Constructs containing or consisting of a peptide sequence capable of inhibiting the interaction of filamin a with ut receptors - pharmaceutical compositions and related products

Abstract

The present invention relates to a construct comprising a cell penetrating peptide sequence coupled to at least a first binding molecule by a peptide bond, said first binding molecule coupled to the N-terminus of an interfering amino acid sequence selected from the following sequences: SEQ ID NO: 1 forming the 2104-2206 domain of the human FlnA protein, and SEQ ID NO: 2 defining the 330-350 domain of the UT receptor, sequences having at least 90% identity and preferably at least 95% identity with the above mentioned sequences.

Description

Technical Field

[0001] This invention relates to synthetic constructs containing peptide sequences that are capable of penetrating cells (through the plasma membrane) and binding to the C-terminal domain amino acid sequence of the urovasopressin II (UII) receptor (i.e., the UT receptor), or capable of penetrating cells and binding to the FlnA domain amino acid sequence involved in the interaction between filament protein A (FlnA) and the UT receptor.

[0002] These constructs can be used as drugs or diagnostic reagents, particularly for the treatment / diagnosis of cancer, and more specifically for the treatment of glioblastoma.

[0003] The present invention also relates to pharmaceutical compositions comprising such constructs and other related products. Background Technology

[0004] The expression of FlnA and its role in tumorigenesis and tumor invasiveness have been extensively described in the literature. Although its expression is not systemically upregulated, changes in FlnA expression are associated with poor prognosis and are related to tumor invasiveness and metastasis. In fact, FlnA is found in breast cancer (see Jiang et al., "Inhibition of Filamin-A reduces cancer metastatic potential," published in the International Journal of Biological Science in 2012, and Tian et al., "Differential expression of filamin A and its clinical significance in breast cancer," published in Oncology Letters in 2012), lung adenocarcinoma (see Uramoto et al., "A positive relationship between filamin and VEGF in patients with lung cancer," published in Anticancer Research in 2010), pancreatic cancer (see Surcel et al., "Targeting mechanoresponsive proteins inpancreatic cancer: 4-hydroxyacetophenone blocks dissemination and invasion by activating MYH14," published in Cancer Research in 2018), and melanoma (see Zhang et al., "Filamin A expression correlates with proliferation and invasive properties of human metastatic melanoma tumors: implications for survival," published in the Journal of Cancer Research and Clinical Oncology in 2014). Overexpression in patients) and the highest levels were systematically associated with poor prognosis, manifested as a larger number of metastases and earlier recurrence.

[0005] In prostate cancer (see Sun et al., "Filamin A regulates MMP-9 expression and suppresses prostate cancer cell migration and invasion," published in Tumor Biology in 2014), colorectal adenocarcinoma (see Tian et al., "New cancer suppressor gene for colorectal adenocarcinoma: Filamin A," published in World Journal of Gastroenterology in 2015), as well as gastric adenocarcinoma, renal cell carcinoma, and nasopharyngeal carcinoma (see Sun et al., "An antiproliferative gene FLNA regulates migration and invasion of gastric carcinoma cell in vitro and its clinical significance," published in Tumor Biology in 2014; "Interactions between Filamin A and MMP-9 Regulate Proliferation and Invasion in Renal Cell Carcinoma," published in Asian Pacific Journal of Cancer Prevention in 2014; and "Absent expression of FLNA is correlated with poor prognosis of nasopharyngeal adenocarcinoma," published in Tumor Biology in 2014; and "Absent expression of FLNA is correlated with poor prognosis of nasopharyngeal adenocarcinoma," published in Tumor Biology in 2014. Decreased FlnA expression levels have been observed in studies such as those published in *Cancer*. In these cases, decreased FlnA expression is often associated with poor prognosis.

[0006] In summary, the impact of changes in FlnA expression in tumors suggests that this protein plays a central role in regulating tumorigenesis-related mechanisms. It appears to constitute a valuable diagnostic and prognostic biomarker and may have applications in tumor management. Therefore, using FlnA as a rapid diagnostic biomarker for treatment response and / or recurrence could also serve as a non-invasive follow-up strategy for monitoring patient treatment response.

[0007] Diffuse gliomas in adults are the most common and aggressive primary brain tumors, particularly anaplastic gliomas (grade III) and glioblastomas (GB). The current first-line standard of care is the Stupp regimen (see Stupp et al., "Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma," published in The New England Journal of Medicine in 2005). This regimen is based on the most complete possible surgical resection of the tumor, followed by concurrent chemoradiotherapy and adjuvant temozolomide (TMZ). However, recurrence is almost inevitable, occurring within 7 to 10 months of diagnosis, which explains the median survival of 15 to 17 months. This is primarily due to the highly diffuse and aggressive nature of GB, making complete tumor resection impossible, as well as pathological angiogenesis and treatment resistance.

[0008] At relapse, no treatment significantly improves patient survival, and chemotherapy has a response rate of approximately 5%. In recent years, despite the development of various treatment strategies, many developed targeted therapies have failed to demonstrate efficacy in overall patient survival. Nevertheless, it is worth emphasizing that anti-angiogenic therapies (bevacizumab, aflibercept, sunitinib, and cedrab), with bevacizumab being an antibody against VEGF-A, have allowed for a 30% response rate at relapse and prolonged progression-free survival and quality of life in GB patients.

[0009] Therefore, despite some progress in the management of high-grade diffuse gliomas or GB, recurrence remains the norm and thus must be treated.

[0010] Invasion of healthy brain parenchyma by GB cells located at or far from the resection margin poses a significant therapeutic challenge. These invasion mechanisms are driven by cell transformation associated with environmental constraints such as hypoxia and angiogenesis, leading to mesenchymal transition (TM), primarily controlled by transcription factors STAT3 and CEBPα, which stimulate 70% of mesenchymal secondary genes. TM-associated hypoxia, in particular, results in the overexpression of chemokine G protein-coupled seven-transmembrane receptors (GCPRs). The most widely studied chemokine GCPR in GB is CXCR4, which delivers pro-migration effects via the Gαi / PI3K / Akt and Gα12 / 13 / Rho / ROCK / myosin-coupled pathways, along with clathrin-dependent endocytosis and β-inhibitor activity-mediated recycling. Published research has also demonstrated that the UT receptor of the neuropeptide UII acts as a chemokine receptor, capable of stimulating the directed migration of GB cells through sequential activation of Gαi / PI3K followed by activation of Gα13 / Rho / ROCK, thereby achieving cell polarization, pseudopodia formation, actin stress fiber polymerization, and cell contraction (see Lecointre et al., "Signaling switch of the urotensin II vasoactive peptide GPCR: prototypic chemotaxic mechanism in glioma," published in Oncogene in 2015, and the abstract of Alexandre Mutel's doctoral dissertation, accessible at the following link: https: / / www.theses.fr / 2019NORMR081).

[0011] Technical problems solved One object of the present invention is to provide a construct comprising one or more peptide sequences capable of penetrating cells and interacting with the UT receptor or FlnA protein of human UII neuropeptides to inhibit the interaction between FlnA and the UT receptor, or (substantially) a construct consisting of one or more peptide sequences capable of penetrating cells and interacting with the UT receptor or FlnA protein of human UII neuropeptides to inhibit the interaction between FlnA and the UT receptor.

[0012] Another object of the present invention is to provide the above-described constructs that can be used as pharmaceuticals or prognostic or diagnostic reagents, particularly for cancer, and more specifically for diffuse gliomas, especially adult diffuse gliomas, anaplastic gliomas (grade III) and glioblastomas.

[0013] Another object of the present invention is to provide the above-described constructs that can reduce or even prevent invasion of healthy parenchyma after resection of diffuse glioma or glioblastoma mediated by UT receptor activation and other chemokine receptors.

[0014] Another object of the present invention is to provide the above-described constructs capable of combating or limiting glioblastoma resistance to current treatments (particularly temozolomide).

[0015] Another objective is to provide constructs that can achieve synergistic effects with other peripheral cancer anticancer drugs, particularly tyrosine kinase inhibitors such as gefitinib or DNA alkylating agents such as temozolomide, especially for the treatment of glioblastoma as well as lung, breast, and prostate cancer.

[0016] Another object of the present invention is to provide a peptide sequence that enables the preparation of the above-described construct. Invention Overview According to a first aspect, the present invention relates to a construct comprising a cell-penetrating peptide sequence coupled to at least a first binding molecule via a peptide bond, the first binding molecule being coupled to the N-terminus of an interfering amino acid sequence selected from: a) A sequence that contains or consists of the following sequences: VTYCPTEPGNYIINIKFADQHVPGSPFSVKVTGEGRVKESITRRRRAPSVANVGSHCDLSLKIPEISIQDMTAQVTSPSGKTHEAEIVEGENHTYCIRFVPAE (SEQ ID NO: 1 or FlnA 2104-2206), which forms the 2104-2206 domain of the human FlnA protein; b) A sequence comprising a fragment of the sequence SEQ ID NO: 1 or a sequence consisting of fragments of the sequence SEQ ID NO: 1, said sequence or said fragment being capable of binding the sequence VRGPGSGGGRGPVPSLQPRAR (SEQ ID NO: 2 or UT-330-350), which defines the 330-350 domain of the UT receptor, or a fragment being capable of binding the sequence SEQ ID NO: 2, particularly a fragment of the sequence GGGRGPVPSLQPRAR (SEQ ID NO: 3 or UT-336-350) which forms the 336-350 domain of the UT receptor, or a fragment of the fragment RGPVPSLQPRAR (SEQ ID NO: 4 or UT-339-350) which defines the 339-350 domain of the UT receptor; c) A sequence containing a fragment of the SEQ ID NO: 2 sequence or a sequence consisting of fragments of the SEQ ID NO: 2 sequence, particularly a fragment forming the 330-350 domain of the UT receptor (SEQ ID NO: 2), or a fragment forming the 336-350 domain of the UT receptor (SEQ ID NO: 3), or a fragment forming the 339-350 domain of the UT receptor (SEQ ID NO: 4), and a fragment capable of binding the SEQ ID NO: 1 sequence; d) A sequence having at least 90% identity, and preferably at least 95% identity, with the sequences defined in items a) to c); and e) A sequence substantially homologous to the sequences defined in items a) to d), and preferably derived from the sequences described above by one or more conservatrices.

[0018] The inventors' contribution lies in demonstrating that certain of the aforementioned constructs can interfere with the UT receptors of filamentin A and UII, and therefore can be used as drugs. These constructs exhibit particular in vitro activity against glioblastoma cells and prevent the spread of tumor cells after glioblastoma resection. Invention Details According to a specific embodiment, the first binding molecule is an amino acid, optionally modified, particularly a β-alanine molecule. The connection between the penetrating sequence and the first binding molecule is a peptide bond, and the connection between the first binding molecule and the interfering sequence is also a peptide bond.

[0020] According to a specific implementation, the interfering amino acid sequence is selected from the amino acid sequence defined in section b), and in particular from the following sequences: - MTAQVTSPSGKTHEAEIVE (SEQ ID NO: 5 or FlnA-D20-CD), GNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 6 or FlnA-D19-FG), THEAEIVEGENHTYCIRF (SEQ ID NO: 7 or FlnA-D20-DE), peptide sequences having at least 90% and preferably at least 95% identity with the said sequences, and substantially homologous peptide sequences, preferably sequences derived from the sequences of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 by one or more conserved substitutions.

