Peptides and pharmaceutical compositions for tumor treatment

Abstract

(Peptides and pharmaceutical compositions for tumor treatment) The present invention relates to peptides comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1, with the proviso that said peptide does not have the amino acid sequence of SEQ ID NO: 2 or 3, and the cysteine ​​at position 6 of SEQ ID NO: 1 remains unchanged. The present invention also relates to pharmaceutical compositions thereof and to their use in the treatment of tumors, in particular in the treatment of cancer and metastases.

Description

[Technical field]

[0001] The present invention is in the field of biomedicine. In particular, the present invention relates to a peptide comprising the amino acid sequence of SEQ ID NO: 1, provided that said peptide does not have the amino acid sequence of SEQ ID NO: 2 or 3. Furthermore, the present invention relates to its use in the treatment of tumors, in particular in the treatment of cancer and metastasis. [Background technology]

[0002] The activity of the c-Src oncoprotein is particularly high in glioblastoma, the most aggressive primary brain tumor (Tabernero, 2022. Oncogene, 41(45): 4917-4928), which has a survival time of less than 2 years after diagnosis. Thanks to c-Src activity, glioblastoma stem cells, responsible for the recurrence and thus lethality of that tumor, can survive, proliferate, maintain a stem cell phenotype, and invade adjacent healthy tissue. c-Src interacts with ligands through its SH3 domain, which binds to proline-rich regions within target proteins.

[0003] Connexin 43 (Cx43) [NCBI accession number AAA52131, version AAA52131.1] is a membrane protein that is widely expressed in various mammalian tissues. In the central nervous system (CNS), Cx43 is mainly localized in astrocytes, the major type of glia, where it forms gap junctions (Gjs), intercellular communication channels that allow astrocytes to act in a coordinated manner. Since Lowenstein proposed that Gjs restricts cell proliferation in the 1960s, the role of Gjs and connexins in controlling cell proliferation has been widely studied.

[0004] Application WO2014191608 A1 discloses a cell-penetrating peptide (TAT-Cx43) in which the 266-283 sequence of connexin-43 (Cx43) is fused to the TAT-penetrating sequence. 266-283 Application WO2014191608 A1 describes the development of TAT-Cx43, which contains a canonical SH3 binding domain. 266-283have demonstrated that IL-1 binds to c-Src and its endogenous inhibitors Csk and PTEN. Binding of c-Src to its inhibitors in this region results in inhibition of c-Src activity (Gonzalez-Sanchez A. et al., Oncotarget, 7(31):49819-49833 (2016); Jaraiz-Rodriguez M. et al., J Vis Exp. (130):56457 (2017)) and subsequent antitumor effects.

[0005] In addition, TAT-Cx43 266-283 Like Cx43, it is able to inhibit c-Src activity and oncogenic processes that depend on the oncogenic activity of c-Src (Herrero-Gonzalez, S. et al., Oncogene, 29(42):5712-23 (2010); Gangoso, E. et al., Cell Death Dis. 5(1):e1023 (2014)), and therefore induces potent antitumor effects in human and mouse glioblastoma stem cells (Jaraiz-Rodriguez, M. et al., Stem Cell Reports 9, 451-463 (2017)), both in vitro and in vivo, without affecting healthy brain cells (Jaraiz-Rodriguez, M. et al., Neuro-Oncology, 22, 493-504 (2020)).

[0006] However, in the development of molecules with therapeutic potential, an important parameter to be considered is their size, since a large size is generally associated with poor membrane permeability, metabolic instability, increased undesirable interactions, increased side effects, reduced specificity, increased cost, or poor bioavailability.Therefore, in view of the above, there is a need in the art to provide alternative compounds with smaller size than existing ones that have antitumor effects and / or can reverse the phenotype of tumor stem cells. Summary of the Invention

[0007] As discussed in the Background, it is known that connexin-43 (Cx43) reverses the phenotype of human glioma stem cells, and that this effect resides in the carboxyl-terminal portion of Cx43.

[0008] In the present invention, the inventors have identified that even shorter amino acid sequences of Cx43 can maintain and even slightly enhance the antitumor effect in tumor stem cells. In particular, the inventors have shown that the sequence 266-275 (SEQ ID NO: 1) of Cx43, and peptides containing such sequences, such as peptides containing SEQ ID NO: 4, 5 and 6, reduce cell proliferation and survival (FIG. 5 and FIG. 6), as well as cell migration (FIG. 7) of tumor stem cells, such as glioblastoma stem cells. The inventors have found that the sequence 266-275 of Cx43 (SEQ ID NO: 1) is sufficient to bind to c-Src and its endogenous inhibitor Csk (FIG. 9). Furthermore, the sequence 266-275 (SEQ ID NO: 1) can bind to albumin, protecting the peptide from proteases and preventing rapid renal clearance (FIG. 8). Without intending to be bound by any theory, it is believed that the cysteine ​​at position 271 is necessary for intracellular interaction and albumin binding, possibly through a disulfide bond.

[0009] The amino acid sequence of the peptide of the present invention is SEQ ID NO: 3 (Cx43 266-283 ), despite the deletion of residues belonging to the canonical proline-rich region of Cx43, contained between amino acids 274-283 and responsible for binding to the SH3 domain of c-Src, it maintains and even slightly increases the antitumor effect.

[0010] These SH3 domains (Src homology 3) are involved in the regulation of important cellular pathways such as cell proliferation or migration, the dysregulation of which is closely related to tumor development. Binding of c-Src to inhibitors located within the 266-283 region of Cx43 leads to inhibition of c-Src activity, resulting in an antitumor effect. However, previous structural studies have demonstrated that the canonical SH3 domain binding region of c-Src (274-283) of Cx43 is not related to TAT-Cx43. 266-283 It has been shown that the antitumor effect of Cx43 is key to its antitumor effect, but the present inventors have shown that the antitumor effect resides in the sequence between amino acids 266-275 of Cx43 (SEQ ID NO: 1). Indeed, the present inventors found that the sequence between 266-275 of Cx43 (SEQ ID NO: 1) is sufficient to bind to c-Src and its endogenous inhibitor Csk (Figure 9). Furthermore, it was shown that cysteine ​​271 is required for this antitumor effect (Figures 1 and 9).

[0011] Developing peptides with shorter amino acid sequences that have anti-proliferative and cell migration reducing abilities (as in the case of the peptides of the present invention) offers the advantages of higher solubility, simpler folding, and improved cellular uptake, which are beneficial for administration to treat diseases involving cell proliferation or involving tumor stem cells, such as cancer, including metastatic cancer. Furthermore, having shorter peptides can prevent the appearance of undesirable effects due to the interaction of the remaining part of Cx43 with other cellular effectors. Finally, an additional advantage associated with the use of the peptides of the present invention is that the peptides can be added with a cellular internalization sequence, so that when administered to a subject, they can be internalized into the cell, and thus exert their effects without the need for gene therapy techniques.

[0012] Based on this, the inventors have developed a number of inventive aspects, which are described below:

[0013] (Peptides of the Invention) One aspect of the present invention is the peptide of SEQ ID NO: 1 (Cx43 266-275) [AYFNGCSSPT], provided that said peptide comprises or consists of an amino acid sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:2 (the carboxyl terminus of Cx43) or SEQ ID NO:3 (the carboxyl terminus of Cx43). 266-283 and the peptide described in SEQ ID NO: 1 does not have the amino acid sequence of the cysteine ​​at position 6 (C 271 ) remains unchanged.

[0014] The peptides of the invention may contain conservative amino acid substitutions in their amino acid sequence that maintain the peptide's ability to reverse tumor stem cell phenotype and / or inhibit cell proliferation, in particular to inhibit tumor stem cell proliferation. Thus, in the context of the present invention, peptides derived from the peptides of the invention, so-called "variants", are also encompassed by the term "peptides of the invention". These variants do not have 100% sequence identity with the peptides of the invention, but retain the ability to inhibit cell proliferation since the amino acids have been replaced by biologically similar ones. Tests to verify whether peptides derived from the peptides of the invention have the ability to reverse tumor cell phenotype, inhibit cell proliferation or reduce cell migration are described in the Examples section. Thus, the peptides of the invention comprise or consist of an amino acid sequence that has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 1, with the proviso that said peptides do not have the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, and do not have a cysteine ​​residue at position 6 of SEQ ID NO: 1 (C 271 In a more particular embodiment of the peptide of the invention, the alanine (A 266 ), tyrosine at position 2 of SEQ ID NO:1 (Y 267 ) and / or a phenylalanine at position 3 of SEQ ID NO:1 (F 268) remain unchanged. In a particular embodiment, the peptide of the invention comprises or consists of an amino acid sequence having 100% sequence identity to SEQ ID NO:1.

[0015] As used herein, the term "sequence identity" or "identity" refers to the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences, i.e.: the percentage of identical nucleotides or amino acids between two nucleotide sequences or polypeptides / proteins compared along the full length sequence. Depending on the number of common residues between the aligned sequences, various degrees of identity expressed as percentages are obtained. The degree of identity between two amino acid or nucleotide sequences can be determined by conventional methods, such as BLAST [Altschul SF et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3): 403-10]. BLAST programs, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN, are in the public domain at the website of The National Center for Biotechonology Information (NCBI). Those skilled in the art understand that mutations in the nucleotide sequence of a gene that result in conservative amino acid substitutions at positions that are not critical to the functionality of a protein are evolutionarily neutral mutations that do not affect its overall structure or functionality.

[0016] SEQ ID NO:2 shows the amino acid sequence and corresponds to the carboxyl terminus of Cx43 (Cx43CT): SPSKDCGSPKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNAKKVAAGHELQPLAIVDQRPSSRASSRASSRPRPDDLEI

[0017] SEQ ID NO: 3 shows the amino acid sequence and peptide Cx43 as described in WO2014191608 A1 266-283 Corresponding to: AYFNGCSSPTAPLSPMSP

[0018] As previously described, SEQ ID NO:1 (Cx43 266-275 Any peptide that contains the amino acid sequence of the above-mentioned nucleotide sequence has the ability to reverse the phenotype of tumor stem cells and / or inhibit cell proliferation, particularly tumor stem cell proliferation, as well as reduce cell migration.

