Inhibitory peptides for cathepsin d and LRP-1 interaction

Abstract

The invention relates to synthetic peptides of no more than 20 amino acids in size useful in the treatment of cancer.

Description

[0001] INHIBITORY PEPTIDES FOR CATHEPSIN D AND LRP-1 INTERACTION

[0002] Background

[0003] In cancers, the tumor microenvironment plays a crucial role in tumorigenesis. Tumor microenvironment is composed of i) an extracellular matrix (ECM), a complex and dynamic structure containing fibrous proteins, glycosaminoglycans, glycoproteins or proteoglycans, and ii) of stromal cells such as fibroblasts, immune cells, endothelial cells or adipocytes (Place et al., 2011 ; Bussard, et al., 2016). Tumor and stromal cells exchange enzymes, growth factors and cytokines that modify the extracellular matrix, stimulate their migration and invasion and promote their proliferation and survival (Place et al., 2011 ). Among these molecules, cathepsin D is a protein overexpressed and secreted by tumor cells in various cancers, such as breast, lung, or ovary cancers (Pranjol, etal., 2020 ; Leto, etal., 2004), and able to stimulate angiogenesis, metastasis formation and fibroblasts growth (Liaudet-Coopman et al., 2006 ; Glondu et al., 2002 ; Berchem et al., 2002 ; Laurent-Matha et al., 2005). These pro-tumoral effects designate the cathepsin D as a recognized marker of poor prognosis, particularly in breast cancer, and as a target of therapeutic interest (Glondu etal., 2002 ; Dubey etal., 2017 ; Ashraf et al., 2019 ; Vetvicka etal., 2012 ; Brouillet etal, 1997).

[0004] Cathepsin D is a soluble aspartic lysosomal endopeptidase of the cathepsin family. In physiological conditions, it cleaves proteins and peptides in the lysosomal compartment (Masson et al., 2010). Cathepsin D is encoded by the CTSD gene as pre-pro-protein. This precursor contains a signal sequence that is cleaved to obtain a 52 kDa precursor, procathepsin D. This 52 kDa form is then transported to the endosomal compartment to be converted to an active 48 kDa intermediate. In the lysosomes, cysteine endopeptidases will cleave the 48 kDa intermediate chain into two chains: a light chain of 14 kDa and a heavy chain of 34 kDa. These two chains will then associate non-covalently to form the mature protease (Masson et al., 2010). In cancer cells, overexpression of the CTSD gene leads to a poor addressing of pro-cathepsin D (52 kDa) to the endosomal compartment and consequently the release of this immature form into the extracellular compartment (Pranjol, et al., 2020 ; Leto, et al., 2004 ; Heylen et al. 2002). The pro-cathepsin D thus secreted into the tumor microenvironment exhibits tumor-promoting activity. Extracellular CathD displays pro-tumor activities through proteolysis at acidic pH, but also non-proteolytic mechanisms. Secreted CathD can, for example, modify the local extracellular matrix by cleaving chemokines, growth factors, various extracellular matrix components (collagens, fibronectin, proteoglycans, ...) or by activating the cathepsin B and L precursors (Hasan et al., 2006, Benes et al., 2008, Alcaraz etal., 2021). It can also promote cancer cells proliferation, induce endothelial cells proliferation and migration and stimulate fibroblasts outgrowth (Vetvicka et al., 1997, Pranjol et al., 2017, Beaujouin etal., 2010). This last effect is mediated by its interaction with the p chain (residues 307-479) of the cell surface receptor LRP-1 (Low density lipoprotein Receptor related Protein- 1 ) (Beaujouin et al., 2010 ; Derocq etal., 2012).

[0005] LRP-1 is a multifunctional membrane receptor belonging to the LDL receptor family with combined endocytosis and cell signaling properties (Etique et al., 2013). The mature LRP-1 receptor is composed of two chains. The extracellular a-chain (515 kDa) can interact with more than 40 distinct extracellular ligands (lipoproteins, proteases, growth factors, toxins, viruses) in order to internalize them and for the most part to direct them to lysosomal catabolism. The P-chain (85 kDa) has an extracellular region of 476 amino acids, a transmembrane region of 25 amino acids and an intracytoplasm ic tail of 100 amino acids. By allowing endocytic clearance of numerous ligands (including proteases and / or their inhibitors), LRP-1 plays a major role in controlling proteolytic activity within the microenvironment (Etique et al., 2013). In addition to its endocytosis function, LRP-1 is also able to modulate some signaling pathways through the intracellular [3-chain domain, mainly via two NPTXY motifS29 and NPTXYes which allow interactions with intracellular scaffolding proteins (Herz et al., 2001 ). Over the last decade, our works, confirmed by other international studies, have demonstrated the pro-tumor activity of LRP-1 in various cancers (Theret et al., 2017 ; Perrot et al., 2012 ; Le et al., 2020; Langlois et al., 2010 ; Appert-Collin et al., 2017). Finally, LRP-1 can be involved in the control of gene expression through a double cleavage process of its [3-chain (Regulated Intramembrane Proteolysis, RIP) (May, et al., 2002). The first proteolysis is performed by metalloproteinases and membrane-associated proteins called sheddases. This first cleavage allows the release of the extracellular part of LRP-1. A membrane-associated fragment, LRP1 p-CTF, then remains and is cleaved by y-secretases at its transmembrane domain (Hass et al., 2009). The intracellular domain, LRP1 [3-ICD, is then released into the cytosol where it can interact with signaling proteins and translocate into the nucleus to play its role as a transcriptional regulator. This mechanism is the one involved in the effect of cathepsin D on the proliferation of fibroblasts. Indeed, cathepsin D prevents the cleavage and release of LRP1 [3-CTF by membrane-associated proteases and consequently blocks the release by a y- secretase of LRP1 |3-ICD that will control fibroblasts growth (Derocq et al., 2012). Thus, cathepsin D, via its interaction with the LRP-1 receptor present at the cell surface of fibroblasts, promotes their proliferation. Fibroblasts are associated with cancer at all stages of disease progression, including metastasis, and are a key component in tumor development (Kalluri et al., 2016 ; Houthuijzen et al., 2018 ; Bhowmick et al., 2004).

[0006] So far, only an interaction region of 45 AA (residues 349 to 394 of LRP-113) has been identified (Beaujouin et al., 2010). However, this 45 AA region, described as “Fragment F4” in W02009 / 043858, has no potential therapeutic application since it is poorly soluble in aqueous solvent, instable when observed in NMR, and difficult to produce and purify due to the presence of numerous cysteines forming multiple disulfide bridges.

[0007] Building on this finding, the Applicants designed and studied a series of novel peptides showing solubility and ability to bind cathepsin D protein.

[0008] Summary

[0009] In a first aspect, the invention relates to a peptide of no more than 20 amino acids in size comprising the following amino acid sequence (I):

[0010] XI-GDR-X2-Q-X3-RQ-X4-SG-X5(SEQ ID NO:1 ) wherein

[0011] Xi is vacant or is an amino acid sequence of one to 4 amino acids;

[0012] X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G);

[0013] X3is tyrosine (Y) or tryptophan (W);

[0014] X4is cysteine (C), valine (V) or tyrosine (Y);

[0015] X5is vacant or is an amino acid sequence of one to 4 amino acids; and wherein the peptide comprises no more than one cysteine (C) residue; or a functional variant deriving from said peptide.

[0016] In embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO:1 wherein: •the amino acid sequence of one to 4 amino acids Xi is a leucine (L) residue, phenylanine- leucine (FL), glycine-phenylalanine-leucine (GFL), or proline-glycine-phenylalanine-leucine (PGFL); and / or

[0017] •the amino acid sequence of one to 4 amino acids X5 is is a tyrosine (Y) residue, tyrosinevaline (YV), tyrosine-cysteine (YC), tyrosine-valine-glutamic acid (YVE), tyrosine-cysteine- glutamic acid (YOE), tyrosine-valine-glutamic acid-asparagine (YVEN), or tyrosine-cysteine- glutamic acid-asparagine (YCEN).

[0018] In embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO:1 wherein:

[0019] If Xi is an amino acid sequence of one to 4 amino acids, X5is vacant

[0020] If Xi vacant, X5 is an amino acid sequence of one to 4 amino acids.

[0021] In embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO:1 wherein:

[0022] Xi is vacant, is a leucine (L) residue, phenylanine-leucine (FL), glycine-phenylalanine- leucine (GFL), or proline-glycine-phenylalanine-leucine (PGFL),

[0023] X3 is Y, and

[0024] X5is vacant.

[0025] Preferably, Xi is proline-glycine-phenylalanine-leucine (PGFL)

[0026] In an embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO:1 wherein the peptide comprises or consists in an amino acid sequence selected from:

[0027] PGFLGDRCQYRQVSG (SEQ ID NO:4),

[0028] PGFLGDRYQYRQVSG (SEQ ID NO:5), and

[0029] PGFLGDRQQYRQCSG (SEQ ID NO:6).

[0030] In embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO:1 wherein:

[0031] Xi is vacant,

[0032] X2is G, and

[0033] X5is vacant, is a tyrosine (Y) residue, tyrosine-valine (YV), tyrosine-cysteine (YC), tyrosine-valine-glutamic acid (YVE), tyrosine-cysteine-glutamic acid (YCE), tyrosine- valine-glutamic acid-asparagine (YVEN), or tyrosine-cysteine-glutamic acid- asparagine (YCEN).

[0034] Preferably, X5 is tyrosine-valine-glutamic acid-asparagine (YVEN) or tyrosine-cysteine- glutamic acid-asparagine (YCEN).

[0035] In embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO:1 wherein the peptide comprises or consists in an amino acid sequence selected from:

[0036] GDRGQYRQYSGYVEN (SEQ ID NO:7),

[0037] GDRGQYRQYSGYCEN (SEQ ID NO:8),

[0038] GDRGQWRQYSGYCEN (SEQ ID NO:9), and

[0039] GDRGQWRQCSGYVEN (SEQ ID NQ:10). In another aspect, the invention relates to a modified peptide deriving from the peptide of amino acid sequence (I) as set forth in SEQ ID NO:1 , by the introduction of one or more chemical modifications which preferably protect the peptide against proteolysis.

[0040] In another aspect, the invention relates to a polynucleotide encoding the peptide of amino acid sequence (I) as set forth in SEQ ID NO:1 .

[0041] In another aspect, the invention relates to a vector comprising the polynucleotide encoding the peptide of amino acid sequence (I) as set forth in SEQ ID NO:1 .

[0042] In another aspect, the invention relates to the modified peptide as described herein, the polynucleotide as described herein, or the vector as described herein, for use as a medicament.

[0043] In another aspect, the invention relates to the modified peptide as described herein, the polynucleotide as described herein, or the vector as described herein, for use in treating a proliferative disorder, in particular a cancer.

[0044] In another aspect, the invention relates to the use of the peptide as described herein or of the modified peptide as described herein in a method for diagnosing and / or staging a disease associated with Cathepsin D overexpression.

[0045] Detailed description

[0046] The peptides useful in the invention are synthetic peptides as described herein.

[0047] In a first aspect, the present invention provides a peptide of no more than 20 amino acids in size comprising or consisting in the following amino acid sequence (I):

[0048] X1-GDR-X2-Q-X3-RQ-X4-SG-X5 (SEQ ID NO:1 ) wherein

[0049] Xi is vacant or is an amino acid sequence of one to 4 amino acids;

[0050] X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G);

[0051] X3 is tyrosine (Y) or tryptophan (W);

[0052] X is cysteine (C), valine (V) or Tyrosine (Y);

[0053] X5is vacant or is an amino acid sequence of one to 4 amino acids; and wherein the peptide comprises no more than one cysteine (C) residue; or a functional variant deriving from said peptide.

[0054] In an embodiment, the peptides comprise or consists in an amino acid sequence of 11 to 20 amino acids in size, preferably of 11 to 15 amino acids in size.

[0055] In an embodiment, the peptide consists in an amino acid sequence of 15 amino acids in size.

[0056] The expression “the peptide comprises no more than one cysteine (C) residue” means that the peptide comprises 0 or 1 cysteine residue. In an embodiment, the peptide comprises 1 cysteine residue. In an embodiment, the peptide comprises 0 cysteine residue, or in other words the peptides does not comprise any cysteine residue.

[0057] In an embodiment, Xi is vacant, a leucine (L) residue, phenylanine-leucine (FL), glycine- phenylalanine-leucine (GFL), or proline-glycine-phenylalanine-leucine (PGFL), Preferably, Xi is glycine-phenylalanine-leucine (GFL) or proline-glycine-phenylalanine-leucine (PGFL).

[0058] In an embodiment, X5is vacant, a tyrosine (Y) residue, tyrosine-valine (YV), tyrosine-cysteine (YC), tyrosine-valine-glutamic acid (YVE), tyrosine-cysteine-glutamic acid (YOE), tyrosinevaline-glutamic acid-asparagine (YVEN), or tyrosine-cysteine-glutamic acid-asparagine (YCEN).

[0059] Preferably, X5 is tyrosine-valine-glutamic acid-asparagine (YVEN) or tyrosine-cysteine- glutamic acid-asparagine (YCEN).

[0060] In an embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO: 1 wherein: the amino acid sequence of one to 4 amino acids X1 is a leucine (L) residue, phenylanine-leucine (FL), glycine-phenylalanine-leucine (GFL), or proline-glycine- phenylalanine-leucine (PGFL); and / or the amino acid sequence of one to 4 amino acids X5 is is a tyrosine (Y) residue, tyrosine-valine (YV), tyrosine-cysteine (YC), tyrosine-valine-glutamic acid (YVE), tyrosine-cysteine-glutamic acid (YCE), tyrosine-valine-glutamic acid-asparagine (YVEN), or tyrosine-cysteine-glutamic acid-asparagine (YCEN).

[0061] In an embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO: 1 wherein:

[0062] If Xi is an amino acid sequence of one to 4 amino acids, X5is vacant, and

[0063] If Xi vacant, X5is an amino acid sequence of one to 4 amino acids.