[0021] - RGPVPSLQPRAR (SEQ ID NO: 4 or UT-339-350) and GGGRGPVPSLQPRAR (SEQ ID NO: 3 or UT-336-350), and peptide sequences having at least 90% and preferably at least 95% identity with the sequences of said SEQ ID NO: 3 and SEQ ID NO: 4, and substantially homologous peptides, preferably sequences derived from the sequences of SEQ ID NO: 3 or SEQ ID NO: 4 by one or more conserved substitutions.

[0022] The cell-penetrating sequence is not limited in this invention. Advantageously, the cell-penetrating sequence is selected from the following sequences: -TCTWLKYH (SEQ ID NO: 8 or Glio) and a peptide sequence having at least 90% and preferably at least 95% identity with the sequence of SEQ ID NO: 8, and a substantially homologous sequence, preferably a sequence derived from the sequence of SEQ ID NO: 8 by one or more conserved substitutions; - A peptide sequence GRRRQRRKKRKCCKRKKRRQRRRG (SEQ ID NO: 9 or CycloGlio), wherein residues 12 and 13 (cysteine) are linked by a disulfide bond bridge, and residues 2 to 10 and 15 to 23 are dextrorotatory (D-type) amino acids, and a peptide sequence having at least 90% and preferably at least 95% identity with the sequence of SEQ ID NO: 9, and substantially homologous sequences, preferably sequences derived from the sequence of SEQ ID NO: 9 by one or more conserved substitutions, wherein each end of the cell-penetrating sequence is connected by a peptide bond to a first binding molecule, the binding molecule being an optionally modified amino acid, the binding molecule itself being connected by a peptide bond to an interfering sequence, the two interfering sequences being identical or different.

[0023] These two penetration sequences exhibit rapid cell penetration kinetics; constructs containing these sequences can penetrate into cells within 5 minutes of the start of in vitro incubation.

[0024] The cell penetration sequence SEQ ID NO: 8 can reduce potential other types of cell penetration, thereby reducing adverse side effects when administered to patients.

[0025] The cell-penetrating sequence SEQ ID NO: 9 or CycloGlio has a U-shaped molecular structure due to the disulfide bond connecting two cysteine ​​molecules. This allows for the synthesis of hairpin-shaped molecules. The two interfering sequences, respectively attached to both ends of the penetrating sequence, can be identical or different. For example, they can be two identical or different fragments of FlnA, or two identical or different fragments of the UT receptor. Therefore, when one arm is attached to FlnA or the UT receptor, the other free arm can repel any molecule approaching FlnA or the UT receptor. Thus, the inhibition of the relationship between FlnA and the UT receptor has a dual effect: on the one hand, due to binding to the construct of the present invention, and on the other hand, due to the steric hindrance effect generated by the U-shaped structure of the construct of the present invention.

[0026] Advantageously, the cell-penetrating sequence is selected from the peptide sequence GRRRQRRKKRKCCKRKKRRQRRRG (SEQ ID NO: 9 or CycloGlio), wherein residues 12 and 13 (cysteine) are linked by disulfide bonds, and wherein residues 2 to 10 and 15 to 23 are dextrorotatory (D-type) amino acids, as well as peptide sequences having at least 90% and preferably at least 95% identity with the SEQ ID NO: 9 sequence, and substantially homologous sequences, preferably sequences derived from the SEQ ID NO: 9 sequence by one or more conserved substitutions, characterized in that the two first binding molecules are identical, particularly the β-alanine molecule; One of the interfering sequences is selected from the sequences defined in claims a) and b) of claim 1, particularly from the following interfering sequences: MTAQVTSPSGKTHEAEIVE (SEQ ID NO: 5 or FlnA-D20-CD), GNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 6 or FlnA-D19-FG), THEAEIVEGENHTYCIRF (SEQ ID NO: 7 or FlnA-D20-DE), and peptide sequences having at least 90% and preferably at least 95% identity with said sequences, as well as substantially homologous sequences, preferably sequences derived from SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 by one or more conserved substitutions, while the other interfering sequence is selected from the sequences defined in claim c) of claim 1, particularly from the following interfering sequences: RGPVPSLQPRAR (SEQ ID NO: 4 or UT-339-350) and GGGRGPVPSLQPRAR (SEQ ID NO: 3 or UT-336-350), and peptide sequences having at least 90% and preferably at least 95% identity with said sequences, as well as sequences substantially homologous to said sequences, preferably sequences derived from SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 by one or more conserved substitutions. The sequences ID NO:3 and SEQ ID NO:4 have at least 90% and preferably at least 95% identity peptide sequences, and substantially homologous sequences, preferably sequences derived from the sequences SEQ ID NO:3 or SEQ ID NO:4 by one or more conserved substitutions.

[0027] Furthermore, the penetrating sequence SEQ ID NO: 9 allows for the release of endosomes from the construct of the present invention, resulting in a cytoplasmic incorporation rate exceeding 90%, thereby enhancing the interference effect of the interfering sequence.

[0028] Advantageously, when the cell-penetrating sequence is selected from the peptide sequence GRRRQRRKKRKCCKRKKRRQRRRG (SEQ ID NO: 9 or CycloGlio), wherein residues 12 and 13 (cysteine) are linked by disulfide bonds, and residues 2 to 10 and 15 to 23 are dextrorotatory (D-type) amino acids, and a peptide sequence having at least 90% and preferably at least 95% identity with the sequence of SEQ ID NO: 9, and substantially homologous sequences, preferably sequences derived from the sequence of SEQ ID NO: 9 by one or more conserved substitutions, lysines at positions 11 and 14 of the cell-penetrating sequence are acetylated. This acetylation avoids the risk of forming peptide bonds with other sequences because lysines still possess free amine function and can still bind even if they are part of a peptide sequence.

[0029] The present invention also relates to interference constructs selected from the following sequence constructs: TVKVSFPSGPVHQDAFKINIIYNGXGrrrqrrkkrKCCKrkkrrqrrrGXGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 10, also known as FlnA-CPPD19-FG-CycloGlio), wherein residues 37 and 38 are connected by disulfide bonds, X is an optional modified amino acid at both positions, preferably β-alanine at both positions, and residues 27 to 35 and 40 to 48 are dextrorotatory (D-type) amino acids, wherein lysines at positions 36 and 39 are acetylated; EVIEAEHTKGSPSTVQATMXGrrrqrrkkrKCCKrkkrrqrrrGXMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 11; also known as FlnA-CPPD20-CD-CycloGlio), wherein residues at positions 32 and 33 are connected by a disulfide bond bridge, X is an optional modified amino acid and preferably β-alanine at both positions, and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids, wherein lysines at positions 31 and 34 are acetylated; FRISYTHNEGEVIEAEHTXGrrrqrrkkrKCCKrkkrrqrrrGXTHEAEIVEGENHTYSIRF (SEQ ID NO: 12; also known as FlnA-CPPD20-DE-CycloGlio), wherein residues 31 and 32 are linked by a disulfide bond, X is an optional modified amino acid, preferably β-alanine, and residues 21 to 29 and 34 to 42 are dextrorotatory (D-type) amino acids; wherein lysines at positions 30 and 33 are acetylated; TCTWLKYHXGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 13; also known as FlnA-CPPD19-FG-Glio), where X is an optional modified amino acid, preferably β-alanine; TCTWLKYHXMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 14; also known as FlnA-CPPD20-CD-Glio), wherein X is an optional modified amino acid, preferably β-alanine; TCTWLKYHXTHEAEIVEGENHTYCIRF (SEQ ID NO: 15; also known as FlnA-CPPD20-DE-Glio), wherein X is an optional modified amino acid, preferably β-alanine; RARPQLSPVPGRGGGXGrrrqrrkkrKCCKrkkrrqrrrGXGGGRGPVPSLQPRAR (SEQ ID NO: 16, also known as UT-CPP336-350-CycloGlio), wherein residues 28 and 29 are linked by a disulfide bond, X is an optional modified amino acid and preferably β-alanine at both positions, and residues 18 to 26 and 31 to 39 are dextrorotatory (D-type) amino acids, wherein lysines at positions 27 and 30 are acetylated; RARPQLSPVPGRXGrrrqrrkkrKCCKrkkrrqrrrGXRGPVPSLQPRAR (SEQ ID NO: 17; also known as UT-CPP339-350-CycloGlio), wherein residues 25 and 26 are linked by a disulfide bond, X is an optional modified amino acid, preferably β-alanine at both positions, and residues 15 to 23 and 28 to 36 are dextrorotatory (D-type) amino acids, wherein lysines at positions 24 and 27 are acetylated. TCTWLKYHXGGGRGPVPSLQPRAR (SEQ ID NO: 18; also known as UT-CPP336-350-Glio), wherein X is an optional modified amino acid, preferably β-alanine; TCTWLKYHXRGPVPSLQPRAR (SEQ ID NO: 19; also known as UT-CPP339-350-Glio), wherein X is an optional modified amino acid, preferably β-alanine; A construct having at least 90% identity, and preferably at least 95% identity, with the aforementioned construct; and The constructs are substantially homologous, and preferably derived from the sequences SEQ ID NO: 10 to SEQ ID NO: 19 by one or more conservative substitutions.

[0030] Sequence constructs SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 13, and SEQ ID NO: 17 have demonstrated particular effectiveness in cell penetration and inhibition of cancer cell migration in various solid tumors (glioblastoma, prostate cancer, lung cancer, and breast cancer). Similarly, marker constructs containing these sequences have also shown similar efficacy.

