Abstract:
The invention relates to the fields of protein chemistry, biology and medicine. More specifically, it relates to the design and preparation of proteinmimics of members of the cystine-knot growth factor superfamily. Further, the invention relates to the use of these proteinmimics as a medicament or prophylactic agent. The invention provides proteinmimics of members of the cystine-knot growth factor superfamily, preferably for use in immunogenic and/or therapeutic compositions.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/NL2010/050053, filed Feb. 5, 2010, published in English as International Patent Publication WO 2010/090523 A1 on Aug. 12, 2010, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 09152305.0, filed Feb. 6, 2009. 
     STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5)—SEQUENCE LISTING SUBMITTED ON COMPACT DISC 
     Pursuant to 37 C.F.R. §1.52(e)(1)(ii), a compact disc containing an electronic version of the Sequence Listing has been submitted concomitant with this application, the contents of which are hereby incorporated by reference. A second compact disc is submitted and is an identical copy of the first compact disc. The discs are labeled “copy 1” and “copy 2,” respectively, and each disc contains one file entitled “P82942US00 seqlist.ST25 DJM.txt,” which is 176 KB and created on Oct. 14, 2011. 
     TECHNICAL FIELD 
     The invention relates to the fields of protein chemistry, biology and medicine. More specifically, it relates to the design and preparation of proteinmimics of members of the cystine-knot growth factor superfamily. Further, the invention relates to the use of these proteinmimics as a medicament or prophylactic agent. 
     BACKGROUND 
     The cystine-knot three-dimensional structure is found in many extracellular molecules and is conserved among divergent species. (ref 4)  The cystine-knot structure is formed by the arrangement of six cysteines which, through their disulfide bonds, form a knot. A typical consensus motif for a cystine-knot structure is: X0-C1-X1-C2-X2-C3-X3-C4-X4-C5-X5-C6-X6 (amino acids of SEQ ID NO:2), wherein cysteines 2, 3, 5 and 6 form a ring that includes X2 and X3, by disulfide bonding of cysteines 2 and 5, and cysteines 3 and 6. The third disulfide bond between cysteines 1 and 4 penetrates the ring, thus forming a knot. (ref 2,3)    FIG. 11  represents a schematic representation of a protein comprising a cystine-knot structure. This cystine-knot folding leads to the formation of three distinct domains, with two distorted beta-hairpin (beta-1 and beta-3) loops protruding from one side of the knot, and a single (beta-2) hairpin loop protruding from the other side of the knot. The beta-1 hairpin loop is formed by the stretch of amino acids between C1 and C2 and is designated “X1” in the above-mentioned consensus motif; the beta-2 (“X3”) and beta-3 (“X4”) hairpin loops are formed by the amino acid stretch between C3 and C4, and between C4 and C5, respectively. 
     Growth factors represent a large group of polypeptides that share the property of inducing cell multiplication both in vivo and in vitro. Although the level of sequence similarity between growth factors is low, they can be classified into subfamilies based on their structural and functional similarities. For instance, the following growth factor subfamilies all show the cystine-knot conformation described above: glycoprotein hormone-beta (GLHB) subfamily, the platelet-derived growth factor (PDGF) subfamily, the transforming growth factor beta (TGF-beta) subfamily, the nerve growth factor (NGF) subfamily, the glycoprotein hormone-alpha (GLHA) subfamily, CTCK subfamily, Noggin-like subfamily, Coagulin subfamily, Mucin-like subfamily, Mucin-like BMP-antagonist subfamily, Mucin-like hemolectin subfamily, Slit-like subfamily, and Jagged-like subfamily. However, the different sub-families have, for instance, different consensus lengths for X1, X2, X3, X4 and/or X5. Further, the different subfamilies have quite different functions and target organs. For instance, the GLHA and GLHB subfamilies are important for physiologic processes involved in reproduction, whereas members of the NGF subfamily exert their function mainly on nerve cells, and members of the PDGF subfamily mainly on endothelial cells. 
     Next to the cysteines involved in cystine-knot formation, other cysteines can be present in a cystine-knot protein, which are normally used to create further disulfide bonds within the cystine-knot, within the protruding domains, or between two proteins, for instance, during dimerization. 
     There has been extensive research on cystine-knot growth factors in health and disease, and therapeutic examples, for instance, are the use of vascular endothelial growth factor-specific antibodies (VEGF; a sub-subfamily of the PDGF subfamily) in the treatment of cancer, Bevacizumab (Avastin™), a monoclonal antibody developed by Genentech was approved in 2004 by the Food and Drug Administration (FDA) for the treatment of colorectal cancer, and the development of a follicle-stimulating hormone (FSH; a member of the GLHA/B subfamily) vaccine as a contraceptive for men. Major drawbacks of the therapeutic VEGF-specific monoclonal antibody Bevacizumab are the high production costs and relatively large amounts needed for treatment, sometimes low tumor penetration and its side effects. Furthermore, the antibody must be administered many times during a few months putting a high burden onto the patient. 
     DISCLOSURE 
     Provided are proteinmimics of members of the cystine-knot growth factor superfamily, which are preferably capable of inducing an immune response against the members. Also provided are alternative means and methods for treatment and/or prophylaxis of cystine-knot protein-related conditions. 
     Provided are proteinmimics of members of the cystine-knot growth factor superfamily, which may or may not be used in immunogenic and/or therapeutic compositions. 
     As said before, cystine-knot proteins have a complex conformation comprising a ring that is constituted of at least two amino acid stretches and two disulfide bonds connecting the amino acid stretches. A third disulfide bond penetrates the ring, forming a knot. All members of the cystine-knot growth factor superfamily further have in common that the amino acid stretches between the first and the second cysteine and the fourth and fifth cysteine form beta-hairpin loops that protrude in one direction, whereas another amino acid stretch, which is situated between cysteines three and four, protrudes from the opposite site of the molecule. ( FIG. 11 .) 
     In a first embodiment, the invention provides a proteinmimic of a member of the cystine-knot growth factor superfamily, the proteinmimic having the motif X0-C1-X1-C2-X2-C3-X3-C4-X4-C5-X5-C6-X6 (SEQ ID NO:1 of the incorporated herein Sequence Listing), wherein C1 to C6 are cysteine residues that form a cystine-knot structure in which C1 is linked to C4, C2 is linked to C5 and C3 is linked to C6; and wherein X0 and X6 represent, independently from each other, an amino acid sequence with a length of zero to ten amino acids, preferably zero to five amino acids, more preferably zero to three amino acids, more preferably zero to two amino acids, even more preferably zero or one amino acid, most preferably zero amino acids; X2 represents an amino acid sequence with a length of 2 to 24 amino acid residues with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to the amino acid sequence located between C2 and C3 of a member of the cystine-knot growth factor superfamily; X5 represents an amino acid sequence with a length of 1 amino acid residue; X1 represents an amino acid sequence with a length of 15 to 50 amino acids with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to the amino acid sequence located between C1 and C2 of a member of the cystine-knot growth factor superfamily; X3 represents an amino acid sequence with a length of 3 to 36 amino acids with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to the amino acid sequence located between C3 and C4 of a member of the cystine-knot growth factor superfamily; and X4 represents an amino acid sequence with a length of 15 to 50 amino acids with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to the amino acid sequence located between C4 and C5 of a member of the cystine-knot growth factor superfamily. Preferably, C2, C3, C5 and C6 form a ring by a bond between C2 and C5, and between C3 and C6, wherein the third bond between C1 and C4 penetrates the ring, thus forming a cystine-knot. 
     In a particular embodiment, a peptidomimetic hereof is provided for which the total number of amino acids equals 130 or less, preferably 110 or less, more preferably 100 or less, even more preferably 90 or less, most preferably 80 or less. 
     In a particular embodiment, a proteinmimic hereonf is provided wherein X1, X2, X3 and X4 each represent an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to an amino acid sequence of the same member of the cystine-knot growth factor superfamily. This thus means that provided is a proteinmimic of a member of the cystine-knot growth factor superfamily, the proteinmimic having the motif X0-C1-X1-C2-X2-C3-X3-C4-X4-C5-X5-C6-X6 (SEQ ID NO:2), wherein C1 to C6 are cysteine residues that form a cystine-knot structure in which C1 is linked to C4, C2 is linked to C5 and C3 is linked to C6; and wherein X0 and X6 represent, independently from each other, an amino acid sequence with a length of zero to ten amino acids, preferably zero to five amino acids, more preferably zero to three amino acids, more preferably zero to two amino acids, more preferably zero or one amino acid, most preferably zero amino acids; X2 represents an amino acid sequence with a length of 2 to 24 amino acid residues with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to the amino acid sequence located between C2 and C3 of the member of the cystine-knot growth factor superfamily; X5 represents an amino acid sequence with a length of one amino acid residue; X1 represents an amino acid sequence with a length of 15 to 50 amino acids with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to the amino acid sequence located between C1 and C2 of the member of the cystine-knot growth factor superfamily; X3 represents an amino acid sequence with a length of 3 to 36 amino acids with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to the amino acid sequence located between C3 and C4 of the member of the cystine-knot growth factor superfamily; and X4 represents an amino acid sequence with a length of 15 to 50 amino acids with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to the amino acid sequence located between C4 and C5 of the member of the cystine-knot growth factor superfamily. Preferably, C2, C3, C5 and C6 form a ring by a bond between C2 and C5, and between C3 and C6, wherein the third bond between C1 and C4 penetrates the ring, thus forming a cystine-knot. 
     In a particular embodiment, a peptidomimetic hereof is provided for which the total number of amino acids equals 130 or less, preferably 110 or less, more preferably 100 or less, even more preferably 90 or less, most preferably 80 or less. 
     A member of the cystine-knot growth factor superfamily is herein defined as any protein that forms a typical cystine-knot three-dimensional structure as described above, thus with at least six cysteines that form a cystine-knot and three hairpin loops protruding from the knot, wherein cysteines 2, 3, 5 and 6 form a ring by a bond between cysteines 2 and 5, as well as between cysteines 3 and 6, and wherein the third bond between cysteines 1 and 4 penetrates the ring, thus forming the knot. A person skilled in the art is able, for instance, by a combination of pattern search and pair-wise alignments, to identify structural motifs, present in members of the cystine-knot growth factor superfamily. A person skilled in the art may be guided in his search, for instance, by known cystine-knot proteins belonging to the cystine-knot growth factor superfamily, for instance, by the non-limiting examples provided in  FIG. 10A-X . 
     The inventors have provided the insight that so-called “truncated cystine-knot proteins” according to the invention are especially useful for treating or preventing cystine-knot protein-related disorders. They have, for instance, shown that a truncated VEGF according to the invention shows negligible hormonal activity, whereas its immunological properties are excellent. One of the advantages of the negligible hormonal activity of truncated VEGF according to the invention is, for instance, that a significant amount of truncated VEGF can be administered to an animal without the hormonal side effects of the whole protein. Another advantage of truncated VEGF in comparison to the native protein or smaller fragments thereof, is that truncated VEGF is immunogenic per se. This is due to the fact that, in contrast to smaller fragments, truncated VEGF is large enough to be immunogenic without being coupled to a carrier protein and, in contrast to the native protein, is “non-native” enough to be seen as non-self by the immune system. With “non-self” is meant that the immune system does not consider the protein or parts of the protein as a self-protein and, therefore, mounts an immune response toward the protein. 
     Without being bound to theory, the fact that a truncated protein according to the invention is seen as “non-self” is explained, for instance, by the concept of “cryptic peptides.” Cryptic peptides are defined as peptides that are part of a (self-)protein, but under normal conditions, are not presented to the immune system. The immune system is “ignorant” of these cryptic peptides. Proteins taken up by antigen-presenting cells are processed, i.e., cut in small peptide fragments. Under normal conditions, these small peptide fragments of a given protein are more or less identical after each processing. These are so-called “dominant peptides”. Each time a given protein is processed, it produces, for instance, peptides x, y and z in sufficient amounts to be effectively presented to the immune system. The immune system, constantly being exposed to peptides x, y and z of self proteins, ignores these dominant peptides of self proteins, whereas dominant peptides of non-self proteins, which are occasionally present, are reacted to. If, however, a self protein is, for instance, truncated according to the invention, the peptide fragments after processing in antigen-presenting cells differ from those of the whole native protein. As a result, so-called “cryptic peptides,” peptides that are not normally presented, are being generated and presented to the immune system in sufficient amounts. Instead of, for instance, the dominant self peptides x, y and z, peptides x, z and w are generated and presented to the immune system. As the immune system has not been exposed to cryptic peptide w previously, the immune system regards peptide w as non-self, and initiates an immune reaction. Without being bound to theory, this phenomenon may explain the enhanced immunogenicity of the truncated protein according to the invention as compared to the native protein. 
     Further shown is that the cystine-knot structure is important for the immunological properties of the protein. This is especially true if the native protein is to be immunologically mimicked. The inventors have, for instance, shown that a truncated VEGF protein in which the cysteines were blocked, disabling cystine-knot formation, is not recognized by the therapeutic VEGF monoclonal antibody Bevacizumab, whereas a truncated VEGF in which a cystine-knot is presented, is recognized by the antibody. What is said above for VEGF is equally true for other members of the cystine-knot growth factor superfamily. If, for instance, a proteinmimic of FSH is used, it is preferred that the biological or hormonal activity is negligible, whereas the proteinmimic is preferably able to induce antibodies, preferably neutralizing antibodies that are capable of cross-reacting with the native protein. The same holds true for other members of the GLHA/GLHB subfamily, or members of other subfamilies. 
     A “truncated cystine-knot protein” is defined herein as a cystine-knot protein in which at least part of the native amino acid sequence has been deleted, preferably N-terminal and/or C-terminal of the cystine-knot sequence. More preferably, the amino acid sequences N-terminal of C1 and C-terminal of C6 have been completely deleted. In a particular embodiment, therefore, provided is a proteinmimic hereof, wherein the proteinmimic has the motif C1-X1-C2-X2-C3-X3-C4-X4-C5-X5-C6 (SEQ ID NO:2). C2, C3, C5 and C6 may form a ring by a bond between C2 and C5, and between C3 and C6, and a third bond between C1 and C4 penetrates the ring, thus forming a cystine-knot. 
     In a more preferred embodiment, a peptidomimetic hereof is provided, for which the total number of amino acids equals 130 or less, preferably 110 or less, more preferably 100 or less, even more preferably 90 or less, most preferably 80 or less so that biological activity, e.g., hormonal side effects, are significantly reduced. 
     In a particular embodiment, a proteinmimic according to the invention is provided wherein X1 represents an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to an amino acid sequence of a member of the cystine-knot growth factor superfamily and wherein X2, X3 and/or X4 represent an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to an amino acid sequence of at least one other member of the cystine-knot growth factor superfamily. This is called a “chimeric proteinmimic” because the proteinmimic contains amino acid sequences with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to sequences of at least two different members of the cystine-knot growth factor superfamily. Such a chimeric proteinmimic preferably comprises loops, at least one of which representing a loop from another member of the cystine-knot growth factor superfamily than the other loops. In a preferred embodiment, each of the loops represents another member of the cystine-knot growth factor superfamily. 
     Also provided is a proteinmimic according to the invention, wherein the proteinmimic comprises the motif C1-X1-C2-X2-C3-X3-C4-X4-C5-X5-C6 (SEQ ID NO:1), wherein each of X1, X2, X3, X4 and X5 represents an amino acid sequence that has at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity with the corresponding part of a sequence selected from any of the sequences 1 to 145 of  FIG. 10A-X . In a most preferred embodiment, each of X1, X2, X3, X4 and X5 represents an amino acid sequence that is identical to the corresponding part of a sequence selected from sequences 1 to 145 of  FIG. 10A-X . 
     It is especially useful to substitute at least one loop of one member of a cystine-knot growth factor superfamily with a loop of another member of a cystine-knot growth factor superfamily, wherein the latter loop is smaller, i.e., comprises lesser amino acids, than the loop that is substituted. One advantage of a substation with a smaller loop is that the proteinmimic is manufactured more easily. In a working example, the invention, for instance, shows that the substitution of the b2 loop (represented by “X3”) of Transforming Growth Factor-B2 (TGFB2) consisting of 29 amino acids with the b2 loop of VEGF consisting of six amino acids provides a proteinmimic that is successfully used to induce antibodies that fully cross-react with the full-length TGFB2 protein. 
     In a preferred embodiment, therefore, provided is a proteinmimic hereof, wherein X3 represents an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to an amino acid sequence of a member of the cystine-knot growth factor superfamily and wherein X1, X2 and/or X4 represent an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to an amino acid sequence of at least one other member of the cystine-knot growth factor superfamily, preferably wherein at least one other member of the cystine-knot growth factor superfamily is a member of the TGF-beta subfamily, more preferably TGFB2. Preferably X1, X2 and X4 each represent an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to an amino acid sequence of the corresponding part of the same cystine-knot growth factor superfamily, whereas X3 represents an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to an amino acid sequence of the corresponding part of another member of the cystine-knot growth factor superfamily. Preferably X1, X2 and X4 represent an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to an amino acid sequence of a member of the TGF-beta subfamily, more preferably to an amino acid sequence of TGFB2. 
     In a particular embodiment, the chimeric proteinmimic consists of the amino acid sequence: C1 ALRPLYIDFKRDLGWKWIHEPKGYNANFC2AGAC3NDEGLEC4VSQDLEPLTILYYIGKTPKIEQLSNMIVKSC5KC6 (SEQ ID NO:35) (TGFB2 15-111/Δ49-77 -VEGF 62-67 ), optionally comprising flanking sequences with a length of at most five amino acids. In a particular embodiment, the flanking sequences have a length of at most two amino acids, preferably at most one amino acid. In a most preferred embodiment, the proteinmimic does not comprise flanking sequences. 
     TGF2B2 is a member of the TGF-beta subfamily. It is a secreted protein (cytokine) that performs many cellular functions and has a vital role during embryonic development. It is also known as Glioblastoma-derived T-cell suppressor factor, G-TSF, BSC-1 cell growth inhibitor, Polyergin, and Cetermin. It is known to suppress the effects of interleukin-dependent T-cell tumors. 
     In another preferred embodiment, provided is a proteinmimichereof, wherein X0 represents acetyl and/or X6 represents amide. In a more preferred embodiment, X0 represents acetyl and X6 represents amide. Acetylation of the N-terminus and/or amidation of the C-terminus has several advantages, for instance, the acetylated and amidated peptide ends are uncharged so they mimic natural peptides, stability toward digestions by aminopeptidases is enhanced and peptide ends are blocked against synthetase activities. 
     In another preferred embodiment, provided is a proteinmimic of a member of the cystine-knot growth factor superfamily, the proteinmimic having an identical sequence as the member, with the exception that the protein is truncated at position 0 to 10, preferably at position 0 to 5, more preferably at position 0 to 3, even more preferably at position 0 to 2, most preferably at position 0 or 1 N-terminal of C1 and at position 0 to 10, preferably at position 0 to 5, more preferably at position 0 to 3, even more preferably at position 0 to 2, more preferably at position 0 or 1, most preferably at position 0 C-terminal of C6. 
     Instead of the native sequence of a given member, consensus sequences of a subfamily can be used for designing a proteinmimic useful in the invention. 
     For the cystine-knot growth factor superfamily, several consensus sequences have been described. (ref 1,3)  For instance, for all but the Noggin-, Coagulin- and NGF-like cystine-knot proteins, X2 consists of two or three amino acids that can be defined as X2a-G-X2b, wherein X2a is any amino acid or none, G is glycine, and X2b is any amino acid. In a preferred embodiment, therefore, a proteinmimic according to the invention is provided wherein X2 has the amino acid sequence X2a-G-X2b, wherein X2a is any amino acid or none, G is glycine, and X2b is any amino acid. Other consensus sequences are known, for instance, for TGF-beta, GLHB, NGF, PDGF, GLHA, and CTCK. Known consensus sequences are depicted for the respective subfamilies in  FIG. 10A-X . 
     In another preferred embodiment, a proteinmimic according to the invention is provided, which comprises at least one of the following consensus sequences: 
                             (SEQ ID NO: 4)           [GSRE]C3[KRL]G[LIVT][DE]XXX[YW]XSXC4;                       (SEQ ID NO: 5)           P[PSR]CVXXXRC2[GSTA]GCC3;                       (SEQ ID NO: 6)           [LIVM]XXPXX[FY]XXXXC2XGXC3;                       (SEQ ID NO: 7)           C2[STAGM]G[HFYL]C3X[ST];                       (SEQ ID NO: 8)           [PA]VAXXC5XC6XXCXXXX[STDAI][DEY]C;                       (SEQ ID NO: 9)           C2XGCC3[FY]S[RQS]A[FY]PTP;           or                       (SEQ ID NOS: 10 and 11)           CC4(X)13C(X)2[GN](X)12C5XC6(X)2,4C;            
wherein C2 to C6 are cysteine residues that are part of a cystine-knot structure;
         X means any amino acid;   [GSRE] means G or S or R or E; [KRL] means K or R or L;   [LIVT] means L or I or V or T; [DE] means D or E; [YW] means Y or W;   [PSR] means P or S or R; [GSTA] means G or S or T or A;   [LIVM] means L or I or V or M; [FY] means F or Y;   [STAGM] means S or T or A or G or M; [HFYL] means H or F or Y or L;   [ST] means S or T; [PA] means P or A; [STDAI] means S or T or D or A or I;   [DEY] means D or E or Y; [GN] means G or N; [RQS] means R or Q or S;   (X)13 means a sequence of 13 amino acids; (X)2 means a sequence of two amino acids;   (X)12 means a sequence of 13 amino acids and (X)2,4 means a sequence of two, three, or four amino acids.       

