WO2013135747A1 - 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 orsaid scaffold, which positions the polypeptide 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 in 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 PROBLEM 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 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: G-W-X₁-X₂-S-X3-W-X4-X5-H (SEQ ID NO: 1); wherein Xi is selected from the group consisting of F and Y; wherein X₂ is selected from the group consisting of L, V and I; wherein X₃ is selected from the group consisting of Q and N; wherein X₄ is selected from the group consisting of A and V; and wherein X₅ 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, or a pharmaceutically acceptable salt thereof.

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 scaffo Id 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 scaffold structures. Figure 1 shows a schematic representation of the miniprotein scaffold Min-23 with its native-like Cys⁴-Cys¹⁶/Cys¹⁰-Cys²² disulfide connectivity. The variable surface-exposed loop is drawn in black. Structural data were created form EETI II (PDB ID: 2IT7) with the PyMol molecular visualization software. Fig. 2: Primary structure and disulfide connectivity of the Min-23 scaffold and of the screening hit DLL-Rib isolated by ribosome display. Random moieties of the Min-23R10 display library are marked as an X, the identified binding sequence RIO-Rib is highlighted in bold.

Fig. 3: HPLC-MS analysis of the synthesis of DLL-Rib. Autonomous oxidative peptide folding results in the formation of two different regioisomers (A). Using an orthogonal protecting group strategy for an consecutive disulfide formation, the native-like Cys⁴- Cys¹⁶/Cys¹⁰-Cys²⁸ disulfide linkage is formed exclusively, as shown in the TIC- chromatogram (B). The different regioisomers show identical mass spectra of the corresponding retention times (C).

Fig. 4: Radio-HPLC analysis of ¹²⁵I-(Tyr¹⁹)DLL-Rib shows the stability in human serum with a half-life of 30 hours. HPLC-conditions: 0-100% MeCN (0.1% TFA) in 10 min, 1 mL/min; XBridge C43.5 μιη (150 x 4.6 mm).

Fig. 5: Binding specificity of DLL-Rib. Surface plasmon resonance senorgrams show the specific binding to immobilized DLL4 of the identified miniprotein DLL-Rib compared to the references DLL-Rib (misfolded), Min-23 or RIO-Rib. Mean values for the binding kinetic parameters were determined for DLL-Rib: k_on = 1.431 x 10⁴ M^'V¹; k₀n = 6.234x 10^"3; Κ_Ό (k_oiilk_on) = 43.56 nM.

Fig. 6: In vitro binding kinetic of ¹²⁵I-(Tyr¹⁹)DLL-Rib to different DLL4-expressing tumor cell lines. HUVECs, AR42J and PC-3 cells were grown for 24 hours. DLL4 expression of the cell lines used was verified by real-time PCR and western blot.

Fig. 7: Biodistribution of ¹³¹I-(Tyr¹⁹)DLL-Rib in AR42J-tumor bearing mice. Radioactivity concentrations (% ID/g) in the organs measured after 10, 30, 60, 120 and 240 min p.i. in mice (n=3).

Fig. 8: Biodistribution of ¹³¹I-(Tyr¹⁹)DLL-Rib, blocking and reference compounds in AR42J- tumor bearing mice. Radioactivity concentrations (% ID/g) in the organs measured after 120 min p.i. in mice (n=3). Fig. 9: Gamma- imaging of ¹²⁵I-(Tyr¹⁹)DLL-Rib in AR42J-tumor bearing rat. The tracer shows rapid accumulation in the tumor lesion already 10 minutes post injection and slow hepatobiliar excretion over 4 hours. Fig. 10: Gamma-imaging of ¹²⁵I-(Tyr¹⁹)DLL-Rib in AR42J-tumor bearing mice. The regioisomer with native-like disulfide connectivity (top) shows accumulation in the tumor lesion as compared to the misfolded disulfide configuration (bottom). Tumor lesions are marked with an arrow.