[0031] In particular, the present invention relates to the following sequence constructs: The present invention also relates to the following sequence constructs: TVKVSFPSGPVHQDAFKINIIYNGAGrrrqrrkkrKCCKrkkrrqrrrGAGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 28, also known as FlnA-CPPD19-FG-CycloGlio-syn), wherein residues 37 and 38 are connected by disulfide bonds, alanine at positions 25 and 50 is β-alanine, and residues 27 to 35 and 40 to 48 are dextrorotatory (D-type) amino acids, and lysine at positions 36 and 39 is acetylated; EVIEAEHTKGSPSTVQATMAGrrrqrrkkrKCCKrkkrrqrrrGAMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 29; also known as FlnA-CPPD20-CD-CycloGlio-syn), wherein residues 32 and 33 are connected by a disulfide bond, alanine at positions 20 and 45 is β-alanine, and residues 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids; and lysine at positions 31 and 34 is acetylated; FRISYTHNEGEVIEAEHTAGrrrqrrkkrKCCKrkkrrqrrrGATHEAEIVEGENHTYSIRF (SEQ ID NO: 30; also known as FlnA-CPPD20-DE-CycloGlio-syn), wherein residues 31 and 32 are connected by a disulfide bond bridge, alanine at positions 19 and 44 is β-alanine, and residues at positions 21 to 29 and 34 to 42 are dextrorotatory (D-type) amino acids; TCTWLKYHAGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 31; also known as FlnA-CPPD19-FG-Glio-syn, where the alanine at position 9 is β-alanine) and the lysines at positions 30 and 33 are acetylated; TCTWLKYHAMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 32; also known as FlnA-CPPD20-CD-Glio-syn), where the alanine at position 9 is β-alanine; TCTWLKYHATHEAEIVEGENHTYCIRF (SEQ ID NO: 33; also known as FlnA-CPPD20-DE-Glio-syn), where the alanine at position 9 is β-alanine; RARPQLSPVPGRGGGAGrrrqrrkkrKCCKrkkrrqrrrGAGGGRGPVPSLQPRAR (SEQ ID NO: 34; also known as UT-CPP336-350-CycloGlio-syn), wherein residues 28 and 29 are connected by a disulfide bond, alanine at positions 16 and 41 is β-alanine, and residues 18 to 26 and 31 to 39 are dextrorotatory (D-type) amino acids, and lysine at positions 27 and 30 is acetylated; RARPQLSPVPGRAGrrrqrrkkrKCCKrkkrrqrrrGARGPVPSLQPRAR (SEQ ID NO: 35; also known as UT-CPP339-350-CycloGlio-syn), wherein residues 25 and 26 are connected by a disulfide bond, alanine at positions 13 and 38 is β-alanine, and residues 15 to 23 and 28 to 36 are dextrorotatory (D-type) amino acids, and lysine at positions 24 and 27 is acetylated; TCTWLKYHAGGGRGPVPSLQPRAR (SEQ ID NO: 36; also known as UT-CPP336-350-Glio-syn), where the alanine at position 9 is β-alanine; TCTWLKYHARGPVPSLQPRAR (SEQ ID NO: 37; also known as UT-CPP339-350-Glio-syn), where the alanine at position 9 is β-alanine; A sequence having at least 90% identity with the above sequence, preferably at least 95% identity, and a sequence substantially homologous to the above sequence, preferably a sequence derived from the above sequence through one or more conservative permutations.

[0032] When the construct contains the Cycloglio sequence (SEQ ID NO: 9) or a sequence that is 90% or 95% identical to that sequence, or a sequence that is substantially homologous and preferably derived from that sequence by one or more conservative substitutions, the lysines on both sides of the disulfide bond are preferably acetylated, i.e., linked to the CH3CO group, when they are not linked to the labeling molecule.

[0033] The SEQ ID NO: 35 or UT-339-350-CycloGlio construct exhibits systemic effects in various glioblastoma cell lines. This construct (peptide) is interfering and penetrating, inhibiting glioblastoma cell motility and chemotactic migration to UII. It reduces glioblastoma cell viability when used alone or in combination with TMZ (temozolomide) chemotherapy, and also reduces the viability of lung cancer cell lines while enhancing the effect of gefitinib chemotherapy.

[0034] The sequence constructs SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 35 and SEQ ID: 45 have demonstrated particular effectiveness in cell penetration and inhibition of cancer cell migration in a variety of solid cancers (glioblastoma, prostate cancer, lung cancer and breast cancer).

[0035] The present invention also relates to constructs that further include a marker capable of penetrating into cells. Specifically, the penetrating sequence is linked to at least one marker. This marker may be a fluorophore.

[0036] The labeling agent is preferably a specific penetrating fluorophore, namely 5-carboxytetramethylrhodamine or 7-diethylaminocoumarin-3-carboxylic acid.

[0037] Advantageously, when the cell-penetrating sequence is selected from the peptide sequence GRRRQRRKKRKCCKRKKRRQRRRG (SEQ ID NO: 9 or CycloGlio), wherein residues 12 and 13 (cysteine) are linked by a disulfide bond bridge, and wherein residues 2 to 10 and 15 to 23 are dextrorotatory (D-type) amino acids, and a peptide sequence having at least 90% and preferably at least 95% identity with the sequence of SEQ ID NO: 9, and substantially homologous sequences, preferably sequences derived from the sequence of SEQ ID NO: 9 by one or more conserved substitutions, the labeling molecule is linked to lysines at positions 11 and 14, and the labeling agent is particularly and preferably 5-carboxytetramethylrhodamine.

[0038] Therefore, the present invention also relates to a construct comprising or composed of peptide sequences selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, a construct having at least 90% identity with the above constructs and preferably at least 95% identity, and a construct substantially homologous to the above sequences and preferably derived from the above sequences by one or more conserved substitutions, wherein the first residue of the penetrating sequence is linked by a peptide bond to a carboxylic acid functional group of β-alanine, which is in turn linked by a peptide bond to a carboxyl group of 7-diethylaminocoumarin-3-carboxylic acid.

[0039] The present invention also relates to a construct comprising or composed of a peptide sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, a construct having at least 90% identity with the above constructs and preferably at least 95% identity, and a construct substantially homologous to the above sequences and preferably derived from the above sequences by one or more conserved substitutions, wherein residues 11 and 14 (K: lysine) of the penetrating sequence are each linked to a carboxylic acid group of 5-carboxytetramethylrhodamine via a peptide bond.

[0040] Therefore, the present invention also relates to the following sequences, which can be used for the treatment and diagnosis / prognosis of the diseases mentioned below: TVKVSFPSGPVHQDAFKINIIYNGAGrrrqrrkkrKCCKrkkrrqrrrGAGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 38, also known as Mq-FlnA-CPPD19-FG-CycloGlio-syn), wherein residues 37 and 38 are connected by disulfide bonds, alanine at positions 25 and 50 is β-alanine, and residues at positions 27 to 35 and 40 to 48 are dextrorotatory (D-type) amino acids, and residues at positions 36 and 39 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bonds; EVIEAEHTKGSPSTVQATMAGrrrqrrkkrKCCKrkkrrqrrrGAMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 39; also known as Mq-FlnA-CPPD20-CD-CycloGlio-syn), wherein residues 32 and 33 are connected by disulfide bonds, alanine at positions 20 and 45 is β-alanine, and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids, and residues at positions 31 and 34 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bonds; FRISYTHNEGEVIEAEHTAGrrrqrrkkrKCCKrkkrrqrrrGATHEAEIVEGENHTYSIRF (SEQ ID NO: 40; also known as Mq-FlnA-CPPD20-DE-CycloGlio-syn), wherein residues 31 and 32 are connected by disulfide bonds, alanine at positions 19 and 44 is β-alanine, and residues at positions 21 to 29 and 34 to 42 are dextrorotatory (D-type) amino acids, and residues at positions 30 and 33 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bonds; ATCTWLKYHAGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 41; also known as Mq-FlnA-CPPD19-FG-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond; ATCTWLKYHAMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 42; also known as Mq-FlnA-CPPD20-CD-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond; ATCTWLKYHATHEAEIVEGENHTYCIRF (SEQ ID NO: 43; also known as Mq-FlnA-CPPD20-DE-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond; RARPQLSPVPGRGGGAGrrrqrrkkrKCCKrkkrrqrrrGAGGGRGPVPSLQPRAR (SEQ ID NO: 44; also known as Mq-UT-CPP336-350-CycloGlio-syn), wherein residues 28 and 29 are connected by disulfide bonds, alanine at positions 16 and 41 is β-alanine, and residues at positions 18 to 26 and 31 to 39 are dextrorotatory (D-type) amino acids, and residues at positions 27 and 30 are each connected to the carboxylic acid group of tetramethylrhodamine via peptide bonds; RARPQLSPVPGRAGrrrqrrkkrKCCKrkkrrqrrrGARGPVPSLQPRAR (SEQ ID NO: 45; also known as Mq-UT-CPP339-350-CycloGlio-syn), wherein residues 25 and 26 are linked by a disulfide bond, the alanine at positions 13 and 38 is β-alanine, and residues at positions 15 to 23 and 28 to 36 are dextrorotatory (D-type) amino acids. Residues at positions 24 and 27 are each linked to the carboxylic acid group of tetramethylrhodamine via peptide bonds. ATCTWLKYHAGGGRGPVPSLQPRAR (SEQ ID NO: 46; also known as Mq-UT-CPP336-350-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond; ATCTWLKYHARGPVPSLQPRAR (SEQ ID NO: 47; also known as Mq-UT-CPP339-350-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond. A sequence having at least 90% identity, and preferably at least 95% identity, with the above sequence; and A sequence that is substantially homologous to the above sequence, and preferably a sequence derived from the above sequence through one or more conservative permutations.

[0041] The present invention also relates to the use of the constructs according to the invention as pharmaceutical or diagnostic / prognostic agents, particularly for cancer, especially solid tumors, breast cancer, lung cancer (especially for the treatment of lung adenocarcinoma), melanoma, prostate cancer, colon cancer (especially colorectal adenocarcinoma), gastric cancer (especially gastric adenocarcinoma), kidney cancer (especially renal cell carcinoma), nasopharyngeal carcinoma (especially nasopharyngeal squamous cell carcinoma), pancreatic cancer, diffuse glioma, and glioblastoma, for patients / subjects in need, particularly humans.

[0042] The present invention also relates to a pharmaceutical composition comprising, as an active ingredient (active substance), at least one construct according to the invention, and a pharmaceutically acceptable solvent, excipient and / or carrier.

[0043] According to a specific embodiment, the pharmaceutical composition of the present invention may further comprise an anticancer agent as a second active substance, said anticancer agent being particularly selected from tyrosine kinase inhibitors, particularly gefitinib, and alkylating agents that cause DNA double-strand breaks and cell death, particularly TMZ, for simultaneous, delayed or sequential use.

[0044] The compositions of the present invention may be in solid form, particularly powder, gel or injectable solution, optionally in colloidal form.

[0045] The composition can be administered orally, intravenously, or intranasally after tumor resection, particularly in the case of glioblastoma, either simultaneously or separately from the injection of chemotherapy and / or radiotherapy products.

[0046] When the pharmaceutical compositions of the present invention are in liquid, gel, or solid form, they can be introduced into the cavity left by surgical resection after tumor removal (particularly in the case of blastoma and glioblastoma) before the patient receives radiotherapy and / or chemotherapy. The constructs and peptides of the present invention will then penetrate surrounding cells and block the invasion of glioblastoma.

[0047] The composition of the present invention can also be injected into tumors.

[0048] The present invention also relates to nucleic acids encoding sequences corresponding to one of the sequences of the present invention, particularly nucleic acids encoding constructs selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.

[0049] The present invention also relates to a vector containing the nucleic acid, preferably an adenovirus vector or a lentivirus vector.

[0050] The present invention also relates to a combination product (kit) comprising at least one construct according to the invention, particularly constructs selected from the following sequence constructs: SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 11, SEQ ID NO: 29, SEQ ID NO: 12, SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 36, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 15, SEQ ID NO: 33, and an anticancer agent, particularly selected from tyrosine kinase inhibitors, particularly gefitinib, and alkylating agents that induce DNA double-strand breaks and cell death, particularly TMZ. The use of the construct and the anticancer agent may be simultaneous, delayed, or sequential.

