Peptide inhibitors of cathepsin D / LRP-1 interaction

Abstract

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

Description

Background of the Invention

[0002] In cancer, the tumor microenvironment plays a crucial role in tumorigenesis. The tumor microenvironment includes: i) the extracellular matrix (ECM), a complex and dynamic structure containing fibrin, glycosaminoglycans, glycoproteins, or proteoglycans; and ii) stromal cells, such as fibroblasts, immune cells, endothelial cells, or adipocytes. et al. , 2011; Bussard, et al (Placement, 2016). Tumor cells exchange enzymes, growth factors, and cytokines with stromal cells. These enzymes, growth factors, and cytokines modify the extracellular matrix, stimulate its migration and invasion, and promote its proliferation and survival (Placement). et al. (Pranjol, 2011). Among these molecules, cathepsin D is a protein that is overexpressed and secreted in tumor cells of various cancers (e.g., breast cancer, lung cancer, or ovarian cancer) (Pranjol, 2011). et al. , 2020; Leto et al. (Liaudet-Coopman, 2004), and can stimulate angiogenesis, metastasis and fibroblast growth (Liaudet-Coopman). et al ., 2006; Glondu et al ., 2002; Berchem et al ., 2002; Laurent-Matha et al (., 2005). These pro-tumorigenic effects have made cathepsin D a recognized marker of poor prognosis, particularly in breast cancer, and a therapeutic target of interest (Glondu). et al ., 2002; Dubey et al ., 2017;Ashraf et al ., 2019; Vetvicka et al ., 2012; Brouillet et al , 1997).

[0003] Cathepsin D is a soluble aspartate lysosomal peptidase belonging to the cathepsin family. Under physiological conditions, it cleaves proteins and peptides (Masson proteins) in lysosomal compartments. et al., 2010 Cathepsin D is composed of... CTSDThe gene encodes a precursor protein. This precursor contains a signal sequence that, upon cleavage, yields a 52 kDa precursor, pro-cathepsin D. This 52 kDa form is then transported to the endosome compartment, where it is converted into a 48 kDa active intermediate. In the lysosome, cysteine ​​endopeptidase cleaves the 48 kDa intermediate chain into two chains: a 14 kDa light chain and a 34 kDa heavy chain. These two chains then non-covalently bind to form the mature protease (Masson). et al (., 2010). In cancer cells, CTSD Overexpression of the gene makes it difficult for cathepsinogen D (52 kDa) to localize to endosomes, thus this immature form is released into the extracellular compartment (Pranjol, et al ., 2020;Leto, et al ., 2004; Heylen et al (2002). The cathepsinogen D secreted by this process can stimulate the growth of fibroblasts, especially mammary fibroblasts. This effect is mediated by its interaction with the β chain (residues 307-479) of the cell surface receptor LRP-1 (low-density lipoprotein receptor-associated protein-1). (Beaujouin) et al ., 2010; Derocq et al. , 2012).

[0004] LRP-1 is a multifunctional membrane receptor belonging to the LDL receptor family, possessing both endocytic and cell signaling functions. et al. (Etiquette, 2013). The mature LRP-1 receptor consists of two chains. The extracellular α chain (515 kDa) interacts with more than 40 different extracellular ligands (lipoproteins, proteases, growth factors, toxins, viruses), internalizing them and primarily guiding them into lysosomes for catabolism. The β chain (85 kDa) has an extracellular region of 476 amino acids, a transmembrane region of 25 amino acids, and a cytoplasmic tail of 100 amino acids. LRP-1 plays an important role in regulating proteolytic activity within the microenvironment by endocytosis to clear a variety of ligands (including proteases and / or their inhibitors) (Etiquette). et al (., 2013). In addition to its endocytic function, LRP-1 can also regulate some signal transduction pathways through its intracellular β-chain domain, mainly through two motifs, NPTXY. 29 and NPTXY 63 Interaction with intracellular scaffold proteins (Herz) et al.(2001). Over the past decade, our work has demonstrated the pro-tumor activity of LRP-1 in various cancers, and this has been confirmed by other international studies (Theret...). et al. , 2017; Perrot et al. , 2012; Le et al. , 2020; Langlois et al. ,2010; Appert-Collin et al. (May, 2017). Finally, LRP-1 can participate in the control of gene expression through a dual cleavage process of its β chain (regulatory intramembrane proteolysis, RIP) (May, 2017). et al. The first proteolysis occurs by metalloproteinases and membrane-bound proteins (called exfoliases). This first cleavage allows the release of the extracellular portion of LRP-1. The membrane-bound fragment LRP1β-CTF is then retained and cleaved in its transmembrane domain by γ-secretase (Hass). et al. (2009). Subsequently, the intracellular domain LRP1β-ICD is released into the cytoplasm, where it can interact with signaling proteins and translocate to the nucleus to function as a transcriptional regulator. This mechanism is related to the effect of cathepsin D on fibroblast proliferation. In fact, cathepsin D prevents membrane-bound proteases from cleaving and releasing LRP1β-CTF, thereby blocking the release of LRP1β-ICD by γ-secretase, which controls fibroblast growth (Derocq. et al. (Kalluri, 2012). Therefore, cathepsin D promotes fibroblast proliferation by interacting with the LRP-1 receptor on the cell surface. Fibroblasts are associated with all stages of cancer development, including metastasis, and are a key component of tumor development (Kalluri). et al. , 2016; Houthuijzen et al. , 2018; Bhowmick et al. , 2004).

[0005] To date, only 45 amino acid interaction regions (residues 349 to 394 of LRP-1β) have been identified (Beaujouin) et al. (2010). However, this 45-amino acid region (described as “fragment F4” in WO2009 / 043858) has no potential therapeutic applications due to its poor solubility in aqueous solvents, instability during NMR observation, and difficulty in preparation and purification due to the presence of a large number of cysteine ​​residues forming multiple disulfide bonds.

[0006] Therefore, in order to develop new targeted therapies for cancer treatment, there is a need for novel drugs that can bind to cathepsin D protein and / or inhibit the interaction between cathepsin D and LRP-1. Summary of the Invention

[0007] In a first aspect, the present invention relates to a peptide having a size of 5 to 20 amino acids, preferably 7 to 15 amino acids, wherein the peptide is (i) a fragment of the amino acid sequence NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:1) comprising at least 5 consecutive amino acids of SEQ ID NO:1, or (ii) a functional variant of the fragment, preferably derived from the fragment by deletion, insertion and / or substitution of one or more amino acids, wherein the fragment or the functional variant derived therefrom comprises at least one of amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO:1.

[0008] In one embodiment, the peptide has at least one activity selected from the following: (a) binding to cathepsin D protein, (b) inhibiting the interaction between cathepsin D protein and LRP-1 protein, (c) inhibiting fibroblast proliferation promoted by cathepsinogen D secreted by cancer cells in the tumor microenvironment, and (d) inhibiting the catalytic activity of cathepsin D.

[0009] In one embodiment, the fragment or functional variants derived therefrom include at least 2, 3, or 4 of the amino acids G16, D17, R18, C19, Q20, Y21, and / or amino acid R9 of SEQ ID NO:1.

[0010] In one embodiment, the peptide is the following fragment: a) The central region of SEQ ID NO:1, whose amino acid sequence is PQCRCLPGFLGDRCQYRQCSGY (SEQ ID NO:2). b) The N-terminal region of SEQ ID NO:1, with the amino acid sequence NQGNQPQCRCLPGFLGDRCQYR (SEQ ID NO:3), c) The C-terminal region of SEQ ID NO:1, with the amino acid sequence RCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:4). d) A region derived from the amino acid sequence in a), b), or c) by the deletion of 1, 2, 3, or 4 amino acids at the N-terminus and / or C-terminus; or Or a functional variant of the fragment, wherein the fragment or a functional variant derived therefrom comprises at least one amino acid corresponding to amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO:1.

[0011] The fragment or functional variants derived therefrom preferably include at least 2, 3 or 4 of the amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1.

[0012] In one embodiment, the fragment or functional variants derived therefrom include amino acids D17, R18, Q20, and Y21 of SEQ ID NO:1, and / or amino acid R9.

[0013] In one embodiment, the fragment or functional variants derived therefrom include amino acids G16, D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1.

[0014] In one embodiment, the fragment or functional variants derived therefrom include amino acids G16, D17, R18, C19, Q20 and / or amino acid R9 of SEQ ID NO:1.

[0015] In one embodiment, the fragment or functional variants derived therefrom include amino acids D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1.

[0016] In one embodiment, the fragment or functional variants derived therefrom include amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1.

[0017] In one embodiment, the fragment or a functional variant derived therefrom comprises at least 8, 9, or 10 consecutive amino acids of SEQ ID NO: 1, 2, 3, or 4.

[0018] In one embodiment, the fragment or functional variant derived therefrom comprises no more than 15, 16, 17, 18, 19 or 20 consecutive amino acids of SEQ ID NO: 1, 2, 3 or 4.

[0019] In one embodiment, the peptide does not contain C residues at either the N-terminus or the C-terminus.

[0020] In one embodiment, the peptide is a fragment selected from SEQ ID NO: 1, 2, 3 or 4: -RCLPGFLGDRCQYRQ (SEQ ID NO:10), -CLPGFLGDRCQYRQC (SEQ ID NO:11), -LPGFLGDRCQYRQCS (SEQ ID NO:12), -PGFLGDRCQYRQCSG (SEQ ID NO:13), -GDRCQYRQCSGYCEN (SEQ ID NO:14), -CRCLPGFLGDRCQYR (SEQ ID NO:15), -PQCRCLPGFLGDRCQ (SEQ ID NO:16), -NQGNQPQCRCLPGFL (SEQ ID NO:17), -DRCQYRQ (SEQ ID NO:25), -DRCQYRQC (SEQ ID NO:26), -GDRCQYRQ (SEQ ID NO:27), -GDRCQYRQC (SEQ ID NO:28), -GDRCQYRQCS (SEQ ID NO:29), -LGDRCQYRQC (SEQ ID NO:30), -LGDRCQYRQCS (SEQ ID NO:31), -LGDRCQYRQCSG (SEQ ID NO:32), -FLGDRCQYRQCS (SEQ ID NO:33), -FLGDRCQYRQCSG (SEQ ID NO:34), -FLGDRCQYRQCSGY (SEQ ID NO:35), -GFLGDRCQYRQCSG (SEQ ID NO:36), -GFLGDRCQYRQCSGY (SEQ ID NO:37), -GDRCQYRQCSGYCE (SEQ ID NO:38), -GDRCQYRQCSGY (SEQ ID NO:39), -GDRCQYRQCSG (SEQ ID NO:40), Or a functional variant of the fragment, preferably derived from the fragment by the deletion, insertion and / or substitution of one or more amino acids.

[0021] In one embodiment, as measured in a cathepsin D / LRP-1 immunoprecipitation assay, the peptide can inhibit the interaction between the LRP-1 receptor and cathepsin D by at least 40%.

[0022] In one embodiment, as determined in a microscale thermophoretic assay, the binding affinity K of the peptide to cathepsin D is... D The value is less than 500 nM.

[0023] In another aspect, the present invention relates to a modified peptide derived from the peptide according to any one of claims 1 to 11, wherein one or more chemical modifications are introduced, preferably said chemical modifications protecting the peptide from proteolysis.

[0024] In another respect, the present invention relates to polynucleotides encoding the peptides of the present invention.

[0025] Another aspect of the invention relates to a carrier comprising the polynucleotides of the invention.

[0026] In another respect, the present invention relates to the peptides, modified peptides, polynucleotides, or carriers described herein, as pharmaceuticals.

[0027] The peptides, modified peptides, polynucleotides, or carriers described herein are preferably used to treat proliferative diseases, particularly cancer.

[0028] Another aspect of the invention relates to the use of the peptides or modified peptides described herein for the diagnosis and / or staging of diseases associated with cathepsin D overexpression. Invention Details

[0030] This disclosure stems at least in part from the inventors’ surprising discovery that small peptides derived from a specific region of the LRP-1 receptor β-chain amino acid sequence can bind to cathepsin D protein and inhibit LRP-1 / cathepsin D interaction, thereby inhibiting fibroblast proliferation in the tumor microenvironment promoted by cathepsin D secreted by cancer cells.

[0031] The inventors have identified a key 30-amino acid region involved in the interaction between LRP-1 and cathepsin D. This key region, located on the β-chain of the LRP-1 receptor, has the amino acid sequence NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:1), which had previously been unidentified and was finally determined after six years of research. Surprisingly, the inventors discovered that small peptides derived from this key interaction region can effectively bind cathepsin D and inhibit the LRP-1 / cathepsin D interaction. They also demonstrated in a model simulating a tumor cell environment that some of these peptides can efficiently inhibit fibroblast proliferation promoted by cathepsin D secreted by cancer cells.

[0032] peptides

[0033] Therefore, in a first aspect, the present invention provides a peptide having a size of 5 to 20 amino acids, preferably 7 to 15 amino acids, and preferably having at least one activity selected from: (a) binding to cathepsin D protein, (b) inhibiting the interaction between cathepsin D protein and LRP-1 protein, (c) inhibiting fibroblast proliferation promoted by cathepsin D secreted by cancer cells in the tumor microenvironment, and (d) inhibiting the catalytic activity of cathepsin D, wherein the peptide is (i) a fragment of the amino acid sequence NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:1) comprising at least 5 consecutive amino acids of SEQ ID NO:1, or (ii) a functional variant of the fragment, preferably derived from the fragment by deletion, insertion and / or substitution of one or more amino acids, wherein the fragment or functional variant derived therefrom comprises at least one of amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO:1.

