WO2013135748A1 - Polypeptides binding to dll4 and uses thereof - Google Patents

Abstract

The present invention relates to a polypeptide, a scaffold protein comprising said polypeptide and a scaffold backbone, which positions the polypeptide at an exposed surface of the scaffold protein, preferably for use in diagnosing, treating or preventing disorders associated with angiogenesis, in particular for diagnosing, treating or preventing tumor angiogenesis, and to a pharmaceutical composition comprising said polypeptide or said scaffold, which positions the polypeptide of the invention at an exposed surface of the scaffold protein.

Description

POLYPEPTIDES BINDING TO DLL4 AND USES THEREOF

The present invention relates to a polypeptide, a scaffold protein comprising said polypeptide and a scaffold backbone, which positions the polypeptide at an exposed surface of the scaffold protein, preferably for use in diagnosing, treating or preventing disorders associated with angiogenesis, in particular for diagnosing, treating or preventing tumor angiogenesis, and to a pharmaceutical composition comprising said polypeptide or said scaffold protein, which positions the polypeptide of the invention at an exposed surface of the scaffold protein.

BACKGROUND OF THE INVENTION

Angiogenesis is a normal physiological process of the human body involving the formation of new blood vessels from pre-existing ones. However, angiogenesis is also a fundamental step in tumor development as well as for metastasis and regarded as one major hallmark of cancer [1]. Consequently, the identification and development of new anti-angiogenic agents is a promising strategy for tumor imaging and therapy. In cancer therapy, several approaches address vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) which are involved in the induction of angiogenesis [2, 3]. It has been shown that the inhibition of VEGF signalling results in tumor growth delay in a wide range of animal models [4]. The application of the humanized monoclonal anti-VEGF antibody bevacizumab, the most prominent anti-angiogenic drug, is approved for treatment of metastatic colon cancer, non-small lung cancer, breast cancer, renal cancer and glioblastome [5, 6]. Unfortunately, several tumors are insensitive or resistant towards VEGF treatment which implicates that new anti-angiogenic drugs are still required [7]. Besides VEGF and bFGF, the membrane protein Delta- like 4 (D114) is involved in the regulation of angiogenesis by binding to a Notch receptor [8, 9]. Studies in zebrafish and mouse indicate that the Notch signalling pathway is involved in coordinating cellular behaviours during angiogenesis [10]. Due to a selective overexpression in tumor vasculature and several tumor cells, D114 represents a high-potential candidate for angiogenesis targeting [11]. The interaction of D114 and its Notch receptor causes the cleavage of the intracellular Notch receptor domain which translocates into the nucleus after cleavage and activates the expression of several transcription factors [12, 7]. In contrast to the VEGF-mediated pro-angiogenic signal, the interaction between D114 and Notch receptor initiates a negative regulatory signalling and decrease of sprouting. In conclusion, the balanced combination of positive and negative signals during angiogenesis leads to the successful generation of new blood vessels [13]. The inhibition of the negative regulatory pathway induced by D114 results in an increased sprouting, the formation of unproductive new blood vessels and as shown in preclinical studies, growth inhibition of tumors [13, 14, 15, 16, 17, 18, 7].

TECHNICAL PROBLEMS UNDERLYING THE PRESENT INVENTION

Antibodies binding specifically to D114 are known from the prior art and were developed to induce the pathological synthesis of endothelial cells in tumors [15, 16, 17]. The high affinity and specificity of antibodies towards their target structure combined with their customizable pharmacokinetic properties (e.g. long half-life) makes antibodies attractive for targeted cancer treatment [19, 20]. However, next to the high costs caused by a time-consuming synthesis, the risk of toxicity and potential allergic reactions, one major disadvantage of antibodies is their large size which slows the diffusion into tissue and results in a low signal-to-noise ratio [20, 21].

Thus, especially in view of a potential clinical use in patients with disorders associated with angiogenesis, there is a need in the art for an alternative molecule to treat said disorders.

Peptides represent a promising alternative to antibodies. Due to their small size their diffusion is not limited and they offer similar binding affinities as antibodies [22]. In addition, the synthesis of peptides is easier and less expensive. Unfortunately, the application of linear peptides is often limited by their low stability in serum caused by enzyme-degradation [23]. Peptide modifications, e.g. the use of unnatural amino acids and chain substitutions, increase the serum stability but decrease simultaneously the target affinity in most cases. The application of scaffold proteins instead of linear peptides solves this problem. Scaffold proteins are small, stable and characterized by a conformational constrained structure [24]. Two representatives are the scaffold proteins Min-23 and the sunflower trypsin inhibitor I (SFTI). The trypsin inhibitor Min-23 is composed of 23 amino acids, stabilized by two disulfide bonds and permissive for loop insertions whereas SFTI consists of 14 amino acids and is stabilized by one disulfide bond (Fig. 1) [25, 26]. In contrast to linear peptides which offer generally a half-live of few minutes in human serum, the half-live of SFTI is 34.5 hours in open and 75.8 hours in cyclic conformation [27].

Quite surprisingly, the inventors of the present invention were able to identify a new D114-specific scaffold polypeptide according to the SEQ ID NO: 1 which is useful for treating or preventing disorders associated with angiogenesis, in particular tumor angiogenesis, as specified throughout the description. SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a polypeptide comprising or consisting of the amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 1); wherein Xi is absent or any amino acid; wherein X₂ is selected from the group consisting of F and Y; wherein X3 or X₄ is any amino acid; wherein X₅ is selected from the group consisting of F and Y; wherein X₆ is selected from the group consisting of L, V, and I; wherein X₇ is selected from the group consisting of F and Y; and wherein X₈ is selected from the group consisting of L, V, and I, or a pharmaceutically acceptable salt thereof.

In a second aspect, the present invention provides a scaffold protein comprising the polypeptide of the first aspect, and a scaffold backbone, which positions the polypeptide of the first aspect at an exposed surface of said scaffold protein.

In a third aspect, the present invention provides a conjugate of

(i) a polypeptide of the first aspect of the invention or the scaffold protein of the second aspect of the invention, and

(ii) a payload, which is covalently or non-covalently linked to the polypeptide or scaffold protein.

In a fourth aspect, the present invention provides a nucleic acid encoding the polypeptide of the first aspect of the invention, the scaffold protein of the second aspect of the invention or the conjugate of the third aspect of the present invention.

In a fifth aspect, the present invention relates to the polypeptide of the first aspect, the scaffold protein of the second aspect, the conjugate of the third aspect, or the nucleic acid of the fourth aspect for medical use.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the polypeptide of the first aspect, the scaffold protein of the second aspect, the conjugate of the third aspect, the nucleic acid of the fourth aspect and a pharmaceutical acceptable excipient, carrier and/or diluent.

In a seventh aspect, the present invention provides the polypeptide of the first aspect, the scaffold protein of the second aspect, the conjugate of the third aspect, the nucleic acid of the fourth aspect or the pharmaceutical composition of the fifth aspect for use in diagnosing, treating or preventing disorders associated with angiogenesis.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: Min-23 and SFTI scaffold structures. Figure 1 shows a schematic representation of the miniprotein scaffolds Min-23 (left) and SFTI (right) scaffold structures, wherein the loops which are colored dark were replaced by variable amino acids and the disulfide bonds are colored light. FIG. 2A to C: Binding analysis between D114 and (a) D11SF3 (0.25, 0.5, 0.75, 1, 1.25 μΜ) and (b) SFTI (0.25, 0.5, 0.75, 1, 1.25 μΜ) using Biacore X100. D114 was immobilized on a CM5 sensor chip (8690 RU) using standard amine coupling method. In contrast to SFTI (Fig. 2A, Panel b)) an specific interaction between D114 and D11SF3 was verified (Fig. 2A, Panel b)) and a Kd value of 22 nM was determined.

In order to analyze the specificity of interaction, the Notch receptor was immobilized on a CM5 sensor Chip (3116 RU) and again both peptides were applied as analytes. Binding analysis of Notch and (c) D11SF3 (0.25, 0.5, 0.75, 1, 1.25 μΜ) and (d) SFTI (0.25, 0.5, 0.75, 1, 1.25 μΜ) were performed using Biacore. As shown in Figure 2B, Panel c), the affinity of D11SF3 and SFTI to Notch- 1 showed only unspecific binding (Fig. 2B, Panel d)). In Figure 2c, the result of binding analysis of D114 and Notch after addition of D11SF3 is shown. D114 was incubated with increasing concentrations of D11SF3 prior to flowing across a chip immobilized with Notch- 1. In contrast to the control, the binding decreased with increasing concentrations of D11SF3. 95% inhibition of binding was observed after addition of 11.42 μΜ D11SF3. FIG. 3A and B: Binding kinetics of D11SF3 and SFTI on (Panel a) PC-3 cells, (Panel b) HUVECs and (Panel c) AR42J. The in vitro binding of D11SF3 and of the native scaffold SFTI to the different cell lines was analyzed after different incubation times.

