WO2024124311A1 - Plp2-derived peptides, pharmaceutical compositions, methods and uses of thereof - Google Patents

Abstract

The present disclosure refers to peptides targeting tumors and having antitumor activity. The peptides are derived from protein PLP2 and have been shown to induce biochemical and morphological alterations characteristic of necrosis and act directly on tumor cells inducing the expression of DAMPs, which trigger the immunoprotective effect in vivo against melanoma cells. Pharmaceutical compositions, methods and uses for treating cancer are also disclosed herein.

Description

PLP2 -DERIVED PEPTIDES, PHARMACEUTICAL COMPOSITIONS, METHODS AND USES OF THEREOF

TECHNICAL FIELD

[1] The present disclosure relates in general to peptides and, specifically, peptides targeting tumors and exhibiting antitumor activity. Pharmaceutical compositions, methods and uses thereof are also disclosed herein.

PRIOR ART

[2] Peptides play an important role in cell biology and in many diseases including cancer. Peptides can be used in early diagnosis, prognostic, and treatment of neoplasms. The main obstacles of chemotherapy are the lack of specificity on tumor targeting, which may cause side effects and resistance to multiple drugs. Hence, a need to develop new therapeutic agents is, therefore, recognized.

[3] Proteolipid protein 2 (PLP2) is a small transmembrane lipoprotein of 152 amino acids that was initially found in human colonic epithelial cells. Breitwieser (1997, p.272) reported that PLP2 multimerizes and functions as an ion channel due to the similarity of its hydropath profile with that of a subunit of the H+ vacuolar ATPase. This hydrophobic lipoprotein is localized to the endoplasmic reticulum and A4/PLP2 has been reported to interact with Bap31 by Wang

(2003, p.278) . PLP2, a four- transmembrane domain protein, has contributed to the tumor formation and metastasis of murine melanoma in a syngeneic B16F10 model as described by Sonoda (2010, p.23) . Ozawa (2012, p.3) disclose reduced PLP2 expression led to growth inhibition of B16BL6 cells in vivo and prevented detectable metastasis from primary tumor cells by decreasing fibronectin and laminin, and by reducing the migratory ability of B16BL6 cells .

GENERAL DESCRIPTION OF THE EMBODIMENTS

[ 4 ] The peptides disclosed herein have been useful to be useful as as antitumor agents due to various characteristics . They have several advantages over some other therapeutic agents such as easy availability by synthesis , convenient puri fication and storage , low molecular weight , ef ficient immunoreactivity, and the ability to speci fically target tumor cells with decreased toxicity . The peptides disclosed herein have been reported to inhibit angiogenesis , to have the ability to target metastases and to elicit cytotoxic T- lymphocyte responses .

[ 5 ] The present disclosure refers to peptides that have been found to be useful in cancer treatment and modulation of the immune system of cancer patients . More speci fically, a N-terminal peptide derived from PLP2 is disclosed herein . Such peptide is known as Rb4 and has been shown to target and have cytotoxic activity against tumors .

[ 6 ] Therefore , the present application refers to peptides that have been found to be useful in cancer treatment and modulation of the immune system of patients under cancer treatment . Rb4 , or similar peptides having a high identity thereto , can induce biochemical and morphological alterations characteristic of necrosis . Such Rb4-induced biochemical and morphological alterations comprise alteration of F-actin dynamics , increase of cytosolic calcium from the endoplasmic reticulum (ER) and the expression of two damage-associated molecular patterns ( DAMPs ) , HMGB1 and calreticulin . [7] In other embodiments, the present application refers to pharmaceutical compositions comprising a Rb4 peptide, methods and uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[8] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[9] Figure 1A shows panels with representative fields of B16F10-Nex2 cells after control, Rb4 or Scr-Rb4 treatment for 1, 6, 12 and 24 h of incubation, wherein loss of morphology and no replication are disclosed for Rb4-treated cells .

[10] Figure IB shows representative panels of B16F10-Nex2 cells treated with Rb4 for 36, 48 and 72 h showing resistant cells, highlighted in red, losing morphology and clustered after prolonged exposure to Rb4 peptide.

[11] Figure 2A shows the effect of Rb4 on the protection against melanoma lung colonization in C57B1/6 mice.

[12] Figure 2B shows the absence of protective activity against melanoma lung colonization in the immunocompromised NOD /SCID mice compared to immunocompetent C57BL/6 mice.

[13] Figure 3A shows the tumor growth curve on Rb4 protection effect against subcutaneous melanoma in C57BL/6 mice .

[14] Figure 3B shows the percent survival of subcutaneous melanoma in C57BL/6 mice cell following Rb4 inoculation.

[15] Figure 4A shows internucleosomal DNA degradation induced by Rb4 peptide detected by TUNEL assay in fluorescence microscopy. Pie charts on the right show the percentage of positive cells (yellow) or negative (blue) of TUNEL assay.

[16] Figure 4B shows ultrastructural characteristics of Rb4-induced cell death, such as the presence of dilated mitochondria (m) , plasma membrane disintegration (pmd) and vacuolization of cytoplasm (vc) in cells treated with Rb4. B16F10-Nex2 cells were either left untreated (control, a) or treated with 0.15 mM Rb4 for 16 hours (b, c, d, 1, 2) followed by transmission electron microscopy.

[17] Figure 4C shows fluorescent staining to detect apoptotic morphology of B16F10-Nex2 cells after treatment with Rb4 at 0.15 mM for 16 h. Arrows indicate cells highlighted in inserts, zoom 200x.

[18] Figure 4D shows Annexin V (Ann) binding in B16F10-Nex2 melanoma cells treated with 0.05 and 0.1 mM Rb4 for 16 h, analyzed by FACS. 7-AAD labeling is also shown.

[19] Figure 5A-5C depict Rb4, but not Scr-Rb4, triggering PARP-l-mediated necrosis independently of RIP-1 in murine melanoma cells.

[20] Figure 5A shows Western blotting of PARP-1 after Rb4 treatment of melanoma cells samples analyzed by immunoblotting showing a cleaved band at 62 kDa when compared to Scr-Rb4 and untreated controls.

[21] Figure 5B shows PARP-1 cleaved band increased ninefold in Rb4 treated cells when normalized by p-actin expression and compared to both controls.

