EP4669663A1

EP4669663A1 - PEPTIDIN HIBITORS OF CATHEPSIN-D/LRP-1 INTERACTION

Info

Publication number: EP4669663A1
Application number: EP24705198.0A
Authority: EP
Inventors: Nicolas ETIQUE; Stéphane Dedieu; Stéphanie BAUD; Tesnim AL-KHALIFA; Bruno Kieffer
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Strasbourg; Universite de Reims Champagne Ardenne URCA
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Strasbourg; Universite de Reims Champagne Ardenne URCA
Priority date: 2023-02-21
Filing date: 2024-02-20
Publication date: 2025-12-31
Also published as: WO2024175592A1; CN121002053A; JP2026507727A

Abstract

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

Description

PEPTIDE INHIBITORS OF CATHEPSIN D/LRP-1 INTERACTION

Background

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

Cathepsin D is a soluble aspartic lysosomal endopeptidase of the cathepsin family. In physiological conditions, it cleaves proteins and peptides in the lysosomal compartment (Masson et al., 2010). Cathepsin D is encoded by the CTSD gene as pre-pro-protein. This precursor contains a signal sequence that is cleaved to obtain a 52 kDa precursor, procathepsin D. This 52 kDa form is then transported to the endosomal compartment to be converted to an active 48 kDa intermediate. In the lysosomes, cysteine endopeptidases will cleave the 48 kDa intermediate chain into two chains: a light chain of 14 kDa and a heavy chain of 34 kDa. These two chains will then associate non-covalently to form the mature protease (Masson et al., 2010). In cancer cells, overexpression of the CTSD gene leads to a poor addressing of pro-cathepsin D (52 kDa) to the endosomal compartment and consequently the release of this immature form into the extracellular compartment (Pranjol, et al., 2020 ; Leto, et al., 2004 ; Heylen et al. 2002). The pro-cathepsin D thus secreted is able to stimulate the growth of fibroblasts and in particular mammary fibroblasts. This effect is mediated by its interaction with the p chain (residues 307-479) of the cell surface receptor LRP-1 (Low density lipoprotein Receptor related Protein-1) (Beaujouin et al., 2010 ; Derocq et al., 2012).

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

So far, only an interaction region of 45 AA (residues 349 to 394 of LRP-1 P) has been identified (Beaujouin et al., 2010). However, this 45 AA region, described as “Fragment F4” in WQ2009/043858, has no potential therapeutic application since it is poorly soluble in aqueous solvent, instable when observed in NMR, and difficult to produce and purify due to the presence of numerous cysteines forming multiple disulfide bridges.

Therefore, to develop new targeted therapeutic strategies for the treatment of cancer, there is a need for new pharmacological agents able to bind cathepsin D protein and/or to inhibit the cathepsin D/LRP-1 interaction.

Brief description

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

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

In an embodiment, the fragment or functional variant derived therefrom includes at least 2, 3, or 4 of the amino acids G16, D17, R18, C19, Q20, Y21 , and/or the amino acid R9 of SEQ ID NO:1.

In an embodiment, the peptide is of a fragment of: a) a central region of SEQ ID NO :1 of the amino acid sequence PQCRCLPGFLGDRCQYRQCSGY (SEQ ID NO:2), b) a N-terminal region of SEQ ID NO:1 of the amino acid sequence

NQGNQPQCRCLPGFLGDRCQYR (SEQ ID NO:3), c) a C-terminal region of SEQ ID NO:1 of the amino acid sequence

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

The fragment or functional variant derived therefrom preferably includes at least 2, 3, or 4 of the amino acids G16, D17, R18, C19, Q20, Y21, and/or the amino acid R9 of SEQ ID NO:1.

In an embodiment the fragment or functional variant derived therefrom includes the amino acids D17, R18, Q20, and Y21 , and/or the amino acid R9 of SEQ ID NO:1.

In an embodiment the fragment or functional variant derived therefrom includes the amino acids G16, D17, R18, Q20, Y21, and/or the amino acid R9 of SEQ ID NO:1.

In an embodiment the fragment or functional variant derived therefrom includes the amino acids G16, D17, R18, C19, Q20, and/or the amino acid R9 of SEQ ID NO:1.

In an embodiment the fragment or functional variant derived therefrom includes the amino acids D17, R18, C19, Q20, Y21, and/or the amino acid R9 of SEQ ID NO:1.

In an embodiment the fragment or functional variant derived therefrom includes the amino acids G16, D17, R18, C19, Q20, Y21, and/or the amino acid R9 of SEQ ID NO:1.

In an embodiment, the fragment or functional variant derived therefrom comprises at least 8, 9 or 10 consecutive amino acids of SEQ I D NO: 1 , 2, 3 or 4.

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

In an embodiment, the peptide does not comprise a C residue both at N- and C- terminal.

In an embodiment, the peptide is a fragment of SEQ ID NO:1, 2, 3, or 4 selected from the group consisting of:

- RCLPGFLGDRCQYRQ (SEQ ID NQ: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 NQ: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), or a functional variant of said fragment, preferably deriving from said fragment by deletion, insertion, and/or substitution of one or more amino acids.

In an embodiment, the peptide can inhibit the interaction between the LRP-1 receptor and cathepsin D by at least 40% as measured in a cathepsin D/LRP-1 co-immunoprecipitation assay.

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

In another aspect, the invention relates to a modified peptide deriving from the peptide according to any of claims 1 to 11 , by the introduction of one or more chemical modifications which preferably protect the peptide against proteolysis.

In another aspect, the invention relates polynucleotide encoding the peptide according to the invention.

Another aspect of the invention relates to a vector comprising the polynucleotide according to the invention.

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

The peptide as described herein, the modified peptide as described herein, the polynucleotide as described herein, or the vector as described herein, is preferably for use in treating a proliferative disorder, in particular a cancer.

Another aspect of the invention relates to the use of the peptide or modified peptide as described herein for diagnosing and/or staging a disease associated with Cathepsin D overexpression.

Detailed description

The present disclosure follows at least in part from the surprising findings by the inventors that small peptides derived from a specific region of the amino acid sequence of the p chain of the LRP-1 receptor are able to bind cathepsin D protein and to inhibit the LRP-1/ cathepsin D interaction, thereby inhibiting the proliferation of fibroblasts promoted by the cathepsin D secreted by cancer cells within a tumor microenvironment.

The inventors have delineated a key region of 30 amino acids involved in the interaction of LRP-1 with cathepsin D. This key region of amino sequence NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:1), located on the p chain of the LRP-1 receptor had not been identified until then and was delimited after 6 years of research by the inventors. The inventors have found that, surprisingly, small peptides derived from this key interaction region were sufficient to bind efficiently to cathepsin D and to inhibit the LRP-1/ cathepsin D interaction. They have also demonstrated the efficacy of some of these peptides to inhibit the proliferation of fibroblasts promoted by the cathepsin D secreted by cancer cells on a model mimicking cell tumor environment. Therefore, in a first aspect, the invention provides a peptide of 5 to 20 amino acids in size, preferably of 7 to 15 amino acids in size, preferably having at least one activity selected from (a) binding cathepsin D protein, (b) inhibiting the interaction between cathepsin D protein and LRP-1 protein, (c) inhibiting the proliferation of fibroblasts promoted by the cathepsin D secreted by cancer cells within a tumor microenvironment, and (d) inhibiting the catalytic activity of cathepsin D, wherein the peptide is (i) a fragment of an amino acid sequence NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:1), said fragment comprising at least 5 consecutive amino acids of SEQ ID NO:1 , or (ii) a functional variant of said fragment, preferably deriving from said fragment by deletion, insertion, and/or substitution of one or more amino acids, wherein the fragment or functional variant derived therefrom includes at least one of the amino acid R9, G16, D17, R18, C19, Q20, and Y21 of SEQ ID NO:1.

In an embodiment, the fragment of SEQ ID NO:1 or functional variant derived therefrom includes at least 2, 3, or 4 of the amino acids G16, D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1.

In an embodiment, the fragment of SEQ ID NO:1 or functional variant derived therefrom includes at least the amino acids G16, D17, R18, C19, Q20 and/or the amino acid R9 of SEQ ID NO:1.

In an embodiment, the peptide is a fragment of: a) a central region of SEQ ID NO :1 of the amino acid sequence PQCRCLPGFLGDRCQYRQCSGY (SEQ ID NO:2), b) a N-terminal region of SEQ ID NO:1 of the amino acid sequence

NQGNQPQCRCLPGFLGDRCQYR (SEQ ID NO:3), c) a C-terminal region of SEQ ID NO :1 of the amino acid sequence

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

In other terms, in this embodiment the peptide is a fragment SEQ ID NO:2, 3 or 4 or a functional variant of said fragment that includes respectively at least one of:

-the amino acid R3, G10, D11 , R12, C13, Q14, Y15 of SEQ ID NO:2;

-the amino acid R9, G16, D17, R18, C19, Q20, Y21 of SEQ ID NO:3;

-the amino acid G8, D9, R10, C11 , Q12, Y13 of SEQ ID NO:4.

In an embodiment, the fragment of SEQ ID NO:2, 3, or 4 or functional variant derived therefrom includes at least 2, 3, or 4 of the amino acids G16, D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1. In other terms, in this embodiment the peptide is a fragment of SEQ ID NO:2, 3 or 4 or a functional variant of said fragment that includes respectively:

-at least 2, 3, 4 of the amino acids G10, D11 , R12, C13, Q14, Y15 and/or the amino acid R3 of SEQ ID NO:2;

-at least 2, 3, 4 of the amino acids G16, D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:3; -at least 2, 3, 4 of the amino acids G8, D9, R10, 011 , Q12, Y13 of SEQ ID NO:4.ln an embodiment, the fragment of SEQ ID NO:2, 3, or 4 or functional variant derived therefrom includes at least the amino acids D17, R18, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1. In other terms, in this embodiment the peptide is a fragment of SEQ ID NO:2, 3 or 4 or a functional variant of said fragment that includes respectively:

- the amino acids D11 , R12, Q14, Y15 and/or the amino acid R3 of SEQ ID NO:2;

- the amino acids D17, R18, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:3;

- the amino acids D9, R10, Q12, Y13 of SEQ ID NO:4.

