylation site-specific ELISA (BioConcept: PathScan® Phospho-Aktl sandwich ELISA kit No. 7160 (Ser473)) was downregulated by protein #21 0.16-fold relative to to PBS compared to other groups, where the negative regulation was in the range of only 0.3-0.7 times. The ELISA results are shown in Table 2.

These results indicate that protein #21 differentially regulates downstream HER2 signaling compared to trastuzumab, pertuzumab, and a combination of both. The greatest differences are found in the AKT-mTOR pathway, a critical pathway for cell survival, which in turn explains why protein #21 induces apoptosis. The phosphoinositide 3-kinase (PI3K/Akt) pathway is considered one critical pathway that maintains cell survival by blocking apoptosis. Its pathological activation, for example by HER2/HER3 heterodimerization, can thus lead to malignant proliferation.

In this experiment, it was additionally found that exposure of BT474 and NCI-N87 cells to protein #21 increased cellular Foxo3a protein levels by 1.88 and 2.8 times, respectively, compared to exposure to PBS alone. The corresponding increase in the content of Foxo3a protein in BT474 and NCI-N87 cells due to exposure to trastuzumab was 1.14 and 1.32 times, respectively; and due to the effect of pertuzumab - by 1.21 and 1.25 times, respectively; and due to the effects of trastuzumab and pertuzumab - by 1.28 and 1.19 times, respectively. Thus, protein #21 results in a much higher increase in cellular levels of the Foxo3a protein compared to trastuzumab or pertuzumab or a combination of both. One of skill in the art knows that Akt1 phosphorylates Foxo3a, resulting in degradation of Foxo3a and promotion of cell survival. Inhibition of Akt phosphorylation results in stabilization of Foxo3a and thus an increase in Foxo3a levels. At elevated levels, Foxo3a can translocate to the nucleus and induce apoptosis.

Example 17: Introduction of concomitant therapy using recombinant binding proteins containing SEQ ID NO: 21 to improve therapy.

Protein #21 and protein #21-His were expressed and purified as described in Example 1. Standard cell proliferation assays were performed using BrdU, well known to those skilled in the art. Briefly, the effects on cell proliferation of #21-His protein, other compounds, or #21-His protein in combination with other compounds were determined by measuring DNA synthesis using BrdU labeling (BrdU, Cell Proliferation ELISA, Roche). Briefly, 10,000 cells were seeded per well in a 96-well plate in 100 μl of complete medium and incubated for 24 hours. Protein #21-His and/or compounds were added for an additional 72 hours. BrdU for cell labeling was added for the last 24 hours. Labeled (proliferating) cells were detected according to the manufacturer's protocol. Data were analyzed using GraphPad prism software (plot of log [s] and OD450-602 nm). Data were fitted using a non-linear regression fit (logarithm (antagonist) versus response curve - variable slope model (four parameters)).

The combination of protein #21-His with eribulin, a microtubule inhibitor: Protein #21-His, eribulin, or a combination of protein #21-His and eribulin was investigated for their ability to inhibit the proliferation of BT474 and MDA-MB175 cells. The dose of eribulin was adjusted starting at 1000 nM in addition to a suboptimal #21-His protein concentration (2 nM in BT474 and 4 nM in MDA-MB175). The combination of both molecules was superior to eribulin alone in both cell lines. Eribulin was not active against BT474 cells alone, but showed strong inhibition when combined with protein #21-His. Eribulin alone showed very low activity on MDA-MB175 cells, but activity improved when combined with protein #21-His. The IC ₅₀ values are shown in Table 3. These data show that protein #21-His can be combined with eribulin and potentiates its activity.

The combination of protein #21-His and GDC-0941, pI3K inhibitor: protein #21-His, GDC-0941 or a combination of protein #21-His and GDC-0941 was investigated for their ability to inhibit the proliferation of BT474 and MDA-MB175 cells. The dose of GDC-0941 was adjusted starting at 20,000 nM in addition to a suboptimal #21-His protein concentration (2 nM in BT474 and 4 nM in MDA-MB175). The combination of both molecules was superior to GDC-0941 alone in both cell lines. GDC-0941 showed activity on its own, but activity was increased when combined with protein #21-His. The activity of GDC-0941 in BT474 was greatly increased, indicating a synergistic effect. Similarly, protein #21-His increased GDC-0941 activity in MDA-MB175 cells, where the effect was additive. The IC ₅₀ values are shown in Table 3. These data show that protein #21-His can be combined with GDC-0941 and potentiates its activity.