[22] Figure 5C shows immunoblotting of RIP-1 having no RIP1 expression after 16 h.

[23] Figure 6A depict Rb4 peptide effect on the morphology and viability of B16F10-Nex2 cells by interference light microscopy is independent of the enhanced level of calcium in the cytosol and RIP1 expression. [24] Figure 6B depict the cell viability determined by MTT assay .

[25] Figure 7A shows endoplasmic reticulum calcium flux increased by Rb4 when B16F10-Nex2 cells were treated with thapsigargin (THG) , digitonin (Dig ) and EGTA.

[26] Figure 7B shows endoplasmic reticulum calcium flux increased by Rb4 when B16F10-Nex2 cells were treated with 0.15 mM Rb4 followed by thapsigargin, digitonin and EGTA.

[27] Figure 7C shows endoplasmic reticulum calcium flux increased by Rb4 when B16F10-Nex2 cells were treated with thapsigargin followed by 0.15 mM Rb4, digitonin and EGTA.

[28] Figures 8A, 8C, 8E depict Rb4 effects on actin polymerization and depolymerization in B16F10-Nex2 cells treated with 0.15 mM Rb4 for 15 h or 20 h, showing lose to full loss of F-actin structure in C and E after Rb4 treatment .

[29] Figures 8B, 8D, 8F shows B16F10-Nex2 cells incubated with Dnase to stain globular actin.

[30] Figure 9A depict expression of DAMPs by Rb4 treatment, showing calreticulin expression of control cells and treated cells detected by flow cytometry.

[31] Figure 9B depict expression of DAMPs by Rb4 treatment, showing the HMGB1 expressions in both control and treated cells by an ELISA assay.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[32] The present disclosure refers to a PLP2 N-terminal- derived peptide. PLP2 is a f our-transmembrane domain protein which has an important role in tumor formation and metastasis. The peptide disclosed herein has the amino acid sequence MADSERLSAPGCWAACTNFSRTRK and will also be referred to herein as Rb4. [33] Therefore, in a first embodiment, the present application refers to a peptide comprising or consisting of the amino acid sequence MADSERLSAPGCWAACTNFSRTRK, or a sequence having, for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, zs, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservative modification" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. Typically conservative substitutions for one another: 1) Alanine (A) , Glycine (G) ; 2) Aspartic acid (D) , Glutamic acid (E) ; 3) Asparagine (N) , Glutamine (Q) ; 4) Histidine (H) , Arginine (R) , Lysine (K) ;

5) Isoleucine (I) , Leucine (L) , Methionine (M) , Valine (V) ;

6) Phenylalanine (F) , Tyrosine (Y) , Tryptophan (W) ; 7) Serine (S) , Threonine (T) ; and 8) Cysteine (C) , Methionine (M) (see, e.g., Creighton, Proteins (1984) ) .

[34] In one specific embodiment, the peptide disclosed herein is a synthetic peptide derived from the PLP2. Peptides can be synthesized and purified through state-of-the-art methods and techniques using conventional peptide synthesis equipment .

[35] In another specific embodiment, the peptide disclosed herein is a peptide isolated from a protein, such as PLP2. Peptides can be isolated and purified through state-of-the- art methods and techniques using conventional peptide isolation equipment. [36] According to such embodiments, the peptides may be purified using well known methods and techniques, for example, reverse-phase HPLC, and/or by gel electrophoresis, excised, dialyzed, lyophylized, resuspended in saline, such as PBS, and filter-sterilized.

[37] The cytotoxicity of the peptide in both tumorigenic and nontumorigenic cells was compared. In vitro, Rb4 exhibits cytotoxic activity against some human and murine cancer cell lines, especially against the murine melanoma B16F10-Nex2 cell line. No activity was observed for the scrambled Rb4 peptide (Scr-Rb4) in all the performed tests. In addition, toxicity of the peptide was analyzed after 24 h in nontumorigenic cells in culture. The murine non-tumorigenic cell line MEF was unaffected by the highest concentration of Rb4 (1 mM) . In a protein microarray assay was determined that the B16F10-Nex2 cell line over-expresses PLP2 , which could justify the cytotoxic effect of Rb4 peptide in this cell lineage by competing with the endogenous protein. On the other side, the expression of PLP2 on A2058 human melanoma cells could be downregulated, decreasing the sensitivity of these cells to Rb4. Ding (2015, p.94) demonstrated that miRNA (miR-664) downregulated PLP2 expression in human melanoma cells by directly targeting the PLP2 untranslated region, mediating direct suppression of PLP2 expression.

[38] In one embodiment, the present disclosure refers to the peptide induction of necrosis in melanoma cells as confirmed by dilated mitochondria, plasma membrane disintegration, and vacuolization of cytoplasm, DNA fragmentation, absence of chromatin condensation, increase of double staining with Annexin V/7-AAD but not with Annexin V only, and absence of caspase-3 and -9 activation. [39] Rb4-treated murine melanoma cells showed necrotic cleavage of PARP-1, which is a known nuclear enzyme characterized by Gobeil (2001, p.8) in Jurkat T cells treated with necrotic cell inducers. Since PARP-l-mediated necrosis might involve RIP1, the inventors have looked for RIP1 expression and found that until 6 h of incubation with Rb4, RIP1 increased slightly, while with 16 h RIP1 expression was not detected, suggesting that the cell death pathway triggered by Rb4 leads to inhibition of RIP1 over time.

[40] In addition, neither the morphological alteration nor the viability of Rb4-treated B16F10-Nex2 melanoma cells was inhibited by Nec-1. These results suggest that PARP1- mediated necrotic death of B16F10-Nex2 cells triggered by Rb4 is independent of RIP1 expression. Luan (2015, p.ll) have shown that RIP1 plays a critical role in survival of human melanoma cells undergoing pharmacological ER stress induced by thapsigargin (THG) . While RIP1 is upregulated in melanoma cells relatively resistant to THG-induced apoptosis, knockdown of RIP1 rendered cells susceptible to be killed by THG.

[41] The results shown hereinbelow provide that Rb4 leads to necrotic cell death of murine melanoma cells mainly through the induction of ER stress and RIP1 inhibition.