[0034] In one embodiment, the fragment of SEQ ID NO:1 or a functional variant derived therefrom includes at least 2, 3, or 4 of the amino acids G16, D17, R18, C19, Q20, Y21, and / or amino acid R9 of SEQ ID NO:1.

[0035] In one embodiment, the fragment of SEQ ID NO:1 or a functional variant derived therefrom includes at least amino acids G16, D17, R18, C19, Q20 and / or amino acid R9 of SEQ ID NO:1.

[0036] In one embodiment, the peptide is a fragment of the following: a) The central region of SEQ ID NO:1, whose amino acid sequence is PQCRCLPGFLGDRCQYRQCSGY (SEQ ID NO:2). b) The N-terminal region of SEQ ID NO:1, with the amino acid sequence NQGNQPQCRCLPGFLGDRCQYR (SEQ ID NO:3), c) The C-terminal region of SEQ ID NO: 1, with the amino acid sequence RCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO: 4). d) A region derived from the amino acid sequence in a), b), or c) by the deletion of 1, 2, 3, or 4 amino acids at the N-terminus and / or C-terminus; or Or (ii) a functional variant of the fragment, preferably derived from the fragment by the deletion, insertion and / or substitution of one or more amino acids, wherein the fragment or the functional variant derived therefrom comprises at least one amino acid corresponding to amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO:1.

[0037] In other words, in this embodiment, the peptide is a fragment SEQ ID NO:2, 3, or 4, or a functional variant of the fragment comprising at least one of the following: - Amino acids R3, G10, D11, R12, C13, Q14, and Y15 of SEQ ID NO:2; - Amino acids R9, G16, D17, R18, C19, Q20, and Y21 of SEQ ID NO:3; - Amino acids G8, D9, R10, C11, Q12, and Y13 of SEQ ID NO:4.

[0038] In one embodiment, the fragment of SEQ ID NO:2, 3, or 4, or a functional variant thereof, comprises at least 2, 3, or 4 of the amino acids G16, D17, R18, C19, Q20, Y21, and / or R9 of SEQ ID NO:1. In other words, in this embodiment, the peptide is a fragment of SEQ ID NO:2, 3, or 4, or comprises, respectively, the following functional variants thereof: - At least 2, 3, or 4 of the amino acids G10, D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO:2; - At least 2, 3, or 4 of the amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:3; - At least 2, 3, or 4 of the amino acids G8, D9, R10, C11, Q12, and Y13 of SEQ ID NO:4.

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

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

[0041] In one embodiment, the fragment of SEQ ID NO:2, 3, or 4, or a functional variant derived therefrom, comprises at least amino acids G16, D17, R18, C19, Q20, and / or amino acid R9 of SEQ ID NO:1. In other words, in this embodiment, the peptide is a fragment of SEQ ID NO:2, 3, or 4, or a functional variant comprising the following fragments respectively: - Amino acids G10, D11, R12, C13, Q14 and / or amino acid R3 of SEQ ID NO:2; - Amino acids G16, D17, R18, C19, Q20 and / or amino acid R9 of SEQ ID NO:3; - Amino acids G8, D9, R10, C11, and Q12 of SEQ ID NO:4.

[0042] In one embodiment, the fragment of SEQ ID NO:2, 3, or 4, or a functional variant derived therefrom, comprises at least amino acids D17, R18, C19, Q20, Y21, and / or amino acid R9 of SEQ ID NO:1. In other words, in this embodiment, the peptide is a fragment of SEQ ID NO:2, 3, or 4, or a functional variant comprising the following fragments respectively: - Amino acids D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO:2; - Amino acids D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:3; - Amino acids D9, R10, C11, Q12, and Y13 of SEQ ID NO:4.

[0043] In one embodiment, the fragment of SEQ ID NO:2, 3, or 4, or a functional variant thereof, comprises at least amino acids G16, D17, R18, C19, Q20, Y21, and / or amino acid R9 of SEQ ID NO:1. In other words, in this embodiment, the peptide is a fragment of SEQ ID NO:2, 3, or 4, or a functional variant thereof comprising the following fragments: - Amino acids G10, D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO:2; - Amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:3; - Amino acids G8, D9, R10, C11, Q12, and Y13 of SEQ ID NO:4.

[0044] Other features of the fragment

[0045] In one embodiment, the peptide is a peptide with a size of 5 to 20 amino acids, preferably a peptide with a size of 7 to 15 amino acids.

[0046] In one embodiment, the peptide is a peptide with a size of 7 to 20 amino acids, preferably a peptide with a size of 7 to 15 amino acids.

[0047] In one embodiment, the peptide contains no more than 15, 16, 17, 18, 19, or 20 amino acids.

[0048] In one embodiment, the peptide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

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

[0050] In one embodiment, the fragment or a functional variant derived therefrom comprises no more than 15, 16, 17, 18, 19 or 20 consecutive amino acids of SEQ ID NO:1.

[0051] In one embodiment, the fragment or functional variant derived therefrom comprises 5 to 20, preferably 5 to 15, consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0052] In one embodiment, the fragment or functional variant derived therefrom comprises 7 to 20, preferably 7 to 15, consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0053] In one embodiment, the fragment or functional variant derived therefrom comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0054] In one embodiment, the peptide does not contain C residues at either the N-terminus or the C-terminus.

[0055] Specific excerpts

[0056] In a particular embodiment, the peptide is a fragment of SEQ ID NO:1, 2, 3, or 4, selected from: - LBC1, amino acid sequence RCLPGFLGDRCQYRQ (SEQ ID NO:10) - LBC2, amino acid sequence CLPGFLGDRCQYRQC (SEQ ID NO:11) - LBC3, amino acid sequence is LPGFLGDRCQYRQCS (SEQ ID NO:12) - LBC4, amino acid sequence is PGFLGDRCQYRQCSG (SEQ ID NO:13) - LBC5, amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO:14) - LBC6, amino acid sequence CRCLPGFLGDRCQYR (SEQ ID NO:15) - LBC7, amino acid sequence is PQCRCLPGFLGDRCQ (SEQ ID NO:16) - LBC8, amino acid sequence NQGNQPQCRCLPGFL (SEQ ID NO:17) - LBC16, amino acid sequence DRCQYRQ (SEQ ID NO:25) - LBC17, amino acid sequence DRCQYRQC (SEQ ID NO:26) - LBC18, amino acid sequence GDRCQYRQ (SEQ ID NO:27) - LBC19, amino acid sequence GDRCQYRQC (SEQ ID NO:28) - LBC20, amino acid sequence GDRCQYRQCS (SEQ ID NO:29) - LBC21, amino acid sequence LDDRCQYRQC (SEQ ID NO:30) - LBC22, amino acid sequence LDDRCQYRQCS (SEQ ID NO:31) - LBC23, amino acid sequence LDDRCQYRQCSG (SEQ ID NO:32) - LBC24, amino acid sequence FLGDRCQYRQCS (SEQ ID NO:33) - LBC25, amino acid sequence FLGDRCQYRQCSG (SEQ ID NO:34) - LBC26, amino acid sequence FLGDRCQYRQCSGY (SEQ ID NO:35) - LBC27, amino acid sequence is GFLGDRCQYRQCSG (SEQ ID NO:36) - LBC28, amino acid sequence is GFLGDRCQYRQCSGY (SEQ ID NO:37) - LBC29, amino acid sequence GDRCQYRQCSGYCE (SEQ ID NO:38) - LBC30, amino acid sequence GDRCQYRQCSGY (SEQ ID NO:39) - LBC31, amino acid sequence GDRCQYRQCSG (SEQ ID NO:40), or functional variants of the fragment, preferably derived from the fragment by deletion, insertion and / or substitution of one or more amino acids, wherein the fragment or functional variant derived therefrom contains at least one amino acid corresponding to amino acids R9, D17, R18, C19, Q20 and Y21 of SEQ ID NO:1.

[0057] In a particular embodiment, the peptide is a fragment of SEQ ID NO:1, 2, 3, or 4, selected from: - LBC4, amino acid sequence is PGFLGDRCQYRQCSG (SEQ ID NO:13) - LBC5, amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO:14) - LBC6, amino acid sequence CRCLPGFLGDRCQYR (SEQ ID NO:15) - LBC16, amino acid sequence DRCQYRQ (SEQ ID NO:25) - LBC31, amino acid sequence is GDRCQYRQCSG (SEQ ID NO:40).

[0058] or functional variants of the fragment, preferably derived from the fragment by deletion, insertion and / or substitution of one or more amino acids, wherein the fragment or functional variants derived therefrom include at least one amino acid corresponding to amino acids R9, G16, D17, R18, C19, Q20 and Y21 of SEQ ID NO:1.

[0059] In one embodiment, the peptide is not a fragment of SEQ ID NO:15.

[0060] peptide activity

[0061] The peptide preferably has at least one activity selected from the following: (a) The ability to bind to cathepsin D protein, (b) The ability to inhibit the interaction between LRP-1 protein and cathepsin D protein. (c) It can inhibit the proliferation of fibroblasts promoted by cathepsin D secreted by cancer cells in the tumor microenvironment.

[0062] Unless otherwise stated, in this disclosure, both cathepsin D and cathepsinogen D are referred to as "cathepsin D". Cathepsin D is derived from the human whole-length... CTSD The protein encoded by the gene (gene ID: 1509; Genbank accession number: NM_001909) is EC: 3.4.23.5. Cathepsinogen D is a 52 kDa, non-catalytically active precursor of cathepsin D.

[0063] The term "LRP1" or "LRP-1" refers to LDL receptor-associated protein 1, well known in the art. LRP1 is a protein composed of a 515 kDa extracellular α chain and an 85 kDa β chain, produced by proteolytic cleavage of a 600 kDa precursor polypeptide in the trans-Golgi compartment. The LRP1 α chain and LRP1 β chain are produced from the same transcript. The full-length, unprocessed human precursor LRP1 corresponds to UniProtKB / SwissProt accession number Q07954.

[0064] (a) Ability to bind cathepsin D

[0065] In one embodiment, the peptide is capable of binding to cathepsin D, particularly in vitro and / or in vivo.

[0066] In a particular embodiment, the peptide is capable of specifically binding to the extracellular portion (LRP-1 βC) of the LRP-1 β chain.

[0067] The statement "capable of specifically binding to the extracellular portion of the LRP-1 β chain" means that the peptide has a higher binding affinity to LRP-1 βC than other parts of the LRP1 protein (especially the LRP-1 α chain) (e.g., not less than about 2, 5, 10, 30, 100, 1000, or 10000 times). In one embodiment, the peptide does not bind to the α chain of LRP-1 (LRP1αC).

[0068] In one embodiment, the peptide has a binding affinity K for cathepsin D. D Values ​​less than 500 nM, such as those determined by microscale thermophoresis.

[0069] In a preferred embodiment, the binding affinity (KD) of the peptide to cathepsin D is less than 400 nM and less than 300 nM, as determined by microscale thermophoresis.

[0070] The binding affinity of microscale thermophoretics is well known to those skilled in the art and can be identified and / or quantified by the following steps: - Provide the test peptide, namely cathepsinogen D labeled with His-Tag. - The labeled cathepsinogen D was serially mixed with the test peptide at 0.5-fold dilutions, ranging from 10 µM to 0.15 µM; - Analyze the mixture using an MST instrument, such as a Monolith NT.115 (NanoTemper) instrument at 25°C with the following instrument parameters: 20% Pico-RED excitation power, 40% medium MST power, and 5 / 20 / 5 laser off / on / off; and analyze the data using, for example, NT MO Affinity Analysis v2.1.3 (NanoTemper); - To identify and / or quantify the binding affinity of the analyte peptide to cathepsinogen D.

[0071] The experimental section below also describes the detailed process.

[0072] (b) Ability to inhibit LRP-1 / cathepsin D interaction

[0073] The term "inhibition" or "inhibitor" refers to the ability of a compound to reduce, slow down, stop, or prevent the activity of a specific biological process. In some embodiments, the term refers to the interaction between LRP-1 and cathepsin D. In some embodiments, the term refers to fibroblast proliferation promoted by cathepsin D secreted by cancer cells in the tumor microenvironment. In some embodiments, the term refers to the catalytic activity of cathepsinogen D.

[0074] In one embodiment, the peptide is capable of inhibiting LRP-1 / cathepsin D interaction.

[0075] In one embodiment, the peptide is capable of inhibiting at least 30% of the interaction between the LRP-1 receptor and cathepsin D, as measured in a cathepsin D / LRP-1 immunoprecipitation assay.

[0076] The peptide is preferably capable of inhibiting at least 40%, preferably at least 50%, and more preferably at least 60% of cathepsin D / LRP-1 interactions, as determined by immunoprecipitation.

[0077] Those skilled in the art can readily determine and quantify the ability of peptides to inhibit cathepsin D / LRP-1 interactions via immunoprecipitation.

