PEPTIDES AND USES THEREOF

Abstract

The present invention relates to CD271-interacting peptides and derivatives thereof and to their use as a medicament for the treatment of CD271-related diseases, in particular for the treatment of melanoma and other CD271-related skin diseases.

Claims

1. A pegylated peptide comprising the amino acid sequence from the N-terminus to the C-terminus: TABLE-US-00004 (SEQ ID NO: 1) R1-R2-Gly-R3-R4-R5-Gly-R6-R7-R8-Gly or of the amino acid sequence from the N-terminus to the C-terminus: TABLE-US-00005 (SEQ ID NO: 2) Gly-R8-R7-R6-Gly-R5-R4-R3-Gly-R2-R1 wherein: R1 is selected from the group consisting of: Met, Ala, Val, Ile, Leu, Phe, Tyr and Trp; R2 is selected from the group consisting of: Leu, Met, Ala, Val, Ile and Phe; R3 is selected from the group consisting of: Ile, Met, Ala, Val, Leu and Phe; R4 is selected from the group consisting of: Ile, Met, Ala, Val, Leu and Phe; R5 is selected from the group consisting of: Ala, Met, Val, Ile, Leu and Phe; R6 is selected from the group consisting of: Lys and Arg; R7 is selected from the group consisting of: Asn, Gln, His, Ser, Thr, Tyr and Cys; R8 is selected from the group consisting of: Ser, Asn, Gln, His, Thr, Tyr and Cys; the N-terminus is W—CO—NH—, where W is a C.sub.1-C.sub.12 alkyl group or a hydrogen atom, or H.sub.2N—; the C-terminus is —CO—N(Z.sub.1)(Z.sub.2), where Z.sub.1 is an hydrogen atom or C.sub.1-C.sub.6 alkyl group and Z.sub.2 is an hydrogen atom or C.sub.1-C.sub.6 alkyl group, or —COOH; and wherein R1 to R8 can be either in their D or L enantiomeric configuration; in which a linear or branched poly-(ethylene glycol) (PEG) polymer is conjugated to at least one of the amino acids of the peptide, and wherein the non-conjugated end of PEG polymer is free or capped.

2. The pegylated peptide according to claim 1 further comprising a D-Cys or L-Cys residue linked to the C-terminus and/or at the N-terminus thereof.

3. The pegylated peptide according to claim 1, wherein the linear or branched PEG polymer is conjugated to either or both the N- and the C-terminus of the peptide.

4. The pegylated peptide according to claim 1, wherein the linear or branched PEG polymer is conjugated to a Cys residue through the sulfur atom of the latter.

5. The pegylated peptide according to claim 4 being Ac-DMet-DLeu-Gly-DIle-DIle-DAla-Gly-DLys-DAsn-DSer-Gly-DCys-NH.sub.2 (SEQ ID NO:5) wherein the linear or branched PEG polymer conjugated to the D-Cys residue through the sulfur atom of the latter is a 20 kDa methoxy-PEG polymer.

6. The pegylated peptide according to claim 5 having the following structure (XYZ): ##STR00005##

7. A peptide according to claim 1, having the following amino acid sequence from the N-terminus to the C-terminus: DMet-DLeu-Gly-DIle-DIle-DAla-Gly-DLys-DAsn-DSer-Gly-DCys (SEQ ID NO:4) wherein the N-terminus of the sequence is W—CO—NH—, where W is CH.sub.3 and the C-terminus of the sequence is —CO—N(Z.sub.1)(Z.sub.2), where Z.sub.1 and Z.sub.2 are hydrogen atoms and functional fragments or derivatives thereof.

8. A bi- or multi-functional, linear or branched PEG polymer, conjugated to at least two peptides according to claim 1.

9. A liposome comprising at least one peptide according to claim 1 and at least one substance selected from the group consisting of a biodegradable and/or biocompatible lipid, cholesterol, a nanoparticle, a polymer or mixtures thereof.

10. A pharmaceutical composition comprising a peptide according to claim 1 and at least one pharmaceutically acceptable excipient and/or carrier, optionally in the form of an injectable pharmaceutical formulation.

11. A method for the treatment of CD271-related diseases, comprising administering a peptide of claim 1 to a patient in need thereof.

12. The method of claim 11, wherein the CD271-related diseases are selected from neuroblastoma, glioblastoma, astrocytoma, head and neck squamous cell carcinoma, psoriasis, Merkel-cell carcinoma, chronic skin ulcers, cutaneous squamous cell carcinoma and melanoma.

13. The pharmaceutical composition of claim 10 further comprising a chemotherapeutic agent and/or non-steroidal anti-inflammatory agent and/or immunotherapeutic agent and/or any treatment increasing the expression of CD271, wherein the chemotherapeutic agent is optionally selected from the group consisting of: dacarbazine, carmustine, cisplatin; the non-steroidal anti-inflammatory agent is optionally selected from the group consisting of: ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen and mixtures thereof; the immunotherapeutic agent is optionally selected from ipilimumab, nivolumab, pembrolizumab; and the treatments increasing the expression of CD271 are optionally selected from the BRAF/MET/KIT inhibitors including vemurafenib, trametinib, cobimetinib.