[42] In other embodiment of the present disclosure, Rb4 increases the cytosolic calcium from the ER as demonstrated in Fig. 6. On one hand, both Rb4 and THG alone increased cytosolic calcium levels considerably. Incubation with each one, however, did not alter the levels of calcium increased by the other. These results suggest that Rb4 might act as an inhibitor of SERCA activity. The cytosolic calcium increase is not central for the Rb4-cytotoxicity in melanoma cells since neither the morphological alteration nor the viability of Rb4-treated B16F10-Nex2 cells was inhibited by BAPTA-AM, an intracellular calcium chelator .

[ 43 ] Actin dynamics has been shown to regulate Ca2+ release from endoplasmic reticulum and is associated with tumor necrosis factor-induced apoptosis . Furthermore , actin depolymeri zation and polymeri zation was described to regulate RIPl-independent necrotic death on U87MG cells expressing Bcl-xL elicited by a non-selective isopeptidase inhibitor . The treatment with Rb4 leads tumor cells to a morphology loss characteri zed by F-actin depolymeri zation and G-actin monomers accumulation . These results demonstrate the peptide interference in the G-actin pool af fecting the conversion to f ilamentous , the stability of F-actin on tumor cells and overall actin dynamics . Similarly, leukemia HL- 60 cells were already shown to respond to UV irradiation with early and transient actin polymeri zation and, later, with actin depolymeri zation on F-actin pools which were shown to be essential elements in the formation and release of apoptotic bodies .

[ 44 ] In other embodiments , the present application refers to pharmaceutical compositions comprising the above- mentioned peptides , as well as methods of treatment and uses thereof . Particularly, a pharmaceutical composition comprising an ef fective amount the Rb4 peptide , or a peptide having a high identity thereto , and pharmaceutical or veterinary acceptable vehicles .

[ 45 ] The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse , allergic, or other untoward reactions when administered to an animal or a human . As used herein, "pharmaceutically acceptable carrier" includes all solvents , dispersion media, coatings , antibacterial and anti fungal agents , isotonic and absorption delaying agents , nanoparticles and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients also can be incorporated into the compositions. In a preferred embodiment, biocompatible nanoparticles are used as pharmaceutical carriers, e.g. mesoporous silica nanoparticles, such as the ones described in Shen et al., 2013 (Curr Pharm Des. 2013; 19 (35) : 6270-89. doi: 10.2174/1381612811319350005. PMID: 23470004) and Stephen et al. 2022 (Drug Deliv Transl Res. 2022 Jan;12 (l) :105-123. doi: 10.1007 / s 13346- 021- 00935-4 ) .

[46] The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. Although, the intravenous route is a preferred embodiment, other routes of administration are contemplated. This includes oral, nasal, buccal, rectal, vaginal, or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.

[47] The active compounds also may be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. [48] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy administration by a syringe is possible. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, using a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and using surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agent, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[49] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

[50] As used herein, "pharmaceutically acceptable carrier" includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions .

[51] The carrier also can be a solvent and/or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and/or liquid polyethylene glycol, and/or the like) , suitable mixtures thereof, and/or vegetable oils. The proper fluidity can be maintained, for example, using a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or using surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and/or the like. In many cases, it will be preferable to include isotonic agents, for example, sugars and/or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate , mesoporous silica nanoparticle and/or gelatin .

[ 52 ] Sterile inj ectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above , as required, followed by filtered sterili zation . Generally, dispersions are prepared by incorporating the various sterili zed active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the required other ingredients from those enumerated above . In the case of sterile powders for the preparation of sterile inj ectable solutions , the preferred methods of preparation are vacuum-drying and/or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- f iltered solution thereof . The preparation of more , and/or highly, concentrated solutions for direct inj ection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area .

[ 53 ] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and/or in such amount as is therapeutically ef fective . The formulations are easily administered in a variety of dosage forms , such as the type of inj ectable solutions described above , but drug release capsules and/or the like can also be employed .

[ 54 ] For parenteral administration in an aqueous solution, for example , the solution should be suitably buf fered, for example with Phosphate-buf f ered saline ( PBS ) , i f necessary and/or the liquid diluent first rendered isotonic with suf ficient saline and/or glucose . In a speci fic embodiment , the pharmaceutical formulation is an aqueous solution that comprises an effective amount of the peptide, water and PBS. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and/or intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art considering the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[55] The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically- acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[56] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art considering the present disclosure. For example, in a specific embodiment, the pharmaceutical composition the pharmaceutical or veterinary compositions disclosed herein comprise an effective amount of the Rb4 peptide, wherein such effective amount is at least 10 mg/kg. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[57] Rb4 peptide when peritoneally injected significantly reduced the number of lung metastatic nodules in the syngeneic B16F10-Nex2 melanoma model. The Rb4 also reduced the volume of subcutaneously grafted B16F10- Nex2 melanoma tumor in syngeneic mice. Moreover, the survival rate of mice treated with Rb4 was significantly prolonged in comparison to that of the vehicle control groups (Fig. 3B) . In vivo activity of Rb4 in murine melanoma depends on the immune system since its protective activity against metastatic melanoma B16F10-Nex2 observed in immunocompetent mice was not observed in immunocompromised (NOD-SCID-IL-2R-gamma null) mice. The expression and release of DAMPs in Rb4- treated melanoma cells could explain the absence of activity of Rb4 in immunocompromised mice. Rb4-treated cells showed 3.54-fold secreted HMGB1 in relation to untreated cells and 1.77-fold increase of cell surface calreticulin . Once present in the extracellular microenvironment, DAMPs function as "danger signals" in the immune system. Calreticulin is a chaperone multifunctional protein, predominantly found in the lumen of the ER, and associated with several physiological and pathological processes in cells. The main functions include chaperone activity and regulation of Ca2+ homeostasis . As a result , calreticulin could be involved in cellular Ca2+ uptake into the ER via SERCA, Ca2+ storage within the ER, and Ca2+ release from the ER24 . On the other side , cell surface calreticulin ensures the phagocytic removal of dying cancer cells by a subset of DCs , being a pre-requisite for the development of adaptive anticancer immunity . The release of high-mobility group protein Bl (HMGB1 ) activates DCs via the inflammasome or Toll-like receptor pathways . The screening for anticancer drugs and treatments in cancer cells revealed that their ability to induce immunogenic cell death ( ICD) depends on the induction of ER stress .

