Technical field

The present invention relates to isolated polypeptides derived from stanniocalcin-2 (STC2), and polypeptide fragments and variants thereof useful for inhibiting proteolytic activity of pregnancy-associated plasma protein-A (PAPP-A), as well as methods for identifying ligands and inhibitors of PAPP-A.

Background

The metzincin metalloproteinase PAPP-A (pregnancy-associated plasma protein-A, pappalysin-1 , EC 3.4.24.79) plays a key role in the regulation of insulin-like growth factor (IGF) signaling by specific cleavage of inhibitory IGF binding proteins (IGFBPs). PAPP- A is ubiquitously expressed in human tissues and is able to cleave insulin-like growth factor binding proteins (IGFBP) -2, -4 and -5 in the extracellular environment. It consequently causes the release of bound bioactive IGF-1 or -2, which are potent growth factors.

The paralogue proteins stanniocalcin (STC)-1 and -2 were recently identified as potent endogenous PAPP-A inhibitors and thus connected with the IGF system. Both STCs are dimers of approximately 250-residue subunits, which do not share sequence similarity with any other human proteins. Except for PAPP-A2, no other target proteinases of the STCs have been identified.

Accumulating data support a model in which the balance between active and STC- inhibited PAPP-A is a major determinant of IGF signalling in tissues. Recognition of the physiological roles of these components has led to the concept of a regulatory STC2 — > PAPP-A — > IGFBP — > IGF axis. The binding of STC2 to PAPP-A has previously been shown to prevent PAPP-A cleavage of insulin-like growth factor-binding protein (IGFBP)-4 and hence release within tissues of bioactive IGF.

Structural studies of PAPP-A have so far been unsuccessful, possibly caused by inherent flexibility of the protein, a high degree of glycosylation, and by difficulties in the expression of isolated domains. Thus, there is a need in the field for a better understanding of the PAPP-A structure and the interaction of PAPP-A-STC2 system leading to inhibition of PAPP-A in order to develop improved inhibitors of PAPP-A able to modulate IGF signalling. Summary

In one aspect, the present invention relates to an isolated polypeptide able to reduce the proteolytic activity of PAPP-A towards IGFBPs, particularly IGFBP4.

The inventors have for the first time elucidated the structural information of the interaction of PAPP-A in complex with STC2 that leads to inhibition of PAPP-A activity towards IGFBP4. The inventors have thus found the specific domains and amino acids of STC2 interacting with PAPP-A, as well as their conformation.

Thus, provided herein is an isolated polypeptide having a length of less than 100 amino acids comprising or consisting of an amino acid sequence selected from the group consisting of: a) the amino acid sequence according to SEQ ID NO.: 2 or SEQ ID NO.: 17; b) a variant of SEQ ID NO.: 2 or SEQ ID NO.: 17, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 2 or SEQ ID NO.: 17, but less than 99% sequence identity to SEQ ID NO.: 2 or SEQ ID NO.: 17; c) a variant of SEQ ID NO.: 2 or SEQ ID NO.: 17, wherein said variant has between 1 and 5 amino acid substitutions relative to SEQ ID NO.: 2 or SEQ ID NO.: 17, such as 1 , 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 2 or SEQ ID NO.: 17; d) a fragment of SEQ ID NO.: 2 or SEQ ID NO.: 17 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 2 or SEQ ID NO.: 17; e) the amino acid sequence according to SEQ ID NO.: 3; f) a variant of SEQ ID NO.: 3, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 3, but less than 99% sequence identity to SEQ ID NO.: 3; g) a variant of SEQ ID NO.: 3, wherein said variant has between 1 and 3 amino acid substitutions relative to SEQ ID NO.: 3, such as 1 , 2, or 3 amino acid substitutions relative to SEQ ID NO.: 3; h) a fragment of SEQ ID NO.: 3 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 3; i) the amino acid sequence according to SEQ ID NO.: 4; j) a variant of SEQ ID NO.: 4, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 4, but less than 99% sequence identity to SEQ ID NO.: 4; k) a variant of SEQ ID NO.: 4, wherein said variant has between 1 and 10 amino acid substitutions relative to SEQ ID NO.: 4, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 4; l) a fragment of SEQ ID NO.: 4 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 4; m) the amino acid sequence according to SEQ ID NO.: 5; n) a variant of SEQ ID NO.: 5, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 5, but less than 99% sequence identity to SEQ ID NO.: 5; o) a variant of SEQ ID NO.: 5, wherein said variant has between 1 and 12 amino acid substitutions relative to SEQ ID NO.: 5, such as 1 , 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 5; p) a fragment of SEQ ID NO.: 5 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 5; q) the amino acid sequence according to SEQ ID NO.: 6; r) a variant of SEQ ID NO.: 6, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 6, but less than 99% sequence identity to SEQ ID NO.: 6; s) a variant of SEQ ID NO.: 6, wherein said variant has between 1 and 9 amino acid substitutions relative to SEQ ID NO.: 6, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 6; t) a fragment of SEQ ID NO.: 6 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 6; u) the amino acid sequence according to SEQ ID NO.: 7; v) a variant of SEQ ID NO.: 7, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 7, but less than 99% sequence identity to SEQ ID NO.: 7; w) a variant of SEQ ID NO.: 7, wherein said variant has between 1 and 13 amino acid substitutions relative to SEQ ID NO.: 7, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 7; x) a fragment of SEQ ID NO.: 7 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 7; y) the amino acid sequence according to SEQ ID NO.: 1; z) a variant of SEQ ID NO.: 1 , wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 1 , but less than 99% sequence identity to SEQ ID NO.: 1; aa) a variant of SEQ ID NO.: 1 , wherein said variant has between 1 and 16 amino acid substitutions relative to SEQ ID NO.: 1, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 1; bb) a fragment of SEQ ID NO.: 1 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 1 ; cc) the amino acid sequence according to SEQ ID NO.: 8; dd) a variant of SEQ ID NO.: 8, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 8, but less than 99% sequence identity to SEQ ID NO.: 8; ee) a variant of SEQ ID NO.: 8, wherein said variant has between 1 and 5 amino acid substitutions relative to SEQ ID NO.: 8, such as 1, 2, or 3 amino acid substitutions relative to SEQ ID NO.: 8; ff) a fragment of SEQ ID NO.: 8 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 8; gg) the amino acid sequence according to SEQ ID NO.: 9; hh) a variant of SEQ ID NO.: 9, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 9, but less than 99% sequence identity to SEQ ID NO.: 9; ii) a variant of SEQ ID NO.: 9, wherein said variant has between 1 and 3 amino acid substitutions relative to SEQ ID NO.: 9, such as 1, 2, or 3 amino acid substitutions relative to SEQ ID NO.: 9; jj) a fragment of SEQ ID NO.: 9 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 9; kk) the amino acid sequence according to SEQ ID NO.: 10;

II) a variant of SEQ ID NO.: 10, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 10, but less than 99% sequence identity to SEQ ID NO.: 10; mm) a variant of SEQ ID NO.: 10, wherein said variant has between 1 and 3 amino acid substitutions relative to SEQ ID NO.: 10, such as 1, 2, or 3 amino acid substitutions relative to SEQ ID NO.: 10; nn) a fragment of SEQ ID NO.: 10 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 10; wherein said isolated polypeptide is capable of reducing Pregnancy-associated plasma protein A (PAPP-A) proteolytic activity, such as PAPP-A proteolytic activity towards Insulin Like Growth Factor Binding Protein 4 (IGFBP4).

In another aspect, the present invention relates to an isolated polynucleotide encoding the polypeptide according to the present disclosure.

In yet another aspect, the present invention relates to a vector comprising the polynucleotide according to the present disclosure.

In another aspect, the present invention relates to a host cell comprising the vector according to the present disclosure.

In a further aspect, the present invention provides a pharmaceutical composition comprising the isolated polypeptide, the polynucleotide, the vector, and/or the host cell according to the present disclosure.

In another aspect, the present invention provides the isolated polypeptide, the polynucleotide, the vector, the host cell, and/or the pharmaceutical composition according to the present disclosure for use as a medicament.

In a further aspect, the present invention provides the isolated polypeptide, the polynucleotide, the vector, the host cell, and/or the pharmaceutical composition according to the present disclosure for use in the treatment and/or prevention of a disease, disorder and/or condition selected from the group consisting of: cancer, cardiovascular disease, such as atherosclerosis, kidney disease, such as diabetic nephropathy, and fibrosis.

In one aspect, the present invention provides the isolated polypeptide, the polynucleotide, the vector, the host cell, and/or the pharmaceutical composition according to the present disclosure for use in in the modulation of insulin growth factor (IGF) signalling.

The novel structural information of PAPP-A in complex with STC2 has the advantage to offer the possibility to develop ligands able to bind PAPP-A and/or inhibit its proteolytic activity using computer-assisted screening technologies.

In one aspect, the present invention provides a computer-readable data storage medium comprising a data storage material encoded with at least a portion of the structure coordinates set forth in figure 6.

In one aspect, the present invention provides for the use of atomic coordinates as presented in figure 6 or atomic coordinates selected from a three-dimensional structure that deviates from the three-dimensional structure as presented in figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A in a method for identifying a ligand capable of binding to one or more of: a) PAPP-A (SEQ ID NO.: 14) b) the C-terminal region of PAPP-A corresponding to residues 1555 to 1627 of SEQ ID NO.: 14 (SEQ ID NO.: 11) c) the LNR3 domain of PAPP-A corresponding to the residues 1555 to 1583 of SEQ ID NO.: 14 (SEQ ID NO.: 12) d) the region of PAPP-A according to amino acid residues 1556 to 1568 of SEQ ID NO.: 14 (SEQ ID NO.: 13) e) the region of PAPP-A according to amino acid residues 766 to 777 of SEQ ID NO.: 14 (SEQ ID NO.: 16) or a fragment or variant of a) through e).

In a further aspect, the present invention provides for a method of identifying a ligand capable of binding to PAPP-A, or a fragment or variant thereof, said method comprising the steps of: a) generating the spatial structure of the binding site on a computer screen using atomic coordinates as presented in figure 6 or atomic coordinates selected from a three-dimensional structure that deviates from the three- dimensional structure presented in figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A, b) generating ligands with their spatial structure on the computer screen, and c) selecting ligands that can bind to at least 1 amino acid residue of the set of binding interaction sites without steric interference.

In another aspect, the present invention provides for a computer-assisted method for identifying a ligand capable of binding to PAPP-A, or a fragment or variant thereof, using a programmed computer comprising a processor, a data storage system, a data input device and a data output device, comprising the following steps: a) inputting into the programmed computer through said input device data comprising: atomic coordinates of a subset of the atoms of said PAPP-A, thereby generating a criteria data set; wherein said atomic coordinates are selected from the three-dimensional structure presented in figure 6 or atomic coordinates selected from a three-dimensional structure that deviates from the three-dimensional structure presented in figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A, b) comparing, using said processor, the criteria data set to a computer data base of low-molecular weight organic chemical structures and peptide fragments stored in the data storage system; and c) selecting from said data base, using computer methods, a chemical structure having a portion that is structurally complementary to the criteria data set and being free of steric interference with the PAPP-A.