[0078] The ability of the peptide to inhibit the interaction between the LRP-1 receptor and cathepsin D can be identified and / or quantified through the following steps: - Provides the target peptide and recombinant cathepsin D labeled with 6His-Tag. - In the absence of the test peptide (control) or in the presence of the test peptide (e.g., 100 µM), incubate 100 ng cathepsin D overnight with an LRP-1 α (LRP1αF) or LRP-1 β fragment (LRP1βF) displaying the myc epitope. - Deposit the mixture onto Ni-NTA beads for 2 hours; - The complex formed was eluted with 2X Laemmli buffer; - SDS-PAGE electrophoresis and Western blotting were performed using antibodies targeting cathepsinogen D and myc tags. - Identify and / or quantify the binding activity of the LRP-1 β fragment to cathepsinogen D in the absence of the test peptide (control) or in the presence of the test peptide.

[0079] The experimental section below also describes the detailed process.

[0080] (c) It can inhibit the proliferation of fibroblasts in the tumor microenvironment promoted by cathepsin D secreted by cancer cells.

[0081] In one embodiment, the peptide is capable of inhibiting fibroblast proliferation promoted by cathepsin D secreted by cancer cells, either in vitro or in vivo, particularly within the tumor microenvironment.

[0082] In one embodiment, the peptide can inhibit at least 30% of fibroblast proliferation promoted by cathepsin D secreted by cancer cells within the tumor microenvironment, as determined in a 3D co-culture model of cancer cells and fibroblasts.

[0083] The peptide is preferably capable of inhibiting at least 40%, preferably at least 50%, and more preferably at least 60% of the proliferation of fibroblasts promoted by cathepsin D secreted by cancer cells in the tumor microenvironment, as determined in a 3D co-culture model of cancer cells and fibroblasts.

[0084] The ability of the peptide to inhibit fibroblast proliferation promoted by cathepsin D secreted by cells, particularly cancer cells within the tumor microenvironment, can be identified and / or quantified through the following steps: - Provides a monolayer of the target peptide, fibroblasts, and COS-7 cells that overexpress and secrete cathepsin D (pCD) or do not overexpress and secrete cathepsin D (control). - In the absence or presence of the test peptide (e.g., 100 µM), fibroblasts embedded in a soluble basement membrane matrix are seeded on top of a pCD or control COS-7 cell monolayer. - In the absence or presence of the test peptide, observe the co-cultures of fibroblast colonies co-cultured with COS-7 control or pCD cells by phase contrast microscopy over 6 days (e.g., on day 1, day 4, and day 6). - To identify and / or quantify the inhibitory activity of the target peptide on fibroblast proliferation promoted by cathepsin D secreted by pCD COS-7 cells.

[0085] Dissolvable basement membranes can be, for example, the dissolvable basement membrane secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma, produced by Corning Life Sciences under the trademark "Matrigel".

[0086] (d) Ability to inhibit cathepsin D activity

[0087] In one embodiment, the peptide is capable of inhibiting the catalytic activity of cathepsin D, particularly in the tumor microenvironment.

[0088] In one embodiment, the peptide is capable of inhibiting at least 20%, preferably 50%, of cathepsin D catalytic activity, particularly in the tumor microenvironment, as determined in assays at pH 6. The pH of the tumor microenvironment is known to be between 5.6 and 6.8. The ability of the peptide to inhibit cathepsin D catalytic activity in the tumor microenvironment can be identified and / or quantified by the following steps: - Provide cathepsinogen D (test peptide) and pepstatin A (control). - Activate cathepsinogen D (20 µg / mL) by pre-incubating it at 37°C in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) for 30 minutes. - Incubate activated cathepsinogen D (0.5 ng / µL) with the test peptide (100 µM) or pepsin A (2 µg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6) for 15 minutes. - Catalytic activity was measured in the presence of a fluorescent substrate (30 µM). - To identify and / or quantify the ability of a target peptide to inhibit the catalytic activity of cathepsin D in a medium with a simulated tumor microenvironment pH.

[0089] Functional variants

[0090] "Functional variant" refers to at least one active amino acid sequence that is derived from the fragment described herein and retains the original fragment.

[0091] Functional variants are derived from the fragments described herein by introducing one or more mutations (deletion, insertion, and / or substitution) at specific amino acid positions, provided that they contain at least some of the amino acids of SEQ ID NO: 1, 2, 3, or 4 as described above.

[0092] Specifically, the functional variations include at least one of the following: - Amino acids R9, G16, D17, R18, C19, Q20, and Y21 of SEQ ID NO:1 - Amino acids R3, G10, D11, R12, C13, Q14, and Y15 of SEQ ID NO:2; - Amino acids R9, G16, D17, R18, C19, Q20, and Y21 of SEQ ID NO:3; - Amino acids G8, D9, R10, C11, Q12, and Y13 of SEQ ID NO:4.

[0093] Functional variants contain amino acid sequences that are preferably “substantially homologous” or “substantially similar” to the reference amino acid sequence from which they originate. Two amino acid sequences are “substantially homologous” or “substantially similar” when one or more amino acid residues are substituted with biologically similar residues or when the sequences are at least 80% identical or 90% similar.

[0094] The amino acid sequence identity / similarity percentage is defined as the percentage of amino acid residues in the compared sequence that are identical or similar to those in the reference sequence after alignment and, if necessary, the introduction of vacancies to achieve maximum sequence identity. The identity percentage is then determined using the following formula: Identity Percentage = 100 x [1 - (C / R)], where C is the number of differences between the reference and compared sequences along the full length of the reference sequence, where (i) each amino acid in the reference sequence that does not have a corresponding compared amino acid in the compared sequence, (ii) each vacancy in the reference sequence, and (iii) each compared amino acid in the reference sequence that is not identical or similar to an amino acid in the compared sequence constitutes a difference; R is the number of amino acids in the reference sequence along the length of the compared sequence, where any vacancy introduced in the reference sequence is also counted as one amino acid.

[0095] To determine the percentage of amino acid sequence identity, comparisons can be made using a variety of methods known to those skilled in the art, such as publicly available computer software, like BLAST (Altschul). et al.The BLASTP program can be used with the following methods: J. Mol. Biol, 1990, 215, 403-; FASTA; GCG (Genetics Computer Group, GCG Software Package Manual, Version 7, Madison, Wisconsin) pileup program; or any program known in the art. When using such software, it is preferable to use the default parameters, such as vacancy penalty and extension penalty. For amino acid sequences, the default word length (W) used by the BLASTP program is 3, and the expected value (E) is 10.

[0096] In one embodiment, the functional variant is derived from the fragment described herein through the deletion, insertion, and / or substitution of one or more amino acids, particularly from the fragment derived from the amino acid sequence SEQ ID NO: 1, 2, 3, or 4 described herein.

[0097] In one embodiment, the functional variant is derived from the fragment described herein by means of deletion, insertion and / or substitution of 1, 2, 3 or 4 amino acids, particularly from the fragment derived from the amino acid sequence SEQ ID NO: 1, 2, 3 or 4 described herein.

[0098] Preferably, the substitution is a conservative substitution.

[0099] Conservative substitution refers to replacing one amino acid with another without altering the overall conformation and function of the peptide. This includes, but is not limited to, replacing one amino acid with another that has similar chemical or physical properties (size, charge, or polarity) that generally do not change the functional characteristics of the peptide. Amino acids with similar properties are well known in the art. Therefore, it should be understood that, in the context of this invention, conservative substitution is considered in the art to be replacing one amino acid with another that has similar properties.

[0100] Examples of conservative substitution are shown in Table 1: Table 1 Examples of conservative substitution

[0101] In one embodiment, the functional variant is derived from the fragment of SEQ ID NO: 1, 2, 3, or 4 as described above by one or more conserved substitutions, preferably by conserved substitutions of 1, 2, 3, or 4 amino acids. In one embodiment, the functional variant is derived from the fragment of SEQ ID NO: 1, 2, 3, or 4 as described above by conserved substitutions of one or more cysteine ​​residues.

[0102] The inventors have demonstrated that the cysteine ​​residue of the LBC4 peptide can form a disulfide bond in an oxidative environment, thereby producing a peptide that cannot block the catalytic activity of cathepsin D. Furthermore, the inventors have demonstrated that replacing the 8th cysteine ​​residue in the LBC4 peptide with a serine residue (C8S) weakens the peptide's ability to block the catalytic activity of cathepsin D. Not bound by theory, the inventors believe that the latter indicates that, at least in some embodiments, this particular cysteine ​​residue is preferably left unchanged.

[0103] In one embodiment, the fragment is a fragment comprising at least two cysteine ​​residues as described above, of SEQ ID NO: 1, 2, 3, or 4, and the functional variant is derived from the fragment by conserved substitution of one or more of these cysteine ​​residues, preferably by conserved substitution of one or more amino acid residues selected from serine (S), valine (V), threonine (T), and selenocysteine, while amino acid C19 of SEQ ID NO: 1, C13 of SEQ ID NO: 2, C19 of SEQ ID NO: 3, or C11 of SEQ ID NO: 4 preferably remain unchanged.

[0104] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, which comprises the following: - Amino acid C19 of SEQ ID NO:1, and at least one of C10 or C24, - Amino acid C14 of SEQ ID NO:2, and at least one of C5 or C19, - Amino acid C19 of SEQ ID NO:3, and at least one of C8 or C10, - Amino acid C11 of SEQ ID NO:4, and at least one of C2 or C16, Furthermore, the functional variant is derived from the fragment by conservatively substituting the following: - One or two of the C10 and C24 amino acids of SEQ ID NO:1, - One or two of the C5 and C19 amino acids of SEQ ID NO:2, - One or two of the C8 and C10 amino acids of SEQ ID NO:3, - One or two of the C2 and C16 amino acids of SEQ ID NO:4, Preferably, it is achieved through conservative substitution of amino acids selected from serine (S), valine (V), threonine (T), and selenocysteine.

[0105] In one embodiment, the fragment is the fragment of SEQ ID NO: 1, 2 or 4 as described above, which comprises the following: - Amino acids C19 and C24 of SEQ ID NO:1 - Amino acids C14 and C19 of SEQ ID NO:2, - Amino acids C11 and C16 of SEQ ID NO:4, Furthermore, the functional variant is derived from the fragment by conservatively substituting the following: - Amino acid C24 of SEQ ID NO:1 - Amino acid C19 of SEQ ID NO:2, - Amino acid C16 of SEQ ID NO:4, Preferably, it is achieved through conservative substitution of amino acids selected from serine (S), valine (V), threonine (T), and selenocysteine.

[0106] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2 or 4 as described above, selected from the following: - LBC4, amino acid sequence is PGFLGDRCQYRQCSG (SEQ ID NO:13) - LBC5, amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO:14) - LBC6, amino acid sequence CRCLPGFLGDRCQYR (SEQ ID NO:15) - LBC31, amino acid sequence GDRCQYRQCSG (SEQ ID NO:40), Furthermore, the functional variant is derived from the fragment by conservatively substituting at least the following: - Amino acid C13 of SEQ ID NO:13 - Amino acid C9 of SEQ ID NO:14 - One or two of the C1 and C3 amino acids in SEQ ID NO:15 - Amino acid C4 of SEQ ID NO:40 Preferably, it is achieved through conservative substitution of amino acids selected from serine (S), valine (V), threonine (T), and selenocysteine.

[0107] In one embodiment, the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence differs from the sequence of the fragment of SEQ ID NO:1, 2, 3 or 4 as described above by no more than 1, 2 or 3 amino acids, preferably no more than 1 or 2 amino acids.

[0108] In one embodiment, the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence is at least 70%, 80%, 85%, 90%, or 95% homologous to the fragments of SEQ ID NO: 1, 2, 3, or 4 as described above.

[0109] In one embodiment, the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the fragment of SEQ ID NO: 1, 2, 3, or 4 as described above.

[0110] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.

[0111] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3 or 4 as described above, and the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, wherein the fragment or functional variant derived therefrom comprises at least one of the following: - Amino acids R9, G16, D17, R18, C19, Q20, and Y21 of SEQ ID NO:1 - Amino acids R3, G10, D11, R12, C13, Q14, and Y15 of SEQ ID NO:2 - Amino acids R9, G16, D17, R18, C19, Q20, and Y21 of SEQ ID NO:3 - Amino acids G8, D9, R10, C11, Q12, and Y13 of SEQ ID NO:4.

[0112] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3, or 4 as described above, and the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, wherein the fragment or functional variant derived therefrom comprises at least 2, 3, or 4 of the following: - Amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1 - Amino acids G10, D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO:2 - Amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:3 - Amino acids G8, D9, R10, C11, Q12, and Y13 of SEQ ID NO:4.

[0113] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3, or 4 as described above, and the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, wherein the fragment or functional variant derived therefrom comprises at least: - Amino acids D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1 - Amino acids D11, R12, Q14, Y15 and / or amino acid R3 of SEQ ID NO:2, - Amino acids D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO:3 - Amino acids D9, R10, Q12, and Y13 of SEQ ID NO:4.

[0114] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3, or 4 as described above, and the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, wherein the fragment or functional variant derived therefrom comprises at least: - Amino acids G16, D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1 - Amino acids G10, D11, R12, Q14, Y15 and / or amino acid R3 of SEQ ID NO:2 - Amino acids G16, D17, R18, Q20, Y21 and / or amino acid R9 of SEQ ID NO:3, - Amino acids G8, D9, R10, Q12, and Y13 of SEQ ID NO:4.

