PEPTIDES DERIVED FROM SORTILIN

Abstract

An isolated polypeptide derived from human sortilin, including an amino acid sequence which has at least 80% identity with the sequence SEQ ID NO 2 Val Leu Ile Val Lys Lys Tyr Val Cys Gly Gly Arg Phe Leu Val Mis Arg Tyr Ser Val Leu Gin Gin Mis Ala Glu Ala Asn Gly Val Asp Gly Val Asp Ala Leu Asp Thr Ala Ser Mis Thr Asn Lys Ser Gly Tyr Mis Asp Asp Ser Asp Glu Asp Leu Leu Glu on condition that the polypeptide does not contain the sequence SEQ ID NO 3 or a sequence having at least 80% identity with the aforementioned sequence SEQ ID NO 3, for use as a drug.

Claims

1. An isolated polypeptide derived from human sortilin comprising: an amino acid sequence having at least 80% identity with sequence SEQ ID NO 2 Val Leu Ile Val Lys Lys Tyr Val Cys Gly Gly Arg Phe Leu Val His Arg Tyr Ser Val Leu Gln Gln His Ala Glu Ala Asn Gly Val Asp Gly Val Asp Ala Leu Asp Thr Ala Ser His Thr Asn Lys Ser Gly Tyr His Asp Asp Ser Asp Glu Asp Leu Leu Glu provided that said polypeptide does not contain sequence SEQ ID NO 3 or a sequence having at least 80% identity with said sequence SEQ ID NO 3 or one of its pharmaceutically acceptable salts for use thereof as a medicament.

2. The polypeptide for use thereof as a medicament according to claim 1, characterized in that said peptide is covalently bound to a cell-penetrating peptide.

3. The polypeptide for use thereof as a medicament according to claim 2, characterized in that said cell-penetrating peptide is selected from the group comprising HIV-1 Tat, penetratin, peptide originating from the homeodomain of the Antennapedia protein, p28 originating from azurin, VP22 from the herpes simplex virus (HSV-1), the synthetic MAP peptide of sequence SEQ ID NO 11 or DPV1047 of sequence SEQ ID NO 12.

4. The polypeptide for use thereof as a medicament according to claim 1 in the treatment of a pathology associated with sortilin deregulation, selected from the group comprising cancers, in particular non-small cell lung cancers, neurological disorders, in particular Parkinson's disease, Alzheimer's disease, coronary diseases and atherosclerosis.

5. The polypeptide for use thereof as a medicament according to claim 1, characterized in that it is presented in a pharmaceutical form suitable for local, regional, systemic or continuous administration.

6. The polypeptide for use thereof as a medicament according to claim 1 in association with at least one second molecule, selected from antineoplastic chemotherapeutic molecules, tyrosine kinase receptor inhibitors, signalling pathway inhibitors used in targeted treatments, antibodies specific for oncogene receptors, T-lymphocyte checkpoint inhibitors, hormones, toxins and radiotherapy agents.

7. An isolated polypeptide derived from human sortilin, comprising; an amino acid sequence having at least 80% identity with sequence SEQ ID NO 2 Val Leu Ile Val Lys Lys Tyr Val Cys Gly Gly Arg Phe Leu Val His Arg Tyr Ser Val Leu Gln Gln His Ala Glu Ala Asn Gly Val Asp Gly Val Asp Ala Leu Asp Thr Ala Ser His Thr Asn Lys Ser Gly Tyr His Asp Asp Ser Asp Glu Asp Leu Leu Glu provided that said polypeptide does not contain sequence SEQ ID NO 3 or a sequence having at least 80% identity with said sequence SEQ ID NO 3; and said peptide is covalently bound to a cell-penetrating peptide.

8. The polypeptide according to claim 7, characterized in that said cell-penetrating peptide is selected from the group comprising HIV-1 Tat, penetratin, peptide originating from the homeodomain of the Antennapedia protein, p28 originating from azurin, VP22 from the herpes simplex virus (HSV-1) or the synthetic peptide MAP of sequence SEQ ID NO 11 or DV.