[ 58 ] In some embodiments , the present disclosure refers to a Rb4 peptide , derived from PLP2 , the Rb4 peptide triggering a necrotic cell death-like in murine melanoma cells , in a dose-dependent manner, through ER stress caused by inhibition of SERCA transporter activity, which leads to alteration of actin dynamics and RIP1 inhibition . Hence , the capacity of Rb4 to lyse tumor cells in a non-physiological (unconventional ) fashion may contribute to its pro-immune ef fects through the liberation of DAMPs such as HMGB1 and calreticulin . These DAMPs would provoke local inflammation and immune reactions in the tissues resulting in a therapeutic ef fect against metastatic and subcutaneous murine melanoma . Since many anticancer drugs signal through mitochondria, ER stress is a potential target for the development of drugs . Therefore , Rb4 peptide is a promising anticancer agent for the treatment of drug-resistant tumors .

[ 59 ] In a specific embodiment , the peptide disclosed herein targets a tumor cell and has an antitumor activity . In view of that , the pharmaceutical compositions disclosed herein are useful for treating and/or preventing cancer . [60] In another specific embodiment, the peptide disclosed herein has an immunomodulation activity, so it has been found to be useful in modulating the immune system of a patient undergoing cancer treatment, as a treatment adjuvant, or in a combination therapy with an anticancer agent.

[61] Potentially, many types of cancers may be treated or prevented with the peptides disclosed herein, a list of which is available at https:// .c ncer.gov/ty es. Examples of the cancers than can be treated by the peptides disclosed herein include but are not limited to solid or soft tissue malignant tumors, sarcomas, carcinomas, lymphomas, leukemias, multiple myeloma, melanoma, brain and spinal cord tumors and head and neck tumors. For example, the cancer may be lymphoma, melanoma, subcutaneous melanoma, breast cancer, uterine cervix cancer, lung cancer, or glioblastoma. In an even more specific embodiment, the cancer is a drug-resistant malignant tumor and/or a metastatic tumor. In a preferred embodiment, the pharmaceutical compositions, medical uses, and methods of treatment disclosed herein disclosed herein may be used for treating the above-mentioned types of cancer.

[62] A method for treating and/or preventing a type of cancer described above in a patient is also described herein. Such method comprises administering to the patient an effective amount of the above-described peptide, or, for example, a pharmaceutical composition comprising a Rb4 peptide, as disclosed herein.

[63] A method of modulating the immune system of a patient undergoing cancer treatment is also described herein. The method comprises administering to the patient an effective amount of a peptide as described hereinabove. In a preferred embodiment, the method comprises administering a pharmaceutical composition comprising a Rb4 peptide, as disclosed herein, intravenously, subcutaneously, or intraperitoneally. Other embodiments disclosed herein include the use of the peptide disclosed herein, such as a Rb4 peptide, for preparing a pharmaceutical composition as described herein for treating and/or preventing cancer or modulating the immune system of a patent under cancer treatment. The cancer can be of any of the types described herein above.

[64]

EXAMPLES

Materials and Methods

Peritumoral treatment of subcutaneously grafted, murine melanoma and of experimental lung metastasis

[65] Six-week-old male C57BL/6 mice obtained from the Center for Development of Experimental Models (CEDEME) , Federal University of Sao Paulo (UNIFESP) (average weight of 25-28g) , were subcutaneously injected with 5xl0⁴ B16F10-Nex2 tumor cells. Peritumoral injections were given starting 24 h after tumor cell graft. Treated groups (5 animals per group) received daily doses of 300 pg of Rb4 and the control group received PBS. Tumor size was measured daily for two weeks with a caliper, using the formula V = 0.52 x DI² * D2, where DI and D2 are the short and long tumor diameters respectively. The maximum allowed tumor volume was 3,000 mm³, before euthanasia. In the Experimental Lung Metastasis model, six-week-old male C57BL/6 mice or NOD/Scid-IL2-Ry^nu11 mice were challenged intravenously with 5xl0⁵ syngeneic B16F10-Nex2 melanoma cells/mouse (0.1 ml) . For protection experiments five groups of five animals received on days 1, 3, 5, 7, 9 after tumor cell challenge, intraperitoneal doses of 300 pg of Rb4 (approximately 10 mg/kg/mice) and the control group received the peptide vehicle. After 20 days, lungs were collected from animals of each group and inspected for metastatic colonization.

Cell lines and Culture

[66] The murine melanoma cell line B16F10 was originally obtained from the Ludwig Institute for Cancer Research (LICR, Sao Paulo, Brazil) and the subline B16F10-Nex2 was isolated at the Experimental Oncology Unit (UNONEX) , Federal University of Sao Paulo, UNIFESP, deposited at Banco de Celulas do Rio de Janeiro (BCRJ-0342) . Human melanoma (A2058) , colon carcinoma (HCT-8) and breast carcinoma (MCF- 7) cell lines were provided by the LICR, Sao Paulo, Brazil. Human cervical carcinoma cell line (HeLa) and human glioblastoma cell line (U87-MG) were provided by Hugo P. Monteiro, UNIFESP, and Osvaldo K. Okamoto, University of Sao Paulo, respectively. Murine syngeneic, colorectal adenocarcinoma (CT26) and murine pancreatic (PANC) cells were provided by Dr. Guillermo Mazzolini from the School of Medicine of Austral University, Buenos Aires, Argentina. The mouse embryonic fibroblasts (MEE) and NIH-3T3 were gifts from Luis F. Lima Reis, Hospital Sirio-Libanes , Sao Paulo. Tumor cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2, in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10 mM N-2-hydroxyethylpiperazine-N' - 2-ethanesulfonic acid (Hepes) (Sigma, St. Louis, MO) , 24 mM sodium bicarbonate (Sigma) , 40mg/l gentamicin (Schering- Plough, Sao Paulo, Brazil) . HeLa, U87-MG, CT26, NIH-3T3 and MEF cells were maintained in DMEM supplemented as well as in the RPMI-1640 medium. All cell lines were checked for mycoplasma using Lonza' s MycoAlertTM Mycoplasma Detection Kit (Catalog #: LT07-118) and were free from the presence of contaminants prior to use.

