Peptide inhibitors of the cathepsin D / LRP-1 interaction

Abstract

The present invention relates to (i) a fragment of SEQ ID NO: 1 comprising at least 5 consecutive amino acids of the amino acid sequence NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 1) or (ii) a functional variant of said fragment, preferably derived from said fragment by deletion, insertion and / or substitution of one or more amino acids, wherein the fragment or functional variant derived therefrom is a peptide 5 to 20 amino acids in size, preferably 7 to 15 amino acids in size, comprising at least one of the amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO: 1, and its use as a pharmaceutical or in diagnostic methods.

Description

[Background technology]

[0001] In cancer, the tumor microenvironment plays an important role in tumor development. The tumor microenvironment is composed of i) the extracellular matrix (ECM), a complex dynamic structure containing fibrous proteins, glycosaminoglycans, glycoproteins, or proteoglycans, and ii) stromal cells such as fibroblasts, immune cells, endothelial cells, and 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, and ovarian cancers (Pranjol et al., 2020; Leto et al., 2004), and can stimulate angiogenesis, metastasis formation, and fibroblast proliferation (Liaudet-Coopman et al., 2006; Glondu et al., 2002; Berchem et al., 2002; Laurent-Matha et al., 2005). These tumor-promoting effects point to cathepsin D as a recognized marker of poor prognosis and a target of therapeutic interest, particularly in breast cancer (Glondu et al., 2002; Dubey et al., 2017; Ashraf et al., 2019; Vetvicka et al., 2012; Brouillet et al., 1997).

[0002] Cathepsin D is a soluble aspartic lysosomal endopeptidase of the cathepsin family. Under physiological conditions, cathepsin D cleaves proteins and peptides in the lysosomal compartment (Masson et al., 2010). Cathepsin D is encoded by the CTSD gene as a preproprotein. This precursor contains a signal sequence, which is cleaved to yield a 52 kDa precursor, pro-cathepsin D. This 52 kDa form is then transported to the endosomal compartment and converted to an active 48 kDa intermediate. In the lysosome, a cysteine ​​endopeptidase cleaves the 48 kDa intermediate chain to generate two chains: a 14 kDa light chain and a 34 kDa heavy chain. These two chains then noncovalently bind to form the mature protease (Masson et al., 2010). In cancer cells, overexpression of the CTSD gene leads to the poor distribution of pro-cathepsin D (52 kDa) in the endosomal compartment, resulting in the release of this immature form into the extracellular compartment (Pranjol et al., 2020; Leto et al., 2004; Heylen et al., 2002). This secreted pro-cathepsin D can stimulate the proliferation of fibroblasts, particularly mammalian fibroblasts. This effect is mediated by its interaction with the β-chain (residues 307–479) of the cell surface receptor LRP-1 (low-density lipoprotein receptor-related protein-1) (Beaujouin et al., 2010; Derocq et al., 2012).

[0003] LRP-1 is a multifunctional membrane receptor belonging to the LDL receptor family that possesses a combination of endocytic and cell signaling properties (Etique et al., 2013). The mature LRP-1 receptor is composed of two chains. The extracellular α-chain (515 kDa) can interact with over 40 distinct extracellular ligands (lipoproteins, proteases, growth factors, toxins, and viruses), internalizing them and, for the most part, targeting them for lysosomal catabolism. The β-chain (85 kDa) has a 476-amino acid extracellular region, a 25-amino acid transmembrane region, and a 100-amino acid cytoplasmic tail. By enabling the endocytic clearance of numerous ligands (including proteases and / or their inhibitors), LRP-1 plays a major role in regulating proteolytic activity within the microenvironment (Etique et al., 2013). In addition to its endocytic function, LRP-1 also contains two main NPTXY motifs that enable interaction with intracellular scaffolding proteins. 29 and NPTXY 63It is also possible that LRP-1 regulates some signaling pathways via the intracellular β-strand domain (Herz et al., 2001). Over the past decade, our research has been confirmed by other international studies, revealing the tumor-promoting 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 may be involved in the regulation of gene expression through a dual cleavage process of its β-strand (Regulated Intramembrane Proteolysis, RIP) (May et al., 2002). The first proteolysis is carried out by membrane-bound proteins called metalloproteinases and sheddases. This first cleavage allows the release of the extracellular portion of LRP-1. The membrane-bound fragment, LRP1β-CTF, then remains and is cleaved by γ-secretase at its transmembrane domain (Hass et al., 2009). The intracellular domain, LRP1β-ICD, is then released into the cytosol, where it can interact with signaling proteins, translocate to the nucleus, and act as a transcriptional regulator. This mechanism is responsible for the effect of cathepsin D on fibroblast proliferation. Indeed, cathepsin D prevents the cleavage and release of LRP1β-CTF by membrane-bound proteases, thereby preventing the release of LRP1β-ICD by γ-secretase, which regulates fibroblast proliferation (Derocq et al., 2012). Thus, cathepsin D promotes fibroblast proliferation through its interaction with the LRP-1 receptor present on the cell surface of fibroblasts. Fibroblasts are associated with cancer at all stages of disease progression, including metastasis, and are key components in tumor development ( Kalluri et al., 2016 ; Houthuijzen et al., 2018 ; Bhowmick et al., 2004 ).

[0004] To date, only a 45AA interacting region (residues 349-394 of LRP-1β) has been identified (Beaujouin et al., 2010). However, this 45AA region, described as "fragment F4" in WO 2009 / 043858, has poor solubility in aqueous solvents, is unstable by NMR, and is difficult to prepare and purify due to the presence of numerous cysteines that form multiple disulfide bridges, making it unlikely for therapeutic applications.

[0005] Therefore, new drugs capable of binding to cathepsin D protein and / or inhibiting the cathepsin D / LRP-1 interaction are needed to develop new targeted therapeutic strategies for the treatment of cancer. Summary of the Invention

[0006] In a first aspect, the present invention relates to a peptide having a size of 5 to 20 amino acids, preferably 7 to 15 amino acids, which is (i) a fragment of SEQ ID NO: 1 comprising at least 5 consecutive amino acids of the amino acid sequence NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 1) or (ii) a functional variant of said fragment, preferably derived from said fragment by deletion, insertion and / or substitution of one or more amino acids (wherein said fragment or functional variant derived therefrom comprises at least one of amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO: 1).

[0007] In one embodiment, the peptide has at least one activity selected from (a) binding to cathepsin D protein, (b) inhibiting the interaction between cathepsin D protein and LRP-1 protein, (c) inhibiting fibroblast proliferation promoted by pro-cathepsin D secreted by cancer cells within the tumor microenvironment, and (d) inhibiting the catalytic activity of cathepsin D.

[0008] In one embodiment, this fragment or a functional variant derived therefrom comprises at least two, three or four of amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1.

[0009] In one embodiment, the peptide comprises: a) the central region of SEQ ID NO: 1, with the amino acid sequence PQCRCLPGFLGDRCQYRQCSGY (SEQ ID NO: 2); b) the N-terminal region of SEQ ID NO: 1, with the amino acid sequence NQGNQPQCRCLPGFLGDRCQYR (SEQ ID NO: 3); c) the C-terminal region of SEQ ID NO: 1, with the amino acid sequence RCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 4); d) a region derived from the amino acid sequence in a), b) or c) by N-terminal and / or C-terminal deletion of 1, 2, 3 or 4 amino acids a fragment of, or A functional variant of the fragment (this fragment or a functional variant derived therefrom comprises at least one of the amino acids corresponding to amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO: 1).

[0010] The fragment or functional variant derived therefrom preferably comprises at least two, three or four of the amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1.

[0011] In one embodiment, this fragment or a functional variant derived therefrom comprises amino acids D17, R18, Q20 and Y21 and / or amino acid R9 of SEQ ID NO:1.

[0012] In one embodiment, this fragment or a functional variant derived therefrom comprises amino acids G16, D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1.

[0013] In one embodiment, this fragment or a functional variant derived therefrom comprises amino acids G16, D17, R18, C19, Q20 and / or amino acid R9 of SEQ ID NO:1.

[0014] In one embodiment, this fragment or a functional variant derived therefrom comprises amino acids D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1.

[0015] In one embodiment, this fragment or a functional variant derived therefrom comprises amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1.

[0016] In one embodiment, the fragment or functional variant derived therefrom comprises at least 8, 9 or 10 consecutive amino acids of SEQ ID NO: 1, 2, 3 or 4.

[0017] In one embodiment, the fragment or functional variant derived therefrom comprises no more than 15, 16, 17, 18, 19 or 20 consecutive amino acids of SEQ ID NO: 1, 2, 3 or 4.

[0018] In one embodiment, the peptide does not contain a C residue at both the N- and C-terminus.

[0019] In one embodiment, the peptide is -RCLPGFLGDRCQYRQ (SEQ ID NO: 10), -CLPGFLGDRCQYRQC (SEQ ID NO: 11), - LPGFLGDRCQYRQCS (SEQ ID NO: 12), -PGFLGDRCQYRQCSG (SEQ ID NO: 13), -GDRCQYRQCSGYCEN (SEQ ID NO: 14), -CRCLPGFLGDRCQYR (SEQ ID NO: 15), -PQCRCLPGFLGDRCQ (SEQ ID NO: 16), -NQGNQPQCRCLPGFL (SEQ ID NO: 17), -DRCQYRQ (SEQ ID NO: 25), -DRCQYRQC (SEQ ID NO: 26), -GDRCQYRQ (SEQ ID NO: 27), - GDRCQYRQC (SEQ ID NO: 28), - GDRCQYRQCS (SEQ ID NO: 29), - LGDRCQYRQC (SEQ ID NO: 30), - LGDRCQYRQCS (SEQ ID NO: 31), -LGDRCQYRQCSG (SEQ ID NO: 32), - FLGDRCQYRQCS (SEQ ID NO: 33), - FLGDRCQYRQCSG (SEQ ID NO: 34), -FLGDRCQYRQCSGY (SEQ ID NO: 35), -GFLGDRCQYRQCSG (SEQ ID NO: 36), -GFLGDRCQYRQCSGY (SEQ ID NO: 37), - GDRCQYRQCSGYCE (SEQ ID NO: 38), -GDRCQYRQCSGY (SEQ ID NO: 39), -GDRCQYRQCSG (SEQ ID NO: 40), a fragment of SEQ ID NO: 1, 2, 3 or 4 selected from the group consisting of: or a functional variant of said fragment, preferably derived from said fragment by deletion, insertion and / or substitution of one or more amino acids.

[0020] In one embodiment, the peptide is capable of inhibiting the interaction between the LRP-1 receptor and cathepsin D by at least 40% as measured by a cathepsin D / LRP-1 co-immunoprecipitation assay.

[0021] In one embodiment, the peptide has a binding affinity K for cathepsin D of less than 500 nM as determined in a microscale thermophoresis assay. D It has a value.

[0022] In another aspect, the present invention relates to modified peptides derived from a peptide according to any of claims 1 to 11 by the introduction of one or more chemical modifications which preferably protect the peptide from proteolytic degradation.

[0023] In another aspect, the present invention relates to a polynucleotide encoding a peptide according to the invention.

[0024] Another aspect of the present invention relates to a vector comprising a polynucleotide according to the present invention.

[0025] In another aspect, the present invention relates to a peptide as described herein, a modified peptide as described herein, a polynucleotide as described herein or a vector as described herein as a medicament.

[0026] A peptide as described herein, a modified peptide as described herein, a polynucleotide as described herein or a vector as described herein is preferably for use in the treatment of a proliferative disorder, in particular cancer.