[0064] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0065] XI-GDR-X2-Q-X3-RQ-X4-SG-X5(SEQ ID NO:18) wherein

[0066] Xi is vacant or proline-glycine-phenylalanine-leucine (PGFL);

[0067] X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G);

[0068] X3is tyrosine (Y) or tryptophan (W);

[0069] X4is cysteine (C), valine (V) or Tyrosine (Y);

[0070] X5 is vacant, tyrosine-valine-glutamic acid-asparagine (YVEN) or tyrosine-cysteine- glutamic acid-asparagine (YCEN); wherein if Xi is PGFL, X5is vacant, and wherein if Xi is vacant, X5is YVEN or YCEN, or a functional variant deriving from said peptide.

[0071] In a preferred embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO: 1 wherein

[0072] Xi is vacant, a leucine (L) residue, phenylanine-leucine (FL), glycine-phenylalanine- leucine (GFL), or proline-glycine-phenylalanine-leucine (PGFL),

[0073] X3is Y, and

[0074] X5is vacant.

[0075] Thus, in an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence (Ila):

[0076] XI-GDR-X2-QYRQ-X4-SG (SEQ ID NO:2) wherein

[0077] Xi is vacant, a leucine (L) residue, phenylanine-leucine (FL), glycine-phenylalanine- leucine (GFL), or proline-glycine-phenylalanine-leucine (PGFL),

[0078] X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G), preferably is cysteine (C), tyrosine (Y), or glutamine (Q);

[0079] X4is cysteine (C), valine (V) or tyrosine (Y), preferably is cysteine (C) or valine (V), or a functional variant thereof.

[0080] In an embodiment, the peptide is a peptide of amino acid sequence (Ila) as set forth in SEQ ID NO: 2 wherein:

[0081] Xi is vacant, a leucine (L) residue, phenylanine-leucine (FL), glycine-phenylalanine- leucine (GFL), or proline-glycine-phenylalanine-leucine (PGFL),

[0082] X2is cysteine (C), tyrosine (Y), or glutamine (Q);

[0083] X4is cysteine (C) or valine (V), or a functional variant thereof.

[0084] Preferably, the peptide is a peptide of amino acid sequence (Ila) as set forth in SEQ ID NO: 2 wherein Xi is glycine-phenylalanine-leucine (GFL), or proline-glycine-phenylalanine-leucine (PGFL).

[0085] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0086] PGFLGDR-X2-QYRQ-X4-SG (SEQ ID NO:14) wherein

[0087] X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G), preferably is cysteine (C), tyrosine (Y), or glutamine (Q);

[0088] X4is cysteine (C), valine (V) or tyrosine (Y), preferably is cysteine (C) or valine (V), or a functional variant thereof.

[0089] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0090] PGFLGDR-X2-QYRQ-X4-SG (SEQ ID NO:15) wherein

[0091] X2is cysteine (C), tyrosine (Y), glutamine (Q);

[0092] X4is cysteine (C) or valine (V), or a functional variant thereof.

[0093] The peptide may be selected from CBP-1 of amino acid sequence PGFLGDRCQYRQVSG (SEQ ID NO:4), CBP-2 of amino acid sequence PGFLGDRYQYRQVSG (SEQ ID NO:5), CBP- 3 of amino acid sequence PGFLGDRQQYRQCSG (SEQ ID NO:6), and a functional variant of amino acid sequence SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0094] A preferred peptide is selected from:

[0095] CBP-1 of amino acid sequence PGFLGDRCQYRQVSG (SEQ ID NO:4),

[0096] CBP-2 of amino acid sequence PGFLGDRYQYRQVSG (SEQ ID NO:5), and

[0097] CBP-3 of amino acid sequence PGFLGDRQQYRQCSG (SEQ ID NO:6). In another preferred embodiment, the peptide is a peptide of amino acid sequence (I) as set forth in SEQ ID NO: 1 wherein

[0098] Xi is vacant,

[0099] X2 is G, and

[0100] X5is vacant, a tyrosine (Y) residue, tyrosine-valine (YV), tyrosine-cysteine (YC), tyrosine- valine-glutamic acid (YVE), tyrosine-cysteine-glutamic acid (YCE), tyrosine-valine-glutamic acid-asparagine (YVEN), or tyrosine-cysteine-glutamic acid-asparagine (YCEN).

[0101] Thus, in an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence (lib):

[0102] GDRGQ-X3-RQ-X4-SG-X5(SEQ ID NO:3) wherein

[0103] X3is tyrosine (Y) or tryptophan (W);

[0104] X4is cysteine (C), valine (V) or tyrosine (Y), preferably is tyrosine (Y) or cysteine (C);

[0105] X5is vacant, a tyrosine (Y) residue, tyrosine-valine (YV), tyrosine-cysteine (YC), tyrosine-valine-glutamic acid (YVE), tyrosine-cysteine-glutamic acid (YCE), tyrosine- valine-glutamic acid-asparagine (YVEN), or tyrosine-cysteine-glutamic acid- asparagine (YCEN), preferably is tyrosine-valine-glutamic acid-asparagine (YVEN) or tyrosine-cysteine-glutamic acid-asparagine (YCEN), or a functional variant thereof.

[0106] In an embodiment, the peptide is a peptide of amino acid sequence (lib) as set forth in SEQ ID NO: 3 wherein

[0107] X3is tyrosine (Y) or tryptophan (W);

[0108] X4is tyrosine (Y) or cysteine (C);

[0109] X5 is vacant, a tyrosine (Y) residue, tyrosine-valine (YV), tyrosine-cysteine (YC), tyrosine-valine-glutamic acid (YVE), tyrosine-cysteine-glutamic acid (YCE), tyrosinevaline-glutamic acid-asparagine (YVEN), or tyrosine-cysteine-glutamic acid- asparagine (YCEN), preferably is tyrosine-valine-glutamic acid-asparagine (YVEN) or tyrosine-cysteine-glutamic acid-asparagine (YCEN).

[0110] Preferably, the peptide is a peptide of amino acid sequence (lib) as set forth in SEQ ID NO: 3 wherein X5is tyrosine-valine-glutamic acid-asparagine (YVEN) or tyrosine-cysteine-glutamic acid-asparagine (YCEN).

[0111] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0112] GDRGQ-X3-RQ-X4-SGY-X5-EN (SEQ ID NO:16) wherein

[0113] X3is tyrosine (Y) or tryptophan (W);

[0114] X4is cysteine (C), valine (V) or tyrosine (Y), preferably is tyrosine (Y) or cysteine (C);

[0115] X5is valine (V) or cysteine (C), or a functional variant thereof.

[0116] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0117] GDRGQ-X3-RQ-X4-SGY-X5-EN (SEQ ID NO: 17) wherein

[0118] X3is tyrosine (Y) or tryptophan (W);

[0119] X is tyrosine (Y) or cysteine (C);

[0120] Xs is valine (V) or cysteine (C), or a functional variant thereof.

[0121] The peptide may be selected from CBP-4 of amino acid sequence GDRGQYRQYSGYVEN (SEQ ID NO:7), CBP-5 of amino acid sequence GDRGQYRQYSGYCEN (SEQ ID NO:8), CBP- 6 of amino acid sequence GDRGQWRQYSGYCEN (SEQ ID NO:9), CBP-7 of amino acid sequence GDRGQWRQCSGYVEN (SEQ ID NQ:10), and a functional variant of amino acid sequence SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0122] A preferred peptide is selected from:

[0123] CBP-4 of amino acid sequence GDRGQYRQYSGYVEN (SEQ ID NO:7),

[0124] CBP-5 of amino acid sequence GDRGQYRQYSGYCEN (SEQ ID NO:8),

[0125] CBP-6 of amino acid sequence GDRGQWRQYSGYCEN (SEQ ID NO:9), and

[0126] CBP-7 of amino acid sequence GDRGQWRQCSGYVEN (SEQ ID NQ:10).

[0127] The peptide may be selected from CBP-1 of amino acid sequence PGFLGDRCQYRQVSG (SEQ ID NO:4), CBP-2 of amino acid sequence PGFLGDRYQYRQVSG (SEQ ID NO:5), CBP- 3 of amino acid sequence PGFLGDRQQYRQCSG (SEQ ID NO:6), CBP-4 of amino acid sequence GDRGQYRQYSGYVEN (SEQ ID NO:7), CBP-5 of amino acid sequence GDRGQYRQYSGYCEN (SEQ ID NO:8), CBP-6 of amino acid sequence

[0128] GDRGQWRQYSGYCEN (SEQ ID NO:9), CBP-7 of amino acid sequence

[0129] GDRGQWRQCSGYVEN (SEQ ID NQ:10), and a functional variant of amino acid sequence SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0130] The peptide may be selected from:

[0131] CBP-1 of amino acid sequence PGFLGDRCQYRQVSG (SEQ ID NO:4), CBP-2 of amino acid sequence PGFLGDRYQYRQVSG (SEQ ID NO:5), CBP-3 of amino acid sequence PGFLGDRQQYRQCSG (SEQ ID NO:6), CBP-4 of amino acid sequence GDRGQYRQYSGYVEN (SEQ ID NO:7), CBP-5 of amino acid sequence GDRGQYRQYSGYCEN (SEQ ID NO:8), CBP-6 of amino acid sequence GDRGQWRQYSGYCEN (SEQ ID NO:9), and CBP-7 of amino acid sequence GDRGQWRQCSGYVEN (SEQ ID NQ:10).

[0132] The peptide may be CBP-1 of amino acid sequence PGFLGDRCQYRQVSG (SEQ ID NO:4) or a functional variant thereof.

[0133] The peptide may be CBP-2 of amino acid sequence PGFLGDRYQYRQVSG (SEQ ID NO:5) or a functional variant thereof.

[0134] The peptide may be CBP-3 of amino acid sequence PGFLGDRQQYRQCSG (SEQ ID NO:6) or a functional variant thereof.

[0135] The peptide may be CBP-4 of amino acid sequence GDRGQYRQYSGYVEN (SEQ ID NO:7) or a functional variant thereof. The peptide may be CBP-5 of amino acid sequence GDRGQYRQYSGYCEN (SEQ ID NO:8) or a functional variant thereof.

[0136] The peptide may be CBP-6 of amino acid sequence GDRGQWRQYSGYCEN (SEQ ID NO:9) or a functional variant thereof.

[0137] The peptide may be CBP-7 of amino acid sequence GDRGQWRQCSGYVEN (SEQ ID NO:10) or a functional variant thereof.

[0138] The peptide may be CBP-1 of amino acid sequence PGFLGDRCQYRQVSG (SEQ ID NO:4). The peptide may be CBP-2 of amino acid sequence PGFLGDRYQYRQVSG (SEQ ID NO:5). The peptide may be CBP-3 of amino acid sequence PGFLGDRQQYRQCSG (SEQ ID NO:6). The peptide may be CBP-4 of amino acid sequence GDRGQYRQYSGYVEN (SEQ ID NO:7). The peptide may be CBP-5 of amino acid sequence GDRGQYRQYSGYCEN (SEQ ID NO:8).

[0139] The peptide may be CBP-6 of amino acid sequence GDRGQWRQYSGYCEN (SEQ ID NO:9). The peptide may be CBP-7 of amino acid sequence GDRGQWRQCSGYVEN (SEQ ID NQ:10).

[0140] In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0141] In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0142] In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 10.

[0143] In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0144] In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 10.

[0145] In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 9.

[0146] In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the peptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 10.

[0147] In some embodiments, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0148] In some embodiments, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0149] In some embodiments, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 10.

[0150] In some embodiments, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0151] In some embodiments, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 10.

[0152] In some embodiments, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. In some embodiments, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 9.

[0153] Peptide solubility

[0154] The peptide preferably is a soluble peptide.

[0155] The expression “soluble peptide” refers to a peptide having the ability to dissolve readily in a solvent such as water, NaCI 0.9% and / or PBS. In an embodiment, the peptide has a maximal solubility of more than 2 mM, preferably of more than 5 mM.

[0156] In an embodiment, the peptide has a maximal solubility comprised between 2 and 10 mM.

[0157] The determination of maximal solubility is well known by the person skilled in the art and can for example be quantified performing the following procedure:

[0158] - providing a stock solution comprising a given known amount of peptide (e.g. 20 mg / ml) in a solvent, e.g. distilled water, NaCI 0.9% or PBS

[0159] - making a series of dilutions from the stock solution (e.g., 15, 10, 5, 2.5, 1 , 0.5, 0.25 mg / mL); optionally, adjusting the pH of each diluted solution to a desired value (e.g. pH 7);

[0160] - incubating at room temperature for 1 hour;

[0161] - reading the optical density of each diluted solution with a spectrophotometer at 205 nm;

[0162] - determining the concentration of each diluted solution considering the molar extinction coefficient of the peptide.

[0163] - determining the maximum solubility identifying the highest concentration where the peptide remains fully dissolved.

[0164] A detailed procedure is also described in the experimental part below.

[0165] Peptide activities

[0166] The peptide preferably has at least one activity selected from:

[0167] (a) the ability to bind cathepsin D protein,

[0168] (b) the ability to inhibit the cathepsin D catalytic activity,

[0169] (c) the ability to inhibit tumorigenesis promoted by the cathepsin D secreted by cancer cells within a tumor microenvironment.

[0170] Unless stated otherwise, cathepsin D and pro-cathepsin D are referred to as “cathepsin D” in the present disclosure. Cathepsin D is the protein (EC:3.4.23.5) encoded by the human full length CTSD gene (Gene ID: 1509;Genbank accession number NM_001909). “Procathepsin D" is the 52 kDa, catalytically inactive, precursor of cathepsin D.

[0171] The term "LRP1" or “LRP-1” refers to LDL receptor-related protein 1 , which is well known in the art. LRP1 is a protein composed of a 515 kDa extracellular a chain and an 85 kDa p chain generated by proteolytic cleavage from a 600 kDa precursor polypeptide in a trans-Golgi compartment. LRP1 a chain and LRP1 p chain are issued from a sole transcript. The human full length of unprocessed precursor LRP1 corresponds to UniProtKB / SwissProt accession number Q07954.