9. An isolated nucleotide sequence selected from the following polynucleotides: a. a polynucleotide coding for the polypeptide according to claim 7 of sequence SEQ ID NO 4 and consisting of sequence SEQ ID NO 6, b. a polynucleotide that has at least 80%, at least 85%, at least 90%, at least 95% homology with the polynucleotide of sequence SEQ ID NO 6 and c. a polynucleotide that is degenerate with respect to polynucleotides a and b.

10. An expression vector comprising a polynucleotide according to claim 9.

11. A host cell comprising a polynucleotide coding for a polypeptide according to claim 9.

12. A pharmaceutical composition comprising a polypeptide according to claim 7 or a pharmaceutically acceptable salt thereof, in combination with at least one pharmaceutically acceptable excipient.

13. The composition according to claim 12, also comprising another active agent selected from chemotherapeutic molecules, tyrosine kinase receptor inhibitors, signalling pathway inhibitors used in targeted treatments, antibodies specific for oncogene receptors, T-lymphocyte checkpoint inhibitors, hormones, toxins and radiotherapy agents.

14. An isolated polypeptide derived from human sortilin, comprising; an amino acid sequence having at least 80% identity with sequence SEQ ID NO 2 Val Leu Ile Val Lys Lys Tyr Val Cys Gly Gly Arg Phe Leu Val His Arg Tyr Ser Val Leu Gln Gln His Ala Glu Ala Asn Gly Val Asp Gly Val Asp Ala Leu Asp Thr Ala Ser His Thr Asn Lys Ser Gly Tyr His Asp Asp Ser Asp Glu Asp Leu Leu Glu provided that said polypeptide does not contain sequence SEQ ID NO 3 or a sequence having at least 80% identity with said sequence SEQ ID NO 3 and pharmaceutically acceptable salts thereof, said peptide being covalently bound or not to a cell-penetrating peptide for use thereof as a marker of pathologies associated with sortilin deregulation, in particular cancers, in particular non-small cell lung cancers, neurological disorders, in particular Parkinson's disease and Alzheimer's disease, coronary diseases and atherosclerosis.

15. A pharmaceutical composition comprising a polynucleotide according to claim 9 or a pharmaceutically acceptable salt thereof, in combination with at least one pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The following FIGS. 1 to 4 and Examples 1 to 3 illustrate the invention:

[0063] FIG. 1.A represents the detection by western blot of the synthetic peptide of sequence SEQ ID NO 7 according to the invention in the intracellular medium according to Example 1. “SICD peptide” corresponds to the increasing concentrations of peptide of SEQ ID NO 7 (SICD peptide coupled with V5 tag and Tat) used according to Example 1. “IB: anti-V5” quantity of intracellular peptide measured with anti-V5 antibody; “IB: anti-SORT-ICD” quantity of intracellular peptide measured with the antibody directed against the intracellular portion of sortilin; “IB: anti-actin” internal control.

[0064] FIG. 1.B represents the visualization of this synthetic peptide at the nucleus level via immunofluorescence according to Example 1. DAPI labels the cell nuclei, while the V5 tag makes it possible to visualize the intracellular location of the synthesized peptide. In fact, when two-dimensional images are taken and the two labels superimposed, the distribution of the V5, and thus that of the synthesized peptide, is superimposed on that of the nucleus. This distribution is punctiform, in the form of spots located at the nucleus level. Three-dimensional imaging makes it possible to assess the distribution of these spots in the depth of the nuclei, along the z axis, and not only on their surface as 2D imaging allows. Thus, as shown in FIG. 1.B, the synthetic peptide forms spots which are distributed within the cell nuclei.

[0065] FIG. 2 represents the cell survival assessment, measured by XTT (Cell Proliferation Kit II-Roche), in the presence of the SICD peptide coupled with the V5 tag and the Tat and corresponding to sequence SEQ ID NO 7 at different concentrations after 24, 48 and 72 hours' incubation according to Example 2.

[0066] FIG. 3 represents the real-time quantification of the fixing of annexin V induced by the pro-apoptotic effect of the SICD peptide coupled with the V5 tag and the Tat corresponding to sequence SEQ ID NO 7 according to Example 3.