Peptides [67] Peptide Rb4 (MADSERLSAPGCWAACTNFSRTRK, corresponding to SEQ ID NO: 1) and Scr-Rb4 (LACTNCRTSDAMWEKFSRPSAGRA, corresponding to SEQ ID NO: 2) were purchased from Peptide 2.0 (Chantilly, VA) . A stock solution of 1 mM was prepared by diluting the peptides, amidated at the C terminus, in RPMI 1640 medium with 10% distilled water.

Time-lapse live cell microscopy

[68] B16F10-Nex2 cells (3xl0⁵) were plated in RPMI 1640 medium on 35 mm glass bottom dishes and kept for 5 hours at 37 °C in a humidified atmosphere containing 5% CO2. A stock solution of Rb4 and Scr-Rb4 peptides were diluted to 0.15 mM in RPMI 1640 and used to replace the medium in the cell plates. B16F10-Nex cells were transferred to a Nikon Biostation IMQ equipped with a 20x objective (NA 0.8) and a high-sensitivity camera for imaging of large bright fields- of-view. Cells were followed for up to 72 h when kept at 37 °C in a CO2 incubation chamber on the equipment. Data acquisition started up to 1 h after placing the specimens into the Biostation IMQ, after system stabilization at 37°C and 5% CO2. Time-lapse phase images were taken every 20 min for 24 to 72 h with five fields for each condition. Images captured were made into a movie using the BioStation IM version 2.12 software at a rate of approximately 10 frames per second and processed using Nikon Elements software.

Cytotoxicity assay in vitro

[69] Rb4 was diluted in supplemented RPMI medium with 0.5% dimethyl sulfoxide (DMSO, SIGMA) and incubated with 5xl0³ or IxlO⁴ murine and human tumor cells in 96-well plates. After a pre-incubation period (16 h) , cell viability was assessed using the Cell Proliferation Kit I (MTT) (Boehringer Mannheim) , a 3- ( 4 , 5-dimethylthiazol-2-yl ) -2 , 5- diphenyltetrazolium bromide-based colorimetric assay. Readings were made in a multiplate reader (SpectraMax) at 570 nm. Necrostatin-1 (Nec-1; methyl-thiohydantoin- tryptophan (MTH-Trp) ) was added Ih before the peptide incubation, in some cytotoxicity assays.

Transmission Electron Microscopy

[70] B16F10-Nex2 cells (5xl0⁴) were cultivated on plastic disks made from Aclar film. Cells were then incubated with 0.15 mM Rb4 for 16 h at 37 °C and fixed in a solution of 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, at room temperature for 20 h. Cells were then washed in the same buffer for 10 min, fixed with 1% osmium tetroxide in 0.1 M cacodylate at pH 7.2 for 30 min, and washed with water for 10 min at room temperature. Subsequently, cells were treated with an aqueous solution of 0.4% uranyl acetate for 30 min and washed with water for 10 min. After fixation, cells were dehydrated in graded ethanol (70, 90, and 100%) , treated quickly with propylene oxide, and embedded in EPON. Semi-thin sections from selected regions were collected on grids and stained in alcoholic 1% uranyl acetate and in lead citrate prior to examination in a Jeol 100 CX electron microscope (Tokyo, Japan) .

Chemi luminescenc -ELISA

[71] A 96-well opaque plate (Nunc, Roskilde, Denmark) was coated with supernatant (0.2 ml) of 0.15 mM Rb4-treated B16F10-Nex2 cells, overnight (16 h) , at 4°C. The plate was washed with 0.05% Tween 20-PBS (T-PBS) and blocked for 4h at RT with 1% BSA. Anti-HMGBl antibody (Abeam) , at 1:500, was then added overnight. After incubation, the plate was washed extensively with T-PBS, and the secondary HRP 1:500 antiRabbit IgG (Invitrogen, Thermo Fisher Scientific) was added and incubated for 2 h. The plate was washed 5 times with T- PBS, and the reaction was evaluated by Absorbance using OPD (Sigma Aldrich) in a multiplate reader (SpectraMax) at 450 nm. Chromatin Condensation

[72] Tumor cells (10⁴) , cultivated overnight on round glass coverslips, were treated with 0.15 mM Rb4 for 16 h, washed with PBS, and fixed for 30 min at room temperature with 2% formaldehyde. The cells were washed with PBS and stained with 2 pM Hoechst 33342 (Invitrogen) for 10 min. Cells were visualized in a Biostation IM-Q (Nikon) fluorescence microscope at 60x magnification. Images were processed with ImageJ. Apoptotic cells are characterized by the presence of chromatin condensation and DNA leakage into the cytoplasm of tumor cells.

TUNEL Assay

[73] TUNEL ( TdT-mediated dUTP-X nick end labeling) was performed according to the manufacturer's instructions (In Situ Cell Death Detection kit, Fluorescein; Roche Applied Science) . Briefly, IxlO⁴ cells were cultivated in round glass coverslips and treated for 2 h with 0.15 mM Rb4 or Scr-Rb4 peptides. Cells were fixed in 2% formaldehyde for 30 min at room temperature. They were then permeabilized with 0.1% Triton X-100 for 30 min at room temperature and incubated with terminal deoxynucleotidyl transferase (TdT) in the reaction buffer with dUTP-f luorescein at 37°C for 1 h, then stained with 10 pg/ml DAPI for 10 min and visualized in an Olympus BX-51 fluorescence microscope with immersion oil, at 60* magnification. Images were processed with ImageJ.

Phosphatidylserine Translocation

[74] Tumor cells (5 x 10⁵) were cultivated in 6-well plates with 0.05 and 0.1 mM Rb4 peptide for 16 h at 37°C and 5% CO2. Treated and untreated cells (1 x 10⁶) were harvested with trypsin and were incubated with binding buffer (10 mM HEPES/NaOH, pH 7.5, 140 mM NaCl, and 2.5 mM CaCl₂) in the presence of 7-AAD and annexin V (Annexin V-PE Apoptosis Detection kit; Sigma) for 10 min at room temperature and analyzed by flow cytometry (Becton-Dickinson FACSCanto II apparatus) with FACSDiva software.