[0027] Another aspect of the present invention relates to the use of a peptide or a modified peptide as described herein for diagnosing and / or staging a disease associated with cathepsin D overexpression. [Brief explanation of the drawings]

[0028] [Figure 1] In silico workflow used to examine LRP-1 residues involved in cathepsin D / LRP-1 interaction. Different conformations of LRP-derived peptides (F4 fragment and EGF1920 fragment) were generated and then selected for docking experiments with cathepsin D (CathD). [Figure 2]In silico analysis of LRP-1 residues involved in cathepsin D / LRP-1 interaction. Figures 2A and 2B: Histograms showing the contact frequency of residues of EGF1920 (Figure 2A) or the F4 fragment (Figure 2B) with cathepsin D during all molecular docking experiments performed. Residues with a contact frequency of 50% or more are indicated with black arrows. Figure 2C: Overlay of histograms A (white bars) and B (black bars) at the level of the common CRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 7) sequence of the F4 and EGF1920 fragments. Residues with a contact frequency of more than 50% for both fragments are indicated with black arrows. The dotted line indicates the 50% frequency. [Figure 3] Affinity study of the F4 fragment and of the LBC4 and LBC5 peptides for cathepsin D by microscale thermophoresis. Dose-response curves represent the binding interaction between cathepsin D and the F4 fragment, the peptide LBC4 or the peptide LBC5. [Figure 4] We tested the ability of LBC1–LBC5 peptides to block the cathepsin D / LRP-1 β-chain interaction by co-immunoprecipitation. 6-His-tagged cathepsin D (100 ng) was incubated overnight with LRP-1α (LRP1αF) or LRP-1β fragment (LRP1βF) expressing the myc epitope in the absence (Figure 4A) or presence (Figure 4B) of LBC peptide (100 μM). The mixture was then precipitated on Ni-NTA beads for 2 h. The formed complex was eluted with 2X Limley buffer and subjected to SDS-PAGE electrophoresis and Western blotting using antibodies against cathepsin D and the myc tag (Figure 4C). The histogram shows the binding of the LRP-1β fragment to cathepsin D in the absence (Ctrl) or presence (Figure 4B) of LBC. [Figure 5]Effect of LBC1-LBC5 peptides on the proliferation of mouse embryonic fibroblasts (MEF-1) cocultured with COS-7 cells overexpressing or not overexpressing cathepsin D. (Figure 5A) Schematic diagram showing the coculture model used. Fibroblasts were seeded in Matrigel on a monolayer of COS-7 cells overexpressing and secreting cathepsin D (pCD) or not overexpressing and secreting cathepsin D (Ctrl). (Figure 5B) Western blot analysis of the amount of cathepsin D present in the coculture medium after 6 days in wells containing COS-7 cells overexpressing and secreting cathepsin D (pCD) or not overexpressing and secreting cathepsin D (Ctrl). (Figure 5C) Phase-contrast microscopy of fibroblast colonies cocultured with COS-7 Ctrl or pCD cells in the absence or presence of 100 μM LBC1-LBC5 peptides at days 1, 4, and 6. (Figure 5D) Histogram showing colonies with an area greater than 5,000 pixels on day 6 (each point equals one colony). n=5, One-way anova ****: p<0.0001, **: p<0.01, Scale bar: 20 μm [Figure 6] Localization of different peptides derived from the β-chain of LRP-1 synthesized to represent the region of high interaction between LRP-1 and cathepsin D. Peptides LBC1-LBC5 were the first peptides studied. Peptides LBC6-LBC8 were used to represent the N-terminus of the high interaction region, and peptides LBC9-LBC15 were used for the C-terminus of the high interaction region. [Figure 7]NMR spectroscopic characterization of the LBC4 peptide under oxidative conditions. The LBC4 peptide was diluted (130 μl) in 30 μl of HCl / NaCl solution containing 20 mM Tris buffer at pH 7.5, followed by the addition of 16 μl of DMSO (oxidative conditions) or 16 μl of water (non-oxidative conditions). (Figure 7A) After 72 h of incubation at room temperature, 1D 1H NMR spectra in the -NH region were measured at 298 Kelvin. (Figure 7B) Determination of the chemical shift in the NH region between spectra obtained in the absence (bottom) and presence (top) of DMSO (oxidative conditions). (Figures 7C and 7D) Determination of the affinity of the oxidized LBC4 peptide for cathepsin D by microscale thermophoresis. Labeled cathepsin D was mixed with the LBC4 peptide lacking the disulfide bridge between its two cysteines (LBC4) or with (oxidized-LBC4) using serial 0.5-fold dilutions ranging from 10 μM to 0.15 nM. [Figure 8] Effect of LBC4, LBC5, and LBC15 peptides on the proliferation of 2D-cultured MCF-7 and MDA-MB-231 breast cancer cells. MDA-MB-231 and MCF-7 cells were seeded at 5,000 and 10,000 cells / well in 96-well plates, respectively, and incubated for 48 hours with or without peptide (NT) or various concentrations (6.25–50 μM) of LBC4, LBC5, or LBC15 peptides. Cell numbers were measured using the cell proliferation reagent WST-1. [Figure 9]Effect of the LBC peptide on cathepsin D catalytic activity at pH 3.5 and pH 6. (Figure 9A) Cathepsin D (2 ng / μL) was preincubated with the LBC peptide (100 μM) or pepstatin A (2 ng / μL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) for 15 min. Activity was then measured in the presence of a fluorogenic substrate (15 μM). (Figure 9B) Cathepsin D (0.5 ng / μL) was preactivated in acidic buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) for 30 min and then preincubated with the peptide (100 μM) or pepstatin A (2 μg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6) for 15 min. Activity was then measured in the presence of a fluorogenic substrate (30 μM). (Figure 9C) Pro-cathepsin D (0.5 ng / μL) was preactivated in acidic buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) for 30 min and then preincubated with various concentrations (10, 50, 100, 250, and 500 μM) of LBC4 peptide or pepstatin A (2 μg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6) for 15 min. Activity was then measured in the presence of fluorogenic substrate (30 μM). [Figure 10] Effect of LBC peptides on a TNBC tumor model (Figure 10A). Experimental procedure: Patient-derived xenografts (PDX from TNBC, Crown Bioscience, Leiden, Netherlands) were seeded in hydrogel in a 384-well plate. Organoids were treated with various concentrations (10, 30, and 100 μM) of LBC4 (Figure 10B), LBC5 (Figure 10C), and LBC8 (Figure 10D) peptides on days 3 and 7, and cultures were maintained until day 10. Cultures were fixed, stained, and analyzed using TIBCO Spotfire software. n=4, Kruskal-Wallis test with Benjamini, Krieger, and Yekutieli corrections. ***: p<0.001, **: p<0.01, *: p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure is based, at least in part, on the surprising discovery by the inventors that small peptides derived from specific regions of the amino acid sequence of the beta chain of the LRP-1 receptor can bind to cathepsin D protein and inhibit the LRP-1 / cathepsin D interaction, thereby inhibiting fibroblast proliferation promoted by cathepsin D secreted by cancer cells within the tumor microenvironment.

[0030] The inventors have identified a critical region of 30 amino acids involved in the interaction between cathepsin D and LRP-1. This critical amino acid sequence, NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 1), located on the β-strand of the LRP-1 receptor, had not previously been identified and was revealed after six years of research by the inventors. The inventors surprisingly found that small peptides derived from this critical interaction region were sufficient to efficiently bind to cathepsin D and inhibit the LRP-1 / cathepsin D interaction. The inventors also demonstrated the efficacy of some of these peptides for inhibiting fibroblast proliferation promoted by cathepsin D secreted by cancer cells in a model mimicking the cellular tumor environment.

[0031] peptide Thus, in a first aspect, the present invention provides a peptide having a size of 5 to 20 amino acids, preferably 7 to 15 amino acids, and having at least one activity selected from (a) binding to cathepsin D protein, (b) inhibiting the interaction between cathepsin D protein and LRP-1 protein, (c) inhibiting fibroblast proliferation promoted by cathepsin D secreted by cancer cells in the tumor microenvironment, and (d) inhibiting the catalytic activity of cathepsin D, wherein the peptide is (i) a fragment of SEQ ID NO: 1 comprising at least 5 consecutive amino acids of the amino acid sequence NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 1), or (ii) a functional variant of said fragment, preferably derived from said fragment by deletion, insertion and / or substitution of one or more amino acids (wherein said fragment or functional variant derived therefrom comprises at least one of amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO: 1).

[0032] In one embodiment, a fragment of SEQ ID NO: 1 or a functional variant derived therefrom comprises at least two, three or four of amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1.

[0033] In one embodiment, a fragment of SEQ ID NO: 1 or a functional variant derived therefrom comprises at least amino acids G16, D17, R18, C19, Q20 and / or amino acid R9 of SEQ ID NO: 1.

[0034] In one embodiment, the peptide comprises: a) the central region of SEQ ID NO: 1, with the amino acid sequence PQCRCLPGFLGDRCQYRQCSGY (SEQ ID NO: 2); b) the N-terminal region of SEQ ID NO: 1, with the amino acid sequence NQGNQPQCRCLPGFLGDRCQYR (SEQ ID NO: 3); c) the C-terminal region of SEQ ID NO: 1, with the amino acid sequence RCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 4); d) a region derived from the amino acid sequence in a), b) or c) by N-terminal and / or C-terminal deletion of 1, 2, 3 or 4 amino acids or (ii) a functional variant of said fragment, preferably derived from said fragment by deletion, insertion and / or substitution of one or more amino acids (this fragment or functional variant derived therefrom comprises at least one of the amino acids corresponding to amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO: 1).

[0035] In another provision, in this embodiment, the peptide is a fragment of SEQ ID NO: 2, 3 or 4, or - amino acids R3, G10, D11, R12, C13, Q14, Y15 of SEQ ID NO: 2; - amino acids R9, G16, D17, R18, C19, Q20, Y21 of SEQ ID NO: 3; amino acids G8, D9, R10, C11, Q12, Y13 of SEQ ID NO: 4 and functional variants of said fragments, each comprising at least one of:

[0036] In one embodiment, the fragment of SEQ ID NO: 2, 3 or 4 or a functional variant derived therefrom comprises at least two, three or four of amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1. In another provision, in this embodiment, the peptide comprises a fragment of SEQ ID NO: 2, 3 or 4 or at least two, three or four of the amino acids G10, D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2; at least two, three or four of the amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 3; - at least 2, 3 or 4 of the amino acids G8, D9, R10, C11, Q12 and Y13 of SEQ ID NO: 4 In one embodiment, the fragment of SEQ ID NO: 2, 3 or 4 or a functional variant derived therefrom comprises at least amino acids D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1. In another provision, in this embodiment, the peptide is a fragment of SEQ ID NO: 2, 3 or 4 or amino acids D11, R12, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2; amino acids D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 3; amino acids D9, R10, Q12, Y13 of SEQ ID NO: 4 and a functional variant of said fragment, each comprising:

[0037] In one embodiment, the fragment of SEQ ID NO: 2, 3 or 4 or a functional variant derived therefrom comprises at least amino acids G16, D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1. In another provision, in this embodiment, the peptide comprises a fragment of SEQ ID NO: 2, 3 or 4 or amino acids G10, D11, R12, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2; amino acids G16, D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 3; amino acids G8, D9, R10, Q12, Y13 of SEQ ID NO: 4 and a functional variant of said fragment, each comprising:

[0038] In one embodiment, the fragment of SEQ ID NO: 2, 3 or 4 or a functional variant derived therefrom comprises at least amino acids G16, D17, R18, C19, Q20 and / or amino acid R9 of SEQ ID NO: 1. In another provision, in this embodiment, the peptide is a fragment of SEQ ID NO: 2, 3 or 4 or amino acids G10, D11, R12, C13, Q14 and / or amino acid R3 of SEQ ID NO: 2; amino acids G16, D17, R18, C19, Q20 and / or amino acid R9 of SEQ ID NO: 3; amino acids G8, D9, R10, C11, Q12 of SEQ ID NO: 4 and a functional variant of said fragment, each comprising:

[0039] In one embodiment, the fragment of SEQ ID NO: 2, 3 or 4 or a functional variant derived therefrom comprises at least amino acids D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1. In another provision, in this embodiment, the peptide is a fragment of SEQ ID NO: 2, 3 or 4 or amino acids D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2; amino acids D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 3; - amino acids D9, R10, C11, Q12, Y13 of SEQ ID NO: 4 and functional variants of said fragments, each comprising:

[0040] In one embodiment, the fragment of SEQ ID NO: 2, 3 or 4 or a functional variant derived therefrom comprises at least amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1. In another provision, in this embodiment, the peptide is a fragment of SEQ ID NO: 2, 3 or 4 or amino acids G10, D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2; amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 3; amino acids G8, D9, R10, C11, Q12, Y13 of SEQ ID NO: 4 and a functional variant of said fragment, each comprising:

[0041] Other characteristics of the fragment In one embodiment, the peptide is a peptide having a size of 5 to 20 amino acids, preferably a peptide having a size of 7 to 15 amino acids.

[0042] In one embodiment, the peptide is a peptide having a size of 7 to 20 amino acids, preferably a peptide having a size of 7 to 15 amino acids.

[0043] In one embodiment, the peptide comprises no more than 15, 16, 17, 18, 19, or 20 amino acids.

[0044] In one embodiment, the peptide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0045] In one embodiment, the fragment or functional variant derived therefrom comprises at least 5, 6, 7, 8, 9 or 10 consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0046] In one embodiment, the fragment or functional variant derived therefrom comprises no more than 15, 16, 17, 18, 19 or 20 consecutive amino acids of SEQ ID NO:1.

[0047] In one embodiment, the fragment or a functional variant derived therefrom comprises 5 to 20, preferably 5 to 15, consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0048] In one embodiment, the fragment or a functional variant derived therefrom comprises 7 to 20, preferably 7 to 15, consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0049] In one embodiment, the fragment or functional variant derived therefrom comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0050] In one embodiment, the peptide does not contain a C residue at either the N-terminus or the C-terminus.