[0067] FIG. 4 represents the real-time quantification of the activation of the caspase 3 and 7 induced by the pro-apoptotic effect of the SICD peptide coupled with the V5 tag and the Tat corresponding to sequence SEQ ID NO 7 according to Example 3.

DETAILED DESCRIPTION

[0068] 1. Cells and Cell Culture

[0069] The embryonic kidney line HEK293T (ATCC® CCL-1573™) and the bronchial adenocarcinoma cell lines A549 (ATCC® .sup.CCL185™) and H1975 (ATCC® CCL-5908™) originate from the American Type Culture Collection (ATCC). The line H3255 was kindly donated by Madame Sylvie Gazzeri (DR2 INSERM, U823, Institut Albert Bonniot, Grenoble). The cells are cultured in a DMEM (Dulbecco's Modified Eagle Medium) culture medium+Glutamax (ref: 10566016, Gibco, ThermoFisher Scientific) made up with 10% fetal calf serum (ref: 9215-50, H2B), 1% pyruvate (ref: 11360070, Gibco, ThermoFisher Scientific) and 1% penicillin/streptomycin (ref: 10378016, Gibco, ThermoFisher Scientific). The DMEM+Glutamax culture medium thus made up will be known as “complete medium” in the present application. The cells are kept in a humid atmosphere in an incubator at 37° C. containing 5% CO2 (Binder, ThermoFisher Scientific). For subculturing and weekly maintenance of the cells, the culture medium containing the dead cells and debris is removed, the cell layer is rinsed with PBS 1×(phosphate buffer saline) (ref: 14190144, Gibco, ThermoFisher) then the cells are detached from their support by addition of trypsin-EDTA (0.05%) (ref: 25300054, Gibco, ThermoFisher) for 5 minutes in a humid atmosphere in an incubator at 37° C. containing 5% CO2. Once detached, the cells are collected in a 15-or 50-mL tube (respectively ref: 62554502; 62547254, Sarstedt) and counted under the microscope (×40) on a counting chamber of the “Malassez” type (ref: 631-0975; VWR). In parallel, the cell viability is estimated on the counting chamber after colouring a sample of the cell suspension quarter-diluted in a solution of trypan blue 0.4% (ref: 15250061, Gibco, ThermoFisher). The cells are then centrifuged at 100 x-g at 20° C. (ref: 75004380, Sorvall, ThermoFisher) for 10 minutes. The cell pellet thus obtained is either returned to suspension so that the cells are at the appropriate density for the various experiments planned, or stored dry at −80° C. for subsequent use. A concentration of 10.10.sup.3 cells/cm.sup.2 is usually used for the seeding of each cell subtype used in this invention. The cells are seeded in culture flasks of 25 or 75 cm.sup.2 (respectively ref: 83.3910.002 and 83.39.11.502, Sarstedt), or even in culture plates with 6 or 24 wells (respectively ref: 83.3920 and 83.3922, Sarstedt) depending on the experiments to be carried out.

[0070] 2. Long-Term Cell Storage: Freezing/Defrosting

[0071] The cultured cells are detached, collected and centrifuged as previously described. They are then taken up in suspension in 1 mL fetal calf serum (FCS) made up with 10% of a cryoprotector, dimethyl sulfoxide (DMSO) (ref: D-8418, Sigma-Aldrich) at a rate of 3×10.sup.6 cells per cryotube (ref: 72380002, Sarstedt). These are placed in a freezing container (ref: 479-0966, VWR) containing isopropanol (ref: 563935, Sigma-Aldrich) for 24 hours to 1 week for progressive freezing, before being stored in a container of liquid nitrogen at −196° C. The defrosting stage must be rapid in order to obtain optimal cell viability. Then, immediately it is removed from the liquid nitrogen, the cryotube is placed in a water bath at 37° C. until complete defrosting of the cell suspension. The cells are taken up in 10 mL of complete medium and centrifuged (300 x-g, 10 minutes at 25° C.) in order to eliminate the DMSO. They are then returned to culture according to the conditions previously described.