     It is preferred to use a proteinmimic that shows a considerable % sequence identity with a native amino acid sequence of the cystine-knot protein in order to produce antibodies and/or T-cells that are capable of cross-reacting towards the native protein. With “considerable % sequence identity” is meant: at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity with the native amino acid sequence of the cystine-knot protein. This is especially true if the proteinmimic is used as a vaccine to induce an immune response that is cross-reactive with a native cystine-knot protein, but also if the proteinmimic is used to induce T-cells and/or antibodies to be used as a medicament. The T-cells and/or antibodies that are raised against the proteinmimic are especially useful if they are able to cross-react with a native cystine-knot protein. However, in another embodiment, it can be especially useful to not generate antibodies against the native protein, for instance, if the proteinmimic is to be used as an antagonist of a cystine-knot protein. In such a case, a proteinmimic according to the invention with a lower sequence identity with the native protein is designed, preferably between 70% and 90%, more preferably between 70% and 80%, most preferably between 70% and 75% sequence identity with the native amino acid sequence of the cystine-knot protein. Administration of such a proteinmimic with antagonistic properties to an individual preferably does not induce a T-cell and/or antibody response in the individual. In order to act as an antagonist, the proteinmimic preferably does not convey protein function to a receptor. 
     “% sequence identity” is defined herein as the percentage of residues in a candidate amino acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. 
     Methods and computer programs for the alignment are well known in the art. One computer program that may be used or adapted for purposes of determining whether a candidate sequence falls within this definition is “Align 2,” authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991. 
     In a particular embodiment, a proteinmimic according to the invention is provided wherein the proteinmimic has an amino acid sequence with at least 70% sequence identity, preferably at least 80%, more preferably at least 85%, most preferably at least 90% sequence identity to the corresponding native amino acid sequence of the member of the cystine-knot growth factor superfamily. In another preferred embodiment, the invention provides a proteinmimic according to the invention, wherein the member of the cystine-knot growth factor superfamily is a member selected from the group consisting of the GLHB subfamily, the PDGF subfamily, the TGF-beta subfamily, the NGF subfamily, the GLHA subfamily, the CTCK subfamily, the Noggin-like subfamily, the Mucin-like subfamily, the Mucin-like BMP antagonist subfamily, the Mucin-like hemolectin subfamily, the Slit-like subfamily, and the Jagged-like subfamily. 
     In another preferred embodiment, a proteinmimic hereof is provided wherein the proteinmimic has an amino acid sequence with between 70% and 90%, more preferably between 70% and 80%, most preferably between 70% and 75% sequence identity to the corresponding native amino acid sequence of the member of the cystine-knot growth factor superfamily. In another preferred embodiment, the invention provides a proteinmimic according to the invention, wherein the member of the cystine-knot growth factor superfamily is a member selected from the group consisting of the GLHB subfamily, the PDGF subfamily, the TGF-beta subfamily, the NGF subfamily, the GLHA subfamily, the CTCK subfamily, the Noggin-like subfamily, the Mucin-like subfamily, the Mucin-like BMP antagonist subfamily, Mucin-like hemolectin subfamily, the Slit-like subfamily, and the Jagged-like subfamily. 
     It is also useful to design a proteinmimic according to the invention with at least 70% sequence identity, preferably at least 80%, more preferably at least 85%, most preferably at least 90% sequence identity to the corresponding native amino acid sequence of the member of the cystine-knot growth factor superfamily, wherein at least one of the amino acid sequences represented by X1, X3, or X4 is at least partly deleted and/or modified. This is, for instance, especially useful if the amino acid sequence comprises an immunodominant peptide, or if the amino acid sequence has no function, for instance, if the sequence it is not part of the immunogenic determinant of the member. Deletion of such an amino acid sequence can, for instance, significantly facilitate the manufacturing process, reduce manufacturing costs or improve solubility of the proteinmimic according to the invention. In a preferred embodiment, therefore, the invention provides a proteinmimic according to the invention, wherein at least one of the amino acid sequences represented by X1, X3, or X4 is at least partly deleted and/or modified. 
     For instance, PDGF plays a role in embryonic development, cell proliferation, cell migration, and angiogenesis. PDGF has also been linked to several diseases such as atherosclerosis, fibrosis and malignant diseases. Especially the VEGF family, a sub-subfamily of the PDGF subfamily, has been linked to angiogenesis related to tumor growth and metastasis. Accordingly, in a preferred embodiment, the invention provides a proteinmimic according to the invention, wherein the member is a member of the PDGF subfamily, and wherein X2 represents an amino acid sequence with a length of three amino acids, X5 represents an amino acid sequence with a length of one amino acid, X1 represents an amino acid sequence with a length of 29 to 32 amino acids, X3 represents an amino acid sequence with a length of six to twelve amino acids, and X4 represents an amino acid sequence with a length of 32 to 41 amino acids. 
     In a more preferred embodiment, a proteinmimic is provided wherein the member is human Vascular Endothelial Growth Factor (hVEGF), and wherein X0 comprises amino acid sequence KFMDVYQRSY (amino acids 1-10 of SEQ ID NO:12), X1 comprises amino acid sequence HPIETLVDIFQEYDPEIEYIFKPSAVPLMR (amino acids 12-41 of SEQ ID NO:12), X2 comprises GGA, X3 comprises NDEGLE (amino acids 47-52 of SEQ ID NO:12), X4 comprises VPTEESNITMQIMRIKPHQGQHIGEMSFLQHNK (amino acids 54-86 of SEQ ID NO:12), X5 comprises E, and X6 comprises RPKKDRARQE (amino acids 90-99 of SEQ ID NO:12). 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of hVEGF, wherein X0-X6 are the respective hVEGF amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of hVEGF. 
     In yet another more preferred embodiment, a proteinmimic according to the invention is provided wherein the member is human Vascular Endothelial Growth Factor (hVEGF), and wherein the proteinmimic consists of the amino acid sequence C1 HPIETLVDIFQEYDPEIEYIFKPSAVPLMRC2GGAC3NDEGLEC4VPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKC5EC6 (SEQ ID NO:26), optionally comprising flanking sequences with a length of at most five amino acids. In a preferred embodiment, the flanking sequences have a length of at most two amino acids, preferably at most one amino acid. In a most preferred embodiment, the proteinmimic does not comprise flanking sequences. 
     Placental growth factor (PLGF) is a member of the PDGF subfamily (subfamily 4) and a key molecule in angiogenesis and vasculogenesis, in particular, during embryogenesis. The main source of PLGF during pregnancy is the placental trophoblast. PLGF is also expressed in many other tissues, including the villous trophoblast. PLGF expression within human atherosclerotic lesions is associated with plaque inflammation and neovascular growth. 
     Serum levels of PLGF and sFlt-l (soluble fms-like tyrosine kinase-1, also known as soluble VEGF receptor-1) are altered in women with preeclampsia. Studies show that in both early and late onset preeclampsia, maternal serum levels of sFlt-l are higher and PLGF lower in women presenting with preeclampsia. In addition, placental sFlt-l levels were significantly increased and PLGF decreased in women with preeclampsia as compared to those with uncomplicated pregnancies. This suggests that placental concentrations of sFlt-l and PLGF mirror the maternal serum changes. This is consistent with the view that the placenta is the main source of sFlt-l and PLGF during pregnancy. 
     In yet another preferred embodiment, a proteinmimic according to the invention is provided wherein the member is human Placental Growth Factor (hPLGF), and wherein X0 comprises amino acid sequence PFQEVWGRSY (amino acids 1-10 of SEQ ID NO:13), X1 comprises amino acid sequence RALERLVDVVSEYPSEVEHMFSPSAVSLLR (amino acids 12-41 of SEQ ID NO:13), X2 comprises TGA, X3 comprises GDENLH (amino acids 47-52 of SEQ ID NO:13), X4 comprises VPVETANVTMQLLKIRSGDRPSYVELTFSQHVR (amino acids 54-86 of SEQ ID NO:13), X5 comprises E, and X6 comprises RHSPGRQSPD (amino acids 90-99 of SEQ ID NO:13). 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of PLGF, wherein X0-X6 are the respective PLGF amino acid sequences depicted in  FIG. 10 . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of PLGF. 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of PLGF, wherein X0-X6 are the respective PLGF amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of PLGF. 
     In yet another preferred embodiment, a proteinmimic according to the invention is provided wherein the member is human Platelet-Derived Growth Factor A (hPDGF-A), and wherein X0 comprises amino acid sequence SIEEAVPAV (amino acids 1-9 of SEQ ID NO:15), X1 comprises amino acid sequence KTRTVIYEIPRSQVDPTSANFLIWPPCVEVKR (amino acids 11-42 of SEQ ID NO:15), X2 comprises TGC, X3 comprises NTSSVK (amino acids 48-53 of SEQ ID NO:15), X4 comprises QPSRVHHRSVKVAKVEYVRKKPKLKEVQVRLEEHLE (amino acids 55-90 of SEQ ID NO:15), X5 comprises A, and X6 comprises ATSLNPDYRE (amino acids 92-103 of SEQ ID NO:15). In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of hPDGF-A, wherein X0-X6 are the respective hPDGF-A amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of hPDGF-A. 
     In yet another preferred embodiment, a proteinmimic according to the invention is provided wherein the member is human Platelet-Derived Growth Factor A (hPDGF-C), and wherein X0 comprises amino acid sequence LLTEEVRLYS (amino acids 1-10 of SEQ ID NO:16), X1 comprises amino acid sequence TPRNFSVSIREELKRTDTIFWPGCLLVKR (amino acids 12-40 of SEQ ID NO:16), X2 comprises GGN, X3 comprises ACCLHNCNECQ (amino acids 46-56 of SEQ ID NO:16), X4 comprises VPSKVTKKYHEVLQLRPKTGVRGLHKSLTDVALEHHEE (amino acids 58-95 of SEQ ID NO:16), X5 comprises D, and X6 comprises VCRGSTGG (amino acids 99-106 of SEQ ID NO:16). 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of hPDGF-C, wherein X0-X6 are the respective hPDGF-C amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of hPDGF-C. 
     In yet another preferred embodiment, a proteinmimic according to the invention is provided wherein the member is human Vascular Endothelial Growth Factor C (hVEGF-C), and wherein X0 comprises amino acid sequence SIDNEWRKTQ (amino acids 1-10 of SEQ ID NO:17), X1 comprises amino acid sequence MPREVAIDVGKEFGVATNTFFKPPCVSVYR (amino acids 12-41 of SEQ ID NO:17), X2 comprises GGC, X3 comprises PDDGLE (amino acids 47-53 of SEQ ID NO:17), X4 comprises VPTGQHQVRMQILMIRYPSSQLGEMSLEEHSQ (amino acids 54-85 of SEQ ID NO:17), X5 comprises E, and X6 comprises RPKKKDSAVK (amino acids 89-98 of SEQ ID NO:17). 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of hVEGF-C, wherein X0-X6 are the respective hVEGF-C amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of hVEGF-C. 
     Other subfamilies of the cystine-knot growth factor superfamily include the GLHA and GLHB subfamily. Members of these subfamilies comprise the glycoprotein hormone-alpha and glycoprotein hormone-beta subunits, respectively, that after dimerization, form luteinizing hormone (LH), thyroid-stimulating hormone (TSH), chorionic gonadotropin (CG) and follicle-stimulating hormone (FSH). These hormones all play a role in reproduction in mammals. For instance, FSH stimulates testicular and ovarian functions through binding to a G-protein-coupled receptor on either Sertoli (male) or granulose (female) cells. Amongst other things, LH stimulates ovulation and sustains the corpus luteum during menstrual cycle, whereas CG, for instance, sustains the corpus luteum during pregnancy. TSH is important for Sertoli cell maturation and ovulatory function. The present invention also provides proteinmimics of this GLHB subfamily. 
     Thus, in another preferred embodiment, the member of the cystine-knot growth factor superfamily is a member of the GLHB subfamily, X2 represents an amino acid sequence with a length of three amino acids, X5 represents an amino acid sequence with a length of one amino acid, X1 represents an amino acid sequence with a length of 23 to 28 amino acids, X3 represents an amino acid sequence with a length of 18 to 20 amino acids, and X4 represents an amino acid sequence with a length of 30 to 33 amino acids. 
     In a more preferred embodiment, a proteinmimic according to the invention is provided wherein the member is human Follicle-Stimulating Hormone (hFSH), and wherein X0 comprises amino acid sequence NS, X1 comprises amino acid sequence ELTNITIAIEKEECRFCISINTTW (amino acids 4-27 of SEQ ID NO:18), X2 comprises AGY, X3 comprises YTRDLVYKDPARPKIQKT (amino acids 33-50 of SEQ ID NO:18), X4 comprises TFKELVYETVRVPGCAHHADSLYTYPVATQ (amino acids 52-81 of SEQ ID NO:18), X5 comprises H, and X6 comprises KCDSDSTDCT (amino acids 85-94 of SEQ ID NO:18). 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of FSH, wherein X0-X6 are the respective FSH amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of FSH. 
     In yet another more preferred embodiment, a proteinmimic according to the invention is provided wherein the member is human Choriogonadotropin (hCG), and wherein X0 comprises amino acid sequence SKEPLRPR (amino acids 1-8 of SEQ ID NO:19), X1 comprises amino acid sequence RPINATLAVEKEGCPVCITVNTTI (amino acids 10-33 of SEQ ID NO:19), X2 comprises AGY, X3 comprises PTMTRVLQGVLPALPQVV (amino acids 39-56 of SEQ ID NO:19), X4 comprises NYRDVRFESIRLPGCPRGVNPVVSYAVALS (amino acids 58-87 of SEQ ID NO:19), X5 comprises Q, and X6 comprises ALCRRSTTDC (amino acids 91-100 of SEQ ID NO:19). 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of hCG, wherein X0-X6 are the respective hCG amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of hCG. 
     In yet another preferred embodiment, the invention provides a proteinmimic according to the invention, wherein the member of the cystine-knot growth factor superfamily is a member of the glycoprotein hormone-alpha (GLHA) subfamily, and wherein X2 represents an amino acid sequence with a length of three amino acids, X5 represents an amino acid sequence with a length of one amino acid, X1 represents an amino acid sequence with a length of 13 to 17 amino acids, X3 represents an amino acid sequence with a length of 27 amino acids, and X4 represents an amino acid sequence with a length of 20 to 21 amino acids. 
     In yet another preferred embodiment, a proteinmimic according to the invention is provided wherein the member of the cystine-knot growth factor superfamily is a member of the nerve growth factor (NGF) subfamily, and wherein X2 represents an amino acid sequence with a length of 9 to 24 amino acids, X5 represents an amino acid sequence with a length of one amino acid, X1 represents an amino acid sequence with a length of 41 to 44 amino acids, X3 represents an amino acid sequence with a length of eleven amino acids, and X4 represents an amino acid sequence with a length of 27 or 28 amino acids. 
     In a more preferred embodiment, a proteinmimic according to the invention is provided wherein the member is human Nerve Growth Factor (hNGF), and wherein X0 comprises amino acid sequence PIFHRGEFSV (amino acids 1-10 of SEQ ID NO:20), X1 comprises amino acid sequence DSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETK (amino acids 12-53 of SEQ ID NO:20), X2 comprises RDPNPVDSG (amino acids 55-63 of SEQ ID NO:20), X3 comprises RGIDSKHWNSY (amino acids 65-75 of SEQ ID NO:20), X4 comprises TTTHTFVKALTMDGKQAAWRFIRIDTA (amino acids 77-103 of SEQ ID NO:20), X5 comprises V, and X6 comprises VLSRKAVRRA (amino acids 107-116 of SEQ ID NO:20). 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of hNGF, wherein X0-X6 are the respective hNGF amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of hNGF. 
     Members of the NGF subfamily play a role in survival and maintenance of sympathetic and sensory neurons and have been associated with Alzheimer disease. NGF plays a role in the repair, regeneration, and protection of neurons, and a proteinmimic of a member of the NGF subfamily according to the invention is thus especially useful for treating or preventing a neurodegenerative disorder. 
     Yet another subfamily of the cystine-knot growth factor superfamily is the TGF-beta subfamily. TGF-beta controls proliferation, cellular differentiation, and other functions in most cells. It plays a role in immunity, cancer, heart disease and in Marfan syndrome, a genetic disorder of the connective tissue. 
     In another preferred embodiment, therefore, the invention provides a proteinmimic according to the invention, wherein the member of the cystine-knot growth factor superfamily is a member of the transforming growth factor beta (TGF-beta) subfamily, and wherein X2 represents an amino acid sequence with a length of three amino acids, X5 represents an amino acid sequence with a length of one amino acid, X1 represents an amino acid sequence with a length of 23 to 41 amino acids, X3 represents an amino acid sequence with a length of 18 to 36 amino acids, and X4 represents an amino acid sequence with a length of 27 to 34 amino acids. 
     In a more preferred embodiment, a proteinmimic according to the invention is provided wherein the member is human Transforming Growth Factor beta2 (hTGF-beta2), and wherein X0 comprises amino acid sequence AYCFRNVQDN (amino acids 1-10 of SEQ ID NO:21), X1 comprises amino acid sequence CLRPLYIDFKRDLGWKWIHEPKGYNANF (amino acids 12-39 of SEQ ID NO:21), X2 comprises AGA, X3 comprises PYLWSSDTQHSRVLSLYNTINPEASASPC (amino acids 45-73 of SEQ ID NO:21), X4 comprises VSQDLEPLTILYYIGKTPKIEQLSNMIVKS (amino acids 75-104 of SEQ ID NO:21), X5 comprises K, and X6 comprises S. 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of hTGF-beta2, wherein X0-X6 are the respective hTGF-beta2 amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of hTGF-beta2. 
     Functional diverse modular proteins share a conserved domain of about 90 amino acids in their C-terminal cysteine-rich region, that has been proposed to be structurally related to the cystine-knot family and that is, therefore, called C-terminal cystine-knot (CTCK). Members of the C-terminal cystine-knot family are, amongst others, von Willebrand factor (vWF), a multifunctional protein that is involved in maintaining homeostasis, mucins, CCN family members (cef-10/cyr61/CTFG/fisp-12/nov protein family), (ref 5)  Drosophila slit protein, which is essential for development of midline glia and commissural axon pathways, Norrie disease protein (NDP), which may be involved in neuroectodermal cell-cell interaction and in a pathway that regulates neural cell differentiation and proliferation, and Silk moth hemocytin, a humoral lectin that is involved in a self-defense mechanism. The teaching of the present invention also encompasses this CTCK family. 