[0051] Specific fragments In certain embodiments, the peptide is LBC1 with the amino acid sequence RCLPGFLGDRCQYRQ (SEQ ID NO: 10), LBC2 with the amino acid sequence CLPGFLGDRCQYRQC (SEQ ID NO: 11), LBC3 with the amino acid sequence LPGFLGDRCQYRQCS (SEQ ID NO: 12), LBC4 with the amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO: 13), LBC5 with the amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO: 14), LBC6 with the amino acid sequence CRCLPGFLGDRCQYR (SEQ ID NO: 15), LBC7 with the amino acid sequence PQCRCLPGFLGDRCQ (SEQ ID NO: 16), LBC8 with the amino acid sequence NQGNQPQCRCLPGFL (SEQ ID NO: 17), LBC16 with the amino acid sequence DRCQYRQ (SEQ ID NO: 25), LBC17 with the amino acid sequence DRCQYRQC (SEQ ID NO: 26), LBC18 with the amino acid sequence GDRCQYRQ (SEQ ID NO: 27), LBC19 with the amino acid sequence GDRCQYRQC (SEQ ID NO: 28), LBC20 with the amino acid sequence GDRCQYRQCS (SEQ ID NO: 29), LBC21 with the amino acid sequence LGDRCQYRQC (SEQ ID NO: 30), LBC22 with the amino acid sequence LGDRCQYRQCS (SEQ ID NO: 31), LBC23 with the amino acid sequence LGDRCQYRQCSG (SEQ ID NO: 32), LBC24 with the amino acid sequence FLGDRCQYRQCS (SEQ ID NO: 33), LBC25 with the amino acid sequence FLGDRCQYRQCSG (SEQ ID NO: 34), LBC26 with the amino acid sequence FLGDRCQYRQCSGY (SEQ ID NO: 35), LBC27 with the amino acid sequence GFLGDPRCQYRQCSG (SEQ ID NO: 36), LBC28 with the amino acid sequence GFLGDRCQYRQCSGY (SEQ ID NO: 37), LBC29 with the amino acid sequence GDRCQYRQCSGYCE (SEQ ID NO: 38), LBC30 with the amino acid sequence GDRCQYRQCSGY (SEQ ID NO: 39), LBC31 with the amino acid sequence GDRCQYRQCSG (SEQ ID NO: 40), A fragment of SEQ ID NO: 1, 2, 3 or 4 selected from the group consisting of or a functional variant of said fragment, preferably derived from said fragment by deletion, insertion and / or substitution of one or more amino acids (this fragment or functional variant derived therefrom comprises at least one of the amino acids corresponding to amino acids R9, D17, R18, C19, Q20 and Y21 of SEQ ID NO: 1).

[0052] In certain embodiments, the peptide is LBC4 with the amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO: 13) LBC5 with amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO: 14) LBC6 with the amino acid sequence CRCLPGFLGDRCQYR (SEQ ID NO: 15) LBC16 with amino acid sequence DRCQYRQ (SEQ ID NO: 25) LBC31 with the amino acid sequence GDRCQYRQCSG (SEQ ID NO: 40) A fragment of SEQ ID NO: 1, 2, 3 or 4 selected from the group consisting of or a functional variant of said fragment, preferably derived from said fragment by deletion, insertion and / or substitution of one or more amino acids (this fragment or functional variant derived therefrom comprises at least one of the amino acids corresponding to amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO: 1).

[0053] In one embodiment, the peptide is not a fragment of SEQ ID NO:15.

[0054] Peptide activity The peptide preferably comprises: (a) the ability to bind to cathepsin D protein; (b) the ability to inhibit the LRP-1 protein / cathepsin D protein interaction; (c) the ability to inhibit fibroblast proliferation promoted by cathepsin D secreted by cancer cells within the tumor microenvironment and (iii) at least one activity selected from the group consisting of:

[0055] Unless otherwise stated, cathepsin D and pro-cathepsin D are referred to as "cathepsin D" in this disclosure. Cathepsin D is a protein (EC: 3.4.23.5) encoded by the human full-length CTSD gene (Gene ID: 1509; Genbank accession number NM_001909). Pro-cathepsin D is a 52 kDa, catalytically inactive precursor of cathepsin D.

[0056] The term "LRP1" or "LRP-1" refers to LDL receptor-related protein 1, which is well known in the art. The LRP1 protein is composed of a 515 kDa extracellular α-chain and an 85 kDa β-chain, which are generated by proteolytic cleavage from a 600 kDa precursor polypeptide in the trans-Golgi compartment. The LRP1 α-chain and LRP1 β-chain arise from a single transcript. The human full-length unprocessed precursor LRP1 corresponds to UniProtKB / SwissProt accession number Q07954.

[0057] (a) Ability to bind to cathepsin D In one embodiment, the peptide is capable of binding to cathepsin D, particularly in vitro and / or in vivo.

[0058] In certain embodiments, the peptide is capable of specifically binding to the extracellular portion of the LRP-1 β chain (LRP-1βC).

[0059] The phrase "capable of specifically binding to the extracellular portion of the LRP-1 β chain" means that the peptide binds to LRP-1 βC with a higher binding affinity (e.g., about 2-fold or more, about 5-fold or more, about 10-fold or more, about 30-fold or more, about 100-fold or more, about 1,000-fold or more, or about 10,000-fold or more) than to another portion of the LRP1 protein, particularly the LRP-1 α chain. In one embodiment, the peptide does not exhibit any binding to the α chain of LRP-1 (LRP1αC).

[0060] In one embodiment, the peptide has a binding affinity K for cathepsin D of less than 500 nM as determined by a microscale thermophoresis assay. D It has a value.

[0061] In preferred embodiments, the peptide has a binding affinity KD value for cathepsin D of less than 400 nM, less than 300 nM, as determined by a microscale thermophoresis assay.

[0062] Binding affinities by microscale thermophoresis are well known to those skilled in the art and can be identified and / or quantified by carrying out the following procedure: providing the peptide to be tested, pro-cathepsin D, labeled with a His-Tag; Mix the peptide to be tested with labeled pro-cathepsin D using 0.5-fold serial dilutions ranging from -10 μM to 0.15 μM; -analyzing the mixture on an instrument for MST, for example using a Monolith NT.115 (NanoTemper) instrument at 25°C with the following instrument parameters: 20% Pico-RED excitation power, 40% medium MST power and 5 / 20 / 5 laser off / on / off; analyzing the 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 for pro-cathepsin D.

[0063] The detailed procedure is also described in the experimental part below.

[0064] (b) Ability to inhibit LRP-1 / cathepsin D interaction The term "inhibit" or "inhibitor" refers to the ability of a compound to attenuate, slow, stop, or prevent the activity of a particular biological process. In some embodiments, the term refers to the interaction between LRP-1 and cathepsin D. In some embodiments, the term refers to the proliferation of fibroblasts promoted by cathepsin D secreted by cancer cells within the tumor microenvironment. In some embodiments, the term refers to the catalytic activity of pro-cathepsin D.

[0065] In one embodiment, the peptide is capable of inhibiting the LRP-1 / cathepsin D interaction.

[0066] In one embodiment, the peptide is capable of inhibiting at least 30% of the interaction between the LRP-1 receptor and cathepsin D as measured by a cathepsin D / LRP-1 co-immunoprecipitation assay.

[0067] The peptide is preferably capable of inhibiting at least 40%, preferably at least 50%, preferably at least 60% of the cathepsin D / LRP-1 interaction as determined by co-immunoprecipitation.

[0068] Determining and quantifying the ability of peptides to inhibit the cathepsin D / LRP-1 interaction by co-immunoprecipitation can be readily performed by one skilled in the art.

[0069] The ability of a peptide to inhibit the interaction between this LRP-1 receptor and cathepsin D can be identified and / or quantified by carrying out the following procedure: -Providing a peptide to be tested tagged with 6His-Tag and recombinant cathepsin D; - Incubating 100 ng of cathepsin D with LRP-1α (LRP1αF) or LRP-1β fragment (LRP1βF) presenting a myc epitope overnight in the absence (control) or presence of the peptide to be tested, e.g., 100 μM; - depositing the mixture on Ni-NTA beads for 2 hours; -Elute the formed complex using 2x Limrie buffer; - SDS-PAGE electrophoresis and Western blotting were performed using antibodies against pro-cathepsin D and the myc tag; - Identifying and / or quantifying the binding activity of LRP-1β fragments to pro-cathepsin D in the absence (Ctrl) or presence of the peptide to be tested.

[0070] The detailed procedure is also described in the experimental part below.

[0071] (c) The ability to inhibit fibroblast proliferation promoted by cathepsin D secreted by cancer cells within the tumor microenvironment. In one embodiment, the peptides may inhibit fibroblast proliferation in vitro or in vivo, particularly that promoted by cathepsin D secreted by cancer cells within the tumor microenvironment.

[0072] In one embodiment, the peptide can inhibit at least 30% of fibroblast proliferation promoted by cathepsin D secreted by cancer cells within the tumor microenvironment, as determined in a 3D co-culture model of cancer cells and fibroblasts.

[0073] The peptides are preferably capable of inhibiting at least 40%, preferably at least 50%, preferably at least 60% of fibroblast proliferation promoted by cathepsin D secreted by cancer cells within the tumor microenvironment, as determined in a 3D co-culture model of cancer cells and fibroblasts.

[0074] The ability of the present peptides to inhibit the proliferation of cells, particularly fibroblasts, promoted by cathepsin D secreted by cancer cells within the tumor microenvironment can be identified and / or quantified by carrying out the following procedure: providing the peptide to be tested, fibroblasts and a monolayer of COS-7 cells overexpressing and secreting cathepsin D (pCD) or not (Ctrl); - seeding fibroblasts embedded in solubilized basement membrane matrix onto a monolayer of pCD or Ctrl COS-7 cells in the presence or absence of the peptide to be tested (e.g., 100 μM); - co-culturing COS-7 Ctrl or pCD cells with fibroblast colonies in the absence or presence of the peptide to be tested and observing the co-cultures by phase contrast microscopy for 6 days, e.g., on days 1, 4 and 6; -pCD To identify and / or quantify the inhibitory activity of the peptides to be tested on the proliferation of fibroblasts stimulated by cathepsin D secreted by COS-7 cells.

[0075] A solubilized basement membrane is, for example, the solubilized form of basement membrane secreted by the Engelbreth-Holm-Swarm (EHS) mouse sarcoma produced by Corning Life Sciences under the trade name "Matrigel."

[0076] (d) the ability to inhibit the activity of cathepsin D In one embodiment, the peptides may inhibit the catalytic activity of cathepsin D, particularly within the tumor microenvironment.

[0077] In one embodiment, the peptides are capable of inhibiting at least 20%, preferably 50%, of the catalytic activity of cathepsin D, particularly within the tumor microenvironment, as determined in an assay at pH 6. It is well known that the tumor microenvironment exhibits a pH of 5.6 to 6.8. The ability of a peptide to inhibit the catalytic activity of cathepsin D within the tumor microenvironment can be identified and / or quantified by carrying out the following procedure: providing pro-cathepsin D, the peptide to be tested and pepstatin A (control), - pre-incubating pro-cathepsin D (20 μg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) for 30 min at 37°C to activate it, - Incubating activated pro-cathepsin D (0.5 ng / μL) with the peptide to be tested (100 μM) or pepstatin A (2 μg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6) for 15 minutes, - measuring catalytic activity in the presence of a fluorogenic substrate (30 μM), - Identifying and / or quantifying the ability of the peptides to be tested to inhibit the catalytic activity of cathepsin D in a medium that mimics the pH of the tumor microenvironment.

[0078] Functional variants "Functional variant" refers to an amino acid sequence derived from a fragment described herein and that retains at least one of the activities of the fragment from which it is derived.

[0079] A functional variant is derived from a fragment as described herein by the introduction of one or more mutations (deletions, insertions and / or substitutions) at specific amino acid positions, provided that it comprises at least some amino acids of SEQ ID NO: 1, 2, 3 or 4 as described above.

[0080] In particular, functional variants include: amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO: 1, - amino acids R3, G10, D11, R12, C13, Q14, Y15 of SEQ ID NO: 2; - amino acids R9, G16, D17, R18, C19, Q20, Y21 of SEQ ID NO: 3; amino acids G8, D9, R10, C11, Q12, Y13 of SEQ ID NO: 4 Contains at least one of the following:

[0081] A functional variant preferably comprises an amino acid sequence that is "substantially homologous" or "substantially equivalent" to the sequence of the reference amino acid sequence from which it is derived. Two amino acid sequences are "substantially homologous" or "substantially equivalent" if one or more amino acid residues are replaced by a biologically equivalent residue, or if the sequences are at least 80% identical or 90% similar.

[0082] The percent amino acid sequence identity / similarity is defined as the percentage of amino acid residues in the comparison sequence that are identical / equivalent to the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum sequence identity. The percent identity is then determined according to the formula: percent identity = 100 x [1 - (C / R)], where C is the number of differences between the reference sequence and the comparison sequence over the entire length of the reference sequence (where (i) each amino acid in the reference sequence that does not have a corresponding aligned amino acid in the comparison sequence, (ii) each gap in the reference sequence, and (iii) each aligned amino acid in the reference sequence that is not identical / equivalent to an amino acid in the comparison sequence constitutes a difference); R is the number of amino acids in the reference sequence over the length of the alignment with the comparison sequence, with any gaps created in the reference sequence also counted as an amino acid).

[0083] Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways known to those of skill in the art, using publicly available computer software such as BLAST (Altschul et al., 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 other program known in the art. When using such software, default parameters are preferably used, e.g., for gap penalties and extension penalties. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3 and an expectation (E) of 10.

[0084] In one embodiment, the functional variant is derived from a fragment as described herein, in particular from a fragment of amino acid sequence number 1, 2, 3 or 4 as described herein by deletion, insertion and / or substitution of one or more amino acids.

[0085] In one embodiment, the functional variant is derived from a fragment as described herein, in particular from a fragment of amino acid sequence SEQ ID NO: 1, 2, 3 or 4 as described herein by deletion, insertion and / or substitution of 1, 2, 3 or 4 amino acids.