Cytosolic Ca²⁺ determinations in spectro fluorimeter

[75] B16F10-Nex2 cells (1 x 10⁶) , washed twice with HBSS buffer supplied with 1.3 mM CaC12, were incubated for 60 min at 37°C in the same buffer and 5 pM of the calcium indicator Fluo-4 AM (Molecular Probes) and 1 mM of probenecid (Sigma) , which minimizes indicator extrusion and compartmentalization. Subsequently, the cells were washed twice with the same buffer, without CaC12, and transferred to a quartz cuvette. Intracellular calcium was determined using a Hitachi F-7000 spectrof luorimeter (Tokyo, Japan) by continuous measurement of the fluorescence variation at Xex = 505 nm and Xem = 530 nm. The intracellular calcium increased with the addition of 10 pM thapsigargin (THG) . Rb4 peptide at 0.15 mM was added before and after THG. Maximal fluorescence (Fmax) was determined after the lysis of cells with 33.3 pM of digitonin (Sigma) , and minimal fluorescence (Fmin) was determined after adding 3 M EGTA in Tris, pH 8,7 until no further decrease in fluorescence was observed. AFU= arbitrary fluorescence units.

Confocal Microscopy for Actin Polymerization and Depolymeriza tion

[76] B16F10-Nex2 cells (1 xlO⁴) were plated on round glass coverslips and incubated overnight previous to incubation with 0.15 mM of Rb4 peptide for 15 and 20 h. After treatment, cells were washed three times with PBS, fixed with 3.7% paraformaldehyde for at least 30 min and permeabilized in 0.1% Triton X-100 for 30 min followed by blocking for 1 h with 150 mM NaCl, 50 mM Tris, and 0.25% BSA (Sigma-Aldrich) . Cells were stained with phalloidin-rhodamine (1:1000) (Invitrogen) for 1 h or with deoxyribonuclease I-Alexa Fluor 594 conjugate (27 pg/ml) (Invitrogen) for 1 h followed by 10 pg/ml DAPI (Invitrogen) for nucleic acid staining for 10 min. The coverslips were mounted on slides with Vectashield (Sigma) and analyzed in a Confocal Leica SP5 microscope, with a 63 X 1.4 oil objective; the Z series was obtained according to sampling criteria built in the software. DAPI was examined at 350-nm excitation and 470-nm emission and the phalloidin-rhodamine, for filamentous actin (F-actin) staining, or deoxyribonuclease I-Alexa Fluor, for globular actin (G-actin) staining, was examined at the excitation/emission at 540/565 nm and at 590/617.

Tumor cell lysate

[77] B16-Nex2 melanoma cells untreated and treated with the peptide were harvested and resuspended in PBS (5x 106 cells) with protease inhibitors. RIPA Lysis Buffer (Sigma-Aldrich) was used for cell disruption. Light microscopy and Trypan blue exclusion staining verified the method's efficiency. The cell lysate was kept at -80 °C for later use.

Western blotting analysis

[78] Western blottings were run with proteins from total cell lysates (40 pg) . They were separated by 10% SDS- polyacrylamide gel electrophoresis and transferred to Immobilon P transfer membrane (Millipore, Darmstadt, Germany) . The membranes were washed in Tris-buffered saline with Tween (lOmM Tris-HCl, pH 8, 150mM NaCl, and 0.05% Tween 20) and blocked overnight at 4°C with 5% nonfat milk in Trisbuffered saline with Tween 20. The blots were probed overnight at 4 °C with mAbs from Cell Signaling, Boston, MA; Bioss-bs336BR Woburn, MA; Santa Cruz, Dallas, TX; ABCAM, Cambridge, UK; as indicated. After 2h incubation with horseradish peroxidase-con ugated secondary antibody, immunoreactive proteins were detected by enhanced chemiluminescence (ECL; Amersham Biosciences, Little Chalfont, UK) . Bands densitometry was obtained using ImageJ software. Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA) .

Statistical Analysis

[79] Statistical significance was examined by the unpaired two-tailed Student's t-test. The Log-rank (Mantel-Cox) test was used to analyze long-term survival curves. The P- value<0.05 was considered as statistically significant. EXAMPLE 1

[80] Investigation on the cell growth and morphology was carried out in B16F10-Nex2 melanoma cells cultivated in RPMI medium with 0.15 mM Rb4 and Scr-Rb4, a scrambled Rb4 peptide, using a Nikon Biostation IMQ, an integrated cell incubator and microscopy system for long-term and multi-point live cell imaging. Pictures in Figure 1 were extracted from timelapse movies as shown in the Supplementary videos. Only the Rb4 sequence was able to interfere with melanoma morphology, replication and association. Rb4-treated cells did not replicate and rapidly formed clusters, absent in the control and Scr-Rb4-treated cells (Figure 1A) . Resistant Rb4 cells after 24 hours of incubation also lost their natural morphology and formed clusters between 36 and 72 hours of treatment (Figure IB) .

[81] Rb4 also inhibited other mouse and human cell lines, with different EC50. After 24 h under normal growth conditions (Table 1) , some human cell lines, uterine cervix cancer and glioblastoma, were more sensitive to Rb4, whereas others such as breast and human melanoma, were less sensitive. Murine colon and pancreatic tumor cell lines did not respond to Rb4. Mouse embryonic fibroblasts (MEF) were resistant to Rb4.

Table 1. Antiproliferative activity of Rb4 peptide on tumor cell lines and non-tumor forming cell lines. ± standard deviation ( s . d . ) .

*EC₅O is the concentration that decreases viability by 50% in a dose-dependent survival curve.