14. A method for the treatment of a CD271 related skin disease, comprising administering a peptide comprising the amino acid sequence from the N-terminus to the C-terminus: TABLE-US-00006 (SEQ ID NO: 1) R1-R2-Gly-R3-R4-R5-Gly-R6-R7-R8-Gly or the sequence from the N-terminus to the C-terminus: TABLE-US-00007 (SEQ ID NO: 2) Gly-R8-R7-R6-Gly-R5-R4-R3-Gly-R2-R1 wherein: R1 is selected from the group consisting of: Met, Ala, Val, Ile, Leu, Phe, Tyr and Trp R2 is selected from the group consisting of: Leu, Met, Ala, Val, Ile and Phe R3 is selected from the group consisting of: Ile, Met, Ala, Val, Leu and Phe R4 is selected from the group consisting of: Ile, Met, Ala, Val, Leu and Phe R5 is selected from the group consisting of: Ala, Met, Val, Ile, Leu and Phe R6 is selected from the group consisting of: Lys and Arg R7 is selected from the group consisting of: Asn, Gln, His, Ser, Thr, Tyr and Cys R8 is selected from the group consisting of: Ser, Asn, Gln, His, Thr, Tyr and Cys the N-terminus of the sequence or of the peptide is W—CO—NH—, where W is a C.sub.1-C.sub.12 alkyl group or a hydrogen atom, or H2N— the C-terminus of the sequence or of the peptide is —CO—N(Z.sub.1)(Z.sub.2), where Z.sub.1 is an hydrogen atom or C.sub.1-C.sub.6 alkyl group and Z.sub.2 is an hydrogen atom or C.sub.1-C.sub.6 alkyl group, or —COOH and wherein R1 to R8 can be either in their D or L enantiomeric configuration and functional fragments or derivatives thereof to a patient in need thereof.

15. The method according to claim 14 wherein the CD271 related skin disease is selected from melanoma, Merkel-cell carcinoma, psoriasis, chronic skin ulcers and cutaneous squamous cell carcinoma.

16. The method according to claim 15 wherein the CD271 related skin disease is melanoma.

17. The method according to claim 14, wherein the peptide comprises sequence: TABLE-US-00008 (SEQ ID NO: 3) DMet-DLeu-Gly-DIle-DIle-DAla-Gly-DLys-DAsn-DSer- Gly wherein the N-terminus of the sequence or of the peptide is W—CO—NH—, where W is CH.sub.3 and the C-terminus of the sequence or of the peptide is —CO—N(Z.sub.1)(Z.sub.2), where Z.sub.1 and Z.sub.2 are hydrogen atoms or functional fragments or derivatives thereof.

18. The method of claim 14, wherein a D-Cys or L-Cys residue is added at the C-terminus and/or at the N-terminus of the sequence.

19. The method according to claim 14, wherein a linear or branched poly-(ethylene glycol) (PEG) polymer is conjugated to at least one of the amino acids of the peptide, wherein the non-conjugated end of PEG polymer is optionally free or capped, preferably it is alkoxylated.

20. (canceled)

21. (canceled)

22. The method according to claim 14, further comprising administering at least one chemotherapeutic agent and/or non-steroidal anti-inflammatory agent and/or immunotherapeutic agent and/or immunotherapeutic agent and/or any treatment increasing the expression of CD271, wherein the chemotherapeutic agent is optionally selected from the group consisting of: dacarbazine, carmustine, cisplatin; the non-steroidal anti-inflammatory agent is optionally selected from the group consisting of: ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen and mixtures thereof; the immunotherapeutic agent is optionally selected from ipilimumab, nivolumab, pembrolizumab; and the treatments increasing the expression of CD271 is are optionally selected from the BRAF/MET/KIT inhibitors.

23. A combination comprising: a. a peptide of claim 1; and b. a chemotherapeutic agent and/or non-steroidal anti-inflammatory agent, and/or immunotherapeutic agent and/or any treatment increasing the expression of CD271, wherein the chemotherapeutic agent is optionally selected from the group consisting of: dacarbazine, carmustine, cisplatin; the non-steroidal anti-inflammatory agent is optionally selected from the group consisting of: ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen and mixtures thereof and/or mixtures of any treatments increasing the expression of CD271; the immunotherapeutic agent is optionally selected from ipilimumab, nivolumab, pembrolizumab; and the treatments increasing the expression of CD271 are optionally selected from the BRAF/MET/KIT inhibitors.

24. A pharmaceutical composition comprising the combination of claim 23 and at least one pharmaceutically acceptable excipient and/or carrier, optionally in the form of an injectable pharmaceutical formulation.

Description

[0137] The present invention is therefore illustrated by means of non-limiting examples in reference to the following figures.

[0138] FIG. 1. The peptide immunoprecipitate with CD271 in two metastatic melanoma cell lines. Protein extracts from two melanoma cell lines were immunoprecipitated with the Pegylated form of the peptide (XYZ) and blotted against CD271. The sepharose beads-protein lysates (without CD271) is used as negative control, while no-immunoprecipitated protein lysate from WM266-4 is used as positive control.