[0086] Preferably, the substitutions are conservative substitutions.

[0087] Conservative substitution refers to the substitution of one amino acid for another amino acid that does not change the overall conformation and function of the peptide, including, but not limited to, the substitution of an amino acid with an amino acid having similar chemical or physical properties (size, charge, or polarity) that does not alter the overall 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 the substitution of one amino acid for another amino acid with similar properties.

[0088] Examples of conservative substitutions are shown in Table 1 below: [Table 1]

[0089] In one embodiment, a functional variant is derived from a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above by one or more conservative substitutions, preferably by conservative substitutions of 1, 2, 3 or 4 amino acids. In one embodiment, a functional variant is derived from a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above by conservative substitutions of one or more cysteine ​​residues.

[0090] The inventors have shown that in an oxidizing environment, the cysteines in the LBC4 peptide are able to form disulfide bridges, resulting in the peptide's inability to block the catalytic activity of cathepsin D. Furthermore, the inventors have also shown that substitution of cysteine ​​8 in the LBC4 peptide with serine (C8S) impairs the peptide's ability to block cathepsin D catalytic activity. Without wishing to be bound by theory, the inventors believe that the latter suggests that, at least in some embodiments, this particular cysteine ​​residue should preferably remain unchanged.

[0091] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above comprising at least two cysteine ​​residues and functional variants derived from said fragments by conservative substitution of one or more of these cysteine ​​residues with one or more amino acid residues, preferably selected from serine (S), valine (V), threonine (T) and selenocysteine, with amino acid C19 of SEQ ID NO: 1, C13 of SEQ ID NO: 2; C19 of SEQ ID NO: 3 or C11 of SEQ ID NO: 4 preferably remaining unchanged.

[0092] In one embodiment, the fragment is - amino acids C19 and at least one of C10 and C24 of SEQ ID NO: 1, - amino acids C14 and at least one of C5 or C19 of SEQ ID NO: 2, - amino acids C19 and at least one of C8 and C10 of SEQ ID NO: 3, - amino acids C11 and at least one of C2 and C16 of SEQ ID NO: 4 a fragment of SEQ ID NO: 1, 2, 3 or 4 as defined above, comprising and: - one or two of the amino acids C10 and C24 of SEQ ID NO: 1, - one or two of the amino acids C5 and C19 of SEQ ID NO: 2, - one or two of the amino acids C8 and C10 of SEQ ID NO: 3, - one or two of amino acids C2 and C16 of SEQ ID NO: 4 of, by conservative substitutions, preferably with amino acids preferably selected from serine (S), valine (V), threonine (T) and selenocysteine, A functional variant derived from said fragment.

[0093] In one embodiment, the fragment is amino acids C19 and C24 of SEQ ID NO: 1, amino acids C14 and C19 of SEQ ID NO: 2, amino acids C11 and C16 of SEQ ID NO: 4 A fragment of SEQ ID NO: 1, 2 or 4 as described above comprising and, amino acid C24 of SEQ ID NO: 1, amino acid C19 of SEQ ID NO: 2, amino acid C16 of SEQ ID NO: 4 of, Preferably, functional variants derived from said fragments by conservative substitution with amino acids selected from serine (S), valine (V), threonine (T) and selenocysteine.

[0094] In one embodiment, the fragment is LBC4 with the amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO: 13), LBC5 with the amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO: 14), LBC6 with the amino acid sequence CRCLPGFLGDRCQYR (SEQ ID NO: 15), LBC31 with the amino acid sequence GDRCQYRQCSG (SEQ ID NO: 40) A fragment of SEQ ID NO: 1, 2 or 4 as described above selected from and amino acid C13 of SEQ ID NO: 13, amino acid C9 of SEQ ID NO: 14, - one or two amino acids C1 and C3 of SEQ ID NO: 15, amino acid C4 of SEQ ID NO: 40 and functional variants derived from said fragments by at least conservative substitutions with amino acids preferably selected from serine (S), valine (V), threonine (T) and selenocysteine.

[0095] In one embodiment, the functional variant comprises or consists of an amino acid sequence that differs from the sequence of a fragment of SEQ ID NO: 1, 2, 3 or 4 as defined above by not more than 1, 2 or 3 amino acids, preferably not more than 1 or 2 amino acids.

[0096] In one embodiment, the functional variant comprises or consists of an amino acid sequence that is at least 70%, 80%, 85%, 90% or 95% homologous to a fragment of SEQ ID NO: 1, 2, 3 or 4 as defined above.

[0097] In one embodiment, the functional variant comprises or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous to a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above.

[0098] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence that shares at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4.

[0099] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence sharing at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, wherein the fragment or functional variant derived therefrom comprises - amino acids R9, G16, D17, R18, C19, Q20, Y21 of SEQ ID NO: 1, - amino acids R3, G10, D11, R12, C13, Q14, Y15 of SEQ ID NO: 2, - amino acids R9, G16, D17, R18, C19, Q20, Y21 of SEQ ID NO: 3, amino acids G8, D9, R10, C11, Q12, Y13 of SEQ ID NO: 4 Contains at least one of the following:

[0100] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence sharing at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, and the fragment or functional variant derived therefrom comprises amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1, amino acids G10, D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2, amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 3, amino acids G8, D9, R10, C11, Q12, Y13 of SEQ ID NO: 4 It includes at least two, three, or four of the following:

[0101] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence that shares at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, and the fragment or functional variant derived therefrom comprises at least: amino acids D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1, amino acids D11, R12, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2, amino acids D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 3, amino acids D9, R10, Q12, Y13 of SEQ ID NO: 4 Includes:

[0102] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence sharing at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, and the fragment or functional variant derived therefrom comprises at least amino acids G16, D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1, amino acids G10, D11, R12, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2, amino acids G16, D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 3, amino acids G8, D9, R10, Q12, Y13 of SEQ ID NO: 4 Includes:

[0103] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence sharing at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, and the fragment or functional variant derived therefrom comprises at least: amino acids G16, D17, R18, C19, Q20 and / or amino acid R9 of SEQ ID NO: 1, amino acids G10, D11, R12, C13, Q14 and / or amino acid R3 of SEQ ID NO: 2, amino acids G16, D17, R18, C19, Q20 and / or amino acid R3 of SEQ ID NO: 3, amino acids G8, D9, R10, C11, Q12 of SEQ ID NO: 4 Includes:

[0104] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence sharing at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, and the fragment or functional variant derived therefrom comprises at least: amino acids D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1, amino acids D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2, amino acids D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 3, amino acids D9, R10, C11, Q12, Y13 of SEQ ID NO: 4 Includes:

[0105] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence sharing at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, and the fragment or functional variant derived therefrom comprises at least: amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO: 1, amino acids G10, D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO: 2, amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R3 of SEQ ID NO: 3, amino acids G8, D9, R10, C11, Q12, Y13 of SEQ ID NO: 4 Includes:

[0106] In one embodiment, the fragment is LBC4 with the amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO: 13), LBC5 with the amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO: 14) and functional variants derived therefrom comprise or consist of an amino acid sequence sharing at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 13 or 14, wherein the fragment or functional variant derived therefrom comprises at least: - amino acids D6, R7, Q9, Y10 of SEQ ID NO: 13, amino acids D2, R3, Q5, Y6 of SEQ ID NO: 14 Includes:

[0107] In one embodiment, the fragment is LBC4 with the amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO: 13), LBC5 with the amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO: 14) and functional variants derived therefrom comprise or consist of an amino acid sequence sharing at least 70% amino acid sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 13 or 14, wherein the fragment or functional variant derived therefrom comprises at least: - amino acids G5, D6, R7, Q9 and Y10 of SEQ ID NO: 13, amino acids G1, D2, R3, Q5, Y6 of SEQ ID NO: 14 Includes:

[0108] Modified peptides In another aspect, the peptides of the present invention are modified peptides derived from the aforementioned peptides by the introduction of one or more amino acid residues, peptide bonds, any modification at the N-terminus and / or C-terminus of the peptide, which retains at least one of the activities of the peptide from which it is derived.

[0109] These modifications, which can be introduced into peptides by conventional methods known to those skilled in the art, include, but are not limited to, the substitution of natural amino acids with non-proteinogenic amino acids (D-amino acids or amino acid analogs); the modification of peptide bonds, in particular with retro- or retro-inverso-type bonds or bonds different from peptide bonds; cyclization and the addition of chemical groups to the side chains or termini of the peptide, in particular for coupling agents of interest to the proteins of the invention.

[0110] These modifications may be used in particular to improve the in vivo stability of the peptide, in particular its resistance to proteolysis.

[0111] Preferably, the peptide comprises one or more chemical modifications, more preferably chemical modifications that protect the peptide from proteolytic degradation.

[0112] The N-terminus and / or C-terminus of the peptide are advantageously protected from proteolysis, for example the N-terminus in the form of an acetyl group and / or the C-terminus in the form of an amide group.

[0113] Alternatively, or in addition, the peptides may be protected from proteolysis by internal modifications such as replacement of at least one -CONH-peptide bond with a (CHNH) reduction bond, a (NHCO) retro-inverso bond, a (CH-O) methylene-oxy bond, a (CH-S) thiomethylene bond, a (CHCH) carba bond, a (CO-CH) cetomethylene bond, a (CHOH-CH) hydroxyethylene bond, a (NN) bond, an E-arsene bond, or a -CH=CH- bond.

[0114] Alternatively, or in addition, the peptides may be modified by acetylation, acylation, amidation, cross-linking, cyclization, disulfide bond formation, covalent cross-link formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristylation, oxidation, phosphorylation, and the like.

[0115] Alternatively or additionally, the peptide is advantageously composed of amino acids in the D configuration, which renders the peptide resistant to proteolysis.

[0116] Alternatively or additionally, the peptides are stabilized by intramolecular cross-linking 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 cross-linking of the chains according to the so-called "stapled-peptide technology" described by Walensky et al., Science, 2004, 305, 1466-1470.

[0117] Alternatively or additionally, the peptides are advantageously stabilized by covalent attachment to polyethylene glycol (PEG) molecules, preferably 1500 Da or 4000 Da PEG, advantageously attached to their C-terminus or lysine residues. Such coupling may have at least one of the following advantages: (i) improved peptide stability in vivo without affecting its affinity, (ii) reduced urinary clearance and therapeutic dose, and / or (iii) increased plasma half-life.

[0118] Alternatively or additionally, the peptides are advantageously stabilized and their half-lives extended by incorporation into biodegradable and biocompatible polymeric materials, such as poly-lactide-co-glycolide (PLGA), for drug delivery systems that form microspheres.

[0119] Alternatively or additionally, the peptides are advantageously fused to one or more other protein / peptide moieties at their N-terminus and / or C-terminus, resulting in fusion or chimeric peptides, including those that enable cellular targeting of the peptides or modified peptides of the present invention and / or improve bioavailability, production in expression systems, and / or stability of the peptides. The length of the chimeric peptide is not critical to the present invention, so long as the peptide remains functional. These protein / peptide moieties can be (i) cell-penetrating moieties or (ii) targeting moieties for directing the chimeric protein to specific cell types or compartments. Cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs), membrane transport sequences (MTSs), transport peptides, carrier peptides, or Trojan peptides, are well known in the art. CPPs can be translocated into cells (including the cytoplasm and organelles such as mitochondria or nuclei) at levels significantly higher than passive diffusion without causing substantial membrane damage, and can be used as vectors for other molecules when linked to them. Furthermore, the peptide or modified peptide may be separated from the peptide / protein moiety by a linker that is long enough to avoid inhibiting the interaction between the peptide or modified peptide and the cell penetrating or targeting moiety.

[0120] In one embodiment, the peptide is advantageously fused at its N- and / or C-terminus to one or more CPPs that allow the peptide to be vectorized into the lysosome. The peptide may for example be fused at its N- and / or C-terminus to a polyhistidine peptide, preferably the H16 peptide of sequence HHHHHHHHHHHHHHHH (SEQ ID NO: 42).

[0121] Polynucleotides Another aspect of the present invention relates to isolated polynucleotides encoding the peptides.

[0122] The polynucleotide may be synthetic or recombinant DNA, RNA, or a combination thereof, either single-stranded and / or double-stranded. The polynucleotide encodes the peptide in an expressible form, i.e., it is a nucleic acid molecule that, upon expression in a cell or cell-free system, produces a functional peptide.

[0123] Preferably, the polynucleotide contains a coding sequence that is optimized for the host in which the peptide is expressed.

[0124] In another preferred embodiment, the polynucleotide is inserted into a vector. Preferably, the recombinant vector is an expression vector capable of expressing the 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 the 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 conventional vectors used in gene modification and gene therapy, including, for example, plasmids and viral vectors, such as lentiviral and adenoviral vectors.

[0125] Therapeutic Uses The inventors have demonstrated that small peptides derived from key interacting regions of LRP1, as described above, are effective in binding to cathepsin D and / or inhibiting the LRP-1 / cathepsin D interaction, demonstrating their efficacy in models that mimic the tumor cell environment.

[0126] Another aspect of the present invention relates to a peptide, modified peptide, polynucleotide and / or vector as described herein as a medicament, which is particularly useful for inhibiting fibroblast proliferation promoted by cancer cells within the tumor microenvironment.