[0019] The present inventors further experimentally demonstrated that other peptides, including SEQ ID NO: 1, also exhibit this effect. Therefore, in a particular embodiment, the peptide of the present invention has the amino acid sequence of SEQ ID NO: 4 (Cx43 266-277 ) [AYFNGCSSPTAP] or consisting of the amino acid sequence.

[0020] In another particular embodiment, the peptide of the present invention has the amino acid sequence of SEQ ID NO: 5 (Cx43 266-279 ) [AYFNGCSSPTAPLS] or consists of the amino acid sequence.

[0021] In another particular embodiment, the peptide of the present invention has the amino acid sequence of SEQ ID NO: 6 (Cx43 266-281 ) [AYFNGCSSPTAPLSPM] or consists of the amino acid sequence.

[0022] In the context of the present invention, a peptide is understood to be a molecule formed by linking 6 to 150 amino acids by peptide bonds. However, in certain embodiments, alone or in combination with the previous specific embodiments, the peptide of the present invention has a length of 10 to 100 amino acids (including the limit), more particularly 10 to 50 amino acids (including the limit), 10 to 40 amino acids (including the limit), 10 to 30 amino acids (including the limit), or 10 to 20 amino acids (including the limit). In a further specific embodiment, the peptide of the present invention has a length of 10 to 16 amino acids (including the limit). As will be appreciated by those skilled in the art, the smaller the peptide, the easier it is to administer and the fewer side effects it will cause.

[0023] The peptides of the present invention can be obtained by techniques widely known in the art. Examples of techniques for obtaining peptides include, but are not limited to, chemical or biological synthesis, genetic recombination or expression of a polynucleotide encoding the peptide of the present invention.

[0024] In addition, the carboxyl and amino termini of the peptides of the present invention may be protected from proteolysis. For example, the amino terminus may be in the form of an acetyl group, and / or the carboxyl terminus may be in the form of an amide group. Internal modifications of the peptides may be made to make them resistant to proteolysis, for example, at least one peptide bridge -CONH- is modified and replaced with a reduced bond (CH2NH), a retro-inverse bond (NHCO), an oxymethylene bond (CH2-O), a thiomethylene bond (CH2-S), a ketomethylene bond (CO-CH2), a hydroxyethylene bond (CHOH-CH2), an (NN) bond, an E-alkene bond, or a -CH=CH- bond. The amino acids of the peptides of the present invention may be in the D-configuration, which may result in the peptides being resistant to proteolysis. It is further contemplated in the present invention that the peptides of the present invention may be associated with a protection system or molecular vehicle, including, but not limited to, albumin. Peptides can also be stabilized by intramolecular cross-linking, for example by modifying at least two amino acid residues with olefinic side chains, preferably C3-C8 alkenyl chains, more preferably pent-2-yl chains, and then cross-linking the chains as described in the so-called "staple" technique (Walensky et al., 2004, Science 205: 1466-1470). All these peptides chemically modified to resist proteolysis are also subject of the present invention.

[0025] Additional modifications to the peptides of the present invention include covalently attaching polyethylene glycol (PEG) molecules to their carboxyl termini or lysine residues to reduce urinary excretion and therapeutic dose and increase the half-life of the peptides in plasma. The half-life of the peptides can also be increased by embedding them in biodegradable and biocompatible polymeric materials to form microspheres used as drug delivery systems. Polymers and copolymers are, for example, poly(D,L-lactide-co-glycolic acid) or PLGA. Techniques and procedures on how to manufacture lipid microspheres or nanocapsules for drug delivery are widely known to those skilled in the art. The peptides of the present invention can be associated with albumin or other systems to reduce urinary excretion, increase half-life, and / or improve pharmacokinetic profile. Any other method of pharmacological administration to selectively target tumor populations can be employed in the context of the present invention.

[0026] Moreover, the use of biotin in the delivery and / or targeting of molecules to tumor cells is well known in the art, since biotin receptors are overexpressed in tumor cells.Therefore, the binding of molecules such as peptides to biotin improves the delivery or targeting of molecules to tumor cells.Therefore, in a preferred embodiment, alone or in combination with all or each of the specific embodiments described above, the peptide of the present invention is covalently bound to biotin.

[0027] As understood by those skilled in the art, in order for the peptide of the present invention to reverse stem cell phenotype and / or inhibit cell proliferation, it is necessary for it to enter cells and interact with corresponding molecules, such as tyrosine kinase c-Src, its activated form Y416-Src, or its endogenous inhibitor Csk. The introduction of peptides into cells can be achieved by methods known in the art, including but not limited to direct injection, electroporation, transfection, etc. However, these are relatively complicated techniques and have limitations in their in vivo application to access the entire tumor cell population. When administering peptides to a subject, they can be introduced into cells by gene therapy techniques using viral vectors or by cell internalization sequences that allow the peptide to cross the cell membrane.

[0028] Thus, in certain embodiments, the peptides of the invention are covalently linked to a cell-internalizing amino acid sequence.

[0029] In the present invention, "cell-internalizing amino acid sequence" or "cell-internalizing sequence" or "cell-penetrating peptides" (CPPs) refers to amino acid sequences that have the ability to transport molecules across the cell membrane without losing its integrity. The most commonly used sequences include TAT, Antennapedia (Antp), and oligoarginine, which share the presence of a cationic amino acid group as a common feature. These internalization sequences allow the direct internalization of peptides into cells.

[0030] Examples of cell-internalizing sequences include RQIKIWFQNRRMKWKK (SEQ ID NO: 12), VKKKKIKREIKI (SEQ ID NO: 13) [Guergnon J, et al. 2006. Mol Pharmacol. 69(4): 1115-24], FFLIPKG (SEQ ID NO: 14) [Ueda et al. 2012. Biomaterials, 35: 9061], SMoCs [Okuyama et al. , 2007. Nature Methods, 4, 153 - 159], YGRKKKRRQRRR (SEQ ID NO: 7), DSLKSYWYLQKFSWR (SEQ ID NO: 15), KLWMRWWSPTTRRYG (SEQ ID NO: 16), RLWMRWYSPWTRRWG (SEQ ID NO: 17), RLIMRIYAPTTRRYG (SEQ ID NO: 18), RLYMRYYSPTTRRYG (SEQ ID NO: 19), RLWMRWYSPRTRAYG (SEQ ID NO: 20), KRPTMRFRYTWNPMK (SEQ ID NO: 21), WKCRRQCFRVLHHWN (SEQ ID NO: 22), WKCRRQAFRVLHHWN (SEQ ID NO: 23), WKARRQAFRVLHHWN (SEQ ID NO: 24), a penetration sequence in glioma cells (Berges et al. Plos One 2012; 7(11):e49436) and Biotin-YSSYSAPVSSSLSVRRSYSSSSGS-CONH2 (SEQ ID NO: 25).

[0031] In certain embodiments of the peptides of the invention, alone or in combination with all or each of the preceding specific embodiments, the cellular internalization sequence comprises or consists of the amino acid sequence (YGRKKRRQRRR) of SEQ ID NO: 7. Throughout this specification, the term "TAT sequence" or "TAT" is also used to refer to SEQ ID NO: 7.

[0032] The cellular internalization sequence may be attached to the peptide of the invention at either the amino or carboxyl terminus of the peptide. However, in certain embodiments of the peptide of the invention, either alone or in combination with all or each of the specific embodiments set forth above, the cellular internalization sequence is attached to the amino terminus of the peptide.

[0033] Therefore, in the present invention, the peptides of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 are found to be linked to a cell-internalizing sequence.

[0034] In certain embodiments, the peptide of the invention comprises or consists of the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11. SEQ ID NO:8-YGRKKRQRRAYFNGCSSPT SEQ ID NO:9 - YGRKKRQRRAYFNGCSSPTAP SEQ ID NO:10-YGRKKRQRRAYFNGCSSPTAPLS SEQ ID NO:11-YGRKKRQRRAYFNGCSSPTAPLSPM

[0035] As explained above, in the present invention, the "peptide of the present invention" refers to a peptide comprising or consisting of an amino acid sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 1, provided that the peptide does not have the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, and does not have a cysteine ​​residue at position 6 of SEQ ID NO: 1 (C 271 ) remain unchanged. Thus, peptides comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, and variants thereof, are encompassed by the terms "peptide of the invention" or "peptides of the invention", which are used interchangeably herein.

[0036] In a particular embodiment, alone or in combination with all or each of the above specific embodiments, the variant of the peptide of the invention comprises or consists of an amino acid sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11. In another particular embodiment, alone or in combination with all or each of the above specific embodiments, the peptide of the invention comprises or consists of an amino acid sequence having 100% sequence identity with SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11. The term "sequence identity" is defined in the preceding paragraph.

[0037] As indicated above, the peptides of the invention can be obtained by techniques well known in the art, such as the intracellular expression of a polynucleotide encoding the peptide of the invention and its subsequent isolation. Therefore, in another aspect, the present invention relates to a polynucleotide encoding the peptide of the invention or a variant thereof as defined above, hereinafter referred to as "polynucleotide of the invention".

[0038] The term "polynucleotide" as used herein refers to a polymer of nucleotides of any length, composed of ribonucleotides and / or deoxyribonucleotides. The term includes both single-stranded and double-stranded polynucleotides, as well as modified (methylated, protected, etc.) polynucleotides. The polynucleotides of the present invention can be DNA, RNA, or cDNA.

[0039] In another aspect, the present invention relates to a genetic construct comprising a polynucleotide of the present invention, hereinafter referred to as the "genetic construct of the present invention".