[0172] (a) ability to bind cathepsin D

[0173] In an embodiment, the peptide is able to bind to cathepsin D, in particular in vitro and / or in vivo.

[0174] In an embodiment, the peptide has a binding affinity KD value of less than 500 nM for cathepsin D, as determined by a microscale thermophoresis assay.

[0175] In a preferred embodiment, the peptide has a binding affinity KD value of less than 400 nM, preferably of less than 300 nM for cathepsin D as determined by microscale thermophoresis assay. In a more preferred embodiment, the peptide has a binding affinity KD value of less than 100 nM, of less than 50 nM for cathepsin D, as determined by microscale thermophoresis assay.

[0176] The determination of binding affinity by microscale thermophoresis is well known by the person skilled in the art and can for example be quantified performing the following procedure:

[0177] - providing a peptide to be tested, pro-cathepsin D labeled with His-Tag mixing the labeled pro-cathepsin D with the peptide to be tested using a 0.5-fold dilution in series ranging from 10 pM to 0.15 pM; analyzing the mixtures with an instrument for MST, for example using a Monolith NT.115 (NanoTemper) instrument at 25°C with instrument parameters as follows : 20% Pico-RED excitation-power, 40% medium MSTpower, and 5 / 20 / 5 laser off / on / off ; and analyzing data for example with NT MO Affinity Analysis v2.1 .3 (NanoTemper); identifying and / or quantifying the binding affinity of the peptide to be tested to procathepsin D.

[0178] A detailed procedure is also described in the experimental part below.

[0179] (b) ability to inhibit the catalytic activity of cathepsin D

[0180] In an embodiment, the peptide can inhibit the catalytic activity of cathepsin D, in particular within a tumor microenvironment.

[0181] In an embodiment, the peptide can inhibit at least 20%, preferably, 50% of the catalytic activity of cathepsin D, in particular within a tumor microenvironment, as determined in an assay at pH 6. It is well known that tumor microenvironment exhibits a pH between 5.6 to 6.8. The capacity of the peptide to inhibit the catalytic activity of cathepsin D within a tumor microenvironment can be identified and / or quantified performing the following procedure:

[0182] - providing pro-cathepsin D, a peptide to be tested, and pepstatin A (control)

[0183] - pre-incubating pro-cathepsin D (20 pg / mL), 30 min at 37°C in Assay Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 3.5) in order to activate it

[0184] - incubating activated pro-cathepsin D (0.5 ng / pL) with the peptide to be tested (100 pM) or pepstatin A (2 pg / mL) 15 min in Assay Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 6).

[0185] - measuring the catalytic activity in the presence of a fluorogenic substrate (30 pM)

[0186] - identifying and / or quantifying the ability of the peptide to be tested to inhibit the catalytic activity of cathepsin D within a medium mimicking the pH of a tumor microenvironment.

[0187] A detailed procedure is also described in the experimental part below.

[0188] (c) ability to decrease cell invasion of overexpressinq cathepsin D breast cancer cells

[0189] In an embodiment, the peptide can decrease the invasion process of breast cancer cells promoted by cathepsin D secretion.

[0190] In an embodiment, the peptide can inhibit at least 5%, preferably 10%, preferably 15% of the invasion in an ex vivo assay. The capacity of the peptide to inhibit the breast cancer cell invasion can be identified and / or quantified performing the following procedure:

[0191] - Cell Line and culture: Use a cancer cell line ( e.g., MDA-MB-231) and culture cells in appropriate conditions, e.g. in a T75 flask with 10 mL of 1% FCS medium at 37°C, 5% CO2, and 95% humidity for 48 hours.

[0192] - Peptides: Prepare peptides at 500 pm. For example, remove peptides from -20°C storage and centrifuge for 5 minutes at 4°C and 10,000g, allow peptides to stand at room temperature for 10 minutes, then solubilize to 500 pM. Let the peptides for one hour at room temperature before use.

[0193] - Invasion Assay Setup: •Matrigel® Coating: Pre-coat ThinCert™ inserts with a 0.3 mg / mL solution of Matrigel® and incubate e.g. for 2 hours at 37°C to allow polymerization.

[0194] •Conditioned Medium preparation : Prepare conditioned medium by supplementing DMEM (1 g / L glucose) with 1% FCS, and centrifuge the medium e.g. for 5 minutes at 1200 rpm.

[0195] •Cell Preparation: Rinse cultured cells with PBS and detach them using trypsin-EDTA, centrifuge cells e.g. for 5 minutes at 1200 rpm, and resuspend the pellet to a concentration of 5 x 104cells / mL in conditioned medium.

[0196] •Seeding and Treatment: Seed 1 x 104cells with or without vehicle / peptides (100 pM) in the insert. Add complete medium (DMEM with 1 g / L glucose and FCS) to the wells and incubate the plate for 16 hours at 37°C, 5% CO2, and 95% humidity.

[0197] - Post-Incubation Procedure:

[0198] •Cell Fixation: Remove the medium and fix the cells in methanol for 10 minutes at room temperature and rinse the cells.

[0199] •Non-invasive Cell Removal: Scrape away non-invasive cells (inside the insert)

[0200] •Preparation for Microscopy: Cut the inserts with a scalpel and place them on an antifade mounting drop with DAPI, and cover with a coverslip.

[0201] •Visualization and Analysis: Take images e.g. using the Evos FL microscope at a 10x objective and count invasive cells e.g. using Imaged software.

[0202] A detailed procedure is also described in the experimental part below.

[0203] (d) ability to reduce in vivo tumor volume of cathepsin D positive murine breast tumor

[0204] In an embodiment, the peptide can reduce tumor growth of a cathepsin D positive breast tumor.

[0205] In an embodiment, the peptide can reduce at least 10%, preferably 20%, preferably 30%, preferably 40% of the tumor volume in an in vivo assay. The capacity of the peptide to reduce the breast cancer tumor volume can be identified and / or quantified performing the following procedure:

[0206] - Animals: Use female mice (e.g., C57BL / 6), typically around 7 weeks old. House mice in pathogen-free, temperature-controlled conditions with a 12-hour lig ht / dark cycle. Provide water and food ad libitum and allow mice to acclimate for at least one week. Ensure compliance with relevant ethical guidelines and protocols.

[0207] -Tumor Induction: Prepare cancer cells (e.g. E0771 murine breast cancer adenocarcinoma cell line) in PBS at a concentration suitable for injection (e.g., 2.5 x 105cells). Inject cells into the mammary fat pad or other relevant tissue of the mice. Allow tumors to develop for a specific period (e.g., between 5 days and 30 days, between 10 days and 20 days, between 12 days and 13 days, or 12 days, or 13 days, or 23 days) before beginning treatments.

[0208] - Treatment Groups: Randomize tumor-bearing mice into different treatment groups (e.g., control, experimental drug treatments). Administer treatments (e.g., intraperitoneal injections) at defined intervals (e.g., three times per week).

[0209] - Statistical Analyses: Present results as scatter plots with medians. Analyze the effects of treatments on tumor volume using appropriate statistical tests (e.g., Kruskal-Wallis test followed by the Benjamini, Krieger, and Yekutieli procedure). Use statistical software (e.g., GraphPad Prism) for analyses, considering P < 0.05 as indicative of statistical significance.

[0210] This protocol can be adapted to various cell lines, animal models, and experimental treatments to assess cell invasion and related phenomena.

[0211] A detailed procedure is also described in the experimental part below. Functional variant

[0212] “Functional variant", refers to an amino acid sequence that derives from a peptide described herein and that retains at least the solubility and / or at least one of the activities of the peptide from which it is derived.

[0213] A functional variant comprises an amino acid sequence which is preferably "substantially homologous" or "substantially similar" to the sequence of the reference amino acid sequence from which it is derived. Two amino acid sequences are "substantially homologous" or "substantially similar" when one or more amino acid residues are replaced by a biologically similar residue or when the sequences are at least 90 % identical 95% or 98 % similar.

[0214] The percent amino acid sequence identity / similarity is defined as the percent of amino acid residues in a Compared Sequence that are identical / similar to the Reference Sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity. The Percent identity is then determined according to the following formula: Percent identity = 100 x [1- (C / R)], wherein C is the number of differences between the Reference Sequence and the Compared sequence over the entire length of the Reference Sequence, wherein (i) each amino acid in the Reference Sequence that does not have a corresponding aligned amino acid in the Compared Sequence, (ii) each gap in the Reference Sequence, and (iii) each aligned amino acid in the Reference Sequence that is not identical / similar to an amino acid in the Compared Sequence constitutes a difference; and R is the number amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as an amino acid.

[0215] Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software such as BLAST (Altschul et ah, J. Mol. Biol, 1990, 215, 403-), FASTA, the GCG (Genetics computer Group, Program Manual for the GCG Package, version 7, Madison, Wisconsin) pileup program, or any of the programs known in the art. When using such software, the default parameters, e.g., for gap penalty and extension penalty, are preferably used. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3 and an expectation (E) of 10.

[0216] In an embodiment, the functional variant derives from a peptide as described herein, in particular from a peptide of amino sequence SEQ ID NO:1 ; 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described herein, by deletion, insertion, and / or substitution of one or more amino acids.

[0217] In an embodiment, the functional variant derives from a peptide as described herein, in particular from a fragment of amino sequence SEQ ID NO: 1 ; 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described herein, by deletion, insertion, and / or substitution of 1 , 2, 3, or 4 amino acids.

[0218] Preferably, the substitution is a conservative substitution.

[0219] Conservative substitution refers to the substitution of one amino acid with another, without altering the overall conformation and function of the peptide, including but not limited to the replacement of an amino acid with one which has similar chemical or physical properties (size, charge or polarity), which generally does not modify the functional properties of the peptide. Amino acids with similar properties are well known in the art. As such, it should be understood that in the context of the present invention, a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.

[0220] Examples of conservative substitutions are set out in the Table below:

[0221] In an embodiment, the functional variant derives from a peptide of SEQ ID NO:1 ; 2, 3, 4, 5, 6, 7, 8, 9 or 10 as described above by one or more conservative substitutions, preferably by conservative substitutions of 1 , 2, 3, or 4 amino acids. In an embodiment, the peptide is a peptide of no more than 20 amino acids in size comprising or consisting in the following amino acid sequence (I):

[0222] XI-GDR-X2-Q-X3-RQ-X4-SG-X5(SEQ ID NO:1 ) wherein

[0223] Xi is vacant or is an amino acid sequence of one to 4 amino acids; X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G);

[0224] X3is tyrosine (Y) or tryptophan (W);

[0225] X4is cysteine (C), valine (V) or tyrosine (Y);

[0226] X5is vacant or is an amino acid sequence of one to 4 amino acids; and wherein the peptide comprises no more than one cysteine (C) residue; or a functional variant deriving from said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0227] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0228] XI-GDR-X2-Q-X3-RQ-X4-SG-X5(SEQ ID NO:18) wherein

[0229] Xi is vacant or proline-glycine-phenylalanine-leucine (PGFL);

[0230] X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G);

[0231] X3is tyrosine (Y) or tryptophan (W);

[0232] X4is cysteine (C), valine (V) or Tyrosine (Y);

[0233] X5is vacant, tyrosine-valine-glutamic acid-asparagine (YVEN) or tyrosine-cysteine- glutamic acid-asparagine (YCEN); wherein if Xi is PGFL, X5is vacant, and wherein if Xi is vacant, X5 is YVEN or YCEN, or a functional variant deriving from said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0234] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence (Ila):

[0235] XI-GDR-X2-QYRQ-X4-SG (SEQ ID NO:2) wherein

[0236] Xi is vacant, a leucine (L) residue, phenylanine-leucine (FL), glycine-phenylalanine- leucine (GFL), or proline-glycine-phenylalanine-leucine (PGFL),

[0237] X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G), preferably is cysteine (C), tyrosine (Y), or glutamine (Q);

[0238] X4is cysteine (C), valine (V) or tyrosine (Y), preferably is cysteine (C) or valine (V), or a functional variant deriving from said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0239] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0240] PGFLGDR-X2-QYRQ-X4-SG (SEQ ID NO: 14) wherein

[0241] X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G), preferably is cysteine (C), tyrosine (Y), or glutamine (Q);

[0242] X4is cysteine (C), valine (V) or tyrosine (Y), preferably is cysteine (C) or valine (V), or a functional variant deriving from said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0243] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0244] PGFLGDR-X2-QYRQ-X4-SG (SEQ ID NO: 15) wherein

[0245] X2is cysteine (C), tyrosine (Y), or glutamine (Q);

[0246] X is cysteine (C) or valine (V), or a functional variant deriving from said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0247] In a particular embodiment, the peptide is selected from:

[0248] CBP-1 of amino acid sequence PGFLGDRCQYRQVSG (SEQ ID NO:4),

[0249] CBP-2 of amino acid sequence PGFLGDRYQYRQVSG (SEQ ID NO:5), and

[0250] CBP-3 of amino acid sequence PGFLGDRQQYRQCSG (SEQ ID NO:6), or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0251] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence (lib):

[0252] GDRGQ-X3-RQ-X4-SG-X5 (SEQ ID NO:3) wherein

[0253] X3is tyrosine (Y) or tryptophan (W);

[0254] X4is cysteine (C), valine (V) or tyrosine (Y), preferably is tyrosine (Y) or cysteine (C);

[0255] X5 is vacant, a tyrosine (Y) residue, tyrosine-valine (YV), tyrosine-cysteine (YC), tyrosine-valine-glutamic acid (YVE), tyrosine-cysteine-glutamic acid (YCE), tyrosine- valine-glutamic acid-asparagine (YVEN), or tyrosine-cysteine-glutamic acid- asparagine (YCEN), preferably is tyrosine-valine-glutamic acid-asparagine (YVEN) or tyrosine-cysteine-glutamic acid-asparagine (YCEN), or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0256] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0257] GDRGQ-X3-RQ-X4-SGY-X5-EN (SEQ ID NO: 16) wherein