In an embodiment, the fragment of SEQ ID NO:2, 3, or 4 or functional variant derived therefrom includes at least the amino acids G16, D17, R18, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1 . In other terms, in this embodiment the peptide is a fragment of SEQ ID NO:2, 3 or 4 or a functional variant of said fragment that includes respectively:

- the amino acids G10, D11 , R12, Q14, Y15 and/or the amino acid R3 of SEQ ID NO:2;

- the amino acids G16, D17, R18, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:3;

- the amino acids G8, D9, R10, Q12, Y13 of SEQ ID NO:4.

In an embodiment, the fragment of SEQ ID NO:2, 3, or 4 or functional variant derived therefrom includes at least the amino acids G16, D17, R18, C19, Q20 and/or the amino acid R9 of SEQ ID NO:1 . In other terms, in this embodiment the peptide is a fragment of SEQ ID NO:2, 3 or 4 or a functional variant of said fragment that includes, respectively:

- the amino acids G10, D11 , R12, C13, Q14 and/or the amino acid R3 of SEQ ID NO:2;

- the amino acids G16, D17, R18, C19, Q20 and/or the amino acid R9 of SEQ ID NO:3;

- the amino acids G8, D9, R10, C11 , Q12 of SEQ ID NO:4.

In an embodiment, the fragment of SEQ ID NO:2, 3, or 4 or functional variant derived therefrom includes at least the amino acids D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1 . In other terms, in this embodiment the peptide is a fragment of SEQ ID NO:2, 3 or 4 or a functional variant of said fragment that includes respectively:

- the amino acids D11 , R12, C13, Q14, Y15 and/or the amino acid R3 of SEQ ID NO:2;

- the amino acids D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:3;

- the amino acids D9, R10, C11 , Q12, Y13 of SEQ ID NO:4.

In an embodiment, the fragment of SEQ ID NO:2, 3, or 4 or functional variant derived therefrom includes at least the amino acids G16, D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1. In other terms, in this embodiment the peptide is a fragment of SEQ ID NO:2, 3 or 4 or a functional variant of said fragment that includes respectively:

- the amino acids G10, D11 , R12, C13, Q14, Y15 and/or the amino acid R3 of SEQ ID NO:2;

- the amino acids G16, D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:3;

- the amino acids G8, D9, R10, C11 , Q12, Y13 of SEQ ID NO:4.

Other characteristics of the fragment

In an embodiment, the peptide is a peptide of 5 to 20 amino acids in size, preferably of 7 to 15 amino acids in size. In an embodiment, the peptide is a peptide of 7 to 20 amino acids in size, preferably of 7 to 15 amino acids in size.

In an embodiment, the peptide comprises no more than 15, 16, 17, 18, 19 or 20 amino acids.

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

In an 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.

In an embodiment, the fragment or functional variant derived therefrom comprises no more than 15, 16, 17, 18, 19 or 20 consecutive amino acids of SEQ I D NO: 1 .

In an embodiment, the fragment or functional variant derived therefrom comprises between 5 and 20, preferably between 5 and 15 consecutive amino acids of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In an embodiment, the fragment or functional variant derived therefrom comprises between 7 and 20, preferably between 7 and 15 consecutive amino acids of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In an 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.

Specific fragments

In a particular embodiment, the peptide is a fragment of SEQ ID NO:1 , 2, 3, or 4 selected from the group consisting of:

LBC1 of amino acid sequence RCLPGFLGDRCQYRQ (SEQ ID NQ:10), LBC2 of amino acid sequence CLPGFLGDRCQYRQC (SEQ ID NO:11), LBC3 of amino acid sequence LPGFLGDRCQYRQCS (SEQ ID NO: 12), LBC4 of amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO:13),

- LBC5 of amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO: 14), LBC6 of amino acid sequence CRCLPGFLGDRCQYR (SEQ ID NO:15), LBC7 of amino acid sequence PQCRCLPGFLGDRCQ(SEQ ID NO:16), LBC8 of amino acid sequence NQGNQPQCRCLPGFL (SEQ ID NO:17), LBC16 of amino acid sequence DRCQYRQ (SEQ ID NO:25), LBC17 of amino acid sequence DRCQYRQC (SEQ ID NO:26), LBC18 of amino acid sequence GDRCQYRQ (SEQ ID NO:27), LBC19 of amino acid sequence GDRCQYRQC (SEQ ID NO:28), LBC20 of amino acid sequence GDRCQYRQCS (SEQ ID NO:29), LBC21 of amino acid sequence LGDRCQYRQC (SEQ ID NQ:30), LBC22 of amino acid sequence LGDRCQYRQCS (SEQ ID NO:31), LBC23 of amino acid sequence LGDRCQYRQCSG (SEQ ID NO:32), LBC24 of amino acid sequence FLGDRCQYRQCS (SEQ ID NO:33), LBC25 of amino acid sequence FLGDRCQYRQCSG (SEQ ID NO:34), LBC26 of amino acid sequence FLGDRCQYRQCSGY (SEQ ID NO:35), LBC27 of amino acid sequence GFLGDRCQYRQCSG (SEQ ID NO:36), LBC28 of amino acid sequence GFLGDRCQYRQCSGY (SEQ ID NO:37), LBC29 of amino acid sequence GDRCQYRQCSGYCE (SEQ ID NO:38), LBC30 of amino acid sequence GDRCQYRQCSGY (SEQ ID NO:39), LBC31 of amino acid sequence GDRCQYRQCSG (SEQ ID NO:40), or a functional variant of said fragment, preferably deriving from said fragment by deletion, insertion, and/or substitution of one or more amino acids, wherein the fragment or functional variant derived therefrom includes at least one of the amino acids corresponding to amino acids R9, D17, R18, C19, Q20, and Y21 of SEQ ID NO:1.

LBC4 of amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO: 13)

- LBC5 of amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO:14)

LBC6 of amino acid sequence CRCLPGFLGDRCQYR (SEQ ID NO: 15) LBC16 of amino acid sequence DRCQYRQ (SEQ ID NO:25) LBC31 of amino acid sequence GDRCQYRQCSG (SEQ ID NQ:40). or a functional variant of said fragment, preferably deriving from said fragment by deletion, insertion, and/or substitution of one or more amino acids, wherein the fragment or functional variant derived therefrom includes at least one of the amino acids corresponding to amino acids R9, G16, D17, R18, C19, Q20, and Y21 of SEQ ID NO:1.

In an embodiment, the peptide is not a fragment of SEQ ID NO:15.

Peptide activities

The peptide preferably has at least one activity selected from:

(a) the ability to bind cathepsin D protein,

(b) the ability to inhibit the LRP-1 protein/ cathepsin D protein interaction,

(c) the ability to inhibit the proliferation of fibroblasts promoted by the cathepsin D secreted by cancer cells within a tumor microenvironment.

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

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

(a) ability to bind cathepsin D

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

In a particular embodiment, the peptide is able to specifically bind the extracellular part of the LRP-1 p chain (LRP-1 PC).

The expression “able to specifically bind the extracellular part of the LRP-1 p chain” means that the peptide binds to LRP-1 pc with a higher binding affinity (e.g., no less than about 2- fold, no less than about 5-fold, no less than about 10-fold, no less than about 30-fold, no less than about 100-fold, no less than about 1 ,000-fold, or no less than about 10,000-fold) than to another part of the LRP1 protein, in particular to LRP-1 a chain. In an embodiment, the peptide does not show any binding with the alpha chain of LRP-1 (LRPIaC).

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

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

The binding affinity by microscale thermophoresis is well known by the person skilled in the art and can be identified and/or quantified performing the following procedure: providing a peptide to be tested, pro-cathepsin D labeled with His-Tag mixing the labeled pro-cathepsin D with the peptide to be tested using a 0.5-fold dilution in series ranging from 10 pM to 0.15 pM; analyzing the mixtures with an instrument for MST, for example using a Monolith NT.115 (NanoT emper) instrument at 25°C with instrument parameters as follows : 20% Pico-RED excitation-power, 40% medium MST-power, and 5/20/5 laser off/on/off ; and analyzing data for example with NT MO Affinity Analysis v2.1.3 (NanoTemper); identifying and/or quantifying the binding affinity of the peptide to be tested to procathepsin D.

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

(b) ability to inhibit the LRP-1/ cathepsin D interaction

The term "inhibit" or "inhibitor" refers to the ability of a compound to reduce, 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 the cathepsin D secreted by cancer cells within a tumor microenvironment. In some embodiments, the term refers to the catalytic activity of procathepsin D

In an embodiment, the peptide is able to inhibit the LRP-1/ cathepsin D interaction.

In an embodiment, the peptide can inhibit at least 30% of the interaction between the LRP-1 receptor and cathepsin D, as measured in a cathepsin D/LRP-1 co-immunoprecipitation assay.

The peptide is preferably able to inhibit at least 40%, preferably at least 50%, preferably at least 60% of the cathepsin D/LRP-1 interaction, as determined by co-immunoprecipitation.

The determination and quantification of the ability of peptides to inhibit the cathepsin D/LRP-1 interaction by co-immunoprecipitation can easily be performed by a skilled person in the art.