[0040] Preferably, the genetic construct comprises a polynucleotide of the invention operably linked to regulatory sequences for the expression of the polynucleotide of the invention. In principle, any promoter can be used in the genetic construct of the invention, provided that it is compatible with the cells in which it is desired to express the polynucleotide.

[0041] A promoter, or promoter region, is a sequence of nucleotides that controls the transcription of a given gene (nucleotide sequence). In the present invention, it refers to a nucleotide sequence that controls the transcription of a polynucleotide of the present invention. A promoter sequence can be unidirectional or bidirectional. A unidirectional promoter is one that controls the transcription of a gene or multiple genes that are located in tandem with it. "Tandem" means that the 3' end of a first gene is followed by the 5' end of a second gene, either consecutively or separated by a specific nucleotide sequence. A bidirectional promoter refers to a promoter region that controls the transcription of two opposite directions, such as the sequence preceding the kivD gene of L. lactis IFPL730, which acts as a bidirectional promoter. That is, a bidirectional promoter directs the transcription of two genes that are divergently or oppositely located, with the 5' ends of both nucleotide sequences being closer to each other than their 3' ends. In the present invention, the terms "promoter" and "promoter region" are used interchangeably. Furthermore, a promoter in the present invention may be constitutive or inducible. As used herein, the term "inducible" refers to the potential for a promoter to have control elements that allow transcription of the regulated gene to be turned on or off (repressed) in the presence of factors external to the promoter.

[0042] Suitable promoters for embodiments of the present invention include, but are not limited to, constitutive promoters derived from the genome of eukaryotic viruses, such as polyomavirus, adenovirus, SV40, CMV, avian sarcoma virus, and hepatitis B virus; constitutive promoters, such as metallothionein gene promoter, herpes simplex virus, and thymidine kinase gene promoter in the LTR region of retrovirus, immunoglobulin gene promoter, actin gene promoter, and EF-1α gene promoter; EF-1α gene promoter and inducible promoters that depend on the expression of proteins, immunoglobulin gene promoter, and actin gene promoter; EF-1α gene promoter and inducible promoters that depend on the addition of exogenous molecules or signals, such as the tetracycline system, the NFKB / VV light system, the Cre / Lox system, and promoters of heat shock genes; RNA polymerase II restricted promoters; and tissue-specific promoters. Glial cell-specific gene promoters include GFAP, nestin, and S-100. Glioma stem cell promoters include Id1 and Sox-2.

[0043] In addition, the genetic construct of the present invention may comprise or contain a marker or tag that allows for isolation of the peptide of the present invention synthesized within the cell.

[0044] On the other hand, the polynucleotide or gene construct of the invention may form part of a vector. Thus, in another aspect, the invention relates to a vector comprising a polynucleotide or gene construct of the invention, hereinafter referred to as "vector of the invention".

[0045] As will be understood by those skilled in the art, the type of vector used can be a cloning vector or an expression vector suitable for propagation.Thus, examples of suitable vectors according to the present invention include expression vectors in prokaryotes such as pUC18, pUC19, Bluescript and its derivatives, mp18, mp19, pBR322, phage and shuttle vectors such as pMB9, CoIEl, pCRl, RP4, pSA3 and pAT28, yeast expression vectors such as 2 micron plasmid type vectors, integrative plasmids, YEP vectors, centromeric and similar plasmids, insect cell expression vectors such as pAC series vectors, pVL series vectors, plant expression vectors such as pIBI series vectors, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and similar, as well as viral vectors (adenovirus, adenovirus-associated virus, retrovirus, lentivirus) and pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg. These include, but are not limited to, expression vectors in higher eukaryotic cells based on non-viral vectors such as pHCMV / Zeo, pCR3.1, pEFVHis, pIND / GS, pRc / HCMV2, pSV40 / Ze02, pTRACER HCMV, pUB6N5-His, pVAXl, pZeoSV2, pCl, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl.

[0046] In a specific embodiment, alone or in combination with all or each of the preceding specific embodiments, the vector of the invention is a viral vector, preferably a retroviral, lentiviral or adenoviral viral vector.

[0047] The vector of the present invention can be used to transform, transfect or infect cells that are susceptible to transformation, transfection or infection by said vector. Such cells may be prokaryotic or eukaryotic. As an example, the vector into which the nucleotide sequence, preferably DNA, is introduced may be a plasmid or vector that, when introduced into a host cell, is integrated into the genome of the cell and replicates together with the chromosome into which it (or they) are integrated. Such vectors can be obtained by conventional methods known to those skilled in the art.

[0048] Therefore, another aspect of the present invention relates to a cell comprising a polynucleotide, a genetic construct or a vector of the present invention, hereinafter referred to as "cell of the present invention", such a cell may be transformed, transfected or infected with a construct or vector provided by the present invention. Transformed, transfected or infected cells can be obtained by conventional methods known to those skilled in the art. In a particular embodiment, the host cell is an animal cell transfected or infected with a suitable vector.

[0049] Suitable host cells for expressing the peptides of the present invention include, but are not limited to, mammalian, plant, insect, fungal and bacterial cells. Bacterial cells include, but are not limited to, gram-positive bacterial cells such as Bacillus, Streptomyces and Staphylococcus species, and gram-negative bacterial cells such as Escherichia and Pseudomonas cells. Fungal cells preferably include yeast cells such as Saccharomyces, Pichia pastoris and Hansenula polymorpha. Insect cells include, but are not limited to, Drosophila cells, Sf9 cells, and the like. Plant cells include, but are not limited to, cells of crop plants such as cereals, medicinal plants, ornamental plants, and bulbous plants. Suitable mammalian cells for use in the present invention include epithelial cell lines (e.g., porcine), osteosarcoma cell lines (e.g., human), neuroblastoma cell lines (e.g., human), epithelial carcinoma cell lines (e.g., human), glial cells (e.g., murine), hepatic cell lines (e.g., monkey), CHO cells (e.g., Chinese homozygous), hepatic cells (e.g., monkey), and Sf9 cells (e.g., human), CHO (Chinese Hamster Ovary) cells, COS cells, BHK cells, HeLa cells, 911, AT1080, A549, 293 or PER.C6 cells, human ECCs 5 NTERA-2 cells, mESCs line D3 cells, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3, 293T, REH, and MCF-7 cells, and hMSCs.

[0050] The inventors have discovered that the peptides of the present invention are capable of reversing the phenotype of tumor stem cells and / or inhibiting cell proliferation, in particular inhibiting proliferation of tumor stem cells, such as glioma stem cells, and reducing their cell migration.Furthermore, the peptides, polynucleotides, gene constructs, vectors or cells of the present invention may be part of a composition, preferably a pharmaceutical composition, as an active agent or ingredient.

[0051] Therefore, another aspect of the present invention relates to a composition comprising a peptide, a polynucleotide, a genetic construct, a vector or a cell of the invention, hereinafter referred to as the "composition of the invention". In a particular embodiment, the composition of the invention is a pharmaceutical composition.

[0052] In certain embodiments, the compositions of the invention comprise or consist of a therapeutically effective amount of a peptide, polynucleotide, genetic construct, vector or cell of the invention, and a vehicle, which in the case of a pharmaceutical composition is a pharma- ceutically acceptable vehicle.

[0053] In the present invention, a "pharmaceutical composition" means any medicinal preparation or form, the formulation of which contains a substance or mixture of substances having a certain weight, volume and percentage expressed in units of the international system, manufactured in a legally established pharmaceutical laboratory, packaged or labeled for distribution, and sold as being effective in the diagnosis, treatment, mitigation and prevention of diseases, physical abnormalities or symptoms, or in restoring balance, correcting or altering the function of organs in humans and animals. The preparation of the pharmaceutical composition can be carried out by any of the methods described in the prior art.

[0054] The expression "effective amount" as used herein means the amount of the compound or pharmaceutical composition of the present invention that produces the desired effect, and is generally determined by, among other things, the properties of the compound or pharmaceutical composition and the therapeutic effect to be achieved. The dosage to obtain a therapeutically effective amount depends on various factors, such as the age, weight, sex or tolerance of the mammal. The "pharmaceutical acceptable adjuvants" and "pharmaceutical acceptable vehicles" that can be used in such compositions are vehicles known in the prior art.

[0055] The term "vehicle" refers to a diluent or excipient used in administering an active substance. Such vehicles may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solutions of saline, dextrose, and glycerol are preferably used as vehicles, particularly for injections. Preferably, for pharmaceutical vehicles, they are approved by state or federal regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans. The vehicles and auxiliary substances required to prepare the desired pharmaceutical dosage form of the pharmaceutical composition of the present invention depend, inter alia, on the pharmaceutical dosage form selected. The pharmaceutical dosage form of such pharmaceutical composition is prepared according to conventional methods known to those skilled in the art.

[0056] Furthermore, in the present invention, it is contemplated that the peptide of the present invention may be associated with a delivery system or molecular vehicle, including but not limited to exosomes and microvesicles.These delivery systems or molecular vehicles, due to their characteristics, can deliver their "cargo", in the present invention, the peptide of the present invention, across cell membranes in a biologically active form, and can also cross biological membranes such as the blood-brain barrier.Therefore, in certain embodiments, alone or in combination with all or each of the above specific embodiments, the composition of the present invention further comprises exosomes and / or microvesicles.

[0057] The compositions of the present invention can be formulated in various forms known in the art for administration to animals, more preferably mammals, including humans. Thus, they can be in aqueous or non-aqueous solutions, emulsions, or suspensions, but are not limited to these. Examples of non-aqueous solutions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, or injectable organic esters, such as ethyl oleate. Examples of aqueous solutions include, but are not limited to, water, alcoholic solutions in water, or saline. Aqueous solutions can be buffered or unbuffered, and can have additional active or inactive ingredients. Additional ingredients can include preservatives, including, but not limited to, salts to adjust ionic strength, antibacterial agents, antioxidants, chelating agents, and the like, or nutrients, including glucose, dextrose, vitamins, minerals, and the like. Alternatively, the compositions can be prepared for administration in solid form. The compositions may be combined with a variety of vehicles or inert excipients, including, but not limited to, binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; and flavorings such as peppermint or methyl salicylate.