     In another preferred embodiment, therefore, the invention provides a proteinmimic according to the invention, wherein the member of the cystine-knot growth factor superfamily is a member of the CTCK subfamily, and wherein X2 represents an amino acid sequence with a length of two to three amino acids, X5 represents an amino acid sequence with a length of one amino acid, X1 represents an amino acid sequence with a length of 22 to 35 amino acids, X3 represents an amino acid sequence with a length of 4 to 28 amino acids, and X4 represents an amino acid sequence with a length of 29 to 41 amino acids. 
     Sclerostin (or SOST) is also a member of the CTCK-subfamily of the cystine-knot growth factor super family. Sclerostin, the product of the SOST gene, was originally believed to be a non-classical bone morphogenetic protein (BMP) antagonist. More recently, sclerostin has been identified as binding to LRP5/6 receptors and inhibiting the Wnt-signaling pathway. Wnt-activation under these circumstances is antagonistic to bone formation. More recently, it has been revealed that the antagonism of BMP-induced bone formation by sclerostin is mediated by Wnt signaling, but not BMP-signaling pathways. The successful synthesis of SOST 67-144  in one of the examples serves to demonstrate that truncated cystine-knot proteins/peptides with an additional SS-bridge between C 71  (loop-1; X1) and C 125  (loop-3; X4) perfectly form the correctly folded cystine-knot structure in the presence of the additional disulfide bond. 
     In a more preferred embodiment, a proteinmimic according to the invention is provided wherein the member is sclerostin, and wherein X0 comprises amino acid sequence FETKDVSEYS (amino acids 1-10 of SEQ ID NO:22), wherein X1 comprises amino acid sequence RELHFTRYVTDGPCRSAKPVTELV (amino acids 12-35 of SEQ ID NO:22), X2 comprises SGQ, X3 comprises GPARLLPNAIGRGKWWRPSGPDFR (amino acids 41-64 of SEQ ID NO:22), X4 comprises IPDRYRAQRVQLLCPGGEAPRARKVRLVAS (amino acids 66-95 of SEQ ID NO:22), X5 comprises K, and X6 comprises KRLTRFHNQS (amino acids 99-108 of SEQ ID NO:22). 
     In another more preferred embodiment, a proteinmimic is provided that has at least 70% sequence identity to X0-X6 of sclerostin, wherein X0-X6 are the respective sclerostin amino acid sequences depicted in  FIG. 10A-X . Preferably, the proteinmimic has at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity to X0-X6 of sclerostin. 
     In yet another more preferred embodiment, a proteinmimic according to the invention is provided wherein the member is sclerostin, and wherein the proteinmimic consists of the amino acid sequence GGGC1RELHFTRYVTDGPCRSAKPVTELVC2SGQC3GPARLLPNAIGRGKWWRPSGPDFRC4IPDRYRAQRVQLLCPGGEAPRARKVRLVASC5KC6 (SEQ ID NO:23), optionally comprising flanking sequences with a length of at most five amino acids. In a preferred embodiment, the flanking sequences have a length of at most two amino acids, preferably at most one amino acid. In a most preferred embodiment, the proteinmimic does not comprise flanking sequences. 
     Members of the Noggin-like subfamily are, for instance, known to inhibit TGF-beta signal transduction by binding to TGF-beta family ligands and preventing them from binding to their corresponding receptors. Noggin plays a key role in neural induction by inhibiting BMP4. A proteinmimic of a member of the Noggin-like subfamily is thus especially useful for regulating TGF-beta and/or BMP4 activity. 
     In another preferred embodiment, therefore, the invention provides a proteinmimic according to the invention, wherein the member of the cystine-knot growth factor superfamily is a member of the Noggin-like subfamily, and wherein X2 represents an amino acid sequence with a length of four to six amino acids, X5 represents an amino acid sequence with a length of one amino acid, X1 represents an amino acid sequence with a length of 22 amino acids, X3 represents an amino acid sequence with a length of seven to nine amino acids, and X4 represents an amino acid sequence with a length of 35 to 98 amino acids. 
     A proteinmimic of a member of the Coagulin-like subfamily is, for instance, especially useful for treating coagulation disorders. Clinical trials have been started, for instance, with gene therapy-based coagulin B supplementation for hemophilia B. However, a proteinmimic of a member of the coagulin-like subfamily as provided herewith is suitable for inhibiting coagulin B, for instance, to reduce blood clotting, thereby preventing thrombosis. 
     In another preferred embodiment, therefore, the invention provides a proteinmimic according to the invention, wherein the member of the cystine-knot growth factor superfamily is a member of the Coagulin-like subfamily, and wherein X2 represents an amino acid sequence with a length of seven amino acids, X5 represents an amino acid sequence with a length of one amino acid, X1 represents an amino acid sequence with a length of 38 amino acids, X3 represents an amino acid sequence with a length of five amino acids, and X4 represents an amino acid sequence with a length of 29 amino acids. 
     Members of the jagged-like subfamily are, for instance, ligands of the Notch family of receptors. The Notch signaling pathway plays a crucial role during embryonic pattern formation, controls many conserved cell determination events and defines a fundamental mechanism controlling cell fate. It is involved in lineage cell decisions in a variety of tissues. It plays a role in hematopoiesis, vascular development and angiogenesis, myogenesis, neurogenesis, somitogenesis, in kidney, eye, ear, and tooth development, etc. Proteinmimics based on jagged-like members are especially useful for controlling the before-mentioned biological processes. 
     In another preferred embodiment, therefore, provided is a proteinmimic, wherein the member of the cystine-knot growth factor superfamily is a member of the Jagged-like subfamily, and wherein X2 represents an amino acid sequence with a length of three amino acids, X5 represents an amino acid sequence with a length of one amino acid, X1 represents an amino acid sequence with a length of 32 amino acids, X3 represents an amino acid sequence with a length of 25 amino acids, and X4 represents an amino acid sequence with a length of 26 amino acids. 
     As said before,  FIG. 10A-X  depicts non-limiting examples of truncated proteins belonging to several cystine-knot growth factor subfamilies. It is especially useful to introduce small mutations, for instance, exchange at least one cysteine, not being one of the conserved cysteines one to six that are necessary for cystine-knot formation, in order to prevent, for instance, dimer formation. In a preferred embodiment, therefore, a proteinmimic according to the invention is provided, wherein X1 represents an amino acid sequence with at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity with any one of the sequences identified as a X1 in  FIG. 10A-X , and/or wherein X3 represents an amino acid sequence with at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity with any one of the sequences identified as X3 in  FIG. 10A-X , and/or wherein X4 represents an amino acid sequence with at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity with any one of the sequences identified as X4 in  FIG. 10A-X , wherein X1, X3 and X4 are taken from a single amino acid sequence of  FIG. 10A-X . 
     In a more preferred embodiment, at least one cysteine in any of the sequences represented by X1, X2, X3, X4, and X6, is replaced by another amino acid, preferably alanine. In another preferred embodiment, X1 represents an amino acid sequence that is identical with any one of the sequences identified as X1 in  FIG. 10A-X , and/or X3 represents an amino acid sequence that is identical with any one of the sequences identified as X3 in  FIG. 10A-X , and/or X4 represents an amino acid sequence that is identical with any one of the sequences identified as X4 in  FIG. 10A-X , wherein X1, X3 and X4 are taken from a single amino acid sequence of  FIG. 10A-X . 
     In another preferred embodiment, a proteinmimic according to the invention is provided wherein X2 represents an amino acid sequence with at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity with any of the sequences identified as X2 in  FIG. 10A-X , and/or wherein X5 represents an amino acid sequence that is identical to any of the sequences identified as X5 in  FIG. 10A-X , wherein X2 and X5 are taken from a single amino acid sequence of  FIG. 10A-X . 
     In a more preferred embodiment, at least one cysteine in any of the sequences represented by X1, X2, X3, X4, and X6, is replaced by another amino acid, preferably alanine. In another more preferred embodiment, X2 represents an amino acid sequence which is identical with a sequence identified as X2 in  FIG. 10 , wherein X2 and X5 are taken from a single amino acid sequence of  FIG. 10A-X . 
     In another preferred embodiment, the invention provides a proteinmimic according to the invention, wherein the proteinmimic comprises the motif C1-X1-C2-X2-C3-X3-C4-X4-C5-X5-C6 (SEQ ID NO:1), wherein the sequence has at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity with a sequence selected from sequences 1 to 145 of  FIG. 10A-X . In a most preferred embodiment, the proteinmimic sequence is identical to a sequence selected from sequences 1 to 145 of  FIG. 10A-X . Such a proteinmimic is especially useful for induction of a cross-reactive, preferably a neutralizing antibody response, because the proteinmimic is identical to a part of the native protein. 
     In a particular embodiment, a proteinmimic hereof is provided wherein C1 is linked to C4 through a disulfide bond and/or C2 is linked to C5 through a disulfide bond, and/or C3 is linked to C6 through a disulfide bond. In a more preferred embodiment, C1 is linked to C4 through a disulfide bond and C2 is linked to C5 through a disulfide bond, and C3 is linked to C6 through a disulfide bond. 
     Now that proteinmimics of members of the cystine-knot growth factor superfamily are provided, also provided is the insight that a proteinmimic hereof is especially useful for inducing an immune response, preferably, the immune response is cross-reactive to a member of the cystine-knot growth factor superfamily. With “cross-reactive” is meant that the antibody produced not only specifically binds the proteinmimic against which the antibody was raised, but also specifically binds to at least one of the members of the cystine-knot growth factor superfamily. In one embodiment therefore, an immunogenic composition is provided, comprising a proteinmimic according to the invention. The immunogenic composition preferably further comprises a therapeutically acceptable carrier, adjuvant, diluent and/or excipient. “Immunogenic composition” is defined herein in its broad sense to refer to any type of biological agent in an administrable form capable of inducing and/or stimulating an immune response in an animal. In one preferred embodiment, an immunogenic composition according to the invention at least comprises a proteinmimic according to the invention and a pharmaceutically acceptable adjuvant. 
     In another preferred embodiment, an immunogenic composition according to the invention is provided wherein the proteinmimic is coupled to an immunogenic carrier, preferably diphtheria toxin (DT) and/or keyhole limpet haemocyanin (KLH). 
     Further provided is a pharmaceutical composition comprising a proteinmimic according to the invention and a pharmaceutically acceptable carrier, diluent and/or excipient. Suitable carriers, diluents, excipients and the like are commonly known in the art of pharmaceutical formulation and may be readily found, and applied by the skilled artisan, in references, for instance, Remmington&#39;s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia Pa., 17th ed. 1985. 
     Members of the cystine-knot growth factor super-family are, as already mentioned before, associated with many diseases, including diseases of the nervous system, hematopoietic development, coagulation disorders, cancer, angiogenesis, etc. In one embodiment, therefore, the invention provides a use of a proteinmimic according to the invention for the preparation of a medicament and/or prophylactic agent for the treatment and/or prevention of a disorder associated with a member of the cystine-knot growth factor superfamily. 
     Thus provided is the use of a proteinmimic in an immunogenic composition. Such immunogenic composition comprising a proteinmimic according to the invention is suitable for inducing an immune reaction in an animal, preferably a human. In a preferred embodiment, a proteinmimic of the invention is used to induce antibodies, which are preferably able to cross-react with the native protein. Even more preferably, the antibodies are neutralizing antibodies, i.e., the function and/or activity of the native cystine-knot protein is diminished, inhibited, or at least reduced after binding of the native cystine-knot protein to the neutralizing antibody. It is possible to induce the antibodies in an individual in need thereof, for instance, by administering a vaccine comprising a proteinmimic according to the invention to the individual. It is also possible to induce the antibodies in a non-human animal by administering an immunogenic composition of the invention to the animal and use antibodies obtained from the animal for the manufacture of a medicament. However, it is also possible to use a proteinmimic according to the invention to directly antagonize the function and/or activity of the native cystine-knot protein. This can, for instance, be achieved if the proteinmimic binds to the receptor but does not or does not fully activate the receptor signal pathway. In one embodiment, the invention provides a use of a proteinmimic according to the invention, or an immunogenic compound comprising a proteinmimic according to the invention, as a partial or full antagonist of a member of the cystine-knot growth factor superfamily. 
     Now that the disclosure provides the insight that a proteinmimic according to the invention is useful as an antagonist and/or agonist for a member of the cystine-knot growth factor superfamily or suitable for raising an immune response against a member of the cystine-knot growth factor superfamily, a method is provided for treating or preventing a disorder associated with a member of the cystine-knot growth factor superfamily, comprising administering a therapeutically effective amount of a proteinmimic according to the invention to a subject suffering from, or at risk of suffering from, the disorder. 
     One subfamily of the cystine-knot growth factor superfamily is the subfamily of vascular endothelial growth factors (VEGF), which is a subfamily of the PDGF subfamily. VEGFs act through a family of cognate receptor tyrosine kinases in endothelial cells to stimulate blood vessel formation. Proteinmimics of, and/or antibodies specific for, VEGF are thus especially useful for treating a disorder related to vascularization. One such disorder is age-related macular degeneration (AMD), which causes rapid and severe visual loss. This loss is due to development of choroidal neovascularization under the macula. Inhibition of VEGF is, therefore, especially useful for the treatment and/or prevention of AMD. Another example of a disease that relates to vascularization is cancer. Tumors need neovascularization in order to grow. Fast-growing tissue needs a continuous supply of oxygen and nutrients and, therefore, the effective inhibition of neovascularization is thought to be one of the promising strategies for cancer therapy. This is, for instance, achieved by inhibiting, for instance, VEGF. As said before, Avastin™, a monoclonal antibody (Bevacizumab, Genentech) was approved in 2004 by the Food and Drug Administration (FDA) for the treatment of colorectal cancer when used with standard chemotherapy. In 2006, the FDA approved Bevacizumab® for the treatment of lung cancer in combination with standard first-line combination therapy. 
     The drawbacks of Bevacizumab®, such as the high production costs and the relative large amounts needed for treatment, sometimes low tumor penetration and frequent administration are reduced when a proteinmimic or an immunogenic composition of the invention is used. For instance, an immunogenic composition comprising a proteinmimic of the invention is administered in a dose of a few mg, preferably 0.1 to 10 mg per subject, in order to induce an immune response. Such an administration is generally repeated two or three times in order to induce a proper protective response. 
     In one embodiment, therefore, the invention provides use of a proteinmimic according to the invention for the preparation of a medicament and/or prophylactic agent for the treatment and/or prevention of a tumor-related disease and/or age-related macular degeneration (AMD), wherein the member of the cystine-knot growth factor superfamily is a member of the VEGF subfamily or the TGF-beta subfamily. 
     Another cystine-knot growth factor subfamily, TGF-beta, is also related to cancer. In normal cells, TGF-beta, acting through its signaling pathway, stops the cell cycle at the G1 stage to stop proliferation, induce differentiation, or promote apoptosis. When a cell is transformed into a cancer cell, parts of the TGF-beta signaling pathway are mutated, and TGF-beta no longer controls the cell. These cancer cells proliferate. The surrounding stromal cells (fibroblasts) also proliferate. Both cells increase their production of TGF-beta. This TGF-beta acts on the surrounding stromal cells, immune cells, endothelial and smooth-muscle cells. It causes immunosuppression and angiogenesis, which makes the cancer more invasive. TGF-beta also converts effector T-cells, which normally attack cancer with an inflammatory (immune) reaction, into regulatory (suppressor) T-cells, which turn off the inflammatory reaction. Inhibiting TGF-beta, for instance, with an antagonistic proteinmimic according to the invention and/or an antibody of the invention or functional part and/or functional equivalent thereof of the invention, wherein the member belongs to the TGF-beta subfamily, is thus especially useful for the treatment of cancer. 
     In a preferred embodiment, therefore, a method according to the invention is provided, wherein the disorder comprises a tumor-related disease and/or age-related macular degeneration (AMD), and wherein the member of the cystine-knot growth factor superfamily is a member of the VEGF subfamily or the TGF-beta subfamily. In a more preferred embodiment, the tumor-related disease is colorectal cancer or non-small cell lung cancer (NSCLC). 