The combination of #21-His protein and everolimus, an mTOR inhibitor: #21-His protein, everolimus, or a combination of #21-His protein and everolimus was investigated for their ability to inhibit proliferation of BT474 and MDA-MB175 cells. The dose of everolimus was adjusted starting at 1 mM in addition to a suboptimal #21-His protein concentration (2 nM in BT474 and 4 nM in MDA-MB175). The combination of both molecules was superior to everolimus alone in both cell lines. Everolimus alone showed activity, but activity was increased when combined with protein #21-His. Everolimus activity in BT474 cells was greatly increased, indicating a synergistic effect. Similarly, protein #21-His increased everolimus activity in MDA-MB175 cells, where the effect was additive. IC ₅₀ values are shown in Table 3. These data show that protein #21-His can be combined with everolimus and potentiates its activity.

The combination of protein #21-His and lapatinib, an inhibitor of any type of HER: protein #21-His, lapatinib, or a combination of protein #21-His and lapatinib was investigated for their ability to inhibit proliferation of BT474 and MDA-MB175 cells. Lapatinib was adjusted starting at 10,000 nM in addition to a suboptimal #21-His protein concentration (2 nM in BT474 and 4 nM in MDA-MB175). The combination of both molecules was superior to lapatinib alone in both cell lines. Lapatinib alone showed activity, but activity was increased when combined with protein #21-His. The activity of lapatinib in BT474 cells was greatly increased, indicating a synergistic effect. Similarly, protein #21-His increased the activity of lapatinib in MDA-MB175 cells, where the effect was additive. The IC ₅₀ values are shown in Table 3. These data show that protein #21-His can be combined with lapatinib and potentiates its activity.

The combination of protein #21 with trastuzumab: protein #21, trastuzumab, or a combination of protein #21 and trastuzumab was investigated for their ability to inhibit the proliferation of BT474 and MDA-MB175 cells. Trastuzumab was adjusted starting at 100 nM in addition to a suboptimal protein #21 concentration (1 nM in BT474 and 10 nM in MDA-MB175). The combination of both molecules was superior to trastuzumab alone in both cell lines. Trastuzumab showed only limited activity on its own and only induced cessation of cell proliferation but not apoptosis, while protein #21 induces apoptosis. The combination of a suboptimal protein #21 concentration with trastuzumab resulted in inhibition of proliferation. This shows that both molecules can be combined and that protein #21 potentiates the activity of trastuzumab. The effect of the combination can also be confirmed by adjusting the dose of protein #21 in addition to a constant concentration of trastuzumab (50 nM).

Similarly, the combination of protein #21 with apitolisib, taselisib, alpelisib, palbociclib, trametinib, cobimetinib, or salirazib was more effective in inhibiting BT474 cell proliferation than either protein #21 alone or apitolisib, taselisib, alpelisib, palbociclib, trametinib, cobimetinib, or salirazib separately (see table 3). For experiments with apitolisib, taselisib, alpelisib, or palbociclib, protein #21 was used at a concentration of 2 nM and the concentration of apitolisib, taselisib, alpelisib, or palbociclib was adjusted to determine IC ₅₀ values. For experiments with trametinib (10 ^-5 M), cobimetinib (10 ^-5 M) or salirazib (3×10 ^-5 M), protein #21 dose was adjusted to determine IC ₅₀ values.

Thus, Protein #21 or Protein #21-His can be combined with several classes of standard breast cancer therapy drugs to potentiate their growth inhibitory effects on cells overexpressing HER2. This indicates the clinical potential for enhanced efficacy and/or dose reduction of standard therapy to reduce toxicity.

--->

Sequence listing

SEQUENCE LISTING

<110> Molecular Partners AG

<120> Recombinant Binding Proteins

<130> P015

<160> 33

<170> BiSSAP 1.3

<210> 1

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> His-tag

<400> 1

Met Arg Gly Ser His His His His His His

1 5 10

<210> 2

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Glyser linker

<400> 2

Gly Gly Gly Gly Ser

fifteen

<210> 3

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Glyser linker

<400> 3

Gly Gly Gly Gly Ser Gly Ser

fifteen

<210> 4

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Glyser linker

<400> 4

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 5

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Glyser linker

<400> 5

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

1 5 10 15

<210> 6

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Glyser linker

<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

twenty

<210> 7

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> ProThr linker

<400> 7

Gly Ser Pro Thr Pro Thr Pro Thr

fifteen

<210> 8

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> ProThr linker

<400> 8

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

1 5 10 15

<210> 9

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> ProThr linker

<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

twenty

<210> 10

<211> 159

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ala Ser Asp Leu Thr Gly Ile Thr Pro Leu His Leu Ala Ala Ala Thr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155