[0058] In addition, the composition of the present invention may contain an adjuvant. By "adjuvant" is meant any substance that enhances the effectiveness of the pharmaceutical composition of the present invention. Examples of adjuvants include adjuvants consisting of aluminum (alum) salts, such as aluminum hydroxide, aluminum phosphate, or aluminum sulfate, oil-in-water or water-in-oil emulsion formulations, such as Freund's complete adjuvant (ACF) and incomplete Freund's adjuvant (AIF), mineral gels, gels, block copolymers, Avridine™, SEAM62, liposaccharides (e.g., lipid A or monophosphoryl lipid A (MLA)), trehalose dimycolate (TDM), and cell wall skeleton components (CWS). adjuvants consisting of bacterial cell wall components, such as adjuvants containing diphtheria toxin (DT), pertussis toxin (PT), cholera toxin (CT), E. coli heat labile toxins (LT1 and LT2), endotoxin A and Pseudomonas exotoxin, B. cereus exotoxin B, B. sphaerieus toxin, C. botulinum toxins C2 and C3, C. limosum exotoxin, and the toxins of C. perfringens, C. spiriforma and C. diffieile, and S.adjuvants derived from ADP-ribosylating bacterial toxins, including toxin mutants such as A. aureus, EDIM, CRM-197, and atoxic mutant diphtheria toxin; saponins such as ISCOMs (immunostimulating complexes), e.g., interleukins (IL-Is); IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, etc.), interferons (e.g., interferon gamma), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), defensin 102, RANTES, MIP1-α, and MEP-2, as well as chemokines and cytokines, such as muramyl peptides, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetyl-muramyl-L-alanyl-disoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3 hydroxyphosphoryloxy)ethylamine (MTP-PE), adjuvants from the CpG family of molecules, synthetic CpG dinucleotides and oligonucleotides containing CpG motifs, lysosomal exoenzyme from C. Limosum, and synthetic adjuvants such as PCPP, cholera toxin, Salmonella toxin, alum and the related aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE, and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 12% / Tween 80 squalene emulsion. Other examples of adjuvants include, but are not limited to, DDA (dimethyldioctadecylammonium bromide), complete and incomplete Freund's adjuvant, QuilA, microvesicles, exosomes, etc.

[0059] The term "pharmaceutical acceptable" means that the vehicle or excipient should allow the activity of the compound of the pharmaceutical composition, in particular the peptide of the present invention, i.e., should be compatible with those components so as not to be harmful to the organism to which it is administered.

[0060] In certain embodiments, alone or in combination with all or each of the preceding specific embodiments, the composition of the invention, more particularly the pharmaceutical composition of the invention, further comprises a chemotherapeutic agent.

[0061] "Chemotherapeutic agent" refers to a substance capable of inhibiting cell proliferation without necessarily killing the cell or capable of inducing cell death. Agents capable of inhibiting cell proliferation without causing cell death are commonly referred to as cytostatic agents, whereas agents capable of inducing cell death, usually by activating apoptosis, are commonly referred to as cytotoxic agents. Non-limiting examples of chemotherapeutic agents suitable for use in the compositions of the invention include: (i) microtubule stabilizing agents such as taxanes, paclitaxel, docetaxel, epothilones, laulimalide, etc.; (ii) cephalosporins, such as Iressa®, Gleevec, Tarceva™, (Erlotinib™, etc.); (iii) kinase inhibitors such as Trastuzumab (Herceptin®), Erlotinib, Gefitinib, Cetuximab (Erbitux®), Bevacizumab (Avastin™), Rituximab (ritusan®), Pertuzumab (Omnitarg™); (iv) mTOR pathway inhibitors such as rapamycin and CCI-778; (v) Apo2L1 Trail; (vi) endostatin, combrestatin, angiostatin, thoron ... (vii) anti-angiogenic agents such as vasospondin, vascular endothelial growth inhibitors (VEGI); (viii) anti-tumor vaccines including activated T cells, non-specific immune enhancers (interferons, interleukins, etc.) or other immunotherapies; (viii) antibiotic cytotoxic agents such as doxorubicin, bleomycin, dactinomycin, daunorubicin, epirubicin, mitomycin, mitoxantrone; (ix) alkylating agents such as Melphalan, Carmustine, Lomustine, cyclophosphamide, ifosfamide, chlorambucil, fotemustine, busulfan, temozolomide, thiotepa;(x) Anticancer hormones such as Nilutamide, Cyproterone acetate, Anastrozole, Exemestane, Tamoxifen, Raloxifene, Bicalutamide, Aminoglutethimide, Leuprorelin acetate, Toremifene citrate, Letrozole, Flutamide, Megestrol acetate, and Goserelin acetate; (xi) Gonadal hormones such as Cyproterone acetate and Medoxyprogesterone acetate; (xii) Cytarabine, Fluorouracil, Gemcitabine, Topotecan, Hydroxyur (xiii) anabolic agents such as nandrolone; (xiv) adrenal steroid hormones such as methylprednisolone acetate, dexamethasone, hydrocortisone, prednisolone, prednisone; (xv) antineoplastic agents such as temozolamide, carboplatin, cisplatin, oxaliplatin, etoposide, and dacarbazine; and (xvi) topoisomerase inhibitors such as topotecan, irinotecan.

[0062] Such compositions and / or their formulations can be administered to animals, including mammals, thus humans, in various forms, including, but not limited to, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraarticularly, oral, enteral, parenteral, intranasal, intraocular or topically.Preferred administration routes of compositions and / or formulations of the compounds of the present invention for preventing or treating cancer are intratumoral and intraperitoneal routes.In certain embodiments, alone or in combination with all or each of the above specific embodiments, the compositions of the present invention are formulated for oral, parenteral, nasal, sublingual or intratumoral administration.

[0063] Therapeutic Uses of the Invention As explained above, the peptides of the invention, as well as their related aspects, the polynucleotides, gene constructs, vectors, cells of the invention, are capable of reversing the phenotype of tumor stem cells and / or inhibiting cell proliferation, in particular inhibiting the proliferation of tumor stem cells, such as glioma stem cells, and reducing their cell migration.Similarly, the peptides, polynucleotides, gene constructs, vectors or cells of the invention may be part of a composition, preferably a pharmaceutical composition, having application in the treatment of tumors or cancers present in a subject.

[0064] In the present invention, the term "subject" refers to any animal, preferably a mammal, more preferably a primate, and particularly a human, regardless of species, sex, or age.

[0065] Therefore, another aspect of the present invention relates to a peptide, a polynucleotide, a genetic construct, a vector, a cell or a composition according to the invention for use as a medicament.

[0066] The term "pharmaceutical product" as used herein refers to any composition / substance used for the prevention, diagnosis, mitigation, treatment or cure of disease in a subject, or that may be administered to a subject for the purpose of restoring, correcting or altering physiological function by exerting a pharmacological, immunological or metabolic effect.

[0067] In another aspect, the present invention relates to a peptide, a polynucleotide, a genetic construct, a vector, a cell or a composition of the invention for use in the treatment of a benign or malignant tumor.

[0068] In the present invention, "treatment" (or "treat" or "treating") refers to a process that includes slowing, inhibiting, arresting, controlling, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, but does not necessarily include the complete elimination of all symptoms, conditions, or disorders associated with the disease. Treatment of a disorder or disease results, for example, in the cessation of the progression of the disorder or disease (e.g., no worsening of symptoms), or in the slowing of the progression of the disorder or disease (if the cessation of progression is transient). "Treatment" of a disorder or disease may also result in a partial response (e.g., improvement of symptoms such that the progression of the disorder or disease is halted or slowed) or a complete response (e.g., disappearance of symptoms) in the subject / patient suffering from the disorder or disease. Such partial or complete responses may subsequently recur. It is understood that a subject / patient may experience a wide range of responses to the treatment (such as the exemplary responses as described herein above). In the context of the present invention, the disease or disorder is a benign or malignant tumor, such as cancer.

[0069] In the present invention, "tumor" refers to a proliferation of tissue resulting from unregulated proliferation of cells. Tumors can be benign or malignant. A tumor is considered benign if the cells forming the tumor do not invade other tissues or metastasize to other parts of the body. Usually, benign tumors are well encapsulated and the cells do not show structural changes. In contrast, a tumor is considered malignant if the tumor cells proliferate rapidly, show undifferentiation, and / or are capable of invading the tissue, invading adjacent tissues, or even spreading to other parts of the body, a process known as metastasis. Generally, malignant tumors are known as cancers. Thus, in certain embodiments, the malignant tumor is a cancer, and in other more specific embodiments, the cancer is a metastatic cancer.

[0070] On the other hand, tumor or cancer (if malignant) can be located or occur in any tissue or organ of the body. Therefore, any tumor or cancer can be treated by the pharmaceutical composition of the present invention, regardless of its development stage, its origin or its location. Examples of cancer include, but are not limited to, lung cancer, colon cancer, skin cancer, pancreatic cancer, gastric cancer, breast cancer, prostate cancer, liver cancer or cervical cancer. However, in certain embodiments, the tumor (benign or malignant) is a central nervous system (CNS) tumor (i.e., located in the CNS), and in more specific embodiments, the tumor is a brain tumor, or a spinal tumor (benign or malignant).

[0071] In another specific embodiment, the tumor is a glioma. The term "glioma" as used herein refers to a type of neoplasm that occurs in the brain or spinal cord. It is called glioma because it originates from glial cells. It most frequently occurs in the brain, but it can also occur in the spinal cord. In the present invention, the composition of the present invention can be used to treat any glioma.