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 or less, 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^AC, more preferably at 37^AC and at 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 and more cysteine residues of the scaffold protein. Preferably, between the discontinuous amino acid chains that are to 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" as used herein 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 a electron dense compound, a paramagnetic compound, a superparamagnetic compound, a fluorophor, a 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. 8: 148- 153, 1997); "Serum free Media" (K. Kitano, Biotechnol. 17:73-106, 1991); and "Suspension Culture of Mammalian Cells" (Birch et al. Bioprocess Technol. 19: 251, 1990).

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 methods. Preferred embodiments

In a first aspect, the present invention provides a polypeptide comprising or consisting of the amino acid sequence: G-W-X₁-X₂-S-X3-W-X4-X5-H (SEQ ID NO: 1); wherein Xi is selected from the group consisting of F and Y, most preferred Y; wherein X₂ is selected from the group consisting of L, V and I, most preferred I; wherein X₃ is selected from the group consisting of Q and N, most preferred N; wherein X₄ is selected from the group consisting of A and V, most preferred A; and wherein X₅ is selected from the group consisting of L, V and I, most preferred 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, more preferably of 10 continuous amino acids. A particularly preferred range of the length of the polypeptide is between 10 to 15 continuous amino acids.

In a preferred embodiment, the polypeptide of the first aspect comprises or consists of the amino acid sequence G-W-X₁-X₂-S-X3-W-X4-X5-H; wherein Xi is Y, X₂ is I, X3 is N, X₄ is A and/or X5 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 Y and X₂ is I; Xi is Y and X3 is N; Xi is Y and X₄ is A; Xi is Y and X₅ is I; X₂ is I and X3 is N; X₂ is I and X₄ is A; X₂ is I and X₅ is I; X3 is N and X₄ is A; X₃ is N and X₅ is I; X₄ is A and X₅ is I; Xi is Y, X₂ is I and X3 is N; Xi is Y, X₂ is I and X₄ is A; Xi is Y, X₂ is I and X₅ is I; Xi is Y, X₃ is N and X₄ is A; Xi is Y, X₃ is N and X₅ is I; Xi is Y, X₄ is A and X₅ is I; X₂ is I, X3 is N and X₄ is A; X₂ is I, X3 is N and X₅ is I; X₂ is I, X₄ is A and X₅ is I; X3 is N, X₄ is A and X₅ is I; Xi is Y, X₂ is I, X3 is N and X₄ is A; Xi is Y, X₂ is I, X3 is N and X₅ is I; Xi is Y, X₃ is N, X₄ is A and X₅ is I; Xi is Y, X₂ is I, X₄ is A and X₅ is I; X₂ is I, X₃ is N, X₄ is A and X₅ is I; or Xi is Y, X₂ is I, X3 is N, X₄ is A and X₅ is I.

Preferably, the polypeptide is selected from the group consisting of: G-W-F-L-S-Q-W-A-L-