FIG. 4A to C: Fluorescent cell staining and flow cyctometry analysis on PC-3 cells. Fig. 4A shows confocal microscopy images of PC-3 cells taken from 5(6)FAM-labeled peptides co- stained with DAPI for nuclei visualization of 5(6)FAM-D11SF3 (left), 5(6)FAM-D11SF3-A9 (middle) and co-incubation with labeled DLLSF3 (right). Both assay systems demonstrated specific binding of 5(6)FAM-D11SF3 to cell line PC-3. Fig. 5: Metabolic stability of D11SF3 was determined. The peptide was incubated in human serum at 37 °C and after defined time points (10 min, 1 h, 2 h, 4 h, 24 h, 48 h, 6 d, 10 d) aliquots were taken, serum proteins precipitated with acetonitrile and analyzed by HPLC. No second peak, indicating the degradation of the peptide, was detected after 10 days. Fig. 6: To determine the biodistribution in mice carrying AR42J tumors, D11SF3 was conjugated with ¹³¹Iod and the biodistribution was analyzed after 120 min. An accumulation of 1.47 % ID/g of D11SF3 in the tumor was detected whereas the peptide SFTI without D114-binding sequence offered an accumulation of only 0.53 % ID/g. As a result of the high amount of lipophilic amino acids in the D114-binding sequence the highest concentration was observed in the liver (29.89 % ID/g).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechno logical terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, and recombinant DNA techniques are employed which are explained in the literature in the field (cf, e.g., Molecular Cloning: A Laboratory Manual, 2^nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Furthermore, conventional methods of clinical cardiology are employed which are also explained in the literature in the field (cf., e.g., Practical Methods in Cardiovascular Research, S. Dhein et al. eds., Springer Verlag Berlin Heidelberg, 2005).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The term "Kd value", is used herein as an indicator of the binding strength between two molecules and refers to the dissociation equilibrium constant of the particular interaction between a binding moiety (e.g. peptide 1, polypeptide 1, protein 1, or a fragment thereof) and a target molecule (e.g. peptide 2, polypeptide 2, protein 2, or a fragment thereof) and is measured in "mol/L", which is sometimes abbreviated as "M".

As used herein, a first compound (e.g. polypeptide 1) is considered to " specifically bind" to a second compound (e.g. polypeptide 2), if it has a dissociation constant Kd to said second compound of 1 mM or less, preferably 100 μΜ or less, preferably 50 μΜ or less, preferably 30 μΜ or less, preferably 20 μΜ or less, preferably 10 μΜ or less, preferably 5 μΜ or less, more preferably 1 μΜ or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, more preferably 200 nM or less, more preferably 100 nM, more preferably 90 nM or less, more preferably 80 nM or less, more preferably 70 nM or less, more preferably 60 nM or less; more preferably 50 nM or less, more preferably 40 nM or less, more preferably 30 nM or less, more preferably 20 nM or less or more preferably 10 nM or less .

The term "amino acid" encompasses naturally occurring amino acids, consisting of Ala,

Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val as well as amino acid derivatives, sometimes called unnatural amino acid analogues that belongs not to one of the 20 common naturally occurring amino acids and which can be D-isomers of the naturally occurring L-isomers, Aad, bAad, bAla, Abu, 4Abu, Acp, Ahe, Aib, bAib, Apm, Dbu, Des, Dpm, Dpr, EtGly, EtAsn, Eth, Hyl, aHyl, 3Hyp, 4Hyp, Ide, alle, MeGly, Melle, MeLys, MeVal, Nva, Nle or Orn, preferably Nle. Techniques for substitutions of amino acids in (polypeptides or proteins with amino acid derivates are well know in the art of protein chemistry (see e.g., [37], [38], [39]). If amino acid derivative are used it is preferred that within a given sequence of naturally amino acids one or more natural amino acids are replaced by amino acid derivates that are isostructural or very closely related to the natural amino acid. For instance Met may be substituted by Nle, since Nle has a methylene group, which has a similar bulk as the naturally occurring sulfur atom in the side chain of Met or L-Ala is substituted by D-Ala.

The term "polypeptide" or "protein" are used interchangeably and refer to a chain of naturally occurring and/or non-naturally occurring amino acid residues linked by peptide bonds.

The term "XX percent identity" means that two peptides or nucleotide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share the respectively indicated level of identity. The percent of sequence identity is preferably determined on the basis of the full length polypeptide or scaffold protein of the present invention specifically indicated, e.g. if a specifically indicated polypeptide has a length of 10 amino acids another polypeptide that has a length of 8 amino acids, which are identical to a stretch of 8 amino acids of the specifically indicated polypeptide the two polypeptides share 80% sequence identity. Thus, when percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the reference sequence, if not specifically indicated otherwise. Preferably, residues which are not identical differ by conservative amino acid substitutions.

Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences [Pearson (2000) supra]. Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See e.g., Altschul et al. [32, 33], each of which is herein incorporated by reference. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art, see e.g., [28]. Examples of groups of amino acids that have side chains with similar chemical properties include

1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine;

2) aliphatic- hydroxyl side chains: serine and threonine;

3) amide-containing side chains: asparagine and glutamine;

4) aromatic side chains: phenylalanine, tyrosine, and tryptophan;

5) basic side chains: lysine, arginine, and histidine;

6) acidic side chains: aspartate and glutamate, and

7) sulfur-containing side chains: cysteine and methionine.

Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine- glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. [29]. A "moderately conservative" replacement is any change having a non-negative value in the PAM250 log-likelihood matrix. Given the known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist can readily construct DNAs encoding conservative amino acid variants.

As used herein, "non-conservative substitutions" or "non-conservative amino acid exchanges" are defined as exchanges of an amino acid by another amino acid listed in a different group of the seven standard amino acid groups 1) to 7) shown above.

The term "scaffold protein" refers to a protein which possesses structural rigidity, i.e. folds into a stable tertiary structure, preferably at 30°C to 40°C; more preferably at 37°C and physiologic conditions. Accordingly, the amino acids of a scaffold protein are likely to occupy a defined three-dimensional position within the scaffold protein. Thus, if one or more of the amino acids of a scaffold protein are replaced by a polypeptide of a suitable length the polypeptide will occupy similar positions as those replaced. This allows positioning a given polypeptide at a defined three-dimensional location and/or orientation within the scaffold protein. Accordingly, scaffold proteins can be used as an alternative to antibodies for molecular recognition [30]. These properties of scaffold proteins were used to integrate the affinity of the polypeptides of the invention, integration of an affinity function into a well-defined, stably folded structural framework by locally reshaping the molecular surface through primary structure modifications, and thus, the design of a small molecule mimetic [31]. The amino acids of the scaffold protein remaining after the replacement or insertion are also referred to as "scaffold backbone". As the insertion/replacement typically is located within the amino acid chain of the scaffold protein the term "scaffold backbone" typically refers to two discontinuous amino acid chains that are located N- and C-terminally to the polypeptide that is inserted or that replaces one or more of the amino acids of the scaffold protein. To ascertain structural rigidity it is preferred that the scaffold protein comprises one, two or more disulfide bonds, which may be formed between two, four or more cysteine residues of the scaffold protein preferably between the discontinuous amino acid chains that are located N- and C-terminally to the polypeptide (see, e.g. Fig. 1). As mentioned above, in an alternative embodiment a polypeptide is inserted into a region of the scaffold protein, i.e. without removing any of the amino acids of the scaffold protein. In either case, it is preferred that the region of the scaffold protein into which the polypeptide is inserted or wherein one or more amino acids are replaced is a region which does not contribute to the structural rigidity of the scaffold protein. Suitable regions are, e.g. loop regions of the scaffold proteins. In the context of the present invention, a distinction is made between the initial scaffold protein which is the basis for the scaffold protein of the present invention, which comprises the polypeptide of the present invention. Unless indicated otherwise, the term "scaffold protein" is used herein to refers to the scaffold protein of the invention comprising the polypeptide of the invention.