[0115] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3, or 4 as described above, and the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, wherein the fragment or functional variant derived therefrom comprises at least: - Amino acids G16, D17, R18, C19, Q20 and / or amino acid R9 of SEQ ID NO:1 - Amino acids G10, D11, R12, C13, Q14 and / or amino acid R3 of SEQ ID NO:2 - Amino acids G16, D17, R18, C19, Q20 and / or amino acid R3 of SEQ ID NO:3 - Amino acids G8, D9, R10, C11, and Q12 of SEQ ID NO:4.

[0116] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3, or 4 as described above, and the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, wherein the fragment or functional variant derived therefrom comprises at least: - Amino acids D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1 - Amino acids D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO:2 - Amino acids D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:3 - Amino acids D9, R10, C11, Q12, and Y13 of SEQ ID NO:4.

[0117] In one embodiment, the fragment is a fragment of SEQ ID NO: 1, 2, 3, or 4 as described above, and the functional variant comprises or consists of an amino acid sequence as described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, wherein the fragment or functional variant derived therefrom comprises at least: - Amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R9 of SEQ ID NO:1 - Amino acids G10, D11, R12, C13, Q14, Y15 and / or amino acid R3 of SEQ ID NO:2 - Amino acids G16, D17, R18, C19, Q20, Y21 and / or amino acid R3 of SEQ ID NO:3 - Amino acids G8, D9, R10, C11, Q12, and Y13 of SEQ ID NO:4.

[0118] In one embodiment, the fragment is the fragment of SEQ ID NO: 1 or 4 as described above, selected from the following: - LBC4, amino acid sequence is PGFLGDRCQYRQCSG (SEQ ID NO:13) - LBC5, amino acid sequence GDRCQYRQCSGYCEN (SEQ ID NO:14), Furthermore, functional variants derived therefrom comprise or consist of the amino acid sequence described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 13 or 14, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, wherein the fragment or functional variant derived therefrom comprises at least: - Amino acids D6, R7, Q9, and Y10 of SEQ ID NO:13 - Amino acids D2, R3, Q5, and Y6 of SEQ ID NO:14.

[0119] In one embodiment, the fragment is the fragment of SEQ ID NO: 1 or 4 as described above, selected from the following: -LBC4, amino acid sequence is PGFLGDRCQYRQCSG (SEQ ID NO:13) -LBC5, the amino acid sequence is GDRCQYRQCSGYCEN (SEQ ID NO:14), Furthermore, functional variants derived therefrom comprise or consist of the amino acid sequence described below: the amino acid sequence has at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 13 or 14, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, wherein the fragment or functional variant derived therefrom comprises at least: - Amino acids G5, D6, R7, Q9, and Y10 of SEQ ID NO:13, - Amino acids G1, D2, R3, Q5, and Y6 of SEQ ID NO:14.

[0120] Modified peptides

[0121] In another aspect, the peptide of the present invention is a modified peptide derived from said peptide by introducing any modification into one or more amino acid residues, peptide bonds, N- and / or C-terminus of the peptide and retaining at least one activity of the peptide from which it is derived.

[0122] These modifications of peptides, introduced by conventional methods known to those skilled in the art, include, but are not limited to: replacing native amino acids with non-protein amino acids (D amino acids or amino acid analogs); peptide bond modification, particularly with reverse or inverse-type bonds or bonds different from peptide bonds; cyclization; and adding chemical groups to the side chains or ends of the peptide, particularly for coupling reagents of interest to the proteins of the present invention.

[0123] These modifications can be used in particular to increase the in vivo stability of peptides, especially their resistance to proteolysis.

[0124] Preferably, the peptide contains one or more chemical modifications, more preferably chemical modifications that protect the peptide from protein hydrolysis.

[0125] Advantageously, the N-terminus and / or C-terminus of the peptide are protected to prevent protein hydrolysis. For example, the N-terminus is in acetyl form and / or the C-terminus is in amide form.

[0126] Optionally or additionally, peptides can be protected from proteolysis through internal modifications, such as replacing at least one -CONH- peptide bond with a (CH2NH) reducing bond, (NHCO) anti-bonding bond, (CH2-O) methylene oxy bond, (CH2-S) thiomethylene bond, (CH2CH2) carbon bond, (CO-CH) cetylmethylene bond, (CHOH-CH2) hydroxyethylidene bond, (NN) bond, E-ene bond, or -CH=CH- bond.

[0127] Optionally or additionally, the peptide may be modified by acetylation, acylation, amidation, cross-linking, cyclization, disulfide bond formation, covalent cross-linking formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristylation, oxidation, phosphorylation, etc.

[0128] Optionally or additionally, the peptide advantageously comprises D-configured amino acids, which makes the peptide resistant to proteolysis.

[0129] Optionally or additionally, the peptide is stabilized by intramolecular crosslinking, by modifying at least two amino acid residues with an olefin side chain (preferably a C3-C8 alkenyl chain, more preferably a penten-2-yl chain), and then according to Walensky et al. Science The so-called "stacking peptide technology" described in 2004, 305, 1466-1470 cross-links the chain.

[0130] Optionally or additionally, the peptide is advantageously stabilized by covalently binding to a polyethylene glycol (PEG) molecule, preferably 1500 Da or 4000 Da, preferably bound to its C-terminus or lysine residue. This coupling may have at least one of the following advantages: (i) improved stability of the peptide in vivo without affecting its affinity; (ii) reduced urinary clearance and therapeutic dose; and / or (iii) prolonged plasma half-life.

[0131] Optionally or additionally, peptides can be advantageously stabilized and their half-life increased by incorporating them into biodegradable and biocompatible polymeric materials, such as poly(lactic-co-glycolic acid) (PLGA), into drug delivery systems that form microspheres.

[0132] Optionally or additionally, the peptide advantageously incorporates the N-terminus and / or C-terminus of its amino acid sequence with one or more other protein / peptide moieties, including those that allow the peptide or modified peptide of the present invention to be cell-targeted and / or improve the bioavailability, yield in the expression system, and / or stability of the peptide, thereby forming a fused peptide or chimeric peptide. The length of the chimeric peptide is not critical to the present invention, as long as the peptide retains its function. These protein / peptide moieties may be (i) cell-penetrating portions or (ii) targeting portions, used to localize the chimeric protein to a specific cell type or cell compartment. Cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs), membrane translocation sequences (MTS), transport peptides, carrier peptides, or Trojan peptides, are well known in the art. CPPs are capable of translocating into the cell (including the cytoplasm and organelles, such as mitochondria or the nucleus) at a level significantly higher than passive diffusion without causing significant membrane damage and can be used as carriers after being linked with other molecules. Furthermore, the peptide or modified peptide can be separated from the peptide / protein portion by a sufficiently long linker to avoid inhibiting the interaction between the peptide or modified peptide and the cell-penetrating or targeting portion.

[0133] In one embodiment, the peptide is advantageously fused to one or more CPPs, thereby allowing the peptide to be carrier-mediated into lysosomes at its N-terminus and / or C-terminus. For example, the peptide may be fused to a polyhistidine peptide, preferably to an H16 peptide with the sequence HHHHHHHHHHHHHHHH (SEQ ID NO:42) at its N-terminus and / or C-terminus.

[0134] Polynucleotides

[0135] Another aspect of the invention relates to isolated polynucleotides encoding said peptides.

[0136] Polynucleotides are synthetic or recombinant DNA, RNA, or combinations thereof, and can be single-stranded or double-stranded. Polynucleotides encode peptides in an expressible form, that is, nucleic acid molecules that, when expressed in cells or cell-free systems, produce functional peptides.

[0137] Preferably, the polynucleotide contains a coding sequence optimized for the host in which the peptide is expressed.

[0138] In another preferred embodiment, the polynucleotide is inserted into a vector. Preferably, the recombinant vector is an expression vector capable of expressing the polynucleotide upon transfection or transformation into host cells (e.g., prokaryotic or eukaryotic cells). The polynucleotide is inserted into the expression vector in the correct orientation and reading frame for expression. Preferably, the polynucleotide is operatively linked to at least one transcriptional regulatory sequence and optionally to at least one translational regulatory sequence. Recombinant vectors include vectors commonly used in genetic engineering and gene therapy, such as plasmids and viral vectors, such as lentiviruses and adenoviruses.

[0139] Treatment

[0140] The inventors have demonstrated that small peptides derived from the aforementioned key interaction region of LRP1 can effectively bind to cathepsin D and / or inhibit the LRP-1 / cathepsin D interaction. Their effectiveness has been demonstrated in models simulating tumor cell environments.

[0141] Another aspect of the invention relates to the use of the peptides, modified peptides, polynucleotides, and / or carriers described herein as pharmaceuticals. These pharmaceuticals are particularly useful for inhibiting cancer cell-promoted fibroblast proliferation in the tumor microenvironment.

[0142] The term "tumor microenvironment" refers to a dynamic mediator containing cells and macromolecules that interact with cancer cells and promote tumor formation, progression, and / or metastasis.

[0143] Therefore, another aspect of the present invention relates to peptides, polynucleotides and / or carriers as described herein for the treatment of proliferative diseases, particularly cancer, preferably in human patients.

[0144] The term "proliferative disorder" refers to a disease caused by abnormal growth or expansion of cells due to proliferation. Proliferative disorders may involve the pathological proliferation of normally resting cells and / or the pathological migration of cells from their normal locations (e.g., tumor cell metastasis). Examples of proliferative disorders include cancer (i.e., "malignant tumors") and benign tumors.

[0145] The term "tumor" refers to an abnormal mass of tissue that grows faster than normal tissue and is not as coordinated in its growth. A growth or tumor can 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 typically well-differentiated, grow significantly slower than malignant tumors, and are confined to the primary site. Furthermore, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant sites. Examples of benign tumors include, but are not limited to, lipomas, chondromas, adenomas, chondromas, senile hemangiomas, seborrheic keratosis, freckles, and sebaceous hyperplasia. In some cases, some "benign" tumors may later develop into malignant tumors, possibly due to additional genetic changes in a subset of the tumor cells; these tumors are called "precancerous tumors." An example of a precancerous tumor is a teratoma. In contrast, "malignant tumors" are typically poorly differentiated (anaplastic), grow significantly faster, and gradually infiltrate, invade, and destroy surrounding tissues. Furthermore, malignant tumors often have the ability to metastasize to distant sites. 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. The term is usually determined by the presence of a “secondary tumor” or “secondary cell cluster” of the tissue type of the primary or original tumor in the organ or tissue where the secondary (metastatic) tumor is located, rather than the tissue type of the organ or tissue in which it is located. For example, prostate cancer that has metastasized to bone is called metastatic prostate cancer, which includes cancerous prostate cancer cells growing in bone tissue.

[0146] In one embodiment, the present invention relates to a method for treating a proliferative disease or cancer in a subject of need using the peptides, polynucleotides, and / or carriers described herein, the method comprising administering an effective amount of the peptides, polynucleotides, and / or carriers.

[0147] The term “effective amount” or “therapeutic effective amount” of an active principal ingredient (such as peptides, polynucleotides and / or carriers as described herein) refers to the amount of an active principal ingredient, alone or in combination with another active principal ingredient (such as in combination with an antitumor agent as described herein), that elicits a biological or medical response in a subject, such as improving symptoms, alleviating symptoms, slowing or delaying disease progression or preventing disease.

[0148] The term "patient" refers to a human or non-human animal, preferably a mammal, who requires treatment, including men, women, adults, and children.

[0149] In one embodiment, the proliferative disease or cancer is a proliferative disease or cancer associated with overexpression of cathepsin D, i.e., in which tumor cells overexpress cathepsin D compared to non-tumor cells, preferably at least 2 times, for example 2 to 50 times.

[0150] In one implementation, proliferative disease or cancer is defined as a proliferative disease or cancer associated with the overexpression and secretion of cathepsin D in biological tissues or fluids, particularly serum and plasma, compared to biological tissues or fluids of a subject without cancer.

[0151] Examples of cancers associated with overexpression of cathepsin D include, but are not limited to, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, kidney cancer, bladder cancer, osteosarcoma, stomach cancer, pancreatic cancer, head and neck cancer, adenoid cystic carcinoma of the salivary gland, squamous cell carcinoma, melanoma, thyroid cancer, lung cancer, liver cancer, malignant glioma, and colorectal cancer.

[0152] In one implementation, a proliferative disease or cancer is a proliferative disease or cancer in which the overexpression of cathepsin D promotes tumor progression.

[0153] Examples of cancers that promote cathepsin D secretion by tumor cells include, but are not limited to, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, kidney cancer, and colorectal cancer.

[0154] The peptides, polynucleotides, and / or carriers described herein can be used to treat tumors, particularly malignant tumors, and especially to prevent or treat tumor metastasis.

[0155] More specifically, the peptides, modified peptides, polynucleotides and / or carriers described herein can be used to treat proliferative diseases or cancers in which tumor cells overexpress cathepsin D (preferably at least 2-fold) compared to non-tumor cells (e.g., non-tumor fibroblasts).

[0156] As used herein, the terms "treatment" or "therapy" include curative treatment and / or preventative treatment. More specifically, curative treatment refers to relieving, improving, and / or eliminating, reducing, and / or stabilizing (e.g., preventing disease progression to a later stage) the symptoms of a particular disease and delaying its progression. Preventative treatment refers to any of the following: preventing the onset of a particular disease, reducing its risk of development, reducing its incidence, delaying its onset, reducing its progression, and prolonging its time until onset. In this invention, "treatment" or "therapy" may specifically refer to reducing or preventing tumor progression.

[0157] Pharmaceutical Composition

[0158] The peptides, modified peptides, polynucleotides, and / or carriers described herein may be administered in the form of pharmaceutical compositions.