H (SEQ ID NO: 6); G-W-Y-L-S-Q-W-A-L-H (SEQ ID NO: 7); G-W-F-V-S-Q-W-A-L-H (SEQ ID NO: 8); G-W-Y-V-S-Q-W-A-L-H (SEQ ID NO: 9); G-W-F-I-S-Q-W-A-L-H (SEQ ID NO: 10); G-W-Y-I-S-Q-W-A-L-H (SEQ ID NO: 11); G-W-F-L-S-N-W-A-L-H (SEQ ID NO: 12); G- W-Y-L-S-N-W-A-L-H (SEQ ID NO: 13); G-W-F-V-S-N-W-A-L-H (SEQ ID NO: 14); G- W-Y-V-S-N-W-A-L-H (SEQ ID NO: 15); G-W-F-I-S-N-W-A-L-H (SEQ ID NO: 16); G-W-Y-I- S-N-W-A-L-H (SEQ ID NO: 17); G-W-F-L-S-Q-W-V-L-H (SEQ ID NO: 18); G-W-Y-L-S-Q- W-V-L-H (SEQ ID NO: 19); G-W-F-V-S-Q-W-V-L-H (SEQ ID NO: 20); G-W-Y-V-S-Q-W-V- L-H (SEQ ID NO: 21); G-W-F-I-S-Q-W-V-L-H (SEQ ID NO: 22); G-W-Y-I-S-Q-W-V-L-H (SEQ ID NO: 23); G-W-F-L-S-N-W-V-L-H (SEQ ID NO: 24); G-W-Y-L-S-N-W-V-L-H (SEQ ID NO: 25); G-W-F-V-S-N-W-V-L-H (SEQ ID NO: 26); G-W-Y-V-S-N-W-V-L-H (SEQ ID NO: 27); G-W-F-I-S-N-W-V-L-H (SEQ ID NO: 28); G-W-Y-I-S-N-W-V-L-H (SEQ ID NO: 29); G-W-F-L-S-Q-W-A-V-H (SEQ ID NO: 30); G-W-Y-L-S-Q-W-A-V-H (SEQ ID NO: 31); G-W-F-V-S-Q-W-A-V-H (SEQ ID NO: 32); G-W-Y-V-S-Q-W-A-V-H (SEQ ID NO: 33); G-W- F-I-S-Q-W-A-V-H (SEQ ID NO: 34); G-W-Y-I-S-Q-W-A-V-H (SEQ ID NO: 35); G-W-F-L-S- N-W-A-V-H (SEQ ID NO: 36); G-W-Y-L-S-N-W-A-V-H (SEQ ID NO: 37); G-W-F-V-S-N-W- A-V-H (SEQ ID NO: 38); G-W-Y-V-S -N-W-A-V-H (SEQ ID NO: 39); G-W-F-I-S-N-W-A-V- H (SEQ ID NO: 40); G-W-Y-I-S-N-W-A-V-H (SEQ ID NO: 41); G-W-F-L-S-Q-W-V-V-H (SEQ ID NO: 42); G-W-Y-L-S-Q-W-V-V-H (SEQ ID NO: 43); G-W-F-V-S-Q-W-V-V-H (SEQ ID NO: 44); G-W-Y-V-S-Q-W-V-V-H (SEQ ID NO: 45); G-W-F-I-S-Q-W-V-V-H (SEQ ID NO: 46); G-W-Y-I-S-Q-W-V-V-H (SEQ ID NO: 47); G-W-F-L-S-N-W-V-V-H (SEQ ID NO: 48); G-W-Y-L-S-N-W-V-V-H (SEQ ID NO: 49); G-W-F-V-S-N-W-V-V-H (SEQ ID NO: 50); G-W-Y-V-S-N-W-V-V-H (SEQ ID NO: 51); G-W-F-I-S-N-W-V-V-H (SEQ ID NO: 52); G-W- Y-I-S-N-W-V-V-H (SEQ ID NO: 53); G-W-F-L-S-Q-W-A-I-H (SEQ ID NO: 54); G-W-Y-L-S- Q-W-A-I-H (SEQ ID NO: 55); G-W-F-V-S-Q-W-A-I-H (SEQ ID NO: 56); G-W-Y-V-S-Q-W- A-I-H (SEQ ID NO: 57); G-W-F-I-S-Q-W-A-I-H (SEQ ID NO: 58); G-W-Y-I-S-Q-W-A-I-H (SEQ ID NO: 59); G-W-F-L-S-N-W-A-I-H (SEQ ID NO: 60); G-W-Y-L-S-N-W-A-I-H (SEQ ID NO: 61); G-W-F-V-S-N-W- A-I-H (SEQ ID NO: 62); G-W-Y-V-S-N-W- A-I-H (SEQ ID NO: 63); G-W-F-I-S-N-W- A-I-H (SEQ ID NO: 64); G-W-Y-I-S-N-W- A-I-H (SEQ ID NO: 65); G- W-F-L-S-Q-W- V-I-H (SEQ ID NO: 66); G-W-Y-L-S-Q-W- V-I-H (SEQ ID NO: 67); G-W-F-V- S-Q-W- V-I-H (SEQ ID NO: 68); G-W-Y-V-S-Q-W- V-I-H (SEQ ID NO: 69); G-W-F-I-S-Q-W- V-I-H (SEQ ID NO: 70); G-W-Y-I-S-Q-W-V-I-H (SEQ ID NO: 71); G-W-F-L-S-N-W-V-I-H (SEQ ID NO: 72); G-W-Y-L-S-N-W- V-I-H (SEQ ID NO: 73); G-W-F-V-S-N-W- V-I-H (SEQ ID NO: 74); G-W-Y-V-S-N-W- V-I-H (SEQ ID NO: 75); G-W-F-I-S-N-W- V-I-H (SEQ ID NO: 76); and G-W-Y-I-S-N-W- V-I-H (SEQ ID NO: 77).