[0258] X3is tyrosine (Y) or tryptophan (W);

[0259] X4is cysteine (C), valine (V) or tyrosine (Y), preferably is tyrosine (Y) or cysteine (C);

[0260] X5is valine (V) or cysteine (C), or a functional variant deriving from said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0261] In an embodiment, the peptide is a peptide comprising or consisting in the following amino acid sequence:

[0262] GDRGQ-X3-RQ-X4-SGY-X5-EN (SEQ ID NO: 17) wherein

[0263] X3is tyrosine (Y) or tryptophan (W);

[0264] X is tyrosine (Y) or cysteine (C);

[0265] Xs is valine (V) or cysteine (C), or a functional variant deriving from said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0266] In a particular embodiment, the peptide is selected from:

[0267] CBP-4 of amino acid sequence GDRGQYRQYSGYVEN (SEQ ID NO:7),

[0268] CBP-5 of amino acid sequence GDRGQYRQYSGYCEN (SEQ ID NO:8),

[0269] CBP-6 of amino acid sequence GDRGQWRQYSGYCEN (SEQ ID NO:9), and

[0270] CBP-7 of amino acid sequence GDRGQWRQCSGYVEN (SEQ ID NQ:10), or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0271] The peptide may be selected from:

[0272] CBP-1 of amino acid sequence PGFLGDRCQYRQVSG (SEQ ID NO:4),

[0273] CBP-2 of amino acid sequence PGFLGDRYQYRQVSG (SEQ ID NO:5),

[0274] CBP-3 of amino acid sequence PGFLGDRQQYRQCSG (SEQ ID NO:6),

[0275] CBP-4 of amino acid sequence GDRGQYRQYSGYVEN (SEQ ID NO:7),

[0276] CBP-5 of amino acid sequence GDRGQYRQYSGYCEN (SEQ ID NO:8),

[0277] CBP-6 of amino acid sequence GDRGQWRQYSGYCEN (SEQ ID NO:9), and

[0278] CBP-7 of amino acid sequence GDRGQWRQCSGYVEN (SEQ ID NQ:10), or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0279] The peptide may be CBP-1 of amino acid sequence PGFLGDRCQYRQVSG (SEQ ID NO:4) or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0280] The peptide may be CBP-2 of amino acid sequence PGFLGDRYQYRQVSG (SEQ ID NO:5) or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution. The peptide may be CBP-3 of amino acid sequence PGFLGDRQQYRQCSG (SEQ ID NO:6) or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0281] The peptide may be CBP-4 of amino acid sequence GDRGQYRQYSGYVEN (SEQ ID NO:7) or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0282] The peptide may be CBP-5 of amino acid sequence GDRGQYRQYSGYCEN (SEQ ID NO:8) or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0283] The peptide may be CBP-6 of amino acid sequence GDRGQWRQYSGYCEN (SEQ ID NO:9) or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0284] The peptide may be CBP-7 of amino acid sequence GDRGQWRQCSGYVEN (SEQ ID NO:10) or a functional variant of said peptide, preferably deriving from said peptide by deletion, insertion, and / or substitution of one or more amino acids, wherein the functional variant derived therefrom preferably derives from said peptide by 1 , 2, or 3 amino acid substitution, preferably by 1 , 2, or 3 amino acid conservative substitution.

[0285] Modified peptide

[0286] In another aspect, the peptide of the invention is a modified peptide derived from the preceding peptides by introduction of any modification into one or more amino acid residues, peptide bonds, N-and / or C- terminal ends of the peptide and retains at least one of the activities of the peptide from which it is derived.

[0287] These modifications which are introduced into the peptide by the conventional methods known to those skilled in the art, include, in a non-limiting manner: the substitution of a natural amino acid with a non-proteinogenic amino acid (D amino acid or amino acid analog); the modification of the peptide bond, in particular with a bond of the retro or retro-inverso type or a bond different from the peptide bond; the cyclization, and the addition of a chemical group to the side chain or the end(s) of the peptide, in particular for coupling an agent of interest to the protein of the invention.

[0288] These modifications may in particular be used to increase the in vivo stability of the peptide, in particular, its resistance to proteolysis.

[0289] Preferably, the peptide comprises one or more chemical modifications, more preferably chemical modification(s) which protect the peptide against proteolysis.

[0290] The N- and / or C-termini of the peptide are advantageously protected against proteolysis. For instance, the N-terminus is in the form of an acetyl group and / or the C-terminus in the form of an amide group. Alternatively or additionally, the peptide may be protected against proteolysis by internal modifications such as the replacement of at least one -CONH- peptide bond by a (CH2NH) reduced bond, a (NHCO) retro-inverso bond, a (CH2-O) methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO- CH) cetomethylene bond, a (CHOH-CH2) hydroxy ethylene bond, a (N-N) bond, a E- alcene bond, or a -CH=CH- bond.

[0291] Alternatively, or additionally, the peptide may be modified by acetylation, acylation, amidation, cross-linking, cyclization, disulfide bond formation, formation of covalent cross-links, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristylation, oxidation, phosphorylation, and the like.

[0292] Alternatively, or additionally, the peptide is advantageously composed of amino acids in D configuration, which renders the peptide resistant to proteolysis.

[0293] Alternatively, or additionally, the peptide is stabilized by intramolecular crosslinking, by modifying at least two amino acid residues with olefinic side chains, preferably C3-C8 alkenyl chains, more preferably penten-2-yl chains, followed by crosslinking of the chains according to the so-called '"stapled-peptide technology" described in Walensky et al., Science, 2004, 305, 1466-1470.

[0294] Alternatively, or additionally, the peptide is advantageously stabilized by covalent binding to a polyethylene glycol (PEG) molecule, preferably a PEG of 1500 Da or 4000 Da, advantageously bound to their C-terminus or a lysine residue. Such coupling may have at least one of the advantages of (i) increasing peptide stability in vivo without affecting its affinity, (ii) decreasing urinary clearance and therapeutic doses and / or (iii) increasing half-life in blood plasma.

[0295] Alternatively, or additionally, the peptide is advantageously stabilized, and its half-life increased by incorporation into a biodegradable and biocompatible polymer material for drug delivery system forming microspheres, such as for instance poly-lactide-co-glycolide (PLGA).

[0296] Alternatively, or additionally, the peptide is advantageously fused to an amino acid sequence to its N-terminal and / or C-terminal end(s) to one or more other protein / peptide moieties including those which allow the cellular targeting of the peptide or modified peptide of the invention, and / or which increase the bioavailability, the production in expression systems and / or stability of said peptide, resulting in a fusion or chimeric peptide . The length of the chimeric peptide is not critical to the invention as long as the peptide remains functional. These protein / peptide moieties may be a (i) cell-penetrating moiety or (ii) a targeting moiety for addressing the chimeric protein to a specific cell type or cell compartment. Cell-penetrating peptides (CPP), also known as protein transduction domains (PTDs), membrane translocation sequences (MTSs), transport peptides, carrier peptides or Trojan peptides are well-known in the art, CPPs are able to translocate into cells (including the cytoplasm and organelles such as mitochondria or the nucleus) at significantly higher levels than passive diffusion, without causing substantial membrane damage, and can be used as vectors of other molecules when linked to them. In addition, the peptide or modified peptide may be separated from the peptide / protein moiety by a linker which is long enough to avoid inhibiting interactions between the peptide or modified peptide and the cell-penetrating or targeting moiety.

[0297] In an embodiment, the peptide is advantageously fused to one or more CPPs allowing to vectorize the peptide to lysosomes to its N-terminal and / or C-terminal end(s). The peptide may for example be fused to a polyhistidine peptide, preferably to a H16 peptide of sequence HHHHHHHHHHHHHHHH (SEQ ID NO:42) to its N-terminal and / or C-terminal end(s). Yet another aspect of the invention relates to an isolated polynucleotide encoding the peptide.

[0298] The polynucleotide is a synthetic or recombinant DNA, RNA or combination thereof, either single- and / or double-stranded. The polynucleotide is encoding the peptide in expressible form, i.e., it is a nucleic acid molecule which, upon expression in a cell or a cell-free system results in a functional peptide.

[0299] Preferably the polynucleotide comprises a coding sequence which is optimized for the host in which the peptide is expressed.

[0300] In another preferred embodiment, the polynucleotide is inserted in a vector. As used herein, the expressions “vector” and “recombinant vector” may be used interchangeably. Thus, another aspect of the invention relates to a recombinant vector comprising the polynucleotide as described herein. Preferably, said recombinant vector is an expression vector capable of expressing said polynucleotide when transfected or transformed into a host cell such as a prokaryotic or eukaryotic cell. The polynucleotide is inserted into the expression vector in proper orientation and correct reading frame for expression. Preferably, the polynucleotide is operably linked to at least one transcriptional regulatory sequence and, optionally to at least one translational regulatory sequence. Recombinant vectors include usual vectors used in genetic engineering and gene therapy including for example plasmids and viral vectors, such as for example lentivirus and adenovirus vectors.

[0301] Therapeutic methods

[0302] The inventors have demonstrated that small peptides derived from a key interaction region of LRP1 as mentioned above were efficient to bind to cathepsin D and / or to inhibit the LRP-1 / cathepsin D interaction. They have demonstrated efficacy on a model mimicking tumor cell environment. They have further demonstrated anticancer and antitumoral effects in vivo.

[0303] Another aspect of the present invention relates to a peptide, modified peptide, polynucleotide, and / or vector as described herein for use as a medicament. The medicament is particularly useful for inhibiting the fibroblast proliferation promoted by cancer cells within tumor microenvironment.

[0304] The term “tumor microenvironment” refers to a dynamic medium containing cells and macromolecules that interact with cancer cells and promotes tumor development, progression and / or metastasis.

[0305] Therefore, another aspect of the present invention relates to a peptide, polynucleotide, and / or vector as described herein, for use in treating a proliferative disorder, in particular a cancer, preferably in a human patient. Another aspect of the invention relates to a peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition as described herein, for use for preventing and / or treating a cancer or a tumor in a subject in need thereof. Another aspect of the invention relates to a peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition as described herein, for use for preventing and / or treating a metastasis in a subject in need thereof.

[0306] As used herein, the terms "patient", "subject" or "individual" are used interchangeably and refer to an animal, preferably a mammal, more preferably a human. In one embodiment, the patient, subject or individual is a man. In another embodiment, the patient, subject or individual is a woman. In one embodiment, the patient, subject or individual is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of a proliferative disorder, such as cancer. In one embodiment, the patient, subject or individual is an adult (for example a subject above the age of 18). In another embodiment, the patient, subject or individual is a child (for example a subject below the age of 18).

[0307] The term "proliferative disorder" refers to a disorder caused by abnormal growth or expansion due to cell proliferation. Proliferative disorders may be associated with pathological proliferation of normal resting stage cells and / or pathological migration of cells from their normal location (e.g., metastasis of tumor cells). Exemplary proliferative disorders include cancer (i.e., "malignant tumors") and benign tumors.

[0308] The terms "tumor" refers to an abnormal mass of tissue in which the growth of the mass exceeds the growth of normal tissue and is not as coordinated as the growth of normal tissue. A neoplasm or tumor may be "benign" or "malignant," depending on the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion and metastasis. "Benign tumors" are generally well differentiated, have significantly slower growth than malignant tumors, and remain localized to the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant locations. Exemplary benign tumors include, but are not limited to, lipoma, chondroma, adenoma, acrochordon, senile hemangioma, seborrheic keratosis, lentigo, and sebaceous hyperplasia. In some cases, some "benign" tumors may later develop into malignant tumors, which may be due to additional genetic changes in the tumor cell subpopulation of the tumor, and these tumors are referred to as "pre-cancerous tumors". An exemplary pre-cancerous tumor is a teratoma. In contrast, "malignant tumors" are generally poorly differentiated (anaplasia) and have significantly rapid growth with progressive infiltration, invasion and destruction of surrounding tissue. In addition, malignant tumors often have the ability to metastasize to distant locations. The terms "metastasis," "metastatic," or "migration" refer to the spread or metastasis of cancer cells from a primary or original tumor to another organ or tissue and is typically determined by: in the organ or tissue where the secondary (metastatic) tumor is located, there is a "secondary tumor" or "secondary cell mass" of the tissue type of the primary or original tumor and not of the organ or tissue where it is located. For example, prostate cancer that has metastasized to bone is referred to as metastatic prostate cancer and includes cancerous prostate cancer cells that grow in bone tissue.

[0309] In an embodiment, the invention relates to peptide, polynucleotide, and / or vector as described herein, for use in a method of treating a proliferative disorder or cancer in a subject in need thereof, the method comprising the administration of an efficient amount of said peptide, polynucleotide, and / or vector.

[0310] The terms “efficient amount” or “therapeutically efficient amount” of an active principle ingredient (for example a peptide, polynucleotide, and / or vector as described herein) refer to an amount of the active principle ingredient that will elicit the biological or medical response of a subject, for example, ameliorate the symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, either alone or in combination with another active principle ingredient (e.g. in combination with anti-tumor agent as described herein).

[0311] The term “patient” refers to a human or non-human animal, preferably a mammal, including male, female, adult and children in need of a treatment.

[0312] In an embodiment, the proliferative disorder or cancer is a proliferative disorder or cancer associated with cathepsin D overexpression, i.e. in which tumor cells overexpress cathepsin D, preferably at least 2-fold, e.g. 2- to 50-fold, compared to non-tumorous cells. In an embodiment, the proliferative disorder or cancer is a proliferative disorder or a cancer associated with cathepsin D overexpression and secretion in biological tissues or fluids, in particular serum, plasma, compared to biological tissues or fluids of a subject not suffering from cancer.

[0313] Examples of cancers associated with cathepsin D overexpression include but are not limited to breast cancer, ovarian cancer, endometrial cancer, prostatic cancer, kidney cancer, bladder cancer, osteosarcoma, gastric cancer, pancreatic cancer, head and neck cancer, salivary adenoid cystic carcinoma, squamous-cell carcinoma, melanoma, thyroid cancer, lung cancer, liver cancer, malignant glioma, colorectal cancer.