[0051] The present invention also discloses the following sequences used as controls: ALQQSRLPPQVSPQDGALAAGrrrqrrkkrKCCKrkkrrqrrrGAALAGDQPSVQPPLRSQQLA (SEQ ID NO: 20 or FlnA-H1a-Cycloglio), wherein residues 32 and 33 are connected by a disulfide bond bridge, the alanine at positions 20 and 45 is β-alanine (at both positions), and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids; WTQQGGQAYTYQPALQQSRAGrrrqrrkkrKCCKrkkrrqrrrGARSQQLAPQYTYAQGGQQTW (SEQ ID NO: 21 or FlnA-H1b-Cycloglio), wherein residues 32 and 33 are connected by a disulfide bond bridge, alanine at positions 20 and 45 is β-alanine (at both positions), and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids; AKTLSDVFVSSTEHLSHNSAGrrrqrrkkrKCCKrkkrrqrrrGASNHSLHETSSVFVDSLTKA (SEQ ID NO: 22 or FlnA-H2a-CycloGlio), wherein residues 32 and 33 are linked by a disulfide bond, alanine at positions 20 and 45 is β-alanine (at both positions), and residues 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids; PGPGPAGHQPASTAKTLSDAGrrrqrrkkrKCCKrkkrrqrrrGADSLTKATSAPQHGAPGPGP (SEQ ID NO: 23 or FlnA-H2b-CycloGlio), wherein residues 32 and 33 are connected by a disulfide bond bridge, the alanine at positions 20 and 45 is β-alanine, and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids; TCTWLKYHAALAGDQPSVQPPLRSQQLA (SEQ ID NO: 24 or FlnA-H1a-Glio), where the alanine at position 9 is β-alanine.

[0052] TCTWLKYHARSQQLAPQYTYAQGGQQTW (SEQ ID NO: 25 or FlnA-H1b-Glio), where the alanine at position 9 is β-alanine.

[0053] TCTWLKYHASNHSLHETSSVFVDSLTKA (SEQ ID NO: 26 or FlnA-H2a-Glio), where the alanine at position 9 is β-alanine.

[0054] TCTWLKYHADSLTKATCAPQHGAPGPGP (SEQ ID NO: 27 or FlnA-H2b-Glio), where the alanine at position 9 is β-alanine.

[0055] This invention discloses the following construct used as a control: ALQQSRLPPQVSPQDGALAAGrrrqrrkkrKCCKrkkrrqrrrGAALAGDQPSVQPPLRSQQLA (SEQ ID NO: 48 or Mq-FlnA-H1a-Cycloglio-syn), wherein residues 32 and 33 are linked by a disulfide bond, the alanines at positions 20 and 45 are β-alanine, and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids, and residues at positions 31 and 34 are each linked by a peptide bond to a carboxylic acid group of 5-carboxytetramethylrhodamine. WTQQGGQAYTYQPALQQSRAGrrrqrrkkrKCCKrkkrrqrrrGARSQQLAPQYTYAQGGQQTW (SEQ ID NO: 49 or Mq-FlnA-H1b-Cycloglio-syn), wherein residues 32 and 33 are connected by a disulfide bond bridge, the alanine at positions 20 and 45 is β-alanine and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids, and residues at positions 31 and 34 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bond; AKTLSDVFVSSTEHLSHNSAGrrrqrrkkrKCCKrkkrrqrrrGASNHSLHETSSVFVDSLTKA (SEQ ID NO: 50 or Mq-FlnA-H2a-CycloGlio-syn), wherein residues 32 and 33 are connected by a disulfide bond bridge, the alanine at positions 20 and 45 is β-alanine and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids, and residues at positions 31 and 34 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bond; PGPGPAGHQPASTAKTLSDAGrrrqrrkkrKCCKrkkrrqrrrGADSLTKATSAPQHGAPGPGP (SEQ ID NO: 51 or Mq-FlnA-H2b-CycloGlio-syn), wherein residues 32 and 33 are connected by a disulfide bond bridge, the alanine at positions 20 and 45 is β-alanine and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids, and residues at positions 31 and 34 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bond; ATCTWLKYHAALAGDQPSVQPPLRSQQLA (SEQ ID NO: 52 or Mq-FlnA-H1a-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond. ATCTWLKYHARSQQLAPQYTYAQGGQQTW (SEQ ID NO: 53 or Mq-FlnA-H1b-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond. ATCTWLKYHASNHSLHETSSVFVDSLTKA (SEQ ID NO: 54 or Mq-FlnA-H2a-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond. ATCTWLKYHADSLTKATCAPQHGAPGPGP (SEQ ID NO: 55 or Mq-FlnA-H2b-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond.

[0056] definition In this application, the term "patient / subject" refers to a mammal, male or female, adult or non-adult (child or adolescent), and in particular a human.

[0057] The term "treatment" includes curative treatment and preventative treatment. Curative treatment includes at least one of the following: relieving, improving, and / or eliminating, reducing, or stabilizing at least one symptom, and delaying the onset or progression of symptoms of deteriorating health. Preventative treatment includes at least one of the following: avoiding deterioration of health, reducing the risk of deterioration, reducing morbidity, developing, and delaying the onset of at least one symptom of deterioration of health.

[0058] The term "penetrating sequence" refers to a peptide or peptide sequence that can be transferred into a cell without causing substantial damage to the cell membrane and can be used as a carrier for other molecules linked to it.

[0059] The term "interference sequence" refers to a peptide sequence that can bind to a given peptide sequence (optionally forming a part of a protein) and thereby alter the mechanism involved in the given peptide sequence or the protein containing that sequence.

[0060] The term "derived from" refers to a peptide sequence that is longer than the sequence from which it is derived, or a sequence that is homologous to the sequence from which it is derived.

[0061] The term "identity percentage" refers to the number of identical residues obtained after optimal alignment of two sequences being compared. This percentage is purely a statistical concept, as the differences between the two sequences are randomly distributed across their entire length. "Optimal alignment" or "best alignment" refers to the alignment that yields the highest identity percentage, as determined below. Comparisons between two sequences are traditionally performed after optimal alignment of these sequences, through segments or "comparison windows" to identify and compare local regions of sequence similarity. For example, the BLOSUM matrix and the local homology algorithm of Smith and Waterman (1981, J. Mol Evol., 18:38-46) can be used. Any other known methods may also be used.

[0062] The term "substitution conservative" refers to the substitution of one amino acid for another without altering the overall conformation and function of the peptide. Amino acids with similar properties are well-known. For example, arginine, histidine, and lysine are interchangeable hydrophilic basic amino acids. Isoleucine can be replaced by leucine, methionine, or valine. Asparagine, glutamine, serine, and threonine are interchangeable.

[0063] The term "essentially composed of peptides" means that more than 50% of the bonds connecting amino acids are peptide bonds, but some residues may be linked to non-amino acid molecules, or to amino acids, for example, via disulfide bridges.

[0064] The abbreviation GB refers to glioblastoma.

[0065] The term "pharmaceutically acceptable" means that a compound, material, excipient, composition, or dosage form is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other related problems or complications, within the bounds of good medical judgment, and has a reasonable benefit / risk ratio.

[0066] The terms "treatment", "therapeutic" or "treatment" (« traiter », « traité » ou « traitement ») in the context of this invention refer to therapeutic treatment aimed at eliminating or alleviating symptoms.

[0067] Beneficial or desired clinical outcomes include, but are not limited to, elimination of symptoms, reduction of symptoms, narrowing of disease range, stable health status (i.e., no deterioration), and delay or slowing of disease progression.

[0068] The effective dose of treatment can be readily determined by a therapeutic diagnostician (as a person skilled in the art) using standard techniques and by observing results obtained in similar circumstances.

[0069] To determine an effective therapeutic dose, a therapeutic diagnostician will consider many factors, including but not limited to: the subject's species; their body size, age, and general health status; the specific disease involved; the extent or severity of the disease; the individual subject's response; the specific compound administered; the route of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; the use of concomitant medications; and other relevant factors.

[0070] The term "active substance" refers to the molecule present in a therapeutically effective amount.

[0071] The binding between peptides and proteins can be measured using any method known to those skilled in the art, such as dual polarization interferometry, static light scattering (SLS), dynamic light scattering (DLS), surface plasmon resonance (SPR), fluorescence polarization / anisotropy, fluorescence correlation spectroscopy, fluorescence intermolecular energy transfer (FRET), isothermal titration calorimetry, and microscale thermophoresis.

[0072] Attached Figure Figure 1 The mechanism of interaction between FlnA and the UT receptor is illustrated in a schematic diagram; Figure 2 and 3 The inhibitory mechanism of FlnA-UT receptor interaction provided by the UT-CPP336-350-Cycloglio family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 16, SEQ ID NO: 34 and SEQ ID NO: 44) and the results obtained are illustrated in schematic form. Figure 4 and 5 The inhibitory mechanism of FlnA-UT receptor interaction provided by the UT-CPP339-350-Cycloglio family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 17, SEQ ID NO: 35 and SEQ ID NO: 45) and the results obtained are illustrated in schematic form. Figure 6 and 7 The inhibitory mechanism of FlnA-UT receptor interaction provided by the FlnA-CPPD19-FG-Cycloglio- family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 10, SEQ ID NO: 28 and SEQ ID NO: 38) and the results obtained are illustrated in schematic form. Figure 8 and 9The inhibitory mechanism of FlnA-UT receptor interaction provided by the FlnA-CPPD20-CD-Cycloglio- family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 11, SEQ ID NO: 29 and SEQ ID NO: 39) and the results obtained are illustrated in schematic form. Figure 10 and 11 The inhibitory mechanism of FlnA-UT receptor interaction provided by the FlnA-CPPD20-DE-Cycloglio- family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 12, SEQ ID NO: 30 and SEQ ID NO: 40) and the results obtained are illustrated in schematic form. Figure 12 and 13 The inhibitory mechanism of FlnA-UT receptor interaction provided by the UTCPP339-350-Glio- family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 19, SEQ ID NO: 37 and SEQ ID NO: 47) and the results obtained are illustrated in schematic form. Figure 14 and 15 The inhibitory mechanism of FlnA-UT receptor interaction provided by the UTCPP336-350-Glio- family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 18, SEQ ID NO: 36 and SEQ ID NO: 46) and the results obtained are illustrated in schematic form. Figure 16 and 17 The inhibitory mechanism of FlnA-UT receptor interaction provided by the FlnACPP-D19-FG-Glio- family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 13, SEQ ID NO: 31 and SEQ ID NO: 41) and the results obtained are illustrated in schematic form. Figure 18 and 19 The inhibitory mechanism of FlnA-UT receptor interaction provided by the FlnACPP-D20-CD-Glio- family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 14, SEQ ID NO: 32 and SEQ ID NO: 42) and the results obtained are illustrated in schematic form. Figure 20 and 21The schematic diagram illustrates the inhibitory mechanism of FlnA-UT receptor interaction provided by the FlnACPP-D20-DE-Glio- family constructs of the present invention (i.e., constructs corresponding to the sequences of SEQ ID NO: 15, SEQ ID NO: 33 and SEQ ID NO: 43) and the results obtained. Example