The capacity of the peptide to inhibit the interaction between the LRP-1 receptor and cathepsin D can be identified and/or quantified performing the following procedure: providing a peptide to be tested and recombinant cathepsin D tagged with 6His-Tag incubating overnight 100 ng of cathepsin D with LRP-1 a (LRPlaF) or LRP-1 p fragments (LRPi F) displaying a myc epitope in the absence (control) or presence of the peptide to be tested, e.g. at 100 pM ; depositing the mixture on Ni-NTA beads for 2h; eluting the complexes formed with 2X Laemmli buffer; performing SDS-PAGE electrophoresis and Western blot using antibodies against procathepsin D and the myc tag; identifying and/or quantifying the binding activity of the LRP-1 fragment to procathepsin D in the absence (Ctrl) or presence of the peptide to be tested. A detailed procedure is also described in the experimental part below.

(c) ability to inhibit proliferation of fibroblasts promoted by the cathepsin D secreted by cancer cells within a tumor microenvironment

In an embodiment, the peptide can inhibit the proliferation of fibroblasts promoted by the cathepsin D secreted by cancer cells, in vitro or in vivo, in particular within a tumor microenvironment.

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

The peptide is preferably able to inhibit at least 40%, preferably at least 50%, preferably at least 60% of the proliferation of fibroblasts promoted by the cathepsin D secreted by cancer cells within a tumor microenvironment, as determined in a 3D co-culture model of cancer cells and fibroblasts.

The capacity of the peptide to inhibit proliferation of fibroblasts promoted by the cathepsin D secreted by cells, especially cancer cells within a tumor microenvironment can be identified and/or quantified performing the following procedure:

- providing a peptide to be tested, fibroblasts, and a monolayer of COS-7 cells overexpressing and secreting (pCD) or not (Ctrl) cathepsin D,

- seeding fibroblasts embedded in a solubilized basement membrane matrix above of a monolayer of pCD or Ctrl COS-7 cells, in presence or not of the peptide to be tested (e.g. at 100 pM);

- observing the co-cultures by phase contrast microscopy during 6 days, for example at Day 1 , Day 4 and Day 6 the fibroblast colonies co-cultured with COS-7 Ctrl or pCD cells in the absence or presence of the peptide to be tested

- identifying and/or quantifying the inhibiting activity of the peptide to be tested on the proliferation of fibroblasts promoted by the cathepsin D secreted by pCD COS-7 cells.

The solubilized basement membrane may for example be a solubilized basement membrane secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma produced by Corning Life Sciences under the trademark “Matrigel”.

(d) ability to inhibit the activity of cathepsin D

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

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

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

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

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

- measuring the catalytic activity in the presence of a fluorogenic substrate (30 pM) - identifying and/or quantifying the ability of the peptide to be tested to inhibit the catalytic activity of cathepsin D within a medium mimicking the pH of a tumor microenvironment.

Functional variant

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

The functional variant is derived from a fragment as described herein by the introduction of one or more mutations (deletion, insertion, and/or substitution) at specific amino acid positions, provided that it includes at least some amino acids of SEQ ID NO:1 , 2, 3 or 4 as described above.

In particular, the functional variant includes at least one of:

- the amino acid R9, G16, D17, R18, C19, Q20, and Y21 of SEQ ID NO:1 ,

- the amino acid R3, G10, D11 , R12, C13, Q14, Y15 of SEQ ID NO:2;

- the amino acid R9, G16, D17, R18, C19, Q20, Y21 of SEQ ID NO:3;

- the amino acid G8, D9, R10, C11, Q12, Y13 of SEQ ID NO:4.

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

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

Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software such as BLAST (Altschul et 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 of the programs known in the art. When using such software, the default parameters, e.g., for gap penalty and extension penalty, are preferably used. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3 and an expectation (E) of 10.

In an embodiment, the functional variant derives from a fragment as described herein, in particular from a fragment of amino sequence SEQ ID NO:1 ; 2, 3 or 4 as described herein, by deletion, insertion, and/or substitution of one or more amino acids. In an embodiment, the functional variant derives from a fragment as described herein, in particular from a fragment of amino 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.

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

Examples of conservative substitutions are set out in the Table 1 below:

Table 1 : Examples of conservative substitutions

In an embodiment, the functional variant derives 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 an embodiment, the functional variant derives from a fragment of SEQ ID NO: 1 , 2, 3 or 4 as described above by the conservative substitution(s) of one or more cysteine residues.

The inventors have shown that the cysteines of the LBC4 peptide are able to form disulfide bridges in oxidizing environment, resulting in a peptide unable to block the catalytic activity of cathepsin D. Besides, the inventors have also shown that the substitution of the cysteine 8 to a serine (C8S) in the LBC4 peptide impairs the ability of the peptide to block the catalytic activity of cathepsin D. Without wanting to be bound by a theory, the inventors believe that the latter suggests that at least in some embodiments, this particular cysteine residue should preferably be left unchanged.

In an embodiment, the fragment is a fragment of SEQ ID NO: 1 , 2, 3 or 4 as described above that includes at least two cysteine residues, and the functional variant derives from said fragment by the conservative substitution of one or more of those cysteine residues, preferably by one or more amino acid residues selected from serine (S), valine (V), threonine (T), and selenocysteine, the 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 remaining preferably unchanged.

In an embodiment, the fragment is a fragment of SEQ ID NO: 1 , 2, 3 or 4 as described above that includes: the amino acid C19 and at least one of C10 or C24 of SEQ ID NO:1 , the amino acid C14 and at least one of C5 or C19 of SEQ ID NO:2, the amino acid C19 and at least one of C8 or C10 of SEQ ID NO:3, the amino acid C11 and at least one of C2 or C16 of SEQ ID NO:4, and the functional variant derives from said fragment by the conservative substitution of:

1 or 2 of the amino acids C10 and C24 of SEQ ID NO:1 ,

1 or 2 of the amino acids C5 and C19 of SEQ ID NO:2,

1 or 2 of the amino acids C8 and C10 of SEQ ID NO:3,

1 or 2 of the amino acids C2 and C16 of SEQ ID NO:4, preferably by an amino acid preferably selected from serine (S), valine (V), threonine (T), and selenocysteine.

In an embodiment, the fragment is a fragment of SEQ ID NO: 1 , 2 or 4 as described above that includes: the amino acids C19 and C24 of SEQ ID NO:1 , the amino acids C14 and C19 of SEQ ID NO:2, the amino acids C11 and C16 of SEQ ID NO:4, and the functional variant derives from said fragment by the conservative substitution of: the amino acid C24 of SEQ ID NO: 1 , the amino acid C19 of SEQ ID NO:2, the amino acid C16 of SEQ ID NO:4, preferably by an amino acid selected from serine (S), valine (V), threonine (T), and selenocysteine.

In an embodiment, the fragment is a fragment of SEQ ID NO: 1 , 2 or 4 as described above selected from:

LBC4 of amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO:13), - LBC5 of amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO: 14), LBC6 of amino acid sequence CRCLPGFLGDRCQYR (SEQ ID NO:15), LBC31 of amino acid sequence GDRCQYRQCSG (SEQ ID NQ:40), and the functional variant derives from said fragment by at least the conservative substitution of: the amino acid C13 of SEQ ID NO:13 the amino acid C9 of SEQ ID NO:14

1 or 2 of the amino acid C1 and C3 of SEQ ID NO:15 the amino acid C4 of SEQ ID NQ:40 preferably by an amino acid selected from serine (S), valine (V), threonine (T), and selenocysteine.

In an embodiment, the functional variant comprises or consists of an amino acid sequence which differs from the sequence of a fragment of SEQ ID NO: 1 , 2, 3 or 4 as described above by no more than 1 , 2, or 3 amino acids, preferably by no more than 1 o 2 amino acids.

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

In an embodiment, the functional variant comprises or consists of an amino acid sequence which 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.

In an 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.

In an 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 includes at least one of:

- the amino acids R9, G16, D17, R18, C19, Q20, Y21 of SEQ ID NO:1 ,

- the amino acids R3, G10, D11 , R12, C13, Q14, Y15 of SEQ ID NO:2,

- the amino acids R9, G16, D17, R18, C19, Q20, Y21 of SEQ ID NO:3,

- the amino acids G8, D9, R10, C11 , Q12, Y13 of SEQ ID NO:4.

In an 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 includes at least 2, 3, or 4 of: the amino acids G16, D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1 , the amino acids G10, D11 , R12, C13, Q14, Y15 and/or the amino acid R3 of SEQ ID NO:2, the amino acids G16, D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:3,

- the amino acids G8, D9, R10, C11 , Q12, Y13 of SEQ ID NO:4.

In an 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 includes at least : the amino acids D17, R18, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1 , the amino acids D11 , R12, Q14, Y15 and/or the amino acid R3 of SEQ ID NO:2, the amino acids D17, R18, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:3,

- the amino acids D9, R10, Q12, Y13 of SEQ ID NO:4.

In an 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 includes at least: the amino acids G16, D17, R18, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1 , the amino acids G10, D11 , R12, Q14, Y15 and/or the amino acid R3 of SEQ ID NO:2, the amino acids G16, D17, R18, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:3,

- the amino acids G8, D9, R10, Q12, Y13 of SEQ ID NO:4.

In an 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 includes at least: the amino acid G16, D17, R18, C19, Q20, and/or the amino acid R9 of SEQ ID NO:1 , the amino acid G10, D11 , R12, C13, Q14, and/or the amino acid R3 of SEQ ID NO:2, the amino acid G16, D17, R18, C19, Q20, and/or the amino acid R3 of SEQ ID NO:3,

- the amino acid G8, D9, R10, C11 , Q12, of SEQ ID NO:4.