[0072] 3. Extraction of the proteins under denaturing conditions Extraction of the whole proteins is carried out directly on the cell layers, after eliminating the supernatants and rinsing the cells in PBS 1×, or even on cell pellets previously stored at −80° C. It is carried out by incubating the cells with the Laemmli lysis buffer (62.5 mM Tris-HCl ref: 11814273001 ROCHE, pH 6.8, 2% SDS ref: L-4509, Sigma-Aldrich, 25% glycerol ref: G5516, Sigma-Aldrich) made up with 1% (v/v) protease inhibitor cocktail (ref: P8340-1ML, Sigma-Aldrich) and 1% (v/v) phosphatase inhibitor cocktail (ref: P0044-1ML, Sigma-Aldrich), in the proportions 100 pL of buffer for 1.10.sup.6 cells for 30 min over ice. In the case of extraction on the cell layer, a scraper (ref: 179693PK, Nunc, ThermoFisher) is used to collect the cells in the lysis buffer. Lysis is then completed by ultrasound parametered at 60 Hz, amplitude 2 s, for 1 minute (ref: SONIVCX-130-220, VWR). The lysates are then centrifuged at 17,000×g (ref: 75002442, Sorvall, ThermoFisher) for 20 min at 4° C., the supernatant containing the proteins is transferred to a sterile 1.5 mL tube (ref: 0030120086, Eppendorf), and the proteins are assayed by the Bradford method.

[0073] 4. Protein Assay

[0074] The protein concentration of the various samples is assessed by the Bradford method (Bradford 1976). The test is conducted by incubating the diluted samples (or the different concentrations of the bovine albumin standard (ref: 5000206, Bio-Rad)) with the reagents from the kit DC™ Protein Assay Kit II (ref: 5000112, Bio-Rad) following the manufacturer's protocol. The absorbance reading is carried out at 595 nm on a spectrophotometer (ref: 51119000, ThermoFisher). The protein concentration of each sample is estimated with respect to the straight line obtained with the BSA concentration range (0; 125; 250; 500; 750; 1000; 1500 and 2000 μg/mL). Once the concentrations of the samples have been determined by this method, the necessary volume of proteins is sampled, then equilibrated as a function of each sample, generally 20 pL. These latter are made up respectively with 0.01% of bromophenol blue and 5% of 3-mercaptoethanol before being denatured at 95° C. for 5 minutes.

[0075] 5. Electrophoresis of the proteins in polyacrylamide gel (SDS-PAGE) The proteins are separated over an SDS-PAGE electrophoresis gel (sodium dodecyl sulfate polyacrylamide gel, mini-PROTEAN system from Bio-Rad). The concentration of the polyacrylamide gels varies from 8 to 15% as a function of the molecular weight of the proteins to be separated and analyzed. All the separation gels are invariably preceded by a 4% concentration gel according to Table 1 below.

TABLE-US-00001 TABLE 1 Composition of the separation and concentration gels. Separation gel (in mL) Concentration For 10 mL 8% 12% 15% gel (in mL) Distilled water 5.3 4.3 3 7.25 40% Acrylamide 2 3 3.75 1.25 1.5M Tris (pH: 8.8) 2.5 2.5 2.5 1.25 1M Tris (pH: 6.8) 10% SDS 0.1 0.1 0.1 0.05 10% APS 0.1 0.1 0.1 0.1 TEMED 0.006 0.006 0.006 0.01

[0076] The migration is carried out for 1 hour 30 mins (150 V) in the 1× migration buffer (ref: 1610732, Bio-Rad). A molecular weight marker is used at each migration (ref: 26619 PageRuler™ Plus Prestained Protein Ladder, Fermantas Life Science). Once separated by electrophoresis, the proteins are transferred on a PVDF membrane (polyvinylidene difluoride) (ref: 10600023) Millipore). The membrane is incubated beforehand for 15 seconds in methanol before being rinsed in water then equilibrated for a few minutes in the transfer buffer (ref: 161-0771, Bio-Rad). The polyacrylamide gel is also equilibrated for a few minutes in the transfer buffer before being placed in contact with the membrane. The transfer is carried out for 1 hour at 20 V with a Transblotting ID Dryer appliance (Bio-Rad), according to a setup using Whatman filter papers. The PVDF membrane and the gel are comprised between 2×4 Whatman papers previously soaked with transfer buffer (FIG. 1). The deposit, migration and transfer qualities are verified by a colouration of the PVDF membrane with Ponceau red (0.2% Ponceau S ref: P3504-50 G, Sigma-Aldrich; 3% TCA ref: T8657-250G, Sigma-Aldrich) for a few seconds before being washed 3 times for 5 min with tert-butyl dimethylsilyl (TBS).