<210> 11

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 12

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 13

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 14

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 15

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 16

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 16

Gly Ser Asp Leu Gly Ile Lys Leu Leu Phe Ala Ala Ala Lys Gly Gln

1 5 10 15

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

20 25 30

Lys Asp Phe Gln Gly Val Thr Pro Leu His Ile Ala Ala Gln Ser Gly

35 40 45

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

50 55 60

Ala Lys Asp Val Thr Gly Asp Thr Pro Leu His Leu Ala Ala Gln His

65 70 75 80

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

85 90 95

Asn Ala Gln Asp Glu Arg Gly Trp Thr Pro Ala Asp Leu Ala Ala Asp

100 105 110

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

115 120 125

<210> 17

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ala Val Asp Ala Ile Gly Phe Thr Pro Leu His Leu Ala Ala Phe Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 18

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 18

Gly Ser Asp Leu Gly Val Lys Leu Leu Trp Ala Ala Ala Arg Gly Gln

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 19

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 19

Gly Ser Asp Leu Gly Val Lys Leu Leu Trp Ala Ala Ala Arg Gly Gln

1 5 10 15

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

20 25 30

Lys Asp Phe Gln Gly Ile Thr Pro Leu His Ile Ala Ala Gln Ser Gly

35 40 45

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

50 55 60

Ala Lys Asp Val Thr Gly Asp Thr Pro Leu His Leu Ala Ala Gln His

65 70 75 80

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

85 90 95

Asn Ala Gln Asp Glu Arg Gly Trp Thr Pro Ala Asp Leu Ala Ala Asp

100 105 110

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

115 120 125

<210> 20

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> Designed ankyrin repeat domain

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ala Val Asp Ala Ile Gly Phe Thr Pro Leu His Leu Ala Ala Phe Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 21

<211> 570

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising four designed ankyrin repeat domains

<400> 21

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

130 135 140

Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu Leu Phe Ala Ala

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Ala Asp Val Asn Ala Gln Asp Glu Arg Gly Trp Thr Pro Ala Asp

245 250 255

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

260 265 270

Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro

275 280 285

Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu

290 295 300

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

305 310 315 320

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro

325 330 335

Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu Ile Val Glu Val Leu

340 345 350

Leu Lys Ala Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr

355 360 365

Pro Leu His Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala 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 Pro Ala Asp Ile Ala Ala Gly Ala Gly Asn Glu Asp Ile Ala Glu

405 410 415

Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro

420 425 430

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu

435 440 445

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

450 455 460

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

465 470 475 480

Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu Lys Ile

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu

545 550 555 560

Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

565 570

<210> 22

<211> 570

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising four designed ankyrin repeat domains

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

130 135 140

Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu Leu Phe Ala Ala

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Ala Asp Val Asn Ala Gln Asp Glu Arg Gly Trp Thr Pro Ala Asp

245 250 255

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

260 265 270

Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro

275 280 285

Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu

290 295 300

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

305 310 315 320

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro

325 330 335

Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu Ile Val Glu Val Leu

340 345 350

Leu Lys Ala Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr

355 360 365

Pro Leu His Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala 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 Pro Ala Asp Ile Ala Ala Gly Ala Gly Asn Glu Asp Ile Ala Glu

405 410 415

Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro

420 425 430

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu

435 440 445

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

450 455 460

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

465 470 475 480

Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu Lys Ile

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu

545 550 555 560

Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

565 570

<210> 23

<211> 570

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising four designed ankyrin repeat domains

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

130 135 140

Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Trp Ala Ala

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro

275 280 285

Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu

290 295 300

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

305 310 315 320

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro

325 330 335

Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu Ile Val Glu Val Leu

340 345 350

Leu Lys Ala Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr

355 360 365

Pro Leu His Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala 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 Pro Ala Asp Ile Ala Ala Gly Ala Gly Asn Glu Asp Ile Ala Glu

405 410 415

Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro

420 425 430

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu

435 440 445

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

450 455 460

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

465 470 475 480

Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu Lys Ile

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu

545 550 555 560

Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

565 570

<210> 24

<211> 570

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising four designed ankyrin repeat domains

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

130 135 140

Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro

275 280 285

Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu

290 295 300

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

305 310 315 320

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

325 330 335

Leu His Ile Ala Ala Gln Ser Gly His Leu Glu Ile Val Glu Val Leu

340 345 350

Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val Thr Gly Asp Thr

355 360 365

Pro Leu His Leu Ala Ala Gln His Gly His Leu Glu Ile Val Glu Val

370 375 380

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

385 390 395 400

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

405 410 415

Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro

420 425 430

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu

435 440 445

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

450 455 460

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

465 470 475 480

Gly Leu Thr Pro Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu Ile

485 490 495

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

500 505 510

Ile Gly Phe Thr Pro Leu His Leu Ala Ala Phe Ile Gly His Leu Glu

515 520 525

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

530 535 540

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

545 550 555 560

Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

565 570

<210> 25

<211> 570

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising four designed ankyrin repeat domains