[0314] In an embodiment, the proliferative disorder or cancer is a proliferative disorder or cancer in which tumor progression is promoted by cathepsin D overexpression.

[0315] Examples of cancers in which cathepsin D is promoted by cathepsin D secreted by tumor cells include, but are not limited to breast cancer, ovarian cancer, endometrial cancer, prostatic cancer, kidney cancer, colorectal cancer, lung cancer, pancreatic cancer, stomach cancer, and esophageal cancer.

[0316] In a particular embodiment, the peptide is for treating a cancer selected from liver hepatocellular carcinomas (LIHC), lung squamous cell carcinomas (NSCLC), esophageal squamous cell carcinomas (ESCA), ovarian cancers (OV), colorectal cancers (CRC), pancreatic cancers (PAAD), and stomach cancers (STAD). In some embodiments, the peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition is for treating a carcinoma. In some embodiments, the peptide is for treating adenocarcinoma or breast cancer.

[0317] The peptide, polynucleotide, and / or vector as described herein are useful for treating tumors, in particular malignant tumors and in particular for preventing or treating tumor metastasis.

[0318] More particularly, the peptide, modified peptide, polynucleotide, and / or vector as described herein is useful in the treatment of a proliferative disorder or cancer in which tumor cells overexpress Cathepsin D, preferably at least 2-fold, compared to non- non-tumorous cells such as non-tumorous fibroblasts.

[0319] As used herein, the term “treatment” or “therapy” includes curative and / or prophylactic treatment. More particularly, curative treatment refers to any of the alleviation, amelioration and / or elimination, reduction and / or stabilization (e.g., failure to progress to more advanced stages) of a symptom, as well as delay in progression of a symptom of a particular disorder. Prophylactic treatment refers to any of: halting the onset, reducing the risk of development, reducing the incidence, delaying the onset, reducing the development, as well as increasing the time to onset of symptoms of a particular disorder. In the present invention, “treating” or “therapy” may in particular refer to the reduction or halting of the tumor progression.

[0320] The present invention further relates to the use of a peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition as described herein, for the manufacture of a medicament for treating a proliferative disorder, preferably a cancer. The present invention further relates to the use of a peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition as described herein, for the manufacture of a medicament for preventing and / or treating a cancer or a tumor in a subject in need thereof. The present invention further relates to the use of a peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition as described herein, for the manufacture of a medicament for preventing and / or treating a metastasis in a subject in need thereof. The present invention further relates to a method of treating and / or reducing the likelihood of occurrence of a proliferative disorder in a subject, comprising administering to said subject a therapeutically effective amount of the peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition as described herein. The present invention further relates to a method of treating and / or reducing the likelihood of occurrence of a cancer in a subject, comprising administering to said subject a therapeutically effective amount of the peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition as described herein. The present invention further relates to a method of treating and / or reducing the likelihood of occurrence of a tumor in a subject, comprising administering to said subject a therapeutically effective amount of the peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition as described herein. The present invention further relates to a method of treating and / or reducing the likelihood of occurrence of a metastasis in a subject, comprising administering to said subject a therapeutically effective amount of the peptide, modified peptide, polynucleotide, vector, and / or pharmaceutical composition as described herein.

[0321] Pharmaceutical composition

[0322] The peptide, modified peptide, polynucleotide, and / or vector as described herein may be administered in the form of a pharmaceutical composition.

[0323] The invention relates also to a pharmaceutical composition, comprising a peptide, modified peptide, polynucleotide, and / or vector as described herein, and at least one pharmaceutically acceptable carrier. The composition may further comprise another active principle, in particle an anti-tumor agent, more preferably a chemotherapeutic agent, as described herein.

[0324] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or vehicle. In some embodiments, the pharmaceutically acceptable carrier, excipient, or vehicle is selected in a group comprising or consisting of a solvent, a diluent, a carrier, an excipient, a dispersion medium, a coating, an antibacterial agent, an antifungal agent, an isotonic agent, an absorption delaying agent and any combinations thereof. The carrier, vehicle, diluent, solvent or excipient must be “acceptable” in the sense of being compatible with the peptide, or derivative thereof, and not be deleterious upon being administered to an individual. Typically, the carrier, excipient, or vehicle does not produce an adverse, allergic or other untoward reaction when administered to an individual, preferably a human individual. For the particular purpose of human administration, the pharmaceutical compositions should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, the Food and Drugs Administration (FDA) Office or the European Medicines Agency (EMA).

[0325] Combination therapy

[0326] The peptide, modified peptide, polynucleotide, and / or vector as described herein may be used in combination with another active principle, in particular an anti-tumor agent.

[0327] The anti-tumor agent may be a chemotherapeutic agent, an immunotherapy agent, a targeted therapy agent, a cell therapy agent, or an hormonal therapy agent such as for example: (i) an inhibitor of DNA replication like DNA binding agents, in particular alkylating or intercalating drugs, (ii) an antimetabolite agent such as DNA polymerase inhibitors or Topoisoraerase I or II inhibitors, (iii) an anti-mitogenic agent such as alkaloids, (iv) a checkpoint modulator in particular a checkpoint inhibitor such as an anti-PD1 , an anti-PDL1 , an anti-CTLA4, an anti- LAG3, or an anti-TIM3 agent; a checkpoint agonist such as an 0X40, 41 BB or GITR agonist; (v) a targeted anti-tumor therapy agent such as anti-EGFR, anti-HER2, anti-VEGF, PARP inhibitors, mTOR inhibitors, (vi) an hormonal therapy agent such as (via) a corticosteroid such as prednisone, dexamethasone, hydrocortisone and methylprednisone (vib) a thyroid hormone, (vic) a somatostatin analogue, (vid) a reproductive hormone drug.

[0328] Chemotherapeutic agents as referred to in (i) to (iii) above may for example be 5-Fll, Oxaliplatin, Cisplatin, Carboplatin, Irinotecan, Docetaxel, or Paclitaxel.

[0329] Targeted therapy agents as referred to in (i) to (iii) above may for example be Cetuximab or Erlotinib.

[0330] Checkpoint modulators and targeted anti-tumor therapy agents as referred to in (iv) and (v) above may for example be antibodies or fragments thereof or small molecules.

[0331] Reproductive hormone drug as referred to in (vid) above may for example be an androgen drug such as Fluoxymesterone; an estrogen drug such as diethylstilbestrol; a progestin drug such as medroxyprogesterone or megestrol; an anti-androgen drug such as Bicalutamide, Flutamide or Nilutamide; an aromatase inhibitor drug such as Anastrozole, Exemestane or Letrozole; a luteinizing hormone-releasing hormone (LHRH) agonist drugs such as Buserelin, Goserelin or Leuprolide; or a gonadotropin-releasing hormone (GnRH) antagonist such as Degarelix.

[0332] The anti-tumor agent as referred to in (i) to (vi) may be in the form of an antibody-drug conjugate (ADC).

[0333] In an embodiment, the invention relates to a peptide, modified peptide polynucleotide, and / or vector as described herein, for use in a method of treating a tumor in a subject in need thereof, the method comprising the combined administration of an efficient amount of said peptide, polynucleotide, and / or vector and of an anti-tumor agent as described herein.

[0334] In some embodiments, a peptide or modified peptide as described herein, in particular a peptide of SEQ ID NO: 1 , 2, 3 or 4, 5, 6, 7, 8 , 9 or 10 or a functional variant derived therefrom as described herein is used in combination with a chemotherapeutic agent as described herein.

[0335] The disclosure also provides the use of the peptide or modified peptide as described herein optionally in association with a pharmaceutically acceptable support and / or one or more active principle as described herein for the manufacture of a medicament, in particular for the treatment of a proliferative disorder, in particular a cancer, preferably in a human patient, as described herein.

[0336] In another embodiment, the present disclosure provides a method of treatment, in particular a method of treatment of a proliferative disorder, in particular a cancer, the method comprising administering to a subject in need thereof an effective amount of peptide or modified peptide optionally in association with a pharmaceutically acceptable support and / or one or more active principle as described herein.

[0337] Administration route

[0338] In an embodiment, the peptide or pharmaceutical composition and optionally the anti-tumor agent is or are administered to the subject using routes selected from systemic e.g. intravenous or oral route to local routes, in particular intra-tumoral (IT) or intraperitoneal (IP) route.

[0339] The peptide or pharmaceutical composition and optionally the anti-tumor agent may for example be administered to a patient having an ovarian cancer using an IP route. In such IP route, chemotherapy is typically administered into the patient’s abdomen as a fluid through a device called an IP port. The chemotherapy goes from the IP port into the patient’s abdomen through an IP catheter. Once the fluid is in the patient’s abdomen, the patient is asked to change position regularly to help the distribution of the fluid all over the abdomen tissue surfaces. The whole procedure usually lasts 3 to 4 hours.

[0340] Diagnostic and / or staging of diseases associated with cathepsin D overexpression

[0341] Another aspect of the present invention relates to the use of a peptide or modified peptide as described herein in a method for diagnosing and / or staging a disease associated with Cathepsin D overexpression in a patient.

[0342] The disease associated with Cathepsin D overexpression may be a proliferative disorder, more particularly a cancer, or Alzheimer’s disease.

[0343] The peptide or modified peptide used in the above-mentioned method is advantageously a labelled peptide or modified peptide, i.e. , a peptide linked to a labeling agent which produces a detectable and / or quantifiable signal, in particular a radioactive, magnetic or luminescent agent. In other terms, the peptide or modified peptide is used as a reagent. The luminescent agent may for example be a fluorophore, e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5). The radioactive agent may for example be a radioactive atom for scintigraphic studies such as I123, I124, In111, Re186, or Re188.

[0344] In an embodiment, the method comprises a step of contacting a biological sample obtained from the patient with a peptide or modified peptide as described which is preferably labeled, wherein said peptide is preferably capable of selectively interacting with a fragment of LRP1 , in particular a LRP1 p chain ecto-domain, present in the biological sample obtained from the patient. The fragment of LRP1 may be then detected and optionally the concentration measured by any known method in the art.

[0345] In an embodiment, the method comprises detecting the presence of a fragment of LRP1 , in particular a LRP1 p chain ecto-domain, in a biological sample obtained from said patient.

[0346] In an embodiment, the method comprises measuring the concentration of a fragment of LRP1 , in particular a LRP1 p chain ecto-domain, in a biological sample obtained from said patient.

[0347] The disclosure also provides the use of peptide or modified peptide as described herein for the manufacture of a diagnosis kit for diagnosing or staging a disease associated with Cathepsin D overexpression, wherein the disease may be a proliferative disorder, more particularly cancer, or Alzheimer's disease.

[0348] In another embodiment, the present disclosure provides a method for diagnosing or staging a disease associated with Cathepsin D overexpression in a patient, comprising applying a peptide or modified peptide as described herein to a sample of the patient.

[0349] Legends of the Figures

[0350] Figure 1 : Docking structure of Pro-Cathepsin-D protein (CatD HM) and LRP1 domain modeled in the present study (LRP-1 HM).

[0351] Figure 2: Dose-response curve for the binding interaction between Pro-Cathepsin D and peptides (CBP or CTL) generated by micro-scale thermophoresis. Briefly, Pro-cathepsin D was labeled with the His-Tag labelling kit Red-Tris NTA (Nanotemper). Labeled procathepsin D was then mixed with the peptides using a 0.5-fold dilution series ranging from 10 pM to 0.15 nM. The 16 mixtures were then analyzed with a Monolith NT.115 (NanoTemper) instrument at 25°C. The instrument parameters were 20% LED power, 40% MST power, and 5 / 30 / 5 laser off / on / off. Dose-response curve were generated with NT MO Affinity Analysis v2.1.3 (NanoTemper).

[0352] Figure 3: Effect of CBP peptides on the cathepsin D catalytic activity at pH 6. Cathepsin D (0.5 ng / pL) was pre-activated 30 min in Acidic Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 3.5) and then pre-incubated with peptide (100 M) or pepstatin A (2 pg / mL) 15 min in Assay Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 6). Activity was then measured in the presence of fluorogenic substrate (30 pM). N=3, Kruskal-Wallis test with Benjamini, Krieger and Yekutieli correction ***: p<0.001 , **: p<0.01 , *: p<0.05.

[0353] Figure 4: Effect of CBP peptides on MDA-MB-231 cells invasion. MDA-MB-231 cells are seeded in inserts pre-coated with Matrigel® with or without CBP peptides (100 pM). After 16 hours at 37°C, non-invasive cells are removed and invasive cells are visualized under Evos microscope by DAPI labeling. Nuclei are counted using Image J software to determine the percentage of invasive cells. N=3, Kruskal-Wallis test with Benjamini, Krieger and Yekutieli correction ***: p<0.001 , **: p<0.01 , *: p<0.05.

[0354] Figure 5: Effect of CBP treatments on tumor volume (first day when a tumor volume reach 2000 mm3(D23)). Scatter plot with median are represented. ‘ P < 0.05 (Kruskal-Wallis followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).

[0355] Figure 6: Impact of Cathepsin D expression on prognostic value in several cancer. Schemes follow the same formatting. Kaplan-Meier overall survival analysis relating to Cathepsin D mRNA levels in patients affected with different tumor types. False discovery rate (FDR), Hazard ratios (HRs), 95% confidence intervals and log rank p-values are indicated in the graphs.

[0356] Figure ?: Proportion of mice reaching a tumor volume of 1500 mm3at Day 28 post-tumor inoculation following peptide treatment. EO771 tumor-bearing mice were i.p. injected with CBP-2, CBP-4, and CBP-6 peptides or D-PBS (control) from D12 to D32 thrice a week for 3 weeks. For each group, data represent the proportion of mice that reached the predefined critical tumor burden (i.e., tumor volume of 1500 mm3). Contingency Fisher Exact test analysis comparing the proportion of animals in each treatment group whose tumor volume reached or not critical human endpoint at Day 28 was performed showing significant difference in CBP-2, CBP-4 and CBP-6 compared to control, (***, p<0.001 ; *, p=0.016; ***, p<0.001 respectively).