[0127] The term "tumor microenvironment" refers to the dynamic medium containing cells and macromolecules that interact with cancer cells and promote tumor initiation, progression, and / or metastasis.

[0128] Thus, another aspect of the present invention relates to a peptide, polynucleotide and / or vector as described herein for use in the treatment of a proliferative disorder, in particular cancer, preferably in a human patient.

[0129] The term "proliferative disorder" refers to a disorder caused by abnormal growth or expansion by cell proliferation. A proliferative disorder can be associated with the pathological proliferation of cells in a normal quiescent state and / or the 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.

[0130] The term "tumor" refers to an abnormal mass of tissue in which the growth of the mass exceeds and is not as coordinated as that of normal tissue. Neoplasms or tumors can be "benign" or "malignant" depending on the following characteristics: degree of cellular differentiation (including morphology and function), growth rate, local invasion, and metastasis. "Benign tumors" are generally well differentiated, grow much slower than malignant tumors, and remain localized at the site of appearance. Furthermore, benign tumors lack the ability to invade, invade, or metastasize to distant sites. Exemplary benign tumors include, but are not limited to, lipomas, chondromas, adenomas, acrochordons, senile hemangiomas, seborrheic keratoses, lentigines, and sebaceous hyperplasia. In some cases, some "benign" tumors can later progress to malignant tumors; this may be due to additional genetic alterations in tumor cell subpopulations of the tumor; these tumors are referred to as "premalignant tumors." An exemplary premalignant tumor is a teratoma. On the other hand, "malignant tumors" are generally poorly differentiated (anaplastic) and grow very rapidly with progressive infiltration, invasion, and destruction of surrounding tissue. Furthermore, malignant tumors often have the ability to metastasize to distant locations. The terms "metastasis," "metastatic," or "migration" refer to the spread or transfer of cancer cells from a primary or original tumor to another organ or tissue, and are generally determined by the presence of a "secondary tumor" or "secondary cell mass" of the tissue type of the primary or original tumor in the organ or tissue in which the secondary (metastatic) tumor is located, but the absence of one of the organ or tissue type in which it is located. For example, prostate cancer that has metastasized to bone is called metastatic prostate cancer and contains cancerous prostate cancer cells growing in bone tissue.

[0131] In one embodiment, the present invention relates to a 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 effective amount of said peptide, polynucleotide and / or vector.

[0132] The term "effective amount" or "therapeutically effective amount" of an active principle (e.g., a peptide, polynucleotide and / or vector as described herein) refers to the amount of the active principle, alone or in combination with another active principle (e.g., in combination with an anti-tumor agent as described herein), that elicits a biological or medical response in a subject, for example, ameliorates symptoms, alleviates a condition, or slows or delays disease progression, or prevents disease.

[0133] The term "patient" refers to a human or non-human animal, preferably a mammal, including males, females, adults and children, in need of treatment.

[0134] In one embodiment, the proliferative disorder or cancer is a proliferative disorder or cancer involving cathepsin D overexpression, i.e., in which the tumor cells overexpress cathepsin D, preferably at least 2-fold, for example 2-50-fold, compared to non-tumor cells.

[0135] In one embodiment, the proliferative disorder or cancer is a proliferative disorder or cancer that is associated with overexpression and secretion of cathepsin D in biological tissues or fluids, particularly serum, plasma, when compared to biological tissues or fluids of subjects not afflicted with cancer.

[0136] Examples of cancers associated with cathepsin D overexpression include, but are not limited to, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, kidney cancer, bladder cancer, osteosarcoma, gastric cancer, pancreatic cancer, head and neck cancer, salivary adenoid cystic carcinoma, squamous cell carcinoma, malignant melanoma, thyroid cancer, lung cancer, liver cancer, malignant glioma, and colorectal cancer.

[0137] In one embodiment, the proliferative disorder or cancer is a proliferative disorder or cancer in which tumor progression is driven by cathepsin D overexpression.

[0138] Examples of cancers promoted by cathepsin D, where cathepsin D is secreted by tumor cells, include, but are not limited to, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, kidney cancer, and colon cancer.

[0139] The peptides, polynucleotides and / or vectors as described herein are useful for treating tumors, particularly malignant tumors, and in particular for preventing or treating tumor metastasis.

[0140] In particular, the peptides, modified peptides, polynucleotides and / or vectors as described herein are useful in the treatment of proliferative disorders or cancers in which tumor cells overexpress cathepsin D, preferably at least two-fold, compared to non-tumor cells, such as non-tumorous fibroblasts.

[0141] As used herein, the term "treatment" or "therapy" includes therapeutic treatment and / or prophylactic treatment. In particular, therapeutic treatment refers to any of the alleviation, remission and / or elimination, reduction and / or stabilization of symptoms (e.g., inability to progress to a more advanced stage), and delay in the progression of symptoms of a particular disorder. Prophylactic treatment refers to any of the arrest of onset, reduction in the risk of development, reduction in incidence, delay in onset, reduction in progression, and extension of the time to onset of symptoms of a particular disorder. In the present invention, "treating" or "treatment" may particularly refer to reducing or stopping tumor progression.

[0142] Pharmaceutical Composition The peptides, modified peptides, polynucleotides and / or vectors as described herein may be administered in the form of a pharmaceutical composition.

[0143] The present invention also relates to a pharmaceutical composition comprising a peptide, modified peptide, polynucleotide and / or vector as described herein and a pharmaceutically acceptable carrier. The composition may further comprise another active ingredient, in particular an anti-tumor agent, more preferably a chemotherapeutic agent as described herein.

[0144] Combination therapy The peptides, modified peptides, polynucleotides and / or vectors as described herein may be used in combination with another active ingredient, in particular an anti-tumor agent.

[0145] Anti-tumor agents include chemotherapeutic, immunotherapeutic, targeted, cellular or hormonal therapeutic agents, such as: (i) inhibitors of DNA replication such as DNA binders, in particular alkylating or intercalating agents; (ii) antimetabolites, such as DNA polymerase inhibitors or topoisomerase I or II inhibitors; (iii) antimitotic agents, such as alkaloids; (iv) checkpoint modulators, in particular checkpoint inhibitors, such as anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3 or anti-TIM; (v) targeted anti-tumor therapeutics such as anti-EGFR, anti-HER2, anti-VEGF, PARP inhibitors, mTOR inhibitors, etc., (vi) hormonal therapy agents such as (via) corticosteroids, e.g., prednisone, dexamethasone, hydrocortisone and methylprednisone, (vib) thyroid hormones, (vic) somatostatin analogs, (vid) reproductive hormone drugs, etc.

[0146] Chemotherapeutic agents such as those referred to in (i) to (iii) above may be, for example, 5-FU, oxaliplatin, cisplatin, carboplatin, irinotecan, docetaxel or paclitaxel.

[0147] The targeted therapeutic agent as referred to in (i)-(iii) above may be, for example, cetuximab or erlotinib.

[0148] Checkpoint modulators and targeted anti-tumor therapeutics, such as those referred to in (iv)-(v) above, can be, for example, antibodies or fragments thereof, or small molecules.

[0149] Reproductive hormone drugs as referred to in (vid) above may be, for example, androgens such as fluoxymesterone; estrogens such as diethylstilbestrol; progestins such as medroxyprogesterone or megestrol; antiandrogens such as bicalutamide, flutamide or nilutamide; aromatase inhibitors such as anastrozole, exemestane or letrozole; luteinizing hormone-releasing hormone (LHRH) agonist drugs such as buserelin, goserelin or leuprolide; or gonadotropin-releasing hormone (GnRH) antagonists such as degarelix.

[0150] The antitumor agents as referred to in (i) to (vi) may be in the form of an antibody-drug conjugate (ADC).

[0151] In one embodiment, the present 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 effective amount of said peptide, polynucleotide and / or vector as described herein and an anti-tumor agent.

[0152] In some embodiments, a peptide or modified peptide comprising a fragment as described herein, in particular a fragment of SEQ ID NO: 1, 2, 3 or 4 as described herein, or a functional variant derived therefrom, is used in combination with a chemotherapeutic agent as described herein.

[0153] The present disclosure also provides the use of a peptide or modified peptide as described herein, optionally in combination with a pharmaceutically acceptable support and / or one or more active ingredients as described herein, in particular for the manufacture of a medicament, preferably as described herein, for the treatment of a proliferative disorder, in particular cancer, in particular in a human patient.

[0154] In another embodiment, the present disclosure provides a method of treatment, particularly a method of treatment of a proliferative disorder, particularly cancer, comprising administering to a subject in need thereof an effective amount of a peptide or modified peptide, optionally in combination with a pharmaceutically acceptable support and / or one or more active ingredients as described herein.

[0155] Route of administration In one embodiment, the peptide or pharmaceutical composition and optionally the anti-tumor agent are administered to the subject using a route selected from systemic, e.g., intravenous or oral, routes to local routes, in particular intratumoral (IT) or intraperitoneal (IP) routes.

[0156] The peptide or pharmaceutical composition and optionally an antitumor agent can be administered to a patient with ovarian cancer, for example, using the IP route. In such an IP route, chemotherapy is typically administered as a liquid into the patient's abdomen through a device called an IP port. The chemotherapy is then introduced into the patient's abdomen through an IP catheter and the IP port. Once the liquid is in the patient's abdomen, the patient is asked to change positions at regular intervals to help distribute the liquid over the surface of the abdominal tissue. The entire procedure typically lasts 3 to 4 hours.

[0157] Diagnosis and / or staging of diseases associated with cathepsin D overexpression Another aspect of the present invention relates to the use of a peptide or a modified peptide as described herein in a method for diagnosing and / or staging a disease associated with cathepsin D overexpression in a patient.

[0158] The disease associated with cathepsin D overexpression may be a proliferative disorder, especially cancer, or Alzheimer's disease.

[0159] The peptides or modified peptides used in the above-mentioned methods are advantageously labeled or modified peptides, i.e., peptides linked to a labeling substance that generates a detectable and / or quantifiable signal, in particular a radioactive, magnetic or luminescent substance. In another provision, the peptides or modified peptides are used as reagents. The luminescent substance can be, for example, a fluorophore, such as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5). The radioactive substance can be, for example, a radioactive atom, such as I, for scintigraphy experiments. 123 , I 124 , In 111 ,Re 186 or Re 188 etc.

[0160] In one embodiment, the method comprises contacting a biological sample obtained from a patient with a peptide or modified peptide as described, preferably labeled, wherein said peptide is preferably capable of selectively interacting with a fragment of LRP1, particularly the LRP1 β-chain ectodomain, present in the biological sample obtained from the patient. The fragment of LRP1 can then be detected, and optionally the concentration can be measured, by any method known in the art.

[0161] In one embodiment, the method comprises detecting the presence of a fragment of LRP1, in particular the LRP1 beta chain ectodomain, in a biological sample obtained from said patient.

[0162] In one embodiment, the method comprises measuring the concentration of a fragment of LRP1, in particular the LRP1 beta chain ectodomain, in a biological sample obtained from said patient.

[0163] The present disclosure also provides the use of a peptide or modified peptide as described herein for the manufacture of a diagnostic kit for diagnosing or staging a disease associated with Cathepsin D overexpression, wherein the disease may be a proliferative disorder, especially cancer, or Alzheimer's disease.

[0164] In another embodiment, the present disclosure provides a method for diagnosing or staging a disease associated with Cathepsin D overexpression in a patient, the method comprising applying a peptide or modified peptide as described herein to a sample from the patient.

[0165] Example The present disclosure is further illustrated by the following examples.

[0166] Example 1: Identification of a key cathepsin D / LRP-1 interacting region and testing of peptides derived from this interacting region 1.1 Materials and Methods Affinity constant determination by microscale thermophoresis Materials and Research PCR tubes and tips: HEPES (H0887-20ml; Sigma Aldrich); Tween® 20 (P1379-25ml; Sigma Aldrich); Monolith His-Tag Labeling Kit RED-Tris-NTA 2nd Generation (MO-L018; Nanotemper); Human Cathepsin D / CTSD Protein, His-Tag (CTD-H5226; Acrobiosystems); Milli-Q® Water; Water (W3500-100ml; Sigma Aldrich); Monolith Capillary (MO-K022; Nanotemper). * Assay buffer: HEPES 50 mM, pH 7.5 supplemented with 0.05% Tween 20® ** Stock solution of cathepsin D at -80°C in aliquots - concentration: 400 μg / mL - 9.2 μM

[0167] Cathepsin D labeling (Monolith His tag labeling kit) Add 8 mL of Milli-Q® water to a vial containing 1.5X PBS-T to obtain 1X PBS-T. Suspend the dye in 2.25 μL of PBS-T to obtain a 5 μM dye solution (2 μL aliquots at −20° C.). 3.2 μL dye (5 μM) and 98 μL assay buffer * Prepare a 100 Nm dye solution by mixing: 4. Cathepsin D ** The concentration is adjusted to 200 nM in a volume of 100 μL. 5. Mix 90 μL of protein (200 nM) with 90 μL of dye (100 nM) in a 1.5 mL tube. 6. Incubate at room temperature for 30 minutes. 7. Centrifuge the sample at 4°C and 15000g for 10 minutes and transfer the supernatant to a fresh tube. 8. The protein is labeled and ready for binding assays.