<400> 25

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

130 135 140

Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro

275 280 285

Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu

290 295 300

Leu Trp Ala Ala Ala Arg Gly Gln Asp Asp Glu Val Arg Glu Leu Leu

305 310 315 320

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

325 330 335

Leu His Ile Ala Ala Thr Asn Gly His Leu Glu Ile Val Glu Val Leu

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro

420 425 430

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu

435 440 445

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

450 455 460

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

465 470 475 480

Gly Leu Thr Pro Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu Ile

485 490 495

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

500 505 510

Ile Gly Phe Thr Pro Leu His Leu Ala Ala Phe Ile Gly His Leu Glu

515 520 525

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

530 535 540

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

545 550 555 560

Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

565 570

<210> 26

<211> 562

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising four designed ankyrin repeat domains

<400> 26

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Gly Ser Gly Ser Asp Leu Gly Ala Lys Leu Leu Ser Asp Leu Gly Val

145 150 155 160

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

165 170 175

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

180 185 190

Thr Pro Leu His Ile Ala Ala Gln Ser Gly His Leu Glu Ile Val Glu

195 200 205

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val Thr Gly

210 215 220

Asp Thr Pro Leu His Leu Ala Ala Gln His Gly His Leu Val Ile Val

225 230 235 240

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

245 250 255

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

260 265 270

Ala Glu Val Leu Glyn Lys Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Ala Ala

<210> 27

<211> 578

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising four designed ankyrin repeat domains

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Gly Ile Thr Pro Leu His Ile Ala Ala Gln Ser Gly His Leu Glu Ile

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr

275 280 285

Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Ala Val Asp Ala Ile Gly Phe Thr Pro Leu His Leu Ala Ala Phe Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

435 440 445

Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Ala Ala

<210> 28

<211> 283

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising two designed ankyrin repeat domains

<400> 28

Gly Ser Asp Leu Gly Ile Lys Leu Leu Phe Ala Ala Ala Lys Gly Gln

1 5 10 15

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

20 25 30

Lys Asp Phe Gln Gly Val Thr Pro Leu His Ile Ala Ala Gln Ser Gly

35 40 45

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

50 55 60

Ala Lys Asp Val Thr Gly Asp Thr Pro Leu His Leu Ala Ala Gln His

65 70 75 80

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

85 90 95

Asn Ala Gln Asp Glu Arg Gly Trp Thr Pro Ala Asp Leu Ala Ala Asp

100 105 110

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

115 120 125

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

130 135 140

Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala

145 150 155 160

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

165 170 175

Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro Leu Tyr Leu Ala

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ala Ala Gly Gly Gly Ser Gly Gly Gly Ser Cys

275 280

<210> 29

<211> 414

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising three designed ankyrin repeat domains

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Ser Arg Ser Asp Leu Gly Ala Lys Leu Leu Ser Asp Leu Gly Val Lys

145 150 155 160

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

165 170 175

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

180 185 190

Pro Leu His Ile Ala Ala Gln Ser Gly His Leu Glu Ile Val Glu Val

195 200 205

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val Thr Gly Asp

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Glu Val Leu Gln Lys Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Ala Val Asp Ala Ile Gly Phe Thr Pro Leu His Leu Ala Ala Phe Ile

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410

<210> 30

<211> 562

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising four designed ankyrin repeat domains

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

275 280 285

Gly Gly Gly Ser Gly Ser Asp Leu Gly Ala Lys Leu Leu Ser Asp Leu

290 295 300

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

305 310 315 320

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

325 330 335

Gly Ile Thr Pro Leu His Ile Ala Ala Gln Ser Gly His Leu Glu Ile

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro Leu Tyr Leu Ala

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Ala Ala

<210> 31

<211> 580

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising four designed ankyrin repeat domains

<400> 31

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

1 5 10 15

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

20 25 30

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser His

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

130 135 140

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

145 150 155 160

Asp Leu Gly Ile Lys Leu Leu Phe Ala Ala Ala Lys Gly Gln Asp Asp

165 170 175

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

180 185 190

Phe Gln Gly Val Thr Pro Leu His Ile Ala Ala Gln Ser Gly His Leu

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gln Asp Glu Arg Gly Trp Thr Pro Ala Asp Leu Ala Ala Asp Trp Gly