[0357] Figure 8: Tumor Necrosis Index (TNI, %) in tumor sections from mice treated with CBP- 6 peptide or vehicle. EO771 tumor-bearing mice were i.p. injected with CBP-6 peptides or D- PBS (control) from D12 to D19 thrice a week. For each group, data represents the necrotic area within tumor section, expressed as histogram with mean + / - SEM. Quantification of necrotic areas within tumor sections is expressed as a percentage of total tumor area. Necrosis was assessed on H&E-stained histological sections using image analysis software Imaged. Higher TNI values reflect decrease in viable tumor tissue and enhanced therapeutic efficacy of the tested treatment, t test performed on these data detected that TNI was significantly different between group (*, p=0.034).

[0358] Examples

[0359] The present disclosure is further illustrated by the following examples.

[0360] Example 1 : Design and assay of novel peptide inhibitors of Cathepsin D / LRP-1 interaction 1. Cathepsin D / LRP-1 interaction characterization by in silico studies

[0361] To identify the exact interaction zone between cathepsin D and LRP-1 and the residues involved, in silico analyses were performed. Briefly, a homology model was built considering LRP1 complete EGF repeat domain from residue numbers 338 to 377 (40 amino acid length) using ICM pro v3.9. This model was docked in CatD homology model generated with a machine learning approach AlphaFold2 [9.12], The Protein-Protein docking modules, available in ICM Pro v3.9 software, were deployed to perform molecular docking. In the docked model generated, LRP1 plausibly binds to both light and heavy chains of CatD and this result is coherent to understand critical residue interaction (Figure 1). In this Model, LRP1 EGF repeat interacts with three regions, 1 , 2 and 3 of Pro-cathepsin-D protein. LRP1 -domain residues Arg- 359, Gln-361 , Arg-363 and Tyr-368 are crucial residues to interact in the hotspot region of CatD protein.

[0362] Materials & Methods

[0363] MolSoft suite of software ICM Pro v3.9 [9.6-9.11] was used to carry out all modeling analysis performed in this study.

[0364] Homology Modeling of Pro-Cathepsin-D protein

[0365] The full length Pro-Cathepsin-D protein along with signal peptide was considered as a model for the first part of the study. This protein system was modelled by deploying a machine learning approach AlphaFold2.

[0366] Protein-Protein docking

[0367] The Protein-Protein docking module, available in ICM Pro v3.9 software, was deployed to perform molecular docking of LRP-1 with pro-CatD. This module uses Fast Fourier Transform (FFT) method to perform the docking. FFT docking method contains two stages: 1) The first stage uses a simplified scoring function representing steric fit and hydrophobic / hydrophilic contact matching. FFT is then used for translational search using systematic search of rotations from 60x27 (coarse) to 256x125 (fine) orientations. 2) The second stage rescores the top 3000-20000 solutions with a more accurate energy function including electrostatics and SAS-based solvation. The conformations are then clustered using contact fingerprints. Finally, the selected complexes in the stack of hits can then be refined with flexible sidechains. The thoroughness used was 3 and generated 10 poses. A grid box was generated around the amino acids identified at the PPI site. The protein was treated as rigid and LRP1 was treated as flexible.

[0368] 2. Design of peptides to block cathepsin D / LRP-1 interaction

[0369] Two peptides of 15 Amino Acids containing crucial residues, show to interact with Pro- cathepsin-D protein (Arg-359, Gln-361 , Arg-363 and Tyr-368), were first identified: FLRP1 -1 of sequence PGFLGDRCQYRQCSG (SEQ ID NO:12) and FLRP1-2 of sequence GDRCQYRQCSGYCEN (SEQ ID NO:13).

[0370] Then, a novel in silico study was performed, first to validate the two peptides FLRP1 -1 and FRLRP1 -2, and then to design peptides with optimized properties, in particular with respect to peptide-Cathepsin D complex stability, peptide stability and peptide solubility.

[0371] Ala and all L-, D-amino acids scan were performed on apo CBP peptides to understand their folding stability after mutations. Subsequently, the stability of CBP peptide-Cathepsin D complexes was assessed by performing ala and L-amino acid scan. Using these data, it was found that cysteines mutation in FLRP1 -1 and FLRP1 -2 peptides with some particular amino acids could possibly result in a greater folding stability, a greater peptide-Cathepsin D complex stability and / or an increased solubility.

[0372] The sequence of mutated FLRP1-1 and FLRP1 -2 showing the greater calculated stability, binding energy and solubility are shown in Table 1 :

[0373] Table 1 : In silica analysis of stability (ddG), Binding energy (ddGBind) and Solubility (dSolubility) of mutated FLRP1-1 and FLRP1-2 peptides. Only cysteines residues were mutated and only mutants with the calculated greater stability, binding energy and solubility are shown. Modified cysteine residues are in bold.

[0374] Based on these studies, the 7 peptides as shown in Table 2 were designed and synthesized:

[0375] Table 2: Sequences of the 7 peptides pre-selected to block cathepsin D / LRP-1 interaction. The amino acids corresponding to mutations are shown in Bold.

[0376] Materials & Methods MolSoft suite of software ICM Pro v3.9 was used to carry out all modeling analysis performed in this study.

[0377] Protein-Peptide docking

[0378] The Protein-Peptide docking modules available in ICM Pro v3.9 software, were deployed to perform molecular docking of peptides with CatD protein. This module uses Fast Fourier Transform (FFT) method to perform the docking. FFT docking method contains two stages: 1 ) The first stage uses a simplified scoring function representing steric fit and hydrophobic / hydrophilic contact matching. FFT is then used for translational search using systematic search of rotations from 60x27 (coarse) to 256x125 (fine) orientations. 2) The second stage rescores the top 3000-20000 solutions with a more accurate energy function including electrostatics and SAS-based solvation. The conformations are then clustered using contact fingerprints. Finally, the selected complexes in the stack of hits can then be refined with flexible sidechains. The thoroughness used was 3 and generated 10 poses for each peptide. A grid box was generated around the amino acids identified at the PPI site. The protein was treated as rigid, and the peptide was treated as flexible.

[0379] Residue Mutation

[0380] Peptide stability is computed using free energy change in peptide folding through mutational analysis. The mutant amino acid e.g., "ala" or "all" for calculation of the energy is calculated for all natural amino acids. The free energy change in protein stability is computed following the principle that: free energy of the unfolded and misfolded states is approximated by a sum of the residue-specific energies. The residue-specific energies are derived empirically using a large set of experimental data. Mutation of a given residue is followed by Monte Carlo simulations with flexible side chains for the mutated residue and its neighboring residues. The rest of the protein structure is considered rigid. A positive energy value indicates that the mutation is likely to be destabilizing. Whereas a negative value indicates stabilization. This method will be deployed to understand the stability of assigned peptide.

[0381] Thermodynamic stability of CBP peptides after mutations.

[0382] Peptide stability is computed using free energy change in protein stability through mutational analysis. The mutant amino acid e.g., "ala" or "all" for calculation of the energy is calculated for all-natural L-amino acids and for D-amino acids. The free energy change in protein stability is computed following the principle that: free energy of the unfolded and misfolded states is approximated by a sum of the residue-specific energies. The residue-specific energies are derived empirically using a large set of experimental data.

[0383] AAG= GMutant - GWild G= ^GfoldedMutant / Wild - Gunf oldedMutant / Wild GPeptide = EIntraPeptide+ GSolvPeptide GSolvPeptide =GNpPeptide+ GPolarPeptide

[0384] GNpPeptide = Non-polar solvation energy calculated using Still’s equation and SASA.

[0385] GPolarPeptide = Polar solvation energy calculated using Generalized Born method.

[0386] Mutation of a given residue is followed by Monte Carlo simulations with flexible side chains for the mutated residue and its neighboring residues. The rest of the protein structure is considered rigid. A positive energy value indicates that the mutation is likely to be destabilizing. Whereas a negative value indicates stabilization.

[0387] Thermodynamic stabilities of Cathepsin-D and peptide complexes

[0388] Cathepsin-D-CBP peptide complexes stability is computed using ICM pro v3.9 software. This method computes the change in binding free energy of Cathepsin-D-Peptide complex upon mutation of a single residue.

[0389] The binding free energy change, AAGbind, is computed as a difference between the binding free energy of mutant and wild type. GbindComp,Mutant=B inding free energy of CathepsinD-mutated CBP peptide GbindComp,Wild=B inding free energy of CathepsinD-Wild CBP peptide

[0390] For calculating free energy of a complex either mutated or wild, Gbind = (EintraComp - EintraParts)+ (GsolvComp - GsolvParts

[0391] ElntraComp=Sum of Energy contribution from Internal terms of CatD-CBP complex ElntraParts=Sum of Energy contribution from Internal terms of apo CathepsinD complex+Sum of Energy contribution from Internal terms of apo CBP peptide GsolvComp=Desolvation energy contribution for CathepsinD-CBP peptide complex GsolvParts=Sum of desolvation energies of individual CatD protein and CBP peptide

[0392] The energy is calculated for fixed backbone and all the side chains except those in the vicinity of the mutable residue. Monte Carlo simulations are carried out to relieve possible atomic clashes created because of mutations to larger amino acid residues.

[0393] A positive energy value indicates that the mutation is likely to be destabilizing. Whereas a negative value indicates stabilization.

[0394] Entropy computation requires conformational sampling of the entire potential energy surface. Here the peptide does not undergo the sampling of all the conformations. Hence, the entropy contribution is negligible.

[0395] 3. CBP peptides Solubility

[0396] These 7 peptides were synthesized by Genepep (Montpellier, France), with no difficulties in terms of synthesis and purification.

[0397] The solubility of the 7 peptides was tested experimentally in first.

[0398] Briefly, the maximal solubility of the peptides was studied in Water, NaCI 0.9% and PBS using optical density (OD) at 205 nm.

[0399] The results are shown in Table 3:

[0400] Table 3: Study of maximal peptides solubility in Water, NaCI 0.9% and PBS using OD at 205 nm

[0401] The results show that all the 7 peptides were soluble in water, NaCI 0.9% and PBS.

[0402] Peptides CBP-1 and CBP-2 show a particularly high solubility in all solvents. Materials & Methods

[0403] Water (W3500-100ML; Sigma Aldrich) - NaCI 0.9% (S8776-100ML; Sigma Aldrich) - PBS (14190-094; Fisher Scientific) - CBP-1 (23571 -001 ; GENEPEP) - CBP-2 (23573-001 ; GENEPEP) - CBP-3 (23574-001 ; GENEPEP) - CBP-4 (23580-001 ; GENEPEP) - CBP-5 (23582-001 ; GENEPEP) - CBP-6 (23584-001 ; GENEPEP) - CBP-7 (23585-001 ; GENEPEP)

[0404] Peptides are removed from -20°C and centrifuged for 5 min at 4°C and 10 000g. After 10 min at room temperature, they are solubilized to 20 mg / mL and then diluted in cascade at the following concentrations: 15 - 10 - 5 - 2.5 - 1 - 0.5 - 0.25 mg / mL.

[0405] Allow to dissolve for one hour at room temperature before reading optical density (OD) at 205 nm with Nanodrop (2pL).

[0406] To determine peptide concentration using the "other proteins" mode, it is necessary to enter the molar extinction coefficient previously calculated using the following tool: http: / / nickanthis.com / tools / a205.html

[0407] 4. CBP peptides affinity for pro-cathepsin D by MST (MicroScale Thermophoresis)

[0408] Then, peptides affinity for pro-cathepsin D was studied by MicroScale thermophoresis (MST). Briefly, Pro-cathepsin D was labeled with the His-Tag labelling kit Red-Tris NTA (Nanotemper). Labeled pro-cathepsin D was then mixed with the peptides using a 0.5-fold dilution series ranging from 10 pM to 0.15 nM. The 16 mixtures were then analyzed with a Monolith NT.115 (NanoTemper) instrument at 25°C. The instrument parameters were 20% LED power, 40% MST power, and 5 / 30 / 5 laser off / on / off. Dose-response curve were generated with NT MO Affinity Analysis v2.1 .3 (NanoTemper).

[0409] The results are shown on Figure 2 and Table 4 below:

[0410] Table 4: Study of peptides affinity for pro-cathespin D by micro-scale thermophoresis. Table with Kd value obtained for each peptide by microscale thermophoresis.

[0411] Microscale thermophoresis (MST) experiments show that all CBP peptides, except control peptide (CTL), can bind pro-cathepsin D (Figure 2) with a strong affinity (Table 4).

[0412] These results show that in silica analyses have identified a key region of LRP-1 involved in the interaction with pro-cathepsin D and that the mutations of the cysteines by the amino acids as designed by the inventors as a result of the in silica studies do not alter the binding capacity. Materials & Methods

[0413] Material & Reagents

[0414] PCR tubes & tips - HEPES (H0887-20ML; Sigma Aldrich) - Tween® 20 (P1379-25ML; Sigma Aldrich) - Monolith His-Tag labeling Kit RED-Tris-NTA 2ndgeneration (MO-L018; Nanotemper) - Pro-cathepsin D (12517-H08H; SinoBiological) - Milli-Q® water - Water (W3500-100ML; Sigma Aldrich) - Monolith capillaries (MO-K022; Nanotemper) - CBP-1 (23571 -001 ; GENEPEP) - CBP-2 (23573-001 ; GENEPEP) - CBP-3 (23574-001 ; GENEPEP) - CBP-4 (23580-001 ; GENEPEP) - CBP-5 (23582-001 ; GENEPEP) - CBP-6 (23584-001 ; GENEPEP) - CBP-7 (23585-001 ; GENEPEP) - CTL (12501 -A; SB-peptide of sequence MAADGSRQCRCTAYF SEQ ID NO:11 )

[0415] 'Assay Buffer: HEPES 50 mM at pH 7,5 supplemented with 0.05% tween®20 (1 mL HEPES 1M + 19 mL Water + 10 / JL Tween® 20)

[0416] "Stock solution of pro-Cathepsin D at -80°C in aliquots - Concentration: 250 pg / mL - 5.7 pM

[0417] Preparation of peptides

[0418] Peptides are removed from -20°C and centrifuged for 5 min at 4°C and 10 000g. After 10 min at room temperature, they are solubilized at a concentration of 500 pM inX ml of water (see table below) and then dissolved for one hour at room temperature before use.