In a preferred embodiment, the polypeptide of the first aspect comprises or consists of the amino acid sequence: G-W-Y-I-S-N-W-A-I-H (SEQ ID NO: 2), wherein preferably maximally 2, and most preferably maximally 1 amino acid is replaced with another amino acid as specified above.

Alternatively or additionally, the polypeptide of the present invention may comprise with respect to SEQ ID NO: 2 one or two amino acid deletions and/or insertions.

In each case indicated above, the polypeptide of the invention based on the amino acid 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.

In a preferred embodiment, the synthesis of the polypeptide comprises both regioisomers, the misfolded regioisomer which has a retention time of 18.04 min and preferably the native regioisomer which has a retention time of 18.22 min, most preferred the native regioisomer. A person skilled in that art can identify such isomers using standard methods which are well known in the art, e.g. using mass spectrometry combined with a HPLC. In particular, the native regioisomer can be identified using an orthogonal protecting group strategy for consecutive disulfide formation, wherein the native-like Cys⁴-Cys¹⁶/Cys¹⁰-Cys²⁸ disulfide linkage is formed exclusively, which can be detected using, for example, a total ion current (TlC)-chromatogram.

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 preferred 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 a peptide or a polypeptide with a native conformation, e.g. via a scaffold protein comprising the polypeptide of the first aspect and/or a scaffold backbone, which positions the polypeptide of the first aspect 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 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 the 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 the art 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 preferred embodiment, the polypeptide according to SEQ ID NO: 1 specifically binds to mammalian D114 protein with an affinity (KD value) 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 or less, more preferred 90 nM or less, more preferred 80 nM or less, more preferred 70 nM or less, more preferred 60 nM or less, more preferred 50 nM or less, more preferred 40 nM or less, more preferred 30 nM or less, more preferred 20 nM or less or more preferably 10 nM or less.

In a preferred embodiment of the present invention, the polypeptide of the first aspect has the ability to accumulate in a tumor and does not accumulate in non-specific compartments, such as heart, lung, spleen, intestine or muscles. A person skilled in the art is well aware of techniques for measurement of the biodistribution of the polypeptide in vivo, e.g. polypeptides or peptides or proteins are labelled with iodine-131 and administered for instance via parenteral route to a mammal, preferably human or rodents, most preferably to a mouse or preferably to a rat. 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 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/or 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-X₆-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-Z-F-C-G; wherein Z designates the polypeptide, and wherein X₆ is selected from the group consisting of an unnatural amino acid derivate, preferably Nle or the amino acid M. In a preferred embodiment, X₆ is Nle.

In a preferred embodiment, the scaffold protein of the second aspect comprises or consists of the amino acid sequence: L-X₆-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-G-W-Xi-X₂-S-X₃- W-X₄-X₅-H-F-C-G according to SEQ ID NO: 3; wherein X X₂, X₃,-X₄, and X₅ have above indicated meaning for the polypeptide; and wherein X₆ is preferably selected from the group consisting of Nle or M, preferably Nle.

In a particularly preferred embodiment, the scaffold protein comprises or consists of the amino acid sequence L-X₆-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-G-W-Y-I-S-N-W-A-I-H-F-C-G according to SEQ ID: NO 4, wherein X₆ is preferably selected from the group consisting of Nle or M, preferably Nle.