[0139] FIG. 2. The peptide or the NON-PEGylated form reduce melanoma cell proliferation. Five primary or metastatic melanoma cell lines were cultured and treated with either the PEGylated and non-PEGylated form of the peptide (Ac-DMet-DLeu-Gly-DIle-DIle-DAla-Gly-DLys-DAsn-DSer-GlyNH2) and evaluated by MTT assay at 48 and 96 hrs.

[0140] FIG. 3. The peptide reduces cell proliferation in primary and metastatic melanoma cell lines. Three primary or metastatic melanoma cell lines were cultured, treated with the peptide (XYZ) at different doses and evaluated by MTT assay at different time points

[0141] FIG. 4. The peptide-induced cell death correlates with CD271 expression levels in melanoma cell lines. Five primary and metastatic melanoma cell lines were cultured and viability measured by Trypan blue staining solution. Flow cytometry was used to evaluate the expression level of CD271 receptor.

[0142] FIG. 5. The peptide significantly increases apoptosis in melanoma cells. WM266-4 melanoma cell line was cultured and the rate of apoptosis measured after treatment with the peptide (XYZ) as compared to diluent at different time points by flow cytometry (A) counting % of cells in sub-G1 with propidium iodide staining or (B) counting % of Annexin positive cells staining. Student's t-test was used for comparison of the means

[0143] FIG. 6. The peptide reduces melanoma cell viability through CD271. WM115 melanoma cell line was transiently transfected with CD271 siRNA, as shwon by western blotting. MTT assay was used to measure cell viability after treatment with the peptide (XYZ) or the diluent in siRNA and scramble cells. SKme128 melanoma cell line was transiently transfected with CD271 mRNA, as shown by western blotting. MTT assay was used to measure cell viability after treatment with the peptide (XYZ) or the diluent in siRNA and scramble cells. Student's t-test was used for comparison of the means.

[0144] FIG. 7. Chemotherapy up-regulates CD271 in primary and metastatic melanoma cell lines. (A) A real-time PCR using primers for intra- and extracellular CD271 receptor was performed on RNA extracts from three melanoma cell lines after treatment with carmustine (BCNU), cisplatin, dacarbazine (DTIC) or diluent. (B) Protein extracts from the three melanoma cell lines treated with different doses of cisplatin, carmustine, dacarbazine or diluent were separated on polyacrylamide gel and transferred onto nitrocellulose membranes. (C) Melanoma cell lines were treated with the chemotherapy or the diluent (see above) stained with anti CD271 mAb and analysed by flow cytometry.

[0145] FIG. 8. The peptide augments dacarbazine (DTIC)-induced reduction of melanoma cell proliferation. Five melanoma cell lines were treated with DTIC, DTIC in combination with the peptide (XYZ) or with diluent alone. Proliferation was measured by MTT assay. Student's t-test was used for comparison of the means.

[0146] FIG. 9. The peptide augments dacarbazine-induced melanoma cell migration. Melanoma cell line SKMel28 plated onto 24-well Boyden/Matrigel chambers was stimulated with the peptide, the peptide in combination with DTIC or the diluent alone. Number of invading cells was measured at 48 hrs by the scratch-wound assay. Student's t-test was used for comparison of the means

[0147] FIG. 10. No toxic effects in 6 mice treated with XYZ. Acute toxicity testing was performed in CD1 mice with different doses of XYZ up to six days after treatment. After one day of fasting, six mice were injected intravenously (28 G needle) through the tail vein with a total dose of 17.5, 175, 550 or 1750 mg/kg/mouse of XYZ in a physiological solution. 50 gr of food were available two hours later. The health status (coat and appearance) of mice was observed from the first day after treatment. (a) Mice were weighed prior to XYZ injection and then every day until the sixth day. The amount of ingested food (b) and drinking water (c) were measured.

[0148] FIG. 11. The peptide reduces initial metastasis formation in zebrafish injected with melanoma cells. Metastatic melanoma cells from a patient with BRAF mutation were treated with the peptide, DTIC, the peptide in combination with DTIC, DTIC alone, BRAF inhibitor or left untreated. They were stained with Vibrant Cell Labelling Solution (red) and injected into the yolk of transparent zebrafish larvae and monitored. The number of embryos with metastasis was counted at 5 days post injection.

[0149] FIG. 12. The peptide, in combination with DTIC, blocks the formation of full metastasis. SKMel28 melanoma cell line was treated with XYZ, DTIC, the peptide in combination with DTIC, or left untreated. They were stained with Vibrant Cell Labelling Solution (red) and injected into the yolk of transparent zebrafish larvae and monitored. The number of embryos with metastasis was counted at 5 days post injection.

[0150] FIG. 13. Non-steroid anti-inflammatory drugs (NSAID) enhance CD271 levels in melanoma cell lines. Three melanoma cell lines were treated with increasing doses of ketoprofen (A) or ibuprofen (B) and the percentage of CD271 positive cells was measured by flow cytometry.