     In another preferred embodiment, a method is provided wherein the disorder comprises a connective tissue disorder, preferably Marfan syndrome. Marfan syndrome is carried by a gene called FBN1, which encodes a connective protein called fibrillin-1. People have a pair of FBN1 genes. Because it is dominant, people who have inherited one affected FBN1 gene from either parent will have Marfan&#39;s. In addition to being a connective protein that forms the structural support for tissues outside the cell, fibrillin-1 binds to another protein, TGF-beta. TGF-beta can cause inflammation. Researchers now believe that the inflammatory effects of TGF-beta, at the lungs, heart valves, and aorta, weaken the tissues and cause the features of Marfan syndrome. A proteinmimic of TGF-beta is thus especially useful for treatment of Marfan syndrome. 
     In contrast, neovascularization (vascular regeneration) is especially useful for the treatment of ischemic disease including, but not limited to, arteriosclerotic occlusion of the lower limbs, angina pectoris/myocardial infarction or cerebral infarction in order to rescue the ischemic tissue by developing collateral circulation. In another preferred embodiment therefore, the disorder comprises an ischemic disorder, preferably, the ischemic disorder is taken from the group consisting of arteriosclerotic occlusion of the lower limbs, angina pectoris, myocardial infarction and cerebral infarction, wherein the member of the cystine-knot growth factor superfamily is a member of the VEGF subfamily. 
     As said before, members of the NGF subfamily are critical for the survival and maintenance of sympathetic and sensory neurons and have been associated with Alzheimer disease. As NGF plays a role in the repair, regeneration, and protection of neurons, a proteinmimic of a member for the NGF subfamily according to the invention is thus especially useful for treating a neurodegenerative disorder. Other possible applications are the use of a proteinmimic of a member of the NGF subfamily according to the invention, for instance, through induction of NGF-specific antibodies, to diminish and/or treat chronic and/or neurodegenerative pain. Further, such NGF-specific antibodies are considered especially useful for the treatment of breast tumors, as NGF is known to be a strong stimulator of breast cancer cell proliferation. 
     In another preferred embodiment, therefore, a method is provided, wherein the disorder comprises a disorder selected from the group consisting of a neurodegenerative disorder, preferably Alzheimer disease, a pain disorder, preferably a chronic and/or neuropathic pain disorder, and cancer, preferably breast cancer. In a more preferred embodiment, a method is provided wherein the member belongs to the NGF subfamily. 
     Further provided is a method for producing antibodies against a member of the cystine-knot growth factor superfamily, comprising administering a proteinmimic according to the invention and or an immunogenic composition according to the invention to a non-human animal, and obtaining antibodies against a member of the cystine-knot growth factor superfamily, which antibodies are produced by the animal. Also provided is the use of a proteinmimic according to the invention in an ex vivo method for producing an antibody, or a functional part or functional equivalent of an antibody, which is specifically directed against a member of the cystine-knot growth factor superfamily. The skilled artisan is aware of the different methods for producing an antibody ex vivo, such as B-cell hybrodima techniques, antibody phage display technologies and the like. 
     A functional part of an antibody is defined herewith as a part that has at least one same property as the antibody in kind, not necessarily in amount. The functional part is preferably capable of binding the same antigen as the antibody, albeit not necessarily to the same extent. A functional part of an antibody preferably comprises a single domain antibody, a single chain antibody, a Fab fragment or a F(ab′) 2  fragment. A functional equivalent of an antibody is defined as an antibody that has been altered such that at least one property—preferably an antigen-binding property—of the resulting compound is essentially the same in kind, not necessarily in amount. An equivalent is provided in many ways, for instance, through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc.), such that the overall functioning is likely not to be seriously affected. 
     The glycoprotein hormone subfamily (GLH), a subfamily of the cystine-knot superfamily of growth factors, comprises the hormones: luteinizing hormone, (LH), thyroid-stimulating hormone (TSH) and chorionic gonadotropin (CG) and follicle-stimulating hormone (FSH). These hormones all comprise an alpha and a beta subunit (GLHA and GLHB, respectively) and they play a role in reproduction in mammals. For instance, FSH stimulates testicular and ovarian functions through binding to a G-protein-coupled receptor on either Sertoli (male) or granulose (female) cells. Amongst other things, LH stimulates ovulation and sustains the corpus luteum during menstrual cycle, whereas CG, for instance, sustains the corpus luteum during pregnancy. TSH is important for Sertoli cell maturation and ovulatory function. 
     In a preferred embodiment, therefore, a method for treating or preventing a disorder associated with the presence of a member of the cystine-knot growth factor superfamily according to the invention is provided, wherein the disorder is a reproductive disorder. Apart from treating a reproductive disorder, a proteinmimic and/or an antibody or functional part or equivalent thereof according to the invention is also especially useful to prevent reproduction, i.e., prevent pregnancy. By inhibition of a GLH, for instance, FSH, CG, LH or TSH, or inhibition of receptor binding and/or signaling of GLH in a female or a male, ovulatory or testicular function is disturbed and the chances of pregnancy are reduced. The invention thus provides a method for preventing pregnancy and/or reducing the chance of pregnancy in a female individual, comprising administering to the female or a sexual partner of the female an effective amount of a proteinmimic according to the invention, an immunogenic composition according to the invention, and/or an antibody obtainable by a method according to the invention or a functional part or functional equivalent of the antibody, wherein the member of the cystine-knot growth factor superfamily is a member of the GLHA or GLHB subfamily. 
     Further provided is a proteinmimic according to the invention, an immunogenic composition according to the invention, and/or an antibody obtainable by a method according to the invention, or a functional part or functional equivalent thereof, for use as a male and/or female contraceptive. 
     Further provided is a method for binding and/or neutralizing an antibody directed to a member of the cystine-knot growth factor superfamily, comprising administering a therapeutically effective amount of a proteinmimic according to any one of claims  1 - 17  to a subject comprising the antibody. Upon binding of the proteinmimic to the antibody, its activity is diminished. Antibodies that are specific for members of the cystine-knot protein are used in treatment protocols. One example thereof is Avastin™ specific for VEGF, which is used to treat metastatic cancer. Antibodies, once administered, have a half-life of several days, even up to several weeks. If, for instance, such an antibody is over-dosed or if the action of such antibody is not desired anymore, a proteinmimic of the invention is especially useful to counteract the action of the antibody by binding and/or neutralizing the antibody. A proteinmimic of the invention is especially useful because the proteinmimic as such is not or to a lesser extent bioactive and, therefore, does not interfere with a condition for which the antibody was initially administered. It is, of course, undesirable to treat a patient receiving, for instance, antibodies against VEGF with bio-active VEGF to neutralize the antibody. Bio-active VEGF administered in excess of the antibody present would exert its biological effect and would undermine the antibody treatment thus far received. An illustrative example that does not limit the invention is the use of a proteinmimic of VEGF that can be used to bind and/or neutralize a monoclonal antibody against VEGF, preferably Avastin™. Avastin™ is a commercially available monoclonal antibody against VEGF, which is administered, for instance, to treat metastatic cancers. Treatment with Avastin™, however, can lead to slow or incomplete wound healing (for example, when a surgical incision has trouble healing or staying closed). In some cases, this event resulted in fatality. It is, therefore, not recommended to start Avastin™ therapy for at least 28 days after surgery and until the surgical wound is fully healed. Of course, during Avastin™ therapy, surgery should be avoided. However, it is sometimes necessary to perform surgery on a person that receives Avastin™ therapy. In such a case, a truncated VEGF, preferably VEGF 26-104 , is preferably administered to neutralize the circulating anti-VEGF antibodies without inducing much biological effect resembling the action of VEGF itself. Shortly after administration of the truncated VEGF and neutralization of the anti-VEGF antibodies, the patient may undergo surgery without the above-mentioned severe side effects that are normally observed after surgery during Avastin™ therapy. 
     In a preferred embodiment, therefore, a method for binding and/or neutralizing an antibody directed to a member of the cystine-knot growth factor superfamily comprising administering a therapeutically effective amount of a proteinmimic according to the invention to a subject comprising the antibody is provided, wherein the antibody is Avastin™ and the proteinmimic is VEGF 26-104 . 
     Further provided is the use of a proteinmimic according to the invention for the manufacture of a medicament for neutralizing an antibody directed to a member of the cystine-knot growth factor superfamily. In a preferred embodiment, the antibody is Avastin™ and the proteinmimic is VEGF 26-104  as explained before. 
     Another member of the cystine-knot growth factor superfamily belonging to the TGF-beta subfamily is sclerostin, the secreted protein product of the SOST gene, which is an osteocyte-derived inhibitor of cultured osteoblasts. Sclerostin deficiency leads to sclerosteosis and van Buchem disease, two closely related, rare sclerosing disorders characterized by substantial increase in bone mass of good quality, which is due to increased bone formation. In contrast, osteoporosis, a disorder in which the density and quality of bone are reduced, leading to weakness of the skeleton and increased risk of fracture, particularly of the spine, wrist, hip, pelvis and upper arm, is possibly caused by an excess production of sclerostin, inhibiting bone formation. An agonistic or antagonistic proteinmimic of sclerostin and/or an antibody specific for sclerostin is thus especially useful for treatment of a bone disorder. 
     In a preferred embodiment, therefore, a method according to the invention is provided wherein the disorder comprises a disorder associated with disturbed bone regulation. In a more preferred embodiment, the disorder comprises osteoporosis or sclerosteosis. 
     The invention is further explained in the following examples that do not limit the scope of the invention, but merely serve to clarify specific aspects of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 . Electro Spray Ionization Mass Spectrum (ESI/MS) of humVEGF 25-107  (Boc) in Panel A) fully reduced form (MW calc =9569.1; MW exp =9566.4), and Panel B) after oxidative folding (MW calc =9563.1; MW exp =9560.7). Folding conditions as described above. 
         FIG. 2 . Panel A) Inhibition of Avastin™-binding to surface-immobilized humVEGF 1-165  (1 μg/mL; GDA-coupling) in ELISA for varying concentrations (125 μM to 2 pM) of oxid-humVEGF 26-104  (▪), humVEGF 1-165  (▴), and a backbone-cyclized peptide covering only the β5-turn-β6 loop of humVEGF (humVEGF 74-98 ) (x). Panel B) Inhibition of Avastin™-binding to surface-immobilized humVEGF 1-165  (1 μg/mL; GDA-coupling) in ELISA for varying concentrations (5 μM to 12.8 pM) of humVEGF 1-165  (▴), oxid-humVEGF 26-104  synthesized via procedure-1 (grey square), or oxid-humVEGF 26-104  synthesized via procedure-2 (white square). 
         FIG. 3 . First neutralization data from BaF3/cell proliferation assay with non-purified rat anti-oxid-humVEGF 26-104  immune sera (I) 50.49 and 50.67 at 1/50 and 1/100 dilution. mAb Avastin™ (anti-humVEGF 1-165 ) was used as positive control, pre-immune (PI) sera (50.49 and 50.67) as negative control. Level of proliferation observed at humVEGF 1-165 =0.6 ng/mL was set by default to 100%, sera proliferation levels were expressed as % of default. Pre-immune sera were taken just before first immunization. Immune sera were taken six weeks after first immunization. In grey: % of proliferation &lt;50; in black: % of proliferation between 50 and 100. 
         FIG. 4 . Neutralization data from BaF3/cell proliferation assay with non-purified anti-oxid-humVEGF 26-104  rat sera Panel A) 50.49 and Panel B) 50.67 from 1/50 and 1/3200 dilution. For further details, see  FIG. 3 . 
         FIG. 5 . Neutralization data from BaF3/cell proliferation assay with protG-purified anti-oxid-humVEGF 26-104  rat sera Panel A) 50.49 and Panel B) 50.67 from 1/50 and 1/3200 dilution. For further details, see  FIG. 3 . 
         FIG. 6 . Neutralization data from BaF3/cell proliferation assay with non-purified mouse anti-oxid-humVEGF 26-104  immune sera (I) 59.01-59.05 (04 died). mAb Avastin™ (anti-humVEGF 1-165 ) and anti-oxid-humVEGF 26-104  rat serum 50.67 were used as positive control; pre-immune (PI) sera as negative control. Level of proliferation observed at humVEGF 1-165 =1.2 ng/mL was set by default to 100%, serum proliferation levels were expressed as % of default. PI: serum taken just before first immunization; I: serum taken six weeks after first immunization. 
         FIG. 7 . Inhibition of Avastin™-binding to surface-immobilized humVEGF 1-165  with non-purified rat immune sera 50.49 and 50.67 at 1/5 and 1/25 dilution. Peptide serum 31.1 (elicited against double-constrained CLIPS/SS-peptide derived from the β3-loop sequence humFSH 56-79  of Follicle-Stimulating Hormone; serum has high neutralizing activity for FSH in cell-based assay) and serum 45.09 (elicited against backbone-cyclized peptide derived from the β5-turn-β6 loop sequence 70-102 of VEGF; serum has neutralizing activity for humVEGF 1-165  in BaF3-cell proliferation assay) were used as negative controls. Minimal concentration of Avastin™ (˜10 ng/mL) was used (OD 450 nm ˜0.4) in order to secure maximal sensitivity for the inhibition experiments. 
         FIG. 8 . Proliferation data from BaF3/cell assay with humVEGF 26-104  at various concentration (0.01-20 ng/mL), either in the absence and presence of humVEGF 1-165 . Level of proliferation observed at humVEGF 1-165 =1.2 ng/mL was set by default to 100%, other proliferation levels were expressed as % of default. 
         FIG. 9 . Schematic overview of the proliferation assay. 
         FIG. 10A-X . Full protein name, species from which the protein was isolated, and amino acid sequence for all proteins known to be part of the cystine-knot growth factor superfamily, subdivided in TGF-beta, GLH-beta, NGF, PDGF, GLHA, Noggin-like, Coagulin-like, and CTCK-like subfamilies (SEQ ID NOS:36-180). Defined consensus sequences per subfamily are projected on top of the listing of sequences for each member. 
         FIG. 11 . Schematic representation of the general structure of the various members of the cystine-knot growth factor superfamily. 
         FIG. 12 . Panel A) Increase of average tumor volume (mm3) per mice in treatment group 1:PBS (           ), 2:anti-oxid-humVEGF 26-104  (Δ), and 3:AVASTIN™ (V). In the PBS group, four out of nine mice were euthanized (#) before the planned day because the estimated volume of the tumors exceeded the (pre-set) maximum volume. Panel B) Total average tumor weight (mgs) per mice in each different treatment group at the end of the experiment. Panel C) Total tumor volume (mm3) of individual mice in each different treatment group at the end of the experiment (mouse 3 in PBS-group died before the start of the experiment).
         FIG. 13 . HPLCs (Panels A/C) and ElectroSpray Ionization Mass Spectra (Panels B/D) of red-ratVEGF 26-104  (Panels A/B) and oxid-ratVEGF 26-104  (Panels C/D). 
         FIG. 14 . Plots of the binding in ELISA of anti-oxid-humVEGF 26-104  rat sera 1+2 (black ------ and - - - lines) and anti-oxid-ratVEGF 26-104  rat sera 3+4 (grey ------ and - - - lines) to both Panel A) oxid-ratVEGF 26-104  and Panel B) oxid-humVEGF 26-104 . 
         FIG. 15 . HPLCs (A/C) and ElectroSpray Ionization Mass Spectra (B/D) of red-humPLGF 34-112  (A/B) and oxid-humPLGF 34-112  (C/D). 
         FIG. 16 . Three-fragment condensation of humSOST 57-144  from fragment humSOST-F1, humSOST-F2, and humSOST-F3 by Native Chemical Ligation. Step a) Ligation of the thiaproline-protected humSOST-F2 to humSOST-F3, generating protected humSOST-F2/3. Step b) Deprotection of humSOST-F2/3 with methoxyamine in at pH 4.0. Step c) Ligation of deprotected humSOST-F2/3 to humSOST-F1 generating humSOST 57-144  at pH 6.5. 
         FIG. 17 . Oxidative refolding of fully red-humSOST 57-144  after ion exchange chromatography. The peptide was folded in 0.4 M Arginine, 1.67 mM Glutathione (red), 0.33 mM Glutathione (ox), 55 mM Tris-HCl, 21 mM sodium chloride, 0.88 mM potassium chloride, pH 8.0, yielding 10.2% of the desired product after 3.5 days at 4° C. 
         FIG. 18 . HPLCs (Panels A/C/E) and ElectroSpray Ionization Mass Spectra (Panels B/D/F) of fully red-humSOST 57-144  (Panels A/B), oxidatively refolded oxid-humSOST 57-144  (Panels C/D), octa-acetamido derivatized humSOST 57-144  (Panels E/F). 
         FIG. 19 . Binding data in ELISA for antibodies selected biotinylated oxid-humSOST 57-144  from a PDL-library. The positive binding to 1. Recombinant humSOST, 2. biotinylated oxid-humSOST 57-144  itself, and the absence of binding to 3. AA 8 -SOST 57-144 , 4. GST, 5) CD33, and finally 6. Bovine Serum Albumin (BSA) illustrate the high-specificity of the antibody binding. 
         FIG. 20 . HPLCs (Panels A/C) and ElectroSpray Ionization Mass Spectra (Panels B/D) of red-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  (Panels A/B) and oxid-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  (Panels C/D). 
         FIG. 21 . (Panel A) Antibody titers in ELISA for 9wpv-rat sera (1 and 2+pre-immune sera) that were elicited via immunization with oxid-humTGFB2 15-111/Δ49-77 -humVEGF 62-67 . Titers were defined as the −10 log [conc] at which the OD in ELISA is equal to 4× the background signal. (Panel B) Antibody binding in ELISA of 9wpv-rat sera to surface-immobilized 1) humTGFB2trunc-1 (with VEGF b2-loop), 2) humTGFB2trunc-2 (with sequence PGGSPA replacing native humTGF-B2 b2-loop), and 3) humVEGFtrunc. 
     