[0073] Description of the implementation plan Synthesis of the construct according to the present invention and the construct used as a control The following sequences, corresponding to the compounds SEQ ID NO: 9, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, were synthesized using a Symphony-X automated synthesizer (GyrosProtein Technologies) on 2-chlorotriphenylmethyl chlororesin according to the Fmoc strategy. Briefly, the C-terminal amino acids of each sequence were anchored to 2-chlorotriphenylmethyl chlororesin, yielding a degree of substitution of approximately 0.25 mmol / g in the presence of diisopropylethylamine in dichloromethane. Subsequently, excess reactive sites were deactivated by treatment with methanol in the presence of diisopropylethylamine. These resins were used for automated synthesis according to the protocol recommended by the Symphony-X manufacturer. For each synthesis cycle, deprotection was performed twice with piperidine in 20% dimethylformamide (DMF) for 5 minutes each time. After washing the resin with DMF (4 times), coupling was performed by treatment with a mixture of Fmoc amino acids (5 equivalents), HATU (5 equivalents), pure oxyma (5 equivalents), and diisopropylethylamine (15 equivalents) for 1 hour. Finally, the resin was washed with DMF (4 times). The last two amino acids to be coupled were Fmoc-Lys(Dde)-OH and Boc-Cys(Trt)-OH. Therefore, the Dde protecting group was cleaved while keeping the peptide on the resin by treatment with hydrazine in 2% DMF three times for 2 minutes each time. The desired carboxytetramethylrhodamine could optionally be coupled to the side chain of the N-terminal lysine residue. Monomeric peptides were lysed and precipitated in diethyl ether after treatment with TFA / water / TIPS / EDT (92.5%, 2.5%, 2.5%, 2.5%) for 5 hours. After centrifugation and decantation, the peptides were dissolved in water and purified by reversed-phase preparative HPLC. The purified monomeric peptides were dissolved in 50 mmol ammonium bicarbonate buffer and adjusted to pH 10 with diluted ammonium hydroxide. The solution was stirred overnight in the presence of atmospheric oxygen to obtain the desired symmetrical dimer. Finally, the solution was lyophilized.

[0074] The following sequences, corresponding to the compounds SEQ ID NO: 8, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 24, SEQ ID NO: 52, SEQ ID NO: 25, SEQ ID NO: 53, SEQ ID NO: 26, SEQ ID NO: 54, SEQ ID NO: 27, and SEQ ID NO: 55, were synthesized using a Symphony-X automated synthesizer (Gyros Protein Technologies) on 2-chlorotriphenylmethyl chlororesin according to the Fmoc strategy. Briefly, the C-terminal amino acids of each sequence were anchored to 2-chlorotriphenylmethyl chlororesin, yielding a degree of substitution of approximately 0.25 mmol / g in the presence of diisopropylethylamine in dichloromethane. Subsequently, excess reactive sites were inactivated by treatment with methanol in the presence of diisopropylethylamine. These resins were used for automated synthesis according to the protocol recommended by the Symphony-X manufacturer. For each synthesis cycle, deprotection was performed twice with piperidine in 20% dimethylformamide (DMF) for 5 minutes each time. After washing the resin (4 times) with DMF, coupling was performed for 1 hour with a mixture of Fmoc amino acids (5 equivalents), HATU (5 equivalents), pure oxyma (5 equivalents), and diisopropylethylamine (15 equivalents). Finally, the resin was washed with DMF (4 times). If desired, the final coupling acid was DEAC. The peptides were cleaved by treatment with TFA / water / TIPS / EDT (92.5%, 2.5%, 2.5%, 2.5%) for 3 hours and precipitated in diethyl ether. After centrifugation and decantation, the peptides were dissolved in water and purified by reversed-phase preparative HPLC.

[0075] Similarly, constructs of the Cycloglio family can be synthesized, which contain two distinct interfering sequences, one that binds to the UT receptor and the other that binds to FlnA.

[0076] In vitro biological effects of the constructs of this invention Example 1: Study on the incorporation of a SEQ ID NO: 35 construct that mimics the FlnA-binding UT sequence into glioblastoma (GB) 8MG cell line, lung cancer A549, breast cancer MDA-MB-231, and prostate cancer LnCAP.

[0077] At t0, 100,000 cells per cell line were seeded in 12-well plates pre-coated with fibronectin (2 µg / ml; 1 h; 37°C). After 24 hours of adhesion, the cells were treated with 100 nM SEQ ID NO: 35 construct, which grafts a 5-carboxytetramethylrhodamine group onto a K2 residue (SEQ ID NO: 35 construct), allowing for peptide incorporation to be tracked during the 12-hour acquisition period using video microscopy.

[0078] Table 1 summarizes the penetration kinetics of the SEQ ID NO: 35 construct in glioblastoma 8MG, lung cancer A549, breast cancer MDA-MB-231 and prostate cancer LnCAP cell lines.

[0079] Table 1 .

[0080] The SEQ ID NO:35 construct, mimicking the FlnA-binding UT sequence, rapidly penetrated glioblastoma 8MG, lung cancer A549, breast cancer MDA-MB-231, and prostate cancer LnCAP cell lines, with an initial penetration time of less than 3 minutes. Furthermore, the SEQ ID NO:35 construct completely diffused in the cytoplasm of the tested solid cancer cells, exhibiting very similar kinetics, ranging from 303 minutes to 416.3 minutes. Therefore, the SEQ ID NO:35 construct rapidly entered the FlnA-UT interaction zone and the subcellular compartments containing FlnA and UT, and could rapidly deliver its interfering effect after administration. Moreover, post-treatment, the SEQ ID NO:35 construct persisted in the cytoplasm of all solid cancer cell lines for at least 774 minutes, maintaining its interfering properties.

[0081] Example 2: The construct of the present invention simulating UT participation in binding to the FlnA fragment (SEQ ID NO: 35) showed positive effects on GB cell lines 8MG, 42MG, U87 and GL15, lung cancer A549, breast cancer MDA-MB-231 and prostate cancer LnCAP cells at 10 -9 The effects of MUUII peptide stimulation on cell migration.

[0082] At time t0, in the presence of 100 nM SEQ ID NO: 35 construct, 30,000 GB cells (8MG, 42MG, U87, and GL15), 75,000 lung cancer A549 cells, 100,000 breast cancer MDA-MB-231 cells, and 75,000 prostate cancer LnCAP cells were seeded onto the upper porous membrane (8 µm) of a Boyden chamber. A uniform concentration of 10... -9MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed and labeled with DAPI (4',6-diamidindo-2-phenylindole), and membranes were then cut and placed between a slide and a coverslip. Eight photomicrographs were taken for each membrane. The number of cells per field of view was semi-automatically quantified using ImageJ image analysis software (8 fields of view analyzed per membrane, 2 membranes per condition). The results (statistical tests: two-way ANOVA; Sidak post-hoc test, 2 membranes per condition, n = 8 fields of view per membrane) are summarized in Table 2 below.

[0083] Table 2 .

[0084] The SEQ ID NO: 35 construct completely and significantly blocked 10 -9 The directed migration induced by UII in GB cell lines 8MG, 42MG, U87, and GL15, as well as in prostate cancer LnCAP cells, showed significantly lower response rates under control conditions compared to the migratory stimulation induced by UII. The construct SEQ ID NO: 35 is indeed a compound capable of effectively inhibiting the directed migration of solid cancer cell lines responding to the chemisorption gradient of the UII peptide.

[0085] Example 3: The present invention simulates the effect of a construct (SEQ ID NO: 35) of UT participating in binding to the FlnA fragment on cell migration of GB cell lines 8MG, 42MG, U87 and GL15, lung cancer A549, breast cancer MDA-MB-231 and prostate cancer LnCAP cells under stimulation with 50 ng / ml EGF (epidermal growth factor).

[0086] At T0, with 100 nM SEQ ID NO: 35 construct present, 30,000 GB cells (8MG, 42MG, U87, and GL15), 75,000 lung cancer A549 cells, 100,000 breast cancer MDA-MB-231 cells, and 75,000 prostate cancer LnCAP cells were seeded onto the upper chamber porous membrane (8 µm) of a Boyden chamber. A uniform concentration of 50 ng / ml EGF was added to the lower chamber. After 24 h, the upper chamber cells were removed, the lower chamber cells were fixed, and the nuclei were labeled with DAPI (4',6-diamidinyl-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (8 fields analyzed per membrane, 2 membranes per condition). The results are summarized in Table 3 below.

[0087] Table 3 .

[0088] The SEQ ID NO: 35 construct potently and significantly inhibited directional migration induced by 50 ng / ml EGF in GB cell lines 8MG, 42MG, and U87, as well as in prostate cancer LnCAP cells, which showed positive responses to control conditions with EGF alone (-40.55%, p=0.0332; -49.48%, p<0.0001; -103.18%, p<0.0001; and -256.54%, p<0.0001, respectively). In GB cell line GL15, lung cancer A549, and breast cancer MDA-MB-231, the SEQ ID NO: 35 construct partially inhibited 50 ng / ml EGF-induced directional migration (-27.61%, p=0.0002; -22.14%, p<0.0001; and -56.54%, p<0.0001, respectively). The SEQ ID NO: 35 construct is indeed a compound that can effectively inhibit the directed migration of solid cancer cell lines that respond to the EGF chemisorption gradient.

[0089] Example 4: The construct of the present invention that mimics FlnA participation in binding to UT fragments (SEQ ID NO: 29) showed positive effects on GB cell lines 8MG, 42MG, U87 and GL15, lung cancer A549, breast cancer MDA-MB-231 and prostate cancer LnCAP cells at 10 -9 The effects of MUUII peptide stimulation on cell migration.

[0090] At T0, with 100 nM SEQ ID NO: 29 construct present, 30,000 GB cells (8MG, 42MG, U87, and GL15), 75,000 lung cancer A549 cells, 100,000 breast cancer MDA-MB-231 cells, and 75,000 prostate cancer LnCAP cells were seeded onto the upper chamber porous membrane (8 µm) of a Boyden chamber. A uniform concentration of 10... -9 MUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed and their nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic quantification of cell count per field was performed using ImageJ image analysis software (8 fields analyzed per membrane, 2 membranes per condition). The results are summarized in Table 4 below. As with all other experiments, statistical tests were performed using two-way ANOVA and Sidak post-hoc tests, with 2 membranes per condition and n = 8 fields per membrane.

[0091] Table 4 .

[0092] The SEQ ID NO: 29 construct completely and significantly blocked 10 -9 MIUII induced directed migration in GB cell lines 8MG, U87, and GL15, as well as in prostate cancer LnCAP cells. These cell lines showed positive responses compared to control conditions with UII alone (-68.41%, p=0.0007; -63.17%, p<0.0001; -91.80%, p<0.0001; and -138.15%, p=0.0022, respectively). Furthermore, it partially inhibited 10 -9 MUII-induced migration in GB 42MG cell line (-26.72%, p=0.0015). The SEQ ID NO: 29 construct is indeed a compound that can effectively inhibit the directed migration of solid cancer cell lines that respond to the chemisorption gradient of the UII peptide.