[0159] This invention also relates to a pharmaceutical composition comprising the peptides, modified peptides, polynucleotides and / or carriers described herein, as well as pharmaceutically acceptable carriers. The composition may also comprise another active ingredient, such as an antitumor agent described herein, more preferably a chemotherapeutic agent.

[0160] Combination therapy

[0161] The peptides, modified peptides, polynucleotides and / or carriers described herein can be used in combination with another active ingredient, particularly an antitumor agent.

[0162] Antitumor agents can be chemotherapeutic agents, immunotherapeutic agents, targeted therapies, cell therapies, or hormonal therapies, such as: (i) DNA replication inhibitors, such as DNA binding agents, especially alkylating agents or insertion agents; (ii) antimetabolites, such as DNA polymerase inhibitors or topoisomerase I or II inhibitors; (iii) antimitotic agents, such as alkaloids; (iv) checkpoint modulators, especially checkpoint inhibitors, such as anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, or anti-TIM3 agents; checkpoint agonists, such as OX40, 41BB, or GITR agonists; (v) targeted antitumor therapies, such as anti-EGFR, anti-HER2, anti-VEGF, PARP inhibitors, mTOR inhibitors; (vi) hormonal therapies, such as (via) corticosteroids, such as prednisone, dexamethasone, hydrocortisone, and methylprednisone; (vib) thyroid hormones; (vic) somatostatin analogs; and (vid) reproductive hormone drugs.

[0163] The chemotherapeutic agents mentioned in (i) to (iii) above may be, for example, 5-FU, oxaliplatin, cisplatin, carboplatin, irinotecan, docetaxel or paclitaxel.

[0164] The targeted therapeutic agents mentioned in (i) to (iii) above can be, for example, cetuximab or erlotinib.

[0165] The checkpoint modulators and targeted antitumor agents mentioned in (iv) and (v) above can be, for example, antibodies or fragments or small molecules thereof.

[0166] The reproductive hormone medications mentioned above (vid) can be, for example, androgen medications such as fluoxytestosterone; estrogen medications such as diethylstilbestrol; progesterone medications such as medroxyprogesterone acetate or megestrol acetate; antiandrogen medications such as bicalutamide, flutamide, or nilumet; aromatase inhibitors such as anastrozole, exemestane, or letrozole; luteinizing hormone-releasing hormone (LHRH) agonists such as buserreline, gosereline, or leuproreline; or gonadotropin-releasing hormone (GnRH) antagonists such as degarelix.

[0167] The antitumor agents mentioned in (i) to (vi) can be in the form of antibody-drug conjugates (ADCs).

[0168] In one embodiment, the present invention relates to a method for treating tumors in a subject of need using peptides, modified peptides, polynucleotides, and / or carriers described herein, the method comprising administering in combination effective amounts of the peptides, polynucleotides, and / or carriers, and the antitumor agents described herein.

[0169] In some embodiments, peptides or modified peptides comprising the fragments described herein (particularly the fragments described herein SEQ ID NO: 1, 2, 3 or 4 or functional variants derived therefrom) are used in combination with the chemotherapeutic agents described herein.

[0170] This disclosure also provides the use of the peptides or modified peptides described herein, optionally in combination with pharmaceutically acceptable supports and / or one or more active ingredients described herein, in the preparation of medicaments, particularly for the treatment of proliferative diseases, especially cancer, preferably in human patients, as described herein.

[0171] In another embodiment, this disclosure provides a treatment method, particularly a method for treating proliferative diseases (especially cancer), comprising administering an effective amount of a peptide or modified peptide to a subject in need, optionally in combination with a pharmaceutically acceptable support and / or one or more active ingredients described herein.

[0172] Application route

[0173] In one embodiment, the peptide or pharmaceutical composition, along with an optional antitumor agent, is administered to a subject via a route selected from systemic (e.g., intravenous or oral) to local (particularly intratumoral (IT) or intraperitoneal (IP)) routes.

[0174] For example, the peptide or pharmaceutical composition, along with optional antitumor agents, can be administered to ovarian cancer patients via the IP route. In this IP route, chemotherapy drugs are typically administered in liquid form through a device called an IP port into the patient's abdomen. The chemotherapy drugs enter the patient's abdomen through the IP port via an IP catheter. After the liquid enters the patient's abdomen, the patient needs to change position periodically to help the liquid distribute across the entire abdominal tissue surface. The entire process typically lasts 3 to 4 hours.

[0175] Diagnosis and / or staging of diseases associated with cathepsin D overexpression

[0176] Another aspect of the invention relates to the use of the peptides or modified peptides described herein in methods for diagnosing and / or staging diseases associated with cathepsin D overexpression in patients.

[0177] Diseases associated with overexpression of cathepsin D can be proliferative disorders, more specifically cancer or Alzheimer's disease.

[0178] The peptides or modified peptides used in the above methods are advantageously labeled peptides or modified peptides, i.e., peptides linked to a labeling agent (particularly a radioactive, magnetic, or luminescent agent) that generates a detectable and / or quantifiable signal. In other words, the peptide or modified peptide serves as a reagent. The luminescent agent can be, for example, a fluorophore, such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), or indocyanine (Cy5). The radioactive agent can be, for example, a radioactive atom used in scintillation imaging studies, such as I... 123 I 124 In 111 Re 186 Or Re 188 .

[0179] In one embodiment, the method includes the steps of contacting a biological sample obtained from a patient with the peptide or modified peptide (preferably labeled), wherein the peptide is preferably capable of selectively interacting with an LRP1 fragment (particularly the LRP1 β-chain extracellular domain) present in the biological sample obtained from the patient. The LRP1 fragment can then be detected by any method known in the art, and optionally its concentration can be measured.

[0180] In one embodiment, the method includes detecting the presence of LRP1 fragments, particularly the LRP1 β-chain extracellular domain, in a biological sample obtained from the patient.

[0181] In one embodiment, the method includes measuring the concentration of LRP1 fragments, particularly the LRP1 β-chain extracellular domain, in a biological sample obtained from the patient.

[0182] This disclosure also provides the use of the peptides or modified peptides described herein in the preparation of diagnostic kits for diagnosing or staging diseases associated with cathepsin D overexpression, wherein said diseases may be proliferative diseases, more specifically cancer or Alzheimer's disease.

[0183] In another embodiment, this disclosure provides a method for diagnosing or staging a disease associated with overexpression of cathepsin D in a patient, comprising applying the peptide or modified peptide described herein to a sample of the patient. Attached Figure Description

[0184] Figure 1 A computer-aided workflow for studying LRP-1 residues involved in the interaction between cathepsin D and LRP-1. LRP-derived peptides of different conformations (F4 fragment and EGF1920 fragment) are generated, and then selected for docking experiments with cathepsin D (CathD).

[0185] Figure 2 Computer analysis of LRP-1 residues involved in cathepsin D / LRP-1 interaction. Figure 2A and Figure 2B The histogram shows that in all molecular docking experiments conducted, EGF1920 ( Figure 2A ) or F4 segment ( Figure 2B The contact frequency of residues with cathepsin D. Residues with a contact frequency greater than or equal to 50% are indicated by a black arrow. Figure 2C Histograms A (white bars) and B (black bars) are superimposed at the level of the common sequence CRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:7) of the F4 and EGF1920 fragments. Residues with a contact frequency greater than 50% between the two fragments are indicated by black arrows. Dashed lines indicate a frequency of 50%.

[0186] Figure 3 The affinity of the F4 fragment and the LBC4 and LBC5 peptides for cathepsin D was investigated using microscale thermophoresis. The dose-response curves represent the binding interactions between cathepsin D and the F4 fragment, peptide LBC4, or peptide LBC5.

[0187] Figure 4 The ability of LBC1 to LBC5 peptides to block the interaction between cathepsin D and LRP-1 β chains was investigated using immunoprecipitation. (In the absence of...) Figure 4 A) or LBC peptide (100 µM) may be present. Figure 4 In case B), cathepsin D (100 ng) with a 6-His tag was incubated overnight with either LRP-1α (LRP1αF) or LRP-1β fragment (LRP1βF) displaying the myc epitope. The mixture was then deposited on Ni-NTA beads for 2 hours. The resulting complex was eluted with 2X Laemmli buffer, followed by SDS-PAGE electrophoresis and Western blotting using antibodies against cathepsin D and the myc tag. Figure 4 C) Histograms show the binding of LRP-1 β fragments to cathepsin D in the absence of LBC (control) or in the presence of LBC.

[0188] Figure 5 Effects of LBC1 to LBC5 peptides on the growth of mouse embryonic fibroblasts (MEF-1) co-cultured with COS-7 cells that overexpress or do not express cathepsin D. Figure 5A A schematic diagram of the co-culture model used. Fibroblasts were seeded on a Matrigel monolayer of COS-7 cells that either overexpressed and secreted cathepsin D (pCD) or did not overexpress and secrete cathepsin D (control). Figure 5BSix days later, Western blot analysis was performed on the levels of cathepsin D in the wells of co-culture medium containing COS-7 cells that overexpressed and secreted cathepsin D (pCD) or did not overexpress and secrete cathepsin D (control). Figure 5C On days 1, 4, and 6, phase-contrast microscopy was used to observe fibroblast colonies co-cultured with COS-7 controls or pCD cells in the absence or presence of 100 µM LBC1 to LBC5 peptides. Figure 5D The histogram shows that the area on day 6 is greater than 5,000 pixels. 2 The number of settlements (each point equals one settlement). n=5, one-way ANOVA. p<0.0001 p < 0.01, scale bar: 20 μm.

[0189] Figure 6 The localization of different peptides derived from the β chain of synthesized LRP-1 was used to depict regions of high interaction between LRP-1 and cathepsin D. LBC1-LBC5 peptides were the first peptides studied. LBC6-LBC8 peptides were used to depict the N-terminus of the high-interaction region, and LBC9-LBC15 peptides were used to depict the C-terminus of the high-interaction region.

[0190] Figure 7 NMR spectral characterization of LBC4 peptide under oxidative conditions. The LBC4 peptide was diluted in 30 μl of HCl / NaCl solution containing 20 mM Tris buffer (pH 7.5, 130 μl), and then 16 μL of DMSO (oxidative conditions) or 16 μL of water (non-oxidative conditions) was added. Figure 7A After incubation at room temperature for 72 hours, the 1D 1H NMR spectrum of the -NH region was measured at 298 Kelvin. Figure 7B Chemical shift determination of the NH region in spectra obtained under conditions of absence of DMSO (lower part) and presence of DMSO (upper part) (oxidation conditions). Figure 7C and Figure 7D The affinity of oxidized LBC4 peptide for cathepsin D was determined by microscale thermophoresis. A 0.5-fold dilution series, ranging from 10 μM to 0.15 nM, was used to mix labeled cathepsin D with either LBC4 peptides without disulfide bonds between their two cysteine ​​residues (LBC4) or LBC4 peptides with disulfide bonds (oxidized-LBC4).

[0191] Figure 8 Effects 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 10000 cells / well in 96-well plates, respectively, and incubated for 48 hours with no Nt (NT) or with different concentrations (6.25 to 50 µM) of LBC4, LBC5, or LBC15 peptides. Cell count was measured using the WST-1 cell proliferation assay.

[0192] Figure 9 Effects of LBC peptides on the catalytic activity of cathepsin D at pH 3.5 and pH 6. Figure 9A Cathepsin D (2 ng / µL) was pre-incubated with LBC peptide (100 µM) or pepsin A (2 ng / µL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) for 15 min. Activity was then measured in the presence of a fluorescent substrate (15 µM). Figure 9B Cathepsin D (0.5 ng / µL) was pre-activated for 30 min in acidic buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5), then pre-incubated with a peptide (100 µM) or pepsin A (2 µg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6) for 15 min. Activity was then measured in the presence of a fluorescent substrate (30 µM). Figure 9C Cathepsinogen D (0.5 ng / µL) was pre-activated for 30 min in acidic buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5), and then pre-incubated for 15 min with different concentrations of LBC4 peptide (10, 50, 100, 250, and 500 µM) or pepsin A (2 µg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6). Activity was then measured in the presence of a fluorescent substrate (30 µM).

[0193] Figure 10 The effect of LBC peptide on a TNBC tumor model. Figure 10A Experimental Procedure: Patient-derived xenografts (from TNBC's PDX, Crown Bioscience, Leiden, Netherlands) were seeded in hydrogels of 384-well plates. On days 3 and 7, different concentrations (10, 30, and 100 µM) of LBC4 (…) were used… Figure 10B LBC5 Figure 10C ) and LBC8 ( Figure 10DOrganoids were treated with peptides and maintained in culture until day 10. Cultures were fixed, stained, and analyzed using TIBCO spotfire software. n=4, Kruskal-Wallis test, and Benjamini, Krieger, and Yekutieli corrections were applied. p<0.001 p<0.01, p<0.05.

[0194] Example

[0195] The present disclosure is further illustrated by the following examples.