    
    
     
       
         
               
             
           
               
                 humTGFB2trunc 1: 
               
               
                 (SEQ ID NO: 24) 
               
               
                 acetyl- C1 ALRPLYIDFKRDLGWKWIHEPKGYNANF C2 AGA C3 NDEGLE 
               
               
                   
               
               
                   C4 VSQDLEPLTILYYIGKTPKIEQLSNMIVKS C5 K C6 -amide 
               
               
                   
               
               
                 humTGFB2trunc 2: 
               
               
                 (SEQ ID NO: 25) 
               
               
                 acetyl- C1 ALRPLYIDFKRDLGWKWIHEPKGYNANF C2 AGA C3 PGGSPA 
               
               
                   
               
               
                   C4 VSQDLEPLTILYYIGKTPKIEQLSNMIVKS C5 K C6 -amide 
               
               
                   
               
               
                 VEGFtrunc: 
               
               
                 (SEQ ID NO: 26) 
               
               
                 acetyl- C1 HPIETLVDIFQEYPDEIEYIFKPSAVPLMR C2 GGA C3 NDEG 
               
               
                   
               
               
                 LE C4 VPTEESNITMQIMRIKPHQGQHIGEMSFLQHNK C5 E C6 -amide 
               
             
          
         
       
     
     DETAILED DESCRIPTION OF THE INVENTION 
     Examples 
     Example 1A 
     Synthesis of Various Forms of VEGF-Truncated 
     Three different forms of VEGF-truncated were synthesized: 
     
       
         
               
             
           
               
                 (SEQ ID NO: 27) 
               
               
                 humVEGF 26-104 ;__ 26 Ac- C1 HPIETLVDIFQEYPDEIEYIFKPS A VP 
               
               
                   
               
               
                 LMR C1 GGA C3 NDEGLE C4 VPTEESNITMQIMRIKPHQGQHIGEMSFLQHN 
               
               
                   
               
               
                 K C5 E C6 # 104   
               
               
                   
               
               
                 (SEQ ID NO: 28) 
               
               
                 humVEGF 25-107 ;__ 25 Ac-Y C1 HPIETLVDIFQEYPDEIEYIFKPS A V 
               
               
                   
               
               
                 PLMR C2 GGA C3 NDEGLE C4 VPTEESNITMQIMRIKPHQGQHIGEMSFLQH 
               
               
                   
               
               
                 NK C5 E C6 RPK# 107   
               
               
                   
               
               
                 (SEQ ID NO: 29) 
               
               
                 humVEGF 25-109 :__ 25 Ac-Y C1 HPIETLVDIFQEYPDEIEYIFKPS A V 
               
               
                   
               
               
                 PLMR C2 GGS C3 NDEGLE C4 VPTEESNITMQIMRIKPHQGQHIGEMSFLQH 
               
               
                   
               
               
                 NK C5 E C6 RPKKD# 109   
               
             
          
         
       