[0077] 6. Incubation of the Membranes with the Antibody

[0078] The membranes are then incubated for 1 hour at ambient temperature in TBS 1×containing 0.1% Tween 20 (ref: P1379) and 5% skimmed milk (Regilait), to saturate the nonspecific fixation sites. Immunolabelling is carried out by incubation with the primary antibody, directed against the specific epitopes of the proteins of interest and according to the conditions described in Table 2.

TABLE-US-00002 TABLE 2 Primary antibodies used in western blot Incubation Dilution Incubation Time Solution PM Antibodies Clones Species Companies WB.sup.1 IF.sup.2 WB IF WB IF Ref. kDa β-actin — rabbit Ozyme 1/1000 — 2-12 h  — a-b — 4970 45 Sortilin rabbit Abcam 1/1000 1/200 12 h 12 h b b Ab16640 95 Anti-V5 2F11F7 mouse Invitrogen, 1/5000 1/200 12 h 12 h b b 37- 5 ThermoFisher 7500

[0079] -: unusable, NA: not reported, (a): TBS 1× Tween 0.1%, Milk 5%, (b): TBS 1×-Tween 0.1%, BSA 3%, (c): TBS 1×, BSA 5%, (d): TBS 1×, BSA 3%, WB: Western Blot, IF: immunofluorescence.

[0080] After washing three times in TBS 1×-Tween 20 buffer (0.1%), the membranes are incubated for 1 hour at ambient temperature with the suitable secondary antibody coupled with the peroxidase (Table 3) (dilution at 1/1000 in the saturation solution). The membranes are then washed twice with TBS 1×-Tween 20 (0.1%) then twice with TBS 1× alone to remove the excess Tween 20.

TABLE-US-00003 TABLE 3 Secondary antibodies used in western blot HRP: Horseradish Peroxidase Secondary antibodies Host Supplier Dilution Anti-mouse IgG HRP Rabbit Dako 1/1000 Anti-rabbit IgG HRP Pig Dako 1/1000

[0081] 7. Visualization by Chemiluminescence Reaction

[0082] Western Blot visualization is carried out by chemiluminescence. This system is based on oxidation of luminol by 02, produced by peroxidase action on H.sub.2O.sub.2. An unstable intermediate light-emitting compound is formed. The membrane is contacted for 1 minute with the mixture of the “ECL” kit (ref: WBULS0500, Millipore) in the proportion 1:1. The chemiluminescence is collected by the G box digital system (Ozyme). The digital images obtained from the western blot visualization are processed using Genesnap (Syngene) and ImageJ (NIH) image analysis software.