In another aspect, the invention provides for a method for identifying a ligand, said method comprising the steps of: a) selecting a ligand using atomic coordinates in conjunction with computer modelling, wherein said atomic coordinates are the atomic coordinates presented in figure 6 or wherein the atomic coordinates are selected from a three-dimensional structure that deviates from the three-dimensional structure presented in figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A, by docking ligands into a set of binding interaction sites in PAPP-A, or a fragment or variant thereof, said binding interaction generated by computer modelling and selecting a ligand capable of binding to at least one amino acid in said set of binding interaction sites of PAPP-A, b) providing said ligand and PAPP-A, c) contacting the ligand with said PAPP-A, and d) detecting the binding of the ligand with said PAPP-A.

In a final aspect, the invention provides for method of identifying a ligand of PAPP-A, or a fragment or variant thereof, said method comprising the steps of: a) introducing into a computer, information derived from atomic coordinates defining a conformation of PAPP-A, or a fragment or variant thereof, based on three-dimensional structure determination, whereby a computer program utilizes or displays on the computer screen the structure of said conformation; wherein said atomic coordinates are selected from the three-dimensional structure as presented in figure 6 or atomic coordinates selected from a three-dimensional structure that deviates from the tree-dimensional structure represented by figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A; b) generating a three-dimensional representation of said PAPP-A, or said fragment or variant thereof by said computer on a computer screen; c) superimposing a model of a ligand on the representation of said three- dimensional representation of said PAPP-A, or said fragment or variant thereof, d) assessing the possibility of binding and/or the absence of steric interference of the ligand with said PAPP-A, or said fragment or variant thereof; e) incorporating said ligand compound in a binding assay and/or enzymatic activity assay of PAPP-A; f) determining whether said ligand binds to PAPP-A, and/or whether said ligand inhibits binding of a competing ligand towards PAPP-A, and/or whether said ligand inhibits the proteolytic activity of PAPP-A. Description of Drawings

Figure 1 : A) Binding to a 415-residue monomeric C-terminal PAPP-A fragment, PA(T1213-G1627), assessed by SPR. PAPP-A mAb PA141 prevents binding of STC2(C120A). B) Similar to A), but carried out in the absence or presence of EDTA. The loss of bound Ca2+ weakens the interactions between STC2 and the C domain. C) Similar to A), but with IGFBP-4 as the analyte in the absence or presence of EDTA to disrupt binding of Ca ²⁺ to LNR3. The loss of bound Ca ²⁺ weakens the interactions between IGFBP-4 and the C domain.

Figure 2: A) Assessment of the ability of PAPP-A variants as indicated above the lanes to form a covalent complex with wild-type STC2. Following incubation (16 h) of separately synthesized proteins, the reaction mixtures were separated by non-reducing SDS-PAGE and visualized by Western blotting for PAPP-A. Only wild-type PAPP-A and PAPP-A(dLNR1-2) show increased molecular weight following incubation with STC2 and thus the ability to form a covalent complex. B) Kinetic analysis of inhibition of PAPP-A cleavage of IGFBP-4 by STC2(C120A). STC2(C120A) cannot bind covalently to PAPP-A. Intact radiolabeled IGFBP-4 was used, and relative initial velocities (v/vO) of the cleavage reaction were determined by quantification of IGFBP-4 and cleavage products following separation by SDS-PAGE for each concentration of inhibitor (the inset shows an example). The inhibition constant (Ki) was determined to be 47.3 + 1.1 x 10-9 M by fitting the Morrison Ki equation (competitive inhibition) to the data.

Figure 3: A) Minimalistic schematic drawing of the PAPP-A STC2 2:2 complex with indication of the intersubunit disulfide bonds (vertical grey bars) and observed (C domain-STC2) and hypothesized (LNR1-STC2) interactions (blue lines). The LNR modules are shown in light grey. Note that because the PAPP-A polypeptides cross at M6, LNR1 and LNR3 of each PAPP-A subunit do not interact with the same STC2 subunit. B) The interface between STC2 and the PAPP-A C domain. Basic residues of STC2 positioned favorably for electrostatic interaction with the negative charge surrounding the Ca2+ ion of LNR3 are indicated. Residues likely to be involved in van der Waal interactions are also indicated (V63 of STC2; Y1566, T1594, and K1592 of PAPP-A). Note that STC2 residues interacts with the C domain of the PAPP-A subunit to which the opposite STC2 subunit is disulfide bound. C) Cartoon representation of PAPP-A LNR1 (grey, top) and the C domain (light bottom), including LNR3 (dark grey top). LNR1 and LNR3 are superimposed. Disulfide bonds are indicated and the position of the two Ca ²⁺ ions (spheres) are shown. D) Maps around the Ca ²⁺ ions of LNR1 and LNR3, respectively.

Figure 4: Inhibition of PAPP-A proteolytic activity on IGFBP-4 in the presence of peptide STC2(86-108) (SEQ ID NO.: 17).

Figure 5: A) Sequence of prepro PAPP-A from human (SEQ ID NO.: 14) (UniProt IDs: Human PAPP-A, Q1321). Domain boundaries and selected motifs are indicated. B) Sequence sequence of preSTC2 from human (SEQ ID NO.: 15) (UniProt IDs: Human STC2, 076061). Cysteine residues are shaded (grey). Regions involved in PAPP-A- STC2 interactions were identified by visual inspection of the structure and are indicated by highlighting and residue numbers in brackets above. PAPP-A and STC2 residues highlighted in bold interact with each other, and PAPP-A and STC2 residues in regions highlighted by underlining interact with each other.

Figure 6: Atomic coordinates of selected regions of PPAP-A in complex with STC2. The regions for which atom coordinates are depicted in the pdb file are in order: PAPP-A LNR1-2 domain (residues 411-473 of SEQ ID NO.: 14), PAPP-A M5 domain (residues 702-883 of SEQ ID NO.: 14), STC2 (residues 44-210 of SEQ ID NO.: 15) and PAPP-A C-term domain (residues 1555-1617 of SEQ ID NO.: 14). The numbering of the amino acid residues and atoms of this pdb file is in accordance with the PDB database entry 8A7D as of the filing date of the present application.

Detailed description

Definitions

Amino acid substitution - The term “amino acid substitution” as used herein refers to the change from one amino acid to a different amino acid in a peptide, polypeptide or protein. The substitution may be a conservative substitution, wherein an amino acid is exchanged into another amino acid that has similar properties. The substitution may also be a nonconservative substitution, wherein an amino acid is exchanged into another amino acid with different properties. Properties of an amino acid include for example the charge, polarity, acidity, size and hydrophobicity of the amino acid. Identity - The term identity, with respect to a polynucleotide or polypeptide, are defined herein as the percentage of nucleic acids or amino acids in the candidate sequence that are identical, to the residues of a corresponding native nucleic acids or amino acids, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity. Methods and computer programs for the alignments are well known in the art.

Treatment The term “treatment” as used herein may refer to any kind of treatment. The treatment may be a curative treatment; it may also be an ameliorating treatment and/or a treatment reducing the effects and/or symptoms of the treated disease, injury and/or disorder. The treatment may also be a treatment that delays progression and/or development of the treated disease, injury and/or disorder. The treatment may also be preventative/prophylactic, i.e. a treatment to eliminate or reduce the risk of developing the diseases, injuries and/or disorders disclosed herein.

Fusion - The term “fursion protein” as used herein refers to a composite polypeptide, i.e., a single contiguous amino acid sequence, made up of two (or more) distinct, heterologous polypeptides which are not normally or naturally fused together in a single amino acid sequence. Thus, a fusion protein may include a single amino acid sequence that contains two entirely distinct amino acid sequences or two similar or identical polypeptide sequences, provided that these sequences are not normally found together in the same configuration in a single amino acid sequence found in nature.

Fusion proteins may generally be prepared using either recombinant nucleic acid methods, i.e., as a result of transcription and translation of a recombinant gene fusion product, which fusion comprises a segment encoding a polypeptide of the invention and a segment encoding a heterologous polypeptide, or by chemical synthesis methods well known in the art. Fusion proteins may also contain a linker polypeptide in between the constituent polypeptides of the fusion protein.

Root mean deviation The term "root mean square deviation" (rmsd) is used as a mean of comparing two closely related structures and relates to a deviation in the distance between related atoms of the two structures after structurally minimizing this distance in an alignment. Related proteins with closely related structures will be characterized by relatively low RMSD values whereas larger differences will result in an increase of the RMSD value. Polypeptide

The present invention relates to a polypeptide derived from stanniocalcin-2 (STC2, SEQ ID NO.: 15). The inventors have shown and identified specific fragments of STC2 that are involved in binding and inhibition of PAPP-A.

Thus, provided herein is an isolated polypeptide having a length of less than 100 amino acids comprising or consisting of an amino acid sequence selected from the group consisting of: a) the amino acid sequence according to SEQ ID NO.: 2 or SEQ ID NO.: 17; b) a variant of SEQ ID NO.: 2 or SEQ ID NO.: 17, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 2 or SEQ ID NO.: 17, but less than 99% sequence identity to SEQ ID NO.: 2 or SEQ ID NO.: 17; c) a variant of SEQ ID NO.: 2 or SEQ ID NO.: 17, wherein said variant has between 1 and 5 amino acid substitutions relative to SEQ ID NO.: 2 or SEQ ID NO.: 17, such as 1 , 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 2 or SEQ ID NO.: 17; d) a fragment of SEQ ID NO.: 2 or SEQ ID NO.: 17 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 2 or SEQ ID NO.: 17; e) the amino acid sequence according to SEQ ID NO.: 3; f) a variant of SEQ ID NO.: 3, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 8, but less than 99% sequence identity to SEQ ID NO.: 3; g) a variant of SEQ ID NO.: 3, wherein said variant has between 1 and 3 amino acid substitutions relative to SEQ ID NO.: 3, such as 1 , 2, or 3 amino acid substitutions relative to SEQ ID NO.: 3; h) a fragment of SEQ ID NO.: 3 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 3; i) the amino acid sequence according to SEQ ID NO.: 4; j) a variant of SEQ ID NO.: 4, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 4, but less than 99% sequence identity to SEQ ID NO.: 4; k) a variant of SEQ ID NO.: 4, wherein said variant has between 1 and 10 amino acid substitutions relative to SEQ ID NO.: 4, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 4; l) a fragment of SEQ ID NO.: 4 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 4; m) the amino acid sequence according to SEQ ID NO.: 5; n) a variant of SEQ ID NO.: 5, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.:

5, but less than 99% sequence identity to SEQ ID NO.: 5; o) a variant of SEQ ID NO.: 5, wherein said variant has between 1 and 12 amino acid substitutions relative to SEQ ID NO.: 5, such as 1 , 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 5; p) a fragment of SEQ ID NO.: 5 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 5; q) the amino acid sequence according to SEQ ID NO.: 6; r) a variant of SEQ ID NO.: 6, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.:

6, but less than 99% sequence identity to SEQ ID NO.: 6; s) a variant of SEQ ID NO.: 6, wherein said variant has between 1 and 9 amino acid substitutions relative to SEQ ID NO.: 6, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 6; t) a fragment of SEQ ID NO.: 6 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 6; u) the amino acid sequence according to SEQ ID NO.: 7; v) a variant of SEQ ID NO.: 7, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.:

7, but less than 99% sequence identity to SEQ ID NO.: 7; w) a variant of SEQ ID NO.: 7, wherein said variant has between 1 and 13 amino acid substitutions relative to SEQ ID NO.: 7, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 7; x) a fragment of SEQ ID NO.: 7 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 7; y) the amino acid sequence according to SEQ ID NO.: 1; z) a variant of SEQ ID NO.: 1 , wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 1 , but less than 99% sequence identity to SEQ ID NO.: 1 ; aa) a variant of SEQ ID NO.: 1 , wherein said variant has between 1 and 16 amino acid substitutions relative to SEQ ID NO.: 1, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 1 ; bb) a fragment of SEQ ID NO.: 1 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 1 ; cc) the amino acid sequence according to SEQ ID NO.: 8; dd) a variant of SEQ ID NO.: 8, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.:

8, but less than 99% sequence identity to SEQ ID NO.: 8; ee) a variant of SEQ ID NO.: 8, wherein said variant has between 1 and 5 amino acid substitutions relative to SEQ ID NO.: 8, such as 1, 2, or 3 amino acid substitutions relative to SEQ ID NO.: 8; ff) a fragment of SEQ ID NO.: 8 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 8; gg) the amino acid sequence according to SEQ ID NO.: 9; hh) a variant of SEQ ID NO.: 9, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.:

9, but less than 99% sequence identity to SEQ ID NO.: 9; ii) a variant of SEQ ID NO.: 9, wherein said variant has between 1 and 3 amino acid substitutions relative to SEQ ID NO.: 9, such as 1, 2, or 3 amino acid substitutions relative to SEQ ID NO.: 9; jj) a fragment of SEQ ID NO.: 9 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 9; kk) the amino acid sequence according to SEQ ID NO.: 10; II) a variant of SEQ ID NO.: 10, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 10, but less than 99% sequence identity to SEQ ID NO.: 10; mm) a variant of SEQ ID NO.: 10, wherein said variant has between 1 and 3 amino acid substitutions relative to SEQ ID NO.: 10, such as 1 , 2, or 3 amino acid substitutions relative to SEQ ID NO.: 10; nn) a fragment of SEQ ID NO.: 10 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 10; wherein said isolated polypeptide is capable of reducing Pregnancy-associated plasma protein A (PAPP-A) proteolytic activity, such as PAPP-A proteolytic activity towards Insulin Like Growth Factor Binding Protein 4 (IGFBP4).

In one embodiment, the length of the peptide is less than 100 amino acids, such as less length of less than 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 or 10 amino acids.

In one embodiment, the polypeptide has a length of less than 100 amino acids In one embodiment, the polypeptide has a length of less than 95 amino acids. In one embodiment, the polypeptide has a length of less than 90 amino acids. In one embodiment, the polypeptide has a length of less than 85 amino acids. In one embodiment, the polypeptide has a length of less than 80 amino acids. In one embodiment, the polypeptide has a length of less than 70 amino acids. In one embodiment, the polypeptide has a length of less than 65 amino acids. In one embodiment, the polypeptide has a length of less than 60 amino acids. In one embodiment, the polypeptide has a length of less than 55 amino acids. In one embodiment, the polypeptide has a length of less than 53 amino acids.

In one embodiment, the polypeptide has a length of less than 50 amino acids. In one embodiment, the polypeptide has a length of less than 45 amino acids. In one embodiment, the polypeptide has a length of less than 40 amino acids. In one embodiment, the polypeptide has a length of less than 35 amino acids. In one embodiment, the polypeptide has a length of less than 30 amino acids.

In one embodiment, the polypeptide has a length of less than 25 amino acids.

In one embodiment, the polypeptide has a length of less than 20 amino acids.

In one embodiment, the polypeptide has a length of less than 15 amino acids.

In one embodiment, the polypeptide has a length of less than 10 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 5 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 6 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 7 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 8 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 9 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 10 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 11 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 12 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 13 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 14 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 15 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 16 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 17 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 18 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 19 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 20 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 21 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 22 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 23 amino acids. In one embodiment, the isolated polypeptide has a length of at least 24 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 25 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 26 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 27 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 28 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 29 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 30 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 31 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 32 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 33 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 34 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 35 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 36 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 37 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 38 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 39 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 40 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 41 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 42 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 43 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 44 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 45 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 46 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 47 amino acids.

In one embodiment, the isolated polypeptide has a length of at least 48 amino acids. In one embodiment, the isolated polypeptide has a length of between 10 and 52 amino acids, such as between 15 and 52 amino acids, such as between 20 and 52 amino acids.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 2, such as at least 61 % identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 2.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 2. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 2. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 2. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 2.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 2.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 2.

In one embodiment, the variant of the polypeptide has between 1 and 5 amino acid substitutions compared to SEQ ID NO.: 2. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 2. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 2. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 2. In one embodiment, the variant of the polypeptide has 4 amino acid substitutions relative to SEQ ID NO.: 2. In one embodiment, the variant of the polypeptide has 5 amino acid substitutions relative to SEQ ID NO.: 2.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 17, such as at least 61% identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 17.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 17. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 17. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 17. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 17.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 17.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 17.

In one embodiment, the variant of the polypeptide has between 1 and 5 amino acid substitutions compared to SEQ ID NO.: 17. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 17. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 17. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 17. In one embodiment, the variant of the polypeptide has 4 amino acid substitutions relative to SEQ ID NO.: 17. In one embodiment, the variant of the polypeptide has 5 amino acid substitutions relative to SEQ ID NO.: 17. In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 3, such as at least 61 % identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 3.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 3. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 3. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 3. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 3.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 3.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 3.

In one embodiment, the variant of the polypeptide has between 1 and 3 amino acid substitutions compared to SEQ ID NO.: 3. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 3. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 3. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 3.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 4, such as at least 61 % identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 4.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 4.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 4.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 4.

In one embodiment, the variant of the polypeptide has between 1 and 10 amino acid substitutions compared to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 4 amino acid substitutions relative to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 5 amino acid substitutions relative to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 6 amino acid substitutions relative to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 7 amino acid substitutions relative to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 8 amino acid substitutions relative to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 9 amino acid substitutions relative to SEQ ID NO.: 4. In one embodiment, the variant of the polypeptide has 10 amino acid substitutions relative to SEQ ID NO.: 4.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 5, such as at least 61 % identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 5.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 5.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 5.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 5.

In one embodiment, the variant of the polypeptide has between 1 and 12 amino acid substitutions compared to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 4 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 5 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 6 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 7 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 8 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 9 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 10 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 11 amino acid substitutions relative to SEQ ID NO.: 5. In one embodiment, the variant of the polypeptide has 12 amino acid substitutions relative to SEQ ID NO.: 5.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 6, such as at least 61 % identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 6.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 6.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 6.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 6.

In one embodiment, the variant of the polypeptide has between 1 and 9 amino acid substitutions compared to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has 4 amino acid substitutions relative to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has 5 amino acid substitutions relative to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has 6 amino acid substitutions relative to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has 7 amino acid substitutions relative to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has 8 amino acid substitutions relative to SEQ ID NO.: 6. In one embodiment, the variant of the polypeptide has 9 amino acid substitutions relative to SEQ ID NO.: 6.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 7, such as at least 61 % identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 7.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 7.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 7.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 7.

In one embodiment, the variant of the polypeptide has between 1 and 13 amino acid substitutions compared to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 4 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 5 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 6 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 7 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 8 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 9 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 10 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 11 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 12 amino acid substitutions relative to SEQ ID NO.: 7. In one embodiment, the variant of the polypeptide has 13 amino acid substitutions relative to SEQ ID NO.: 7.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 1 , such as at least 61 % identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 1.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 1 . In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 1.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 1.

In one embodiment, the variant of the polypeptide has between 1 and 16 amino acid substitutions compared to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 4 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 5 amino acid substitutions relative to SEQ ID NO.: 1 . In one embodiment, the variant of the polypeptide has 6 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 7 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 8 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 9 amino acid substitutions relative to SEQ ID NO.: 1 . In one embodiment, the variant of the polypeptide has 10 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 11 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 12 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 13 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 14 amino acid substitutions relative to SEQ ID NO.: 1. In one embodiment, the variant of the polypeptide has 15 amino acid substitutions relative to SEQ ID NO.: 1 . In one embodiment, the variant of the polypeptide has 16 amino acid substitutions relative to SEQ ID NO.: 1.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 8, such as at least 61 % identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 8.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 8. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 8. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 8. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 8.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 8.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 8.

In one embodiment, the variant of the polypeptide has between 1 and 5 amino acid substitutions compared to SEQ ID NO.: 8. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 8. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 8. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 8. In one embodiment, the variant of the polypeptide has 4 amino acid substitutions relative to SEQ ID NO.: 8. In one embodiment, the variant of the polypeptide has 5 amino acid substitutions relative to SEQ ID NO.: 8.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 9, such as at least 61 % identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 9.

In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 9. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 9. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 9. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 9.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 9.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 9.

In one embodiment, the variant of the polypeptide has between 1 and 3 amino acid substitutions compared to SEQ ID NO.: 9. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 9. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 9. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 9.

In one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO.: 10, such as at least 61% identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72% identity, such as at least 73% identity, such as at least 74% identity, such as at least 75% identity, such as at least 76% identity, such as at least 77% identity, such as at least 78% identity, such as at least 79% identity, such as at least 80% identity, such as at least 81 % identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91 % identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as at least 100% identity to SEQ ID NO.: 10. In one embodiment, the variant of the polypeptide has at least 80% sequence identity to SEQ ID NO.: 10. In one embodiment, the variant of the polypeptide has at least 90% sequence identity to SEQ ID NO.: 10. In one embodiment, the variant of the polypeptide has at least 95% sequence identity to SEQ ID NO.: 10. In one embodiment, the variant of the polypeptide has at least 98% sequence identity to SEQ ID NO.: 10.

In one embodiment, the variant of the polypeptide has less than 99% sequence identity to SEQ ID NO.: 10.

In one embodiment, the isolated polypeptide is according to SEQ ID NO.: 10.