[0093] Example 5: The effect of the construct of the present invention that simulates FlnA participating in the binding of UT fragment (SEQ ID NO: 29) on cell migration of GB cell lines 8MG, 42MG, U87 and GL15, lung cancer A549, breast cancer MDA-MB-231 and prostate cancer LnCAP cells under stimulation with 50 ng / ml EGF (epidermal growth factor).

[0094] At T0, with 100 nM SEQ ID NO: 29 construct present, 30,000 GB cells (8MG, 42MG, U87, and GL15), 75,000 lung cancer A549 cells, 100,000 breast cancer MDA-MB-231 cells, and 75,000 prostate cancer LnCAP cells were seeded onto the upper chamber porous membrane (8 µm) of a Boyden chamber. A uniform concentration of 50 ng / ml EGF was added to the lower chamber. After 24 h, the upper chamber cells were removed, the lower chamber cells were fixed, and the nuclei were labeled with DAPI (4',6-diamidinyl-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (8 fields analyzed per membrane, 2 membranes per condition). The results are summarized in Table 5 below.

[0095] Table 5 .

[0096] The SEQ ID NO: 29 construct partially inhibited directed migration induced by 50 ng / ml EGF in GB cell lines U87 and GL15, which exhibited positive responses under control conditions with EGF alone (-63.59%, p=0.0022 and -37.75%, p<0.0001, respectively). The SEQ ID NO: 29 construct is indeed a compound capable of effectively partially inhibiting the directed migration of solid cancer cell lines responding to the chemisorption gradient of EGF.

[0097] Example 6: The construct of the present invention simulating UT participation in binding to the FlnA fragment (SEQ ID NO: 37) showed positive effects on GB cell lines 8MG, 42MG, U87, and U251 at 10 -9 The effects of MUUII peptide stimulation on cell migration.

[0098] At T0, with 100 nM SEQ ID NO: 37 construct present, 30,000 GB cells (8 mg, 42 mg, U87, and U251) were seeded onto the upper chamber porous membrane (8 µm) of a Boyden chamber. A uniform concentration of 10... -9 MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed, and nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields were analyzed per membrane, two membranes per condition). The results are summarized in Table 6 below.

[0099] Table 6 .

[0100] The SEQ ID NO: 37 construct completely and significantly blocked 10 -9 MIUII induced directed migration in GB cell lines 8MG, 42MG, U87, and U251, which showed positive responses to the control condition of UII alone (-123.98%, p<0.0001; -115.36%, p<0.0001; -110.96%, p<0.0001; and -60.50%, p<0.0001, respectively). The construct SEQ ID NO: 37 is indeed a compound that effectively inhibits the directed migration of GB cell lines responding to the chemisorption gradient of the UII peptide.

[0101] Example 7: The construct of the present invention simulating UT participation in binding to the FlnA fragment (SEQ ID NO: 36) showed positive effects on GB cell lines 8MG, 42MG, U87, and U251 at 10 -9 The effects of MUUII peptide stimulation on cell migration.

[0102] At T0, with 100 nM SEQ ID NO: 36 construct present, 30,000 glioblastoma cells (8MG, 42MG, U87, and U251) were seeded onto the upper chamber porous membrane (8 µm) of a Boyden chamber. A uniform concentration of 10... -9 MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed and their nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields were analyzed per membrane, two membranes per condition). The results are summarized in Table 7 below.

[0103] Table 7 .

[0104] The SEQ ID NO: 36 construct completely and significantly blocked 10 -9 MIUII induced directed migration in GB cell lines 8MG and 42MG, which showed positive responses compared to control conditions with UII alone (-155.78%, p<0.0001 and -100.85%, p<0.0001, respectively). Furthermore, the SEQ ID NO: 36 construct partially inhibited 10 -9Migration induced by MUII in GB U87 cell line (-66.45%, p=0.0211). The construct SEQ ID NO: 36 is indeed a compound that can effectively partially inhibit the directed migration of GB cell lines in response to the chemisorption gradient of the UII peptide.

[0105] Example 8: The construct of the present invention simulating FlnA participation in binding to UT fragments (SEQ ID NO: 32) showed efficacy against GB cell lines 8MG, 42MG, U87, and U251 at 10 -9 The effects of MUUII peptide stimulation on cell migration.

[0106] At T0, with 100 nM SEQ ID NO: 32 construct present, 30,000 GB cells (8MG, 42MG, U87, and U251) were seeded onto the upper chamber porous membrane (8 µm) of a Boyden chamber. A uniform concentration of 10... -9 MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed and their nuclei were labeled with DAPI (4',6-diamidinyl-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields were analyzed per membrane, two membranes per condition). The results are summarized in Table 8 below.

[0107] Table 8 .

[0108] The SEQ ID NO: 32 construct completely and significantly blocked 10 -9 MIUII induced directed migration in GB cell lines 8MG, 42MG, U87, and U251, which showed positive responses to the control condition of UII alone (-116.32%, p<0.0001; -97.40%, p=0.0009; -119.71%, p<0.0001; and -46.12%, p<0.0001, respectively). The construct SEQ ID NO: 32 is indeed a compound that effectively inhibits the directed migration of GB cell lines responding to the chemisorption gradient of the UII peptide.

[0109] Example 9: The construct of the present invention simulating FlnA participation in binding to UT fragments (SEQ ID NO: 33) showed efficacy against GB cell lines 8MG, 42MG, U87, and U251 at 10 -9 The effects of MUUII peptide stimulation on cell migration.

[0110] At T0, with 100 nM SEQ ID NO: 33 construct present, 30,000 GB cells (8 mg, 42 mg, U87, and U251) were seeded onto the upper chamber porous membrane (8 µm) of a Boyden chamber. A uniform concentration of 10... -9 MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed, and nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields were analyzed per membrane, two membranes per condition). The results are summarized in Table 9 below: Table 9 .

[0111] The SEQ ID NO: 33 construct completely and significantly blocked 10 -9 MIUII induced directed migration in GB cell lines 8MG, 42MG, and U251, which showed positive responses compared to control conditions with UII alone (-105.48%, p<0.0001; -104.29%, p=0.0037; and -58.53%, p<0.0001, respectively). Furthermore, the SEQ ID NO: 33 construct partially inhibited 10 -9 Migration induced by MUII in GB U87 cell line (-24.83%, p=0.0037). The SEQ ID NO: 33 construct is indeed a compound that can effectively inhibit the directed migration of GB cell lines in response to the chemisorption gradient of the UII peptide.

[0112] Example 10: The construct of the present invention simulating FlnA participation in binding to UT fragments (SEQ ID NO: 31) showed positive effects on GB cell lines 8MG, 42MG, U87, and U251 at 10 -9 The effects of MUUII peptide stimulation on cell migration.

[0113] At T0, with 100 nM SEQ ID NO: 31 construct present, 30,000 GB cells (8MG, 42MG, U87, and U251) were seeded onto the upper chamber porous membrane (8 µm) of a Boyden chamber. A uniform concentration of 10... -9MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed, and nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields were analyzed per membrane, two membranes per condition). The results are summarized in Table 10 below.

[0114] Table 10 .

[0115] The SEQ ID NO: 31 construct completely and significantly blocked 10 -9 MIUII induced directed migration in the GBU251 cell line, which produced a positive response (-93.60%, p<0.0001) under control conditions with UII alone. Furthermore, the SEQ ID NO: 31 construct partially inhibited 10 -9 MUII-induced migration in GB U87 cell line (-37.17%, p=0.0164). The SEQ ID NO: 31 construct is indeed a compound that can effectively partially inhibit the directed migration of GB cell lines in response to the chemisorption gradient of the UII peptide.

[0116] Example 11: The construct of the present invention simulating FlnA participation in binding to UT fragments (SEQ ID NO: 53) showed efficacy against GB cell lines 8MG and U87 at 10 -9 Effects of MUUII stimulation on cell migration.

[0117] At T0, with 10 µM of the SEQ ID NO: 53 construct present, 30,000 GB cells (8 mg) and U87 cells were seeded onto the 8 µm porous membrane in the upper chamber of a Boyden chamber. A uniform concentration of 10 µM of the SEQ ID NO: 53 construct was added to the lower chamber. -9 MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed, and nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields of view per membrane, two membranes per condition). The results are summarized in Table 11 below: Table 11 .

[0118] The SEQ ID NO: 53 construct significantly blocked 10 -9MIUII induced directed migration in GB cell lines 8MG and U87, which showed a positive response compared to the control condition with UII alone (-127.06%, p<0.0001 and -93.65%, p<0.0001, respectively). The SEQ ID NO: 53 construct is indeed a compound that effectively inhibits the directed migration of GB cell lines responding to the chemisorption gradient of the UII peptide, independent of UT receptor binding.

[0119] Example 12: The construct of the present invention simulating FlnA participation in binding to UT fragments (SEQ ID NO: 54) showed efficacy against GB cell lines 8MG and U87 at 10 -9 Effects of MUUII stimulation on cell migration.

[0120] At T0, with 10 µM of the SEQ ID NO: 54 construct present, 30,000 GB cells (8 mg) and U87 cells were seeded onto the 8 µm porous membrane in the upper chamber of a Boyden chamber. A uniform concentration of 10 µM of the construct was added to the lower chamber. -9 MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed and their nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields were analyzed per membrane, two membranes per condition). The results are summarized in Table 12 below.

[0121] Table 12 .

[0122] The SEQ ID NO: 54 construct significantly blocked 10 -9 M UII induced directed migration in GB cell lines 8MG and U87, which showed a positive response compared to the control condition with UII alone (-192.90%, p<0.0001 and -106.12%, p=0.0002, respectively). The SEQ ID NO: 54 construct is indeed a compound that effectively inhibits the directed migration of GB cell lines responding to the chemisorption gradient of the UII peptide, independent of UT receptor binding.

[0123] Example 13: The construct of the present invention simulating FlnA participation in binding to UT fragments (SEQ ID NO: 55) showed efficacy against GB cell lines 8MG and U87 at 10 -9 The effects of MUUII peptide stimulation on cell migration.

[0124] At T0, with 10 µM of the SEQ ID NO: 55 construct present, 30,000 GB cells (8 mg) and U87 cells were seeded onto the 8 µm porous membrane in the upper chamber of a Boyden chamber. A uniform concentration of 10 µM of the construct was added to the lower chamber. -9 MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed and their nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields of view analyzed per membrane, two membranes per condition). The results are summarized in Table 13 below.

[0125] Table 13 .

[0126] The SEQ ID NO: 55 construct significantly blocked 10 -9 MIUII induced directed migration in GB cell lines 8MG and U87, which showed a positive response compared to the control condition with UII alone (-173.77%, p<0.0001 and -96.41%, p<0.0001, respectively). The SEQ ID NO: 55 construct is indeed a compound that effectively inhibits the directed migration of GB cell lines responding to the chemisorption gradient of the UII peptide, independent of UT receptor binding.

[0127] Example 14: The construct of the present invention simulating FlnA participation in binding to UT fragments (SEQ ID NO: 48) showed positive effects on the GB U87 cell line at 10 -9 Effects of MUUII stimulation on cell migration.