[0196] Example 1: Identification of key cathepsin D / LRP-1 interaction regions and study of the interactions derived from them region peptides

[0197] 1.1. Materials and methods

[0198] Affinity constant determined by microscale thermophoresis

[0199] Materials and reagents

[0200] PCR tubes and pipette tips – HEPES (H0887-20ML; Sigma Aldrich) – Tween® 20 (P1379-25ML; Sigma Aldrich) – Monolith His-Tag Labeling Kit RED-Tris-NTA Generation 2 (MO-L018; Nanotemper) – Human cathepsin D / CTSD protein, His Tag (CTD-H5226; Acrobiosystems) – Milli-Q® Water – Water (W3500-100ML; Sigma Aldrich) – Monolith capillary tubes (MO-K022; Nanotemper) Assay buffer: HEPES 50 mM, pH 7.5, supplemented with 0.05% Tween® 20. Cathepsin D stock solution, aliquoted at -80°C - Concentration: 400 µg / mL – 9.2 µM Cathepsin D labeling (Monolith His-Tag labeling kit) 1. Add 8 mL of Milli-Q® water to a vial containing 5X PBS-T to obtain 1X PBS-T. 2. Suspend the dye in 25 µL PBS-T to obtain a 5 µM dye solution (2 µL aliquots at -20°C). 3. Mix 2 µL of dye (5 µM) with 98 µL of assay buffer. Mix to prepare a 100 Nm dye solution 4. [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] The concentration was adjusted to 200 nM, and the volume was 100 µL. 5. Mix 90 µL of protein (200 nM) with 90 µL of dye (100 nM) in a 1.5 mL tube. 6. Incubate at room temperature for 30 minutes. 7. Centrifuge the sample at 4°C and 15,000 g for 10 minutes, and transfer the supernatant to a new tube. 8. Label the protein and prepare for binding assay. Peptide preparation 1. Remove the peptide from -20°C and centrifuge at 4°C and 10,000 g for 5 minutes. 2. Let the peptide sit at room temperature for 10 minutes. 3. Add X µL of Milli-Q® water to obtain a 500 µM concentration. 4. Allow the peptides to dissolve at room temperature for 60 minutes, then place them at 4°C. Combined measurement All reagents should be equilibrated to room temperature before use.

[0201] 1. Prepare 50 µL of an aqueous solution containing 20 µM peptide (2 µL qsp 50 µL water).

[0202] 2. Add 10 µL of Milli-Q® water to PCR tube 2-16.

[0203] 3. Transfer 20 µL of peptide to PCR tube 1

[0204] 4. Using a pipette (P20), transfer 10 µL from tube 1 to tube 2, and pipette up and down several times. Repeat this operation for tubes 3-16, and discard any excess contents in tube 16.

[0205] 5. Add 10 µL of labeled protein to each well (16 to 1) and mix using a pipette.

[0206] 6. Incubate at room temperature for 30 minutes.

[0207] 7. Load the capillary tube (NT.115 capillary tube) and measure the sample using the Monolith NT.115 system with the following settings: 20% Pico-RED excitation power and medium MST power.

[0208] Investigating cathepsinogen D / LRP-1 interaction using co-immunoprecipitation

[0209] Materials and reagents

[0210] Cos-7 cells – Lipofectamine 2000 transfection reagent (11668027; Invitrogen) – pSectag SD1 – pSectag F0 Ø6His – 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) – Cathepsin D Monoclonal Antibody (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-Conjugated Antibody (7076S; Cell Signaling) – Human Cathepsin D / 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) – Trypsin-EDTA (0.05%) (11580626; Gibco) – TG-SDS powder (865984; Interchim) – Tris-glycine buffer 10X solution (UP91493A; Interchim) – Water (W3500-100ML; Sigma Aldrich).

[0211] Cos-7 transfection

[0212] 1. Inoculate Cos-7 onto B10 until confluence reaches 80%.

[0213] 2. transfection : 3. Tube 1: Mix 900 µL of OptiMEM with 30 µL of Lipofectamine 2000 to obtain a B10. 4. Tube 2: Mix 15 µg DNA with 900 µL OptiMEM. pSectag SD1 (LRP1α) 781 ng / µL / 587 ng / µL: 25.5 µL 5. pSectag F0 (LRP1β): 1.15 µg / mL. Then add "tube 2" dropwise into "tube 1". 6. Incubate at room temperature for 5 minutes. 7. Add this mixture dropwise to the cells. 8. Incubate at 37°C, 5% CO2, and 95% humidity for 48 hours. 9. Collect the supernatant and centrifuge at 1500 rpm for 10 minutes at 4°C. 10. Store at 4°C for 24–72 hours, and at -20°C for long-term storage. Peptide preparation 1. Remove the peptide from -20°C and centrifuge at 4°C and 10,000 g for 5 minutes. 2. Let the peptide sit at room temperature for 10 minutes. 3. Add X µL of water to obtain a 500 µM concentration. 4. Allow the peptides to dissolve at room temperature for 60 minutes, then place them at 4°C. Immunoprecipitation condition: SD1 + cathepsin D – F0 + cathepsin D – F0 + cathepsin D + peptide (100µM).

[0214] On the ice: 1. Prepare 50 µL of cathepsin D (2 ng / µL) in PBS + / - 100 µM peptide and incubate gently by vortexing at 4°C for 1 hour. 2. Add 500 µL of supernatant "SD1 or F0" to each tube and incubate overnight at 4°C with gentle rotation. 3. Prepare 25 µL of NiNTA agarose (50 µL) for use under certain conditions. 4. Wash the resin three times with 1 mL PBS-T, centrifuge (1000g – 4°C – 1 min) and remove the supernatant. 5. Incubate with 1 mL of 2% BSA at 4°C for 30 minutes. 6. Wash 3 times with 1 mL PBS-T. 7. Centrifuge at 1000g for 1 minute at 4°C to remove the supernatant. 8. Resuspend the resin in 25 µL of PBS-T. 9. Add 50 µL to each tube. 10. Incubate gently by rotating for 4 hours at 4°C. 11. Wash 5 times with 1 mL PBS-T (centrifuge at 1000g – 4°C – 1 minute between each wash). 12. Remove the supernatant and elute the sample with 30 µL of Laemmli 2X and β-mercaptoethanol. 13. Place the sample at -20°C Western blot analysis 1. Heat the sample in a 100°C dry bath for 10 minutes. 2. Centrifuge the sample and transfer the supernatant to a 10% agarose gel. 3. Migrate to 70V (stacking gel), then migrate to 120V (separating gel). 4. Transfer the protein from the nitrocellulose membrane to 100V, 4°C, for 60 minutes. 5. Place the membrane on a TBS-T containing 5% milk. 6. Preparation of antibodies using 5% TBS-T in milk. 7. Incubate overnight at 4°C with gentle stirring. 8. Clean the membrane with TBS-T for 10 minutes (x3) 9. Incubate with the secondary antibody at room temperature for 1 hour. 10. Repeat the cleaning process with TBS-T, then clean once more with TBS. 11. Exposure using the Li-Cor Odyssey FC 2800 imaging system. Investigating the role of cathepsin D in a 3D co-culture model of cancer cells and fibroblasts. Materials and reagents Cos-7 cells – MEF1 cells – Lipofectamine 2000 transfection reagent (11668027; Invitrogen) – pCDNA 3.1 CTL – pCDNA 3.1 pCD – OptiMEM (31985-070; Gibco); DMEM 4.5 g / L glucose (31966-021; Gibco) – FCS (F7524; Sigma Aldrich) – PBS (14190-094; Gibco) – 48-well plate (353078; Falcon®) – Trypsin-EDTA (0.05%) (11580626; Gibco) – Water (W3500-100 mL; Sigma Aldrich) – EVOS FL microscope (Thermofisher).

[0215] COS 7 inoculation and transfection

[0216] 1. Seed 20,000 cells per well in 300 µL of DMEM supplemented with 10% FCS at 4.5 g / L glucose (48-well plate).

[0217] 2. Incubate at 37°C, 5% CO2, and 95% humidity for 24 hours.

[0218] 3. Transfection was performed using plasmids and Lipofectamine 2000.

[0219] 4. Tube 1: Mix 20 µL of OptiMEM with 0.862 µL of Lipofectamine 2000 per well.

[0220] 5. Tube 2: Mix 20 µL of OptiMEM with 0.287 µg of DNA.

[0221] pCDNA3.1 CTL: 1.22 µg / µL

[0222] pCDNA3.1 pCD: 1.26 µg / µL

[0223] 6. Then add "tube 2" dropwise into "tube 1".

[0224] 7. Incubate at room temperature for 5 minutes.

[0225] 8. Add this mixture dropwise to the cells.

[0226] 9. Incubate at 37°C, 5% CO2, and 95% humidity for 24 hours.

[0227] Place Matrigel on ice and thaw overnight at 4°C. Place pipette tips (200 µL and 1000 µL) at -20°C.

[0228] Peptide preparation

[0229] 1. Remove the peptide from -20°C and centrifuge at 4°C and 10,000 g for 5 minutes.

[0230] 2. Let the peptide sit at room temperature for 10 minutes.

[0231] 3. Add X µL of water to obtain a 500 µM concentration.

[0232] 4. Allow the peptides to dissolve at room temperature for 60 minutes, then place them at 4°C.

[0233] MEF-1 inoculated onto Matrigel

[0234] 1. Remove COS7 medium

[0235] 2. Add 100 µL of Matrigel to each well. (Use a cold nozzle)

[0236] 3. Polymerize for 1 hour at 37°C, 5% CO2, and 95% humidity.

[0237] 4. Rinse MEF1 with 5 mL PBS

[0238] 5. Add 1 mL of trypsin-EDTA (For a B10) Incubate at 37°C for 5 minutes

[0239] 6. Add culture medium containing 10% FCS and count the cells.

[0240] 7. Take a volume sample to obtain 50,000 cells per well.

[0241] 8. Centrifuge at 1200g for 5 minutes

[0242] 9. Resuspend cells in 300 µL of Matrigel per well: 300 µL per well, 50,000 cells.

[0243] 10. Inoculate MEF1 on COS7 cells.

[0244] 11. Polymerize for 1 hour at 37°C, 5% CO2, and 95% humidity.

[0245] 12. Add 200 µL of culture medium + / - 100 µM peptide to each well.

[0246] 13. Take photographs under a microscope at D+1, D+4, and D+6.

[0247] Effects of LBC peptide on breast cancer cell proliferation

[0248] Materials and reagents

[0249] MDA-MB-231 cells – MCF7 cells – DMEM 1g / L glucose (21885-025; Gibco) – FCS (F7524; Sigma Aldrich) – PBS (14190-094; Gibco) – Trypsin-EDTA (0.05%) (11580626; Gibco) – 96-well plate (83.3924; Sarstedt) – Cell proliferation reagent WST-1 (11644807001; Sigma Aldrich) – Water (W3500-100ML; Sigma Aldrich) – Infinite 200® pro (TECAN).

[0250] Peptide preparation

[0251] 1. Remove the peptide from -20°C and centrifuge at 4°C and 10,000 g for 5 minutes.

[0252] 2. Let the peptide sit at room temperature for 10 minutes.

[0253] 3. Add X µL of water to obtain a 500 µM concentration.

[0254] 4. Allow the peptides to dissolve at room temperature for 60 minutes, then place them at 4°C.

[0255] Measurement Procedure

[0256] 1. For MDA-MB-231 and MCF7, 5,000 and 10,000 cells per well were seeded in 100 µL DMEM 1 g / L glucose (96-well plate) supplemented with 10% FCS, respectively. The two plates were used for WST-1 assays at 24 and 48 hours post-treatment.

[0257] 2. Incubate at 37°C, 5% CO2, and 95% humidity for 24 hours.

[0258] 3. Treat cells with peptides of 50 µM, 25 µM, 12.5 µM, and 6.25 µM in DMEM supplemented with 10% FCS and 1 g / L glucose.

[0259] 4. Incubate at 37°C, 5% CO2, and 95% humidity for 24 hours / 48 hours.

[0260] 5. Add 10 µL / well of cell proliferation reagent WST-1

[0261] 6. Incubate the cells at 37°C, 5% CO2, and 95% humidity for 2 hours.

[0262] 7. Shake thoroughly on the shaker for 1 minute.

[0263] 8. Measure the absorbance of the sample at 450 nm using an ELISA reader, with the background control as a blank. The reference wavelength is >600 nm.

[0264] Analysis of cathepsin D catalytic activity at pH 3.5

[0265] Materials and reagents

[0266] PCR tubes and pipette tips – Recombinant human proteaseogen D protein (ab286010; Abcam) – Water (W3500-100ML; Sigma Aldrich) – Anhydrous sodium acetate (W302406; Sigma Aldrich) – Sodium chloride (207790010; ThermoScientific) – Cathepsin D and E substrate (fluorescent) (BML-P145-0001; EnzoLife Sciences) – 96-well ELISA plate (82.1581.120; Sarstedt) – PBS (14190-094; Gibco) – BSA (Bovine serum albumin) 04-100-812-E; Euromedex) – P5318-5MG; SigmaAldrich – Infinite 200® pro (TECAN).

[0267] Peptide preparation

[0268] 1. Remove the peptide from -20°C and centrifuge at 4°C and 10,000 g for 5 minutes.

[0269] 2. Let the peptide sit at room temperature for 10 minutes.

[0270] 3. Add X µL of water to obtain a 500 µM concentration.

[0271] 4. Allow the peptides to dissolve at room temperature for 60 minutes, then place them at 4°C.

[0272] Measurement Procedure

[0273] Equilibrate the assay buffer to room temperature before use.

[0274] 1. Prepare the assay buffer: 0.1 M NaOAc, 0.2 M NaCl, pH 3.5

[0275] 2. Dilute cathepsinogen D to 20 µg / mL in assay buffer.