In an 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 includes at least : the amino acids D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:1 , the amino acids D11 , R12, C13, Q14, Y15 and/or the amino acid R3 of SEQ ID NO:2, the amino acids D17, R18, C19, Q20, Y21 and/or the amino acid R9 of SEQ ID NO:3, - the amino acids D9, R10, C11 , Q12, Y13 of SEQ ID NO:4.

In an 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 includes at least: the amino acid G16, D17, R18, C19, Q20, Y21 and/or the amino acid R9 of

SEQ ID NO:1 , the amino acid G10, D11 , R12, C13, Q14, Y15 and/or the amino acid R3 of

SEQ ID NO:2, the amino acid G16, D17, R18, C19, Q20, Y21 and/or the amino acid R3 of

SEQ ID NO:3,

- the amino acid G8, D9, R10, C11 , Q12, Y13 of SEQ ID NO:4.

In an embodiment, the fragment is a fragment of SEQ ID NO: 1 or 4 as described above selected from:

LBC4 of amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO:13),

- LBC5 of amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO:14). and the functional variant derived therefrom 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: 13 or 14, wherein the fragment or functional variant derived therefrom includes at least: the amino acids D6, R7, Q9, Y10 of SEQ ID NO:13, the amino acids D2, R3, Q5, Y6 of SEQ ID NO: 14.

LBC4 of amino acid sequence PGFLGDRCQYRQCSG (SEQ ID NO:13),

- LBC5 of amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO:14). and the functional variant derived therefrom 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: 13 or 14, wherein the fragment or functional variant derived therefrom includes at least: the amino acids G5, D6, R7, Q9, and Y10 of SEQ ID NO:13, the amino acids G1 , D2, R3, Q5, Y6 of SEQ ID NO:14.

Modified peptide

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

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

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

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

The N- and/or C-termini of the peptide are advantageously protected against proteolysis. For instance, the N-terminus is in the form of an acetyl group and/or the C-terminus in the form of an amide group.

Alternatively, or additionally, the peptide may be protected against proteolysis by internal modifications such as the replacement of at least one -CONH- peptide bond by a (CH2NH) reduced bond, a (NHCO) retro-inverso bond, a (CH2-O) methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO- CH) cetomethylene bond, a (CHOH-CH2) hydroxy ethylene bond, a (N-N) bond, a E- alcene bond, or a -CH=CH- bond.

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

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

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

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

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

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

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

Polynucleotide

Yet another aspect of the invention relates to an isolated polynucleotide encoding the peptide.

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

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

In another preferred embodiment, the polynucleotide is inserted in a vector. Preferably, said recombinant vector is an expression vector capable of expressing said polynucleotide when transfected or transformed into a host cell such as a prokaryotic or eukaryotic cell. The polynucleotide is inserted into the expression vector in proper orientation and correct reading frame for expression. Preferably, the polynucleotide is operably linked to at least one transcriptional regulatory sequence and, optionally to at least one translational regulatory sequence. Recombinant vectors include usual vectors used in genetic engineering and gene therapy including for example plasmids and viral vectors, such as for example lentivirus and adenovirus vectors.

Therapeutic method of use

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

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

The term “tumor microenvironment” refers to a dynamic medium containing cells and macromolecules that interact with cancer cells and promotes tumor development, progression and/or metastasis. Therefore, another aspect of the present invention relates to a peptide, polynucleotide, and/or vector as described herein, for use in treating a proliferative disorder, in particular a cancer, preferably in a human patient.

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

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

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

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

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

In an embodiment, the proliferative disorder or cancer is a proliferative disorder or cancer associated with cathepsin D overexpression, i.e. in which tumor cells overexpress cathepsin D, preferably at least 2-fold, e.g. 2- to 50-fold, compared to non-tumorous cells.

In an embodiment, the proliferative disorder or cancer is a proliferative disorder or a cancer associated with cathepsin D overexpression and secretion in biological tissues or fluids, in particular serum, plasma, compared to biological tissues or fluids of a subject not suffering from cancer.

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

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

Examples of cancers in which cathepsin D is promoted by cathepsin D secreted by tumor cells include, but is not limited to breast cancer, ovarian cancer, endometrial cancer, prostatic cancer, kidney cancer, colorectal cancer.

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

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

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

Pharmaceutical composition

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

The invention relates also 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 principle, in particle an anti-tumor agent, more preferably a chemotherapeutic agent, as described herein.

Combination therapy

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

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

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

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

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

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

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

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

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 or a functional variant derived therefrom as described herein is used in combination with a chemotherapeutic agent as described herein.

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

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

Administration route

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

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

Diagnostic and/or staging of diseases associated with cathepsin D overexpression.

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

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

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

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

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

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

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

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

Legend of the Figures

Figure 1 : In silico workflow used to study LRP-1 residues involved in the cathepsin D/LRP-1 interaction. Different conformations of LRP-derived peptides (F4 Fragment and EGF1920 fragments) were generated and then selected to performed docking experiments with Cathepsin D (CathD).

Figure 2: In silico analysis of LRP-1 residues involved in the cathepsin D/LRP-1 interaction. Figure 2A and Figure 2B: Histograms representing the contact frequency of the residues of the EGF1920 (Figure 2A) or F4 fragment (Figure 2B) with cathepsin D during all the molecular docking experiments performed. Residue with contact frequencies greater than or equal to 50% are pointed with a black arrow. Figure 2C: Superposition of the histogram A (white bar) and B (black bar) at the level of the common CRCLPGFLGDRCQYRQCSGYCEN (SEQ I D NO:7) sequence of the F4 and EGF 1920 fragments. Residue with contact frequencies greater than 50% for both fragments are pointed with a black arrow The dotted line materializes a frequency of 50%.

Figure 3: Affinity study of the F4 Fragment and of the LBC4 and LBC5 peptides for cathepsin D by microscale thermophoresis. Dose-response curves representing the binding interaction between cathepsin D and the F4 Fragment, the peptide LBC4 or the peptide LBC5.

Figure 4: Study of the ability of LBC1 to LBC5 peptides to block the cathepsin D/LRP-1 P-chain interaction by co-immunoprecipitation. Cathepsin D (100 ng) with a 6-His tag is incubated overnight with LRP-1 a (LRPlaF) or LRP-1 fragments (LRPipF) displaying a myc epitope in the absence (Figure 4A) or presence of LBC peptides (100 pM) (Figure 4B). The mixture was then deposited on Ni-NTA beads for 2h. The complexes formed were eluted with 2X Laemmli buffer and subjected to SDS-PAGE electrophoresis and Western blot using antibodies against cathepsin D and the myc tag. (Figure 4C) Histograms represent the binding of the LRP-1 p fragment to cathepsin D in the absence (Ctrl) or presence of LBC.

Figure 5: Effect of LBC1 to LBC5 peptides on the growth of murine embryonic fibroblasts (MEF-1) co-cultured with COS-7 cells overexpressing or not cathepsin D. (Figure 5A) Schematic representing the co-culture model used. Fibroblasts are seeded in Matrigel above of a monolayer of COS-7 cells overexpressing and secreting (pCD) or not (Ctrl) cathepsin D. (Figure 5B) Western blot analysis of the amount of cathepsin D present, after 6 days, in the co-culture medium of wells with COS-7 cell overexpressing and secreting (pCD) or not (Ctrl) cathepsin D (Figure 5C) Phase contrast microscopy observation at Day 1 , Day 4 and Day 6 of fibroblast colonies co-cultured with COS-7 Ctrl or pCD cell in the absence or presence of LBC1 to LBC5 peptides at 100 pM. (Figure 5D) Histogram representing colonies with area greater than 5,000 pixels² at Day 6 (each point equals one colony). n=5, one-way anova ****: p<0.0001 , **: p<0.01 , Scale bar: 20 pm

Figure 6: Localization of the different peptides derived from the p-chain of LRP-1 synthesized to delineate the region of high interaction between LRP-1 and cathepsin D. LBC1-LBC5 peptides are the peptides first studied. The LBC6-LBC8 peptides were used to delineate the N-terminal of the high interaction region and the LBC9-LBC15 peptides for the C-terminal of the high interaction region.

Figure 7: NMR spectroscopic characterization of the LBC4 peptide under oxidative conditions. The LBC4 peptide was diluted in HCI/NaCI solution (30 pl) containing 20 mM Tris Buffer at pH 7.5 (130 pl,) then 16 pL of DMSO (oxidizing condition) or 16 pL of water (nonoxidizing condition) were added. (Figure 7A) After, 72 h incubation at room temperature, 1 D 1 H NMR spectra in the -NH region was measured at 298 Kelvin. (Figure 7B) Determination of chemical shifts in the NH region between spectra obtained in the absence (bottom) and presence (top) of DMSO (oxidizing condition). (Figure 7C and Figure 7D) Affinity determination for cathespin D of the oxidized LBC4 peptide by microscale thermophoresis. Labeled cathepsin D was mixed with the LBC4 peptide without a disulfide bridge between its 2 cysteines (LBC4) or with (oxydised-LBC4) using a 0.5-fold dilution series, ranging from 10 pM 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 5000 and 10 000 cells/well respectively in a 96-well plate and incubated during 48 hours without (NT) or with LBC4, LBC5 or LBC15 peptides at various concentration (6.25 to 50 pM). Cell number was measured using Cell proliferation Reagent WST-1.