[0419] Pro-cathepsin D Labeling (Monolith His-Tag Labeling kit)

[0420] All products are reconstituted according to the instructions supplied with the kit. Pro-cathepsin D” at 200 nM and Dye at 100 nM are mixed in Assay Buffer* (1 / 1 v / v) and the mix is incubated 30 min at room temperature. Labeled protein (100 nM) is centrifuged 10 min at 4°C and 15 000g and transferred to a fresh tube.

[0421] Binding Assay

[0422] All reagents are equilibrated to room temperature prior to use. A serial dilution of the peptide from 20 pM to 0.15 pM (from tube 1 to tube 16) in water is prepared. Next, 10 pL of 20 nM proCathepsin D is added to each tube from 16 to 1 and is mixed by pipetting. The mixture is incubated 25 minutes at room temperature. Capillaries are loaded with sample and are incubated 5 min at 25°C prior to measurement using a Monolith NT.115 system with the following settings: 20% Pico-RED excitation power and medium MST-power, 25°C.

[0423] 5. Effect of CBP peptides on pro-cathepsin D catalytic activity

[0424] Extracellular CathD displays pro-tumor activities through proteolysis at acidic pH and also non- proteolytic mechanisms. Secreted CathD can, for example, modify the local extracellular matrix by cleaving chemokines, growth factors, various extracellular matrix components (collagens, fibronectin, proteoglycans, ...) or by activating the cathepsin B and L precursors (Hasan et al., 2006, Benes etal., 2008, Alcaraz et al., 2021 ).

[0425] Cathepsin D is an aspartic protease. Like most lysosomal aspartic proteases, cathepsin D has a maximal catalytic activity at an acidic pH between 2.4 to 5. At higher pH values of 5.0, the activity of cathepsin D decreases, and none is detectable at a pH of 7.0. In the extracellular compartment (where the secreted form of pro-cathepsin D is found), and particularly in the tumor microenvironment, a decrease of pH around 5.6 to 6.8 is observed and is a hallmark of malignant tumor cells and is due to glycolysis in tumor cells, hypoxia, and insufficient blood perfusion.

[0426] The ability of the peptides to inhibit the activity of cathepsin D at a pH of 6 was therefore tested.

[0427] Briefly, Cathepsin D (0.5 ng / pL) was pre-activated 30 min in Acidic Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 3.5) and then pre-incubated with peptide (100 pM) or pepstatin A (2 pg / mL) 15 min in Assay Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 6). Activity was then measured in the presence of fluorogenic substrate (30 pM).

[0428] The results are shown in Figure 3.

[0429] At a pH of 6, peptides CBP-2, CBP-3, CBP-4; CBP-6 and CBP-7 (100 pM) significantly blocked cathepsin D activity but only to a limited extent. Pepstatin A, a potent inhibitor of Cathepsin D, totally blocks cathepsin D activity.

[0430] Interestingly, the CBP-1 and CBP-5 peptides, which can bind (pro-)cathepsin D, show no effect on the catalytic activity of cathepsin D.

[0431] Materials & Methods

[0432] Material and reagents

[0433] PCR tubes & tips - Recombinant Human Cathepsin D protein (1014-AS; R&D systems) - Water (W3500-100ML; Sigma Aldrich) - Sodium acetate anhydrous (NaOAc) (W302406; Sigma Aldrich) - Sodium chloride (NaCI) (207790010; ThermoScientific) - Cathepsin D & E substrate (fluorogenic) (BML-P145-0001 ; Enzo Life Sciences) - 96-well ELISA plate (82.1581.120; Sarstedt) - PBS (14190-094; Gibco) - BSA (Bovine Serum Albumin) (04-100- 812-E; Euromedex) - Pepstatin A (P5318-5MG; Sigma Aldrich) - Infinite 200® pro (TECAN) - - CBP-1 (23571-001 ; GENEPEP) - CBP-2 (23573-001 ; GENEPEP) - CBP-3 (23574-001 ; GENEPEP) - CBP-4 (23580-001 ; GENEPEP) - CBP-5 (23582-001 ; GENEPEP) - CBP-6 (23584-001 ; GENEPEP) - CBP-7 (23585-001 ; GENEPEP)

[0434] Preparation of peptides

[0435] Peptides are removed from -20°C and centrifuged for 5 min at 4°C and 10 000g. After 10 min at room temperature, they are solubilized to 500 pM (see table below) and then dissolved for one hour at room temperature before use.

[0436] Assay procedure

[0437] Equilibrate Assay Buffer to room temperature prior to use. Assay Buffer (0.1 M NaOAc ; 0.2M NaCI) is prepared in water and pH is adjusted with hydrochloric acid (HCI). 96 well-plate is saturated overnight at 4°C with 1% BSA and rinsed with PBS before use. Cathepsin D (stock concentration: 440 pg / mL) is diluted to 20 pg / mL in Assay Buffer at pH 3.5 and incubated 30 min at 37°C for activation. Then, cathepsin D is diluted to 5 ng / pL in Assay Buffer pH 6. In each well, 5 pL of cathepsin D 0.5 ng / pL, 10 pL of peptide (500 pM) or pepstatin (10 pg / mL) and 35 pL of Assay Buffer pH 6 are loaded and incubated

[0438] 15 min at 37°C. The reaction starts by the addition of 60 pM of substrate (stock concentration 1 mM) per well. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 7 min (Readings every 40 seconds).

[0439] 6. Effect of CBP peptides on MDA-MB-231 cells invasion

[0440] Cell invasion, or directed migration of tumor cells into adjacent tissues, is one of the hallmarks of cancer and the first step towards metastasis. The effects of the CBP peptides on the invasion of MDA-MB-231 breast cancer cells were tested using boyden chamber assays. These triplenegative breast cancer (TNBC) cells are known to overexpress and secrete pro-cathepsin D.

[0441] Briefly, MDA-MB-231 cells were seeded in inserts pre-coated with Matrigel® with or without (vehicle) CBP-1 , CBP-2, CBP-3, CBP-4, CBP-5, CBP-6, or CBP-7 peptides at 100 pM. After 16 hours at 37°C, the non-invasive cells were removed, and invasive cells were visualized under Evos microscope by DAPI labeling.

[0442] The results are shown in Figure 4.

[0443] The presence of CBP-3, CBP-5, CBP-6 and CBP-7 peptides at 100 pM significantly decreases the invasive capacity of these cells up to 20%. In contrast, CBP-1 , CBP-2 and CBP-4 peptides had no significant effect on this particular property of tumor cells.

[0444] Materials & Methods:

[0445] Material & reagents

[0446] Water (W3500-100ML; Sigma Aldrich) - Sodium acetate anhydrous (W302406; Sigma Aldrich)

[0447] - Sodium chloride (207790010; ThermoScientific) - PBS (14190-094; Gibco) - Matrigel® (354230; Corning) - DMEM 1 g / L glucose (2773772; Fisher Scientific) - FCS (ATCC-30-2025; ATCC) - - CBP-1 (23571 -001 ; GENEPEP) - CBP-2 (23573-001 ; GENEPEP) - CBP-3 (23574-001 ; GENEPEP) - CBP-4 (23580-001 ; GENEPEP) - CBP-5 (23582-001 ; GENEPEP) - CBP-6 (23584-001 ; GENEPEP) - CBP-7 (23585-001 ; GENEPEP) - ProlongGold Antifade Mounting with DAPI (15260719 ; Invitrogen) - Microscopy slide (631 - 0909 ; VWR) - Cover slips (631 -1577P ; VWR) - FL EVOS microscope - Methanol (M3950 / 21 ; Fisher Scientific) - Distilled water - ThinCert™ - 24 well (662638 ; Greiner bio- one) - Trypsine-EDTA (0,05%) (11580626; Gibco) - TC20 Automated cell counter (Bio-Rad)

[0448] - Cotton-swab (115-0504 ; VWR).

[0449] Preparation of peptides

[0450] Peptides are removed from -20°C and centrifuged for 5 min at 4°C and 10 000g. After 10 min at room temperature, they are solubilized to 500 pM (see table below) and then dissolved for one hour at room temperature before use.

[0451] Assay procedure:

[0452] MDA-MB-231 cells are cultured in 10 mL of 1% FCS medium (T75 flask) at 37°C, 5% CO2, 95% humidity for 48h.

[0453] ThinCert™ inserts are pre-coated with 0.3 mg / mL solution of Matrigel® and incubated for 2 hours at 37°C to allow polymerization.

[0454] Conditioned medium (DMEM 1 g / L of glucose supplemented with 1% FCS) is collected and centrifuged for 5 min at 1200 rpm. Cells are rinsed with PBS, detached with trypsin-EDTA and centrifuged for 5 min at 1200 rpm. The pellet is resuspended at 5.104cells / mL in conditioned media. 1 .104cells with or without vehicle / peptides (100 pM) are seeded in the insert, complete medium (DMEM 1 g / L of glucose supplemented with FCS) is added to the wells and the plate is incubated for 16 hours at 37°C, 5% CO2 and 95 % humidity. Medium is then removed and cells are fixed in methanol for 10 minutes at room temperature. After 2 rinses, non-invasive cells (inside the insert) are scraped away with a cotton-swab. The inserts are cut with a scalpel and placed on an antifade mounting drop with DAP I covered with a coverslip.

[0455] Images are taken with the Evos FL microscope at x10 objective and cells are counted with Image J software.

[0456] 7. In vivo effect of CBP peptides on Breast cancer tumor volume

[0457] Finally, the in vivo effects of two representative CBP peptides (CBP-2 derived from FLRP1-1 and CBP-6 derived from FLRP1 -2) were tested on a Triple-Negative Breast Cancer (TNBC) syngenic mouse model. The efficacy of CBP-2 and -6 peptides was evaluated on tumor growth. Tumor volumes of treated vs. control animals were compared at D23, the first day on which tumor volume reach 2000 mm3. Tumor volume is estimated by the following formula: Tumor Volume = (width2* length) / 2. 23 days after tumor induction (D23), median tumor volumes were 1347.06, 828.33 and 909.82 mm3in Control, CBP-2, and CBP-6 groups, respectively. Kruskal- Wallis test followed by a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli detected that tumor volumes were significantly different between groups (*, P = 0.0487) with a detected difference in both CBP-2 and CBP-6 compared to control (*, P = 0.0472 and *, P = 0.0236 respectively).

[0458] The results are shown in Figure 5.

[0459] These data demonstrate that both CBP-2 and CBP-6 peptides induce tumor growth inhibition after 10 days of treatment compared to control group in the E0771 TNBC syngeneic mouse model. Interestingly, the CBP-2 peptide, which had no significant effect on cell invasion (Figure 4), now inhibits tumor growth, suggesting that it acts on another cathepsin D-induced process.

[0460] Materials & Methods:

[0461] Cancer cell line:

[0462] E0771 were cultured in DMEM (High Glucose, L-Glutamine, Sodium Pyruvate) (Gibco) with 10% foetal calf serum (FCS) (ATCC), 1% Penicillin / streptomycin (Gibco) and 20 mM HEPES, pH=7.5 (Dominique Dutscher). E0771 is a murine breast cancer adenocarcinoma cell line originally isolated as a spontaneous tumor from C57BL / 6 mouse and is most often presented as a TNBC syngeneic model used by orthotopic injection.

[0463] Animals:

[0464] Seven weeks old female C57BL / 6 mice were purchased from Janvier Labs (France) and housed in a pathogen-free, temperature-controlled environment with 12-hour day and night cycles. The animals received water and food ad libitum and were allowed to acclimate for at least 1 week. Animal experiments were conducted in compliance with the EU Directive 63 / 2010 and with protocol # 41322 by the local Ethical Committee (‘Reims Champagne Ardenne' registered at the French Ministry of Research).

[0465] Tumor induction:

[0466] E0771 cells (2.5x 105in PBS) were injected into the fourth right fat pad mammary glands of 8- week-old female C57BL / 6N mice (Janvier Labs, France). After 13 days, tumor-bearing mice with similar tumor volume were randomized in three treatment groups: control (PBS), CBP-2 (10 mg / kg), or CBP-6 (10 mg / kg) (all by intraperitoneal injection three times per week until day 32).

[0467] Statistical analyses:

[0468] Results are expressed as scatter plot with median. The effect of treatments on tumor volume was analysed with a non-parametric methodology using Kruskal-Wallis test followed by two- stage linear step-up procedure of Benjamini, Krieger and Yekutieli. Statistical analysis were performed using GraphPad Prism 10 (GraphPad Software). P <0.05 values were considered indicative of statistical significance.

[0469] 8. Cathepsin D / LRP-1 interaction is a relevant target in oncology

[0470] The pro-cathespin D / LRP-1 interaction and its deleterious effects on tumor growth are well described in breast cancer, particularly in TNBC (Beaujouin etal., 2010, Ashraf et al., 2019).

[0471] The effects of CBP-2 and CBP-6 peptides on tumor growth observed in Figure 5 support these datas. In order to broaden the field of application, other cancers have been identified as potential targets for CBP peptides. The expression of Cathepsin D expression is associated with a poor prognosis in several cancer types.

[0472] We evaluated the correlation between cathepsin D expression and prognosis in tumor samples of patients affected with cancer, analyzing samples from 25 cancer types. Only cancers with a false discovery rate (FDR) below 50%, a hazard ratio (HR) above 1 , and a p-value below 0.05 were considered for the rest of the analysis. Thus, the analysis of combined transcriptomic data (www.kmplot.com and www.proteinatlas.org, accessed on May 29, 2024) showed that the mRNA level of Cathepsin D, encoded by the CTSD gene, was significantly associated with reduced survival in liver hepatocellular carcinomas, lung squamous cell carcinomas, esophageal squamous cell carcinomas, ovarian cancers, colorectal cancers (colon cancers and rectum adenocarcinomas), pancreatic cancers, and stomach cancers (Figure 6).