[0151] FIG. 14. NSAID reduce melanoma cell line proliferation. Three melanoma cell lines were treated with increasing concentrations of either ketoprofene (A) or ibuprofene (B). Proliferation was measured by MTT assay at 48 hrs.

[0152] FIG. 15 The peptide, in combination with NSAID, further reduces melanoma cell line proliferation Two melanoma cell lines were treated with the peptide alone, ibuprofene or ketoprofene alone or in combination with the peptide. Proliferation was measured by MTT assay at 48 hrs. Student's t-test was used for comparison of the means

[0153] FIG. 16. The combination of the peptide with NSAID decreases cell migration in melanoma cell lines. SKMel 28 melanoma cell line was treated with the peptide alone, ibuprofene or ketoprofene alone or in combination with the peptide. Number of invading cells was measured at 48 hrs by the scratch-wound assay. Student's t-test was used for comparison of the means. * 0.01<p<0.05; ** p<0.01

EXAMPLES

Materials and Methods

Peptide synthesis and PEGylation

[0154] The synthesis of the different peptides was performed at NeoMPS/PolyPeptide Laboratories (Strasbourg, France) using Solid-Phase Peptide Synthesis with standard tBoc chemistry and preparative Reversed-Phase Chromatography purification.

[0155] Peptide indicated as XYZ corresponds to the following structure:

##STR00004##

[0156] The PEGylation of the DCys-containing peptide to give XYZ was performed as per the following protocol.

[0157] Materials:

[0158] Ac-dM-12-dC-NH.sub.2 peptide, MW 1204Da (NeoMPS/PolyPeptide laboratories, p/n SP081269 lot n AW11015); mPEG20 kDa-maleimide (MW 21721Da: NOF Co., SUNBRIGHT ME-200MA lot M60519); Sodium acetate buffer 20 mM pH 5.0; Ammonium acetate 20 mM

[0159] Procedure:

[0160] In a 50 ml polypropylene Falcon tube with magnetic stirrer, 10.6 mg (8.3×10.sup.−3 mmol) of peptide are dissolved in 10 ml of acetate buffer at room temperature. Then, 162.4 mg (7.47×10.sup.−3 mmol, 0.9 eq) of PEG are added. After 2 h the HPLC (see analytical method) shows complete conversion of mPEG. The buffer of the reaction mixture is then exchanged to 20 mM ammonium acetate (see buffer exchange method) and lyophilized. Purification of the resulting solid by RP-HPLC (see preparative HPLC method) with further lyophilization of the fractions of interest yields 99 mg of conjugate.

[0161] Analytical Method:

[0162] Column: Phenomenex Jupiter C18, 5 μm, 300 Å, 4.6×250 mm

[0163] Flow: 1 ml/min

[0164] Eluents: A) water 0.1% trifluoroacetic acid (vol/vol) [0165] B) acetonitrile 0.1% trifluoroacetic acid (vol/vol)

[0166] Detector: UV 214 and 280 nm, ELSD (mPEG-maleimide is detectable at UV)

[0167] Elution: 5 min at A/B 90/10 [0168] Gradient to 45/55 in 45 min [0169] Wash step at 100% B for 7 min [0170] Re-equilibration at 90/10 for 7 min

[0171] Retention times: peptide ˜29.5 min [0172] peptide dimer ˜34.0 min [0173] mPEG-maleimide ˜36.2 min [0174] XYZ ˜36.8 min

[0175] Buffer Exchange Method:

[0176] Column: GE Healthcare HiPrep 26/10 Desalting

[0177] Flow: 10 ml/min

[0178] Eluant: Ammonium acetate 20 mM

[0179] Preparative HPLC Method:

[0180] Column: Phenomenex Jupiter C18, 5 μm, 300 Å, 15×250 mm

[0181] Flow: 10 ml/min

[0182] Sample injection: 1 ml of 50 mg/ml solution in eluent A/ eluent B 90/10

[0183] Eluents and elution program as per the analytical method.

Melanoma Cell Lines and Primary Human Cells

[0184] WM-115 (Part of the Wistar Special Collection, Catalog N. CRL-1675), WM-266-4 (Part of the Wistar Special Collection, Catalog N. CRL-1676), and SK-MEL-28 (Catalog N. HTB-72) melanoma cell lines (American Type Culture Collection, Manassas, Va., USA) were maintained in BME Melanoma Medium composed by: BME (Lonza, Basel, Switzerland) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 1% penicillin/streptomycin and 1.5 g/L sodium bicarbonate. WM793-B (Part of the Wistar Special Collection, Catalog N. CRL-2806), and 1205Lu (Part of the Wistar Special Collection, Catalog N. CRL-2812), melanoma cell lines (American Type Culture Collection, Manassas, Va., USA) were cultured in Tumor Medium 2% containing: MCDB154CF medium (Thermo Fisher Scientific Inc, Waltham, Mass., USA) supplemented with 2% FBS, 1.5 g/L sodium bicarbonate, Leibovitz L-15 medium, 2 mM L-glutamine, 200 mM CaCl2, 5 mg/ml insulin and 1%. penicillin/streptomycin (Lonza, Basel, Switzerland).