[0072] The new 2021 WHO classification of tumors of the central nervous system places at least as much emphasis on molecular changes as on histopathological features in classifying gliomas. Gliomas may be named according to the particular cell type they most closely resemble, such as ependymoma (ependymal cells), astrocytoma (astrocytic cells), or oligodendroglioma (oligodendrocytes), and may be graded by grade from 1 to 4, with 4 being the most aggressive. Thus, in certain embodiments, the glioma is a glioma selected from the group consisting of astrocytoma, oligodendroglioma, glioblastoma, and ependymoma. In even more particular embodiments, the tumor is a glioblastoma, the most aggressive (grade 4) and frequent primary brain tumor.

[0073] Tumor stem cells or cancer stem cells are known in the art to have typical properties of stem cells, i.e., the ability to self-renew and differentiate into multiple cell types, as well as to be resistant to conventional treatments and persist in tumors as a separate population, leading to tumor recurrence and metastasis through the growth or development of new tumors. Thus, in certain embodiments, alone or in combination with all or each of the above specific embodiments, the tumor is composed of stem cells, preferably tumor stem cells.

[0074] In more particular embodiments, the tumor stem cell is a glioma stem cell, and more particularly, the glioma stem cell is an astrocytoma stem cell, an oligodendroglioma stem cell, a glioblastoma stem cell or an ependymoma stem cell. Examples of glioblastoma stem cells include, but are not limited to, G166 cells, G179 cells, GliNS2 cells or G144 cells (Pollard SM et al., Cell Stem Cell, 4, 568-580 (2009)), primary glioma stem cells obtained from glioblastomas of patients generally immediately after surgery, or mouse glioblastoma stem cells. Therefore, in particular embodiments, the glioblastoma stem cell is selected from the list consisting of G166 cells, G179 cells, GliNS2 cells, G144 cells or other primary human glioblastoma stem cells. Preferably, the glioblastoma stem cell is a G166 cell.

[0075] As explained above, the peptides of the invention and their related aspects (polynucleotides, polynucleotides, genetic constructs, vectors or cells of the invention) which form part of a composition, preferably a pharmaceutical composition, and which may be the active agent or active ingredient thereof, are capable of reversing the phenotype of tumor stem cells and / or inhibiting cell proliferation, in particular inhibiting the proliferation of tumor stem cells, such as glioma stem cells, as well as reducing their cell migration.

[0076] Therefore, one aspect of the present invention relates to a peptide, a polynucleotide, a genetic construct, a vector, a cell or a composition of the present invention for use in reversing the phenotype of tumor stem cells and / or inhibiting cell proliferation, in particular for inhibiting the proliferation of stem cells, more particularly tumor stem cells, even more particularly glioma stem cells. In another particular embodiment, the glioma stem cells are astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells or ependymoma stem cells.

[0077] Moreover, as shown in the examples illustrating the invention, the peptides of the invention reduce the migration of human glioma stem cells, which are responsible for the invasion (metastasis) of other surrounding tissues or other parts of the body, increasing the chances of escaping surgery and causing the recurrence of these tumors.

[0078] Therefore, another aspect of the present invention relates to a peptide, a polynucleotide, a gene construct, a vector, a cell or a composition of the present invention for use in preventing the proliferation, migration and / or metastasis of tumor cells. In a particular embodiment, the tumor cell is a stem cell. In another more particular embodiment, the stem cell is a glioma stem cell. In another particular embodiment, the glioma stem cell is an astrocytoma stem cell, an oligodendroglioma stem cell, a glioblastoma stem cell or an ependymoma stem cell.

[0079] In certain embodiments, the glioblastoma stem cells are selected from the list consisting of G166 cells, G179 cells, GliNS2 cells and G144 cells.Preferably, the glioblastoma stem cells are G166 cells.

[0080] Use of the peptides of the invention and embodiments derived therefrom in the manufacture of pharmaceuticals. The inventors have shown that the peptides of the invention are capable of reversing the phenotype of tumor stem cells and / or inhibiting cell proliferation, in particular tumor stem cells such as glioma stem cells, and reducing their cell migration, which allows the peptides of the invention to be used in the treatment of tumors or cancers present in a subject.

[0081] Therefore, in another aspect, the present invention relates to the use of a peptide, a polynucleotide, a genetic construct, a vector, a cell or a composition of the present invention in the preparation / manufacture of a medicament / pharmaceutical composition. Techniques and procedures for producing pharmaceutical compositions have been described herein above.

[0082] In another aspect, the present invention relates to the use of a peptide, a polynucleotide, a genetic construct, a vector, a cell or a composition of the invention in the preparation of a medicament / pharmaceutical composition for the treatment of a benign or malignant tumor.

[0083] The terms "treatment", "tumor", "benign tumor" or "malignant tumor" have already been defined or explained above, and both their preferred embodiments are applicable to aspects of the present invention.

[0084] Thus, in one particular embodiment, the malignancy is a cancer, and in another more particular embodiment, a metastatic cancer.

[0085] In particular embodiments, the tumor (benign or malignant) is a central nervous system (CNS) tumor (i.e., located in the CNS), and in more particular embodiments, the tumor is a brain tumor, or a spinal tumor (benign or malignant).

[0086] In another particular embodiment, the tumor is a glioma. In a more particular embodiment, the glioma is a glioma selected from the group consisting of astrocytoma, oligodendroglioma, glioblastoma, and ependymoma. More particularly, the glioblastoma is glioblastoma multiforme.

[0087] In another particular embodiment, the tumor comprises stem cells, preferably tumor stem cells, more preferably glioma stem cells, even more preferably glioma stem cells, wherein the glioma stem cells are astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells or ependymoma stem cells.

[0088] As explained above, the peptides of the invention, as well as related embodiments thereof, the polynucleotides, genetic constructs, vectors, cells or compositions of the invention, are capable of reversing the phenotype of tumor stem cells and / or inhibiting cell proliferation, in particular inhibiting the proliferation of tumor stem cells, more particularly glioma stem cells, even more particularly astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells or ependymoma stem cells.

[0089] Therefore, in another aspect, the present invention relates to the use of a peptide, a polynucleotide, a genetic construct, a vector or a cell of the present invention in the preparation / manufacture of a medicament / pharmaceutical composition for reversing the phenotype of tumor stem cells and / or inhibiting cell proliferation, in particular stem cells, more particularly tumor stem cells, even more particularly glioma stem cells, even more particularly glioma stem cells being astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells or ependymoma stem cells.

[0090] Moreover, as shown in the examples illustrating the invention, the peptides of the invention reduce the proliferation and migration of human glioma stem cells. Therefore, another aspect of the invention relates to the use of the peptides, polynucleotides, gene constructs, vectors, cells or compositions of the invention in the preparation / manufacture of a medicament / pharmaceutical composition for preventing the proliferation, migration and / or metastasis of tumor cells. In a particular embodiment, the tumor cells are stem cells. In another more particular embodiment, the stem cells are glioma stem cells. In a more particular embodiment, the glioma stem cells are astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells or ependymoma stem cells.

[0091] (In vitro use of the peptides of the present invention) In addition to the above therapeutic applications, the peptides of the present invention and the inventive aspects derived therefrom can also be applied to in vitro assays.Therefore, another aspect of the present invention relates to the use of the peptides, polynucleotides, gene constructs, vectors, cells or compositions of the present invention for inhibiting cell proliferation in vitro and / or for reversing the phenotype of tumor stem cells in vitro.In a particular embodiment, the tumor stem cells are glioma tumor stem cells.In a more particular embodiment, the tumor stem cells are astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells or ependymoma stem cells.

[0092] In the present invention, "reversal of tumor stem cell phenotype" means that tumor stem cells lose their phenotypic characteristics, i.e., high tumorigenicity (ability to give rise to malignant tumors) and resistance to conventional therapies in tumor treatment. Molecularly, tumor stem cells are characterized by high expression of Id1, Sox2 and N-cadherin, low expression of Cx43 and E-cadherin, and high activity of c-Src. Therefore, in the present invention, "a peptide has the ability to reverse tumor stem cell phenotype" means that the peptide causes the tumor stem cells to lose the phenotypic characteristics when it comes into contact with the tumor stem cells. Examples of assays for checking whether tumor stem cell phenotype has been reversed include immunocytochemical, PCR or Western blot investigations of the expression of Id1, Sox2, N-cadherin, E-cadherin or other stem cell markers.

[0093] In the present invention, "inhibition of cell proliferation" refers to a reduction, decrease, attenuation or blockage of cell division or cell cycle. An assay for checking inhibition of cell proliferation may, for example, be the MTT colorimetric assay.

[0094] In another aspect, the present invention relates to the use of a peptide, a polynucleotide, a genetic construct, a vector, a cell or a composition of the invention for the in vitro identification of compounds that regulate or modulate tumorigenicity.

[0095] In a particular embodiment of the in vitro use of the peptide of the invention, alone or in combination with all or each of the particular embodiments above, the concentration of the peptide of the invention is between 1 and 220 μM. In another more particular embodiment, the concentration of the peptide of the invention is between 10 and 200 μM. In yet another more particular embodiment, the concentration of the peptide of the invention is between 40 and 60 μM, including the values ​​40 and 60 μM. More particularly, the concentration of the peptide of the invention is between 45 and 55 μM, including the values ​​45 and 55 μM. More particularly, the concentration of the peptide of the invention is between 46, 47, 48, 49, 49, 50, 51, 52, 53, 54 or 55 μM. Preferably, the concentration of the peptide is 50 μM.

[0096] (Kit of the present invention and its uses) The administration of a peptide, polynucleotide, genetic construct, vector, cell or pharmaceutical composition of the invention may require a number of components, which may be arranged together in the form of a kit.

[0097] Therefore, in another aspect, the present invention relates to a kit comprising a peptide, a polynucleotide, a genetic construct, a vector, a cell or a composition of the invention, hereinafter referred to as a "kit of the invention".