In one embodiment, the variant of the polypeptide has between 1 and 3 amino acid substitutions compared to SEQ ID NO.: 10. In one embodiment, the variant of the polypeptide has 1 amino acid substitution relative to SEQ ID NO.: 10. In one embodiment, the variant of the polypeptide has 2 amino acid substitutions relative to SEQ ID NO.: 10. In one embodiment, the variant of the polypeptide has 3 amino acid substitutions relative to SEQ ID NO.: 10.

In one embodiment, the amino acid substitutions are conservative substitutions. A conservative amino acid substitution is a replacement of an amino acid in a polypeptide to a given amino acid with similar biochemical properties, such as for example similar size, charge, hydrophobicity and/or polarity. Such substitutions often have a smaller effect on polypeptide function compared to non-conservative substitutions. Examples of conservative amino acid substitutions can be seen in the table below.

Examples of conservative amino acid substitutions.

Using the elucidated three-dimensional structure of PAPP-A in complex with STC2 the inventors have identified that the regions of STC2 corresponding to the amino acid residues 86 to 107 of SEQ ID NO.:15 (corresponding to SEQ ID NO.: 2) and amino acid residues 60 to 66 of SEQ ID NO.: 15 (corresponding to SEQ ID NO.: 3) interact with the C-terminal domain of PAPP-A (SEQ ID NO.: 11), such as the LNR3 domain of PAPP-A (SEQ ID NO.: 12), such as the amino acid residues 1556 to 1665 of PAPP-A (SEQ ID NO.: 13).

Thus, in one embodiment the isolated polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO.: 2 and/or the amino acid sequence according to SEQ ID NO.: 3.

In one embodiment, the isolated polypeptide comprises or consists of the amino acid sequence of SEQ ID NO.: 17.

Furthermore, the inventors have shown that the residues 45 to 58 of SEQ ID NO.: 15 and the residues 118 to 124 of SEQ ID NO.:15 (corresponding to SEQ ID NO.: 9 and SEQ ID NQ.:10 respectively) interact with the amino acids of PAPP-A corresponding to positions 766 to 777 of SEQ ID NO.: 14, (SEQ ID NO.: 16).

In one embodiment, the isolated polypeptide described herein comprises a variant of SEQ ID NO.: 2 or a variant of a fragment of SEQ ID NO.: 2, wherein said variant comprises: an M residue at position 1 of SEQ ID NO.: 2; and/or an L residue at position 4 of SEQ ID NO.: 2; and/or an H residue at position 5 of SEQ ID NO.: 2; and/or an A residue at position 7 of SEQ ID NO.: 2; and/or G residue at position 8 of SEQ ID NO.: 2; and/or an A residue at position 12 of SEQ ID NO.: 2; and/or a K residue at position 15 of SEQ ID NO.: 2; and/or an S residue at position 16 of SEQ ID NO.: 2; and/or a I residue at position 18 of SEQ ID NO.: 2; and/or a K residue at position 19 of SEQ ID NO.: 2; and/or a L residue at position 22 of SEQ ID NO.: 2.

In one embodiment, the isolated polypeptide described herein comprises a variant of SEQ ID NO.: 3 or a variant of a fragment of SEQ ID NO.: 3, wherein said variant comprises: a G residue at position 2 of SEQ ID NO.: 3; and/or a D residue at position 3 of SEQ ID NO.: 3; and/or a V residue at position 4 of SEQ ID NO.: 3; and/or a G residue at position 6 of SEQ ID NO.: 3. In one embodiment, the isolated polypeptide described herein comprises a variant of SEQ ID NO.: 9 or a variant of a fragment of SEQ ID NO.: 9, wherein said variant comprises: an L residue at position 1 of SEQ ID NO.: 9, and/or an S residue at position 2 of SEQ ID NO.: 9, and/or a T residue at position 6 of SEQ ID NO.: 9, and/or a Q residue at position 10 of SEQ ID NO.: 9, and/or a H residue at position 11 of SEQ ID NO.: 9 and/or a V residue at position 14 of SEQ ID NO.9.

In one embodiment, the isolated polypeptide described herein comprises a variant of SEQ ID NO.: 10 or a variant of a fragment of SEQ ID NO.: 10, wherein said variant comprises: a F residue at position 1 of SEQ ID NO.: 10, and/or a G residue at position 2 of SEQ ID NO.: 10; and/or a C residue at position 3 of SEQ ID NO.: 10, and/or a I residue at position 4 of SEQ ID NO.: 10, and/or a S residue at position 5 of SEQ ID NO.: 10, and/or an R residue at position 6 of SEQ ID NO.: 10, and/or K residue at position 7 of SEQ ID NO.: 10.

Using the elucidated three-dimensional structure of PAPP-A in complex with STC2 the inventors have identified that specific amino acids in STC2 interact with PAPP-A. For example, the amino acid residues corresponding to positions 61 to 64, 66, 86, 89, 90, 92, 93, 97, 100, 101 , 103, 104, 107 and 108 of SEQ ID NO.: 15 and/or the amino acid residues corresponding to residues 45, 47, 50, 54, 55, 58 and 118 to 124 of SEQ ID NO.: 15.

Thus, in one embodiment the present disclosure refers to the isolated polypeptide as described herein, wherein one or more of the amino acid residues corresponding to positions 61 to 64, 66, 86, 89, 90, 92, 93, 97, 100, 101 , 103, 104, 107 and 108 of SEQ ID NO.: 15 are not substituted or modified.

In one embodiment the present disclosure refers to the isolated polypeptide as described herein, wherein one or more of the amino acid residues corresponding to positions 45, 47, 50, 54, 55, 58, and 118 to 124 of SEQ ID NO.: 15 are not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 60 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 61 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 62 of SEQ ID NO.: 15 is not substituted or modified. In one embodiment, the amino acid residue corresponding to position 63 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 64 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 66 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 86 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 89 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 90 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 92 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 93 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 97 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 100 of SEQ ID

NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 101 of SEQ ID

NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 103 of SEQ ID

NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 104 of SEQ ID

NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 107 of SEQ ID

NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 108 of SEQ ID

NO.: 15 is not substituted or modified. In one embodiment, the amino acid residue corresponding to position 45 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 47 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 50 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 54 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 55 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 58 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 118 of SEQ ID

NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 119 of SEQ ID

NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 120 of SEQ ID

NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 121 of SEQ ID

NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 122 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 123 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residue corresponding to position 124 of SEQ ID NO.: 15 is not substituted or modified.

In one embodiment, the amino acid residues corresponding to positions 63 and 104 of SEQ ID NO.: 15 are not substituted or modified.

In one embodiment, the present disclosure refers to an isolated polypeptide as described herein, wherein one or more of the amino acid residues corresponding to positions 61 to 64, 66, 86, 89, 90, 92, 93, 97, 100, 101 , 103, 104, 107 and 108 of SEQ ID NO.: 15 are conservatively substituted.

In one embodiment, the present disclosure refers to an isolated polypeptide as described herein, wherein one or more of the amino acid residues corresponding to positions 45, 47, 50, 54, 55, 58 and 118 to 124 of SEQ ID NO.: 15 are conservatively substituted.

In one embodiment, the present disclosure provides a polypeptide as described herein, wherein the C corresponding to position 120 of SEQ ID NO.: 15 is substituted by an A.

In one embodiment, the isolated polypeptide described herein is capable of binding one or more of: a) PAPP-A (SEQ ID NO.: 14), b) the C-terminal region of PAPP-A corresponding to residues 1555 to 1627 of SEQ ID NO.: 14 (SEQ ID NO.: 11), c) the LNR3 domain of PAPP-A corresponding to the residues 1555 to 1583 of SEQ ID NO.: 14 (SEQ ID NO.: 12), d) the region of PAPP-A according to amino acid residues 1556 to 1568 of SEQ ID NO.: 14 (SEQ ID NO.: 13), e) the region of PAPP-A according to amino acid residues 766 to 777 of SEQ ID NO.: 14 (SEQ ID NO.: 16), or a fragment or variant of a) through e).

In one embodiment, the isolated polypeptide binds PAPP-A (SEQ ID NO.: 14). In one embodiment, the isolated polypeptide binds the C-terminal region of PAPP-A corresponding to residues 1555 to 1627 of SEQ ID NO.: 14 (SEQ ID NO.: 11). In one embodiment, the isolated polypeptide binds the LNR3 domain of PAPP-A corresponding to the residues 1555 to 1583 of SEQ ID NO.: 14 (SEQ ID NO.: 12). In one embodiment, the isolated polypeptide binds the region of PAPP-A according to amino acid residues 1556 to 1568 of SEQ ID NO.: 14 (SEQ ID NO.: 13). In one embodiment, the isolated polypeptide binds the region of PAPP-A according to amino acid residues 766 to 777 of SEQ ID NO.: 14 (SEQ ID NO.: 16).

The polypeptide disclosed herein may be further modified, for example by the attachment of one or more moieties. Such modifications may improve the properties of the polypeptide, hereunder the in vivo stability, membrane permeability, and/or the half-life of the polypeptide. Thus, in one embodiment, the polypeptide comprises one or more moieties conjugated to said polypeptide, optionally wherein the polypeptide and the one or more moieties are conjugated to each other by a linker.

In one embodiment, the polypeptide described herein reduces the proteolytic activity of PAPP-A towards IGFBP4.

In one embodiment, the polypeptide described herein is not one of: SEQ ID NO.: 18 or SEQ ID NO.: 19.

In one aspect, the invention provides a fusion protein comprising the polypeptide presented herein and a further polypeptide.

In one embodiment, the fusion protein comprises SEQ ID NO.: 2 and a further polypeptide. In one embodiment, the fusion protein comprises SEQ ID NO.: 3 and a further polypeptide.

In one embodiment said further polypeptide is not derived from STC2 (SEQ ID NO.:15). In one embodiment, the fusion protein is not according to SEQ ID NO.: 15.

In one embodiment, the fusion protein described herein reduces the proteolytic activity of PAPP-A towards IGFBP4.

Nucleic acid, Vector and Host cell

In one aspect, the invention provides an isolated polynucleotide encoding the polypeptide and/or the fusion protein presented herein in the section “Polypeptide”.

In one embodiment, the polypeptide is selected from the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 1 , SEQ ID NO.: 8, SEQ ID NO.: 9 and SEQ ID NO.: 10.

Also provided herein is a vector comprising the polynucleotide presented herein. The vector may be any type of vector. In one embodiment, the vector is an expression vector, such as an expression vector selected from the group consisting of bacterial expression vectors, mammalian expression vectors, and insect expression vectors. .

Further provided is a host cell comprising the polynucleotide and/or vector presented herein. The host cell may be any type of host cell capable of expressing and secreting the polypeptide encoded by the polynucleotide disclosed herein. Pharmaceutical composition

In one embodiment, the present invention provides a pharmaceutical composition comprising the polypeptide, the fusion protein, the polynucleotide, the vector and/or the host cell as described herein.