Cell Lineage

B16F10-Nex2 0.263 ± 0.585

HeLa 0.525 ± 0.160

U87 0.427 ± 0.053

A2058 0.744 ± 0.208

HCT-8 > 1

MCF7 0.724 ± 0.340

PANC02 > 1

CT26 > 1

MEF > 1

EXAMPLE 2

[82] The Rb4 peptide significantly (p<0.01) reduced the number of lung metastatic nodules in the syngeneic B16F10- Nex2 melanoma model (Fig. 2A) . Melanoma cells were injected intravenously in C57B1/ 6 mice and treatment consisted of 5 i.p. injections of 300 pg of Rb4 peptide or vehicle (10% water and 90% PBS) , in alternate days, starting the day after tumor cell challenge. The Rb4 protective activity was also evaluated using subcutaneous B16F10-Nex2 melanoma grafted syngeneic mice. The peptide was injected i.p., with 5 doses of 300 pg/animal in alternate days, which delayed the tumor growth up to 40 days (Fig. 3A) . Moreover, the survival rate of mice treated with Rb4 was significantly (p=0.0019) greater than that of vehicle control groups (Fig. 3B) , increasing group survival more than 25% and up to 10 days. In addition, this protective activity was absent in the immunocompromised NOD/SCID (Fig. 2B) . Like treatment in the immunocompetent C57B1/6 mice, immunocompromised NOD/SCID mice were inoculated intravenously with melanoma cells and treatment consisted in 5 i.p. injections of 300 pg of Rb4 peptide or vehicle (10% water and 90% PBS) , in alternate days, starting the day after tumor cell challenge. It should be noted that in all in vivo experiments, mice showed healthy physical appearance, normal activity levels and normal weight throughout the study period, demonstrating no toxic effects of peptides.

EXAMPLE 3

[83] Cellular phenotypic alterations induced by Rb4 peptide were observed in B16F10-Nex2 cells treated with 0.15 mM Rb4 but not with the scrambled-Rb4 (Scr-Rb4) peptide. TUNEL assay was used to measure internucleosomal DNA degradation (Fig. 4A) . Actinomycin (ACT) treatment, as positive control, stained positive in more than 90% of cells and the Rb4 peptide was also stained positive in approximately 75% of the B16F10-Nex2 cells. Vehicle treatment (PBS) or mock peptide (Scr-Rb4) were inactive, with a positive signal in less than 1% of total cells (Fig. 4A) . Along with this experiment, Rb4-treated melanoma cells were also examined by transmission electron microscopy (Fig. 4B) . Treated cells exhibited features of necrosis, such as the loss of plasma membrane integrity and absence of nuclear condensation. Transmission electron microscopy did not reveal morphological signs of apoptosis since nuclei appeared largely intact and major chromatin condensation was absent. The vast majority of cells adopted a necrotic morphology with plasma membrane disintegration (Fig. 4B-b,2) , swelling of mitochondria (Fig. 4B-c,l) with many hollowed areas, and vacuolated cytoplasm (Fig. 4B-d) . The cells were brightly fluorescent, Hoechst-stained nuclei appeared independent of chromatin condensation. Treatment of B16F10-Nex2 cells with 0.15 mM for 16 h led to rounded cells in clusters, with no chromatin condensation (Fig. 4C) . In addition, double staining with both Annexin V-PE and 7-AAD or 7-AAD alone identified these necrotic cells (Fig. 4D) . Briefly, melanoma cells were treated with 0.05 and 0.1 mM Rb4 during 16 h and stained with 7-AAD and/or Annexin V-PE. While the number of early apoptotic cells (annexin V positive, 7-AAD negative) was not considerably increased with the treatment (0.1 mM Rb4, 9.11 %; 0.05 mM Rb4, 7.64 %; untreated, 6.52 %, white bars) , the necrotic cells (annexin V positive, 7-AAD positive, black bars) did increase (0.1 mM Rb4, 22.3%; 0.05 mM Rb4, 18.6%; untreated 9.48%) in a dose-dependent manner. Altogether, these results revealed that Rb4 causes necrosis after 16 h in melanoma tumor cells.

EXAMPLE 4

[84] A necrotic cleavage of Poly (ADP-ribose) polymerase (PARP-1) has been characterized by Gobeil (2001, p.8) in Jurkat T cells treated with necrotic inducers such as O2, EtOH or HgC12. In our model, melanoma cells showed a degradation band (approximately 62 kDa) of cleaved PARP-1 when incubated with 0.15 mM Rb4 for 16 h and 18 h but not for 2, 4 or 6 h (Fig. 5A, 5B and supplementary data) . No signal of degradation was observed after Scr-Rb4 or PBS treatment for 16 h. The densitometry of degradation bands at 62 kDA showed that Rb4 increased more than 9-fold the signal when compared to PBS-mock treatment or Scr-Rb4 peptide incubation (Fig. 5B) . In addition, caspase-3 and -9 activity was measured using colorimetric and Western blotting (WB) assays; no caspase activation was detected under the same experimentation conditions (data not shown) . Since PARP-1- mediated necrosis might involve RIP1, we also investigated the protein expression by WB . While the expression of RIP1 was little increased at 6 h of incubation with Rb4, cells incubated 16 h with Rb4 surprisingly did not show RIP1 expression (Fig. 5C) . These results explain the results obtained with B16F10-Nex2 cells incubated with Rb4, pretreated with 100 pM of necrostatin-1 (Nec-1) , a RIP1 activity inhibitor. These cells continued to present the typical morphology of Rb4 treatment, which is clusters of round cells (Fig. 6A) . In addition, the cytotoxicity of Rb4 in melanoma cells was not prevented by preincubation with Nec-1 for 1 h (Fig. 6B) . These results showed that melanoma cells treated with Rb4 undergo PARP-l-mediated necrosis independently of RIP1.

EXAMPLE 5

[85] The endoplasmic reticulum (ER) plays a key role in maintaining Ca²⁺ homeostasis within the cell. This Ca²⁺ resource is vital for numerous signaling pathways including cell death. The ER has three ubiquitously expressed Ca²⁺ transporters: IP3-R (inositol 1 , 4 , 5-trisphosphate-receptor ) which releases Ca²⁺ from the ER, RyR (ryanodine receptor) also involved in releasing Ca²⁺ from ER, and SERCA ( sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase) transporter which acquires Ca²⁺ from cytoplasm, transferring it into the ER.