Figure 9: Effect of LBC peptides on the cathepsin D catalytic activity at pH 3.5 and pH 6. (Figure 9A) cathepsin D (2 ng/pL) with LBC peptides (100 pM) or pepstatin A (2 ng/pL) were pre-incubated 15 min in Assay Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 3.5). Activity was then measured in the presence of a fluorogenic substrate (15 pM). (Figure 9B) Cathepsin D (0.5 ng/pL) was pre-activated 30 min in Acidic Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 3.5) and then pre-incubated with peptide (100 pM) or pepstatin A (2 pg/mL) 15 min in Assay Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 6). Activity was then measured in the presence of fluorogenic substrate (30 pM). (Figure 9C) Pro-cathepsin D (0.5 ng/pL) was pre-activated 30 min in Acidic Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 3.5) and then pre-incubated with LBC4 peptide at various concentration (10 - 50 - 100 - 250 and 500 pM) or pepstatin A (2 pg/mL) 15 min in Assay Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 6). Activity was then measured in the presence of fluorogenic substrate (30 pM).

Figure 10: Effect of LBC peptides on TNBC tumoroid model. (Figure 10A) Experimental procedure: patient Derived Xenograft (PDX from TNBC, Crown Bioscience, Leiden, Netherlands) were seeded in hydrogel in 384-wells plates. Organoid were treated at days 3 and 7 with LBC4 (Figure 10B), LBC5 (Figure 10C) and LBC8 (Figure 10D) peptides at various concentrations (10, 30 and 100 pM) and cultures were maintained until day 10. Cultures were fixed and stained and analyzed using TIBCO spotfire software. n=4, Kruskal-Wallis test with Benjamini, Krieger and Yekutieli correction ***: p<0.001 , **: p<0.01 , *: p<0.05.

Examples

The present disclosure is further illustrated by the following examples.

Example 1 : Identification of key cathespin D/LRP-1 interaction region and study of peptides derived from this interaction region

1.1. MATERIALS AND METHODS

Affinity constant determination by microscale thermophoresis

Materials & Reagents

PCR tubes & tips - HEPES (H0887-20ML; Sigma Aldrich) - Tween® 20 (P1379-25ML; Sigma Aldrich) - Monolith His-Tag labeling Kit RED-Tris-NTA 2^nd generation (MO-L018; Nanotemper)

- Human Cathepsin D / CTSD protein, His Tag (CTD-H5226; Acrobiosystems) - Milli-Q® water

- Water (W3500-100ML; Sigma Aldrich) - Monolith capillaries (MO-K022; Nanotemper)

‘Assay Buffer: HEPES 50 mM at pH 7,5 supplemented with 0,05% tween®20

"Stock solution of cathepsin D at -80°C in aliquots - Concentration: 400 pg/mL - 9.2 pM

Cathepsin D Labeling (Monolith His-Tag Labeling Kit)

1 . Add 8 mL of Milli-Q® water to the vials containing 5X PBS-T to obtain 1X PBS-T

2. Suspend the dye in 25 pL of PBS-T to obtain 5 pM dye solution (Aliquots of 2 pL at - 20°C)

3. Prepare a 100 Nm dye solution by mixing 2 pL of dye (5 pM) and 98 pL of Assay Buffer*

4. Adjust the cathepsin D" concentration to 200 nM in a volume of 100 pL

5. Mix 90 pL of protein (200 nM) with 90 pL of dye (100 nM) in a 1 .5 mL tube

6. Incubate for 30 min at room temperature

7. Centrifuge the sample for 10 min at 4°C and 15 000 g and transfer the supernatant to a fresh tube

8. The protein is labeled and ready for binding assay

Preparation of peptides 1 . Remove the peptides of -20°C and centrifuge them for 5 min at 4°C and 10 000 g

2. Put them at room temperature for 10 min

3. Add X pL of Milli-Q® water to obtain a 500 pM concentration

4. Let them dissolve 60 min at RT and put them at 4°C

Binding Assay

Equilibrate all reagents to room temperature prior to use.

1 . Prepare 50 pL of peptide at 20 pM in water (2 pL qsp 50 pL of water)

2. Add 10 pL of Milli-Q® water into the PCR-tubes 2-16

3. Transfer 20 pL of peptide into PCR-tube 1

4. Transfer 10 pL from tube 1 to tube 2 with a pipette (P20) and mix by pipetting up-and- down multiple times. Repeat the procedure for tube 3-16 and discard the extra from tube 16

5. Add 10 pL of labeled protein to each well (16 to 1) and mix by pipetting

6. Incubate 30 min at RT

7. Load the capillaries (NT.115 capillary) and measure the sample with a Monolith NT.115 system with the following settings: 20% Pico-RED excitation power and medium MST- power.

Pro-cathespin D/LRP-1 interaction study by co-immunoprecipitation

Materials & Reagents

Cos-7 cells - Lipofectamine 2000 Transfection reagent (11668027; Invitrogen) - pSectag SD1

- pSectag F0 06His - OptiMEM (31985-070; Gibco); DMEM 4.5g/L glucose (31966-021 ; Gibco) - FCS (F7524; Sigma Aldrich) - PBS (14190-094; Gibco) - Tween (BP337-500; Fisher Scientific) - BSA (Bovine Serum Albumin) (04-100-812-E; Euromedex) - NiNTA Agarose (30210; Qiagen) - Monoclonal Antibody, cathepsin D (AF4G5) (MA517236; Invitrogen) - Myc- Tag (9B11) Antibody (#2276; Cell Signaling) - Goat anti-rabbit IgG Dylight 800 4X PEG (SA535571 ; Fisher Scientific) - Anti-Mouse IgG HRP-linked Antibody (7076S; Cell signaling)

- Human Cathepsin D I CTSD Protein, His Tag (CTD-H5226; Acrobiosystems) (12517-H08H; SinoBiological) - Acrylamide 30% (EU0088-C; Euromedex) - SDS (Sodium Dodecyl Sulfate) (L4509; Sigma Aldrich) - APS (Ammonium PerSulfate) (A3678; Sigma Aldrich) - TEMED (T7024; Sigma Aldrich) - Tris (BP152-1 ; Fisher Scientific) - Trypsine-EDTA (0.05%) (11580626; Gibco) - TG-SDS Powder (865984; Interchim) - Tris-Glycine Buffer 10X solution (UP91493A; Interchim) - Water (W3500-100ML; Sigma Aldrich).

Cos-7 T ransfection

1 . Seed Cos-7 on B10 and let them until 80% confluence

2. Transfection:

3. Tube 1 : Mix 900 pL of OptiMEM with 30 pL Lipofectamine 2000 for one B10

4. Tube 2: Mix 15 pg of DNA with 900 pL of OptiMEM pSectag SD1 (LRP1a) 781 ng/pL/587 ng/pL : 25.5 pL

5. pSectag F0 (LRP1 p): 1.15 pg/mL Then add “Tube 2” in “Tube 1” in droplet

6. Incubate 5 min at RT 7. Add this mix on cells in droplet

8. Incubate for 48h at 37°C, 5% CO2, 95% humidity

9. Collect supernatant and centrifuge 10 min at 1 500 rpm at 4°C

10. Store at 4°C for 24 - 72h and -20°C for long term storage

Preparation of peptides

1. Remove the peptides of -20°C and centrifuge them for 5 min at 4°C and 10 000 g

2. Put them at room temperature for 10 min

3. Add X pL of water to obtain a 500 pM concentration

4. Let them dissolve 60 min at RT and put them at 4°C

Conditions: SD1 + cathepsin D F0 + cathepsin D F0 + cathepsin D + Peptides (100pM).

On ice:

1. Prepare 50 pL of cathepsin D at 2 ng/pL in PBS +/- 100 pM of peptides and incubate 1h a 4°C under gentle rotation

2. Add 500 pL of supernatant “SD1 or F0” per tube and incubate at 4°C under gentle rotation overnight

3. Prepare 25 pL of NiNTA agarose (50pL) for one condition

4. Wash resin 3 times with 1 mL PBS-T, centrifuge (1000g - 4°C - 1 min) and eliminate supernatant

5. Incubate with 1 mL of 2% BSA 30 min at 4°C

6. Wash 3 times with 1 mL PBS-T

7. Centrifuge at 4°C, 1000g for 1 min and eliminate supernatant

8. Resuspend resin with 25 pL PBS-T

9. Put 50 pL per tube

10. Incubate 4 hours at 4°C under gentle rotation

11. Wash 5 times with 1 mL PBS-T (centrifuge between each wash at 1000g - 4°C - 1 min)

12. Eliminate supernatant and elute sample with 30 pL of Laemmli 2X with - mercaptoethanol

13. Put samples at -20°C

Western Blot analysis

1. Heat samples 10 min at 100°C on dry bath

2. Centrifuge samples and transfer supernatants on a 10% agarose gel

3. Migrate to 70V (stacking gel) and then 120V (separating gel)

4. Transfer protein on nitrocellulose membrane to 100V at 4°C for 60 min

5. Put the membrane on TBS-T with 5% milk 6. Prepare Antibody on milk 5% TBS-T

7. Incubate overnight at 4°C under gentle agitation

8. Wash membrane in TBS-T for 10 min (x3)

9. Incubate with secondary antibody for 1 hour at room temperature

10. Repeat wash in TBS-T and one in TBS

11 . Revelation with Li-Cor Odyssey FC 2800 Imaging system

Study of cathepsin D effect in a 3D co-cultured models of cancer cells and fibroblast

Materials & Reagents

Cos-7 cells - MEF1 cells - Lipofectamine 2000 Transfection reagent (11668027; Invitrogen) - PCDNA3.1 CTL - pCDNA3.1 pCD - OptiMEM (31985-070; Gibco); DMEM 4.5g/L glucose (31966-021 ; Gibco) - FCS (F7524; Sigma Aldrich) - PBS (14190-094; Gibco) - 48-well plates (353078; Falcon®) - Trypsine-EDTA (0.05%) (11580626; Gibco) - Water (W3500-100ML; Sigma Aldrich) - EVOS Fl Microscope (Thermofisher).