[0083] 8. Indirect Immunofluorescence

[0084] The cells are seeded on a 24-well plate on 14 mm diameter glass slides. After each treatment condition, the cells are fixed, either with the complete medium containing paraformaldehyde (PFA) at 3.7% for 20 min at 4° C., or with methanol at −20° C. for 5 min. With methanol the fixation is carried out by dehydration (denaturing of the proteins), while with the aldehydes (formaldehyde) the 3D structure of the protein is preserved (fixing by bridging). The phosphorylations are analyzed after fixing with PFA. After fixing, the cell layer is washed 3 times for 5 minutes with 500 pL PBS. In the case of fixing with PFA, the cells are permeabilized with Triton X-100 at 0.1% in PBS for 10 min at 4° C., then washed again. With regard to fixing with methanol, this confers the advantage of fixing and permeabilizing the cells at the same time. Saturation of the nonspecific sites is carried out with 1% PBS-serum (of the same origin as the animal from which the secondary antibody originates) or with 5% PBS-BSA for 1 hour at ambient temperature. Starting from the fixing stage, all the solutions are filtered beforehand over a 0.2 μm filter in order to eliminate the fluorescence artefacts and the background noise. The primary antibody directed against the protein of interest, (i.e., the V5 tag (allowing visualization of the synthetic peptide)) is incubated overnight at 4° C. in a PBS solution containing 3% BSA (Table 2). Then, three washes of 5 min in PBS are carried out, before incubation of the secondary antibody coupled with an Alexa fluor 488® (green fluorescence) (Table 4) for 1 hour at ambient temperature in the PBS-BSA solution. After three washes of 5 min in PBS, the nuclei are labelled with a PBS 1× solution containing 1 μM fluorochrome DAPI for 5 min. At the end of this stage, the cells are rinsed again with three washes of 5 min in PBS. The slides are then placed on object slides using an aqueous mounting medium (Dako) and placed overnight at 4° C. The preparations are visualized with a confocal microscope of the LSM 880 type (Zeiss) equipped with a helium/neon and argon laser (×63 or ×100 magnification). The images are processed with Zen (Zeiss) or Image J (NIH) software.

TABLE-US-00004 TABLE 4 Secondary antibodies used in immunofluorescence Secondary antibodies Host Supplier Dilution Anti-mouse IgG Alexa Fluor ® Goat Invitrogen 1/1000 488

[0085] 9. Cell Death Analysis with Real-Time Imaging Via the IncuCyteZOOM® System

[0086] In order to study cell death by apoptosis, the cells are seeded at a concentration of 2000 cells per well in 100 μL of culture medium, in 96-well plates. 24 hours later, the cell medium is removed, then the cells are rinsed once with sterile PBS 1× before adding the culture medium containing the different treatments, including the peptide according to the invention at 600 nM, and the reagents necessary for analysis of cell death. These latter are: IncuCyte® Caspase-3/7 Green Reagent (ref: 4440; EssenBio) diluted to 1:1000 in the culture medium; IncuCyte® Annexin V Green Reagent (ref: 4642; EssenBio) diluted to 1:200 diluted in the culture medium. Image acquisition is then carried out by positioning the plates in the IncuCyte Zoom® system (Ref: EssenBio) for 96 hours. Four images per well are then obtained, in high definition, every two hours, with the ×10 objective and the phase-contrast and green channels. These acquisitions are carried out automatically and non-invasively. The data are processed automatically with the IncuCyte Zoom® Live-Cell Analysis System (ref: EssenBio).

Example 1: Cell Penetration Test Via Western Blot and Immunofluorescence

[0087] This is conducted according to the technique described above. The synthetic peptide of sequence SEQ ID NO 2 (SICD or sortilin intracellular domain) is rendered competent to penetrate the cells by addition of a Tat sequence (trans-activator of transcription), SEQ ID NO 8 belonging to the family of cell-penetrating peptides of the human immunodeficiency virus (HIV). Furthermore, a V5 tag (SEQ ID NO 10), epitope known for easy detection via a specific antibody or a fluorescence molecule, is grafted on the peptide, then leading to the synthetic peptide of sequence SEQ ID NO 7. The cells are treated with a concentration range of the SICD peptide coupled with the V5 tag and the Tat of sequence SEQ ID NO 7, ranging from 0 to 500 nM diluted in complete culture medium. After 24 hours' treatment, the cells are lysed and the peptide is detected in the cell lysate (intracellular medium) with an anti-V5 antibody. Following the different stages of western blot, a progressive increase in the quantity of intracellular peptide is detected with a maximum at the highest concentration (500 nM) thus suggesting a dose-dependent peptide penetration, as illustrated in FIG. 1.A line IB: anti-V5. Actin acts as internal control of deposit, demonstrating that an identical quantity of protein has been deposited in each protein track presented (FIG. 1.A line IB: anti-actin). A similar profile is obtained by using an antibody directed against the intracellular portion of sortilin (FIG. 1.A line IB: anti-SORT ICD). These results confirm on the one hand that the peptide is in fact internalized by the cells, and on the other hand that this is indeed a peptide derived from the intracellular portion of sortilin.