The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients and/or other additives.

The pharmaceutical composition may further contain one or more additional active ingredients suitable for the treatment of the indications disclosed herein.

Medical uses

The data presented herein indicates that the isolated polypeptide as described herein, as well as the fragments and variants thereof, can bind to PAPP-A and inhibit the proteolytic activity of PAPP-A. Thus, the isolated polypeptide, as well as fragments or variants thereof as described herein are believed to be effective in the modulation of IGF signalling and in the treatment or prevention of diseases and/or disorders involving dysregulated and/or increased IGF signalling, such as cancer, cardiovascular disease, such as atherosclerosis, kidney disease, such as diabetic nephropathy, and fibrosis.

Thus, provided herein is an isolated polypeptide, a fusion protein, a polynucleotide, a vector, a host cell and/or a pharmaceutical composition according to the present disclosure for use as a medicament.

In one embodiment, the isolated polypeptide, the fusion protein, the polynucleotide, the vector, the host cell and/or the pharmaceutical composition according to the present disclosure is for use in the modulation of insulin growth factor (IGF) signalling. In one embodiment, the IGF is insulin growth factor-1 (IGF-1) and/or insulin growth factor-2 (IGF-2). In one embodiment, said modulation is a reduction of IGF signalling.

In one embodiment, the isolated polypeptide, the fusion protein, the polynucleotide, the vector, the host cell and/or the pharmaceutical composition according to the present disclosure is for use in the treatment and/or prevention of a disease, disorder and/or condition selected from the group consisting of: cancer, cardiovascular disease, such as atherosclerosis, kidney disease, such as diabetic nephropathy, and fibrosis.

In one embodiment, the isolated polypeptide, the fusion protein, the polynucleotide, the vector, the host cell and/or the pharmaceutical composition according to the present disclosure is for use in the treatment and/or prevention of kidney disease. In one embodiment, the kidney disease is diabetic nephropathy. In one embodiment, the diabetic nephropathy is caused by hyperglycemia. In one embodiment, the hyperglycemia is caused by diabetes 1 or diabetes 2.

Also provided herein is the use of the isolated polypeptide, the fusion protein, the polynucleotide, the vector, the host cell and/or the pharmaceutical composition according to the present disclosure, in the manufacture of a medicament for the treatment and/or prevention of a disease, disorder and/or condition selected from the group consisting of: cancer, cardiovascular disease, such as atherosclerosis, kidney disease, such as diabetic nephropathy, and fibrosis.

Further provided herein is a method for the treatment of a disease, disorder and/or condition selected from the group consisting of: cancer, cardiovascular disease, such as atherosclerosis, kidney disease, such as diabetic nephropathy, and fibrosis; wherein said method comprises administering the isolated polypeptide, the fusion protein, the polynucleotide, the vector, the host cell and/or the pharmaceutical composition according to the present disclosure to an individual or a subject in need thereof.

The polypeptide, the fusion protein, the polynucleotide, the vector, the host, and/or the pharmaceutical composition is administered in a therapeutically effective amount.

In one embodiment, the individual or subject is a mammal, preferably a human being.

Method of identifying a ligand of STC2

The inventors have determined the first known structure of PAPP-A in complex with STC2. The results highlight the regions of PAPP-A and STC2 interacting with each other as well as their conformation. This structure and binding analysis is useful in future modelling and computational studies of the interaction of PAPP-A with other molecules and its role in signalling pathways.

Thus, the invention relates to a computer-readable data storage medium comprising a data storage material encoded with at least a portion of the structure coordinates set forth in figure 6. In one embodiment, the computer-readable data storage medium comprises a data storage material encoded with the structure coordinates set forth in figure 6.

In a further aspect, the invention relates to the use of atomic coordinates as presented in figure 6 or atomic coordinates selected from a three-dimensional structure that deviates from the three-dimensional structure as presented in figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A in a method for identifying a ligand capable of binding to one or more of: a) PAPP-A (SEQ ID NO.: 14) b) the C-terminal region of PAPP-A corresponding to residues 1555 to 1627 of SEQ ID NO.: 14 (SEQ ID NO.: 11) c) the LNR3 domain of PAPP-A corresponding to the residues 1555 to 1583 of SEQ ID NO.: 14 (SEQ ID NO.: 12) d) the region of PAPP-A according to amino acid residues 1556 to 1568 of SEQ ID NO.: 14 (SEQ ID NO.: 13) e) the region of PAPP-A according to amino acid residues 766 to 777 of SEQ ID NO.: 14 (SEQ ID NO.: 16) or a fragment or variant of a) through e).

The present invention provides methods for identification and design of ligands capable of binding PAPP-A, specifically to any of the regions corresponding to the C-terminal domain (SEQ ID NO.: 11), the LNR3 domain (SEQ ID NO.: 13), a region of PAPP-A according to amino acid residues 1556 to 1568 of SEQ ID NO.: 14 (SEQ ID NO.: 13), a region of PAPP-A according to amino acid residues 766 to 777 of SEQ ID NO.: 14 (SEQ ID NO.: 16) or a fragment or variant thereof.

In a preferred embodiment, the identified and/or designed ligands are inhibitors of the proteolytic activity of PAPP-A towards IGFBP4.

A potential inhibitor may then be designed de novo in conjunction with computer modelling. Models of chemical structures and/or peptides, or fragments thereof, may be generated on a computer screen using information derived from known low-molecular weight organic chemical structures and/or peptides stored in a computer data base or are built using the general knowledge of an organic chemist regarding bonding types, conformations etc. Suitable computer programs may aid in this process in order to build chemical structures of realistic geometries. Chemical structures or molecule fragments may be selected and/or used to construct a potential inhibitor such that favourable interactions to said subset or criteria data set become possible. The more favourable interactions become possible, the stronger the potential inhibitor will bind to PAPP-A. Preferably, favourable interactions to at least one amino acid residue should become possible. Such favourable interactions may occur with any atom of the amino acid residue e.g. atoms of the peptide back-bone or/and atoms of the side chains.

Favourable interactions are any non-covalent attractive forces which may exist between chemical structures such as hydrophobic or van-der-Waals interactions and polar interactions such as electrostatic interactions, hydrogen bonding, salt-bridges etc. Unfavourable interactions such as hydrophobic-hydrophilic interactions should be avoided but may be accepted if they are weaker than the sum of the attractive forces. Steric interference such as clashes or overlaps of portions of the inhibitor being selected or constructed with protein moieties will prevent binding unless resolvable by conformational changes. The binding strength of a potential inhibitor thus created may be assessed by comparing favourable and unfavourable interactions on the computer screen or by using computational methods implemented in commercial computer programs.

A inhibitor is at least partially complementary to at least a portion of PAPP-A in terms of shape; and/or in terms of hydrophilic or hydrophobic interactions and/or in terms of electrostatic interactions or hydrogen bonding.

Exemplary programs for computer modelling include Quanta (Molecular Simulations, Inc.) and Sibyl (Tripos Associates). Other useful programs are Autodock (Scripps Research Institute, La Jolla, described in Goodsell and Olsen (1990) Proteins: Structure, Function and Genetics, 8, 195-201), Dock (University of California, San Francisco, described in: Kuntz et al. (1982) J. Mol. Biol. 161 ,269-288.

In a further aspect, the invention relates to a method of identifying a ligand capable of binding to PAPP-A, or a fragment or variant thereof, said method comprising the steps of: a) generating the spatial structure of the binding site on a computer screen using atomic coordinates as presented in figure 6 or atomic coordinates selected from a three-dimensional structure that deviates from the three- dimensional structure presented in figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A, b) generating ligands with their spatial structure on the computer screen, and c) selecting ligands that can bind to at least 1 amino acid residue of the set of binding interaction sites without steric interference. In another aspect, the invention relates to a computer-assisted method for identifying a ligand capable of binding to PAPP-A, or a fragment or variant thereof, using a programmed computer comprising a processor, a data storage system, a data input device and a data output device, comprising the following steps: a) inputting into the programmed computer through said input device data comprising: atomic coordinates of a subset of the atoms of said PAPP-A, thereby generating a criteria data set; wherein said atomic coordinates are selected from the three-dimensional structure presented in figure 6 or atomic coordinates selected from a three-dimensional structure that deviates from the three-dimensional structure presented in figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A, b) comparing, using said processor, the criteria data set to a computer data base of low-molecular weight organic chemical structures and peptide fragments stored in the data storage system; and c) selecting from said data base, using computer methods, a chemical structure having a portion that is structurally complementary to the criteria data set and being free of steric interference with the PAPP-A.

In yet another aspect, the present invention relates to a method for identifying a ligand, said method comprising the steps of: a) selecting a ligand using atomic coordinates in conjunction with computer modelling, wherein said atomic coordinates are the atomic coordinates presented in figure 6 or wherein the atomic coordinates are selected from a three-dimensional structure that deviates from the three-dimensional structure presented in figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A, by docking ligands into a set of binding interaction sites in PAPP-A, or a fragment or variant thereof, said binding interaction generated by computer modelling and selecting a ligand capable of binding to at least one amino acid in said set of binding interaction sites of PAPP-A, b) providing said ligand and PAPP-A, c) contacting the ligand with said PAPP-A, and d) detecting the binding of the ligand with said PAPP-A. In one embodiment of the present invention the docking of ligand molecules is performed by employing a three-dimensional structure defined by atomic coordinates of the three dimensional structure presented in figure 6 and such that said ligand is capable of binding to at least three amino acids of PAPP-A, or a fragment or variant thereof.

In a further aspect, the invention relates to a method of identifying a ligand of PAPP-A, or a fragment or variant thereof, said method comprising the steps of: a) introducing into a computer, information derived from atomic coordinates defining a conformation of PAPP-A, or a fragment or variant thereof, based on three-dimensional structure determination, whereby a computer program utilizes or displays on the computer screen the structure of said conformation; wherein said atomic coordinates are selected from the three-dimensional structure as presented in figure 6 or atomic coordinates selected from a three-dimensional structure that deviates from the tree-dimensional structure represented by figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A; b) generating a three-dimensional representation of said PAPP-A, or said fragment or variant thereof by said computer on a computer screen; c) superimposing a model of a ligand on the representation of said three- dimensional representation of said PAPP-A, or said fragment or variant thereof, d) assessing the possibility of binding and/or the absence of steric interference of the ligand with said PAPP-A, or said fragment or variant thereof; e) incorporating said ligand compound in a binding assay and/or enzymatic activity assay of PAPP-A; f) determining whether said ligand binds to PAPP-A, and/or whether said ligand inhibits binding of a competing ligand towards PAPP-A, and/or whether said ligand inhibits the proteolytic activity of PAPP-A.