Seeding of COS 7 & Transfection

1. Seed 20 000 cells per well on 300 pL of DMEM 4.5 g/L glucose supplemented with 10% FCS (48-Well plate)

2. Incubate for 24h at 37°C, 5% CO2 and 95% humidity

3. Transfection with plasmide & Lipofectamine 2000

4. Tube 1 : Mix 20 pL of OptiMEM with 0.862 pL of Lipofectamine 2000 for one well

5. Tube 2: Mix 20 pL of OptiMEM with 0.287 pg of DNA pCDNA3.1 CTL: 1.22 pg/pL pCDNA3.1 pCD: 1.26 pg/pL

6. Then add “Tube 2” in “Tube 1” in droplet

7. Incubate 5 min at RT

8. Add this mix on cells in droplet

9. Incubate for 24h at 37°C, 5% CO2, 95% humidity

Put the Matrigel on ice and let it thaw overnight at 4°C. Put tips (200 & 1000 pL) at -20°C.

2. Put them at room temperature for 10 min

3. Add X pL of water to obtain a 500 pM concentration

4. Let them dissolve 60 min at RT and put them at 4°C

Seeding MEF-1 on matrigel

1 . Remove culture medium of COS7

2. Put 100 pL of Matrigel per well (with cold tips) 3. Allow polymerize for 1h at 37°C, 5% CO2 and 95% humidity

4. Rinse MEF1 with 5 mL of PBS

5. Put 1 mL of trypsin-EDTA (for one B10), incubate 5 min at 37°C

6. Add medium with 10% FCS and count cells

7. Take the volume to obtain 50 000 cells per well

8. Centrifuge at 1200g for 5 min

9. Resuspend cells with 300 pL of Matrigel for one well: 50 000 cells on 300 pL per well

10. Seed MEF1 on COS7

11. Allow polymerize for 1 h at 37°C, 5% CO2 and 95% humidity

12. Add 200 pL of medium +/- 100 pM of peptide per well

13. Take pictures under microscope at D+1 , D+4 and D+6

Effect of LBC peptides on breast cancer cells proliferation

Materials & Reagents

MDA-MB-231 cells - MCF7 cells - DMEM 1g/L glucose (21885-025; Gibco) - FCS (F7524; Sigma Aldrich) - PBS (14190-094; Gibco) - Trypsine-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).

Preparation of peptides

2. Put them at room temperature for 10 min

3. Add X pL of water to obtain a 500 pM concentration

4. Let them dissolve 60 min at RT and put them at 4°C

Assay procedure

1. Seed 5 000 and 10 000 cells per well for respectively MDA-MB-231 and MCF7 on 100 pL of DMEM 1 g/L glucose supplemented with 10% FCS (96-Well plate): Two plates for WST-1 assay 24h and 48h after treatment

2. Incubate for 24h at 37°C, 5% CO2 and 95% humidity

3. Treat cells with peptide at 50 pM - 25 pM - 12.5 pM and 6.25 pM in DMEM 1 g/L glucose supplemented with 10% FCS.

4. Incubate for 24h 148h at 37°C, 5% CO2 and 95% humidity

5. Add 10 pL/well Cell Proliferation Reagent WST-1

6. Incubate the cells for 2 hours at 37°C, 5% CO2 and 95% humidity

7. Shake thoroughly for 1 min on a shaker

8. Measure the absorbance of the samples against a background control as blank using a microplate reader at 450 nm. Use reference wavelength > 600 nm Cathepsin D catalytic activity analysis at pH 3.5

Materials & Reagents

PCR tubes & tips - Recombinant Human pro-cathepsin D protein (ab286010; Abeam) - Water (W3500-100ML; Sigma Aldrich) - Sodium acetate anhydrous (W302406; Sigma Aldrich) - Sodium chloride (207790010; ThermoScientific) - cathepsin D & E substrate (fluorogenic) (BML-P145-0001; Enzo Life Sciences) - 96-well ELISA plate (82.1581.120; Sarstedt) - PBS (14190-094; Gibco) - BSA (Bovine Serum Albumin) (04-100-812- E; Euromedex) - Pepstatin A (P5318-5MG; Sigma Aldrich) - Infinite 200® pro (TECAN).

Preparation of peptides

1 . Remove the peptides of -20°C and centrifuge them for 5 min at 4°C and 10 000 g

2. Put them at room temperature for 10 min

3. Add X pL of water to obtain a 500 pM concentration

4. Let them dissolve 60 min at RT and put them at 4°C

Assay procedure

Equilibrate Assay Buffer to room temperature prior to use.

1. Prepare Assay Buffer: 0.1 M NaOAc, 0.2 M NaCI, pH 3.5

2. Dilute pro-cathepsin D to 20 pg/mL in Assay Buffer

3. Put 5 pL of pro-cathepsin D +/- peptide at 100 pM in Assay Buffer (Final volume: 50pL)

4. Incubate 15 min at room temperature

5. Dilute substrate to 30 pM in Assay Buffer

6. Load 50 pL of pro-cathepsin D +/- peptide (100 pM) or pepstatin (2 pg/mL) in a plate (pre-saturated with 1 % BSA)

7. Start the reaction by adding 50 pL of 30 pM substrate

8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 1 h

Cathepsin D catalytic activity analysis at pH 6

Materials & Reagents

PCR tubes & tips - Recombinant Human Cathepsin D protein (1014-AS; R&D systems) - Water (W3500-1 OOM L; Sigma Aldrich) - Sodium acetate anhydrous (W302406; Sigma Aldrich) - Sodium chloride (207790010; ThermoScientific) - Cathepsin D & E substrate (fluorogenic) (BML-P145-0001; Enzo Life Sciences) - 96-well ELISA plate (82.1581.120; Sarstedt) - PBS (14190-094; Gibco) - BSA (Bovine Serum Albumin) (04-100-812- E; Euromedex) - Pepstatin A (P5318-5MG; Sigma Aldrich) - Infinite 200® pro (TECAN).

Preparation of peptides

2. Put them at room temperature for 10 min

3. Add X pL of Milli-Q® water to obtain a 500 pM concentration

4. Let them dissolve 60 min at RT and put them at 4°C

Assay procedure Equilibrate Assay Buffer to room temperature prior to use.

1. Prepare Assay Buffer: 0.1 M NaOAc, 0.2 M NaCI. pH 3.5 / 0.1 M NaOAc, 0.2 M NaCI, pH 6

2. Dilute cathepsin D to 20 pg/mL in Assay Buffer at pH 3.5

3. Incubate at 37°C for 30 min to allow protein activation

4. Dilute incubated cathepsin D to 0.5 ng/pL in Assay Buffer pH 6

5. Dilute substrate to 60 pM in Assay Buffer pH 6

6. Load 50 pL of cathepsin D +/- peptide (100 pM) or pepstatin (2 pg/mL) in a plate (presaturated with 1% BSA)

7. Incubate 15 min at 37°C

8. Start the reaction by adding 50 pL of 60 pM substrate

9. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 45 min

1 .2. RESULTS AND DISCUSSION

Cathepsin D/LRP-1 interaction characterization by in s/7/'co studies

To identify the exact interaction zone between cathepsin D and LRP-1 and the residues involved, we performed in silico analyses (bioinformatics strategies, molecular modeling and dynamics) according to the workflow presented in Figure 1.

Briefly, representative structures and conformations of the F4 Fragment were generated using structural prediction methods (MODELLER software, l-TASSER online servers) and molecular dynamics tools (GROMACS software). Another longer fragment from this region of LRP-1 p, which takes into consideration structural elements identified in these sequences, was also modeled. Indeed, this region of LRP-1 is rich in EGF-like domain and the F4 fragment partially overlaps with EGF-like domains 19 and 20. We therefore proposed a fragment that contains the entirety of these two domains (EGF1920 fragment) in order to respect structural coherence.

The amino acid sequence of F4 fragment and EGF1920 are as given in Table 2 below:

Table 2: Amino acid sequence of F4 and EGF1920 fragments of LRP-1

The same methods that we used for the F4 fragment was used for the EGF1920 fragment and allowed us to propose representative structures and conformations of this new fragment. For each of these fragments (F4 fragment (SEQ ID NO:5) and EGF1920 fragment (SEQ ID NO:6)), the same molecular docking protocol was performed. The structure resulting from the energy minimization and the first two clusters extracted from the molecular dynamics were kept and submitted to a rigid/rigid docking (HEX software) on the cathepsin D structure available on the PDB server (1 LYW). Each docking experiment generates 100 solutions and the total analysis generated 1500 solutions. For each of the fragments, the number of contacts was normalized so that the residue that statistically forms the largest number of contacts has a frequency value of 1 . A cutoff of 0.5 was used to separate the key residues from the others (Figure 2A and Figure 2B). Analysis of the results obtained identified within the F4 (Figure 2A) and EGF1920 (Figure 2B) fragments several residues likely to interact with cathepsin D. 5 residues with high frequencies for both fragments have been identified (Figure 2C). An isolated R residue and four residues (DR_QY) in a five- residues region (DRCQY (SEQ ID NO:9). This region clearly emerges and served as a starting point for the design of blocking peptides.

Design of 5 peptides (LBC1 to LBC5) to block cathepsin D/LRP-1 interaction

From the sequence (DRCQY) we pre-selected a series of 5 peptides (Table 3) with a length of only 15 amino acids in order to optimize the synthesis (compatibility with a standard Fmoc procedure on solid support, low cost, high yield and purification rate), but also to meet biocompatibility requirements. Indeed, for this pre-selection, we were particularly attentive to determine sequences with a high predictive solubility in aqueous solvent (water, NaCI 0.9%) in projection of clinical applications. interaction. The sequence of 5 amino acids identified by in silica studies is shown in Bold. The predictive isoelectric point, Net charge at pH 7 and solubility in NaCI 0.9% are also indicated.