The term "payload", as used in the present invention, comprises any compound that may be delivered by the polypeptides of scaffold proteins of the present invention to their site of binding. Preferably, the payload is a therapeutic and/or diagnostic compound, wherein the therapeutic compound is selected from the group consisting of cytostatic compound, cytotoxic compound, anti-angiogenic compound, an alkylating agent, an anti-metabolite compound, a natural product, a hormone, an oncogenic inhibitor, a therapeutic isotope or a therapeutic antibody, and the diagnostic compound is selected from the group consisting of an electron dense compound, a paramagnetic compound, a superparamagnetic compound, a fluorophor, radioactive compound, a fluorescent compound, a non-radioactive isotope, a diagnostic isotope, paramagnetic label and fusion protein which are well known in the art.

The term "carrier", when used herein, refers to an adjuvant or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

A pharmaceutically composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The compounds of the invention can be formulated as neutral or salt forms. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

Generally known and practiced methods in the fields of molecular biology, cell biology, protein chemistry and antibody techniques are fully described in the continuously updated publications "Molecular Cloning: A Laboratory Manual", (Sambrook et al, Cold Spring Harbor); Current Protocols in Molecular Biology (F. M. Ausubel et al. Eds., Wiley & Sons); Current Protocols in Protein Science (J. E. Colligan et al. eds., Wiley & Sons); Current Protocols in Cell Biology (J. S. Bonifacino et al, Wiley & Sons) and Current Protocols in Immunology (J. E. Colligan et al, Eds., Wiley & Sons). Known techniques relating to cell culture and media are described in "Large Scale Mammalian Cell Culture (Hu et al, Curr. Opin. Biotechnol. 1997, 8: 148-153); "Serum free Media" (Kitano K., Biotechnology 1991, 17:73-106); and "Suspension Culture of Mammalian Cells" (Birch et al., Bioprocess Technol. 1990, 19: 251).

As used herein, "treat", "treating" or "treatment" of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity and/or duration of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).

As used herein, "prevent", "preventing", "prevention", or "prophylaxis" of a disease or disorder means preventing that a disorder occurs in subject.

As used herein, the expression "is for administration" and "is to be administered" have the same meaning as "is prepared to be administered". In other words, the statement that an active compound "is for administration" has to be understood in that said active compound has been formulated and made up into doses so that said active compound is in a state capable of exerting its therapeutic activity.

The terms "therapeutically effective amount" or "therapeutic amount" are intended to mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, a system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. As used herein, a therapeutically effective amount of the polypeptide is an amount that is sufficient to treat or prevent disorders associated with angiogenesis, in particular tumor angiogenesis. The term "prophylactically effective amount" is intended to mean that amount of a pharmaceutical drug that will prevent or reduce the risk of occurrence of the biological or medical event that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician. Dosage levels are based on a variety of factors, including age, weight, sex, medical condition of the individual, severity of the condition, administration route, and the particular compound employed. Therefore, the dosage regimen may vary but can be determined routinely by a physician using standard metho ds .

Preferred embodiments

In a first aspect, the present invention provides a polypeptide comprising or consisting of the amino acid sequence: X1-X2-X3-X4- 5- 6- 7- 8 (SEQ ID NO: 1), wherein Xi is absent or any amino acid, preferably Xi is selected from the group consisting of L, V, and I, more preferably is L; wherein X₂ is selected from the group consisting of F and Y, preferably is F; wherein X₃ or X₄ is any amino acid, preferably X₃ is H and X₄ is preferably selected from the group consisting of L, V, and I, more preferred X₄ is L; wherein X₅ is selected from the group consisting of F and Y, preferably is F; wherein X₆ is selected from the group consisting of L, V, and I, preferably is I; wherein X₇ is selected from the group consisting of F and Y, preferably is Y; and wherein X₈ is selected from the group consisting of L, V, and I, preferably is I, or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the polypeptide of the first aspect has a length of 50 amino acids or less then 50, less than 45, less then 40, less then 35, less then 30, less then 25 continuous amino acids. More preferably the polypeptide has a length of 20 continuous amino acids, more preferably of 19 continuous amino acids, more preferably of 18 continuous amino acids, more preferably of 17 continuous amino acids, more preferably of 16 continuous amino acids, more preferably of 15 continuous amino acids, more preferably of 14 continuous amino acids, more preferably of 13 continuous amino acids, more preferably of 12 continuous amino acids, more preferably of 11 continuous amino acids, acids, more preferably of 10 continuous amino acids, more preferably of 9 continuous amino acids, more preferably of 8 continuous amino acids. A particular preferred range of the length of the polypeptide is between 7 and 10 continuous amino acids, most preferably of 8 continuous amino acids.

In a preferred embodiment, the polypeptide of the first aspect comprises or consists of the amino acid sequence X1-X2-X3-X4- 5- 6- 7- 8, wherein X₂ is F, X₅ is F, X₆ is I, X₇ is Y and/or Xs is I. Thus, in preferred combinations the following amino acids have the following meanings (in each case those amino acids not further designated have the above indicated preferred and particularly preferred meanings): Xi is L and X₂ is F; Xi is L and X₅ is F; Xi is L and X₆ is I; Xi is L and X₇ is Y; Xi is L and Xs is I; X₂ is F and X₅ is F; X₂ is F and X₆ is I; X₂ is F and X₇ is Y; X₂ is F and Xs is I; X₅ is F and X₆ is I; X₅ is F and X₇ is Y; X₅ is F and Xs is I; X₆ is I and X₇ is Y; X₆ is I and Xs is I; X₇ is Y and Xs is I; Xi is L, X₂ is F and X₅ is F; Xi is L, X₂ is F and X₆ is I; Xi is L, X₂ is F and X₇ is Y; Xi is L, X₂ is F, and Xs is I; Xi is L, X₅ is F, X₆ is I; Xi is L, X₅ is F and X₇ is Y; Xi is L, X₅ is F, and Xs is I; Xi is L, X₆ is I and X₇ is Y; Xi is L, X₆ is I, and Xs is I; X₂ is F, X₅ is F and X₆ is I; X₂ is F, X₅ is F and X₇ is Y; X₂ is F, X₅ is F, and Xs is I; X₂ is F, X₆ is I and X₇ is Y; X₂ is F, X₆ is I and Xs is I; X₂ is F, X₇ is Y and Xs is I; X₅ is F, X₆ is I and X₇ is Y; X₅ is F, X₆ is I and Xs is I; X₅ is F, X₇ is Y and Xs is I; X₆ is I, X₇ is Y and Xs is I; Xi is L, X₂ is F, X₅ is F and X₆ is I; Xi is L, X₂ is F, X₅ is F and X₇ is Y; Xi is L, X₂ is F, X₅ is F, and Xs is I; Xi is L, X₅ is F, X₆ is I, X₇ is Y; Xi is L, X₅ is F, X₆ is I, and Xs is I; Xi is L, X₆ is I, X₇ is Y and Xs is I; X₂ is F, X₅ is F, X₆ is I and X₇ is Y; X₂ is F, X₅ is F, X₆ is I and Xs is I; X₂ is F, X₅ is F, X₇ is Y and Xs is I; X₂ is F, X₆ is I, X₇ is Y and Xs is I; X₅ is F, X₆ is I, X₇ is Y and Xs is I; Xi is L, X₂ is F, X₅ is F, X₆ is I and X₇ is Y; Xi is L, X₂ is F, X₅ is F, X₆ is I and Xs is I; Xi is L, X₅ is F, X₆ is I, X₇ is Y and Xs is I; Xi is L, X₂ is F, X₆ is I, X₇ is Y and Xs is I; Xi is L, X₂ is F, X₅ is F, X₇ is Y and Xs is I; X₂ is F, X₅ is F, X₆ is I, X₇ is Y and Xs is I or Xi is L, X₂ is F, X₅ is F, X₆ is I, X₇ is Y and Xs is I.