[0276] 3. Add 5 µL of 100 µM cathepsinogen D+ / - peptide to the assay buffer (final volume: 50 µL).

[0277] 4. Incubate at room temperature for 15 minutes.

[0278] 5. Dilute the substrate to 30 µM in assay buffer.

[0279] 6. Load 50 µL of cathepsinogen D+ / - peptide (100 µM) or pepsin (2 µg / mL) into a plate (pre-saturated with 1% BSA).

[0280] 7. Add 50 µL of 30 µM substrate to begin the reaction.

[0281] 8. In kinetic mode, readouts were performed for 1 hour at excitation and emission wavelengths of 320 nm and 405 nm (top readout), respectively.

[0282] Analysis of cathepsin D catalytic activity at pH 6

[0283] Materials and reagents

[0284] PCR tubes and pipette tips – Recombinant human cathepsin D protein (1014-AS; R&D systems) – Water (W3500-100ML; Sigma Aldrich) – Anhydrous sodium acetate (W302406; Sigma Aldrich) – Sodium chloride (207790010; ThermoScientific) – Cathepsin D and E substrates (fluorescent) (BML-P145-0001; EnzoLife Sciences) – 96-well ELISA plate (82.1581.120; Sarstedt) – PBS (14190-094; Gibco) – BSA (Bovine serum albumin)04-100-812-E; Euromedex) – P5318-5MG; SigmaAldrich – Infinite 200® pro (TECAN).

[0285] Peptide preparation

[0286] 1. Remove the peptide from -20°C and centrifuge at 4°C and 10,000 g for 5 minutes.

[0287] 2. Let the peptide sit at room temperature for 10 minutes.

[0288] 3. Add X µL of Milli-Q® water to obtain a 500 µM concentration.

[0289] 4. Allow the peptides to dissolve at room temperature for 60 minutes, then place them at 4°C.

[0290] Measurement Procedure

[0291] Equilibrate the assay buffer to room temperature before use.

[0292] 1. Preparation Assay buffer :0.1 M NaOAc, 0.2 M NaCl, pH 3.5 / 0.1 M NaOAc, 0.2 M NaCl, pH 6

[0293] 2. Dilute cathepsin D to 20 µg / mL in assay buffer at pH 3.5.

[0294] 3. Incubate at 37°C for 30 minutes to activate the protein.

[0295] 4. Dilute the incubated cathepsin D to 0.5 ng / µL in assay buffer at pH 6.

[0296] 5. Dilute the substrate to 60 µM in assay buffer at pH 6.

[0297] 6. Load 50 µL of cathepsin D+ / - peptide (100 µM) or pepsin (2 µg / mL) into a plate. (Pre-saturated with 1% BSA)

[0298] 7. Incubate at 37°C for 15 minutes.

[0299] 8. Add 50 µL of 60 µM substrate to begin the reaction.

[0300] 9. In kinetic mode, readouts were performed for 45 minutes at excitation and emission wavelengths of 320 nm and 405 nm (top readout), respectively.

[0301] 1.2. Results and Discussion

[0302] Characterizing cathepsin D / LRP-1 interaction using computer studies

[0303] To determine the exact interaction region between cathepsin D and LRP-1 and the residues involved, we based on Figure 1 The workflow shown was subjected to computer analysis (bioinformatics strategies, molecular modeling, and dynamics).

[0304] In summary, we generated representative structures and conformations of the F4 fragment using structure prediction methods (MODELLER software, I-TASSER online server) and molecular dynamics tools (GROMACS software). We also modeled another longer fragment of this region of LRP-1β, which takes into account the structural elements identified in these sequences. In fact, this region of LRP-1 is rich in EGF-like domains, and the F4 fragment partially overlaps with EGF-like domains 19 and 20. Therefore, to maintain structural consistency, we proposed a fragment containing both of these domains (the EGF1920 fragment).

[0305] The amino acid sequences of the F4 fragment and EGF1920 are shown in Table 2.

[0306] Table 2: Amino acid sequences of the F4 and EGF1920 fragments of LRP-1

[0307] We used the same method as with the F4 fragment to analyze the EGF1920 fragment, and derived a representative structure and conformation of this new fragment. The same molecular docking scheme was performed for each of the two fragments (F4 fragment (SEQ ID NO:5) and EGF1920 fragment (SEQ ID NO:6)).

[0308] The structure obtained by minimizing energy, along with the first two clusters extracted from molecular dynamics, was preserved and submitted to the available cathepsin D structure on the PDB server (1LYW) for rigid / rigid docking (HEX software). Each docking experiment generated 100 solutions, for a total of 1500 solutions. For each fragment, the contact number was normalized so that the frequency of residues statistically forming the maximum contact number was 1. A cutoff value of 0.5 was used to distinguish key residues from other residues. Figure 2A and Figure 2B The analysis of the results is in F4 (). Figure 2A) and EGF1920 ( Figure 2B Several residues were identified in the fragment that may interact with cathepsin D. Five residues with high frequency values ​​were identified in both fragments. Figure 2C Within a five-residue region (DRCQY(SEQ ID NO:9)), there is an isolated R residue and four residues (DR_QY). This region is clearly visible and serves as the starting point for designing blocking peptides.

[0309] Five peptides (LBC1 to LBC5) were designed to block the interaction between cathepsin D and LRP-1.

[0310] We preselected five peptides (Table 3) of only 15 amino acids in length from the DRCQY sequence to optimize synthesis (compatible with the standard Fmoc synthesis process on solid-phase support, low cost, high yield, and high purification rate) and meet biocompatibility requirements. In fact, during the preselection process, we paid particular attention to identifying sequences with high predicted solubility in aqueous solvents (water, NaCl 0.9%) to predict clinical applications.

[0311] Table 3: Sequences of five peptides pre-selected for blocking cathepsin D / LRP-1 interaction. The sequences of the five amino acids identified by computer analysis are shown in bold. The predicted isoelectric point, net charge at pH 7, and solubility in 0.9% NaCl are also indicated in the figure.

[0312]

[0313] These five peptides were synthesized under a service contract with SB-Peptide (Grenoble, France), and no difficulties were encountered in the synthesis and purification (synthesis reports are available upon request).

[0314] To verify the ability of these peptides to block cathepsin D / LRP-1 interaction, three tests were performed on these peptides: 1- The constant affinity (Kb) of these peptides for cathepsin D was determined using microscale thermophoresis (MST). D (See the Materials and Methods section above) ) 2- The ability to block the interaction between cathepsin D and LRP-1 in an in vitro co-immunoprecipitation assay using a soluble fragment of the LRP-1 β chain and recombinant cathepsin D (see Materials and Methods section above). 3- The ability to block cathepsin D / LRP-1 interaction and its proliferative effect in a mouse fibroblast model (MEF-1 cells, expressing LRP-1) (see Materials and Methods section above). The affinity of LBC1 to LBC5 peptides for cathepsin D was studied using microscale thermophoresis. We first investigated the affinity of LBC1 to LBC5 peptides for cathepsin D. Microscale thermophoresis (MST) experiments showed that only peptides LBC1, LBC3, LBC4, and LBC5 could bind to cathepsin D.

[0315] In summary, cathepsinogen D (Abcam) was labeled using the His-Tag labeling kit Red-Tris NTA (NanoTemper). The labeled cathepsinogen D was then mixed with F4 fragments or peptides LBC1, LBC2, LBC3, LBC4, and LBC5, using a 0.5-fold dilution series ranging from 50 μM to 1.5 nM. These 16 mixtures were subsequently analyzed using a Monolith NT.115 (NanoTemper) instrument at 256°C. Instrument parameters included 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper).

[0316] The results are as follows Figure 3 As shown in Table 4.

[0317] Table 4: K values ​​of LBC1 to LBC5 peptides obtained by microscale thermophoresis (MST) D value

[0318] The results showed that the affinities of LBC1 (52.5 nM), LBC3 (340 nM), LBC4 (4.3 nM), and LBC5 (8.7 nM) were all higher than those of fragment F4 (2.37 µM), which corresponds to 45 amino acid residues from positions 349 to 394 of LRP-1β. These results indicate that computer simulation analysis has identified a key region in LRP-1 that is indeed involved in the interaction with cathepsin D. The primary amino acid sequence flanking this region appears to play a role in the affinity of these peptides.

[0319] The blocking properties of LBC1 to LBC5 peptides on cathepsin D / LRP-1 were investigated by immunoprecipitation.

[0320] The ability of the peptide to block the interaction between cathepsin D and LRP-1 was then investigated using an immunoprecipitation assay (n=5). Figure 4 We first demonstrated that cathepsin D can bind to the extracellular portion of the LRP-1 β chain (LRP1β-F). Figure 4A). This interaction is specific, as we did not observe binding to the α-chain fragment of LRP-1 (LRP1αF). In the presence of different peptides, 100 µM LBC3, LBC4, and LBC5 significantly blocked this interaction by approximately 60% to 70%, while peptide LBC1 showed less blocking (40%). Figure 4 B). These results confirm the findings observed in the MST.

[0321] In a 3D co-culture model of cancer cells and fibroblasts, the effects of LBC1 to LBC5 peptides on cathepsin D / LRP-1's blocking properties

[0322] Finally, we tested the ability of peptides LBC1 to LBC5 to block cathepsin D / LRP-1 interaction in a previously validated cell model (Beaujouin). et al. , 2010; Derocq et al. (2012).

[0323] Figure 5A Histograms show a 3D co-culture model of mouse embryonic fibroblasts naturally expressing LRP-1 and COS-7 cancer cells that overexpress and secrete cathepsin D or do not overexpress and secrete cathepsin D.

[0324] Six days later, Western blot analysis was performed on the amount of cathepsin D present in co-culture wells of COS-7 cells that overexpressed and secreted cathepsin D (pCD) or did not overexpress and secrete cathepsin D (control). The results are as follows: Figure 5B As shown.

[0325] Phase-contrast microscopy was performed on days 1, 4, and 6 on fibroblast colonies co-cultured with COS-7 controls or pCD cells, with or without 100 µM LBC 1 to 5 peptides. Results are as follows: Figure 5C As shown.

[0326] Figure 5D This means that the area on day 6 is greater than 5,000 pixels. 2 Histogram of colonies (each point equals one colony). n=5, one-way ANOVA. p<0.0001 p < 0.01, scale bar: 20 μm.

[0327] As expected (positive control), the presence of cathepsin D significantly stimulated fibroblast growth and the colony size observed at 4 and 6 days post-incubation. Figure 5C , Figure 5DSimilar experiments using LRP-1 KO mouse embryonic fibroblasts (LRP1 - / -, PA-13 cells) validated the specificity of this effect, as the presence of cathepsin D had no effect on growth in these cells (negative control, data not shown). Therefore, we investigated the effect of cathepsin D on fibroblast growth in the presence of different LBC peptides (100 µM). This effect of cathepsin D was significantly inhibited by peptides LBC2 and LBC5, strongly inhibited by peptide LBC4 (90% to 100% inhibition), while peptides LBC1 and LBC3 had no significant effect. Figure 5C and Figure 5D ).

[0328] After performing three tests on these different peptides, we identified at least two peptides, LBC4 and LBC5, both of which exhibited good affinity and were able to block cathepsin D / LRP-1 interaction in vitro and intracellularly in a mouse model. 。 Other peptides cannot be excluded either, as they exhibit binding and / or blocking abilities in vitro or intracellularly.

[0329] The study investigated a set of peptide sequences derived from the F4 LRP-1 fragment that can bind cathepsin D.

[0330] In vitro and intracellular data obtained for each of the LBC1 to LBC5 peptides suggest that we should not limit ourselves to single peptide sequences, but rather study larger sequences. To characterize these sequences, we decided to synthesize 10 new peptides (LBC6 to LBC15) to explore the N-terminal and C-terminal sequences of the analyzed peptides.

[0331] Figure 6 Different peptides derived from the β chain of LRP-1 are shown, which were synthesized to depict regions of high interaction between LRP-1 and cathepsin D.

[0332] Figure 6 The region of the LRP-1 β chain with the amino acid sequence STCTVNQGNQPQCRCLPGFLGDRCQYRQYRQCSGYCENFGTCQMAADGSRQCRCTAYFEGSRC (SEQ ID NO:8) is shown. This region includes, but is not limited to, the LRP-1 region referred to as “fragment F4”, from which peptides LBC6 (SEQ ID NO:15) to LBC15 (SEQ ID NO:24) were designed.

[0333] Peptides LBC1 (SEQ ID NO:10) to LBC5 (SEQ ID NO:14) were the first peptides studied. Peptides LBC6 (SEQ ID NO:15) to LBC8 (SEQ ID NO:17) were used to describe the N-terminus of the highly interacting region of the amino acid sequence (SEQ ID NO:1), and peptides LBC9 (SEQ ID NO:18) to LBC15 (SEQ ID NO:24) were used to describe the C-terminus of the highly interacting region of the amino acid sequence (SEQ ID NO:1).

[0334] These ten novel peptides were synthesized by SB-peptide (purity >90%, acetate neutralized). For each of these peptides, we performed solubility tests and affinity tests for cathepsin D using microscale thermophoresis.