[0168] Preparation of peptides 1. Remove the peptide at -20°C and centrifuge at 4°C and 10000g for 5 minutes. 2. Let them sit at room temperature for 10 minutes. Add 3.X μL of Milli-Q® water to obtain a 500 μM concentration. 4. Let them thaw for 60 minutes at RT and place them at 4°C.

[0169] Binding assay All reagents are equilibrated to room temperature before use. 1. Prepare 50 μL of peptide at 20 μM in water (2 μL qsp 50 μL water). 2. Add 10 µL of Milli-Q® water to PCR tubes 2-16. 3. Transfer 20 μL of peptide to PCR-tube 1. 4. Pipette 10 µL from tube 1 to tube 2 (P20) and mix by pipetting up and down multiple times. Repeat for tubes 3-16 and discard excess from tube 16. 5. Transfer 10 μL of labeled protein to each well (1 to 16) and mix by pipetting. 6. Incubate at RT for 30 minutes. 7. Load the capillary (NT.115 capillary) and measure the sample using a Monolith NT.115 system with the following settings: 20% Pico-RED excitation power and medium MST power.

[0170] Pro-cathespin D / LRP-1 interaction assay by co-immunoprecipitation Materials and Reagents

number

[0171] Cos-7 transfection 1. Seed Cos-7 on B10 until they reach 80% confluence. 2. Gene transfer: 3. Tube 1: For one B10, mix 900 μL of OptiMEM with 30 μL Lipofectamine 2000. 4. Tube 2: Mix 15 μg of DNA with 900 μL of OptiMEM. pSectag SD1(LRP1α)781ng / μL / 587ng / μL:25.5μL 5. pSectag F0 (LRP1β): 1.15 μg / mL. Then add "Tube 2" dropwise in "Tube 1". 6. Incubate at RT for 5 minutes. 7. Add this mixture dropwise onto the cells. 8. Incubate at 37°C, 5% CO2, and 95% humidity for 48 hours. 9. Collect the supernatant and centrifuge at 1500 rpm for 10 minutes at 4°C. Store at 10.4°C for 24 to 72 hours, or at -20°C for longer storage.

[0172] Preparation of peptides 1. Take the peptides at -20°C and centrifuge them at 10000g for 5 minutes at 4°C. 2. Let them sit at room temperature for 10 minutes. Add 3.X μL of water to give a concentration of 500 μM. 4. Let them dissolve for 60 minutes at RT and place them at 4°C.

[0173] Co-immunoprecipitation Conditions: SD1 + cathepsin D-F0 + cathepsin D-F0 + cathepsin D + peptide (100 μM). On ice: 1. Prepare 50 μL of cathepsin D at 2 ng / μL in PBS + / - 100 μM peptide and incubate for 1 hour at 4 °C with gentle rotation. 2. Add 500 μL of supernatant "SD1 or F0" per tube and incubate overnight at 4°C with gentle rotation. 3. Prepare 25 μL of NiNTA agarose (50 μL) per condition. 4. Wash the resin three times with 1 mL of PBS-T, centrifuge (1000 g, -4°C, 1 minute), and remove the supernatant. 5. Incubate with 1 mL of 2% BSA at 4°C for 30 minutes. Wash three times with 6.1 mL PBS-T. 7. Centrifuge at 1000 g for 1 minute at 4°C and remove the supernatant. Resuspend the resin with 8.25 μL PBS-T. 9. Place 50 μL per tube. 10. Incubate for 4 hours at 4°C with gentle rotation. 11. Wash five times with 1 mL PBS-T (centrifugation at 1000 g, 4°C, 1 min between each wash). 12. Remove the supernatant and elute the sample with 30 μL of Limly 2X with β-mercaptoethanol. 13. Place the sample at -20°C.

[0174] Western blot analysis 1. Heat the sample in a dry bath at 100°C for 10 minutes. 2. Centrifuge the sample and run the supernatant on a 10% agarose gel. Shift to 3.70V (stacking gel) and then 120V (separating gel). The proteins are transferred onto a nitrocellulose membrane at 100V for 60 minutes at 4.4°C. 5. Place the membrane in TBS-T with 5% milk. 6. Prepare antibody in milk 5% TBS-T. 7. Incubate overnight at 4°C under gentle agitation. 8. Wash membrane in TBS-T for 10 minutes (x3). 9. Incubate with secondary antibody for 1 hour at room temperature. 10. Repeat wash in TBS-T and once in TBS. 11. Develop using a Li-Cor Odyssey FC 2800 Imaging system.

[0175] Examining the effects of cathepsin D in a 3D co-culture model of cancer cells and fibroblasts Materials and Reagents COS-7 cells - MEF1 cells - Lipofectamine 2000 transfection reagent (11668027; Invitrogen) - pCDNA3.1 CTL - pCDNA3.1 pCD - OptiMEM (31985-070; Gibco); DMEM 4.5 g / L glucose (31966-021; Gibco) - FCS (F7524; Sigma-Aldrich) - PBS (14190-094; Gibco) - 48-well plates (353078; Falcon®) - Trypsin - EDTA (0.05%) (11580626; Gibco) - Water (W3500-100ML; Sigma-Aldrich) - EVOS Fl microscope (Thermofisher).

[0176] COS7 seeding and gene transfer 1. Seed 20,000 cells / well (48-well plate) on 300 μL of DMEM 4.5 g / L glucose supplemented with 10% FCS. 2. Incubate at 37°C, 5% CO2 and 95% humidity for 24 hours. 3. Transfection using plasmids and Lipofectamine 2000. 4. Tube 1: For one well, mix 20 μL of OptiMEM with 0.862 μL of Lipofectamine 2000. 5. Tube 2: Mix 20 μL of OptiMEM with 0.287 μg of DNA. pCDNA3.1 CTL: 1.22 μg / μL pCDNA3.1 pCD: 1.26 μg / μL 6. Next, add "Tube 2" dropwise into "Tube 1". 7. Incubate at RT for 5 min. 8. Add this mixture dropwise onto the cells. 9. Incubate at 37°C, 5% CO2, and 95% humidity for 24 hours. Place the Matrigel on ice and freeze-thaw it overnight at 4° C. Place chips (200 and 1000 μL) at −20° C.

[0177] Preparation of peptides 1. Remove the peptides at -20°C and centrifuge them for 5 minutes at 4°C and 10000g. 2. Let them sit at room temperature for 10 minutes. Add 3.X μL of water to obtain a 500 μM concentration. 4. Let them dissolve for 60 minutes at RT and put them at 4°C.

[0178] Seeding of MEF-1 on Matrigel 1. Remove the COS7 culture medium. 2. Add 100 μL of Matrigel per well (using ice-cold tips). 3. Polymerize for 1 hour at 37°C, 5% CO2 and 95% humidity. 4. Rinse MEF1 with 5 mL of PBS. Add 5.1 mL of trypsin-EDTA (for 1 B10) and incubate at 37°C for 5 minutes. 6. Add medium containing 10% FCS and count the cells. 7. Adjust the volume to 50,000 cells / well. 8. Centrifuge at 1200g for 5 minutes. 9. Resuspend cells with 300 μL of Matrigel per well: 50,000 cells in 300 μL / well 10. Seed MEF1 on COS7. 11. Polymerize for 1 hour at 37°C, 5% CO2 and 95% humidity. 12. Add 200 μL of medium + / - 100 μM peptide / well. 13. Take photographs under a microscope on D+1, D+4 and D+6.

[0179] Effect of LBC peptide on breast cancer cell proliferation Materials and Reagents MDA-MB-231 cells - MCF7 cells - DMEM 1 g / L glucose (21885-025; Gibco) - FCS (F7524; Sigma Aldrich) - PBS (14190-094; Gibco) - Trypsin - EDTA (0.05%) (11580626; Gibco) - 96-well plates (83.3924; Sarstedt) - Cell proliferation reagent WST-1 (11644807001; Sigma Aldrich) - Water (W3500-100ML; Sigma Aldrich) - Infinite 200® pro (TECAN).

[0180] Preparation of peptides 1. Take the peptides at -20°C and centrifuge them at 10000g for 5 minutes at 4°C. 2. Let them sit at room temperature for 10 minutes. Add 3.X μL of water to give a concentration of 500 μM. 4. Let them thaw for 60 minutes at RT and place them at 4°C.

[0181] Assay procedure 1. Seed 5000 and 10000 cells / well for MDA-MB-231 and MCF7, respectively, on 100 μL of DMEM 1 g / L glucose supplemented with 10% FCS (96-well plates): two plates for WST-1 assay 24 and 48 hours after treatment. 2. Incubate at 37°C, 5% CO2 and 95% humidity for 24 hours. 3. Treat cells with peptides at 50 μM-25 μM-12.5 μM and 6.25 μM in DMEM 1 g / L glucose supplemented with 10% FCS. 4. Incubate at 37°C, 5% CO2 and 95% humidity for 24 / 48 hours. 5. Add 10 μL / well of cell proliferation reagent WST-1. 6. Incubate the cells for 2 hours at 37°C, 5% CO2 and 95% humidity. 7. Shake well on a shaker for 1 minute. 8. Measure the absorbance of the samples against the background control as a blank using a microplate reader at 450 nm. Use a reference wavelength >600 nm.

[0182] Cathepsin D catalytic activity assay at pH 3.5 Materials & Reagents PCR tubes and tips - recombinant human pro-cathepsin D protein (ab286010; Abcam) - water (W3500-100ML; Sigma-Aldrich) - sodium acetate anhydrous (W302406; Sigma-Aldrich) - sodium chloride (207790010; ThermoScientific) - cathepsin D and E substrate (fluorogenic) (BML-P145-0001; Enzo Life Sciences) - 96-well ELISA plates (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).

[0183] Preparation of peptides 1. Take the peptides at -20°C and centrifuge them at 10000g for 5 minutes at 4°C. 2. Let them sit at room temperature for 10 minutes. Add 3.X μL of water to give a concentration of 500 μM. 4. Let them thaw for 60 minutes at RT and place them at 4°C.

[0184] Assay procedure Assay buffer is equilibrated to room temperature before use. 1. Prepare assay buffer: 0.1 M NaOAc, 0.2 M NaCl, pH 3.5. 2. Dilute pro-cathepsin D to 20 μg / mL in assay buffer. 3. Add 5 μL of 100 μM pro-cathepsin D + / - peptide in assay buffer (final volume: 50 μL). 4. Incubate at room temperature for 15 minutes. 5. Dilute substrate to 30 μM in assay buffer. 6. Load 50 μL of pro-cathepsin D + / - peptide (100 μM) or pepstatin (2 μg / mL) in plates (pre-saturated with 1% BSA). 7. The reaction is initiated by adding 50 μL of 30 μM substrate. 8. Read in kinetic mode for 1 hour, excitation and emission wavelengths at 320 nm and 405 nm (top read), respectively.

[0185] Cathepsin D catalytic activity assay at pH 6 Materials and Reagents PCR tubes and tips - recombinant human cathepsin D protein (1014-AS; R&D systems) - water (W3500-100ML; Sigma Aldrich) - sodium acetate anhydrous (W302406; Sigma Aldrich) - sodium chloride (207790010; ThermoScientific) - cathepsin D and E substrate (fluorogenic) (BML-P145-0001; Enzo Life Sciences) - 96-well ELISA plates (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).

[0186] Preparation of peptides 1. Take the peptides at -20°C and centrifuge them at 10000g for 5 minutes at 4°C. 2. Let them sit at room temperature for 10 minutes. Add 3.X μL of Milli-Q® water to obtain a concentration of 500 μM. 4. Let them thaw for 60 minutes at RT and place them at 4°C.

[0187] Assay procedure Assay buffer is equilibrated to room temperature before use. 1. Prepare assay buffer: 0.1 M NaOAc, 0.2 M NaCl, pH 3.5 / 0.1 M NaOAc, 0.2 M NaCl, pH 6. 2. Dilute cathepsin D to 20 μg / mL in assay buffer at pH 3.5. 3. Incubate at 37°C for 30 minutes to activate the protein. 4. Dilute the incubated cathepsin D to 0.5 ng / μL in assay buffer, pH 6. 5. Dilute substrate to 60 μM in assay buffer pH 6. 6. Load 50 μL of Cathepsin D+ / - peptide (100 μM) or pepstatin (2 μg / mL) in the plate (pre-saturated with 1% BSA). 7. Incubate at 37°C for 15 minutes. 8. The reaction is initiated by adding 50 μL of 60 μM substrate. 9. Read excitation and emission wavelengths at 320 nm and 405 nm (top read), respectively, in kinetic mode for 45 minutes.

[0188] 1.2. Results and Discussion Characterization of the cathepsin D / LRP-1 interaction by in silico studies To identify the precise interaction zone between cathepsin D and LRP-1 and the residues involved, we performed in silico analyses (bioinformatics strategies, molecular modeling, and dynamics) following the workflow shown in Figure 1.