Preferably, the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: X₁-F-X₃-X₄-F-I-Y-I (SEQ ID NO: 8); X₁-Y-X₃-X₄-F-I-Y-I (SEQ ID NO: 9); X1-F-X3-X4-Y-I-Y-I (SEQ ID NO: 10); X1-Y-X3-X4-Y-I-Y-I (SEQ ID NO: 11); ¾- F-X₃-X₄-F-L-Y-I (SEQ ID NO: 12); X₁-Y-X₃-X₄-F-L-Y-I (SEQ ID NO: 13); X₁-F-X₃-X₄-Y-L- Y-I (SEQ ID NO: 14); X₁-Y-X3-X4-Y-L-Y-I (SEQ ID NO: 15); X₁-F-X₃-X₄-F-V-Y-I (SEQ ID NO: 16); X1-Y-X3-X4-F-V-Y-I (SEQ ID NO: 17); X1-F-X3-X4-Y-V-Y-I (SEQ ID NO: 18); ¾- Y-X3-X₄-Y-V-Y-I (SEQ ID NO: 19); X₁-F-X₃-X4-F-I-F-I (SEQ ID NO: 20); X₁-Y-X₃-X4-F-I-F-I (SEQ ID NO: 21); X₁-F-X₃-X4-Y-I-F-I (SEQ ID NO: 22); X₁-Y-X₃-X4-Y-I-F-I (SEQ ID NO: 23); X1-F-X3-X4-F-L-F-I (SEQ ID NO: 24); X1-Y-X3-X4-F-L-F-I (SEQ ID NO: 25); X1-F-X3- X₄-Y-L-F-I (SEQ ID NO: 26); X₁-Y-X₃-X4-Y-L-F-I (SEQ ID NO: 27); X₁-F-X₃-X4-F-V-F-I (SEQ ID NO: 28); X₁-Y-X₃-X4-F-V-F-I (SEQ ID NO: 29); X₁-F-X₃-X4-Y-V-F-I (SEQ ID NO: 30); X₁-Y-X3-X₄-Y-V-F-I (SEQ ID NO: 31); X₁-F-X₃-X4-F-I-Y-L (SEQ ID NO: 32); X₁-Y-X₃- X₄-F-I-Y-L (SEQ ID NO: 33); X₁-F-X₃-X4-Y-I-Y-L (SEQ ID NO: 34); X₁-Y-X₃-X4-Y-I-Y-L (SEQ ID NO: 35); X₁-F-X₃-X₄-F-L-Y-L (SEQ ID NO: 36); X₁-Y-X₃-X₄-F-L-Y-L (SEQ ID NO: 37); X₁-F-X₃-X₄-Y-L-Y-L (SEQ ID NO: 38); X₁-Y-X₃-X₄-Y-L-Y-L (SEQ ID NO: 39); ¾- F-X₃-X₄-F-V-Y-L (SEQ ID NO: 40); X₁-Y-X₃-X₄-F-V-Y-L (SEQ ID NO: 41); X₁-F-X₃-X₄-Y- V-Y-L (SEQ ID NO: 42); X₁-Y-X₃-X₄-Y-V-Y-L (SEQ ID NO: 43); X₁-F-X₃-X4-F-I-F-L (SEQ ID NO: 44); X₁-Y-X₃-X4-F-I-F-L (SEQ ID NO: 45); X₁-F-X₃-X4-Y-I-F-L (SEQ ID NO: 46); ¾- Y-X₃-X₄-Y-I-F-L (SEQ ID NO: 47); X₁-F-X₃-X₄-F-L-F-L (SEQ ID NO: 48); X₁-Y-X₃-X4-F-L- F-L (SEQ ID NO: 49); X₁-F-X₃-X₄-Y-L-F-L (SEQ ID NO: 50); X₁-Y-X₃-X₄-Y-L-F-L (SEQ ID NO: 51); X₁-F-X₃-X₄-F-V-F-L (SEQ ID NO: 52); X₁-Y-X₃-X₄-F-V-F-L (SEQ ID NO: 53); ¾- F-X₃-X₄-Y-V-F-L (SEQ ID NO: 54); X₁-Y-X₃-X₄-Y-V-F-L (SEQ ID NO: 55); X₁-F-X₃-X4-F-I- Y-V (SEQ ID NO: 56); X1-Y-X3-X4-F-I-Y-V (SEQ ID NO: 57); X1-F-X3-X4-Y-I-Y-V (SEQ ID NO: 58); X₁-Y-X₃-X4-Y-I-Y-V (SEQ ID NO: 59); X₁-F-X₃-X₄-F-L-Y-V (SEQ ID NO: 60); ¾- Y-X₃-X₄-F-L-Y-V (SEQ ID NO: 61); X₁-F-X₃-X₄-Y-L-Y-V (SEQ ID NO: 62); X₁-Y-X₃-X₄-Y- L-Y-V (SEQ ID NO: 63); X₁-F-X₃-X₄-F-V-Y-V (SEQ ID NO: 64); X₁-Y-X₃-X₄-F-V-Y-V (SEQ ID NO: 65); X₁-F-X₃-X₄-Y-V-Y-V (SEQ ID NO: 66); X₁-Y-X₃-X₄-Y-V-Y-V (SEQ ID NO: 67); X1-F-X3-X4-F-I-F-V (SEQ ID NO: 68); X1-Y-X3-X4-F-I-F-V (SEQ ID NO: 69); X1-F-X3- X₄-Y-I-F-V (SEQ ID NO: 70); X₁-Y-X₃-X4-Y-I-F-V (SEQ ID NO: 71); X₁-F-X₃-X₄-F-L-F-V (SEQ ID NO: 72); X₁-Y-X₃-X₄-F-L-F-V (SEQ ID NO: 73); X₁-F-X₃-X₄-Y-L-F-V (SEQ ID NO: 74); X₁-Y-X₃-X₄-Y-L-F-V (SEQ ID NO: 75); X₁-F-X₃-X₄-F-V-F-V (SEQ ID NO: 76); X₁-Y-X₃- X₄-F-V-F-V (SEQ ID NO: 77); X₁-F-X₃-X₄-Y-V-F-V (SEQ ID NO: 78); and X₁-Y-X₃-X4-Y-V- F-V (SEQ ID NO: 79).

In a preferred embodiment, the polypeptide of the first aspect comprising or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8, wherein Xi is absent or selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, V, preferably L, V and I, most preferably L; wherein X₃ is selected from the group consisting of A, R, N, D, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, preferably H and K, most preferred H; and wherein X₄ is selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, preferably L, V and I, most preferred L.

In a preferred embodiment Xi is not M. In a further preferred embodiment X₃ is not Q. In a preferred embodiment is Xi not M and X₃ is not Q.

In a preferred embodiment, the polypeptide of the present invention comprises or consists of the amino acid sequence: L-F-H-L-F-I-Y-I (SEQ ID NO: 2), wherein optionally no more than 2, and preferably no more than 1 amino acid is/are replaced with another amino acid as specified above. Alternatively or additionally, the polypeptide of the invention may comprise with respect to SEQ ID NO: 2 no more than 1 or 2 amino acid deletions and/or insertions, preferably no more than 1 amino acid deletions and/or insertions.

In each case indicated above, the polypeptide of the invention based on the amino acid sequence according to SEQ ID NO: 2 maintain their ability to specifically bind to D114.

A person skilled in the art is well aware of methods for producing polypeptides according to the present invention. For example, the polypeptide of any aspect can be synthesized by general chemical synthesis methods or may be genetically engineered using recombinant DNA techniques and a cellular expression system, e.g. bacteria or induced viruses. Methods for chemical peptide synthesis are well known in the art and can be performed for instance, via solid phase synthesis method (e.g. Merrifield synthesis [34]) or via liquid phase synthesis method. Polypeptide can be identified for instance using phage display technology as selection method. Peptide selection methods like phage display technology are well known in the art [35, 36]. Recombinant expression can be accomplished using standard methods in the art, generally involving the cloning of nucleic acid sequences capable to direct the expression of the polypeptide into an expression vector, which can be used to transfect or transduce a host cell in order to provide the cellular machinery to carry out the expression of said polypeptide. The polypeptide of the present invention can further be derivatized, e.g. to provide enhanced half- life, for example by linking to polyethylene glycol or other for a person well known in the art compounds, as long as such derivatization does not interfere with the formation of the polypeptide. Furthermore, the polypeptide of the present invention can be joined with other compounds, e.g. with radioactive isotopes.

It is preferred that the polypeptide of the first aspect specifically binds to D114 protein, preferably to mammalian D114 protein, more preferred to rat D114 protein (Accession No. NP 001101230), more preferred to mouse D114 protein (Accession No. NP 062327.2) or more preferred to human D114 protein (Accession No. AAQ89253 or Version AAQ89253.1), most preferably to human D114 protein. Preferably, the polypeptide of the present invention exhibits the ability of binding to D114 protein in vitro as well in vivo. Preferably, the in vivo effect is observed when the polypeptide is administered via a parenteral administration route, e.g. intravenous, intra-arterial, intraosseous infusion, intra-muscular, intracerebral, intracerebroventricular or subcutaneous. In a preferred embodiment, the binding of the polypeptide to the D114 protein includes preferably, a peptide or a polypeptide with a native conformation, e.g. via a scaffold protein comprising the polypeptide and/or a scaffold backbone, which positions the polypeptide at an exposed surface of the scaffold protein, most preferably a polypeptide with a native conformation, e.g. via a scaffold protein comprising the polypeptide and/or a scaffold backbone, which positions the polypeptide at an exposed surface of the scaffold protein.