[0473] Cathepsin D is expressed by tumor and immune cells.

[0474] Using TISCH2 database (www.tisch.comp-genomics.org, accessed on May 29, 2024), we analyzed Cathepsin D expression and cellular infiltrations in tumor samples from patients with the various cancers for which Cathepsin D mRNA levels were significantly associated with reduced survival. Data from 7, 16, 3, 9, 11 , 9, and 2 prospective cohorts were respectively analyzed for liver hepatocellular carcinomas (LIHC), lung squamous cell carcinomas (NSCLC), esophageal squamous cell carcinomas (ESCA), ovarian cancers (OV), colorectal cancers (CRC), pancreatic cancers (PAAD), and stomach cancers (STAD). Except for colorectal and stomach cancers, we found a positive correlation between Cathepsin D expression by tumor cells and, for all analyzed cancers, a positive correlation between Cathepsin D expression and tumor infiltration by immune cells, mostly monocytes / macrophages (data not presented here).

[0475] LRP-1 is mostly expressed by fibroblasts

[0476] Using TISCH2 database (www.tisch.comp-genomics.org, accessed on May 29, 2024), we analyzed LRP-1 expression and cellular infiltrations in tumor samples from patients with the various cancers for which Cathepsin D mRNA levels were significantly associated with reduced survival. Data from the same prospective cohorts as for Cathepsin D were analyzed for liver hepatocellular carcinomas (LIHC), lung squamous cell carcinomas (NSCLC), esophageal squamous cell carcinomas (ESCA), ovarian cancers (OV), colorectal cancers (CRC), pancreatic cancers (PAAD), and stomach cancers (STAD). For all cancers analyzed, we found a positive correlation of LRP-1 expression by fibroblasts and a positive correlation between LRP-1 expression and tumor infiltration by monocyte / macrophage type immune cells (data not presented here).

[0477] Materials & Methods:

[0478] A global "pan-cancer" analysis was conducted using the Kaplan Meier Plotter database, an online public database combining the analysis of over 35,000 samples from 21 tumor types, to determine tumor types for which cathepsin D (CTSD) mRNA expression appeared to be related to overall survival (OS) of patients (www.kmplot.com, accessed on May 29, 2024). This was supplemented by the analysis of 4 additional cancers using The Human Protein Atlas database (www.proteinatlas.org, accessed on May 29, 2024).

[0479] Seven tumor types were selected for more comprehensive analyses using "The Human Protein Atlas" and "Kaplan-Meier Plotter." Databases. Both databases were used to generate Kaplan- Meier survival plots for the seven selected cancers and to determine the relevance of cathepsin D (CTSD) mRNA expression to the overall survival (OS) of patients with one of these seven cancers. Using the "Tumor Immune Single-cell Hub 2 (TISCH2)" database (www.tisch.comp- genomics.org, accessed on May 29, 2024), an scRNA-seq (single-cell RNA sequencing) database focusing on tumor microenvironment (TME) and enabling the exploration of TME across different cancer types, the expression of cathepsin D in tumor samples from patients with one of the seven selected cancers was analyzed to determine the source of cathepsin D for each of these cancers.

[0480] For each of the seven considered cancers, using the "Tumor Immune Single-cell Hub 2 (TISCH2)" database (www.tisch.comp-genomics.org, accessed on May 29, 2024), we analyzed the expression of LRP-1 in tumor samples from patients to determine its cellular origin.

[0481] Example 2: Effect of CBP peptides on time to reach a critical tumor burden

[0482] Results

[0483] Treatment with cathepsin binding peptides optimized from both fragments (FLRP1 -1 & FLRP 1-2) significantly delayed the time required to reach the critical tumor volume of 1500 mm3.

[0484] At day 28, the proportion of mice reaching the predefined endpoint was 75%, in the control group, compared to 10%, 37% and 42 % in the CBP-2 (SEQ ID NO:5), CBP-4 (SEQ ID NO:7), and CBP-6 (SEQ ID NO:9) groups, respectively (Fig. 7). The differences were significant (***, p<0.001 ; *, p=0.016; ***, p<0.001 respectively).

[0485] Furthermore, the median time to reach the critical tumor burden threshold was respectively, 25 days (control), 30 days (CBP-2) and 28 days (CBP-4 and CBP-6), confirming that CBP treatment delayed tumor progression.

[0486] These results thus show that treatment with CBP peptides prolonged time to reach the predefined critical tumor burden in treated groups indicating a tumor growth delay effect and anticancer effect of CBP peptides.

[0487] Materials and methods

[0488] Cancer cell line

[0489] The murine EO771 mammary adenocarcinoma cell line was cultured in DMEM (High Glucose, L-Glutamine, Sodium Pyruvate) (Gibco) with 10% foetal calf serum (FCS) (ATCC), 1% Penicillin / streptomycin (Gibco) and 20 mM HEPES, pH=7.5 (Dominique Dutscher). EO771 is spontaneously derived tumor from C57BL / 6 mouse, frequently used as a TNBC syngeneic model.

[0490] Animals

[0491] Seven-week-old female C57BL / 6 mice were purchased from Janvier Labs (France) and housed in a pathogen-free, temperature-controlled environment with 12-hour day and night cycles. The animals received water and food ad libitum and were allowed to acclimate for at least 1 week before starting experiments. Animal experiments were conducted in compliance with the EU Directive 63 / 2010 and with protocol # 41322 by the local Ethical Committee (‘Reims Champagne Ardenne’ registered at the French Ministry of Research).

[0492] Tumor induction EO771 cells (2.5 x 105in PBS) were orthotopically injected into the fourth right fat pad mammary glands of eight-week-old female C57BL / 6 mice. After 12 days, tumor-bearing mice with comparable tumor volume were randomized in four treatment groups: control (PBS), CBP- 2 (SEQ ID NO:5), CBP-4 (SEQ ID NO:7), or CBP-6 (SEQ ID NO:9) (each at 10 mg / kg), administrated intraperitoneally (i.p.) three times per week until day 32. Tumor volume was estimated, three times per week until day 32, by the following formula: Tumor Volume = (width2x length) / 2.

[0493] Statistical analyses

[0494] Data were summarized for each animal and condition to conclude on CBP peptide efficacy.

[0495] The comparison of the efficacy of CBP peptides was assessed by measuring the time required to reach a predefined critical tumor burden endpoint (i.e. tumor volume > 1500 mm3). The percentage of mice reaching this critical tumor burden was calculated at day 28 (D28) and compared between groups using Fisher’s exact test (GraphPad Prism software). Difference between groups were considered significant when p<0.05.

[0496] Example 3: Effect of CBP-6 peptide on tumor necrosis

[0497] Results

[0498] After one week treatment (D19), the mean TNI was 6.43% in the control group and 11 .15% in the CBP-6 (SEQ ID NO:9) group. The increase in TNI in the CBP-6 group was statistically significant (*, p=0.034). This data presented on Figure 8 demonstrated that CBP-6 peptide treatment was associated with enhanced tumor necrosis. Furthermore, increase in TNI was observed inversely correlated (r=-0.6) with tumor volume.

[0499] Therefore, the CBP-6 treatment increased the T umor Necrosis Index, reflecting a higher extent of tumor necrosis and anticancer effect of the CBP peptides compared to control.

[0500] Materials and methods

[0501] Cancer cell line

[0502] EO771 were cultured in DMEM (High Glucose, L-Glutamine, Sodium Pyruvate) (Gibco) with 10% foetal calf serum (FCS) (ATCC), 1% Penicillin / streptomycin (Gibco) and 20 mM HEPES, pH=7.5 (Dominique Dutscher). EO771 is a murine breast cancer adenocarcinoma cell line originally isolated as a spontaneous tumor from C57BL / 6 mouse and is most often presented as a TNBC syngeneic model used by orthotopic injection.

[0503] Animals

[0504] Seven-week-old female C57BL / 6 mice were purchased from Janvier Labs (France) and housed in a pathogen-free, temperature-controlled environment with 12-hour day and night cycles. The animals received water and food ad libitum and were allowed to acclimate for at least 1 week before starting experiments. Animal experiments were conducted in compliance with the EU Directive 63 / 2010 and with protocol # 41322 by the local Ethical Committee (‘Reims Champagne Ardenne’ registered at the French Ministry of Research).

[0505] Tumor induction EO771 cells (2.5 x 105in PBS) were orthotopically injected into the fourth right fat pad mammary glands of eight-week-old female C57BL / 6 mice (Janvier Labs, France). Following tumor induction as described above, mice were randomized in two treatment groups: control (PBS), and CBP-6 (10 mg / kg), administered intraperitoneally (i.p.) three times per week until day 19.

[0506] Oman collection & preparation for histolooical analysis

[0507] After one week of treatment, animals were euthanized and tumors collected for histological analysis. Tumor of mice were sampled and fixed after 1 to 3 days incubation into formalin solution at room temperature. Organs were preserved into D-PBS at 4°C until paraffin embedding. Tumors were cut in slides and stained using hematoxylin and eosin (H&E). Then stained slides were analyzed considering the necrotic tumor area relative to the total tumor area estimated using Imaged software.

[0508] Statistical analyses

[0509] The Tumor Necrosis Index (TNI, %) correspond to the percentage of necrotic tissue within tumor: necrotic tumor area (mm2) / total tumor area (mm2) X 100. Higher values indicate improved antitumor efficacy.

[0510] Results are expressed as histogram with mean (+ / - SEM). The effect of treatments on Tumor Necrosis Index (TNI) was analyzed using a parametric t-test. Statistical analysis was performed using GraphPad Prism 10 (GraphPad Software). p<0.05 values were considered indicative of statistical significance.

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Claims

CLAIMS1 . A peptide of no more than 20 amino acids in size comprising the following amino acid sequence (I):XI-GDR-X2-Q-X3-RQ-X4-SG-X5(SEQ ID NO:1) whereinXi is vacant or is an amino acid sequence of one to 4 amino acids;X2is cysteine (C), tyrosine (Y), glutamine (Q) or glycine (G);X3is tyrosine (Y) or tryptophan (W);X4is cysteine (C), valine (V) or tyrosine (Y);Xs is vacant or is an amino acid sequence of one to 4 amino acids; and wherein the peptide comprises no more than one cysteine (C) residue; or a functional variant deriving from said peptide.

2. The peptide according to claim 1 , wherein:•the amino acid sequence of one to 4 amino acids Xi is a leucine (L) residue, phenylanine-leucine (FL), glycine-phenylalanine-leucine (GFL), or proline-glycine- phenylalanine-leucine (PGFL); and / or•the amino acid sequence of one to 4 amino acids X5is is a tyrosine (Y) residue, tyrosine-valine (YV), tyrosine-cysteine (YC), tyrosine-valine-glutamic acid (YVE), tyrosine-cysteine-glutamic acid (YCE), tyrosine-valine-glutamic acid-asparagine (YVEN), or tyrosine-cysteine-glutamic acid-asparagine (YCEN).

3. The peptide according to claim 1 or 2, wherein:If Xi is an amino acid sequence of one to 4 amino acids, X5is vacantIf Xi vacant, X5is an amino acid sequence of one to 4 amino acids.

4. The peptide according to any one of claims 1 to 3, whereinXi is vacant, is a leucine (L) residue, phenylanine-leucine (FL), glycine-phenylalanine- leucine (GFL), or proline-glycine-phenylalanine-leucine (PGFL),X3 is Y, andX5is vacant.

5. The peptide according to claim 4, wherein Xi is proline-glycine-phenylalanine-leucine (PGFL)6. The peptide according to any one of claim 1 to 5, wherein the peptide comprises or consists in an amino acid sequence selected from:PGFLGDRCQYRQVSG (SEQ ID NO:4),PGFLGDRYQYRQVSG (SEQ ID NO:5), andPGFLGDRQQYRQCSG (SEQ ID NO:6)7. The peptide according to any one of claims 1 to 3, whereinXi is vacant,X2is G, andX5is vacant, is a tyrosine (Y) residue, tyrosine-valine (YV), tyrosine-cysteine (YC), tyrosine-valine-glutamic acid (YVE), tyrosine-cysteine-glutamic acid (YCE), tyrosine- valine-glutamic acid-asparagine (YVEN), or tyrosine-cysteine-glutamic acid- asparagine (YCEN).

8. The peptide according to claim 7, wherein X5is tyrosine-valine-glutamic acid- asparagine (YVEN) or tyrosine-cysteine-glutamic acid-asparagine (YCEN).

9. The peptide according to claim 7 or 8, wherein the peptide comprises or consists in an amino acid sequence selected from:GDRGQYRQYSGYVEN (SEQ ID NO:7), GDRGQYRQYSGYCEN (SEQ ID NO:8), GDRGQWRQYSGYCEN (SEQ ID NO:9), and GDRGQWRQCSGYVEN (SEQ ID NQ:10).

10. The peptide according to any one of claim 1 to 3, wherein the peptide has at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

11. The peptide according to claim 10, wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

12. The peptide according to claim 11 , wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

13. A modified peptide deriving from the peptide according to any one of claims 1 to 12, by the introduction of one or more chemical modifications which preferably protect the peptide against proteolysis.

14. A polynucleotide encoding the peptide according to any one of claims 1 to 12.

15. A vector comprising the polynucleotide according to claim 14.

16. The peptide according to any one of claims 1 to 12, the modified peptide according to claim 13, the polynucleotide according to claim 14, or the vector according to claim 15, for use as a medicament.

17. The peptide according to any one of claims 1 to 12, the modified peptide according to claim 13, the polynucleotide according to claim 14, or the vector according to claim 15, for use in treating a proliferative disorder, in particular a cancer.

18. Use of the peptide according to any one of claims 1 to 12 or of the modified peptide according to claim 13 in a method for diagnosing and / or staging a disease associated with Cathepsin D overexpression.

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