     
     Amino acids are indicated by the single-letter codes; “Ac” refers to N-terminal acetylation; “#” indicates C-terminal amidation; Cysteines (C1-C6) in boldface indicate cysteines involved in formation of the cystine-knot fold; alanines in boldface indicate native cysteines that were replaced by Ala. 
     Three different synthetic procedures were used: 
     I. Direct synthesis (Fmoc) of full-length peptide; only used for humVEGF 26-104 . 
     II. Peptide-thioester synthesis using Fmoc-chemistry. Subsequent Native Chemical Ligation (NCL) of peptide fragments humVEGF 26-67 (thioester)+humVEGF 68-104 (free N-terminal cysteine) for humVEGF 26-104 , humVEGF 25-67 (thioester)+humVEGF 68-107 (free N-terminal cysteine) for humVEGF 25-107 , and humVEGF 25-67 (thioester)+humVEGF 68-109 (free N-terminal cysteine) for humVEGF 25-109 . 
     III. Peptide-thioester synthesis using Boc-chemistry. Subsequent Native Chemical Ligation (NCL) of peptide fragments humVEGF 25-67 (thioester)+humVEGF 68-107 (free N-terminal cysteine) for humVEGF 25-107  and humVEGF 26-67 (thioester)+humVEGF 68-104 (free N-terminal cysteine) or humVEGF 26-60 (thioester)+humVEGF 61-104 (free N-terminal cysteine) for humVEGF 26-104 . 
     Procedure I: 
     General Procedure (A) for Fmoc-Synthesis of Peptides: 
     Peptides were synthesized on solid-phase using a 4(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy (RinkAmide) resin (BACHEM, Germany) on a Symphony (Protein Technologies Inc., USA), Voyager (CEM GmbH, Germany), or SyroII (MultiSyntech, Germany) synthesizer. All Fmoc-amino acids were purchased from Biosolve (Netherlands) or Bachem GmbH (Germany) with side-chain functionalities protected as N-t-Boc (KW), O-t-Bu (DESTY), N-Trt (HNQ), S-Trt (C), or N-Pbf (R) groups. A coupling protocol using a five-fold excess of HBTU/HOBt/amino acid/DIPEA (1:1:1:2) in NMP with a 20-minute activation time using double couplings was employed for every amino acid coupling step. Acetylation (Ac) of the peptide was performed by reacting the resin with NMP/Ac 2 O/DIEA (10:1:0.1, v/v/v) for 30 minutes at room temperature. The acetylated peptide was cleaved from the resin by reaction with TFA (40 mL/mmol resin) containing 13.3% (w) phenol, 5% (v) thioanisole, 2.5% (v) 1,2-ethanedithiol, and 5% (v) milliQ-H 2 O for 2 hours at room temperature, unless indicated otherwise. Precipitation with ice-cold Et 2 O+lyophilization of the precipitated material afforded the crude peptide. 
     humVEGF 26-104  was synthesized in one step following this procedure (resin-loading 0.88 mmol/g) on a Symphony synthesizer (Protein Technologies Inc., USA). In the first coupling step, a 4:1 (w/w) mixture of Ac-Cys(Trt)-OH and Fmoc-Cys(Trt)-OH was used. The acylated peptide was cleaved from the resin by reaction with a slightly different mixture: TFA (40 mL/mmol resin) containing 5% (v) TES, 2.5% (v) 1,2-ethanedithiol, and 2.5% (v) milliQ-H 2 O. Finally, the peptide was purified by HPLC and folded by oxidation following procedure G. 
     The fragment peptides humVEGF 68-104 , humVEGF 68-107 , and humVEGF 68-109  (free N-terminal cysteine for NCL; see procedure II) were also synthesized following this procedure as described above for humVEGF 26-104  on a Rink-Made resin (loading 0.5 mmol/g) using a Liberty-synthesizer (CEM GmbH, Germany). 
     Procedure II: 
     Fmoc-Synthesis of Peptide Thioesters: 
     The fragment peptides humVEGF 25-67  and humVEGF 26-67  (free C-terminus) were synthesized on a SASRIN-resin (loading 0.5 mmol/g; Bachem GmbH, Germany) following the general procedure for Fmoc-synthesis of peptides as described in procedure I. The peptides were cleaved from the resin by repetitive treatment (20 cycles) with 1% TFA (40 mL/mmol resin) in DCM. The combined fractions were neutralized with pyridine, whereafter DCM was removed by evaporation under reduced pressure. Finally, the peptides were precipitated by addition of excess of H 2 O, followed by centrifugation and lyophilization. The crude lyophilized peptides were dissolved in DCM (2.0 mM), twelve equivalents of 4-acetamidothiophenol in DCM (0.334 mg/mL, 2.0 mM), three equivalents of PyBOP in DCM (1.040 mg/mL, 2.0 mM), and 2.6 equivalents of DIPEA in DCM (1 vol %) were subsequently added and the mixture was stirred at room temperature for six hours. Then, another twelve equivalents of 4-acetamidothiophenol in DCM (0.334 mg/mL, 2.0 mM) were added and the mixture was stirred overnight at room temperature. Finally, the mixture was neutralized with ˜2.6 equivalents of TFA and DCM was removed by evaporation under reduced pressure. The crude fragment peptide thioesters were then deprotected and purified by RP-HPLC following general procedures. 
     Native Chemical Ligation (NCL) of Fragment Peptides: 
     Condensation of fragment peptides humVEGF 68-104 , humVEGF 68-107 , or humVEGF 68-109  (A) with either fragment peptide thioesters humVEGF 25-67  or humVEGF 26-67  (B) by native chemical ligation was performed by mixing almost equimolar (1:1.2) solutions of A (10 mg/mL; ˜2.0 mM) and B (10 mg/mL; ˜2.0 mM) in working buffer (6 M guanHCl/20 mM TCEP/200 mM MPAA in 0.2 M phosphate buffer pH 8.0) and overnight stirring at room temperature. After mixing of the solutions (acidic!), the pH was adjusted to 6.5 by addition of 10 M NaOH (μL of NaOH is roughly equal to mg of MPAA used). Excess of MPAA was removed by Amicon filtration using working buffer (without MPAA!!) in the washing steps. Finally, the crude humVEGF 26-104 , humVEGF 25-107 , or humVEGF 25-109  in reduced form were purified by RP/HPLC following the standard procedure. 
     Oxidative Folding of Red-humVEGF 26-104 , Red-humVEGF 25-107 , and Red-humVEGF 25-109 : 
     Fully reduced red-humVEGF 26-104 , red-humVEGF 25-107 , or red-humVEGF 25-109  were dissolved in 0.1 M Tris-buffer (pH 8.0), with or without 1 M guanidine.HCl, containing 1.0 mM cystine (SS-form) and 8.0 mM cysteine (SH-form) in a final concentration of 0.1 mg/mL and stirred at room temperature. Immediately, a sharp peak appears at a lower retention time (more polar) in addition to some broad peaks that correspond to incomplete or incorrectly folded peptide. When HPLC-analysis showed no further change in peak intensities (usually after ˜4 hours), the mixture was loaded onto a preparative RP/C 18  column and purified following our standard procedure (see below). 
     Procedure III: 
     General Procedure for tBoc-Synthesis of Peptides: 
     Fragment peptides were prepared by manual solid phase peptide synthesis (SPPS) typically on a 0.25 mmol scale using the in situ neutralization/HBTU activation procedure for Boc chemistry as previously described. Each synthetic cycle consisted of Nα-Boc-removal by a one- to two-minute treatment with neat TFA, a one-minute DMF-flow wash, a ten- to twenty-minute coupling time with 1.0 mmol preactivated Boc-amino acid in the presence of excess DIEA, followed by a second DMF-flow wash. Nα-Boc amino acids (1.1 mmol) were preactivated for 3 minutes with 1.0 mmol HBTU (0.5 M in DMF) in the presence of excess DIEA (3 mmol). After coupling of Gln residues, a DCM flow wash was used before and after deprotection using TFA, to prevent possible high-temperature (TFA/DMF)-catalyzed pyrrolidonecarboxylic acid formation. Side-chain protected amino acids were: Boc-Arg (p-toluenesulfonyl)-OH, Boc-Asn(xanthyl)-OH, Boc-Asp(O-cyclohexyl)-OH, Boc-Cys(4-methylbenzyl)-OH, Boc-Glu(O-cyclohexyl)-OH, Boc-His(dinitrophenyl)-OH, Boc-Lys(2-Cl—Z)—OH, Boc-Ser(benzyl)-OH, Boc-Thr(benzyl)-OH, and Boc-Tyr(2-Br—Z)—OH. Other amino acids were used without side-chain protection. Nα-acetylation of peptides was performed by treatment with acetic anhydride (0.1 M)/Pyridine (0.1 M) in DMF for 2×2 minutes). After chain assembly was completed, the peptides were deprotected and cleaved from the resin by treatment with anhydrous HF for one hour at 0° C. with 4% p-cresol as a scavenger. In all cases, the imidazole side chain-dinitrophenyl (Dnp) protecting groups remained on His residues because the Dnp-removal procedure is incompatible with C-terminal thioester groups. However, Dnp is gradually removed by thiols during the ligation reaction yielding unprotected His. After cleavage, the peptide fragments were precipitated with ice-cold diethylether, dissolved in aqueous acetonitrile and lyophilized. 
     Preparation of Thioester-Generating (-COSR) Resin: 
     1.1 mmol Nα-Boc Leu was activated with 1 mmol HBTU in the presence of 3 mmol DIEA and coupled for 10 minutes to 0.25 mmol MBHA resin. Next, 1.1 mmol S-trityl mercaptopropionic acid was activated with 1 mmol HBTU in the presence of 3 mmol DIEA and coupled for 30 minutes to Leu-MBHA resin. The resulting trityl-mercaptopropionic acid-leucine resin can be used as a starting resin for polypeptide chain assembly following removal of the trityl protecting group with 2×1-minute treatments with 2.5% triisopropylsilane and 2.5% H 2 O in TFA. The thioester bond was formed with the desired amino acid using standard peptide coupling protocols. Treatment of the final peptide with anhydrous HF yielded the C-terminal activated mercaptopropionic acid-leucine (MPAL) thioester (-COSR) peptides for participation in the native chemical ligation reaction. 
     Native Chemical Ligation (NCL) of Fragment Peptides: 
     The ligation of fully deprotected fragment peptide thioesters humVEGF 26-60 , humVEG 26-67 , and humVEGF 25-67  with either the fragment peptides humVEGF 61-104 , humVEGF 68-104 , or humVEGF 68-107  was performed as follows: peptide fragments were dissolved in a ˜1:1 molar ratio at 10 mg/ml in 0.1 M tris buffer, pH 8.0, containing 6 M guanidine. Benzylmercaptan and thiophenol were added to 2% (v/v) resulting in a final peptide concentration of 1-3 mM at a pH˜7 (lowered due to addition of thiols and TFA from the lyophilized peptide). The ligation reaction was performed in a heating block at 37° C. and was vortexed periodically to equilibrate the thiol additives. The reaction was monitored by HPLC and ESI-MS until completion. Respective NCLs (humVEGF 26-60 +humVEGF 61-104 ; humVEGF 26-67 +humVEGF 68-104 ) yielded reduced VEGF 26-104  with identical HPLC and ESI-MS specifications. 
     Oxidative Folding of Red-humVEGF 26-104  and Red-humVEGF 25-107 : 
     Fully reduced red-humVEGF 26-104  and red-humVEGF 25-107  were dissolved in 0.1 M Tris-buffer (pH 8.0), with or without 1 M guanidin.HCl, containing 1.0 mM cystine (SS-form) and 8.0 mM cysteine (SH-form) in a final concentration of 0.1 mg/mL and stirred at room temperature. Immediately, a sharp peak appears at a lower retention time (more polar) corresponding to the correctly folded cysknot structure, in addition to some broad peaks that correspond to incomplete or incorrectly folded peptide. When HPLC-analysis showed no further change in peak intensities (usually after ˜4 hours), the mixture was loaded onto a preparative RP/C 18  column and purified following our standard procedure (see below). 
     General Procedure for Purification by HPLC: 
     Crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC), either on a “DeltaPack” (25×100 or 40×210 mm inner diameter, 15 μm particle size, 100 Å pore size; Waters, USA) or on a “Atlantis” (10×100 mm inner diameter, 5 μm particle size (Waters, USA) RP-18 preparative C 18  column with a linear AB gradient of 1-2% B/minute where solvent A was 0.05% TFA in water and solvent B was 0.05% TFA in ACN. Alternatively, analytical reversed-phase HPLC was performed on a Varian Prostar system using Vydac C-18 columns (5 μm, 0.46×15 cm) and preparative reversed-phase HPLC was performed on a Waters system using Vydac C-18 columns (10 μm, 1.0/2.5×25 cm). Linear gradients of acetonitrile in water/0.1% TFA were used to elute bound peptides. The flow rates used were 1 ml/minute (analytical), and 5/10 ml/minute (preparative). 
     Analysis by RP-HPLC/ESI-MS: 
     Analysis of the purified peptide was performed by reversed-phase high-performance liquid chromatography (RP-HPLC) on an “Acquity” UPLC (Waters, USA) using a RP-18 preparative “BEH” column (2.1×50 inner diameter, 1.7 mm particle size, Waters, USA) with a linear AB gradient (5-55% B, 25% B/minute), where solvent A was 0.05% TFA in water and solvent B was 0.05% TFA in ACN. The primary ion molecular weight of the peptides was determined by electron-spray ionization mass spectrometry. 
     Analysis by ESI-MS: 
     Electrospray ionization mass spectrometry (ESI-MS) of HPLC samples was performed on an API-150 single quadrupole mass spectrometer (Applied Biosystems). Peptide masses were calculated from the experimental mass to charge (m/z) ratios from all the observed protonation states of a peptide using Analysis software. 
     For each peptide the following characteristics were determined: 
                                                 Oxidation                       state   Retention   MW   MW       Peptide   (RED/OX)   (% ACN)   calculated   experimental                   Red-humVEGF 26-104     RED   48.5   9065.6   9064.4       Oxid-humVEGF 26-104     OX   42.5   9059.6   9058.5       Red-humVEGF 25-107     RED   45.8   9569.1   9566.4       (Boc)       Oxid-humVEGF 25-107     OX   40.5   9563.1   9560.7       (Boc)       Red-humVEGF 25-107     RED   45.8   9569.1   9568.8       (Fmoc)       Oxid-humVEGF 25-107     OX   40.5   9563.1   9561.7       (Fmoc)       Red-humVEGF 25-109     RED   43.8   9869.5   9869.6       Oxid-humVEGF 25-109     OX   38.2   9863.5   9863.8                    
These data and  FIG. 1  show that the various forms of humVEGF trunc  can be synthesized in various different ways with identical outcomes.
 
     Example 1B 
     Inhibitory Activity of Oxid-humVEGF 26-104  in Avastin™-Binding to Surface-Immobilized Oxid-humVEGF 1-165    
     Binding ELISA: Binding of various mAbs (Avastin™, mAb 293, PDL-antibody) to oxid-humVEGF 26-104  and humVEGF 1-165  was determined in ELISA. Therefore, polystyrene 96-well plates (Greiner, Germany) were treated with 100 μL/well of 0.2% glutaric dialdehyde in phosphate-buffer (0.1 M, pH=5) for four hours at room temperature while shaking, following by washing (3×10 minutes) with phosphate-buffer (0.1 M, pH=8). Then, the wells were coated with 100 μL/well of a 1 μg/mL solution of oxid-humVEGF 26-104 /humVEGF 1-165  in phosphate-buffer (0.1 M, pH=8) for three hours at 37° C., followed by overnight standing at room temperature. After washing with 1% TWEEN®-80 (3×), the plates were incubated with the antibody at various different dilutions in horse serum (4% in PBS/1% TWEEN®-80/3% NaCl), starting with 1/10 dilution in the first well and three-fold dilution steps in subsequent wells. Incubation was performed for one hour at 37° C., followed by washing with 1% TWEEN®-80 (3×). Then, the plates were incubated with 100 μL/well of peroxidase-labeled Goat-anti-rat serum (1/1000 dilution in 4% horse serum, see above) for one hour at 25° C., followed by washing with 1% TWEEN®-80 (4×). Finally, the plates were incubated with a 0.5 μg/mL solution of ABTS (2,2′-azine-di(ethylbenzthiazoline sulfonate)) containing 0.006% H 2 O 2  in citric acid/phosphate-buffer (0.1 M each, pH=4). OD 405 nm -values were measured after 45 minutes standing at room temperature in the dark. 
     Competition ELISA: ELISA binding competition studies were carried out largely following the procedure as described for binding in ELISA (see above). Incubation with antibody was carried out at one fixed antibody-concentration (10 ng/mL of Avastin™; OD 405 nm  between 1.0-1.5) in the presence of decreasing amounts of oxid-humVEGF 26-104  (start at 5 μM; 1/5 dilution steps) and humVEGF 1-165  (positive control; start at 500 nM; 1/5 dilution steps). 
     The data in  FIG. 2  show that oxid-humVEGF 26-104  binds with less than five-fold difference in affinity (as compared to humVEGF 1-165 ) to Avastin™, while the (cyclic) peptide-mimic derived from the beta3-loop of humVEGF is &gt;10,000-fold less active in binding to Avastin™. This illustrates the big step forward in reconstruction of the discontinuous Avastin™ binding site on humVEGF using this novel technology of the present invention. 
     Example 1C 
     Use of Oxid-humVEGF 26-104  for Generating VEGF-Neutralizing Antibodies and Sera in Rats and Mice 
     Immunization experiments using oxid-humVEGF 26-104  (not-conjugated to a carrier protein!!) were carried out both in female Wistar rats and female Balb/C mice. The antisera were analyzed for:
         A) binding to surface-immobilized humVEGF 1-165  (titer determination)   B) ability to inhibit the binding of Avastin™ to surface-immobilized humVEGF 1-165      C) neutralizing activity for humVEGF 1-165  in a BaF3-cell proliferation assay       

     The results of these studies are shown below and in  FIGS. 3-6 . 
     Immunization Protocols: 
     Wistar rats: Female Wistar rats were immunized with anti-humVEGF 26-104  at day 0 with 400 μL (intramuscular+subcutaneous, 200 μL each) of a 375 μg/mL solution of humVEGF 26-104  in PBS/CoVaccine 1:1 (v/v) (PBS=Phosphate-Buffered Saline), followed by a booster (same quantity and concentration) at two and four weeks. Subsequently, the rats were bled after six weeks and the antisera collected. Anti-VEGF titers were determined as described as below. 
     Balb/C mice: Immunization with oxid-humVEGF 26-104  was performed in female Balb/C mice, using two different formulations, i.e., with a CFA/IFA adjuvant (group 1: two animals), and with a CoVaccine adjuvant (group 2: three animals). The animals (2) in group 1 were immunized intraperitoneal (i.p.) at day 0 with 250 μL of a 1.0 mg/mL solution of oxid-humVEGF 26-104  in PBS/CFA 2:3 (v/v) (PBS=Phosphate-Buffered Saline, CFA=Complete Freund&#39;s Adjuvance), followed by a booster (same quantity, method and concentration; Incomplete Freund&#39;s Adjuvance (IFA) instead of CFA) at four weeks. The animals (3) in group 2 were immunized at day 0 with 210 μL (intramuscular+subcutaneous, 105 μL each) of a 1.25 mg/mL solution of VEGF 26-104  in PBS/CoVaccin 1:1 (v/v) (PBS=Phosphate-Buffered Saline), followed by a booster (same quantity, method and concentration) at two and four weeks. Subsequently, all five mice were bled after six weeks and the antisera collected. Anti-VEGF titers were determined as described as below. 
     ELISA Titer Determination: 
     Titers were calculated by determining the serum dilution for which OD 405 nm  is equal to 4×OD 405 nm  that of a buffer solution (see “ELISA-binding studies, example 1B”). The titer defines the negative  10  log-value of the dilution factor (1/10=1, 1/100=2, 1/1000=3, 1/10000=4, etc.). 
                                                           humVEGF 1-165     humVEGF 1-165             Titer 0 wpv   Titer 6 wpv                                    Animal               50.49 (Wistar rat 1; CoVaccine)   &lt;&lt;2   4.8       50.67 (Wistar rat 2; CoVaccine)   &lt;&lt;2   5.4       59.01 (Balb/C mouse 1, CFA/IFA)   &lt;&lt;2   5.3       59.02 (Balb/C mouse 2, CFA/IFA)   &lt;&lt;2   5.2       59.03 (Balb/C mouse 3, CoVaccine)   &lt;&lt;2   5.4       59.04 (Balb/C mouse 4, CoVaccine)   &lt;&lt;2   †       59.05 (Balb/C mouse 5, CoVaccine)   &lt;&lt;2   5.3       Control Abs       Avastin ™ (500 ng/mL start)   —   4.4       BioVision ™ (5000 ng/mL)   —   4.2                    
ELISA Competition Studies of Rat Antisera with Avastin™:
 