These 5 peptides were synthesized by a service contract with SB-Peptide (Grenoble, France), which did not encounter any difficulty in terms of synthesis and purification (synthesis report available upon request).

In order to validate the ability of these peptides to block the cathepsin D/LRP-1 interaction, three tests were performed on these peptides:

1- Determination of the constant affinity (KD) of these peptides for the cathepsin D using microscale thermophoresis (MST) (see section Material and methods above)

2- Ability to block the cathepsin D/LRP-1 interaction in vitro in co-immunoprecipitation experiments using soluble fragments of the LRP-1 p-chain and recombinant cathepsin D (see section Material and methods above) 3- Ability to block the cathepsin D/LRP-1 interaction and its proliferation effects in a murine fibroblast model (MEF-1 cells, expressing LRP-1) (see section Material and methods above)

Study of LBC1 to LBC5 peptides affinity for cathepsin D by Microscale Thermophoresis

We first studied LBC1 to LBC5 peptides affinity for cathepsin D. Microscale thermophoresis (MST) experiments show that only peptides LBC1 , LBC3, LBC4 and LBC5 can bind cathepsin D.

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

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

Table 4: KD value obtained for LBC1 to LBC5 peptides by microscale thermophoresis (MST).

The results show that respective affinities of LBC1 (52.5nM), LBC3 (340 nM), LBC4 (4.3 nM) and LBC5 (8.7 nM) are higher than that obtained with Fragment F4 (2.37 pM) of 45 AA and corresponding to residues 349 to 394 of LRP-1 p. These results show that the in silico analyses have identified a key region of LRP-1 that is indeed involved in the interaction with cathepsin D. The Amino acid primary sequences located on both sides of this region seem to play a role in the affinity of these peptides.

Study of the blocking properties of LBC1 to LBC5 peptides against cathepsin D/LRP- 1 by co-immunoprecipitation

The ability of peptides to block the cathepsin D/LRP-1 interaction was then investigated by coimmunoprecipitation experiments (n=5) (Figure 4). We first verified that the cathepsin D was able to bind the extracellular part of the LRP-1 chain (LRP1 p-F) (Figure 4A). This interaction is specific since we do not observe binding with a fragment of the alpha chain of LRP-1 (LRPlaF). In the presence of the different peptides, at 100 pM, LBC3, LBC4 and LBC5 can significantly block this interaction by about 60 to 70%, and to a lesser extent, the peptide LBC1 (40%) (Figure 4B). These results confirm the results observed in MST. Study of the blocking properties of LBC1 to LBC5 peptides against cathepsin D/LRP- 1 in a 3D co-culture model of cancer cells and fibroblast

Finally, we tested the ability of the peptides LBC1 to LBC5 to block the cathepsin D/LRP-1 interaction in a previously validated cell model (Beaujouin et al., 2010 ; Derocq et al., 2012).

Figure 5A illustrates a 3D co-culture model of mouse embryonic fibroblasts naturally expressing LRP-1 and COS-7 cancer cells overexpressing and secreting or not -cathepsin D.

A western blot analysis of the amount of cathepsin D present, is performed after 6 days, in the co-culture medium of wells with COS-7 cell overexpressing and secreting (pCD) or not (Ctrl) cathepsin D. The results are shown in Figure 5B.

A phase contrast microscopy observation is performed at Day 1 , Day 4 and Day 6 of fibroblast colonies co-cultured with COS-7 Ctrl or pCD cell in the absence or presence of LBC 1 to 5 peptides at 100 pM. The results are shown in Figure 5C.

Figure 5D is a histogram representing colonies with area greater than 5,000 pixels² at Day 6 (each point equals one colony). n=5, one-way anova ****: p<0.0001 , **: p<0.01 , Scale bar: 20 pm.

As expected (positive control), the presence of cathepsin D significantly stimulates fibroblast growth and consequently the size of the colonies observed 4 and 6 days after incubation (Figure 5C, Figure 5D). Similar experiments performed with LRP-1 KO mouse embryonic fibroblasts (LRP1 -/-, cell PA-13) validate the specificity of this effect since in these cells the presence of cathepsin D has no effect on growth (negative control, data not shown). We thus studied the effect of cathepsin D on the growth of fibroblasts in the presence of different LBC peptides (100 pM). This effect of cathepsin D is significantly inhibited with the peptides LBC2 and LBC5 and strongly with the peptide LBC4 (90 to 100% inhibition) while the peptides LBC1 and LBC3 have no significant effect (Figure 5C and Figure 5D).

At the end of the three tests carried out on these different peptides, we have identified at least two peptides LBC4 peptides and LBC5 which both have a good affinity and the ability to block the cathepsin D/LRP-1 interaction in vitro and in cellulo in a murine model. The other peptides cannot be discarded since they show a binding capacity and/or a capacity to block the interaction either in vitro or in cellulo.

Study of the set of peptide sequences derived from the F4 LRP-1 fragment capable of binding cathepsin D

The in vitro and in cellulo data obtained for each of the peptides LBC1 to LBC5 suggested not to limit to a single peptide sequence but investigate a larger sequence. To delineate this sequence, we decided to synthesize 10 new peptides (LBC6 to LBC15) allowing us to explore the N- and C-terminal sequences of the peptides already analyzed.

The different peptides derived from the p-chain of LRP-1 synthesized to delineate the region of high interaction between LRP-1 and cathepsin D are illustrated in Figure 6.

Figure 6 shows the region of LRP-1 chain of amino acid sequence STCTVNQGNQPQCRCLPGFLGDRCQYRQYRQCSGYCENFGTCQMAADGSRQCRCTAYF EGSRC (SEQ ID NO:8), that includes but is not limited to the region of LRP-1 known as “fragment F4”, from which the peptides LBC6 (SEQ ID NO:15) to LBC15 (SEQ ID NO:24) were designed.

The peptides LBC1 (SEQ ID NQ:10) to LBC5 (SEQ ID NO:14) are the peptides first studied. The peptides LBC6 (SEQ ID NO: 15) to-LBC8 (SEQ ID NO: 17) were used to delineate the N- terminal of the high interaction region and the peptides LBC9 (SEQ ID NO:18) to LBC15 (SEQ ID NO:24) peptides for the C-terminal of the high interaction region of amino acid sequence (SEQ ID NO:1).

These ten new peptides were synthesized by the SB-peptide company (>90% purity, neutralization in acetate salts). For each of these peptides, solubility tests as well as affinity tests for cathepsin D were performed by micro-scale thermophoresis.

Briefly, cathepsin D (Acrobiosystems) was labeled with the His-Tag labelling kit Red-Tris NTA (NanoTemper). Labeled cathepsin D was then mixed with the LBC peptides using a 0.5-fold dilution series ranging from 10 pM to 0.15 nM. The 16 mixtures were then analyzed with a Monolith NT.115 (NanoTemper) instrument at 25°C. The instrument parameters were 20% Pico-RED excitation power, medium MST-power, and 5/20/5 laser off/on/off. Data were analyzed with NT MO Affinity Analysis v2.1.3 (NanoTemper). The results are shown in Table 5 below:

Table 5 Study of LBC1 to LBC15 peptides affinity for cathepsin D by micro-scale thermophoresis. Table with Kd value obtained for each peptide by microscale thermophoresis. Briefly, cathepsin D (Acrobiosystems) was labeled with the His-Tag labelling kit Red-Tris-NTA (NanoTemper). Labeled cathepsin D was then mixed with the the LBC peptides (1 to 15) using a 0.5-fold dilution series ranging from 10 pM to 0.15 nM. The 16 mixtures were then analyzed with a Monolith NT.115 (NanoTemper) instrument at 25°C. The instrument parameters were 20% Pico-RED excitation power, medium MST-power, and 5/20/5 laser off/on/off. Data were analyzed with NT MO Affinity Analysis v2.1 .3 (NanoTemper). The solubility tests allowed us to identify some peptide sequences (peptides LBC9, LBC11, and LBC14) that could not be used in cellulo and in vivo because they are insoluble in water.

The equilibrium dissociation constants (KD) obtained for soluble peptides allowed to i) identify peptide sequences able to interact with the cathespin D and ii) to delimit the high interaction region. This high interaction region was delimited by the peptides LBC8 and LBC9 and is constituted of 30 Amino acids: NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:1).

LBC4 peptide structure characterization by Nuclear Magnetic Resonance

To better characterize the LBC4 peptide, which presents the best results according to our in vitro and in cellulo tests, Nuclear Magnetic Resonance (NMR) spectroscopic analyses were performed by Prof. Bruno Kieffer (UMR CNRS 7104, Strasbourg, France).

The LBC4 peptide was diluted in HCI/NacI solution (30 pl) containing 20 mM Tris Buffer at pH 7.5 (130 pl,) then 16 pL of DMSO (oxidizing condition, spectra in red) or 16 pL of water (nonoxidizing condition, spectra in blue) were added.

The results are shown in Figure 7:

- Figure 7A: After, 72 h incubation at room temperature, 1D 1H NMR spectra in the -NH region was measured at 298 Kelvin.

- Figure 7B: Determination of chemical shifts in the NH region between spectra obtained in the absence (blue) and presence (red) of DMSO (oxidizing condition).

- Figure 7C and Figure 7D: Affinity determination for cathespin D of the oxidized LBC4 peptide by microscale thermophoresis Labeled cathepsin D was mixed with the LBC4 peptide without a disulfide bridge between its 2 cysteines (LBC4) or with (oxydised-LBC4) using a 0.5-fold dilution series, ranging from 10 pM to 0.15 nM. The 16 mixtures were then analyzed with a Monolith NT.115 (NanoTemper) instrument at 25°C. The instrument parameters were 20% Pico-RED excitation-power, medium MST-power, and 5/20/5 laser off/on/off. Data were analyzed with NT MO Affinity Analysis v2.1.3 (NanoTemper).