[0189] Briefly, using structure prediction methods (MODELLER software, I-TASSER online server) and molecular dynamics tools (GROMACS software), we generated representative structures and conformations of the F4 fragment. Taking into account the structural elements identified in these sequences, we also modeled another, longer fragment from this region of LRP-1β. Indeed, this region of LRP-1 is rich in EGF-like domains, and the F4 fragment partially overlaps with EGF-like domains 19 and 20. Therefore, to ensure structural integrity, we proposed a fragment containing the entire two domains (EGF1920 fragment).

[0190] The amino acid sequences of the F4 fragment and EGF1920 are given in Table 2 below: [Table 2]

[0191] The same method we used for the F4 fragment can be used for the EGF1920 fragment, allowing us to propose a representative structure and conformation for this new fragment. The same molecular docking protocol was performed for each of these fragments (F4 fragment (SEQ ID NO: 5) and EGF1920 fragment (SEQ ID NO: 6)).

[0192] The first two clusters extracted from the structures and molecular dynamics obtained from energy minimization were retained and subjected to rigid / rigid docking (HEX software) on cathepsin D structures available on the PDB server (1LYW). Each docking experiment generated 100 solutions, and the overall analysis generated 1500 solutions. For each fragment, the number of contacts was normalized, and the residue statistically producing the maximum number of contacts was assigned a frequency of 1. A cutoff of 0.5 was used to separate important residues from others (Figures 2A and 2B). Analysis of the results identified several residues in the F4 (Figure 2A) and EGF1920 (Figure 2B) fragments that likely interact with cathepsin D. Five frequently occurring residues were identified for both fragments (Figure 2C). A region of five residues (DRCQY (SEQ ID NO: 9)) was isolated, including the R residue and four residues (DR_QY). This region appears to serve as a starting point for the design of blocking peptides.

[0193] Design of five peptides (LBC1-LBC5) to block cathepsin D / LRP-1 interaction From the sequence (DRCQY), the inventors preselected a series of 5 peptides (Table 3) only 15 amino acids long in order to optimize the synthesis (compatibility with standard Fmoc procedures on solid support, low cost, high yield and purification speed), but also to meet the requirement of biocompatibility. Indeed, for this preselection, the inventors paid particular attention to determining sequences with high predicted solubility in aqueous solvents (water, NaCl 0.9%) in planned clinical applications. [Table 3] Table 3: Sequences of five preselected peptides for blocking cathepsin D / LRP-1 interaction. The sequences of the five amino acids identified by in silico testing are shown in bold. The predicted isoelectric point, net charge at pH 7, and solubility in NaCl 0.9% are also shown.

[0194] These five peptides were synthesized by contract with SB-Peptide (Grenoble, France), which did not encounter any difficulties with regard to synthesis and purification (synthesis report available upon request).

[0195] To verify that these peptides are capable of blocking cathepsin D / LRP-1 interaction, three tests were performed on these peptides: 1- The affinity constants (K) of these peptides for cathepsin D using microscale thermophoresis (MST) D ) determination (see Materials and Methods section above) 2-Ability to block the cathepsin D / LRP-1 interaction in vitro in co-immunoprecipitation experiments using soluble fragments of LRP-1 β-chain and recombinant cathepsin D (see Materials and Methods section above). 3-Ability to block the cathepsin D / LRP-1 interaction and its proliferative effects in a mouse fibroblast model (MEF-1 cells, expressing LRP-1) (see Materials and Methods section above).

[0196] Examining the affinity of LBC1-LBC5 peptides for cathepsin D by microscale thermophoresis The inventors first tested the affinity of the LBC1 to LBC5 peptides for cathepsin D. Microscale thermophoresis (MST) experiments showed that only peptides LBC1, LBC3, LBC4, and LBC5 could bind to cathepsin D.

[0197] Briefly, pro-cathepsin D (Abcam) was labeled using the His-Tag labeling kit Red-Tris NTA (NanoTemper). The labeled pro-cathepsin D was then mixed with the F4 fragment or peptides LBC1, LBC2, LBC3, LBC4, and LBC5 using a 0.5-fold dilution series ranging from 50 μM to 1.5 nM. The 16 mixtures were then analyzed on a Monolith NT.115 (NanoTemper) instrument at 256 °C. Instrument parameters were 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper).

[0198] The results are shown in Figure 3 and Table 4 below: [Table 4]

[0199] These results indicate that the individual affinities of LBC1 (52.5 nM), LBC3 (340 nM), LBC4 (4.3 nM), and LBC5 (8.7 nM) are higher than those obtained with fragment F4 (2.37 μM), which is 45 AA and corresponds to residues 349–394 of LRP-1β. These results demonstrate that in silico analysis has indeed identified a critical region of LRP-1 involved in its interaction with cathepsin D. The primary amino acid sequences flanking this region likely contribute to the affinity of these peptides.

[0200] Examining blocking properties of LBC1-LBC5 peptides against cathepsin D / LRP-1 by co-immunoprecipitation Next, we examined the ability of peptides to block the cathepsin D / LRP-1 interaction by co-immunoprecipitation experiments (n = 5) (Figure 4). We first verified that cathepsin D can bind to the extracellular portion of the LRP-1 β chain (LRP1β-F) (Figure 4A). This interaction is specific, as we did not observe binding to the LRP-1 α chain fragment (LRP1αF). In the presence of different peptides at 100 μM, LBC3, LBC4, and LBC5 were able to significantly block this interaction by approximately 60–70%, while peptide LBC1 had a lower blocking capacity (40%) (Figure 4B). These results confirm the results observed with MST.

[0201] Examining the blocking properties of LBC1-LBC5 peptides against cathepsin D / LRP-1 in a 3D co-culture model of cancer cells and fibroblasts Finally, we tested the ability of peptides LBC1-LBC5 to block the cathepsin D / LRP-1 interaction in previously validated cellular models (Beaujouin et al., 2010; Derocq et al., 2012).

[0202] FIG. 5A illustrates a 3D co-culture model of mouse embryonic fibroblasts that naturally express LRP-1 and COS-7 cancer cells that overexpress and secrete, or do not, cathepsin D.

[0203] Western blot analysis of the amount of cathepsin D present in the co-culture medium of wells containing COS-7 cells that either overexpress and secrete (pCD) or do not (Ctrl) cathepsin D was performed after 6 days. The results are shown in Figure 5B.

[0204] Fibroblast colonies co-cultured with COS-7 Ctrl or pCD cells in the absence or presence of 100 μM LBC1-5 peptide were observed under a phase-contrast microscope on days 1, 4, and 6. The results are shown in Figure 5C.

[0205] Figure 5D shows 5,000 pixels on day 6. 2Histogram showing colonies with larger area (each point equals one colony). n=5, one-way anova **** :p<0.0001, ** : p<0.01, Scale bar: 20 μm.

[0206] As expected (positive control), the presence of cathepsin D significantly stimulated fibroblast proliferation, thus increasing colony size after 4 and 6 days of incubation (Figure 5C, Figure 5D). Similar experiments performed with LRP-1 KO mouse embryonic fibroblasts (LRP1- / -, PA-13 cells) validated the specificity of this effect, since the presence of cathepsin D did not affect proliferation (negative control, data not shown). Thus, we tested the effect of cathepsin D on fibroblast proliferation in the presence of different LBC peptides (100 μM). This effect of cathepsin D was significantly inhibited by peptides LBC2 and LBC5 and strongly inhibited by peptide LBC4 (90-100% inhibition), whereas peptides LBC1 and LBC3 had no effect (Figure 5C, Figure 5D).

[0207] At the end of three studies performed on these different peptides, the inventors identified at least two peptides, LBC4 and LBC5, that both have good affinity and blocking capacity for the Cathepsin D / LRP-1 interaction in vitro and in cellulo in a mouse model. Other peptides cannot be discarded because they show binding and / or interaction blocking capacity either in vitro and in cellulo.

[0208] Testing a set of peptide sequences derived from the F4 LRP-1 fragment capable of binding to cathepsin D The in vitro and in cellulo data obtained for each peptide LBC1 to LBC5 suggested that we were not limited to a single peptide sequence, but rather explored a larger sequence. To define this sequence, we decided to synthesize 10 new peptides (LBC6 to LBC15) that would allow us to explore the N- and C-terminal sequences of the peptides we had already analyzed.

[0209] Different peptides derived from the β-chain of LRP-1 that were synthesized to define the region of high interaction between LRP-1 and cathepsin D are shown in FIG.

[0210] FIG. 6 shows a region of the LRP-1 β chain with the amino acid sequence STCTVNQGNQPQCRCLPGFLGDRCQYRQYRQCSGYCENFGTCQMAADGSRQCRCTAYFEGSRC (SEQ ID NO: 8), including but not limited to the region of LRP-1 known as "fragment F4," from which peptides LBC6 (SEQ ID NO: 15) to LBC15 (SEQ ID NO: 24) were designed.

[0211] Peptides LBC1 (SEQ ID NO: 10) to LBC5 (SEQ ID NO: 14) were the first peptides tested. Peptides LBC6 (SEQ ID NO: 15) to LBC8 (SEQ ID NO: 17) were used to define the N-terminus of the highly interacting region, and peptides LBC9 (SEQ ID NO: 18) to LBC15 (SEQ ID NO: 24) were used to define the C-terminus of the highly interacting region of amino acid sequence (SEQ ID NO: 1).

[0212] These 10 new peptides were synthesized (>90% purity, neutralized in acetate) by SB-Peptide, Inc. Solubility and affinity tests for cathepsin D were performed on each of these peptides by microscale thermophoresis.

[0213] Briefly, cathepsin D (Acrobiosystems) was labeled using the His-Tag labeling kit Red-Tris NTA (NanoTemper). The labeled cathepsin D was then mixed with the LBC peptide using a 0.5-fold dilution series ranging from 10 μM to 0.15 nM. The 16 mixtures were then analyzed using a Monolith NT.115 (NanoTemper) instrument at 25°C. Instrument parameters were 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper). The results are shown in Table 5 below: [Table 5] Table 5 Testing the affinity of LBC1–LBC15 peptides for cathepsin D by microscale thermophoresis. Table showing the Kd values ​​obtained for each peptide by microscale thermophoresis. Briefly, cathepsin D (Acrobiosystems) was labeled with the His-Tag labeling kit Red-Tris-NTA (NanoTemper). The labeled cathepsin D was then mixed with the LBC peptides (1–15) using a 0.5-fold dilution series ranging from 10 μM to 0.15 nM. The 16 mixtures were then analyzed using a Monolith NT.115 (NanoTemper) instrument at 25°C. Instrument parameters were 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper).

[0214] Solubility studies allowed the inventors to identify several peptide sequences (peptides LBC9, LBC11 and LBC14) that could not be used in cellulo and in vivo due to their insolubility in water.

[0215] The equilibrium dissociation constants (KD ) made it possible to i) identify peptide sequences capable of interacting with cathepsin D and ii) determine the region of high interaction, which is defined by peptides LBC8 and LBC9 and consists of 30 amino acids: NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 1).

[0216] Characterization of the LBC4 peptide structure by nuclear magnetic resonance. To investigate in more detail the characteristics of the LBC4 peptide, which gave the best results according to our in vitro and in cellulo studies, nuclear magnetic resonance (NMR) spectroscopic analysis was performed by Prof. Bruno Kieffer (UMR CNRS 7104, Strasbourg, France).

[0217] The LBC4 peptide was diluted (130 μl) in HCl / NaCl solution (30 μl) containing 20 mM Tris buffer at pH 7.5, followed by the addition of 16 μl of DMSO (oxidative conditions, red spectrum) or 16 μl of water (non-oxidative conditions, blue spectrum).

[0218] The results are shown in Figure 7: - Figure 7A: 1D 1H NMR spectrum in the -NH region measured at 298 Kelvin after 72 h incubation at room temperature. - Figure 7B: Determination of the chemical shift in the NH region between the spectra obtained in the absence (blue) and presence (red) of DMSO (oxidizing conditions). Figures 7C and 7D: Affinity determination of oxidized LBC4 peptide for cathepsin D by microscale thermophoresis. Labeled cathepsin D was mixed with LBC4 peptide without (LBC4) or with (oxidized-LBC4) a disulfide bridge between its two cysteines using a 0.5-fold dilution series ranging from 10 μM to 0.15 nM. Sixteen mixtures were then analyzed using a Monolith NT.115 (NanoTemper) instrument at 25 °C. Instrument parameters were 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper).

[0219] Although the LBC4 peptide appears unstructured, in an oxidizing environment, the LBC4 cysteines can form disulfide bridges and modify the peptide conformation (Figures 7A and 7B). MST analysis shows that this cyclization impaired binding of the LBC4 peptide to cathepsin D (Figures 7C and 7D).

[0220] Effect of LBC4 and LBC5 peptide sequence minimization We then tested the possibility of using smaller peptides: we synthesized 16 peptides of 7-15 AA length derived from the LBC4 and LBC5 peptides and containing the DRCQY (SEQ ID NO: 8) sequence (previously identified by our in silico studies as detailed above).

[0221] The affinity constants of these peptides were determined by microscale thermophoresis.

[0222] The results are shown in Table 6. [Table 6] Table 6: Examination of LBC16-LBC31 peptide affinity for cathepsin D by microscale thermophoresis. Table containing Kd values ​​obtained for each peptide by microscale thermophoresis. Briefly, cathepsin D (Acrobiosystems) was labeled with the His-Tag labeling kit Red-Tris-NTA (NanoTemper). Next, LBC peptides (16-31) and labeled cathepsin D were mixed using a 0.5-fold dilution series ranging from 10 μM to 0.15 nM. The 16 mixtures were then analyzed using a Monolith NT.115 (NanoTemper) instrument at 25 °C. Instrument parameters were 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper).