In a further embodiment of the present invention, the information derived from the atomic coordinates of one or more amino acid residues corresponding to the C-terminal region of PAPP-A corresponding to residues 1555 to 1627 of SEQ ID NO.: 14 (SEQ ID NO.: 11), such as more specifically to the LNR3 domain corresponding to residues 1555 to 1583 of SEQ ID NO.: 14 (SEQ ID NO.: 12), such as more specifically to the region corresponding to residues 1556 to 1568 of SEQ ID NO.: 14 (SEQ ID NO.:13). are used for ligand prediction and/or design.

In one embodiment, the information derived from the atomic coordinates of one or more amino acid residues corresponding to the region corresponding to residues 766 to 777 of SEQ ID NO.: 14 (SEQ ID NO.: 16) is used for ligand prediction and/or design.

In one embodiment, the atomic coordinates are determined to a resolution of at least 5 A, such as 4.5, 4, 3.5, or 3 A. In one embodiment, the atomic coordinates are determined to a resolution of at least 5 A. In one embodiment, the atomic coordinates are determined to a resolution of at least 4.5 A. In one embodiment, the atomic coordinates are determined to a resolution of at least 4 A. In one embodiment, the atomic coordinates are determined to a resolution of at least 3.4 A. In one embodiment, the atomic coordinates are determined to a resolution of at least 3 A.

In one embodiment, assessing or identifying the binding interaction and/or spatial fit of the ligand to PAPP-A includes scoring the energy of interaction between one or more atoms of the ligand with one or more atoms of PAPP-A.

In one embodiment, the ligand is selected from the group consisting of: peptides and peptides analogues, organic compounds and inorganic compounds.

In one embodiment, the peptide or peptide analogue is a non-hydrolysable peptide. Non-hydrolysable generally refers to peptides or peptide analogues that do not decompose by reacting with water under physiological conditions. For example, they do not decompose by reacting with water in the bloodstream or in the digestive tract compartments, such as the stomach. These peptides do not react with water to an extent that prevents their effect or function, for example binding or inhibition of other proteins.

In one embodiment, a library of small organic molecules are screened. In one embodiment, a library of peptide ligands are screened.

In one embodiment, the ligand is capable of inhibiting the proteolytic activity of PAPP-A. In one embodiment, the ligand is an inhibitor of the proteolytic activity of PAPP-A towards IGFBP4.

Sequence overview

Items

1. An isolated polypeptide having a length of less than 100 amino acids comprising or consisting of an amino acid sequence selected from the group consisting of: a) the amino acid sequence according to SEQ ID NO.: 2; b) a variant of SEQ ID NO.: 2, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 2, but less than 99% sequence identity to SEQ ID NO.: 2; c) a variant of SEQ ID NO.: 2, wherein said variant has between 1 and 5 amino acid substitutions relative to SEQ ID NO.: 2, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 2; d) a fragment of SEQ ID NO.: 2 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 2; e) the amino acid sequence according to SEQ ID NO.: 3; f) a variant of SEQ ID NO.: 3, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 3, but less than 99% sequence identity to SEQ ID NO.: 3; g) a variant of SEQ ID NO.: 3, wherein said variant has between 1 and 3 amino acid substitutions relative to SEQ ID NO.: 3, such as 1, 2, or 3 amino acid substitutions relative to SEQ ID NO.: 3; h) a fragment of SEQ ID NO.: 3 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 3; i) the amino acid sequence according to SEQ ID NO.: 4; j) a variant of SEQ ID NO.: 4, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 4, but less than 99% sequence identity to SEQ ID NO.: 4; k) a variant of SEQ ID NO.: 4, wherein said variant has between 1 and 10 amino acid substitutions relative to SEQ ID NO.: 4, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 4; l) a fragment of SEQ ID NO.: 4 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 4; m) the amino acid sequence according to SEQ ID NO.: 5; n) a variant of SEQ ID NO.: 5, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 5, but less than 99% sequence identity to SEQ ID NO.: 5; o) a variant of SEQ ID NO.: 5, wherein said variant has between 1 and 12 amino acid substitutions relative to SEQ ID NO.: 5, such as 1 , 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 5; p) a fragment of SEQ ID NO.: 5 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 5; q) the amino acid sequence according to SEQ ID NO.: 6; r) a variant of SEQ ID NO.: 6, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 6, but less than 99% sequence identity to SEQ ID NO.: 6; s) a variant of SEQ ID NO.: 6, wherein said variant has between 1 and 9 amino acid substitutions relative to SEQ ID NO.: 6, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 6; t) a fragment of SEQ ID NO.: 6 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 6; u) the amino acid sequence according to SEQ ID NO.: 7; v) a variant of SEQ ID NO.: 7, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 7, but less than 99% sequence identity to SEQ ID NO.: 7; w) a variant of SEQ ID NO.: 7, wherein said variant has between 1 and 13 amino acid substitutions relative to SEQ ID NO.: 7, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 7; x) a fragment of SEQ ID NO.: 7 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 7; y) the amino acid sequence according to SEQ ID NO.: 1; z) a variant of SEQ ID NO.: 1 , wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 1 , but less than 99% sequence identity to SEQ ID NO.: 1 ; aa) a variant of SEQ ID NO.: 1 , wherein said variant has between 1 and 16 amino acid substitutions relative to SEQ ID NO.: 1, such as 1, 2, 3, 4 or 5 amino acid substitutions relative to SEQ ID NO.: 1 ; bb) a fragment of SEQ ID NO.: 1 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 1 ; cc) the amino acid sequence according to SEQ ID NO.: 8; dd) a variant of SEQ ID NO.: 8, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 8, but less than 99% sequence identity to SEQ ID NO.: 8; ee) a variant of SEQ ID NO.: 8, wherein said variant has between 1 and 5 amino acid substitutions relative to SEQ ID NO.: 8, such as 1, 2, or 3 amino acid substitutions relative to SEQ ID NO.: 8; ff) a fragment of SEQ ID NO.: 8 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 8; gg) the amino acid sequence according to SEQ ID NO.: 9; hh) a variant of SEQ ID NO.: 9, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 9, but less than 99% sequence identity to SEQ ID NO.: 9; ii) a variant of SEQ ID NO.: 9, wherein said variant has between 1 and 3 amino acid substitutions relative to SEQ ID NO.: 9, such as 1, 2, or 3 amino acid substitutions relative to SEQ ID NO.: 9; jj) a fragment of SEQ ID NO.: 9 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 9; kk) the amino acid sequence according to SEQ ID NO.: 10;

II) a variant of SEQ ID NO.: 10, wherein said variant has at least 80%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO.: 10, but less than 99% sequence identity to SEQ ID NO.: 10; mm) a variant of SEQ ID NO.: 10, wherein said variant has between 1 and 3 amino acid substitutions relative to SEQ ID NO.: 10, such as 1, 2, or 3 amino acid substitutions relative to SEQ ID NO.: 10; nn) a fragment of SEQ ID NO.: 10 having a length of at least 5 amino acids, or a variant of said fragment having 1 or 2 amino acid substitutions relative to SEQ ID NO.: 10; wherein said isolated polypeptide is capable of reducing Pregnancy-associated plasma protein A (PAPP-A) proteolytic activity, such as PAPP-A proteolytic activity towards Insulin Like Growth Factor Binding Protein 4 (IGFBP4). The isolated polypeptide according to item 1 , wherein the polypeptide comprises the amino acid sequence according to SEQ ID NO.: 2 and the amino acid sequence according to SEQ ID NO.: 3. The isolated polypeptide according to according to any one of the preceding items item 1 , wherein the length of the peptide is less than 100 amino acids, such as less length of less than 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 or 10 amino acids. The isolated polypeptide according to any one of the preceding items, wherein one or more of the amino acid residues corresponding to positions 61 to 64, 66, 86, 89, 90, 92, 93, 97, 100, 101 , 103, 104, 107 and 108 of SEQ ID NO.: 15 are not substituted or modified. The isolated polypeptide according to any of the preceding items, wherein the amino acid substitutions are conservative substitutions. The isolated polypeptide according to any one of the preceding items, wherein said isolated polypeptide is capable of binding to PAPP-A, such as to the region corresponding to residues 766 to 777 of SEQ ID NO.: 14 (SEQ ID NO.: 16), such as the C-terminal region of PAPP-A corresponding to residues 1555 to 1627 of SEQ ID NO.: 14 (SEQ ID NO.: 11), such as more specifically to the LNR3 domain corresponding to residues 1555 to 1583 of SEQ ID NO.: 14 (SEQ ID NO.: 12), such as more specifically to the region corresponding to residues 1556 to 1568 of SEQ ID NO.: 14 (SEQ ID NO.:13). A fusion protein comprising the polypeptide according to any one of the preceding items, and a further polypeptide, wherein said further polypeptide is not derived from STC2.

8. An isolated polynucleotide encoding the polypeptide according to any one of items 1 to 6, or the fusion protein according to item 7.

9. A vector comprising the polynucleotide according to item 8.

10. A host cell comprising the polynucleotide according to item 8, and/or the vector according to item 9.

11. A pharmaceutical composition comprising the polypeptide according to any one of items 1 to 6, the fusion protein according to item 7, the polynucleotide according to item 8, the vector according to item 9, and/or the host cell according to item 10 and one or more pharmaceutically acceptable excipients.

12. A polypeptide according to any one of items 1 to 6, the fusion protein according to item 7, the polynucleotide according to item 8, the vector according to item 9, the host cell according to item 10 and/or the pharmaceutical composition according to item 11 for use as a medicament.

13. A polypeptide according to any one of items 1 to 6, the fusion protein according to item 7, the polynucleotide according to item 8, the vector according to item 9, the host cell according to item 10 and/or the pharmaceutical composition according to item 11 for use in the treatment and/or prevention of a disease, disorder and/or condition selected from the group consisting of: cancer, cardiovascular disease, such as atherosclerosis, kidney disease, such as diabetic nephropathy, and fibrosis.

14. A computer-readable data storage medium comprising a data storage material encoded with at least a portion of the structure coordinates set forth in figure 6.

15. A method of identifying a ligand of PAPP-A, or a fragment or variant thereof, said method comprising the steps of: a) introducing into a computer, information derived from atomic coordinates defining a conformation of PAPP-A, or a fragment or variant thereof, based on three-dimensional structure determination, whereby a computer program utilizes or displays on the computer screen the structure of said conformation; wherein said atomic coordinates are selected from the three-dimensional structure as presented in figure 6 or atomic coordinates selected from a three-dimensional structure that deviates from the tree-dimensional structure represented by figure 6 by a root mean square deviation over protein backbone atoms of not more than 3 A; b) generating a three-dimensional representation of said PAPP-A, or said fragment or variant thereof by said computer on a computer screen; c) superimposing a model of a ligand on the representation of said three- dimensional representation of said PAPP-A, or said fragment or variant thereof, d) assessing the possibility of binding and/or the absence of steric interference of the ligand with said PAPP-A, or said fragment or variant thereof; e) incorporating said ligand compound in a binding assay and/or enzymatic activity assay of PAPP-A; f) determining whether said ligand binds to PAPP-A, and/or whether said ligand inhibits binding of a competing ligand towards PAPP-A, and/or whether said ligand inhibits the proteolytic activity of PAPP-A.