[0185] The use of melanoma biopsies was approved by the ethical committee (Comitato Etico dell'Area Vasta Emilia Nord). Biopsies were taken from the University Hospital Policlinico of Modena and patient gave inform consent. Immediately after surgical resection, melanoma cells were dissociated into single cells with collagenase (1 mg/ml; Biochem, Nuoro, Italy) as previously indicated (Civenni et al., 2011). The single cell suspension was filtered and single cells were harvested and seeded as spheroids according to the liquid overlay method. Cells were maintained in RPMI, 10% decomplemented FBS, 2 mM L-glutamine and 1% penicillin/streptomycin and used to perform Zebrafish xenotransplant.

Immunoprecipitation

[0186] 1205Lu and WM266-4 melanoma cell lines were treated with or without XYZ. 24 h later cells were harvested with RIPA buffer (10 mM Tris, pH 8.0; 150 mM NaCl; 1% Nonidet P-40, 0.5% deoxycholate; 0.1% SDS) containing protease inhibitors. Nuclei were removed by centrifugation (11,000 rpm for 3 min). Monoclonal antibody anti-p75NTR (Upstate) was bound to sepharose beads lh at 4° C. under rotation. Then, pre-cleared lysates were conjugated to sepharose beads-antibody complex or to sepharose beads alone as negative control, overnight at 4° C. under rotation. Immunocomplexes were washed with Port Buffer (10 mM Tris-HCl, pH 8.5; 150 mM NaCl; 0.1% Renex 30; 0.01% BSA; 2.5% NaN3) six times and with PT Buffer (10 mM Tris-HCl, pH 8.5; 150 mM NaCl; 0.5% Tween 20; 2.5% NaN3) three times. Samples were eluted with 1× Laemnli sample buffer for 5° C. at 90° C. and Western Blotting for p75NTR was performed.

MTT Assay

[0187] 5×103 cells/well were seeded in 96-well culture plates. At different time points, cells were incubated with 0.5% MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 4 h at 37° C. and then dissolved with 100 μl Isopropanol with 0.04N HCl. The plate was read at 560 nm with a reference filter of 650 nm. Results are expressed as viability percentage, as compared to control.

CD271 Expression by Flow Cytometry Analysis

[0188] Melanoma cell lines were treated with the chemotherapy or the diluent for 48 h, then were harvested and incubated with anti CD271 antibody (1:100 in PBS, Lab Vision Corporation, Thermo Fisher Scientific, Fremont, Calif., USA) for 20 min at 4° C. Cells were labeled with secondary antibody Alexa Fluor anti-mouse 488 (1:50, Thermo Fisher Scientific) for 20 min at 4° C. and analyzed with Epics XL flow cytometer (Beckman Coulter).

Cell Death Analysis by Flow Cytometry

[0189] WM266-4 cell line was treated with the peptide (XYZ) or diluent at different time points. After 24 and 48 h, cells were trypsinised and resuspended in hypotonic fluorochrome solution: 50 μg/ml propidium iodide containing 0.1% sodium citrate and 0.5% Triton X-100 (Sigma Aldrich, Milano, Italy). After 15 min, cells were analyzed using an Epics XL flow cytometer (Coulter Electronics Inc., Hialeah, Fla., USA). Apoptosis was detected by evaluating the reduced fluorescence of the DNA-binding dye PI in the apoptotic nuclei.

Annexin V Staining

[0190] WM266-4 cell line was treated with the peptide (XYZ) or diluent at different time points. After 24 and 48 h, cells were trypsinised, collected by centrifugation and resuspended in 500 μL of binding buffer containing annexin V-FITC and propidium iodide (PI). After incubation at room temperature for 5 min in the dark, annexin V-FITC binding was analyzed by flow cytometry using FITC signal detector and PI staining by the phycoerythrin emission signal detector.

Melanoma Cells Transfection with siRNA

[0191] Melanoma cells were plated in 2D for 24 h in antibiotic free medium. WM115 cell line was then transfected with scrambled or CD271 siRNA (50 nM and 100 nM, respectively) (Dharmacon Inc, Lafayette, Colo., USA) in antibiotic/FBS free medium supplemented with 0.1% BSA and 48 h later cells were seeded for MTT assay or lysed for western blotting.

Melanoma Cells Infection

[0192] Skme128 cell line was plated according to the liquid overlay method. After 24 h, cells were transduced by infection with viral supernatant generated by CD271-LNSN packaging cells or by LNSN packaging cells (kindly provided from F. Mavilio) in appropriate medium for melanoma, as described previously in the presence of polybrene (8 μg/ml). 48 h after infection cells were lysed for WB analysis or seeded for MTT assay.