In a preferred embodiment, the polypeptide of the first aspect has the ability to reduce the binding of D114 protein to the Notch receptor. Preferably, the polypeptide of the present invention exhibits the ability to reduce the binding of D114 protein to the Notch receptor in vitro as well as in vivo. Preferably, the in vivo effect is observed when the polypeptide is administered via a parenteral administration route, e.g. intravenous, intra-arterial, intraosseous infusion, intramuscular, intracerebral, intracerebroventricular or subcutaneous. "Reducing" in this context preferably means that in an experimental set up, wherein equimolar amounts of the D114 or a Notch receptor binding fragment thereof and the polypeptide or the scaffold protein of the present invention are contacted with Notch receptor and the binding of D114 protein to the Notch receptor is at least 15%, more preferably at least 25%, more preferably at least 35%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% or more preferably at least 100% reduced in comparison to the reaction without the polypeptide or the scaffold protein of the present invention. In such assay it is preferred that at least one of the binding components, preferably the Notch receptor is immobilized and the other two components are in solution. Immobilization can occur, e.g. on the surface of a biacore chip or on a liposome. In the later embodiment it is preferred that the Notch receptor is integrated into the membrane of the liposome. It is preferred that D114 or the Notch receptor binding fragment thereof is preincubated with the polypeptide or the scaffold protein of the present invention prior to being contacted with the immobilized Notch receptor.

A person skilled in that is well aware of techniques for the measurement of the interaction or the specific binding of two peptides, more precisely for the measurement of the Kd value using, e.g. Biacore measurement. In this context, the term "binding" preferably relates to a specific binding between two polypeptides or two proteins or two fragments thereof.

In a second aspect, the present invention relates to a scaffold protein comprising the polypeptide of the first aspect and a scaffold backbone, which positions the polypeptide of the first aspect at an exposed surface of the scaffold protein or a pharmaceutically acceptable salt thereof. The term "exposed surface" refers to an amino acid domain of the scaffold protein, which is free to form intermolecular interactions, e.g. to D114. Three-dimensional structures of many scaffold proteins are known and, thus, the skilled person can easily determine regions of a given scaffold protein that are exposed and are, thus, suitable for insertion of the polypeptide of the present invention or of regions where one or more amino acids can be replaced with the polypeptide of the present invention. Such regions can also be determined indirectly by using the natively folded scaffold protein to elicit an immune response in an animal. The anti-scaffold protein antibodies formed will specifically detect those regions of the scaffold protein that are on an exposed surface of the scaffold protein. It is no burden for the skilled person to test such regions on whether the replacement or insertion of amino acids into these regions alters the three-dimensional structure of a given scaffold protein.

Suitable scaffold backbones have to meet several requirements. In general, scaffold backbones can be designed on the basis of either a previously characterised three-dimensional topology or de novo. The scaffold proteins suitable to be used as the source for the scaffold backbone used in the context of the present invention have one or more of the following properties:

(i) they are monomeric;

(ii) they are small proteins chains, e.g. typically in the range of 10 to 100, preferably in the range of 10 to 50 and more preferably in the range of 10 to 30 amino acids;

(iii) they are highly stable against enzymatic degradation;

(iv) they can be efficiently produced by recombinant expression or solid-phase peptide synthesis;

(v) they form a defined, rigid three-dimensional topology by secondary structural elements, e.g. a-helices, β-sheet or β-turn motifs;

(vi) they tolerate sequence variations or insertions within the recognition site, i.e. without compromising protein folding or stability;

(vii) they have a surface domain or binding pocket, which is accessible as a recognition site; and/or

(viii) they have a well-defined hydrophobic core that contributes to the high free energy of folding.

An optimal scaffold backbone should include all these characteristics to ensure that the polypeptide of the first aspect of the invention, is exposed on the surface of the structure formed from, e.g. a-helices, β-sheet or β-turn motifs. These secondary structural elements promote the formation of a rigid protein-like architecture, even within small polypeptides with less then 30 amino acids, and constitute a high proteolytic stability, which can be further increased by disulfide linkages. Nevertheless, the accommodation of the randomised binding epitope within the protein template has to be achieved without changing the overall topology and folding capability of the scaffold. In a preferred embodiment of the second aspect, the scaffold backbone is preferably selected from the group consisting of Min-23, SFTI, EETI-II, Scorpion toxin, Z domain, Zinc finger, CBD, DX-88, fibronectin III, lipocalin, apolipoprotein D and Tendamistat, preferably Min-23, SFTI, EETI-II and Scorpion toxin, most preferred Min-23 and SFTI. In these proteins one or more of the natural amino acids of the scaffold backbone may be replaced with amino acid derivates that do not disrupt the overall three-dimensional structure of the respective scaffold protein. Each of these scaffold backbones are known to be capable of accommodating a heterologous polypeptide of a given length. Thus, if the polypeptide of the first aspect is inserted into the scaffold backbone the length of the polypeptide is chosen in such to be within the minimal and maximal length constraints of the respective scaffold backbone.

Disulfide bonds are typically formed in a polypeptide between two appropriately spaced Cys-residues under non-reducing conditions. Such covalent bonds can "lock" a polypeptide in a particular conformation, e.g. exposing a certain loop region on the exposed surface of the polypeptide, and/or can stabilize the fold of polypeptide, e.g. render the polypeptide more stable against denaturation. It is, thus, preferred that the scaffold backbone comprises at least one disulfide bond formed between two appropriately spaced amino acid residues. More preferably, it comprises at least two, preferably four or six appropriately spaced Cys-residues and, accordingly, one, two or three disulfide bonds between these Cys-residues.

In a preferred embodiment, the scaffold protein of the second aspect comprises or consists of the amino acid sequence: Y₁-C-Y₂-Z-Y₃-C-Y4, wherein Z designates the polypeptide; and wherein Yi has a length of 1 to 50 continuous amino acids, preferably of 1 to 20 continuous amino acids, Y₂ has a length of 0 to 30 continuous amino acids, preferably of 0 to 5 continuous amino acids, Y₃ has a length of 0 to 30 continuous amino acids, preferably of 0 to 5 continuous amino acids, and wherein Y₄ has a length of 1 to 50 continuous amino acids, preferably of 1 to 20 continuous amino acids. In order to meet above mentioned property (ii) of scaffold backbones it is preferred that the sum of the amino acids of Yi, Y₂, Y3 and Y₄ is between 10 to 100, preferably between 10 to 50 and more preferably between 10 to 30 amino acids.

It is preferred that all amino acids are naturally occurring amino acids. However, in certain embodiments naturally occurring amino acids can be substituted by amino acid derivatives to increase, e.g. stability or binding affinity to D114 or improve pharmacokinetics of the scaffold protein. The skilled person is well aware, which natural amino acids may be substituted by amino acid derivatives to achieve these improved properties. It is, for example, known that Met residues may destabilize a polypeptide and, accordingly, it is preferred that one or more Met residues of the scaffold backbone is(are) replaced by ethionine (Eth) or norleucine (Nle), preferably by Nle. Preferably, 1 to 5 natural amino acids of a given scaffold backbone are replaced by amino acid derivates. It is mandatory that such derivatization does not interfere with the ability to specifically bind to D114.

In a particular preferred embodiment, Yi and/or Y₄ comprise 1 to 5, preferably 1 , 2, 3, 4, or 5 amino acid derivates. Preferred amino acid derivates are selected from the group consisting of Aad, bAad, bAla, Abu, 4Abu, Acp, Ahe, Aib, bAib, Apm, Dbu, Des, Dpm, Dpr, EtGly, EtAsn, Eth, Hyl, aHyl, 3 Hyp, 4Hyp, Ide, alle, MeGly, Melle, MeLys, MeVal, Nva, Nle or Orn, preferably Nle.

In a preferred embodiment the scaffold protein comprises or consists of the amino acid sequences: Yi-C-Y₂-Z-Y₃-C-Y₄, Yi-C-Z-Y₃-C-Y₄, Yi-C-Y₂-Z-C-Y₄ or Yi-C-Z-C-Y₄, wherein in each case Yi, Y₂, Y₃ and Y₄ have the above indicated meanings and preferred meanings.

In a preferred embodiment, the scaffold protein of the second aspect comprises or consists of the amino acid sequence: L-X₉-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-Z-F-C-G or G-R- C-T-Z-C-Xio-P-D, wherein Z designates the polypeptide of the first aspect; and wherein X₉ is selected from the group consisting of Nle or M, preferably Nle and Xio is preferably selected from Y and F, preferably Y.