The LBC4 peptide does not seem to be structured, however in oxidizing environment, LBC4 cysteines are able to form disulfide bridges and to modify peptide conformation (Figure 7A and 7B). MST analysis shows that this cyclization impaired the binding of the LBC4 peptide to the cathepsin D (Figure 7C and 7D).

Effect of the LBC4 and LBC5 peptides sequences minimization

Then, we studied the possibility of using smaller peptides. Sixteen peptides of 7 to 15 AA derived from LBC4 and LBC5 peptides and containing the DRCQY (SEQ ID NO:8) sequence (previously identified by our in silico study as detailed above) were synthesized.

The affinity constants of these peptides were determined by microscale thermophoresis

The results are shown in Table 6:

Table 6: Study of LBC16 to LBC31 peptides affinity for cathepsin D by micro-scale thermophoresis. Table with Kd value obtained for each peptide by microscale thermophoresis. Briefly, cathepsin D (Acrobiosystems) was labeled with the His-Tag labelling kit Red-Tris-NTA (NanoTemper). Labeled cathepsin D was then mixed with the LBC peptides (16 to 31) using a 0.5-fold dilution series ranging from 10 M to 0.15 nM. The 16 mixtures were then analyzed with a Monolith NT.115 (NanoTemper) instrument at 25°C. The instrument parameters were 20% Pico-REDexcitationpower, medium MST-power, and 5/20/5 laser off/on/off. Data were analyzed with NT MO Affinity Analysis v2.1.3 (NanoTemper).

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 the peptides display a good affinity for the cathepsin D except the peptides LBC27 (SEQ ID NO:36) and LBC30 (SEQ ID NO:39). These results show that it is possible to decrease the size of our blocking peptides up to 7 AA (e.g. LBC16 (SEQ ID NO:25)).

Direct effect of the LBC peptides on breast cancer cell proliferation

The LBC peptides target the cathepsin D secreted by cancer cells and thus their communication with fibroblasts. We determined if the peptides have an effect in the cancer cells themselves and particularly their ability to block proliferation in MCF-7 and MDA-MB-231 breast cancer cells.

MDA-MB-231 and MCF-7 cells were seeded at 5000 and 10000 cells/well respectively in a 96- well plates and incubated during 48 hours without (NT) or with LBC4 (SEQ ID NO:13), LBC5 (SEQ ID NO:14) or LBC15 (SEQ ID NO:15) peptides at various concentration (6.25 to 50 pM). Cell number was measured using Cell proliferation Reagent WST-1.

The results are shown in Figure 8.

All LBC peptides tested have no significant effect on the proliferation of these tumor cells even LBC4 (SEQ ID NO: 13) and LBC5 (SEQ ID NO: 14) peptides which display the strongest effects on fibroblasts (Figure 5D). These results seem to confirm the mechanism of action of LBC peptides which target stromal cells within the tumor microenvironment. Moreover, these results suggest that our peptides do not have cytotoxic effect at up to 50 pM which is an interesting result in the perspective of a therapeutic use.

Effect of the LBC peptides on the cathepsin D catalytic activity

Besides its ability to bind to LRP-1 and induce cellular effects, cathepsin D is an aspartic protease able to cleave various substrates. Like most lysosomal aspartic proteases, cathepsin D has a maximal catalytic activity at an acidic pH between 2.4 to 5. At higher pH values of 5.0, the activity of cathepsin D decreases, and none is detectable at pH 7.0.

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

Thus, we tested the ability of our peptides to inhibit cathepsin D activity at pH 3.5 and pH 6.

Experiments at pH 3.5: Pro-cathepsin D (2 ng/pL) with LBC peptides (100 pM) or Pepstatin A (2 ng/pL) were pre-incubated 15 min in Assay Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 3.5). Activity was then measured in the presence of a fluorogenic substrate (15 pM).

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

The results are shown in Figure 9. The results at pH 3.5 are shown in Figure 9A and those at pH 6 in Figure 9B.

Results at pH 3.5:

Pepstatin A, an inhibitor of cathepsin D, totally blocks cathepsin D activity. In contrast, 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 pM) can significantly block the activity of the enzyme (Figure 9A).

Results at pH 6:

At pH 6, the peptide LBC4 (100 pM) significantly block cathepsin D activity (about 67%) (Figure 9B). A mutation of the cysteine 8 to a serine (C8S) in the LBC4 peptide sequence (peptide LBC4C8S PGFLGDRSQYRQCSG (SEQ ID NO:41)) impairs the ability of the peptide to block the catalytic activity of cathepsin D showing the specificity of this interaction.

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 effects were 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.

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

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

All these results show that LBC peptides able to antagonize the cathepsin D/LRP-1 interaction do not show similar effects regarding the catalytic activity of the enzyme. These results allow to consider the future use of LBC peptides that may or may not possess both activities. These differential properties of the LBC peptides allow i) to differentiate the LBC peptides from other existing technologies such as the anti-cathD monoclonal antibody ii) to play on one or the other of these properties depending on the effects of the different peptides observed in vivo on tumorigenesis.

Dose-dependant effect of LBC4 peptide:

Pro-cathepsin D (0.5 ng/pL) was pre-activated 30 min in Acidic Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 3.5) and then pre-incubated with LBC4 peptide at various concentration (10 - 50 - 100 - 250 and 500 pM) or pepstatin A (2 pg/mL) 15 min in Assay Buffer (0.1 M NaOAc, 0.2 M NaCI, pH 6). Activity was then measured in the presence of fluorogenic substrate (30 pM).

The results are shown in Figure 9C. LBC4 inhibits cathepsin D catalytic activity in a dosedependent manner up to 250 pM.

Example 2: Effect of LBC peptides on triple-negative breast cancer (TNBC) patient- derived tumoroids model

2.1. MATERIAL AND METHODS

An overview of the experimental procedure is presented in Figure 10A.

This study was carried out on BR20001 B PDX models (TNBC model) from Crown Biosicience, Leiden (Biopartner Center Leiden, J.H. Oortweg 21 n 2333 CH Leiden, Netherlands). As used herein, "PDX" stands for "Patient-Derived Xenograft." Patient-Derived Xenografts are models in cancer research where tumor tissues from patients are collected and cultivated for further study. 450 PDX were homogeneously seeded in hydrogel (Matrigel® and collagen based, Crown Bioscience, Leiden) in 384-wells plates. After 3 days, organoids were treated with LBC4, LBC5 and LBC8 peptides at 3 doses: 10, 30 and 100 pM or vehicle.

After 7 days, organoids were retreated and after 10 days, supernatant was collected and cultures were fixed and stained to make High Content Imaging analysis (HCI) and determined:

- The number of organoid

-Total nuclei count in organoid

-The volume of organoid

-Total branch of organoid

TIBCO Spotfire was utilized for data analysis and visualization.

2.2. RESULTS

The results for LBC4, LBC5 and LBC8 are respectively shown in Figure 10B, Figure 10C and Figure 10D.

The results show that LBC4, LBC5 and LBC8 peptides display antitumor activity in TNBC tumoroid models in a dose dependant manner. More especially the results show that these peptides result in:

- a decrease nuclei count in organoids,

- a decrease in total organoid volume,

- a decrease in total branch of organoids. REFERENCES

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Claims

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

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

3. The peptide according to any of claims 1 or 2, wherein the fragment or functional variant derived therefrom includes at least 2, 3, or 4 of the amino acids G16, D17, R18, C19, Q20, Y21 , and/or the amino acid R9 of SEQ ID NO:1.

4. The peptide according to any of claims 1 to 3, wherein the peptide is of a fragment of: a) a central region of SEQ ID NO :1 of the amino acid sequence PQCRCLPGFLGDRCQYRQCSGY (SEQ ID NO:2), b) a N-terminal region of SEQ ID NO:1 of the amino acid sequence NQGNQPQCRCLPGFLGDRCQYR (SEQ ID NO:3), c) a C-terminal region of SEQ ID NO :1 of the amino acid sequence RCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO :4), d) a region deriving from an amino acid sequence in a), b) or c) by a N- and/or C- terminal deletion of 1 , 2, 3 or 4 amino acids; or or a functional variant of said fragment, wherein the fragment or functional variant derived therefrom includes at least one of the amino acids corresponding to amino acids R9, G16, D17, R18, C19, Q20, and Y21 of SEQ ID NO:1.

5. The peptide according to claim 4, wherein the fragment or functional variant derived therefrom includes at least 2, 3, or4 of the amino acids G16, D17, R18, C19, Q20, Y21 , and/or the amino acid R9 of SEQ ID NO:1 .

6. The peptide according to any 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. The peptide according to any 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 of claims 1 to 7, wherein the peptide does not comprise a C residue both at N- and C- terminal.

9. The peptide according to any of claims 1 to 8, wherein the peptide is a fragment of SEQ ID NO:1 , 2, 3, or 4 selected from the group consisting of:

- RCLPGFLGDRCQYRQ (SEQ ID NQ: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 NQ: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 NQ:40), or a functional variant of said fragment, preferably deriving from said fragment by deletion, insertion, and/or substitution of one or more amino acids.

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

11. The peptide according to any of claims 1 to 10, wherein the peptide has a binding affinity KD value of less than 500 nM for cathepsin D, as determined in a microscale thermophoresis assay.

12. A modified peptide deriving from the peptide according to any of claims 1 to 11 , by the introduction of one or more chemical modifications which preferably protect the peptide against proteolysis.

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

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

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

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

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