[0098] Components that are useful for administration and may be included in the kit include, but are not limited to, buffers, dissolving solutions, sterile materials (such as syringes, swabs or forceps), distilled water or alcohol (ethanol). In addition, the kit may include instructions or directions to guide a skilled artisan in its administration.

[0099] Kits of the invention that have utility for use in the administration of the peptides, polynucleotides, genetic constructs, vectors, cells or compositions of the invention may also be used for in vitro assays.

[0100] Therefore, another aspect of the present invention relates to the use of the kit of the present invention in the in vitro determination of the effect of peptides on the tumorigenicity of cell lines, for inhibiting cell proliferation in vitro and / or for reversing the tumor stem cell phenotype.

[0101] In certain embodiments of the kit of the present invention, the inhibition of cell proliferation comprises the inhibition of cell proliferation of tumor stem cells, preferably glioma stem cells, more preferably the glioma stem cells are astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells or ependymoma stem cells.

[0102] The terms used to define the kits of the invention and the uses of the kits have been explained above, and both of them and their preferred embodiments are applicable to the various uses of the kits of the invention.

[0103] (Treatment / Prevention Method of the Present Invention) In another aspect, the present invention relates to a method for the treatment and / or prevention of tumors, both benign and malignant (cancer), in a subject, comprising administering to said subject a peptide, polynucleotide, genetic construct, vector, cell or composition of the invention.

[0104] Another aspect of the invention relates to a method for preventing tumor cell proliferation, migration and / or metastasis in a subject, comprising administering to said subject a peptide, polynucleotide, genetic construct, vector, cell or composition of the invention.

[0105] The terms defined and explained for the other aspects of the invention and its preferred embodiments are also applicable to the therapeutic and / or prophylactic methods of the invention. [Brief description of the drawings]

[0106] [Figure 1]Effect of cysteine ​​271 of Cx43 sequence 266-283 on survival and proliferation of human glioblastoma stem cells. G166 human glioblastoma stem cells were treated in the absence (control) or presence of TAT-Cx43 266-283 or the same peptide with cysteine ​​271 substituted with alanine (TAT-Cx43 266-283, C / A), both at a concentration of 50 μM. 48 hours after the start of treatment, micrographs were taken and cell viability was analyzed by MTT assay. Results are expressed as percentage of control, mean ± SEM (n=3). Significance of difference from control is expressed as ***p<0.001 (ANOVA). [Diagram 2] Comparison of the effects of TAT-Cx43271-287 vs. TAT-Cx43266-283 on human glioblastoma stem cell survival and proliferation. G166 human glioblastoma stem cells were treated in the absence (control) or presence of TAT-Cx43266-283 or TAT-Cx43271-287, both at a concentration of 50 μM. 72 hours after the start of treatment, micrographs were taken and cell viability was analyzed by MTT assay. Results are expressed as percentage of control, mean ± SEM (n=3). Significance of difference from control is expressed as ***p<0.001 (ANOVA). [Diagram 3] Comparison of the effects of TAT-Cx43268-283 vs. TAT-Cx43266-283 on human glioblastoma stem cell survival and proliferation. G166 human glioblastoma stem cells were treated in the absence (control) or presence of TAT, TAT-Cx43266-283 or TAT-Cx43268-283, all at a concentration of 50 μM. 72 hours after the start of treatment, cell viability was analyzed by MTT assay. Results are expressed as percentage of control and are mean ± SEM (n=3). Significance of difference from control condition is expressed as ***p<0.001, significance of difference from TAT condition is expressed as ###p<0.001 (ANOVA). [Figure 4]Effect of phenylalanine 268 of Cx43 sequence 266-283 on survival and proliferation of human glioblastoma stem cells. G166 human glioblastoma stem cells were treated in the absence (control) or presence of TAT-Cx43266-283 or the same peptide with phenylalanine 268 substituted with alanine (TAT-Cx43266-283 F / A), all at a concentration of 50 μM. Photomicrographs were taken 48 hours after the start of treatment and cell viability was analyzed by MTT assay. Results were normalized to the control (value 1) and presented as mean ± SEM (n=3). Significance of difference from control condition is expressed as ***p<0.001, and significance of difference between TAT-Cx43266-283 vs. TAT-Cx43266-283 F / A is expressed as ###p<0.001 (ANOVA). [Diagram 5] Representative images of the effect of Cx43-based penetrating peptides with reduced amino acids in the consensus SH3 binding region in human glioblastoma stem cells. G166 human glioblastoma stem cells were treated in the absence (control) or presence of TAT, TAT-Cx43274-291, TAT-Cx43266-283, TAT-Cx43266-281, TAT-Cx43266-279, TAT-Cx43266-277, TAT-Cx43266-275, all at a concentration of 50 μM. Representative phase contrast images of cells 48 hours after the start of treatment are shown. It should be noted that peptides containing the Cx43 sequences 266-283, 266-281, 266-279, 266-277, 266-275 reduce the viability and proliferation of G166 human glioblastoma stem cells, whereas the TAT peptide and TAT-Cx43274-291 have no effect on the viability and proliferation of these cells compared to controls. [Figure 6]Effect of Cx43-based penetrating peptides with reduced amino acids in the consensus SH3 binding region on survival and proliferation of human glioblastoma stem cells. G166 human glioblastoma stem cells were treated in the absence (control) or presence of TAT, TAT-Cx43274-291, TAT-Cx43266-283, TAT-Cx43266-281, TAT-Cx43266-279, TAT-Cx43266-277, and TAT-Cx43266-275, all at a concentration of 50 μM. Cell viability was analyzed by MTT assay at 0, 24, 48, and 72 hours after the start of treatment. Results were normalized to the value obtained at 0 hours (dotted value 1) and presented as mean ± SEM (n=3). Significant differences relative to the control condition are expressed as ***p<0.001; **p<0.01, significant differences relative to the TAT-Cx43 condition 266-283 are expressed as ##p<0.01 and #p<0.05 (ANOVA). [Figure 6-1] Continued from Figure 6. [Figure 7] Representative images of the effect of Cx43-based penetrating peptides on human glioblastoma stem cell migration. G166 human glioblastoma stem cells were treated in the absence (control) or presence of TAT-Cx43266-283 or TAT-Cx43266-275, both at a concentration of 50 μM. Phase-contrast images of cells were taken using a live cell microscope. Three representative frames taken every 100 min in cells under control conditions, TAT-Cx43266-283, and TAT-Cx43266-275 are shown. Three cells were selected and numbered in each condition. Note that in the control condition, the change in cell position was greater than in cells treated with peptides TAT-Cx43266-283 or TAT-Cx43266-275, indicating that these peptides affect glioma stem cell migration. [Figure 8]TAT-Cx43266-283 and TAT-Cx43266-275 bind albumin via a cysteine-dependent mechanism. TAT, TAT-Cx43274-291, TAT-Cx43266-283, TAT-Cx43266-283 F / A, TAT-Cx43266-283 C / A, and TAT-Cx43266-275 were fused with biotin to allow analysis by Western blot. Solutions of each peptide and albumin in PBS were analyzed under native (N) or denaturing and reducing conditions (DR) to identify interactions with albumin. A) Ponceau staining shows the electrophoretic profile of albumin under native (N) or denaturing and reducing conditions (DR). Note that the sensitivity of Ponceau staining is insufficient to detect peptides of approximately 10 kDa, and therefore only albumin bands are shown. B) To visualize the biotinylated peptides, the membrane was incubated with streptavidin. This allowed us to identify albumin-bound peptides under native conditions with the same profile (50-150 kDa) as albumin showed by Ponceau staining, and unbound peptides under native or denatured and reduced conditions below about 10 kDa. Note that TAT-Cx43266-283, and TAT-Cx43266-275 appear to bind to albumin under native conditions, while denaturation and reduction promote the release of these peptides. Substitution of phenylalanine with alanine (TAT-Cx43266-283 F / A) did not affect the albumin-peptide interaction. However, substitution of cysteine ​​with alanine (TAT-Cx43266-283 C / A) disrupted the albumin-peptide interaction, indicating that the cysteine ​​at position 271 mediates this interaction. [Figure 8-1] Continued from Figure 8. [Figure 9]Intracellular interactions of biotinylated TAT-Cx43266-283, TAT-Cx43266-283 C / A and TAT-Cx43266-275 in human glioblastoma stem cells. Human E22 glioblastoma stem cells were incubated with 50 μM biotinylated TAT-Cx43266-283, TAT-Cx43266-283 C / A and TAT-Cx43266-275. After 30 min, cells were lysed and biotinylated peptides bound to their intracellular partners were pulled down with NeutrAvidin beads. Eluted proteins were imported and analyzed by Western blot to investigate the levels of c-Src, Y416-Src and Csk. Note that c-Src, Y416-Src, and Csk preferentially interact with TAT-Cx43266-283 and TAT-Cx43266-275, but showed no significant interaction with TAT-Cx43266-283 C / A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] In the following pages, the present invention will be explained and its effectiveness will be demonstrated by experiments carried out by the inventors.

[0108] 1. Materials and Methods 1.1. Cultivation of human glioblastoma stem cells The human glioblastoma stem cell line G166 was obtained from Biorep and deposited and characterized in Pollard et al., Cell Stem Cell. 4(6):568-80 (2009). The human glioblastoma stem cell line E22 was also obtained from the Pollard laboratory (U.Edinburgh). These cells were obtained from a glioblastoma patient after surgery and display characteristics of human glioblastoma stem cells, including the ability to self-renew, differentiate into different neural lineages, and tumorigenicity.

[0109] In all these cases, cells were grown on laminin-coated culture plates in neurobasal medium supplemented with 2% B27, 1% N2, 20 ng / mL EGF, and 20 ng / mL bFGF to generate adherent cell cultures. Thanks to their self-renewal potential, these cells maintained the above-mentioned properties, i.e., characteristic glioblastoma stem cell phenotype and genotype, including tumorigenicity, for more than 20 passages when grown adherently under these culture conditions.