     ELISA binding competition studies were carried out largely following the procedure as described for binding in ELISA (see above). Incubation with antibody was carried out at a fixed Avastin™-concentration (10 ng/mL; OD 405 nm  between 1.0-1.5) in the presence of decreasing amounts of rat antisera (start at 1/5; further 1/3 dilution steps). 
     Neutralization in BaF3-Cell Proliferation Assay: 
     The cells that are used in the assay are murine pre-B lymphocytes stable expressing human (h) humVEGF-Receptor 2 (Makinen et al., 2001). These recombinant cells survive/proliferate only in the presence of IL-3 (natural cytokine required for the survival of the parental cells) or humVEGF. For the experiment, IL-3 has to be washed off the medium so that proliferation capability in dependence of humVEGF can be tested. 
     Ba/F3 R2 cells were grown in DMEM (Gibco #31885) containing 10% fetal bovine serum (Perbio #CH30160.03), 2 mM L-glutamine (Sigma #G7513), 2 ng/ml mIL-3 (Calbiochem #407631) and 500 μg/ml Zeocin (Invitrogen #450430). Cells were grown at 37° C. in a humidified incubator with an atmosphere of 5% CO2/95% air. 
     Differently concentrated humVEGF (+humVEGF) or medium (−humVEGF) was either added directly to the cells (to test the proliferation efficiency) or pre-incubated for one hour with different concentrations of Avastin™ (positive control), different concentrations of rat or mouse sera and then added to the cells (in case of inhibition experiments). Two days later, cell proliferation was measured by adding WST-1 (Roche #1644807). See  FIG. 9  for a graphical representation of the assay. 
     The WST-1 assay is based on the measurement of the mitochondrial succinate dehydrogenase activity. To function correctly, this enzyme requires the integrity of this organelle and is a good indicator of the number of proliferating cells present in the culture. A tetrazolium salt (WST-1) is used as substrate since it generates a soluble dark metabolic (formazon) through the action of the enzyme, which can then be quantified by measuring the absorbance (450 nm) in an ELISA reader. The higher the absorbance measured in the assay, the stronger the proliferation. Absorbance is positively correlated with proliferation. Experiments were repeated three times in triplicate showing overall similar results. 
     The data obtained proves that high levels of antibodies were successfully generated via immunization with oxid-humVEGF 26-104  (not-conjugated to a carrier protein!!), both in female Wistar rats and female Balb/C mice. The antisera generated in this way exhibit strong neutralizing activity for humVEGF 1-165  in a BaF3-cell proliferation assay ( FIGS. 3-6 ), and the ability to inhibit binding of Avastin™ to humVEGF ( FIG. 7 ). 
     Example 1D 
     Oxid-humVEGF 26-104  does not Induce BaF3-Cell Proliferating by Itself 
     In order to check whether oxid-humVEGF 26-104 , the truncated form of humVEGF 1-165 , is also able to induce BaF3-cell proliferation, we measured cell proliferation in the presence of varying amounts of oxid-humVEGF 26-104  (0.01-20 ng/mL). In order to check if oxid-humVEGF 26-104  was able to enhance or inhibit the proliferative capacity of humVEGF 1-165 , itself, the experiments with varying amounts of oxid-humVEGF 26-104  were also run in the presence of humVEGF 1-165 =1.2 ng/mL. 
     The results shown in  FIG. 8  clearly demonstrate no activity for oxid-humVEGF 26-104  in BaF3-cell proliferation nor any affect on the proliferating ability of humVEGF 1-165 . 
     Example 1E 
     Passive Immunization Study with Anti-humVEGF 26-104  Rat-Antisera in Swiss Nu/Nu Mice Inoculated with Human LS174T Tumor Cells: In Vivo Proof of Principle of the Tumor-Reducing Potential of Anti-humVEGF 26-104  Antisera 
     In order to demonstrate the tumor-reducing potential of anti-humVEGF 26-104  antisera, the following immunization experiment was carried out in 30 male Swiss nu/nu mice (Charles river), six weeks of age at the beginning of the study. The animals were divided in the following three treatment groups: 
     Group 1: PBS (n=10; negative control group): intraperitoneal (i.p.) PBS injections (500 μl) after tumor cell inoculation. 
     Group 2: oxid-humVEGF 26-104  (n=10): i.p. injections (500 μl) with IgG-purified anti-VEGF peptide rat-antiserum after tumor cell inoculation. 
     Group 3: AVASTIN™ (n=10; positive control group): i.p. injections (500 μl) with anti-humVEGF mAb AVASTIN™ following tumor cell inoculation. 
     On day 1 of the study, all 30 mice were injected subcutaneously (right flank) with 10 million human LS174T tumor cells suspended in a 100 μL solution. Tumor-take was ˜100%. Subsequently, the mice were given on days 1, 8, and 15, i.p. injections (500 μl) with either A) PBS (group 1), B) anti-oxid-humVEGF 26-104  rat-antiserum (5× conc. rat serum; group 2), and C) AVASTIN™ (group 3). Anti-oxid-humVEGF 26-104  rat serum was obtained by immunizing a total number of 20 male Whistar rats in a separate experiment 4× with 250-microgram doses of humVEGF 26-104  using CoVaccine adjuvant (inoculations at days 0, 14, 28, and 49; bled on day 63). The resulting rat sera were purified by affinity chromatography (ProtG-column) and concentrated 5×. The ten most potent antisera (based on in vitro neutralization data in BaF3 assay; see previous Example) of these were pooled and used for inoculation of the ten mice in treatment group 2. Lengths and breadths of the tumors were measured every other day, starting on the first day after tumor cell inoculation. Tumor volumes were estimated using the formula (breadth2×length)/2. (ref 6)  The data are shown in  FIG. 12 . 
     The data presented above lead to the following conclusions: 
     1. anti-oxid-humVEG F 26-104  antisera have the ability to strongly reduce tumor growth in mice. 
     2. in this experimental setting, the observed effect of treatment with anti-oxid-humVEGF 26-104  antisera was visibly more pronounced than that for AVASTIN™. 
     3. treatment of nude mice with anti-oxid-humVEGF 26-104  antibodies was received well by all animals and is thus not toxic! 
     Example 1F 
     Immunogenicity of Oxid-ratVEGF 26-104  in Rats 
     
       
         
               
             
           
               
                 (SEQ ID NO: 30) 
               
               
                 Peptide sequence oxid-ratVEGF 26-104 : Acetyl- C1 RPIE 
               
               
                   
               
               
                 TLVDIFQEYPDEIEYIFKPSAVPLMR C2 AGA C3 NDEALE C4 VPTSESNVT 
               
               
                   
               
               
                 MQIMRIKPHQSQHIGEMSFLQHSR C5 E C6 -amide. 
               
             
          
         
       
     
     Solid-phase synthesis of ratVEGF 26-104 . ratVEGF 26-104  was synthesized by normal solid-phase synthesis on a Rink-amide resin (downloaded to 0.1 mmol/g) following standard procedures as described for humVEGF 26-104  (see Example 1). Subsequent oxidative refolding was carried out exactly as described for humVEGF 26-104 . Purification of both red-ratVEGF 26-104  and oxid-ratVEGF 26-104  was carried out by preparative High Performance Liquid Chromatography (HPLC). Characterization of both peptides was carried out by analytical HPLC and ElectroSpray Ionization Mass Spectrometry (ESI-MS). 
     The successful refolding of red-ratVEGF 26-104  was evidenced by the characteristic shift to lower Rf-values (from 48.5% to 41.3% ACN, see Table below), normally observed when proteins or fragments thereof are oxidative refolded. The characteristic narrow shape of the new peak at lower R f -value provides evidence that an intact cystine-knot structure is indeed formed upon oxidative refolding of red-ratVEGF 26-104 . 
     Also, the ESI-MS spectrum undergoes a significant change upon oxidative refolding. First of all, the overall mass goes down by six mass units (formation of three disulfide bonds releases a total of 6H). Moreover, there is a very characteristic shift of MS-signals to higher m/z-values. For example, the MS-spectrum for red-ratVEGF 26-104  gives the most intense signals for the M 9+  and M 10+  charged species, whereas these signals disappear and a much weaker signal at M 5+  remains (see  FIG. 13 ) that is much less intense. Also, this shift is characteristic for folding of proteins into their oxidized native structure and shows that oxidative refolding of red-ratVEGF 26-104  has been successful. The reason is that the protein or protein fragment adopts a more condensed structure that is no longer able to pick up so many charges. In contrast to this, the flexible and extended structure of the reduced protein is able to accommodate many more charges. 
     
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Oxidation 
                   
                   
                   
               
               
                   
                 state 
                 Retention 
                 MW 
                 MW 
               
               
                 Peptide 
                 (RED/OX) 
                 (% ACN) 
                 calculated 
                 experimental 
               
               
                   
               
             
             
               
                 red-ratVEGF 26-104   
                 RED (SH) 6   
                 48.5 
                 9087.5 
                 9085.3 
               
               
                 oxid-ratVEGF 26-104   
                 OX (SS) 3   
                 41.3 
                 9081.5 
                 9080.0 
               
               
                   
               
             
          
         
       
     
     This example describes the results of an immunization study in male Whistar rats with both oxid-hum-VEGF 26-104  and oxid-ratVEGF 26-104  with an intact cystine-knot fold (oxid-form). The data unequivocally show that oxid-ratVEGF 26-104  is equally immunogenic and potent as compared to oxid-humVEGF 26-104  in generating antibodies in rats. The use of truncated VEGF as described in this patent can thus be used to bypass immune tolerance to “self proteins,” like, for example, the full-length homodimeric VEGF protein in this particular case. 
     A total of four Wistar rats (2×2) were immunized on day 0 with 250 micrograms each of either oxid-ratVEGF 26-104  (two rats) or oxid-humVEGF 26-104  (two rats) using CoVaccine as adjuvant, followed by booster inoculations at day 14, 28, and 42. The rats were finally bled at day 56, and the sera were analyzed for antibody titers against ratVEGF 1-165 , humVEGF 1-165 , oxid-ratVEGF 26-104 , and oxid-humVEGF 26-104 . (Part of) the antibody-binding data are shown in Table 1 and  FIG. 14 . 
     The data in Table 1 and  FIG. 14  do not show any detectable difference in binding between antisera elicited with oxid-ratVEGF 26-104  and those elicited with oxid-humVEGF 26-104  in rats, which strongly suggests that oxid-ratVEGF 26-104  is equally immunogenic in rats (homologous species) as compared to oxid-humVEGF 26-104  (heterologous species), and is able to elicit comparable amounts of antibodies that even show cross-reactivity with the homodimeric VEGF 1-165  protein (Table 1C). 
     Furthermore, the experiment provides a very strong basis for the fact that oxid-humVEGF 26-104  can be used to elicit anti-VEGF in humans, and that oxid-humVEGF 26-104  will not suffer from lack of immunogenicity as a result of immune tolerance to self proteins. 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 List of the binding of rat-antisera in ELISA to A) oxid-ratVEGF 26-104 , 
               
               
                 B) oxid-humVEGF 26-104 , C) humVEGF 1-165  homodimer (recombinant full-length 
               
               
                 humanVEGF), and D) ratVEGF 1-165  homodimer (recombinant full-length ratVEGF). For 
               
               
                 comparison, the binding data to the humanized anti-humVEGF mAb AVASTIN ™ are included. 
               
             
          
           
               
                   
                 titers 
                 1/100 
                 1/300 
                 1/1000 
                 1/3000 
                 1/10000 
                 1/30000 
                 1/100000 
                 1/300000 
                 titer endblood 
               
               
                   
                   
               
             
          
           
               
                 ratVEGF26-104 
               
             
          
           
               
                 A 
                 rat 1 (a-oxid-ratVEGF26-104) 
                 3298 
                 3263 
                 3123 
                 3028 
                 2357 
                 1214 
                 514 
                 225 
                 5.1 
               
               
                   
                 rat 2 (a-oxid-ratVEGF26-104) 
                 3597 
                 3424 
                 3262 
                 3197 
                 2516 
                 1241 
                 532 
                 237 
                 5.1 
               
               
                   
                 rat 3 (a-oxid-humVEGF26-104) 
                 3376 
                 3172 
                 3209 
                 3176 
                 2910 
                 1951 
                 861 
                 355 
                 5.3 
               
               
                   
                 rat 4 (a-oxid-humVEGF26-104) 
                 3200 
                 3263 
                 3465 
                 3060 
                 2895 
                 1736 
                 754 
                 349 
                 5.3 
               
             
          
           
               
                 humanVEGF26-104 
               
             
          
           
               
                 B 
                 rat 1 (a-oxid-ratVEGF26-104) 
                 3334 
                 3148 
                 3210 
                 3174 
                 2989 
                 1929 
                 811 
                 366 
                 5.3 
               
               
                   
                 rat 2 (a-oxid-ratVEGF26-104) 
                 3297 
                 3121 
                 3564 
                 3329 
                 2801 
                 1871 
                 728 
                 332 
                 5.2 
               
               
                   
                 rat 3 (a-oxid-humVEGF26-104) 
                 3263 
                 3098 
                 3385 
                 3300 
                 2908 
                 2188 
                 898 
                 409 
                 5.3 
               
               
                   
                 rat 4 (a-oxid-humVEGF26-104) 
                 3229 
                 3174 
                 3289 
                 3298 
                 3051 
                 2166 
                 873 
                 373 
                 5.3 
               
               
                   
                 Avastin (a-humVEGF mAb) 
                 4037 
                 3033 
                 1839 
                 736 
                 333 
                 158 
                 116 
                 97 
                  15-25 ng/mL 
               
             
          
           
               
                 humanVEGF1-165 
               
             
          
           
               
                 C 
                 rat 1 (a-oxid-ratVEGF26-104) 
                 3404 
                 3320 
                 3449 
                 2681 
                 1305 
                 548 
                 280 
                 158 
                 4.6 
               
               
                   
                 rat 2 (a-oxid-ratVEGF26-104) 
                 3245 
                 3216 
                 3672 
                 2955 
                 1588 
                 955 
                 301 
                 166 
                 4.7 
               
               
                   
                 rat 3 (a-oxid-humVEGF26-104) 
                 3456 
                 3406 
                 3334 
                 3078 
                 1776 
                 739 
                 351 
                 176 
                 4.7 
               
               
                   
                 rat 4 (a-oxid-humVEGF26-104) 
                 3758 
                 3282 
                 3604 
                 3313 
                 2508 
                 1374 
                 510 
                 235 
                 5.1 
               
               
                   
                 Avastin (a-humVEGF mAb) 
                 3261 
                 3016 
                 2493 
                 1322 
                 528 
                 222 
                 129 
                 100 
                   5-10 ng/mL 
               
             
          
           
               
                 ratVEGF1-165 
               
             
          
           
               
                 D 
                 rat 1 (a-oxid-ratVEGF26-104) 
                 2993 
                 2519 
                 1481 
                 731 
                 346 
                 172 
                 122 
                 98 
                 3.8 
               
               
                   
                 rat 2 (a-oxid-ratVEGF26-104) 
                 3032 
                 3055 
                 2717 
                 1568 
                 753 
                 315 
                 179 
                 122 
                 4.2 
               
               
                   
                 Avastin (a-humVEGF mAb) 
                 236 
                 148 
                 103 
                 89 
                 93 
                 89 
                 91 
                 88 
                 &lt;1000 ng/mL 
               
               
                   
               
             
          
         
       
     
     Example 1G 
     Synthesis of humPLGF 34-112  (humPLGFtrunc) 
     
       
         
               
             
           
               
                 (SEQ ID NO: 14) 
               
               
                 Peptide sequence of humPLGF 34-112 : Acetyl- C1 RALERL 
               
               
                   
               
               
                 VDVVSEYPSEVEHMFSPSAVSLLR C2 TGA C3 GDENLH C4 VPVETANVTMQ 
               
               
                   
               
               
                 LLKIRSGDRPSYVELTFSQHVR C5 E C6 -amide. 
               
               
                   
               
               
                 X0 = acetyl 
               
               
                   
               
               
                 (amino acids 2-31 of SEQ ID NO: 14) 
               
               
                 X1 = RALERLVDVVSEYPSEVEHMFSPS A VSLLR (A-mutation 
               
               
                   
               
               
                 for native C) 
               
               
                   
               
               
                 X2 = TGA (A-mutation for native C) 
               
               
                   
               
               
                 (amino acids 37-42 of SEQ ID NO: 14) 
               
               
                 X3 = GDENLH 
               
               
                   
               
               
                 (amino acids 44-76 of SEQ ID NO: 14) 
               
               
                 X4 = VPVETANVTMQLLKIRSGDRPSYVELTFSQHVR 
               
               
                   
               
               
                 X5 = E 
               
               
                   
               
               
                 X6 = amide 
               
             
          
         
       
     
     Solid-phase synthesis of red-PLGF 34-112 . Red-PLGF 34-112  was synthesized by normal solid-phase synthesis on a Rink-amide resin (downloaded to 0.1 mmol/g) following standard procedures as described for red-humVEGF 26-104  (see Example 1E). Subsequent oxidative refolding was carried out exactly as described for oxid-humVEGF 26-104 . 
     Purification of both red-humPLGF 34-112  and oxid-humPLGF 34-112  was carried out by preparative High Performance Liquid Chromatography (HPLC). Characterization of both red-humPLGF 34-112  and oxid-humPLGF 34-112  was carried out by analytical HPLC and ElectroSpray Ionization Mass Spectrometry (ESI-MS). 
     The successful refolding of red-humPLGF 34-112  was evidenced by the characteristic shift to lower Rf-values (from 49% to 38.3% ACN, see Table below) that is normally observed when proteins or fragments thereof are oxidative refolded. The characteristic narrow shape of the new peak at lower Rf-value provides evidence that an intact cystine-knot structure is indeed formed upon oxidative refolding of red-humPLGF 34-112 . 
     Also the ESI-MS spectrum undergoes a significant change upon oxidative refolding. First of all, the overall mass goes down by six mass units (formation of three disulfide bonds releases a total of 6H). Moreover, there is a very characteristic shift of MS-signals to higher m/z-values. For example, the MS-spectrum for red-humPLGF 34-112  gives clear signals for the M 6+  to M 10+  charged species, whereas these signals disappear and a much weaker signal at M 5+  remains (see  FIG. 15 ) that is much less intense. Also this shift is characteristic for folding of proteins into their oxidized native structure and shows that refolding of red-humPLGF 34-112  was successful. The reason is that the protein or protein fragment adopts a more condensed structure that is no longer able to pick up so many charges. In contrast to this, the flexible and extended structure of the reduced protein is able to accommodate many more charges. 
     