Real Time PCR

[0193] Total cellular RNA was extracted from the three melanoma cell lines after chemotherapy treatment using TRI Reagent method performed according to manufacturer instructions (Sigma-Aldrich). Quantitative Real time PCR was performed with an ABI 7500 (Applied Biosystems, Foster City, Calif., USA) for sortilin. As an internal control, housekeeping gene beta-actin mRNA expression was measured in separated tube. RNAse-free H2O was used as a negative control. lmicrog of RNA was subjected to retro-transcription and amplification in a 50 μl reaction mixture using the One-Step RT-PCR Master Mix Reagents kit (Applied Biosystems). Both sortilin and beta-actin real time PCR were performed using Pre-Developed TaqMan Assay Reagents (CD271 MGB probe was FAM dye labelled; beta-actin MGB probe was VIC dye labelled, Applied Biosystems). Thermal cycling conditions for One-Step RT-PCR were: initial reverse transcription at 48° C. for 30 min, then DNA Polymerase activation at 95° C. for 10 min, followed by 40 cycles of denaturation at 95° C. for 15 s, annealing/extension at 60° C. for 1 min. Data from each sample were compared with CD271 expression of no-treated cells, as calibrators, using the Sequence Detection Software Version 1.2.3 according to the Relative Quantification (ddCt) Study method (Applied Biosystems). Results were obtained as the mean from three independent experiments. Double sided Student's t-test was performed between samples and calibrator.

Western Blotting

[0194] Melanoma spheroids were disaggregated and harvested for CD271 in lysis buffer pH 7.5 (150 mM NaCl, 15 mMMgCl, 1 mM EGTA, 50 mM Hepes, 10% Glycerol, 1% Triton). Membranes were first incubated in blocking buffer and then overnight at 4° C. with primary antibodies: anti-human CD271 mouse monoclonal antibody (1:1000, Upstate, Lake Placid, NY, USA),), anti-human TrkB rabbit polyclonal antibody (1:750, Upstate), anti-human TrkC goat polyclonal antibody (1:750, Upstate), anti-human TrkA rabbit polyclonal antibody (1:1000, Upstate) and anti-human beta-actin monoclonal antibody (1:5000, Sigma-Aldrich). Membranes were then incubated for 45 min at room temperature with the following peroxidase-conjugated secondary antibodies: goat anti-mouse (1:3000, Biorad, Hercules, Calif., USA) for p75NTR and beta-actin; goat anti-rabbit (1:3000, Biorad) for TrkA and TrkB; donkey anti-goat (1:1000, Santa Cruz Biotechnology Inc Santa Cruz, Calif.) for TrkC. Membranes were washed and developed using the ECL chemiluminescent detection system (Amersham Biosciences UK Limited, Little Chalfont Buckinghamshire, England). The band intensity was quantitatively determined using ImageJ software (Wayne Rasband).

Scratch-Wound Assay

[0195] 50,000 cells were plated on six-well tissue culture plates and were treated with 10 microg/ml mitomycin C for 2 hours. 24 hours later, cells were washed three times in serum-free medium and three lines for each well were drawn along the cell monolayer with a sterile plastic tip. Plates were washed twice with serum-free medium to remove all detached cells and incubated in serum-free medium with XYZ, chemotherapy, ibuprofen or ketoprofen alone or in combination with the peptide. Cells were monitored at 48 hours from stimulation. The result of each experiment was expressed as the mean of migrated cells from six different areas. The final results are expressed as the mean±SD of three different experiments. Student's T-test was used for comparison of the means.

Zebrafish Handling for Xeno-Transplantation

[0196] Zebrafish handling was performed at the Zebrafish Centre of the University of Padova, Italy, under ethical committee (OPBA) authorization 407/2015-PR. Zebrafish embryos were obtained from natural spawning of albino adults, raised following standard protocols (Westerfield, M et al., 2000) and staged according to Kimmel et al. (1995). For xeno-transplantation, embryos were mechanically dechorionated at 2 days post-fertilization (dpf), anesthetized with tricaine 0.16 mg/ml and placed along plastic lanes immersed in 2% methylcellulose/PBS. Melanoma human cells were stained with Vybrant Cell-Labeling Solution (5 ug/ml, Molecular Probes) or CFSE (2 uM, Invitrogen) for 20 minutes at 37° C. according to the manufacturer. Stained cells were loaded in a glass capillary needle and microinjected into the yolk (about 50 cells/embryo), using a WPI PicoPump apparatus. Xenotransplanted embryos were grown at 33° C., treated with was treated with XYZ, DTIC, the peptide in combination with DTIC, or left untreated. Zebrafishes were monitored daily and documented from 1 day post injection (dpi) up to 1 week (experimental endpoint). Imaging was performed using a Leica MZFLIII dissecting microscope equipped with a Leica DFC7000T camera. Panels were assembled using Adobe Photoshop CC v. 14.0×64.

RESULTS

[0197] To demonstrate that the peptide is able to directly bind CD271, total protein lysates of WM266-4 and Lu1205 melanoma cell lines were immunoprecipitated with the peptide conjugated to sepharose beads. Immunocomplexes were washed with Port Buffer and with PT Buffer, then eluted with Laemnli sample buffer for 5′ at 90° C. The samples were run onto 7% polyacrylamide gel, transferred onto nitrocellulose membrane and blotted with anti-CD271 monoclonal antibody. The sepharose beads-peptide complex without protein lysates is used as negative control, while total protein lysate from WM266-4 without sepharose beads-peptide complex is used as positive control (FIG. 1).