260 265 270

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

275 280 285

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

435 440 445

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Gln Lys Ala Ala

580

<210> 32

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising two designed ankyrin repeat domains

<400> 32

Gly Ser Asp Leu Gly Ile Lys Leu Leu Phe Ala Ala Ala Lys Gly Gln

1 5 10 15

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

20 25 30

Lys Asp Phe Gln Gly Val Thr Pro Leu His Ile Ala Ala Gln Ser Gly

35 40 45

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

50 55 60

Ala Lys Asp Val Thr Gly Asp Thr Pro Leu His Leu Ala Ala Gln His

65 70 75 80

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

85 90 95

Asn Ala Gln Asp Glu Arg Gly Trp Thr Pro Ala Asp Leu Ala Ala Asp

100 105 110

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

115 120 125

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

130 135 140

Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala

145 150 155 160

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

165 170 175

Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro Leu Tyr Leu Ala

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ala Ala

<210> 33

<211> 422

<212> PRT

<213> Artificial Sequence

<220>

<223> Protein comprising three designed ankyrin repeat domains

<400> 33

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr

130 135 140

Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu Leu Phe Ala Ala

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Ala Asp Val Asn Ala Gln Asp Glu Arg Gly Trp Thr Pro Ala Asp

245 250 255

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

260 265 270

Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro

275 280 285

Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu

290 295 300

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

305 310 315 320

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro

325 330 335

Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu Ile Val Glu Val Leu

340 345 350

Leu Lys Ala Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr

355 360 365

Pro Leu His Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala 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 Pro Ala Asp Ile Ala Ala Gly Ala Gly Asn Glu Asp Ile Ala Glu

405 410 415

Val Leu Gln Lys Ala Ala

420

<---

Claims (15)

1. Recombinant binding protein for binding human epidermal growth factor receptor 2 (HER2) and serum albumin, containing an amino acid sequence that is characterized by at least 97% amino acid sequence identity with respect to SEQ ID NO: 21, and the specified recombinant binding protein contains two engineered ankyrin repeat domains with binding specificity for HER2; and two engineered ankyrin repeat domains with binding specificity for serum albumin. 2. The recombinant binding protein of claim 1, wherein said recombinant binding protein contains an amino acid sequence that has at least 99% amino acid sequence identity according to SEQ ID NO: 21. 3. The recombinant binding protein of claim 1, wherein each of said two engineered ankyrin repeat domains with binding specificity for serum albumin contains an amino acid sequence according to SEQ ID NO: 14. 4. The recombinant binding protein of claim 3, wherein each of said engineered ankyrin repeat domains with binding specificity for serum albumin exhibits improved storage stability in PBS compared to the engineered ankyrin repeat domain with binding specificity for serum albumin, containing the amino acid sequence according to SEQ ID NO: 13. 5. The recombinant binding protein of claim 1, wherein said recombinant binding protein contains the amino acid sequence according to SEQ ID NO: 21. 6. The recombinant binding protein of claim 1, wherein said recombinant binding protein consists of the amino acid sequence according to SEQ ID NO: 21. 7. The recombinant binding protein of claim 1, wherein said recombinant binding protein inhibits the proliferation of BT474 cells with an inhibition constant below 10 ^-7 M. 8. The recombinant binding protein of claim 1, wherein said recombinant binding protein at a concentration of 100 nM exhibits stronger downregulation of AKT-S473 phosphorylation in BT474 cells than does trastuzumab at a concentration of 100 nM. 9. The recombinant binding protein of claim 1, wherein said recombinant binding protein contains an amino acid sequence that has at least 98% amino acid sequence identity according to SEQ ID NO: 21. 10. The recombinant binding protein of claim 1, wherein said binding protein binds to human HER2 in PBS with a dissociation constant (Kd) below 10 ^-7 M, and wherein said binding protein binds to human serum albumin in PBS with a dissociation constant (Kd) below ^10-7M . 11. Nucleic acid encoding the recombinant binding protein of claim 1. 12. A pharmaceutical composition for the treatment of HER2-expressing cancer, comprising a therapeutically effective amount of the recombinant binding protein of claim 1 and a pharmaceutically acceptable carrier and/or diluent. 13. A method of treating a disease in a subject in need of such treatment, comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition according to claim 12, wherein the disease is a cancer expressing HER2. 14. The method of treatment according to claim 13, wherein the HER2-expressing cancer is HER2 overexpressing cancer, HER2 addictive cancer, HER2 partial addictive cancer, or HER2 amplifying cancer. 15. The method of treatment according to claim 13, wherein the cancer is breast cancer, ovarian cancer, gastrointestinal cancer, or gastric cancer.

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