In a preferred embodiment, the scaffold protein comprises or consists of the amino acid sequence: L-X₉-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-Xi-X₂-X₃-X₄-X₅-X₆-X7-X8-F-C-G (SEQ ID NO: 3) or G-R-C-T-Xi-X₂-X₃-X₄-X₅-X₆-X₇-X8-C-Xio-P-D (SEQ ID NO: 4), wherein X_u X₂, X₃, X₄, X₅, X₆, X₇, and Xs have the meaning indicated above for the polypeptide of the invention; and wherein X₉ is preferably selected from the group consisting of Nle or M, preferably Nle and Xio is preferably selected from Y and F, preferably Y

The presence of at least one Y amino acid is preferred in the scaffold proteins of the present invention as Y is susceptible to radio labelling, e.g. with radioactive iodine. If no Y is naturally occurring in the scaffold protein it is preferred that another bulky amino acid is replaced, e.g. F. It is also ascertained that the substitution does not significantly alter the properties of the resulting scaffold protein.

In a preferred embodiment, the scaffold protein of the second aspect comprises or consists of the amino acid sequence: L-X₉-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-L-F-H-L-F-I-Y-I- F-C-G (SEQ ID NO: 5), wherein X₉ is preferably selected from the group consisting of Nle or M, preferably Nle; or the scaffold protein of the second aspect comprises or consists of the amino acid sequence: G-R-C-T-L-F-H-L-F-I-Y-I-C-Y-P-D (SEQ ID NO: 6) or G-R-C-T-L-F-H- L-F-I-Y-I-C-Y-P-D (SEQ ID NO: 80).

In a preferred embodiment, the scaffold protein comprises or consists of the amino acid sequence: L-Nle-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-L-F-H-L-F-I-Y-I-F-C-G (SEQ ID NO: 7). While in some embodiments the polypeptide of the invention or the scaffold protein of the invention can elicit an effect alone, it is preferred that the ability to specifically bind to D114 is used to deliver a payload attached to the polypeptide of the invention or the scaffold protein of the invention to a desired site within a subject. Accordingly, in a third aspect the present invention relates to a conjugate of

(i) a polypeptide of the first aspect of the invention or the scaffold protein of the second aspect of the invention, and

(ii) a payload, which is covalently or non-covalently, preferably covalently linked to the polypeptide or scaffold protein.

In a preferred embodiment, the payload is selected from the group consisting of a therapeutic compound or a diagnostic compound.

The term "diagnostic compound" refers to a chemical moiety which is directly or indirectly detectable by analytical methods including measurement of fluorescence, nuclear magnetic resonance, computer tomography or scintigrams. In preferred embodiments, the diagnostic agent is selected from the group consisting of an electron dense molecule, a paramagnetic molecule, a superparamagnetic molecule, a radioactive molecule like, for example,

13- Nvr, O, 18 F, 51 ^ Gr, 54 Fe, 60^ Co, 67^ Ga, 75_c Se, ^ Tc, 1 11 _T In, 1 12m A_A g, 1 13m_T In, 123_T I, 133 X_ve, 148 A _Λ u, 35_c S, 33_D P,

32 11 2 13

P, or C, non-radioactive isotopes, which include, for example, H and C, and a fluorescent molecule or a molecule generating fluorescence or light emission like, for example, green fluorescent protein, luciferase, and a variety of fluorescent dyes all of which are well known to someone of skill in the art. If the payload is a protein, it is preferred that the scaffold protein and the payload is a fusion protein, i.e. that both parts are connected via a peptide bond.

In a preferred embodiment, the therapeutic compound is selected from the group consisting of a cytostatic compound, cytotoxic compound, anti-angiogenic compound, an anti- metabolite compound, a natural product, a hormone, a therapeutic isotope or a therapeutic antibody. Preferred examples of a cytostatic or cytotoxic drug, however, from the known cytostatic and cytotoxic drugs are the following: alkylating substances, anti-metabolites, antibiotics, epothilones, nuclear receptor agonists and antagonists, anti-androgens, anti- estrogens, platinum compounds, hormones and antihormones, interferons and inhibitors of cell cycle-dependent protein kinases (CDKs), inhibitors of cyclooxygenases and/or lipoxygenases, biogenic fatty acids and fatty acid derivatives, including prostanoids and leukotrienes, inhibitors of protein kinases, inhibitors of protein phosphatases, inhibitors of lipid kinases, platinum coordination complexes, ethyleneimenes, methylmelamines, trazines, vinca alkaloids, pyrimidine analogs, purine analogs, alkylsulfonates, folic acid analogs, anthracendiones, substituted urea, methylhydrazin derivatives, in particular acediasulfone, aclarubicine, ambazone, aminoglutethimide, L-asparaginase, azathioprine, bleomycin, busulfan, calcium folinate, carboplatin, carpecitabine, carmustine, celecoxib, chlorambucil, cis-platin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin dapsone, daunorubicin, dibrompropamidine, diethylstilbestrole, docetaxel, doxorubicin, enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphate, estrogen, ethinylestradiole, etoposide, flavopiridol, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide fosfestrol, furazolidone, gemcitabine, gonadotropin releasing hormone analog, hexamethylmelamine, hydroxycarbamide, hydroxymethylnitrofurantoin, hydroxyprogesteronecaproat, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon a, irinotecan, leuprolide, lomustine, lurtotecan, mafenide sulfate olamide, mechlorethamine, medroxyprogesterone acetate, megastrolacetate, melphalan, mepacrine, mercaptopurine, methotrexate, metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone, mithramycin, nalidixic acid, nifuratel, nifuroxazide, nifuralazine, nifurtimox, nimustine, ninorazole, nitrofurantoin, nitrogen mustards, oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine, phthalylsulfathiazole, pipobroman, prednimustine, prednisone, preussin, procarbazine, pyrimethamine, raltitrexed, rapamycin, rofecoxib, rosiglitazone, salazosulfapyridine, scriflavinium chloride, semustine streptozocine, sulfacarbamide, sulfacetamide, sulfachlopyridazine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfaethidole, sulfafurazole, sulfaguanidine, sulfaguanole, sulfamethizole, sulfamethoxazole, co-trimoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfaperin, sulfaphenazole, sulfathiazole, sulfisomidine, staurosporin, tamoxifen, taxol, teniposide, tertiposide, testolactone, testosteronpropionate, thioguanine, thiotepa, imidazole, topotecan, triaziquone, treosulfan, trimethoprim, trofosfamide, UCN-01, vinblastine, vincristine, vindesine, vinblastine, vinorelbine, and zorubicin, or their respective derivatives or analogs thereof.

In a fourth aspect, the present invention provides a nucleic acid encoding the polypeptide of the present invention, the scaffold protein of the present invention or the conjugate of the present invention. Preferably, such an encoding nucleic acid is comprised in a vector, which is configured to express the polypeptide, the scaffold protein or the conjugate of the present invention in a suitable expression system, e.g. a cell. To that end, it is preferred that the vector comprises transcriptional control sequences, e.g. promoter sequences, wherein the promoter comprises a constitutive promoter, an inducible promoter, a tissue-specific promoter, a synthetic promoter, preferably a constitutive or inducible promoter.

In a preferred embodiment, the vector includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculo viral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

If the payload is a protein, it is preferred that the payload is connected to the polypeptide or the scaffold protein of the invention via a peptide link. In this preferred embodiment, the polypeptide or the scaffold protein of the invention and the payload can be encoded as one fusion protein, i.e. the conjugate can be encoded by a nucleic acid.

In a fifth aspect, the present invention provides a polypeptide of the first aspect, a scaffold protein of the second aspect, the conjugate of the third aspect, or a nucleic acid of the fourth aspect for medical use.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the polypeptide of the first aspect, the scaffold protein of the second aspect, the conjugate of the third aspect, or the nucleic acid of the fourth aspect and a pharmaceutical acceptable excipient, carrier and/or diluent.

The term "pharmaceutically acceptable salt" refers to a salt of the polypeptide of the present invention, or to the scaffold protein comprising or consisting of the polypeptide of the present invention. Suitable pharmaceutically acceptable salts include acid addition salts which may, for example, be formed by mixing a solution of the polypeptide of the present invention, or the scaffold protein comprising or consisting of the polypeptide of the present invention, with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the polypeptide or the scaffold protein comprising or consisting of the polypeptide carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, S. M. Berge et al, "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)).

The term "excipient" when used herein is intended to indicate all substances in a pharmaceutical formulation which are not active ingredients such as, e.g., binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, or colorants.

The pharmaceutical composition contemplated by the present invention may be formulated in various ways well known to one of skill in the art. For example, the pharmaceutical composition of the present invention may be in liquid form such as in the form of solutions, emulsions, or suspensions. Preferably, the pharmaceutical composition of the present invention is formulated for parenteral administration, preferably for intravenous, intramuscular, subcutaneous, transdermal, intrapulmonary, intraperitoneal, intracardiac administration, or administration via mucous membranes, preferably for intravenous, subcutaneous, or intraperitoneal administration. Preferably, the pharmaceutical composition of the present invention is in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9, more preferably to a pH of from 5 to 7), if necessary.