Examples

General protocol for preparation of recombinant proteins:

Recombinant proteins were expressed in human embryonic kidney 293T cells (293tsA1609neo, ATCC) cultured in DM EM (Thermo Fisher) supplemented with 10% fetal bovine serum (Sigma-Aldrich) or serum free CD 293 Medium (Thermo Fisher). Cells stably expressing active site-inactivated human PAPP-A (293T_PA_E563Q_Hygro) were previously reported ^{1 2} . Cells stably expressing human STC2 (293T_STC2_Hygro) were generated by transfection of 293T cells with linearized pSTC2_Hygro, obtained by cloning the Xhol/Hindl 11 fragment of pSTC220 into pcDNA3.1/Hygro(+) (Invitrogen). The transfected cells were cultured with hygromycin B (Invitrogen) and stably expressing clones were selected. To generate the PAPP-A STC2 complex, 293T_PA_E563Q_Hygro cells and 293T_STC2_Hygro cells were mixed in a 1 :1 ratio and co-cultured in serum free medium. All other proteins were produced by transient transfection of 293T cells using calcium phosphate co-preci pitation and plasmids encoding wild-type human PAPP-A (pcDNA3.1-PAPP-A21 , a C-terminal fragment of PAPP-A truncated at SCR1 (PA_T1213-G162717), a PAPP-A variant with C732 replaced by alanine (PA_C732A54), a PAPP-A variant with D1564 replaced by alanine (PA_D1564A18), C-terminally truncated variants of PAPP-A (PA_E82-S1030, PA_E82- A1209, PA_E82-G155718), deletion variants of PAPP-A (PA_d[C414-C473] and PA_d[C1558-C1583]18), an STC2 variant with C120 replaced by alanine (pSTC2(C120A)20), or human IGFBP-4 (pBP4mH12). Culture supernatants were harvested 48 h post-transfection and cleared by centrifugation, or the cells were further cultured in serum-free medium to facilitate purification.

PAPP-A(T1213-G1627) and dimeric PAPP-A was purified by nickel-affinity and heparin- affinity chromatography ³ , and IGFBP-4 was purified by nickel-affinity, size exclusion and reversed-phase chromatography ⁴ , as previously described. The PAPP-A- STC2 complex for cryo-EM analysis was purified by nickel-affinity chromatography using a 1 mL HisTrap HP column (Sigma-Aldrich). Serum free culture supernatant was diluted 1 :1 with 50 mM NaH2PC>4, 150 mM NaCI, pH 7.4 (buffer A) and loaded onto the column, which was then washed with 50 mM NaH2PC>4, 1 M NaCI, 20 mM imidazole, 0.05% Tween 20, pH 7.4, equilibrated with buffer A and eluted in 1 mL fractions with 50 mM NaH2PC>4, 500 mM imidazole, pH 7.4. Following dialysis into 20 mM HEPES, 100 mM NaCI, 1 mM CaCI2, pH 7.4 (buffer B), the eluate was passed through a 1 mL HiTrap NHS-activated HP column (Sigma-Aldrich) coupled with mAb PA141 ⁵ , which specifically recognizes dimeric PAPP-A. The unbound PAPP-A STC2 was finally concentrated by ultrafiltration using Amicon Ultracel 100K (Millipore) and further purified by size exclusion chromatography on a Superose 6 Increase 10/300 GL (Sigma-Aldrich) equilibrated with buffer B. Throughout, fractions were analyzed by 3-8% non-reducing or reducing SDS-PAGE (NuPAGE, Thermo Fisher). To confirm that protein subunits were not truncated during processing, peptide coverage was assessed by mass spectrometry following tryptic digestion on a TripleTOF 6600 system (AB Sciex). The purified PAPP-A STC2 complex was stored at 4°C.

The sequence of constructs encoding wild-type PAPP-A or STC2 were in agreement with UniProt entries Q13219 and 076061 , respectively. The PAPP-A sequence of some earlier databases and publications erroneously has a valine inserted between R107 and L108.

Cryo-electron microscopy (cryo-EM)

Samples of PAPP-A STC2 were diluted to a concentration of 0.6 mg/mL. C-flat holey carbon grids, CF-2.2-4C (Protochips), were glow-discharged on a Quorum GloQube Plus glow discharge system at 15 mA for 45 s. 3 pL of protein sample were added to the grids and vitrified at 4 °C and 100% humidity with a blotting time of 4 s on an EM GP2 automatic plunge freezer (Leica). Movies were collected on a Titan Krios G3i (EMBION, Danish National Cryo-EM Facility, Aarhus node) with X-FEG operated at 300 kV and equipped with a Gatan K3 camera and a Bioquantum energy filter using a 20 eV slit width. Movies were collected using aberration-free image shift (AFIS) data collection with the EPU data acquisition software (Thermo Fisher Scientific) at a pixel size of 0.507 A/pixel (corresponding to a magnification of x 165,000). Two datasets were collected. Dataset I contained 10,073 movies with 59 dose fractions over a 0.8 s exposure and a total dose of ~58 e- per ki. Defocus range was 0.6-1.6 over six exposures in each hole. Dataset II contained 32,115 movies with 67 dose fractions over a 0.91 s exposure and a total dose of ~59 e- per A2. Defocus range was 0.6-1.8 over seven exposures in each hole). Data processing:

Data processing was carried out by using cryoSPARC v3 ⁶ based on datasets I and II. Three maps were generated. MAPI : Patch-motion-correction and Patch CTF determination was performed. Micrographs were manually inspected before initial manual particle picking. Particles were used to create 2D classes for template picking on all movies, with a dynamic mask of 180-220 A, followed by inspection of the picked particles. Particles were extracted in a 440-pixel box and Fourier cropped to a 128-pixel box (1.74 A/pixel). Extracted particles (6,677,370) were used for iterative 2D classification, resulting in a set of particles used for ab initio 3D construction. Several rounds of heterogeneous refinement was performed to sort out junk particles and keeping one good class. Selected particles (278,982) were re-extracted in a 220-pixel box (1.01 A/pixel) and subjected to non-uniform refinement followed by local refinement. MAP2: Initially, particles were picked using templates made from dataset I. After several rounds of 2D classification, six classes comprising approximately 3,000 particles were used for Topaz training ⁷ . Topaz picking resulted in 601 ,179 particles. After particle cleanup with 2D classification, five initial models were built and heterogeneously refined. One model was refined with imposed C2 symmetry. In order to enhance the resolution, two more rounds of heterogenous, homogenous and non-uniform refinement was performed (the final number of particles was 114,226). MAP3: Particles from the MAP2 was subjected to C2 symmetry expansion that was used with MAP2 volume for 3D variability analysis in C1 symmetry. Four conformations identified were used as seeds in heterogenous refinement. Refinement in C1 was performed on all four conformations, yielding volumes with clearly distinct conformations, and the best one was picked as MAP3 (the final number of particles was 31 ,119). A composite map (MAPI -3) was generated by using PHENIX ¹⁰ .

Example 1: Model construction

Aim

To prepare a structural model of the PAPP-A and STC2 complex.

Materials and Methods

Initial PAPP-A (UniProt Q13219) and STC2 (UniProt 076061) models were calculated separately using AlphaFold2 (AF2) ⁸ . A partial dimer model was created from the LG, CD, M1-4, SCR2-SCR3, and STC2 domains from the AF2 models, comprising domains that appear to move as a rigid body. These models were placed in the 3.05 A map (MAPI) and subjected to multiple cycles of manual adjustments and rebuilding in Coot ⁹ and real space refinement in PHENIX ¹⁷ . The model (PDB ID 8A7D) contains chain C (PAPP-A S94-Y1014) from one PAPP-A subunit, chain Q (PAPP-A P1282-P1412 + PAPP-A G1476-S1617 from the other PAPP-A subunit, and chain P corresponding to one STC2 subunit (STC2 R44-F210). Thus, PAPP-A regions 1015-1281 (M5, M6, SCR1) and 1413- 1475 (SCR4), and STC2 regions 22-43 and 210-302 were omitted due to poor map coverage and pronounced flexibility in the SCR region. Subsequently, a full PAPP- A STC2 heterotetrametic model (PDB ID 8A7E) was constructed by using the regions mentioned above for the partial PAPP-A dimer joined with the remaining regions (1018- 1283 and 1413-1475) extracted from the AF2 model. These were placed and refined (real space rigid body refinement and minimization) within a 2-fold symmetric composite map consisting of a 4.0 A resolution overall map and a focused map of 5.0 A resolution covering residues 1018-1283 (the PAPP-A dimerization interface) from both monomers. In order to construct a model of the PAPP-A dimer interface region, formed by M5 and M6, AF2 Multimer was used to calculate a dimeric M5-M6 structure, that subsequently docked well into the derived 5.0 A PAPP-A STC2 dimer density (MAP3). The M5 domains were refined as rigid bodies in PHENIX and the M6 dimer was further refined using Namdinator ¹¹ , as especially the positions of the two 2-strand p-sheets needed adjustment. SCR1-3 were docked into the dimer density using ChimeraX 1.3 ¹² and subsequently subjected to rigid body refinement in PHENIX. SCR5 and the C-terminal region were fitted to the density as a rigid body followed by manual rebuilding and PHENIX refinement. An AF2 Multimer model of the STC2 dimer could readily be docked into the density and subsequently be manually rebuilt and refined in PHENIX. Structural figures were prepared by using ChimeraX 1.3 or PyMOL (https://www.pymol.org).

Results

The model of the protein complex shows for the first time the three-dimensional structure of PAPP-A in complex with STC2.

The STC2 dimer is suspended in the core of the PAPP-A STC2 complex. In addition to the disulfide bond formed with the M2 domain, the STC2 dimer interacts noncovalently with the C domain, including electrostatic interactions between the C domain and four basic residues of STC2, and via van der Waal interactions between STC2 V63 and a hydrophobic pocket formed by PAPP-A residues Y1566, T1594, and K1592 (Fig. 4B). In particular, K104 is positioned favourably for interaction with the negative charge surrounding the Ca2+ ion of LNR3. Within the PAPP-A STC2 complex, the C domains and the LNR3 modules of each PAPP-A subunit do not interact with the STC2 subunit to which the C120-C732 disulfide is formed, but rather the opposite subunit of the STC2 dimer. This reflects the crossing of the PAPP-A polypeptides at the PAPP-A dimerization disulfide between M6 and SCR1 (Fig. 4A).