[0128] At T0, with 10 µM of the SEQ ID NO: 48 construct present, 30,000 GBU87 cells were seeded onto the 8 µm porous membrane in the upper chamber of the Boyden chamber. A uniform concentration of 10 µM of the SEQ ID NO: 48 construct was added to the lower chamber. -9 MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed and their nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields of view analyzed per membrane, two membranes per condition). The results are summarized in Table 14 below.

[0129] Table 14 .

[0130] SEQ ID NO: 48 The construct partially blocked 10-9 MIUII induced directed migration in the GB U87 cell line, which showed a positive response under control conditions with UII alone (-19.08%, p=0.3674). The construct SEQ ID NO: 48 is indeed a compound ineffective against directed migration in GB cell lines that respond to the chemisorption gradient of the UII peptide, independent of UT receptor binding.

[0131] Example 15: The construct of the present invention simulating FlnA participation in binding to UT fragments (SEQ ID NO: 51) showed positive effects on the GB U87 cell line at 10 -9 Effects of MUUII stimulation on cell migration.

[0132] At T0, with 10 µM of the SEQ ID NO: 51 construct present, 30,000 GBU87 cells were seeded onto the upper porous membrane (8 µm) of the Boyden chamber. A uniform concentration of 10 µM of the SEQ ID NO: 51 construct was added to the lower chamber. -9 MIUII. After 24 hours, cells in the upper chamber were removed, cells in the lower chamber were fixed and their nuclei were labeled with DAPI (4',6-diamidindo-2-phenylindole). Membrane slides were then cut and placed between a slide and a coverslip, and eight photomicrographs were taken for each slide. Semi-automatic cell count per field was performed using ImageJ image analysis software (eight fields of view analyzed per membrane, two membranes per condition). The results are summarized in Table 15 below.

[0133] Table 15 .

[0134] SEQ ID NO: 51 The construct partially blocked 10 -9 M UII induced directed migration in the GB U87 cell line, which showed a positive response under control conditions with UII alone (-40.16%, p=0.3674). The SEQ ID NO: 51 construct is indeed a compound ineffective against directed migration in GB cell lines that respond to the chemisorption gradient of the UII peptide, independent of UT receptor binding.

[0135] Example 16: Effects of the UT-binding FlnA sequence construct SEQ ID NO: 35 on the viability of GB cell lines U251, 8MG, 42MG, U87 and GL15, lung cancer cell lines A549, HCC827 and PC9, breast cancer cell line MDA-MB-231 and prostate cancer LnCAP cells.

[0136] At time T0, 2×10 3Cells were seeded in 96-well plates, and the SEQ ID NO: 35 construct was present at progressively increasing concentrations, with the tested concentration range being 10-1. -5 M to 10 -12 M. Cell density was assessed at 24h, 48h, and 72h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 16.

[0137] Table 16 .

[0138] The SEQ ID NO: 35 construct exhibited enhanced inhibitory effects over time in all the aforementioned solid cancer cell lines, with particularly significant inhibitory effects at 72 h. The inhibitory effect was greatest at a concentration of 10 µM, with the following percentages of inhibition on the viability of U251, 8MG, 42MG, U87, GL15, A549, HCC827, PC9, MDA-MB-231 and LnCAP cell lines: 61.14% (p<0.0001), 74.99% (p<0.0001), 58.07% (p=0.0021), 72.57% (p<0.0001), 79.60% (p=0.0006), 58.16% (p=0.0008), 77.72% (p<0.0001), 66.92% (p<0.0001), 60.26% (p<0.0001) and 74.63% (p<0.0093). In the U251, 8MG, 42MG, U87, GL15, A549, HCC827, and MDA-MB-231 cell lines, minimal inhibitory effects were observed at a concentration of 1 µM, specifically 33.18% (p=0.0073), 48.87% (p<0.0001), 35.04% (p=0.0128), 41.36% (p=0.0011), 56.63% (p=0.0059), 39.22% (p=0.0034), 21.48% (p=0.0006), and 35.92% (p=0.0004), respectively. In the PC9 cell line, the inhibition rate at a concentration of 0.01 µM was 34.46% (p=0.0104). At 72 h, the IC50 of the SEQ ID NO: 35 construct... 50 The values ​​were in the micromolar range, between 1.06 and 6.23 µM. The SEQ ID NO: 35 construct showed an inhibitory effect on the viability of GB, lung cancer, and breast cancer cell lines.

[0139] Example 17: Effects of the construct SEQ ID NO: 29, which simulates the binding of FlnA to the UT sequence, on the viability of GB cell lines U251, 8MG, 42MG, U87 and GL15, lung cancer cell lines A549, HCC827 and PC9, breast cancer cell line MDA-MB-231 and prostate cancer LnCAP cells.

[0140] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 29 construct was present at progressively increasing concentrations, with the tested concentration range being 10-1. -5 M to 10 -12 M. Cell density was assessed at 24h, 48h, and 72h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 17. The SEQ ID NO: 29 construct showed enhanced inhibitory effects over time in all the above solid cancer cell lines, with a particularly significant inhibitory effect at 72h. The inhibitory effect was greatest at a concentration of 10 µM, with inhibition percentages of 49.48% (p=0.0036), 46.28% (p<0.0001), 44.39% (p=0.0035), 46.41% (p=0.0209), 43.73% (p=0.0014), 53.19% (p<0.0001), 49.25% (p=0.002), and 61.68% (p=0.0024) in U251, 8MG, U87, A549, HCC827, PC9, MDA-MB-231, and LnCAP cell lines, respectively. However, this construct showed no effect in 42MG and GL15 cell lines after 72 h. At a concentration of 1 µM, significant inhibition was observed in 8MG, PC9, and LnCAP cell lines at 21.94% (p=0.0084), 18.76% (p=0.0007), and 19.77% (p=0.0251), respectively. At 72 h, the IC50 of the SEQ ID NO:29 construct against the reactive cell lines... 50 The values ​​were in the tens of micromolar range, between 5.17 and 25.18 µM. Table 17 summarizes the experimental results of the effect of the SEQ ID NO: 29 construct on the viability of different GB cell lines (U251, 8MG, 42MG, U87, GL15), lung cancer (A549, HCC827, PC9), breast cancer (MDA-MB-231), and prostate cancer (LnCAP). Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells per cell line.

[0141] Table 17 .

[0142] Example 18: Effect of the UT-binding FlnA sequence construct SEQ ID NO: 37 on the viability of GB cell lines U251, 8MG, 42MG, U87 and GL15, and lung cancer A549 cells.

[0143] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 37 construct was present at increasing concentrations, with the tested concentration range being 10-1. -5 M to 10 -12 M. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 18. The SEQ ID NO: 37 construct showed an increasing inhibitory effect over time only in the GBU251 cell line, with the maximum effect at 72 h, inhibiting cell viability by 89.40% at a concentration of 10 µM (p = 0.0017). The inhibitory properties of the SEQ ID NO: 37 construct were relatively low specificity, suggesting that the fusion of the SEQ ID NO: 4 and SEQ ID NO: 8 constructs was less effective than its homolog SEQ ID NO: 35 (generated by the fusion of the SEQ ID NO: 4 and SEQ ID NO: 9 constructs). In fact, the SEQ ID NO: 9 penetration sequence appears to be decisive in conveying the inhibitory effect of this construct on cell viability.

[0144] Table 18 summarizes the experimental results on the effect of the SEQ ID NO: 37 construct on the viability of different GB cell lines (U251, 8MG, 42MG, U87, GL15) and lung cancer (A549) cells. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells for each cell line.

[0145] Table 18 .

[0146] Example 19: Effect of the construct SEQ ID NO: 32 simulating FlnA binding to UT sequence on the viability of GB cell lines U251, 8MG, 42MG, U87 and GL15 and lung cancer A549 cells.

[0147] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 32 construct was present at progressively increasing concentrations, with the tested concentration range being 10-1. -5M to 10 -12 M. Cell density was assessed at 24h, 48h, and 72h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 19. The SEQ ID NO: 32 construct did not show significant inhibitory effects in any of the tested cell lines. The lack of inhibitory properties in the SEQ ID NO: 32 construct suggests that it is a fusion of the SEQ ID NO: 5 and SEQ ID NO: 8 constructs and is less effective than its homolog SEQ ID NO: 29 (generated by the fusion of the SEQ ID NO: 5 and SEQ ID NO: 9 constructs). In fact, the penetration sequence of SEQ ID NO: 9 appears to be decisive for conveying the inhibitory effect of this construct on cell viability. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells per cell line.

[0148] Table 19 .

[0149] Example 20: Effect of the construct SEQ ID NO: 30 simulating FlnA binding to UT sequence on the viability of GB cell lines U251, 8MG and 42MG.

[0150] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 30 construct was present at increasing concentrations, with the tested concentration range being 10. -5 M to 10 -12 M. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 20. The SEQ ID NO: 30 construct showed an enhanced inhibitory effect over time only in the GB U251 cell line, with the maximum effect at 72 h, inhibiting cell viability by 73.67% at a concentration of 10 µM (p = 0.0017). The lack of potency of the SEQ ID NO: 30 construct suggests that the interfering sequence SEQ ID NO: 5 contained in its structure is insufficient to convey the inhibitory effect on GB cell line viability.

[0151] Table 20 summarizes the experimental results on the effect of the SEQ ID NO: 30 construct on the cell viability of different GB cell lines (U251, 8MG, 42MG). Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells for each cell line.

[0152] Table 20 .

[0153] Example 21: Effect of the SEQ ID NO: 34 construct simulating UT binding to FlnA sequence on the viability of GBU251 cells.

[0154] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 34 construct was present at increasing concentrations, with the tested concentration range being 10. -5 M to 10 -12 M. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 21. The SEQ ID NO: 34 construct exhibited an early inhibitory effect on the viability of the U251 cell line, which was observed at a concentration of 10 µM at 24 h (50.15%, p=0.0095). However, unlike other effective compounds of this invention, its effect diminished to no longer being significant at 72 h (p=0.0513).

[0155] Table 21 summarizes the experimental results regarding the effect of the SEQ ID NO: 34 construct on the viability of GBU251 cells. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=3 independent experiments, triplet wells.

[0156] Table 21 .

[0157] Example 22: Effect of the SEQ ID NO: 36 construct simulating UT binding to FlnA sequence on the viability of GBU251 cells.

[0158] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 36 construct was present at progressively increasing concentrations, with the tested concentration range being 10-1. -5 M to 10 -12 M. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 22. The SEQ ID NO: 36 construct showed an increasing inhibitory effect on GBU251 cell viability over time, with the maximum effect at 72 h, inhibiting cell viability by 82.41% at a concentration of 10 µM (p=0.0025).

[0159] Table 22 summarizes the experimental results regarding the effect of the SEQ ID NO: 36 construct on the viability of GBU251 cells. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=3 independent experiments, triplet wells.

[0160] Table 22 .

[0161] Example 23: Effects of a construct SEQ ID NO: 35 simulating the binding of the UT to the FlnA sequence in combination with clinically used standard therapies on the viability of GB cell lines U251, 8MG, 42MG, U87 and GL15, lung cancer cell lines A549, HCC827 and PC9, breast cancer cell line MDA-MB-231 and prostate cancer LnCAP cells.