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 MW 
               
               
                   
                 Oxidation state 
                 Retention 
                 MW 
                 experi- 
               
               
                 Peptide 
                 (RED/OX) 
                 (% ACN) 
                 calculated 
                 mental 
               
               
                   
               
             
             
               
                 red-humPLGF 34-112   
                 RED (SH) 6   
                 48.5 
                 8855.2 
                 8855.3 
               
               
                 oxid-humPLGF 34-112   
                 OX (SS) 3   
                 38.3 
                 8849.2 
                 8847.5 
               
               
                   
               
             
          
         
       
     
     Example 1H 
     Synthesis of humSOST 57-144  (humSOSTtrunc) 
     
       
         
               
             
           
               
                 (SEQ ID NO: 31) 
               
               
                 Peptide sequence for humSOST 57-144 : Biotine-GGG C1 R 
               
               
                   
               
               
                 ELHFTRYVTDGPCRSAKPVTELV C2 SGQ C3 GPARLLPNAIGRGKWWRPSG 
               
               
                   
               
               
                 PDFR C4 IPDRYRAQRVQLL C PGGEAPRARKVRLVAS C5 K C6 # 
               
               
                   
               
               
                 X0 = biotine-GGG 
               
               
                   
               
               
                 (amino acids 5-28 of SEQ ID NO: 31) 
               
               
                 X1 = RELHFTRYVTDGP C RSAKPVTELV 
               
               
                   
               
               
                 X2 = SGQ 
               
               
                   
               
               
                 (amino acids 34-57 of SEQ ID NO: 31) 
               
               
                 X3 = GPARLLPNAIGRGKWWRPSGPDFR 
               
               
                   
               
               
                 (amino acids 59-88 of SEQ ID NO: 31) 
               
               
                 X4 = IPDRYRAQRVQLL C PGGEAPRARKVRLVAS 
               
               
                   
               
               
                 X5 = K 
               
               
                   
               
               
                 X6 = amide 
               
             
          
         
       
     
     Synthesis of red-humSOST 57-144  could not be performed directly on solid-phase on a downloaded resin, as described for humVEGF 26-104 . Therefore, the shorter fragments humSOST-F1/3 were synthesized and subsequently ligated by Native Chemical Ligation (NCL) as described below. Also, the subsequent oxidative refolding of fully red-humSOST 57-144  was carried out as described below. Solid-phase synthesis of the fragments humSOST-F1/3 was carried out following standard procedures as described for humVEGF 26-104 . 
     Fragment Condensation of humSOST-F1/3 by NCL to give Red-humSOST 57-144  (for a Schematic Overview see  FIG. 16 ) 
     First, humSOST-F2 and humSOST-F3 were dissolved (2 mg/ml) in NCL reaction mixture (6 M guanidine, 20 mM TCEP, 200 mM MPAA, 0.2 M disodium hydrogenphosphate, adjusted with 10 M sodium hydroxide to pH 6.5) in a 1.2:1 ratio, and reacted for 24 hours at room temperature. The thiaproline-protected humSOST-F2/3 was obtained in 66.5% yield after reversed phase HPLC purification. Subsequently, the thiaproline was deprotected with 0.02 M methoxyamine in NCL buffer at pH 4.0 for 60 hours. Then, the pH was adjusted to 6.5 and 1.2 equivalents of humSOST-F1 was added and reacted for 1.5 day. The reaction was monitored by RPLC/MS and each day 40 mM TCEP was added to completely reduce all reagents. After completion of the reaction, crude red-humSOST 57-144  was purified using ion exchange chromatography, and subsequently by reversed phase HPLC giving pure red-humSOST 57-144  in 24.2% yield (overall 16.1%). 
     Structure of peptide fragments used for the fragment condensation of reduced SOST 67-144   
                                 Name   Peptide Sequence                   humSOST 57-144     Biotine-GGG   C   RELHFTRYVTDGP C RSAKPVTELV               C   SGQ   C   GPARLLPNAIGRGKWWRPSGPDFR   C   IPDRYR           AQRVQLL C PGGEAPRARKVRLVAS   C   K   C    (SEQ ID           NO: 31)-amide               humSOST-F1   Biotine-GGG   C   RELHFTRYVTDGP C RSAKPVTELV               C   SGQ (SEQ ID NO: 32)-thioester               humSOST-F2   BocNH-   C   ( Thz )GPARLLPNAIGRGKWWRPSGPDFR           (SEQ ID NO: 33)-thioester               humSOST-F3   Amine-   C   IPDRYRAQRVQLL C PGGEAPRARKVRLVA           S   C   K   C   (SEQ ID NO: 34)-amide                   C    = cysteines involved in cystine-knot formation;  C  = cysteines forming SS-bond between loop-1 and loop-3 of humSOST            
Oxidate Refolding of Red-humSOST 57-144  to give Oxid-humSOST 57-144 .
 
     Subsequently, red-humSOST 57-144  was natively refolded by dissolving the peptide (2 mg/ml) in a pH 8.0 buffer solution, containing 55 mM Tris-HCl, 21 mM sodium chloride, 0.88 mM potassium chloride, 0.48 L-arginine, 20 mM Glutathion-SH, and 4 mM Glutathion-SS. The peptide was oxidized over time and yielded 10.2% of oxid-humSOST 57-144  after 3.5 days at 4° C. (see  FIG. 17 ). 
     Purification of both red-humSOST 57-144  and oxid-humSOST 57-144  was carried out by preparative High Performance Liquid Chromatography (HPLC). Characterization of both compounds was carried out by analytical HPLC and ElectroSpray Ionization Mass Spectrometry (ESI-MS; see below). 
     
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Oxidation 
                   
                   
                 MW 
               
               
                   
                 state 
                 Retention 
                 MW 
                 experi- 
               
               
                 Peptide 
                 (RED/OX) 
                 (% ACN) 
                 calculated 
                 mental 
               
               
                   
               
             
             
               
                 red-humSOST 57-144   
                 RED (SH) 8   
                 35.0 
                 10237.2 
                 10235.0 
               
               
                 oxid-humSOST 57-144   
                 OX (SS) 4   
                 30.0 
                 10229.2 
                 10229.8 
               
               
                 AA 8 -humSOST 57-144   
                 RED 
                 33.0 
                 10694.1 
                 10692.5 
               
               
                   
                 (S—AcNH 2 ) 8   
               
               
                   
               
             
          
         
       
     
     The successful refolding of humSOST 57-144  was evidenced by the characteristic shift to lower Rf-values (from 35% to 30% ACN, see Table below) that is normally observed when proteins or fragments thereof are oxidative refolded. The characteristic narrow shape of the new peak at lower Rf-value provides evidence that an intact cystine-knot structure is indeed formed upon oxidative refolding. 
     Also, the ESI-MS spectrum undergoes a significant change upon oxidative refolding. First of all, the overall mass goes down by eight mass units (formation of four disulfide bonds releases a total of 8H). Moreover, there is a very characteristic shift of MS-signals to higher m/z-values. For example, the MS-spectrum for the red-humSOST 57-144  gives clear signals for the M 8+  to M 12+  charged species, whereas these signals disappear and a much weaker signal at M 6+  and M 7+  remains (see  FIG. 18D ) that is much less intense. Also, this shift is characteristic for folding of proteins into their oxidized native structure and shows that refolding of red-humSOST 57-144  was successful. The reason is that the protein or protein fragment adopts a more condensed structure that is no longer able to pick up so many charges. In contrast to this, the flexible and extended structure of the reduced protein is able to accommodate many more charges. 
     In order to prove further that oxid-humSOST 57-144  adopts a native cystine-knot fold, we present binding data of a series of three mAbs that were selected from phage-display libraries using oxid-humSOST 57-144 . It was shown that all three anti-oxid-humSOST 57-144  antibodies:
         bind strongly to oxid-humSOST 57-144  in ELISA.   bind strongly to recombinant full length humSOST/sclerostin in ELISA.   do not bind at all to AA 8 -humSOST 57-144  in ELISA.   do not bind at all to three other, non-related proteins in ELISA.       

     Altogether, these data show that oxid-humSOST 57-144  can be used instead of full-length humSOST/sclerostin to select antibodies from phage-display libraries (PDLs), that show full selectivity and specificity to full-length humSOST/sclerostin with respect to non-related proteins, and that oxid-humSOST 57-144  can, therefore, be used as an “easy-available” protein mimic of full-length humSOST/sclerostin for purposes of antibody generation and selection. 
     Example 1I 
     Synthesis of humTGFB2 15-111/Δ49-77 -humVEGF 62-67  (Chimeric humTGFB2-humVEGFtrunc) 
     In this example, we demonstrate the synthesis of the truncated protein mimic of oxid-humTGFB2 15-111 , in which the beta2-loop (28 amino acids long; X3 in general sequence) was replaced by the humVEGF beta2-loop (aa 62-67). The successful synthesis and oxidative (cystine-knot) folding of this TGFB2 15-111/Δ49-77 -humVEGF 62-67  mainly serves as an example to demonstrate that interchange of beta2-loop sequences amongst different cystine-knot proteins in general leads to chimeric peptides that retain the ability to form an intact cystine-knot fold, just like that observed for the fully homologous trunc-peptides (see other examples). 
     
       
         
               
             
           
               
                 (SEQ ID NO: 35) 
               
               
                 Peptide sequence of humTGFB 15-111/Δ49-77 - 
               
               
                   
               
               
                 humVEGF 62-67 : Acetyl- C1 ALRPLYIDFKRDLGWKWIHEPKGYNAN 
               
               
                   
               
               
                 F C2 AGA C3 NDEGLE C4 VSQDLEPLTILYYIGKTPKIEQLSNMIVKS C5 K 
               
               
                   
               
               
                   C6 -amide. 
               
               
                   
               
               
                 X0 = acetyl 
               
               
                   
               
               
                 (amino acids 2-29 of SEQ ID NO: 35) 
               
               
                 X1 =    A   LRPLYIDFKRDLGWKWIHEPKGYNANF (A-mutation for 
               
               
                   
               
               
                 native C) 
               
               
                   
               
               
                 X2 = AGA 
               
               
                   
               
               
                 (amino acids 35-40 of SEQ ID NO: 35) 
               
               
                 X3 =  NDEGLE  (beta2-loop sequence of humVEGF-A; aa 
               
               
                   
               
               
                 62-67) 
               
               
                   
               
               
                 (amino acids 42-71 of SEQ ID NO: 35) 
               
               
                 X4 = VSQDLEPLTILYYIGKTPKIEQLSNMIVKS 
               
               
                   
               
               
                 X5 = K 
               
               
                   
               
               
                 X6 = amide 
               
             
          
         
       
     
     Solid-phase synthesis of red-humTGFB2 15-111/Δ49-77 -humVEGF 62-67 . Red-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  was synthesized by normal solid-phase synthesis on a Rink-amide resin (downloaded to 0.1 mmol/g) following standard procedures as described for humVEGF 26-104  (see Example 1). Subsequent oxidative refolding was carried out exactly as described for humVEGF 26-104 . Purification of both red- and oxid-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  was carried out by preparative High Performance Liquid Chromatography (HPLC). Characterization of both the red- and oxid-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  was carried out by analytical HPLC and ElectroSpray Ionization Mass Spectrometry (ESI-MS). 
     The successful refolding of red-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  was evidenced by the characteristic shift to lower Rf-values upon oxidative refolding (from 46.8% to 42.0% ACN, see Table below) (see other examples). The characteristic narrow shape of the new peak at lower Rf-value provides evidence that an intact cystine-knot structure is indeed formed. Also, the ESI-MS spectrum undergoes a significant change upon oxidative refolding. First of all, the overall mass goes down by six mass units (formation of three disulfide bonds releases a total of 6H). Moreover, there is a very characteristic shift of MS-signals to higher m/z-values. For example, the MS-spectrum for the red-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  gives clear signals for the M 6+  to M 11+  charged species, whereas these signals completely disappear and a much weaker signal at M 5+  remains (see  FIG. 20 ) that is much less intense. Also this shift is characteristic for folding of proteins into their oxidized native structure and shows that refolding of humTGFB2 15-111/Δ49-77 -humVEGF 62-67  was successful. The reason is that the protein or protein fragment adopts a more condensed structure that is no longer able to pick up so many charges. In contrast to this, the flexible and extended structure of red humTGFB2 15-111/Δ49-77 -humVEGF 62-67  is able to accommodate many more charges. 
     
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Oxidation state 
                 Retention 
                 MW  
                   
               
               
                 Peptide 
                 (RED/OX) 
                 (%ACN) 
                 calc. 
                 MW exper. 
               
               
                   
               
             
             
               
                 red-humTGFB2 15-111/Δ49-77 -hum 
                 RED 
                 46.8 
                 8498.1 
                 8500.2 
               
               
                 VEGF 62-67   
               
               
                 oxid-humTGFB2 15-111/Δ49-77 -hum 
                 OX 
                 42.0 
                 8492.1 
                 8490.5 
               
               
                 VEGF 62-67   
               
               
                   
               
             
          
         
       
     
     In order to prove that oxid-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  can be used to generate anti-TGF-B2 antibodies via immunization, we carried out an immunization experiment in two rats. Each animal received four inoculations (0, 2, 4, and 7.5 wks) with 2×450+2×130 microgram of oxid-humTGFB2 15-111/Δ49-77 -humVEGF 62-67 . Analysis of the nine weeks post vaccination (wpv) antisera ( FIG. 21 ) showed strong binding in ELISA to full-length TGF-B2 (titers 3.8 and 4.1) compared to those of the pre-immune sera (≦2.1) indicating that antibodies specific for TGF-B2 were generated upon immunization. Moreover, it was observed that the majority of antibodies in the sera were directed towards the TGFB2-part of the peptide in oxid-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  rather that to the VEGF-part (humVEGF 62-67 ). This indicates the humVEGF 62-67  sequence is a good substitute for the much longer b2-loop of humTGFB2 (28 amino acids), but that it does not disturb the making of humTGF-B2-specific antibodies, nor the oxidative refolding of red-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  into oxid-humTGFB2 15-111/Δ49-77 -humVEGF 62-67 . 
     These data prove that oxid-humTGFB2 15-111/Δ49-77 -humVEGF 62-67  can be used as a substitute for TGF-B2 for eliciting anti-humTGFB2 antibodies that are fully cross-reactive with the native protein humTGF-B2. 
     REFERENCES 
     
         
         1. Vitt U. A., Y. H. Sheau, and A. J. W. Hsueh, “Evolution and Classification of Cystine Knot-containing Hormones and Related Extracellular Signaling Molecules,”  Mol. Endocrin . (2001) 15:681-94. 
         2. Tamaoki H., R. Miura, M. Kusunoki, Y. Kyogoku, Y. Kobayashi, and L. Moroder, “Folding motifs induced and stabilized by distinct cystine frameworks,”  Prot. Engin . (1998) 11:649-59. 
         3. Isaacs N. W., “Cystine Knots,”  Curr. Opin. Struct. Biol . (1995) 5:391-5. 
         4. McDonald N., and W. A. Hendrickson, “A structural superfamily of growth factors containing a cystine-knot motif,”  Cell  (1993) 73:421-4. 
         5. Bork P., “The modular architecture of a new family of growth regulators related to connective tissue growth factor,”  FEBS  (1993) 327:125-130. 
         6. Tomayko M. M., and C. P. Reynolds, “Determination of subcutaneous tumor size in athymic (nude) mice,”  Cancer Chemother. Pharmacol . (1989) 24:148-156.