In a particularly 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-G-W-Y-I-S-N-W-A-I-H-F-C-G according to SEQ ID: NO 5.

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 5 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.

10 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,

1 15 13- Nvr, 15 O, 18 F, 51 ^ G, 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_n 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 0 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 alkylating agent, an anti-metabolite compound, a natural product, a hormone, an oncogenic inhibitor, a therapeutic isotope or a therapeutic antibody. Preferred examples of cytostatic or 5 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, interferones and inhibitors of cell cycle-dependent protein kinases (CDKs), inhibitors of cyclooxygenases and /or lipogygenases, biogenic fatty acids and fatty acid derivates, including

30 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,

35 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 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 preferably comprises a constitutive promoter, preferably an inducible promoter, preferably a tissue-specific promoter, preferably a synthetic promoter, most preferred 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 baculoviral 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 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, 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 or of the scaffold protein comprising 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 or the scaffold protein comprising 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 the polypeptide of the present invention carries 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 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 is selected from the group consisting of hematologic tumors, solid tumors, metastasis, proliferative retinopathy, diabetic retinopathy, rheumatoid arthritis, Crohn^'s disease, psoriasis, endopetriosis, 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 and the hematologic tumors are selected from the group consisting of leukemias, lymphomas and myelomas.

For treating or preventing a disorder associated with angiogenesis as specified in the fourth and fifth aspect of the present invention, the peptide 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 : Isolation of an anti-DLL4 miniprotein scaffold using ribosome display screening.

Ribosome-display has been conducted using a combinatorial designed scaffold library based on the Min-23 scaffold structure. The miniprotein Min-23 was rationally designed and comprises a cysteine-stabilized β-sheet (CSB) motif that forms an autonomous folding unit by three β-sheets and a short a-helix (Fig. 1). This structural arrangement presents a single surface-exposed β-turn, which is highly tolerant to substitutions and insertion and of up to 10 amino acids. These characteristics classify Min-23 as suitable peptide scaffold for ribosome display library construction bioactive epitopes to generate novel binding specificities.

The anti-D114 binding sequence RIO-Rib exposed on the surface loop of the Min-23 scaffold was isolated after the several biopanning cycles of ribosome display screening against the immobilized DLL4 protein. This approach yielded in the lead structure DLL-Rib (Fig. 2).

Example 2: Peptide engineering of the screening hits. The synthesis of the primary structure of DLL-Rib has been achieved by fully automated solid- phase peptide synthesis (SPPS). The formation of the disulfide linkages has been performed by DMSO-mediated oxidative peptide folding in ammonium acetate buffer containing 50% (v/v) isopropanol and 0.01 M guanidium hydrochloride as denaturation agents to increase peptide solubility.

Conducting an autonomous oxidative folding approach, only 39% of the native-like Cys⁴- Cys¹⁶/Cys¹⁰-Cys²⁸ regioisomer was formed within 4 days. As consecutive folding approach using the orthogonal Cys(Acm)/Cys(Trt) protecting group strategy was performed to verify the native disulfide connectivity. This stepwise cysteine- formation resulted in one regioisomer exclusively (Fig. 3).

Example 3 : Serum stability of DLL-Rib

The radio labelled miniprotein ¹²⁵I-(Tyr¹⁹)DLL-Rib demonstrated an high stability in human serum. HPLC analysis verified a half-life in human serum of 30 hours (Fig. 4). Prolonged incubation leaded to proteolytic fragmentation. No release of the radio label was detected over a period of ten days. This stability revealed the suitability of DLL-Rib for in vivo applications.

Example 4: Surface Plasmon resonance analysis.

The binding specifity of DLL-Rib to DLL4 has been verified by surface plasmon resonance spectrometry. The miniprotein showed specific binding to immobilized DLL4 with an affinity (Kd) of43.56 nM (Fig. 5).

Example 5: Cell binding assays.