LNR3 follows immediately after SCR5 and continues its downward direction to a position at level with LNR1-2 of the CD. The three approximately 30-residue LNR modules are similar to one another in structure with limited secondary structure in the form of short helical turns along a spiral-shaped path of the backbone, stabilized by disulfide bonds (Fig 3C). Map density corresponding to a Ca ²⁺ ion in the space surrounded by the side chains of these residues, showing that a Ca ²⁺ ion is also present in the PAPP-A LNR modules (Fig 3D). The very C-terminal residues following LNR3 (C1584-G1627) continue one turn of the LNR3 spiral and then points further downward with no map density for the last C-terminal 10 residues. Except for a small two-strand sheet structure formed with LNR3, the C domain contains almost no secondary structure (Fig. 3C).

The interacting regions and amino acids between the two proteins have been identified. These are highlighted in Figure 4, and include STC2 (SEQ ID NO.: 15) amino acid residues 45 to 58 (corresponding to SEQ ID NO.: 9) and 118 to 124 (corresponding to SEQ ID NO.: 10), which interact with PAPP-A (SEQ ID NO.: 14) residues 766 to 777 (corresponding to SEQ ID NO.: 16) and STC2 (SEQ ID NO.: 15) amino acid residues 60 to 66 (corresponding to SED ID NO.: 3) and 86 and 107 (corresponding to SEQ ID NO.: 2), which interact with PAPP-A residues at the C-terminal domain, such as the residues at the LNR3 domain, such as the residues 1556 to 1568 of PAPP-A.

Conclusion

The three-dimensional structure of the PAPP-A and STC2 complex has been determined, as well as the relevant domains and amino acid residues involved in the interaction.

Example 2: Surface plasmon resonance (SPR).

Aim

To determine the binding affinity between PAPP-A or PAPP-A variants and STC2 or STC2 variants under different conditions.

Materials and Methods SPR experiments were carried out using a Biacore 3000 instrument (Cytiva) running at 25°C. The running buffer was 10 mM HEPES pH 7.5, 150 mM NaCI, 2 mM CaCh, and 0.05% Tween-20. In some experiments, the bufferwas supplemented with 10 mM EDTA. Data were collected at a rate of 1 Hz, and analyzed using the BIAevaluation 4.1.1 software (Cytiva) and GraphPad Prism 9. Double referencing was applied, i.e. the signal from the in-line reference flow cell was subtracted, as was the signal from a blank run (0 nM analyte). For the analysis of interactions between monoclonal antibodies and PAPP- A(E563Q) or PAPP-A:STC-2, an anti-PAPP-A chip was prepared by direct immobilization of antibody 234-5 ²⁰ to a level of 10,000 Response Units (RU) in flow cells 1 and 2 of a CM5 chip (Cytiva), which had been activated with a 1 :1 mixture of 0.5 M 1- Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.1 M N-Hydroxysuccinimide (NHS). Remaining active groups were blocked by a 7 min injection of 1 M ethanolamine pH 8.5. For binding analysis, PAPP-A(E563Q) or PAPP-A:STC-2 were captured at a level of approximately 600 RU in flow cell 2 only, using flow cell 1 as the in-line reference. Purified monoclonal PAPP-A antibodies (mAb PAIA or mAb PA141 ⁵ ) were injected at a concentration of 100 nM for 2 min, at a 30 pl/min flow rate, followed by a 180 s dissociation phase. At the end of each cycle, the surfaces of both flow cells were regenerated by a 90 s injection of 10 mM glycine pH 2.2.

Interactions with PA_T1213-G1627 were analyzed in a similar manner. Briefly, PAPP-A mAb (PA6 ¹³ ) was immobilized on a CM5 chip to a final response of 12,000 RU (active and reference cell), using standard amine chemistry as described above. PA_T1213- G1627 was then captured in one flow cell only (active flow cell), using an adjacent flow cell for in-line reference. Next, STC2_C120A was injected in both flow cells for 2 minutes at 10 pl/min. Regeneration between successive rounds was performed by a 2 min injection of 10 mM glycine pH 2.5. For the analysis of IGFBP-4 binding, PA_T1213- G1627 was immobilized on a CM5 chip (active cell only, using a blank cell as reference) to a level of 7,000 RU (standard amine chemistry as above). IGFBP-4 was then injected in both reference and active flow cells for 5 min, at a 10 pl/min flow rate. Surfaces were regenerated by a 60 s injection of 10 mM glycine pH 1.7. All sensorgrams shown are representative of at least three independent experiments.

Results

Results show that STC2 is able to bind an isolated monomeric C-terminal fragment of PAPP-A containing the C domain, and that this interaction can be abrogated by the PA141 antibody (Fig. 1A), thus suggesting that the inhibitory mechanism of mAb PA141 is based on mimicking of the endogenous inhibitor, STC2. Removal of bound Ca ²⁺ from LNR3 of the C-terminal fragment did not completely abolish STC2 binding (Fig. 1 B), indicating that residues C-terminally to LNR3 are likely to be involved in the binding. Finally, we similarly find that the substrate, IGFBP-4, is capable of binding to the isolated C-terminal fragment, and that this interaction is diminished by chelation of Ca ²⁺ (Fig. 1 C), demonstrating that LNR3 is involved in the binding of both inhibitor and substrate.

Conclusion

LNR3 domain of PAPP-A is involved in the binding of STC2 and IGFBP4.

Example 3: Proteolytic activity assays

Aim

To assess the ability of PAPP-A or mutated variants to form complexes with wild-type STC2.

Materials and methods

Conditioned media containing PAPP-A or mutated variants were mixed (1 :1) with wild-type STC2 and incubated for 16h at 37°C as described2o. The mixtures were then separated by 3-8% non-reducing SDS-PAGE and analyzed by Western blotting using polyclonal rabbit anti(PAPP-A) ¹⁴ . Blots shown are representative of at least three independent experiments.

Results

C-terminally truncated variants of PAPP-A, PAPP-A lacking LNR3, or PAPP-A in which a Ca ²⁺ -coordinating residue of LNR3 is mutated to alanine, cannot form a covalent complex with STC2. In contrast, in the absence of LNR1-2, STC2 is still able to form the PAPP-A STC2 complex with PAPP-A (Fig. 2A).

Conclusion

PAPP-A C-terminal domains are critically involved in the formation of the PAPP-A:STC2 complex. Example 4: Proteolytic activity assays

Aim

To determine the proteolytic activity of an STC2 variant (STC2(C120A)) not able to form covalent complex with PAPP-A.

Materials and methods

Determination of inhibitory potency of STC2(C120A) towards PAPP-A cleavage of intact IGFBP-4 was carried essentially as described ¹⁵ ’ ¹⁶ for STC1 using radiolabeled substrate ( ¹²⁵ l-IGFBP-4, 10nM) under the assumption of competitive inhibition. The concentration of PAPP-A and IGF2 were 50 pM and 100 nM, respectively.

Results

As shown in Fig. 2B, STC2(C120A) variant, which cannot irreversibly bind to PAPP-A, is still a relatively potent competitive inhibitor of the proteolytic activity against IGFBP4.

Conclusion

STC2 variant STC2(C120A) inhibits PAPP-A proteolytic activity against IGFBP4.

Example 5: Inhibition of PAPP-A activity with STC-2 fragments Aim

To assess the inhibitory effect on the proteolytic activity of PAPP-A of the peptides according to the present disclosure.

Materials and Methods

The following conditions for the cleavage reaction were used to asses inhibitory activity of a peptide STC2(86-108): MTFLHNAGKFDAQGKSFIKDALK (SEQ ID NO.: 17) on the proteolytic activity of PAPP-A towards IGFBP-4:

IGF-I (375 nM) and IGFBP-4 (300 nM) were pre-incubated for 30 min at 37 °C. STC2 peptide was added (200 nM), and immediately after PAPP-A (1nM) was added. The reaction was carried out in 50 mM Tris-HCI, 100 mM NaCI, 1 mM CaCI2, pH 7.5 and incubated at 37°C. Samples were taken out for analysis 0, 15 and 30 minutes, and the reaction was terminated by the addition of 0.1 M EDTA. As a control, a solution without the presence of an inhibitor was used. The degree of cleavage of IGFBP-4 was determined by using a standard ELISA setup with an antibody specific for an epitope, which is created as a result of PAPP-A cleavage. Briefly, for measurement N-terminal IGFBP-4 cleavage fragment concentration 96-well plates (Maxisorp, Nunc) were coated with 4 ug/mL of catching antibody (mAb IBP3cc) diluted in 100 pL 137 mM NaCI, 2.7 mM KCI, 10 mM Na2HPO4, KH2PO4, pH 7.4 (PBS) pr. well by incubation overnight at room temperature (RT). After a wash in Mil liQ water, the wells were blocked with 250 pL 2% bovine serum albumin (BSA) in 30 mM Tris-HCI, 300 mM NaCI, 2 mM CaCI2, pH 7.4 (TBS) for 1 hour at RT. Following a wash in TBS-T (TBS supplemented with 0.05% Tween-20), samples were diluted 5 times in TBS-T with 1 % BSA an loaded in 100 pL pr. well before incubating for 1 hour at RT with light shaking. Biotinylated mouse monoclonal anti-IGFBP-4 N-terminal cleavage epitope (Hytest, mAb IBP180) was used as detecting antibody. IBP180 was dialyzed against PBS, pH 7.5 and 2.8 pM antibody was incubated at room temperature for 30 min. with 100-fold molar excess biotin from the EZ-Link™ Micro Sulfo-NHS-LC-Biotinylation Kit (Thermo Scientific). After a wash in TST-T, the wells were incubated with 100 pL biotinylated detecting antibody diluted in TBS-T with 1% BSA to 2 ug/mL for 1 hour at RT under shaking. Another TBS-T wash and wells were incubated for 1 hour at RT with avidine peroxidase (Sigma) diluted 1 :4000 in TBS-T with 1 % BSA under dark conditions and shaking. After a final TBS-T wash, the plate was developed by adding 100 pL 3, 3', 5,5'- Tetramethylbenzidin (TMB) (Sigma Aldrich) to each well and subsequently quenching the color development with H2SO4. Absorbance was measured at 490 nm on an EnSpire Multimode Plate Reader (Perkin Elmer). Standard row points were based on fully cleaved purified IGFBP-4 diluted in TBS-T with 1% BSA. Eight concentrations in the range of 2000-15.625 ng/mL N-terminal cleavage fragment were used. Blank values were subtracted from all measured values and data was analyzed using linear curve fitting.

Cleavage was expressed as a percentage relative to 100% cleaved IGFBP-4 (obtained after 4h incubation in the absence of the STC2 peptide).

Results

Figure 4 shows that the STC2 peptide fragment 86-108 (SEQ ID NO.: 17) inhibits the proteolytic activity of PAPP-A on IGFBP-4. References

1. Boldt HB, Overgaard MT, Laursen LS, Weyer K, Sottrup-Jensen L, Oxvig C. Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin. Biochem J 358, 359-367 (2001).

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