[0110] 1.2. Treatment with Cx43-based penetrating peptides. Different peptides based on the 266-283 sequence of Cx43 fused to the N-terminal TAT were used in culture, all at a concentration of 50 μM, and their effects were analyzed at different treatment times. In parallel, these cells were left untreated (control), or treated with the antitumor peptide TAT-Cx43 266-283 (YGRKKRRQRRRAYFNGCSSPTAPLSPMSP; corresponding to the peptide described in patent WO2014191608 A1 bound to the cell-internalizing sequence TAT; SEQ ID NO: 35), as well as in the presence of peptides without antitumor activity, TAT (YGRKKRRQRRR; SEQ ID NO: 7) and TAT-Cx43 274-291 (YGRKKRRQRRR PTAPLSPMSPPGYKLVTG; SEQ ID NO: 26). For pull-down assays and visualization, the peptides were fused to biotin via the C-terminal lysine.

[0111] The sequences investigated are as follows: [ka]

[0112] In all cases, these sequences were probed in combination with the penetrating peptide YGRKKRRQRRR (SEQ ID NO: 7): [ka]

[0113] 1.3.MTT Chromogenic Viability Assay For this study, cells were seeded at low density and the number of viable cells was measured after different days of treatment. For this purpose, the medium was removed and the cells were incubated with 300 μL of PBS in the presence of MTT (0.5 mg / mL) for 75 min in the dark at 37°C in a CO2 incubator. The medium was then removed and 500 μL of DMSO was added. The cells were shaken for 10 min in the dark. Finally, the absorbance was measured at 570 nm.

[0114] 1.4.Western Blotting Western blotting was performed as previously described (Jaraiz-Rodriguez, M. et al., Neuro-Oncology, 22, 493-504 (2020)). Briefly, 2.5 μM biotinylated peptides in PBS plus 0.1% (w / v) albumin were analyzed by Western blot under native (N) or denaturing and reducing (DR) conditions. For native conditions, samples were analyzed without further treatment. For denaturing and reducing conditions, samples were treated with 0.1 M β-mercaptoethanol and 4% SDS and heated at 99 °C for 5 min. Samples were subjected to electrophoresis on NuPAGE Novex Bis-Tris 4-12% Midi gels (Life Technologies) at room temperature and constant voltage. Proteins were transferred to nitrocellulose membranes (iBlot Gel Transfer Stacks Nitrocellulose) using the iBlot Dry Blotting System (Life Technologies). The membrane was incubated with Ponceau S stain to stain proteins red. After washing, the membrane was blocked with 5% nonfat dry milk in PBS and then incubated with HRP-Streptavidin (1:5000; Dako P0397) for 30 min at room temperature. After extensive washing, the membrane was developed in a MicroChemi imaging system (Bioimaging Systems) with a chemiluminescent substrate (Western Blotting Luminol Reagent; Santa Cruz Biotechnology).

[0115] 1.5.Pull-down experiments Pull-down experiments were performed as described in Gonzalez-Sanchez A. et al., Oncotarget, 7(31):49819-49833 (2016); Jaraiz-Rodriguez M. et al., J Vis Exp. (130):56457 (2017). 2 Confluent cells grown in flasks were incubated with 50 μM biotinylated peptide for 30 min. Proteins were then collected in 0.5 ml of lysis buffer (20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% IGEPAL, 1 mM PMSF, protease cocktail (1:100; Cocktail III, Calbiochem), 1 mM NaF, and 0.1 mM Na3VO4). Lysates were centrifuged at 11000×g for 10 min at 4°C, and the supernatants were collected. A 50 μl portion of each lysate was used for protein content analysis, and the remaining lysates were incubated with NeutrAvidin-Agarose (Thermo Scientific, Rockford, IL, USA; Ref. 29200) for 12 h at 4°C with gentle shaking. Avidin beads bound to peptides were collected by centrifugation (3000×g, 1 min, 4°C). The beads were then washed five times with lysis buffer and bound proteins were eluted and analyzed by Western blotting under denaturing and reducing conditions. The membranes were blocked with 5% nonfat dry milk in PBS-T and then incubated overnight at 4°C with primary antibodies against rabbit CSK (1:250; Cell Signaling Technology, Ref. 4980), rabbit Y416 Src (1:250, Cell Signaling Technology, Ref. 2101) and mouse total c-Src (1:500, Cell Signaling Technology, Ref. 2110). To detect biotinylated peptides, the membranes were incubated for 30 min with HRP-labeled streptavidin (1:5000; Dako Ref. P0397) in PBS and then developed with a chemiluminescent substrate.

[0116] 2.Results The following results indicate that the amino acids important for the antitumor effect of Cx43 are located in the 266-274 region.

[0117] In particular, cysteine ​​(C) at position 271 is essential for this effect. Therefore, replacement of this cysteine ​​with alanine (A) causes the loss of the antitumor effect (Figure 1). [ka] The morphology and viability of glioblastoma stem cells treated with TAT-Cx43 were very similar to those of untreated (control) cells, whereas TAT-Cx43 266-283 We show that Cx43 significantly reduces the viability of these tumor cells. These results demonstrate the relevance of cysteine ​​271 in the antitumor effect of the 266-283 sequence of Cx43.

[0118] To determine whether the presence of cysteine ​​alone is sufficient for activity, we analyzed the effect of the 271-287 sequence, which includes cysteine ​​271. Cysteine ​​271 has been previously reported to have the effect of inhibiting the acidification of gap junctions formed by Cx43 (Calero G., Circ Res. Circ Res., 82(9): 929-35 (1998)). Figure 2 shows the TAT-Cx43 271-287 Treatment with peptide (SEQ ID NO: 32) showed a slight decrease in glioblastoma stem cell proliferation, but the effect was not observed in TAT-Cx43 266-283 These results indicate that cysteine ​​271 is necessary but not sufficient for the antitumor effect.

[0119] Indeed, deletion of amino acids 266 and 267 affects the antitumor effect (Figure 3). TAT-Cx43 peptide 268-283 (TAT-FNGCSSPTAPLSPMSP (SEQ ID NO: 33)) is TAT-Cx43 266-283This peptide had a lower ability to reduce proliferation than (TAT-AYFNGCSSPTAPLSPMSP), indicating that amino acids 266 and 267 are important for the antitumor effect of this peptide.

[0120] Substitution of phenylalanine (F) at position 268 with alanine (A) [ka] Figure 4 shows that the penetrating peptide TAT-Cx43 266-283 We show how F / A affects tumor cell morphology and viability, but to what extent TAT-Cx43 peptide 266-283 Much lower.

[0121] On the other hand, we analyzed how amino acids in the proline-rich consensus region are related to the antitumor effect. For this purpose, we used different peptides with truncated sequences from the C-terminus: TAT-Cx43 266-281 (TAT-AYFNGCSSPTAPLSPM (SEQ ID NO: 11)), TAT-Cx43 266-279 (TAT-AYFNGCSSPTAPLS (SEQ ID NO: 10)), TAT-Cx43 266-277 (TAT-AYFNGCSSPTAP (SEQ ID NO: 9)) and TAT-Cx43 266-275 (TAT-AYFNGCSSPT (SEQ ID NO: 8)). As shown in FIG. 5, the results showed that the penetrating peptide: TAT-Cx43 266-281 ,TAT-Cx43 266-279 ,TAT-Cx43 266-277 and TAT-Cx43 266-275 is TAT-Cx43 266-283 The peptide TAT-Cx43 was shown to reduce the proliferation and survival of human glioblastoma stem cells, similar to that observed with the 266-275 The peptide showed significantly higher antitumor effects than the other peptides (Figure 6). In contrast, the cell-penetrating peptide TAT-Cx43 274-291 (SEQ ID NO:26) or TAT show no significant effect on G166 glioblastoma stem cells (Figures 5 and 6).

[0122] These same peptides were fused to biotin to generate TAT-Cx43 266-281 , TAT-Cx43 266-279 , TAT-Cx43 266-277 , TAT-Cx43 266-275 However, TAT-Cx43 266-283 Using a similar method, we confirmed that it can be internalized into human glioblastoma stem cells.

[0123] Furthermore, images taken at various times using a live cell microscope showed that TAT-Cx43 266-275 The peptide is TAT-Cx43 266-283 It was shown to reduce the migration of human glioblastoma stem cells (Figure 7), similar to that observed in. Migration is what allows these tumor cells to invade the surrounding parenchyma, escape surgery, and cause the recurrence of these tumors, which is the main cause of mortality.

[0124] One of the main advantages of peptide-based drugs as described in this invention is their low toxicity and high specificity (Otvos, L. and Wade, J. 2014. Frontiers in Chemistry, volume 2; doi: 10.3389 / fchem.2014.00062). However, peptide-based drugs have rapid renal clearance and degradation by endogenous proteases leading to poor pharmacokinetic profiles, limiting their therapeutic use. Importantly, our results show that TAT-Cx43 266-283 and TAT-Cx43 266-275 We have shown that peptides can bind to albumin, the most abundant protein in plasma, and strongly improve their pharmacokinetic properties (Figure 8). Indeed, albumin binding protects peptide-based drugs from endogenous proteases, reduces renal clearance, and improves the pharmacokinetic profile of these peptides (Liu and Xiaoyuan Chen. 2016. Chem Soc Rev. 2016 March 07; 45(5): 1432-1456. doi:10.1039 / c5cs00158g.).