The cell binding capability of the radio labelled DLL-Rib peptide derivative was characterized on different cell lines. The native folded DLL-Rib demonstrated the highest binding potential to all cell lines used compared to the misfolded regioisomer of the disulfide-constrained peptide. On the other hand, the Min-23 scaffold without the incorporated binding sequence RIO-Rib and the binding sequence by itself did not show any specific binding capability (Fig. 6). Example 6: Biodistribution of radiolabeled DLL-Rib.

To investigate the biodistribution of the identified DLL-Rib in vivo, the peptides were labelled with iodine-131 and administrated intravenously to AR42J tumor-bearing mice. The tracer showed a rapid accumulation in the tumor with 3.3 ± 0.9 %ID/g already 10 min post injection (p.i.) and reached a maximum of 3.9 ± 0.9 %ID/g after 60 min. No accumulation in the non- specific compartment, such as the heart, lung, spleen, intestine or the muscles was observed (Fig. 7). The fast blood-pool-clearance via the kidneys and the hepatobiliar excretion into the duodenum resulted in a maximum tumor-to-muscle ratio of 9.7 ± 1.2 and a tumor-to-blood ratio of 2.2 ± 0.3 after 120 min (Table 1).

In an in vivo blocking experiment, the accumulation of the tracer has been reduced to only 1.7 ± 0.4 %ID/g 120 min p.i. (Fig. 8). For comparison, the misfolded regioisomer of DLL-Rib and the Min-23 scaffold, that does not present any binding sequence, showed only insignificant tumor- to-organ contrast at this time point (Table 2).

Table 1 : Tumor-to-organ ratios of ¹³¹I-(Tyr¹⁹)DLL-Rib in AR42J-tumor bearing mice (n = 3).

10 min 30 min 60 min 120 min 240 min tumor/heart 0.90 ± 0.01 1.83 ± 0.60 2.06 ± 0.30 5.30 ± 0.91 2.47 ± 0.98 tumor/lung 0.11 ± 0.03 0.21 ± 0.05 0.40 ± 0.13 1.63 ± 1.28 0.38 ± 0.19 tumor/spleen 0.39 ± 0.05 0.95 ± 0.46 1.14 ± 0.17 3.48 ± 1.23 1.48 ± 0.64 tumor/liver 0.11 ± 0.01 0.52 ± 0.18 0.77 ± 0.03 2.30 ± 1.51 1.07 ± 0.44 tumor/kidney 0.19 ± 0.01 0.48 ± 0.17 0.59 ± 0.08 1.09 ± 0.35 0.67 ± 0.25 tumor/duodenum 0.48 ± 0.04 0.52 ± 0.16 0.35 ± 0.06 1.06 ± 0.37 0.43 ± 0.23 tumor/intestine 1.06 ± 0.30 1.82 ± 0.88 1.77 ± 0.80 2.70 ± 0.69 2.75 ± 0.93 tumor/blood 0.30 ± 0.01 0.83 ± 0.43 0.74 ± 0.08 2.18 ± 0.32 1.06 ± 0.55 tumor/muscle 1.76 ± 0.06 3.92 ± 1.65 3.69 ± 0.43 9.74 ± 1.19 4.39 ± 1.64

Table 2: tumor-to-organ ratios of ¹³¹I-(Tyr¹⁹)DLL-Rib, blocking and reference compounds

AR42J-tumor bearing mice (n = 3).

DLL-Rib DLL-Rib DLL-Rib Min-23

(blocking) (misfolded)

tumor/heart 5.30 ± 0.91 1.83 ± 0.04 1.52 ± 0.09 1.64 ± 0.39 tumor/lung 1.63 ± 1.28 0.73 ± 0.04 0.57 ± 0.06 0.66 ± 0.21 tumor/spleen 3.48 ± 1.23 0.93 ± 0.09 0.98 ± 0.11 0.89 ± 0.26 tumor/liver 2.30 ± 1.51 0.47 ± 0.06 0.37 ± 0.01 1.21 ± 0.42 tumor/kidney 1.09 ± 0.35 0.42 ± 0.05 0.49 ± 0.03 0.20 ± 0.07 tumor/duodenum 1.06 ± 0.37 0.55 ± 0.05 0.28 ± 0.12 0.17 ± 0.07 tumor/intestine 2.70 ± 0.69 1.03 ± 0.07 1.04 ± 0.06 1.61 ± 0.39 tumor/blood 2.18 ± 0.32 0.66 ± 0.00 0.56 ± 0.06 0.51 ± 0.14 tumor/muscle 9.74 ± 1.19 2.96 ± 0.18 2.11 ± 0.66 2.49 ± 0.65 Example 7: Molecular imaging studies.