[0198] Five melanoma cell lines were used for testing the efficacy of XYZ or its non-PEGylated peptide component: WM115 and WM266-4, which are respectively the primary and metastatic melanoma derived from the same patient and expressing high levels of CD271, and SKme128, a metastatic melanoma cell line expressing low levels of CD271, WM793B, a primary melanoma, and 1205Lu derived from lung metastatic melanoma. The viability of the cell culture is assessed by MTT conversion. MTT solution is applied to the plates and placed into a cell culture incubator for 4 hours. The blue formazan metabolite produced by viable cells is then extracted into isopropanol and absorbance is measured at 570 nm using a spectrophotometer. XYZ or the non-PEGylated peptide reduce cell proliferation, as shown by MTT assay, in all melanoma cells 48 h and 96 h after treatment, with no significant difference (FIG. 2), indicating that PEG conjugation does not alter peptide efficacy.

[0199] In addition, XYZ diminishes cell proliferation, as shown by MTT assay, in a dose-dependent manner in WM115, WM266-4 and SKme128 cell lines (FIG. 3).

[0200] Because CD271 mediates apoptosis in many cell systems, we analysed the effect of XYZ in melanoma cell lines in relation to the levels of the receptor. After treatment, melanoma cell lines were harvested and incubated with Trypan Blue staining solution or incubated with CD271 antibody for flow cytometer analysis. XYZ is more efficacious in cell lines expressing higher levels of CD271, as shown by flow cytometry analysis and under a microscope viable cell counting (FIG. 4).

[0201] In particular, XYZ triggers melanoma apoptosis, a most well-known type of programmed cell death in culture. Indeed, XYZ induces a significantly higher rate of apoptosis than diluent alone in two apoptosis assays (Propidium iodide, subG1 analysis; Annexin staining) (FIG. 5). In details, melanoma cell lines, gently were trypsinised and washed once with serum-containing media before incubation with annexin V-FITC or with propidium iodide. 24 h and 48 h later, FITC signal detector or sub G0/G1 peak for annexin or PI staining respectively, were observed by flow cytometry.

[0202] Moreover, XYZ triggers melanoma cell death in culture through CD271. XYZ fails to reduce cell viability (MTT assay) in the WM115 CD271 silenced (siRNA) melanoma cell line, while it induces cell death to a greater extent in SKMe128 melanoma cell lines overexpressing CD271 (CD271 viral vector) (FIG. 6). This indicates that CD271 mediates XYZ effects in melanoma.

[0203] It was also found that chemotherapeutic agents dose-dependently upregulate CD271 expression at the mRNA (Real-time PCR) and protein level (Western blotting and flow cytometry) (FIG. 7). Therefore, total cellular RNA was extracted from melanoma cell lines using TRI Reagent method.

[0204] To verify the quality of the RNA, we ran an agarose minigel in RNase-free conditions. 1 ml of RNA was subjected to retro-transcription and amplification in a 50 μl reaction mixture using the One-Step RT-PCR Master Mix Reagents kit. Quantitative Real Time PCR for CD271 receptor was performed by an ABI 7500 Real-Time PCR System. Regarding protein expression, melanoma cells were harvested in lysis buffer pH 7.5, run onto 7% polyacrylamide gel and transferred onto nitrocellulose membrane. Membranes were first incubated in blocking buffer and with primary CD271 antibody, then with secondary antibody. Finally, membranes were washed and developed using the ECL chemiluminescent detection system.

[0205] Indeed, XYZ (180 mM) combined with DTIC (200 mg/ml) is significantly more effective in reducing cell proliferation (MTT assay) than the chemotherapeutic agent alone 48 h after treatment (FIG. 8). Moreover, XYZ combined with DTIC, inhibits melanoma cell migration significantly more than either DTIC or XYZ alone as shown by scratch-wound assay with SKmel28 cell line (FIG. 9).

[0206] XYZ did not show any acute toxic effect (appearance, weight, water and food intake), as shown by the up and down procedure. Intravenous injections were performed in six CD1 mice (Charles River, Calco (MI) Italy) tails for administration from 17.5 mg/kg to 1750 mg/kg XYZ (FIG. 10).

[0207] Zebrafish model represents an alternative xenotransplant model in vivo that offers a rapid and efficient approach for assessing drug effects in human cancer cells. (Haldi M, Ton C, Seng W L, McGrath P: Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish. Angiogenesis 2006; 9:139-151). Zebrafish embryos are particularly useful for microscopic analysis as they are translucent, thus offering the opportunity to visualize the metastatic process at high resolution (Stoletov K, Montel V, Lester R D, Gonias S L, Klemke R: High-resolution imaging of the dynamic tumour cell vascular interface in transparent zebrafish; PNAS 2007; 104:17406-17411). We have recently shown that CD271 down-regulation promotes melanoma progression and metastasis in zebrafish (Saltari A, Truzzi F, Quadri M, Lotti R, Palazzo E, Grisendi G, Tiso N, Marconi A, Pincelli C: CD271 down-regulation promotes melanoma progression and invasion in three-dimensional models and in zebrafish; J Invest Dermatol 2016; 136:2049-2058).