[0223] The results obtained show that all peptides derived from LBC4 (SEQ ID NO: 13) and LBC5 (SEQ ID NO: 14) are soluble in water. All peptides show good affinity for cathepsin D, except for peptides LBC27 (SEQ ID NO: 36) and LBC30 (SEQ ID NO: 39). These results show that it is possible to reduce the size of our blocking peptides down to 7 AA (e.g., LBC16 (SEQ ID NO: 25)).

[0224] Direct effects of LBC peptide on breast cancer cell proliferation LBC peptides target cathepsin D secreted by cancer cells and, therefore, their communication with fibroblasts. We determined whether these peptides have an effect on cancer cells themselves and their ability to block proliferation, particularly in MCF-7 and MDA-MB-231 breast cancer cells.

[0225] MDA-MB-231 and MCF-7 cells were seeded at 5,000 and 10,000 cells / well in 96-well plates, respectively, and incubated for 48 hours with either no peptide (NT) or various concentrations (6.25-50 μM) of LBC4 (SEQ ID NO: 13), LBC5 (SEQ ID NO: 14), or LBC15 (SEQ ID NO: 15) peptides. Cell numbers were measured using the cell proliferation reagent WST-1.

[0226] The results are shown in Figure 8.

[0227] All of the LBC peptides tested, even the LBC4 (SEQ ID NO: 13) and LBC5 (SEQ ID NO: 14) peptides, which show the strongest effect on fibroblasts, had no significant effect on the proliferation of these tumor cells (Figure 5D). These results appear to confirm the mechanism of action of the LBC peptides, which targets stromal cells within the tumor microenvironment. Furthermore, these results suggest that our peptides have no cytotoxic effect up to 50 μM, which is intriguing for their potential therapeutic applications.

[0228] Effect of LBC peptides on cathepsin D catalytic activity In addition to its ability to bind to LRP-1 and induce cellular effects, cathepsin D is an aspartic protease capable of cleaving a variety of substrates. Like most lysosomal aspartic proteases, cathepsin D has maximal catalytic activity at an acidic pH between 2.4 and 5. At higher pH values ​​of 5.0, the activity of cathepsin D decreases, and none is detectable at pH 7.0.

[0229] In the extracellular compartment (where the secreted form of pro-cathepsin D is found) and especially in the tumor microenvironment, a decrease in pH around 5.6-6.8 is observed, which is characteristic of malignant tumor cells and is due to glycolysis, hypoxia, and insufficient blood perfusion in tumor cells.

[0230] Therefore, we tested the ability of our peptides to inhibit cathepsin D activity at pH 3.5 and pH 6.

[0231] pH 3.5 experiments: Pro-cathepsin D (2 ng / μL) was preincubated with LBC peptide (100 μM) or pepstatin A (2 ng / μL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) for 15 min. Activity was then measured in the presence of fluorogenic substrate (15 μM).

[0232] pH 6 experiments: Pro-cathepsin D (0.5 ng / μL) was preactivated for 30 min in acidic buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) and then preincubated for 15 min with peptide (100 μM) or pepstatin A (2 μg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6). Activity was then measured in the presence of fluorogenic substrate (30 μM).

[0233] The results are shown in Figure 9. The results at pH 3.5 are shown in Figure 9A, and the results at pH 6 are shown in Figure 9B.

[0234] pH 3.5 results: Pepstatin A, an inhibitor of cathepsin D, completely blocks cathepsin D activity, whereas none of the peptides LBC4 (SEQ ID NO: 13), LBC5 (SEQ ID NO: 14), LBC15 (SEQ ID NO: 15), LBC7 (SEQ ID NO: 16), and LBC8 (SEQ ID NO: 17) (at 100 μM) was able to significantly block the activity of the enzyme ( FIG. 9A ).

[0235] pH6 result: At pH 6, peptide LBC4 (100 μM) significantly blocks (approximately 67%) the activity of cathepsin D (FIG. 9B). Mutation of cysteine ​​8 to serine (C8S) in the LBC4 peptide sequence (peptide LBC4C8S PGFLGDRSQYRQCSG (SEQ ID NO: 41)) impairs the peptide's ability to block the catalytic activity of cathepsin D, demonstrating the specificity of this interaction.

[0236] Interestingly, the LBC5 (SEQ ID NO: 14) peptide, which can block the cathepsin D / LRP-1 interaction, has no effect on the catalytic activity of cathepsin D. The same effect was observed for the LBC2 (SEQ ID NO: 11), LBC6 (SEQ ID NO: 15), LBC9 (SEQ ID NO: 18), and LBC15 (SEQ ID NO: 24) peptides.

[0237] Peptides LBC7 (SEQ ID NO: 16) and LBC8 (SEQ ID NO: 17), which have good affinity for cathepsin D, have a weaker effect on the catalytic activity of the protein.

[0238] Finally, peptides LBC16 (SEQ ID NO: 25) and LBC21 (SEQ ID NO: 30), which are only 7 and 10 AA peptides derived from peptide LBC4, still block catalytic activity, but only by 30%.

[0239] All these results indicate that LBC peptides capable of antagonizing cathepsin D / LRP-1 interaction do not have the same effect on the catalytic activity of the enzyme. These results allow us to consider future uses of LBC peptides that may or may not retain both activities. These distinct properties of LBC peptides allow us to i) distinguish them from other existing technologies, such as anti-cathD monoclonal antibodies, and ii) stimulate one or the other of these properties, depending on the effects of different peptides observed in vivo during tumor development.

[0240] Dose-dependent effects of LBC4 peptide: Pro-cathepsin D (0.5 ng / μL) was preactivated for 30 min in acidic buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) and then preincubated for 15 min with various concentrations (10, 50, 100, 250, and 500 μM) of LBC4 peptide or pepstatin A (2 μg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6). Activity was then measured in the presence of fluorogenic substrate (30 μM).

[0241] The results are shown in Figure 9C. LBC4 inhibits cathepsin D catalytic activity in a dose-dependent manner up to 250 μM.

[0242] Example 2: Effect of LBC peptides in tumor models derived from triple-negative breast cancer (TNBC) patients

[0243] 2.1 Materials and Methods An outline of the experimental procedure is shown in Figure 10A.

[0244] This study was performed on the BR20001B PDX model (TNBC model) from Crown Bioscience, Leiden (Biopartner Center Leiden, JHOortweg 21n 2333 CH Leiden, Netherlands). As used herein, "PDX" stands for "patient-derived xenograft." Patient-derived xenografts are models in cancer research in which tumor tissue from patients is harvested and cultured for further testing. 450 PDX were uniformly seeded in a hydrogel (Matrigel® and collagen-based, Crown Bioscience, Leiden) in a 384-well plate. After 3 days, the organoids were treated with three doses: 10, 30, and 100 μM of LBC4, LBC5, and LBC8 peptides or vehicle.

[0245] After 7 days, organoids were reprocessed, and after 10 days, supernatants were collected, cultures were fixed, stained, and subjected to high-content imaging (HCI) analysis. - Number of organoids - Total number of nuclei in organoids -Organoid volume - Total branching of organoids Determine TIBCO Spotfire was used for data analysis and visualization.

[0246] 2.2.Results The results for LBC4, LBC5 and LBC8 are shown in Figures 10B, 10C and 10D, respectively.

[0247] These results demonstrate that the LBC4, LBC5, and LBC8 peptides exhibit antitumor activity in a dose-dependent manner in a TNBC tumor model. -Decreased number of nuclei in organoids, -Total organoid volume is reduced, -Decreased total branching of organoids This indicates that...

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Claims

1. (i) a fragment of SEQ ID NO: 1, comprising at least 5 consecutive amino acids of the amino acid sequence NQGNQPQCRCLPGGFLGDRCQYRQCSGYCEN (SEQ ID NO: 1); or (ii) a functional variant of said fragment, preferably derived from said fragment by deletion, insertion and / or substitution of one or more amino acids, wherein said fragment or functional variant derived therefrom is a peptide 5-20 amino acids in size, preferably 7-15 amino acids in size, comprising at least one of amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO:

1.

2. (a) binding to cathepsin D protein, (b) inhibiting the interaction between cathepsin D protein and LRP-1 protein, (c) inhibiting the proliferation of fibroblasts promoted by pro-cathepsin D secreted by cancer cells in the tumor microenvironment, and (d) inhibiting the catalytic activity of cathepsin D. The peptide of claim 1, having at least one activity selected from the group consisting of:

3. 3. The peptide of claim 1, wherein the fragment or functional variant derived therefrom comprises at least two, three or four of the amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:

1.

4. a) the central region of SEQ ID NO: 1, with the amino acid sequence PQCRCLPGGFLGDRCQYRQCSGY (SEQ ID NO: 2); b) the N-terminal region of SEQ ID NO: 1, with the amino acid sequence NQGNQPQCRCLPGGFLGDRCQYR (SEQ ID NO: 3); c) the C-terminal region of SEQ ID NO: 1, with the amino acid sequence RCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 4) d) a region derived from the amino acid sequence in a), b) or c) by N- and / or C-terminal deletion of 1, 2, 3 or 4 amino acids a fragment of; or or a functional variant of said fragment, wherein said fragment or functional variant derived therefrom comprises at least one of amino acids corresponding to amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO:

1.

5. 5. The peptide of claim 4, wherein the fragment or functional variant derived therefrom comprises at least two, three or four of the amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:

1.

6. The peptide of any one of claims 1 to 5, wherein the fragment or functional variant derived therefrom comprises at least 8, 9 or 10 consecutive amino acids of SEQ ID NO: 1, 2, 3 or 4.

7. 7. The peptide of any one of claims 1 to 6, wherein the fragment or functional variant derived therefrom comprises no more than 15, 16, 17, 18, 19 or 20 consecutive amino acids of SEQ ID NO: 1, 2, 3 or 4.

8. The peptide according to any one of claims 1 to 7, which does not contain any C residue at either the N-terminus or the C-terminus.

9. -RCLPGFLGDRCQYRQ (SEQ ID NO: 10), -CLPGFLGDRCQYRQC (SEQ ID NO: 11), - LPGFLGDRCQYRQCS (SEQ ID NO: 12), -PGFLGDRCQYRQCSG (SEQ ID NO: 13), - GDRCQYRQCSGYCEN (SEQ ID NO: 14), - CRCLPGFLGDRCQYR (SEQ ID NO: 15), - PQCRCLPGFLGDRCQ (SEQ ID NO: 16), - NQGNQPQCRCLPGFL (SEQ ID NO: 17), -DRCQYRQ (SEQ ID NO: 25), -DRCQYRQC (SEQ ID NO: 26), - GDRCQYRQ (SEQ ID NO: 27), - GDRCQYRQC (SEQ ID NO: 28), - GDRCQYRQCS (SEQ ID NO: 29), - LGDRCQYRQC (SEQ ID NO: 30), - LGDRCQYRQCS (SEQ ID NO: 31), - LGDRCQYRQCSG (SEQ ID NO: 32), - FLGDRCQYRQCS (SEQ ID NO: 33), - FLGDRCQYRQCSG (SEQ ID NO: 34), - FLGDRCQYRQCSGY (SEQ ID NO: 35), - GFLGDRCQYRQCSG (SEQ ID NO: 36), - GFLGDRCQYRQCSGY (SEQ ID NO: 37), - GDRCQYRQCSGYCE (SEQ ID NO: 38), - GDRCQYRQCSGY (SEQ ID NO: 39), - GDRCQYRQCSG (SEQ ID NO: 40), A fragment of SEQ ID NO: 1, 2, 3 or 4 selected from the group consisting of or a functional variant of said fragment, preferably derived from said fragment by deletion, insertion and / or substitution of one or more amino acids. The peptide according to any one of claims 1 to 8,

10. The peptide of any one of claims 1 to 9, which can inhibit the interaction between the LRP-1 receptor and cathepsin D by at least 40% when measured in a cathepsin D / LRP-1 co-immunoprecipitation assay.

11. A binding affinity K for cathepsin D of less than 500 nM as determined in a microscale thermophoresis assay Ｄ 11. The peptide according to claim 1, having a value of

12. A modified peptide derived from a peptide according to any of claims 1 to 11 by the introduction of one or more chemical modifications which preferably protect said peptide from proteolytic degradation.

13. A polynucleotide encoding the peptide according to any one of claims 1 to 11.

14. A vector comprising the polynucleotide according to any one of claims 1 to 13.

15. A peptide according to any one of claims 1 to 11, a modified peptide according to claim 12, a polynucleotide according to claim 13 or a vector according to claim 14 for use as a pharmaceutical.

16. A peptide according to any one of claims 1 to 11, a modified peptide according to claim 12, a polynucleotide according to claim 13 or a vector according to claim 14 for use in the treatment of a proliferative disorder, in particular cancer.

17. Use of a peptide according to any one of claims 1 to 11 or a modified peptide according to claim 12 in a method for diagnosing and / or staging a disease associated with cathepsin D overexpression.

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