The beneficial tumor-to-organ ratios evaluated in biodistribution studies facilitated high contrast molecular imaging in AR42J-tumor bearing rats (Fig. 9) and in mice (Fig. 10). Immediately after i.v. administration of the tracer, the tumor lesion showed an increased uptake of the radioactivity. Hepatobiliar and also renal excretion of the tracer could be visualized 60 min p.i.

As a reference, the misfolded regioisomer of DLL-Rib did not show tumor targeting capability in vivo (Fig. 10). These findings clarify the potential of the ribosome display-derived miniprotein DLL-Rib as diagnostic agent for angiogenesis in vivo using molecular imaging techniques.

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Claims

Claims

A polypeptide comprising or consisting of the amino acid sequence:

G-W-X₁-X₂-S-X₃-W-X4-X5-H (SEQ ID NO: 1), wherein

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

X₂ is selected from the group consisting of L, V and I;

X₃ is selected from the group consisting of Q and N;

X₄ is selected from the group consisting of A and V; and

X₅ is selected from the group consisting of L, V and I,

or a pharmaceutically acceptable salt thereof.

The polypeptide according to claim 1, wherein the polypeptide has a length of 15 amino acids or less.

The polypeptide according to claim 1 or 2, wherein Xi is Y, X₂ is I, X₃ is N, X₄ is A, and/or X₅ is I, preferably comprises or consists of the amino acid sequence: G-W-Y-I-S- N-W-A-I-H (SEQ ID NO: 2)

The polypeptide according to any of claims 1 to 3, which specifically binds to DLL4.

A scaffold protein comprising the polypeptide according to any of claims 1 to 4 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) Y₁-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-X₆-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-Z-F-C-G, wherein Z designates the polypeptid; and wherein X₆ is selected from the group consisting of Nle or M, preferably L-X₆-R-C-K-Q-D-S-D-C-L-A-G-S-V-C-G-W-Xi-X₂-S-X₃-W-X₄-X₅-H- F-C-G (SEQ ID NO: 3), wherein X X₂, X₃, ¾, X₅ and X₆ have above indicated meaning.

8. A conjugate of

(i) a polypeptide any of claims 1 to 4 or the scaffold protein of any of claims 5 to 7, and

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

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

10. 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 antimetabolite 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, radioactive compound, non-radioactive isotopes, a fluorescent compound, a diagnostic isotope, paramagnetic label or fusion protein.

1 1. A nucleic acid encoding the polypeptide of any of claims 1 to 4, the scaffold protein of any of claims 5 to 7 or the conjugate of any of claims 8 to 10.

12. A polypeptide of any of claims 1 to 4, a scaffold protein of any of claims 5 to 7, a conjugate of any of claims 8 to 10 or a nucleic acid according to claim 1 1 for medical use.

13. A pharmaceutical composition comprising the polypeptide of any of claims 1 to 4, a scaffold protein of any of claims 5 to 7, a conjugate of any of claims 8 to 10 or a nucleic acid according to claim 11 and a pharmaceutical acceptable excipient, carrier and/or diluent.

14. A polypeptide of any of claims 1 to 4, a scaffold protein of any of claims 5 to 7, a conjugate of any of claims 8 to 10, a nucleic acid according to claim 11 or a pharmaceutical composition of claim 13 for use in diagnosing, treating or preventing disorders associated with angiogenesis.

15. 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, endopetriosis, adiposity and neointimal formation.