[0125] As shown in Figure 8, biotinylated peptides (TAT, TAT-Cx43 274-291 , TAT-Cx43 266-283 , TAT-Cx43 266-283 F / A, TAT-Cx43 266-283 C / A or TAT-Cx43 266-275 ) was subjected to Western blot analysis under native (N) or denatured and reduced (DR) conditions to determine the binding ability to albumin. 266-283 and TAT-Cx43 266-275 showed the same profile as albumin in Ponceau staining (various bands from 50 to 150 kDa), indicating that these peptides bind to albumin under native conditions. Interestingly, the substitution of phenylalanine for alanine (TAT-Cx43 266-283 F / A) did not affect the interaction with albumin. However, the substitution of cysteine ​​with alanine (TAT-Cx43 266-283 C / A) disrupted albumin-peptide interactions as judged by the absence of peptide signals of 50–150 kDa under native conditions. These results suggest that cysteine ​​at position 271 mediates the interaction of these peptides with albumin, possibly through a disulfide bond with a free cysteine ​​residue in albumin (Nakashima, F., Shibata, T., Kamiya, K. et al. 2018. Sci Rep 8, 932. https: / / doi.org / 10.1038 / s41598-018-19610-9). To confirm these results, samples were heat denatured with SDS under reducing conditions (DR). Western blot showed the presence of a peptide of approximately 10 kDa, which was released from binding to albumin under denaturing and reducing conditions (DR). Taken together, these results suggest that TAT-Cx43 266-283 and TAT-Cx43 266-275The peptide was shown to bind to albumin through a disulfide bond between the cysteine ​​at position 271 and a free cysteine ​​residue in albumin.

[0126] Previous results showed that the sequence 266-283 from Cx43 recruits c-Src, its active form Y416-Src, and its endogenous inhibitor Csk, promoting the inhibition of the oncogenic activity of c-Src (Gonzalez-Sanchez A. et al., Oncotarget, 7(31):49819-49833 (2016); Jaraiz-Rodriguez M. et al., J Vis Exp. (130):56457 (2017)). In this study, we demonstrated that the region 266-275 is sufficient to recruit c-Src, its active form Y416-Src, and its endogenous inhibitor Csk. Figure 9 shows the biotinylated TAT-Cx43 266-283 , TAT-Cx43 266-283 C / A and TAT-Cx43 266-275 The results of pull-down assays after incubation of TAT-Cx43 with human glioblastoma stem cells are shown. As previously described, the 266-283 region recruits c-Src, Y416-Src, and Csk. Importantly, the substitution of cysteine ​​at position 271 with alanine (TAT-Cx43) 266-283 C / A), these intracellular interactions were significantly reduced. Moreover, the region 266-275 was sufficient to recruit c-Src, Y416-Src, and its endogenous inhibitor, Csk.

[0127] 3. Conclusion 1. As can be seen from Figure 1, the effect achieved by the peptide containing the C271A substitution was not significantly different from the control, indicating that cysteine ​​at position 271 in the sequence 266-283 of Cx43 is necessary for the antitumor effect of this sequence, but substitution with alanine abolishes this effect.

[0128] 2. Cysteine ​​at position 271 in the sequence 266-283 of Cx43 is not sufficient for antitumor effect, and the effect of the sequence 271-287 is much lower than that shown by the sequence 266-283.

[0129] 3. Deletion of amino acids at positions 266 and 267 in the sequence 266-283 of Cx43 reduces the antitumor effect.

[0130] 4. Substitution of phenylalanine at position 268 in the sequence 266-283 of Cx43 with alanine reduces the antitumor effect of this sequence.

[0131] 5. The amino acids from positions 276 to 283 in the Cx43 sequence 266-283 are not necessary for the antitumor effect of this sequence, and their removal does not affect this effect.

[0132] 6. TAT-Cx43 266-275 The antitumor effect of the peptide was 266-283 This is significantly higher than the antitumor effect observed in

[0133] 7. TAT-Cx43 266-283 and TAT-Cx43 266-275 binds to albumin, protecting the peptide from proteases and preventing rapid renal clearance. Cysteine ​​271 is required for peptide-albumin binding, presumably through a disulfide bond.

[0134] 8. The region 266-275 is sufficient to recruit c-Src, its activated form Y416-Src, and the Src inhibitor Csk. These interactions require the cysteine ​​at position 271.

[0135] Therefore, these results indicate that the antitumor effect resides in the sequence 266-275 of Cx43 and that cysteine ​​271 is required for this function.

Claims

1. A peptide consisting of an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 1, provided that said peptide does not have the amino acid sequence of either SEQ ID NO: 2 or 3, said peptide does not consist of the amino acid sequence AYFNGCSSPTAPLDP (SEQ ID NO: 36), and the cysteine ​​at position 6 of SEQ ID NO: 1 remains unchanged.

2. The peptide of claim 1, comprising an amino acid sequence having 100% sequence identity with SEQ ID NO:

1.

3. The peptide of claim 1, comprising the amino acid sequence of SEQ ID NO:

4.

4. The peptide of claim 1, comprising the amino acid sequence of SEQ ID NO:

5.

5. The peptide of claim 1, comprising the amino acid sequence of SEQ ID NO:

6.

6. The peptide of claim 1, wherein the peptide has a length of 10 to 100 amino acids.

7. The peptide of claim 1 , wherein the peptide is covalently linked to a cell-internalizing amino acid sequence.

8. The peptide of claim 7, wherein the cell-internalizing amino acid sequence is attached to the amino terminus of the peptide.

9. The peptide of claim 7, wherein the cell-internalization sequence comprises the amino acid sequence of SEQ ID NO:

7.

10. The peptide of claim 1 , wherein the peptide comprises the amino acid sequence of SEQ ID NO: 8, 9, 10, or 11.

11. The peptide of claim 1 , wherein the peptide is covalently bound to biotin.

12. A polynucleotide encoding the peptide of claim 1.

13. A genetic construct comprising the polynucleotide of claim 12.

14. A vector comprising the polynucleotide of claim 12 or the genetic construct of claim 13.

15. The vector according to claim 14, wherein the vector is a viral vector, preferably a retroviral, lentiviral or adenoviral viral vector.

16. A cell comprising the polynucleotide of claim 12, the genetic construct of claim 13, or the vector of claim 14.

17. A composition comprising the peptide according to any one of claims 1 to 11, the polynucleotide according to claim 12, the gene construct according to claim 13, the vector according to claim 14, or the cell according to claim 16.

18. 18. The composition of claim 17, further comprising a vehicle and / or a chemotherapeutic agent.

19. 18. The composition of claim 17, wherein the composition is formulated for oral, parenteral, nasal, sublingual, or intratumoral administration.

20. The composition of claim 17, wherein the composition is a pharmaceutical composition.

21. A peptide according to any one of claims 1 to 11, a polynucleotide according to claim 12, a gene construct according to claim 13, a vector according to claim 14, a cell according to claim 16, or a composition according to claim 17, for use as a pharmaceutical.

22. A peptide according to any one of claims 1 to 11, a polynucleotide according to claim 12, a gene construct according to claim 13, a vector according to claim 14, a cell according to claim 16, or a composition according to claim 17 for use in treating a benign or malignant tumor.

23. 23. The peptide, polynucleotide, gene construct, vector, cell, or composition for use according to claim 22, wherein the malignant tumor is cancer.

24. 23. The peptide, polynucleotide, gene construct, vector, cell, or composition for use according to claim 22, wherein the tumor is a brain tumor.

25. 23. The peptide, polynucleotide, gene construct, vector, cell, or composition for use according to claim 22, wherein the tumor is a glioma.

26. 26. The peptide, polynucleotide, gene construct, vector, cell, or composition for use according to claim 25, wherein the glioma is a glioma selected from the group consisting of astrocytoma, oligodendroglioma, glioblastoma, and ependymoma.

27. 23. The peptide, polynucleotide, gene construct, vector, cell, or composition for use according to claim 22, wherein the tumor comprises stem cells.

28. 28. The peptide, polynucleotide, gene construct, vector, cell, or composition for use according to claim 27, wherein said stem cells comprise tumor stem cells.

29. A peptide according to any one of claims 1 to 11, a polynucleotide according to claim 12, a gene construct according to claim 13, a vector according to claim 14, a cell according to claim 16, or a composition according to claim 17, for use in preventing the proliferation, migration and / or metastasis of tumor cells.

30. 30. The peptide, polynucleotide, gene construct, vector, cell, or composition for use according to claim 29, wherein the tumor cells are stem cells.

31. 31. The peptide, polynucleotide, gene construct, vector, cell, or composition for use according to claim 30, wherein said stem cells are glioma stem cells, preferably said glioma stem cells are astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells, or ependymoma stem cells.

32. Use of a peptide according to any one of claims 1 to 11, a polynucleotide according to claim 12, a genetic construct according to claim 13, a vector according to claim 14 or 15, a cell according to claim 16 or a composition according to claim 17 for inhibiting cell proliferation in vitro and / or for reversing the phenotype of tumor stem cells.

33. 33. The use according to claim 32, wherein said tumor stem cells are glioma stem cells, preferably said glioma stem cells are astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells or ependymoma stem cells.

34. Use of a peptide according to any one of claims 1 to 11, a polynucleotide according to claim 12, a gene construct according to claim 13, a vector according to claim 14, a cell according to claim 16, or a composition according to claim 17 for in vitro identification of tumorigenicity controlling or modulating compounds.

35. A kit comprising the peptide according to any one of claims 1 to 11, the polynucleotide according to claim 12, the gene construct according to claim 13, the vector according to claim 14, the cell according to claim 16, or the composition according to claim 17.

36. Use of the kit according to claim 35 for the in vitro determination of the effect of a peptide according to any one of claims 1 to 11 on the tumorigenicity of a cell line or for the in vitro inhibition of cell proliferation and / or reversal of the tumor stem cell phenotype.

37. 37. The use of claim 36, wherein inhibiting cell proliferation comprises inhibiting cell proliferation of tumor stem cells, preferably glioma stem cells, more preferably astrocytoma stem cells, oligodendroglioma stem cells, glioblastoma stem cells or ependymoma stem cells.

Images