[0088] The subcellular distribution of the synthesized peptide was visualized by immunofluorescence (FIG. 1.B). The images show a punctiform nuclear distribution of the synthetic peptide of sequence SEQ ID NO 7 by using an antibody directed against the V5 tag. This distribution is also confirmed by using the peptide of sequence SEQ ID NO 7 and an anti-sortilin antibody. The superposition of the labels (DAPI labelling the cell nuclei and V5 labelling the synthesized peptide) confirms the specific presence of the peptide in the cell nuclei. The images taken in 3D confirm this location within the nuclei, and not on the surface thereof. The presence of the synthetic peptide in this cellular compartment is evidence of the action thereof at the level of the nucleus.

Example 2: Measurement of the Antineoplastic Effect by Measuring Cell Survival

[0089] The antineoplastic effect of the synthetic peptide, comprising Tat and a V5 tag of sequence SEQ ID NO 7 was tested on the A549 cell line (adenocarcinoma line having no mutation on the EDFR). A concentration range (0 to 10 μM) of the synthetic peptide of sequence SEQ ID NO 7 according to the invention was incubated for 24, 48 and 72 hours in the cell medium and the dehydrogenase activity thereof (proxy for viability) was measured (Cell Proliferation Kit II (XTT)-Roche) at the end of each incubation, i.e. at 24, 48 and 72 hours. The results are given in FIG. 2. Analysis of the graph representing the activity as a function of the log of the synthetic peptide concentration made it possible to establish CI.sub.50 values corresponding to the concentrations for which the peptide results in 50% inhibition of cell activity. These latter are respectively equal to 1.42, 1.52 and 0.57 μM for incubation times of 24, 48 and 72 hours.

Example 3: Measurement of the Pro-Apoptotic Effect of the Synthetic Peptide

[0090] The pro-apoptotic effect of the peptide of sequence SEQ ID NO 7 at the concentration of 1 μM was measured in real time using the IncuCyte Zoom® system for 96 hours on the A549 line, on human fibroblasts (non-cancerous cells), as well as on two lines of adenocarcinomas H3255 and H1975, respectively presenting mutations of EGFR that are sensitive (L858R) and resistant (L858R/T790M) to the tyrosine kinase activity receptor inhibitors (TKI) used in the clinic via activation of two markers of apoptosis, namely annexin V and caspases 3 and 7. The results show that the peptide does not seem to lead to activation of these two markers of apoptosis in the human fibroblast culture (annexin V FIG. 3 and caspases 3 and 7 FIG. 4). In fact, the levels of expression of these markers are identical under the conditions treated with the peptide and the control conditions. The peptide could therefore have a specific toxicity to cancer cells.

[0091] Conversely, on the three adenocarcinoma lines tested, an increase is observed over time in the apoptotic process in the cells. In fact, the levels of expression of annexin V (FIG. 3) or of caspases 3 and 7 (FIG. 4) increase in the three adenocarcinoma lines tested, reflecting an action mechanism of the peptide with respect to the antineoplastic effect thereof.

[0092] Unlike the molecules available in conventional chemotherapy, the synthetic peptide according to the invention results from an amino acid assembly to form a peptide that is naturally present in human cells and not from chemical molecules. Therefore, improved tolerance by the organism is envisaged. Owing to the ease of synthesis thereof, at reasonable cost, and its specific intracellular target, different from those of the TKIs, the peptide according to the invention could be a good candidate for use in bitherapy. In this case, it would allow a reduction in the doses of conventional antineoplastics administered, and a reduction in the associated treatment costs.

[0093] These results show that after administration thereof, the peptide according to the invention is in fact present inside the nuclei (FIG. 1) and that it is capable of inhibiting the activity of tumour cells (FIG. 2) and has a pro-apoptotic effect (FIG. 3). Therefore, this peptide can be used in diseases associated with sortilin deregulation, in particular in the case where sortilin is suppressed