[0208] For xenotransplantation, albino embryos were mechanically dechorionated at 2 days post-fertilisation, anesthetized with tricaine 0.16 mg/ml and placed along plastic lanes immersed in 2% methylcellulose/PBS. SKmel28 cells or melanoma cells from patient metastasis were stained with Vybrant Cell-Labeling Solution. Stained cells were loaded in a glass capillary needle and microinjected into the yolk (about 50 cells/embryo), Xenotransplanted embryos were monitored daily and documented from one day post injection up to one week.

[0209] XYZ alone reduces metastasis formation in zebrafish injected with melanoma cells derived from patient with BRAF mutation, as compared to control. Moreover, XYZ, in combination with DITC, further enhances this effect (FIG. 11).

[0210] Moreover, in xenotransplanted zebrafish with SKmel 28 cell line from primary melanoma DTIC or XYZ reduces full metastasis, while XYZ in combination with DTIC blocks the formation of full metastasis (FIG. 12).

[0211] There is growing evidence that inflammation may exacerbate cancer metastasis and several clinical studies show that taking nonsteroidal anti-inflammatory drugs (NSAIDs) appears to reduce metastases. TNF-alpha upregulates malignant melanoma migration in vitro and this could be reduced by ibuprofen both in solution and delivered from a hydrogel (Redpath M, Marques C M, Dibden C, Waddon A, Lalla R, Macneil S: Ibuprofen and hydrogel-released ibuprofen in the reduction of inflammation-induced migration in melanoma cells; Br J Dermatol. 2009; 161:25-33).

[0212] Khwaja et al. observed that at higher concentrations, some NSAIDs inhibit proliferation and induce apoptosis of cancer cells. They also observed that p75NTR is an important upstream modulator of the anticancer effects of NSAIDs and that ibuprofen induction of the p75NTR protein establishes an alternate mechanism by which ibuprofen may exert an anticancer effect (Khwaja F 1, Allen J, Lynch J, Andrews P, Djakiew D: Ibuprofen inhibits survival of bladder cancer cells by induced expression of the p75NTR tumor suppressor protein; Cancer Res. 2004; 64:6207-13)

[0213] Epidemiologic studies show that patients chronically consuming NSAIDs for arthritis exhibit a reduced incidence of prostate cancer. In addition, some NSAIDs show anticancer activity in vitro. NSAIDs exert their anti-inflammatory effects by inhibiting cyclooxygenase (COX) activity; however, evidence suggests that COX-independent mechanisms mediate decreased prostate cancer cell survival. R-flurbiprofen and ibuprofen have been found to selectively induce p75NTR-dependent decreased survival of prostate cancer cells independently of COX inhibition (Quann E J, Khwaj a F, Zavitz K H, Djakiew D: The aryl propionic acid R-flurbiprofen selectively induces p75NTR-dependent decreased survival of prostate tumor cells; Cancer Res. 2007; 67:3254-62).

[0214] Ketoprofen is a widely used NSAID that also exhibits cytotoxic activity against various cancers. A carboranyl analogue prodrug ester of ketoprofen exhibited high cytostatic activity against melanoma and colon cancer cell lines, with the most pronounced activity was found in cell lines that are sensitive to oxidative stress (Buzharevski A, Paskas S, Laube M, Lönnecke P, Neumann W, Murganic B, Mijatovic S, Maksimovic-Ivanic D, Pietzsch J, Hey-Hawkins E: Carboranyl Analogues of Ketoprofen with Cytostatic Activity against Human Melanoma and Colon Cancer Cell Lines; ACS Omega. 2019; 4:8824-8833).

[0215] We found that 2D melanoma cell cultures (WM115, WM266-4 and SKMEL28) treated with ketoprofen or ibuprofen from 1 to 2.5 mM show increased CD271 protein levels after 48 h treatment (FIG. 13).

[0216] We also demonstrated that treatment with ketoprofen and ibuprofen dose-dependently decreases proliferation in CD271-positive melanoma cell cultures (WM115, WM266-4 and SKMEL28) (FIG. 14).

[0217] In our experiments, two different 2D melanoma cell cultures (WM266-4 and SKMEL28), treated with a combination of XYZ with either ibuprofen or ketoprofen, clearly showed a statistically significant decrease in cell survival (i.e. increased efficacy) when compared to the treatment with the anti-inflammatory agent alone (MTT assay), suggesting a synergistic effect between the intrinsic cytotoxic effect of the anti-inflammatory agent and the targeting of CD271 by XYZ (FIG. 15).

[0218] The combination of XYZ with anti-inflammatory treatments such as ibuprofen and ketoprofen was also shown to decrease cell migration in 2D melanoma cell cultures, as shown by scratch-wound assay with SKme128 cell line (FIG. 16).