The pharmaceutical composition is preferably in unit dosage form. In such form the pharmaceutical composition is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of pharmaceutical composition such as vials or ampoules.

In a seventh aspect, the present invention provides a polypeptide of the present invention, a scaffold protein of the present invention, a conjugate of the present invention, a nucleic acid of the present invention or a pharmaceutical composition of the present invention for use in diagnosing, treating or preventing disorders associated with angiogenesis.

In a preferred embodiment, the disorders associated with angiogenesis are selected from the group consisting of hematologic tumors, solid tumors, metastasis, proliferative retinopathy, diabetic retinopathy, rheumatoid arthritis, Crohn^'s disease, psoriasis, endometriosis, adiposity and neointimal formation.

In a preferred embodiment, the solid tumors are selected from the group consisting of CNS-tumors, neuroblasoma, Wilms-tumor, hepatoblastoma, colon tumors, lung tumors, prostate tumors, breast tumors, pancreas tumors, glioblastoma and retinoblastoma retinoblastoma and the hematologic tumors are selected from the group consisting of leukemias, lymphomas and myelomas.

For treating or preventing a disorder associated with angiogenesis the polypeptide, the scaffold protein, the conjugate, the nucleic acid or the pharmaceutical composition according to the present invention can be administered to an animal patient, preferably a mammalian patient, preferably a human patient, preferably via a parenteral administration route, for example, intravenously, intramuscularly, subcutaneously, transdermally, intrapulmonary, intraperitoneally, intracardiacally, or via mucous membranes, preferably intravenously, subcutaneously, or intraperitoneally. Administration may be by infusion or classical injection, for example, using cannulas, or by needleless injection techniques.

EXAMPLES

The Examples are designed in order to further illustrate the present invention and serve a better understanding. They are not to be construed as limiting the scope of the invention in any way.

Example 1 : Identification and synthesis of a D114-binding peptide

For identification of a D114-specific scaffold-peptide a phage display technology was used as selection method as previously described [35, 36]. The scaffold protein Min-23 was chosen as scaffold structure and the amino acids gly-pro-asn-gly of the Min-23-loop were replaced by eight variable amino acids using random oligonucleotides. The new peptide library (~3xl0⁷ variable sequences) was fused to the N-terminus of the minor coat protein pill, displayed on Ml 3 phages and incubated for selection of D114-specific binders with recombinant target protein D114-Fc in solution. Due to the biotinylation of D114-Fc the target protein and bound peptides were captured by use of streptavidin magnetic beads. Negative selections with beads and the recombinant protein FGFR-Fc in each panning round impeded the identification of beads- and Fc-binding peptides. After four selection rounds the phagemid vectors were isolated and 24 single clones were sequenced. Two of the sequenced clones offered the Min-23 sequence with the identical amino acids in the variable loop region: leu-phe-his-leu-phe-ile-tyr-ile. This peptide was called DUMin3.

DllMin3 was synthesized by solid phase synthesis, but the yield of DllMin3 synthesis did not fulfil the expectations. To circumvent the problem of DllMin3 peptide synthesis, the identified sequence was grafted into the loop of the scaffold peptide SFTI-I (Fig. 1). Therefore, the amino acids lys-ser-ile-pro-pro-ile of the SFTI-loop were replaced by the identified amino acids and this peptide was called D11SF3.

Example 2: Analysis of interaction between D114 and D11SF3 using Biacore XI 00.

In order to validate the target specificity, SPR analysis of designed peptide was performed against D114 and Notch- 1 as control. Both ligands were immobilized on CM5 sensor chip to facilitate direct binding measurements. A specific binding kinetics was only verified for D11SF3 against D114 showing a K_D value of 22 nM. As control, acyclic SFTI-I was applied resulting in no specific interaction with both ligands (Figure 2).

To estimate the competence of a pharmacological activity of D11SF3 caused by an inhibition of the D114/Notch-interaction was also investigated by SPR. Therefore, D114 was preincubated with different concentrations of the peptide dissolved in DMSO. Subsequently, the binding of D114 to immobilized Notch- 1 was analyzed to quantify an induced reduction of the D114/Notch-binding. As control, D114 was also incubated with equivalent amounts of DMSO without D11SF3 and assayed to exclude solvent effects on SPR signaling. A reduction of D114- binding to the Notch-1 receptor was observed with increasing concentrations of D11SF3. 95% inhibition of binding was achieved after incubation with 22 μg/mL D11SF3 (Figure 2). These findings indicate that the generated D11SF3 blocks the specific interaction between D114 and the Notch receptor.

Example 3 : Binding kinetics

In preparation of in vitro cell binding assays and in vivo experiments the human prostate cancer cell line PC-3, rat pancreatic tumor cells (AR42J) and endothelial cells (HUVECs) were tested for D114-expression by RT-PCR and Western Blot (data not shown). Since all tested cell lines produced D114 these cell lines were applied in the following cell binding as well as in vivo experiments.

The in vitro binding of D11SF3 and of the native scaffold SFTI to the different cell lines was analyzed after different incubation times. As shown in Figures 3a, b, c, D11SF3 offers a binding of 11.2% (PC-3), 7.0% (AR42J), and 27.6% (HUVECs) after 10 min which increases in the course of time and reaches 21.9% (PC-3), 13.5% (AR42J) and 52.9% (HUVECs) after 60 min. The binding is then stable until 240 min.

In contrast to D11SF3 the negative control peptide SFTI did not show any binding to the different cell lines and the binding rates did not exceed 0.2% (PC-3, AR42J) or 1.6% (HUVECs) (Fig. 3a, b, c).

Example 4: Alanine-Scan

In order to probe the contributions of the individual amino acid side chains to the D114- targeting properties, the individual positions of the binding domain comprised by the residues Thr⁴ and Cys¹³ of the SFTI scaffold was substituted sequentially by alanine. The D114- binding specificity of the resulting eight disulfide- stabilized peptides (D11SF-Ax) were assayed using SPR and by binding to PC-3 cells. In both systems, the replacement by alanine at all positions scanned demonstrated an influence on the binding capability (Table 1). Whereas the substitution of Leu⁵, His⁷ and Leu⁸ still demonstrated specific binding to D114 by SPR with a modest reduction of cellular binding capability to 51%, the modification of aromatic functions such as Phe⁶'⁹ and the lipophilic residues He¹⁰'¹² resulted in unspecific binding with modest 38% of cell binding potential compared to the non-substituted peptide.

Table 1 : Alanine scan

Summarized results of SPR analysis (n = 2) and binding ratio on PC-3 cells. Binding domains are comprised by the residues Thr⁴ and Cys¹³ of the disulfide-stabilized SFTI scaffold. For cell binding assays (n = 3), the iodine-125 labeled peptides were applied (except D11SF3-A11 due to the loss of tyrosine as labeling position), n.d.: not dedicated as specific interaction with immobilized D114 by the Biacore evaluation software.

binding binding ratio on PC-3 compound K_on [1/Ms] K_off [l/s] K_D [nM]

domain (D11SF3-AX / D11SF3)

D11SF3 LFHLFIYI 5.56x 10" 0.0121 22 1.00

D11SF3-A5 AFHLFIYI 5.34x l0⁴ 0.0273 511 0.51

D11SF3-A6 LAHLFIYI n.d. n.d. n.d. 0.38

D11SF3-A7 LFALFIYI 7.25x l0⁴ 0.0121 167 1.08

D11SF3-A8 LFHAFIYI 3.22x l0⁴ 0.0214 665 0.58

D11SF3-A9 LFHLAIYI n.d. n.d. n.d. 0.27

D11SF3-A10 LFHLFAYI n.d. n.d. n.d. 0.32

D11SF3-A11 LFHLFIAI n.d. n.d. n.d. -/-

D11SF3-A12 LFHLFIYA n.d. n.d. n.d. 0.42

SFTI-I KSIPPI n.d. n.d. n.d. 0.01

5(6)FAM-D11SF3 LFHLFIYI 1.60x l0⁴ 0.0014 88 0.70 Example 5 : Fluorescent cell staining and and flow cyctometry analysis.