[86] Rb4 it is a synthetic peptide derived from the protein PLP2, a f our-transmembrane domain protein, which has been described to exhibit ion channel characteristics at the endoplasmic reticulum. We hypothesized that Rb4 treatment, added as a single peptide, might interfere in the calcium flux to the ER. We, therefore, examined the peptide effect on the cytosolic Ca²⁺ in B16F10-Nex2 cells (Fig. 7) . By using the fluo-4AM dye, we monitored the cytosolic Ca²⁺ levels in Ca²⁺-free medium after thapsigargin (THG) addition (Fig. 7A) , thus reflecting the kinetics of Ca²⁺ release after sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibition²⁵. In addition, B16F10-Nex2 cells were incubated with 0.15 mM Rb4 and pre-incubated or post-incubated with THG. Immediately after the incubation of Rb4, the Ca²⁺ peak increased and the cytosolic Ca²⁺ did not change in presence of THG (Fig. 7B) . The same was observed when Rb4 was incubated after THG (Fig. 7C) . These results suggest that the ER supplies the calcium and that Rb4 could function as a competitor of THG activity in ER calcium flux (inhibitor of SERCA transporter activity) resulting in the blocking of cytoplasmic Ca²⁺ uptake into ER. Altogether, the results suggest that Rb4 increases calcium in the cytoplasm of murine melanoma cells, which originates from ER.

[87] The importance of calcium for Rb4-cytotoxicity was also evaluated in B16F10-Nex2 cells pretreated for 1 h with 5 pM BAPTA-AM (an intracellular calcium chelator) before Rb4 incubation. B16F10-Nex2 cells treated with 0.15 mM for 24 h, preincubated with BAPTA-AM showed the same cellular morphological alterations caused by Rb4 (Fig. 6A) , and the decrease of Rb4-treated melanoma cells viability was not prevented by the calcium chelator (Fig. 6B) . This result demonstrates that calcium does not play a central role in Rb4-cytotoxicity in B16F10-Nex2 cells.

EXAMPLE 6

[88] The synthetic peptide Rb4 significantly reduced B16F10-Nex2 melanoma growth by triggering PARP-l-mediated tumor-cell necrosis. Furthermore, Rb4-treated melanoma cells show increased cytosolic Ca²⁺ released by the ER. Actin dynamics have been shown to regulate Ca²⁺ released from the ER. Furthermore, actin depolymerization and polymerization was described to regulate RIPl-independent necrotic death on U87MG cells expressing Bcl-xL elicited by a non-selective isopeptidase inhibitor.

[89] To address whether Rb4 influences actin polymerization, melanoma cells were evaluated by confocal microscopy after 15 h and 20 h of 0.15 mM Rb4 treatment with F-actin and G-actin staining. The complete loss of morphology was shown to be accompanied by depolymerization of almost all F-actin and the accumulation of G-actin on treated cells (Figure 8) suggesting that Rb4 significantly alters actin dynamics, polymerization, and the F-actin stabilization.

EXAMPLE 7

[90] Dying and stressed cells secrete, release, or undergo surface expression of DAMPs. Particular DAMPs serve as powerful immunological adjuvants and mediate immunogenic cell death (ICD) . The exposure and release of calreticulin and HMGB1 has been described on pro-apoptotic, post- apoptotic and/or necrotic cells.

[91] In our model, calreticulin surface expression was assessed by flow cytometry (Fig. 9A) . B16F10-Nex2 cells treated with Rb4 showed 1.77-fold increase of calreticulin on the cell surface as compared to non-treated cells. In parallel, we also examined the effects of the peptide on the HMGB1 release and surface exposure of calreticulin. With this purpose, B16F10-Nex2 cells were treated with 0.15 mM Rb4 for 16 h. ELISA was used to measure HMGB1 released into the medium (Fig. 9B) . The results showed that HMGB1 significantly (p<0.001) increased 3.54-fold in the medium of Rb4-treated B16F10-Nex2 cells compared with non-treated cells. The data shows Rb4 treatment could also promote increase in the expression of two DAMPs.

Claims

1. A peptide comprising or consisting of the amino acid sequence MADSERLSAPGCWAACTNFSRTRK (SEQ ID NO: 1) , or a sequence having at least 70% identity thereto.

2. The peptide of claim 1 wherein said peptide targets a tumor cell.

3. The peptide of claim 2 wherein said peptide has an antitumor activity.

4. The peptide of claim 1, wherein it is a synthetic peptide derived from the Proteolipid protein 2 (PLP2) .

5. The peptide of claims 1-4 for use in cancer treatment.

6. A pharmaceutical composition comprising an effective amount the peptide described in any one of claims 1-4 and pharmaceutical or veterinary acceptable vehicles.

7. The pharmaceutical composition according to claim 6, wherein the effective amount of the Rb4 peptide is at least 10 mg/kg.

8. The pharmaceutical composition according to claim 5 or 6, wherein the vehicles are selected from water and saline.

9. A method for treating and/or preventing cancer in a patient, the method comprising administering to the patient an effective amount of a peptide of claim 1 or a pharmaceutical composition of claim 6.

10. A method of modulating the immune system of a cancer patient, the method comprising administering to the patient an effective amount of a peptide of claim 1 or a pharmaceutical composition of claim 6.

11. The method of claim 9 or 10, wherein the pharmaceutical composition is for intravenous, subcutaneous or intraperitoneal administration.

12. The method of claim 9 or 10, wherein the cancer is selected from solid or soft tissue malignant tumors, sarcomas, carcinomas, lymphomas, leukemias, multiple myeloma, melanoma, brain and spinal cord tumors and head and neck tumors , optionally metastatic melanoma, subcutaneous melanoma, breast cancer, uterine cervix cancer, lung cancer and glioblastoma .

13 . The method of any one of claims 9- 12 , wherein the cancer is a drug-resistant cancer .

14 . The pharmaceutical composition of claim 6 for use in treating and/or preventing cancer, or for modulating the immune system of a patient under cancer treatment .

15 . The pharmaceutical composition of claim 6 for use in treating a cancer selected from metastatic and subcutaneous melanoma, breast cancer, uterine cervix cancer, lung cancer and glioblastoma .

16 . Use of the peptide of claim 1 for preparing a pharmaceutical composition as defined in claim 5 for treating and/or preventing cancer or modulating the immune system of a patient under cancer treatment .

17 . The use of claim 16 , wherein the cancer is selected from solid or soft tissue malignant tumors , sarcomas , carcinomas , lymphomas , leukemias , multiple myeloma, melanoma, brain and spinal cord tumors and head and neck tumors , optionally metastatic melanoma, subcutaneous melanoma, breast cancer, uterine cervix cancer, lung cancer and glioblastoma .

18 . The use of any one of claims 16 or 17 , wherein the cancer is a drug-resistant cancer .