[0335] In summary, cathepsin D (Acrobiosystems) was labeled using the His-Tag labeling kit Red-Tris NTA (NanoTemper). The labeled cathepsin D was then serially diluted 0.5-fold, ranging from 10 μM to 0.15 nM, and mixed with LBC peptides. These 16 mixtures were subsequently analyzed at 25°C using a Monolith NT.115 (NanoTemper) instrument. Instrument parameters included 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper). The results are shown in Table 5 below. Table 5: The affinity of LBC1 to LBC15 peptides for cathepsin D was studied using microscale thermophoresis. The Kd values ​​for each peptide obtained by microscale thermophoresis are listed in the table. Briefly, cathepsin D (Acrobiosystems) was labeled using the His-Tag labeling kit Red-Tris-NTA (NanoTemper). The labeled cathepsin D was then mixed with LBC peptides (1 to 15) using a 0.5-fold dilution series, ranging from 10 μM to 0.15 nM. These 16 mixtures were then analyzed at 25°C using a Monolith NT.115 (NanoTemper) instrument. Instrument parameters included 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper).

[0336]

[0337] Through solubility tests, we identified some peptide sequences (peptides LBC9, LBC11, and LBC14) that cannot be used intracellularly or in vivo because they are insoluble in water.

[0338] The equilibrium dissociation constant (K) of the obtained soluble peptide D This can be used to i) identify peptide sequences that can interact with cathepsin D; and ii) define highly interactive regions. These highly interactive regions are defined by peptides LBC8 and LBC9 and consist of 30 amino acids: NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:1).

[0339] The structure of the LBC4 peptide was characterized by nuclear magnetic resonance.

[0340] To better characterize the LBC4 peptide, which presented optimal results according to our in vitro and intracellular assays, nuclear magnetic resonance (NMR) spectroscopy analysis was performed by Professor Bruno Kieffer (UMR CNRS 7104, Strasbourg, France).

[0341] The LBC4 peptide was diluted in 20 mM Tris buffer (pH 7.5, 130 μl) containing HCl / Nacl solution (30 μl), and then 16 μL of DMSO (oxidation conditions, red spectrum) or 16 μL of water (non-oxidation conditions, blue spectrum) was added.

[0342] The results are shown in Figure 7: - Figure 7A After incubation at room temperature for 72 hours, the 1D 1H NMR spectrum of the -NH region was measured at 298 Kelvin.

[0343] - Figure 7B : Determine the chemical shift between the spectra obtained in the NH region in the absence of DMSO (blue) and in the presence of DMSO (oxidation conditions) (red).

[0344] - Figure 7C and Figure 7DThe affinity of oxidized LBC4 peptide for cathepsin D was determined by microscale thermophoresis. Labeled cathepsin D was mixed with either LBC4 peptides without disulfide bonds between the two cysteine ​​residues (LBC4) or LBC4 peptides with disulfide bonds (oxidized LBC4) using a 0.5-fold dilution series, ranging from 10 μM to 0.15 nM. These 16 mixtures were then analyzed at 25°C using a Monolith NT.115 (NanoTemper) instrument. Instrument parameters included 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper).

[0345] The LBC4 peptide appears to be unstructured; however, in an oxidative environment, the LBC4 cysteine ​​residue can form disulfide bonds and alter the peptide conformation. Figure 7A and 7B MST analysis showed that this cyclization disrupted the binding of LBC4 peptide to cathepsin D. Figure 7C and 7D ).

[0346] The effect of LBC4 and LBC5 peptide sequence minimization

[0347] We then explored the possibility of using smaller peptides. We synthesized 16 peptides ranging in length from 7 to 15 amino acids, derived from LBC4 and LBC5 peptides, and containing the DRCQY (SEQ ID NO:8) sequence (which we previously determined using computer analysis, as detailed above).

[0348] The affinity constants of these peptides were determined by microscale thermophoresis.

[0349] The results are shown in Table 6.

[0350] Table 6The affinity of LBC16 to LBC31 peptides for cathepsin D was investigated by microscale thermophoresis. The Kd values ​​for each peptide obtained by microscale thermophoresis are listed in the table. In summary, cathepsin D (Acrobiosystems) was labeled using the His-Tag labeling kit Red-Tris-NTA (NanoTemper). The labeled cathepsin D was then mixed with LBC peptides (16 to 31) using a 0.5-fold dilution series, ranging from 10 μM to 0.15 nM. These 16 mixtures were then analyzed at 25°C using a Monolith NT.115 (NanoTemper) instrument. Instrument parameters included 20% Pico-RED excitation power, medium MST power, and 5 / 20 / 5 laser off / on / off. Data were analyzed using NT MO Affinity Analysis v2.1.3 (NanoTemper).

[0351]

[0352] The results showed that all peptides derived from LBC4 (SEQ ID NO:13) and LBC5 (SEQ ID NO:14) were water-soluble. Except for peptides LBC27 (SEQ ID NO:36) and LBC30 (SEQ ID NO:39), all peptides exhibited good affinity for cathepsin D. These results indicate that the blocking peptides can be reduced to a maximum of 7 amino acids (e.g., LBC16 (SEQ ID NO:25)).

[0353] Direct effects of LBC peptide on breast cancer cell proliferation

[0354] LBC peptides target cathepsin D secreted by cancer cells and its communication with fibroblasts. We determined whether these peptides have effects on the cancer cells themselves, specifically whether they can inhibit the proliferation of MCF-7 and MDA-MB-231 breast cancer cells.

[0355] MDA-MB-231 and MCF-7 cells were seeded at 5000 and 10000 cells / well in 96-well plates, respectively, and incubated for 48 hours with no (NT) or with different concentrations (6.25 to 50 µM) of LBC4 (SEQ ID NO:13), LBC5 (SEQ ID NO:14), or LBC15 (SEQ ID NO:15) peptides. Cell count was determined using the WST-1 cell proliferation assay.

[0356] The results are as follows Figure 8 As shown.

[0357] All tested LBC peptides had no significant effect on the proliferation of these tumor cells, even the LBC4 peptide (SEQ ID NO:13) and LBC5 peptide (SEQ ID NO:14), which showed the strongest effect on fibroblasts. Figure 5D These results appear to confirm the mechanism of action of the LBC peptide in targeting stromal cells in the tumor microenvironment. Furthermore, these results indicate that our peptide does not exhibit cytotoxicity at concentrations up to 50 µM, which is a noteworthy finding from a therapeutic application perspective.

[0358] Effect of LBC peptide on cathepsin D catalytic activity

[0359] Besides binding to LRP-1 and inducing cellular effects, cathepsin D is also an aspartic protease capable of cleaving a variety of substrates. Like most lysosomal aspartic proteases, cathepsin D exhibits maximum catalytic activity at acidic pH values ​​between 2.4 and 5. At higher pH values ​​(5.0), the activity of cathepsin D decreases, and at pH 7.0, its activity is undetectable.

[0360] In the extracellular compartment (where secreted cathepsinogen D is found), particularly in the tumor microenvironment, a pH decrease of approximately 5.6 to 6.8 was observed, a hallmark of malignant tumor cells, resulting from glycolysis, hypoxia, and insufficient blood perfusion within the tumor cells.

[0361] Therefore, we tested the ability of our peptide to inhibit cathepsin D activity at pH 3.5 and pH 6.

[0362] Experiment under pH 3.5 conditions Cathepsinogen D (2 ng / µL) was pre-incubated with LBC peptide (100 µM) or pepsin A (2 ng / µL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) for 15 min. Activity was then measured in the presence of a fluorescent substrate (15 µM).

[0363] Experiment under pH 6 conditions Cathepsinogen D (0.5 ng / µL) was pre-activated for 30 min in acidic buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5), then pre-incubated with a peptide (100 µM) or pepsin A (2 µg / mL) in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6) for 15 min. Activity was then measured in the presence of a fluorescent substrate (30 µM).

[0364] The results are shown in Figure 9. The results at pH 3.5 are as follows... Figure 9A As shown, the results at pH 6 are as follows: Figure 9B As shown.

[0365] Results at pH 3.5 : The inhibitor of cathepsin D, pepsin A, completely blocked cathepsin D activity. In contrast, 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) (100 µM) did not significantly block the enzyme's activity. Figure 9A ).

[0366] Results at pH 6 : At pH 6, peptide LBC4 (100 µM) significantly blocked cathepsin D activity (approximately 67%). Figure 9B The 8th cysteine ​​residue in the LBC4 peptide sequence is mutated to a serine residue (C8S) (peptide LBC4C8S PGFLGDR). S QYRQCSG (SEQ ID NO:41) disrupts the ability of the peptide to block the catalytic activity of cathepsin D, thus demonstrating the specificity of this interaction.

[0367] Interestingly, the LBC5 (SEQ ID NO:14) peptide, which blocks the interaction between cathepsin D and LRP-1, had no effect on the catalytic activity of cathepsin D. The same effect was also observed in the LBC2 (SEQ ID NO:11), LBC6 (SEQ ID NO:15), LBC9 (SEQ ID NO:18), and LBC15 (SEQ ID NO:24) peptides.

[0368] Peptides LBC7 (SEQ ID NO:16) and LBC8 (SEQ ID NO:17) have good affinity for cathepsin D, but have only a weak effect on the catalytic activity of the protein.

[0369] Finally, peptides LBC16 (SEQ ID NO:25) and LBC21 (SEQ ID NO:30), which are peptides derived from peptide LBC4 with lengths of only 7 and 10 amino acids respectively, still blocked catalytic activity, but only by 30%.

[0370] All these results indicate that LBC peptides capable of antagonizing cathepsin D / LRP-1 interaction do not exhibit similar effects in terms of catalytic activity against the enzyme. These results allow us to consider the future use of LBC peptides that may or may not possess both activities. These differential properties of LBC peptides enable i) the differentiation of LBC peptides from other existing technologies (e.g., anti-cathepsin D monoclonal antibodies); and ii) the ability to exert one or more of these properties based on the different effects of the peptides on tumorigenesis observed in vivo.

[0371] Dose-dependent effects of LBC4 peptide: Cathepsinogen D (0.5 ng / µL) was pre-activated for 30 min in acidic buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5), and then pre-incubated for 15 min in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 6) with different concentrations (10, 50, 100, 250, and 500 µM) of LBC4 peptide or with pepsin A (2 µg / mL). Activity was then measured in the presence of a fluorescent substrate (30 µM).

[0372] The results are as follows Figure 9C As shown, LBC4 inhibits cathepsin D catalytic activity in a dose-dependent manner at up to 250 µM.

[0373] Example 2: Effects of LBC peptide on a tumor-like model derived from triple-negative breast cancer (TNBC) patients

[0374] 2.1. Materials and Methods

[0375] Figure 10A An overview of the experimental procedure is provided.

[0376] This study was conducted on the BR20001B PDX model (TNBC model) from Crown Bioscience, Leiden (Biopartner Center Leiden, JHOortweg 21n 2333 CH Leiden, Netherlands). The term "PDX" as used in this article stands for "patient-derived xenograft." Patient-derived xenograft is a model used in cancer research where tumor tissue from patients is collected and cultured for further study. 450 PDX units were uniformly seeded into hydrogels (based on Matrigel® and collagen, Crown Bioscience, Leiden) in 384-well plates. After 3 days, the organoids were treated with three doses of LBC4, LBC5, and LBC8 peptides (10, 30, and 100 µM) or a mediator.

[0377] Seven days later, the organoids were reprocessed. Ten days later, the supernatant was collected, the culture was fixed and stained for high-content imaging analysis (HCI), and measurements were taken. - Number of organoids - Total number of cell nuclei in organoids - Volume of organoids - The main branch of organoids TIBCO Spotfire is used for data analysis and visualization.

[0378] 2.2. Results

[0379] The results for LBC4, LBC5, and LBC8 are as follows: Figure 10B , Figure 10C and Figure 10D As shown.

[0380] The results showed that LBC4, LBC5, and LBC8 peptides exhibited dose-dependent antitumor activity in a TNBC-like tumor model. More specifically, the results indicated that these peptides could lead to: - The number of cell nuclei is reduced in organoids. - The overall volume of organoids has decreased. - The total number of branches in organoids has decreased.

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Claims

1. A peptide having a size of 5 to 20 amino acids, preferably 7 to 15 amino acids, wherein the peptide is (i) a fragment of the amino acid sequence NQGNQPQCRCLPGFLGDRCQYRQCSGYCEN (SEQ ID NO:1) comprising at least 5 consecutive amino acids of SEQ ID NO:1, or (ii) a functional variant of the fragment, preferably derived from the fragment by deletion, insertion and / or substitution of one or more amino acids, wherein the fragment or the functional variant derived therefrom comprises at least one of amino acids 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 to cathepsin D protein, (b) inhibiting the interaction between cathepsin D protein and LRP-1 protein, (c) inhibiting fibroblast proliferation promoted by cathepsinogen D secreted by cancer cells in the tumor microenvironment, and (d) inhibiting the catalytic activity of cathepsin D.

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

1.

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

1.

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

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

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

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

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

11. The peptide according to any one of claims 1 to 10, wherein the peptide has a binding affinity K for cathepsin D. D Values ​​less than 500 nM, such as those measured in microscale thermophoresis.

12. A modified peptide, which is derived from the peptide of any one of claims 1 to 11 by introducing one or more chemical modifications, wherein the chemical modifications preferably protect the peptide from proteolysis.

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

14. A vector comprising the polynucleotide as described in the preceding claims.

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 carrier according to claim 14, used as a drug.

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 carrier according to claim 14, for the treatment of proliferative diseases, particularly cancer.

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

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