Fluorescent staining on PC-3 cells clearly visualize the cell surface binding of 5(6)FAM- D11SF3 compared to the single mutated derivative 5(6)FAM-D11SF3-A9. Inhibition of the binding by blocking with unlabeled D11SF3 caused a significant reduction of the green fluorescence intensity (Figure 4). These microscopy results were confirmed by flow cyctometry using identically labeled peptide concentrations for incubation with PC-3 cells. The highest cell- bound fluorescence signals were determined for 5(6)FAM-D11SF3. As compared, 5(6)FAM- D11SF3-A9 showed a signal reduction to 52=1=1%. Competition with unlabeled D11SF3 led to 39±5% inhibition of cellular binding. These findings verified the specificity of D11SF3 binding to the D114-expressing cell line

Example 6: Serum stability.

In preparation of in vivo experiments the metabolic stability of D11SF3 was determined. The peptide was incubated in human serum at 37 °C and after defined time points (10 min, 1 h, 2 h, 4 h, 24 h, 48 h, 6 d, 10 d) aliquots were taken, serum proteins precipitated with acetonitrile and analyzed by HPLC. No second peak, indicating the degradation of the peptide, was detected after 10 days (Fig. 5). Example 7: Biodistribution.

To determine the biodistribution in mice carrying AR42J tumors D11SF3 was conjugated with ¹³¹Iod and the biodistribution after 120 min was analyzed. An accumulation of 1.47 % ID/g of D11SF3 in the tumor was detected whereas the peptide SFTI without D114-binding sequence offered an accumulation of only 0.53 % ID/g (Fig. 6). As a result of the high amount of lipophilic amino acids in the D114-binding sequence the highest concentration was observed in the liver (29.89 % ID/g).

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Claims

Claims

A polypeptide comprising or consisting of the amino acid sequence:

X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 1), wherein

Xi is absent or any amino acid;

X₂ is selected from the group consisting of F and Y;

X₃ or X₄ is any amino acid;

X₅ is selected from the group consisting of F and Y;

X₆ is selected from the group consisting of L, V, and I;

X₇ is selected from the group consisting of F and Y; and

Xs is selected from the group consisting of L, V, and I,

or a pharmaceutically acceptable salt thereof,

wherein the polypeptide has a length of 50 amino acids or less.

The polypeptide according to claim 1, wherein the polypeptide has a length of 10 amino acids or less.

The polypeptide according to claim 1 or 2, wherein

(a) X₂ is F, X₅ is F, X₆ is I, X₇ is Y and/or X₈ is I and/or

(b) Xi is selected from the group consisting of L, V and I, preferably L; wherein X₃ is selected from the group consisting of H and K, preferably H; and/or wherein X₄ is selected from the group consisting of L, V and I, preferably L, preferably comprising or consisting of the amino acid sequence: L-F-H-L-F-I-Y-I (SEQ ID NO: 2).

The polypeptide according to any of claims 1 to 3, which specifically binds to D114.

A scaffold protein comprising a polypeptide comprising or consisting of the amino acid sequence:

X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 1), wherein Xi is absent or any amino acid; X₂ is selected from the group consisting of F and Y;

X₃ or X₄ is any amino acid;

X₅ is selected from the group consisting of F and Y;

X₆ is selected from the group consisting of L, V, and I;

X₇ is selected from the group consisting of F and Y; and

Xs is selected from the group consisting of L, V, and I,

and a scaffold backbone, which positions the polypeptide at an exposed surface of the scaffold protein or a pharmaceutically acceptable salt thereof.

The scaffold protein according to claim 5, wherein the scaffold backbone

(a) is selected from the group consisting of Min-23, SFTI, EETI-II, Scorpion toxin, Z domain, Zinc finger, CBD, DX-88, fibronectin III, lipocalin, apo lipoprotein D and Tendamistat, preferably Min-23, SFTI, EETI-II and Scorpion toxin, most preferred Min-23 and SFTI and/or

(b) comprises at least one disulfide bond.

The scaffold protein according to claim 6, comprising or consisting of the amino acid sequence:

(a) Yi-C-Y₂-Z-Y₃-C-Y₄, wherein Z designates the polypeptide; and wherein Yi has a length of 1 to 50 amino acids, Y₂ has a length of 0 to 30 amino acids, Y₃ has a length of 0 to 30 amino acids and wherein Y₄ has a length of 1 to 50 amino acids,

.(b) L-Xg-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-Z-F-C-G or G-R-C-T-Z-C-Xio-P-D, wherein Z designates the polypeptide; and wherein X₉ is selected from the group consisting of Nle or M;

(c) L-X₉-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-Xi-X₂-X₃-X₄-X₅-X6-X7-X8-F-C-G (SEQ ID NO: 3) or G-R-C-T-Xi-X₂-X₃-X₄-X₅-X₆-X₇-X₈-C- Xio-P-D (SEQ ID NO: 4), wherein X_{l s} X₂, X₃, X₄, X₅, X₆, X₇, and Xs have the meaning indicated in claims 1 to 5, and wherein X₉ is selected from the group consisting of Nle and M and Xio is selected from the group consisting of F and Y and/or

(d) L-Nle-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-L-F-H-L-F-I-Y-I-F-C-G (SEQ ID NO:

7), G-R-C-T-L-F-H-L-F-I-Y-I-C-Y-P-D (SEQ ID NO: 6) or G-R-C-T-L-F-H-L-F- I-Y-I-C-F-P-D (SEQ ID NO: 80).

A conjugate of

(i) a polypeptide comprising or consisting of the amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 1), wherein

Xi is absent or any amino acid;

X₂ is selected from the group consisting of F and Y;

X₃ or X₄ is any amino acid;

X₅ is selected from the group consisting of F and Y;

X₆ is selected from the group consisting of L, V, and I;

X₇ is selected from the group consisting of F and Y; and

Xs is selected from the group consisting of L, V, and I,

or the scaffold protein according to any of claims 7 to 13 and

(ii) a payload, which is covalently or non-covalently linked to the polypeptide or scaffold protein.

The conjugate according to claim 8, wherein the payload is selected from the group consisting of a therapeutic compound or a diagnostic compound.

The conjugate according to claim 9, wherein the therapeutic compound is selected from the group consisting of a cytostatic compound, cytotoxic compound, anti-angiogenic compound, an alkylating agent, an anti-metabolite compound, a natural product, a hormone, an oncogenic inhibitor, a therapeutic isotope or an therapeutic antibody and the diagnostic compound is selected from the group consisting of an electron dense compound, a paramagnetic compound, a superparamagnetic compound, a fluorophor, a radioactive molecule, non-radioactive isotopes, a fluorescent compound or a molecule generating fluorescence or light emission or a fusion protein.

A nucleic acid encoding the polypeptide of any of claims 1 to 4, the scaffold protein according to any of claims 6 to 7 or the conjugate of claims 8 to 10.

A polypeptide comprising or consisting of the amino acid sequence:

X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 1), wherein

Xi is absent or any amino acid;

X₂ is selected from the group consisting of F and Y; X₃ or X₄ is any amino acid;

X₅ is selected from the group consisting of F and Y;

X₆ is selected from the group consisting of L, V, and I;

X₇ is selected from the group consisting of F and Y; and

Xs is selected from the group consisting of L, V, and I;

a scaffold protein of any of claims 6 to 7, a conjugate of any of claims 8 to 10 or a nucleic acid according to claim 11 for medical use.

A pharmaceutical composition comprising a polypeptide comprising or consisting of the amino acid sequence:

X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 1), wherein

Xi is absent or any amino acid;

X₂ is selected from the group consisting of F and Y;

X₃ or X₄ is any amino acid;

X₅ is selected from the group consisting of F and Y;

X₆ is selected from the group consisting of L, V, and I;

X₇ is selected from the group consisting of F and Y; and

Xs is selected from the group consisting of L, V, and I;

a scaffold protein of any of claims 6 to 7, a conjugate of claims 8 to 10 or a nucleic acid according to claim 11 and a pharmaceutical acceptable excipient, carrier and/or diluent.

A polypeptide, a scaffold protein, a conjugate or a nucleic acid of claim 12 or a pharmaceutical composition of claim 13 for use in diagnosing, treating or preventing disorders associated with angiogenesis.

The polypeptide, scaffold protein, conjugate, nucleic acid or pharmaceutical composition of claim 14, wherein the disorders associated with angiogenesis is selected from the group consisting of hematologic tumors, preferably selected from the group consisting of leukemias, lymphomas and myelomas, solid tumors, preferably selected from the group consisting of CNS-tumors, neuroblasoma, Wilms-tumor, hepatoblastoma, colon tumors, lung tumors, prostate tumors, breast tumors, pancreas tumors, glioblastoma and retinoblastoma, metastasis, proliferative retinopathy, diabetic retinopathy, rheumatoid arthritis, Crohn^'s disease, psoriasis endometriosis, adiposity and neointimal formation.