[0162] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 35 construct was present at progressively increasing concentrations, with the tested concentration range being 10-1. -5 M to 10 -12 M. In addition, GB cell lines U251, 8MG, 42MG, U87, and GL15 were treated with 950 µM TMZ; lung cancer cell lines A549, HCC827, PC9, and breast cancer cell line MDA-MB-231 were treated with 20 µM, 0.008 µM, 0.020 µM, and 20 µM gefitinib, respectively. Finally, prostate cancer cell line LnCAP was treated with 1 nM docetaxel. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). The results are summarized in Table 23. The SEQ ID NO: 35 construct at a concentration of 10 µM showed a significant additive effect relative to TMZ in GB cell lines U251, 8MG, and U87, which was particularly significant at 72 h, increasing the effect of TMZ alone from 38.3% to 86.51% (p<0.0001); from 80.11% to 92.80% (p=0.0199); and from 79.95% to 91.86% (p=0.0398).

[0163] Table 23 summarizes the experimental results on the effects of the SEQ ID NO: 35 construct on the viability of different GB cell lines (U251, 8MG, 42MG, U87, GL15), lung cancer (A549, HCC827, PC9), breast cancer (MDA-MB-231), and prostate cancer (LnCAP) cells, as well as the effects of combination therapy with corresponding conventional therapies. GB cell lines U251, 8MG, 42MG, U87, and GL15 were treated with 950 µM TMZ; lung cancer cell lines A549, HCC827, PC9, and breast cancer cell line MDA-MB-231 were treated with 20 µM, 0.008 µM, 0.020 µM, and 20 µM gefitinib, respectively; finally, the prostate cancer cell line LnCAP was treated with 1 nM docetaxel. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells per cell line.

[0164] Table 23 .

[0165] In lung cancer cell lines HCC827 and PC9, this construct also exhibited an additive effect relative to gefitinib at 72 h, increasing the gefitinib-induced inhibition of activity from 5.58% to 76.47% (p=0.0028) and from 76.00% to 91.44% (p=0.0009). The additive effect of compound SEQ ID NO: 35 with TMZ and gefitinib is particularly significant for clinical applications in combination with chemotherapy or targeted therapy.

[0166] Example 24: Effects of a construct SEQ ID NO: 29 that mimics the binding of FlnA to the UT sequence, in combination with clinically used standard therapies, on the viability of GB cell lines U251, 8MG, 42MG, U87, and GL15, lung cancer cell lines A549, HCC827, and PC9, breast cancer cell line MDA-MB-231, and prostate cancer LnCAP cells.

[0167] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 29 construct was present at progressively increasing concentrations, with the tested concentration range being 10-1. -5 M to 10 -12M. In addition, GB cell lines U251, 8MG, 42MG, U87, and GL15 were treated with 950 µM TMZ; lung cancer cell lines A549, HCC827, PC9, and breast cancer cell line MDA-MB-231 were treated with 20 µM, 0.008 µM, 0.020 µM, and 20 µM gefitinib, respectively. Finally, prostate cancer cell line LnCAP was treated with 1 nM docetaxel. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). The results are summarized in Table 24. The 10 µM concentration of the SEQ ID NO: 29 construct exhibited a significant additive effect relative to TMZ in GB cell lines U251 and 8 MG, particularly at 72 h, increasing the effect of TMZ alone from 41.68% to 80.07% (p=0.0137) and from 78.26% to 88.86% (p=0.0108). In the lung cancer cell line PC9, the construct also showed an additive effect relative to gefitinib at 72 h, increasing the gefitinib-induced inhibition of activity from 76.00% to 89.80% (p=0.0168). The additive effect of the SEQ ID NO: 29 compound with TMZ and gefitinib was not systematically observed in GB and lung cancer cell lines.

[0168] Table 24 summarizes the experimental results on the effects of the SEQ ID NO: 29 construct on the viability of different GB cell lines (U251, 8MG, 42MG, U87, GL15), lung cancer (A549, HCC827, PC9), breast cancer (MDA-MB-231), and prostate cancer (LnCAP) cells, as well as the effects of combination therapy with corresponding conventional therapies. GB cell lines U251, 8MG, 42MG, U87, and GL15 were treated with 950 µM TMZ; lung cancer cell lines A549, HCC827, PC9, and breast cancer cell line MDA-MB-231 were treated with 20 µM, 0.008 µM, 0.020 µM, and 20 µM gefitinib, respectively; finally, the prostate cancer cell line LnCAP was treated with 1 nM docetaxel. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells per cell line.

[0169] Table 24 .

[0170] Example 25: Effect of a construct SEQ ID NO: 37 simulating the binding of the UT to the FlnA sequence in combination with a clinically used standard therapy (i.e., TMZ) on the viability of GB cell lines U251, 8MG, 42MG, U87 and GL15.

[0171] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 37 construct was present at increasing concentrations, with the tested concentration range being 10-1. -5 M to 10 -12 M. In addition, GB cell lines U251, 8MG, 42MG, U87, and GL15 were treated with 950 µM TMZ. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 25. The SEQ ID NO: 37 construct showed a significant additive effect relative to TMZ only at 72 h at a concentration of 10 µM on the U251 cell line, increasing the effect of TMZ from 61.65% to 94.36% (p=0.0066). This effect was not reproduced in other cell lines 8MG, 42MG, U87, and GL15, suggesting that the SEQ ID NO: 37 construct is not the optimal choice for use in combination with TMZ in clinical patients.

[0172] Table 25 summarizes the experimental results of the effect of the SEQ ID NO: 37 construct on the cell viability of different GB cell lines (U251, 8MG, 42MG, U87, GL15), and the effects of combination therapy with corresponding conventional therapies (TMZ). GB cell lines U251, 8MG, 42MG, U87, and GL15 were treated with 950 µM TMZ. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells for each cell line.

[0173] Table 25 .

[0174] Example 26: Effect of a construct SEQ ID NO: 32 that mimics the binding of FlnA to the UT sequence in combination with a clinically used standard therapy (i.e., TMZ) on the viability of GB cell lines U251, 8MG, 42MG, U87 and GL15.

[0175] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 32 construct was present at progressively increasing concentrations, with the tested concentration range being 10-1. -5 M to 10 -12M. In addition, GB cell lines U251, 8MG, 42MG, U87, and GL15 were treated with 950 µM TMZ. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 26. The SEQ ID NO: 32 construct did not show an additive effect relative to TMZ in any of the tested cell lines, suggesting that it is not suitable for use in combination with TMZ in clinical patients. Table 26 summarizes the experimental results of the effect of the SEQ ID NO: 32 construct on cell viability of different GB cell lines (U251, 8MG, 42MG, U87, GL15) and its effect in combination with the corresponding conventional therapy (TMZ). GB cell lines U251, 8MG, 42MG, U87, and GL15 were treated with 950 µM TMZ. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells per cell line.

[0176] Table 26 .

[0177] Example 27: Effect of a construct SEQ ID NO: 30 that mimics the binding of FlnA to the UT sequence in combination with a clinically used standard therapy (i.e., TMZ) on the viability of GB cell lines U251, 8MG, and 42MG.

[0178] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 30 construct was present at increasing concentrations, with the tested concentration range being 10. -5 M to 10 -12M. In addition, GB cell lines U251, 8MG, and 42MG were treated with 950 µM TMZ. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). The results are summarized in Table 27. The SEQ ID NO: 30 construct showed a transient but significant additive effect on the U251 cell line at 48 h at a concentration of 10 µM, increasing its effect from 22.12% to 59.01% (p=0.0193). This weak but transient effect was not reproduced in the other two GB cell lines, 8MG and 42MG, therefore the combination of the SEQ ID NO: 30 construct and TMZ cannot be considered for clinical use. Table 27 summarizes the experimental results of the effect of the SEQ ID NO: 30 construct on cell viability of different GB cell lines (U251, 8MG, and 42MG), and the effects of its combination with the corresponding conventional therapy (TMZ). GB cell lines U251, 8MG, and 42MG were treated with 950 µM TMZ. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells per cell line.

[0179] Table 27 .

[0180] Example 28: Effect of a construct SEQ ID NO: 34 simulating the binding of the UT to the FlnA sequence in combination with the clinically used standard therapy (i.e., TMZ) on the viability of G U251 glioblastoma cells.

[0181] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 34 construct was present at increasing concentrations, with the tested concentration range being 10. -5 M to 10 -12M. In addition, the G U251 cell line was treated with 950 µM TMZ. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 28. The 10 µM concentration of the SEQ ID NO: 34 construct showed a significant additive effect relative to TMZ in the G U251 cell line, particularly at 72 h, increasing the effect of TMZ alone from 55.82% to 83.33% (p=0.0045). Therefore, the combination of the SEQ ID NO: 34 construct and TMZ could be considered for clinical treatment of patients carrying the same GB subtype as the U251 cell line. Table 28 summarizes the experimental results of the effect of the SEQ ID NO: 34 construct on the viability of G U251 cells (in combination with the corresponding conventional therapy TMZ). The G U251 glioblastoma cell line was treated with 950 µM TMZ. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells per cell line.

[0182] Table 28 .

[0183] Example 29: Effect of a construct SEQ ID NO: 36 simulating the binding of the UT to the FlnA sequence in combination with a clinically used standard therapy (i.e., TMZ) on the viability of GBU251 cells.

[0184] At time T0, 2×10 3 Cells were seeded in 96-well plates, and the SEQ ID NO: 36 construct was present at progressively increasing concentrations, with the tested concentration range being 10-1. -5 M to 10 -12M. In addition, the G U251 cell line was treated with 950 µM TMZ. Cell density was assessed at 24 h, 48 h, and 72 h using the CellTiter-Glo 3D kit (Promega). Results are summarized in Table 29. The 10 µM concentration of the SEQ ID NO: 36 construct showed a significant additive effect relative to TMZ in the G U251 cell line, particularly at 72 h, increasing the effect of TMZ alone from 69.77% to 95.83% (p = 0.0031). Therefore, the combination of the SEQ ID NO: 36 construct and TMZ could be considered for clinical treatment of patients carrying the same GB subtype as the U251 cell line. Table 29 summarizes the experimental results of the effect of the SEQ ID NO: 36 construct on the viability of G U251 cells (in combination with the corresponding conventional therapy TMZ). The G U251 cell line was treated with 950 µM TMZ. Statistical tests: Kruskal-Wallis test; Dunn post-hoc test, n=2 or 3 independent experiments, triplet wells per cell line.

[0185] Table 29 .

Claims

1. A construct comprising a cell-penetrating peptide sequence coupled to at least a first binding molecule via a peptide bond, the first binding molecule being coupled to the N-terminus of an interfering amino acid sequence via a peptide bond, said interfering amino acid sequence being selected from: a) A sequence that contains or consists of the following sequences: VTYCPTEPGNYIINIKFADQHVPGSPFSVKVTGEGRVKESITRRRRAPSVANVGSHCDLSLKIPEISIQDMTAQVTSPSGKTHEAEIVEGENHTYCIRFVPAE (SEQ ID NO: 1 or FlnA 2104-2206), which forms the 2104-2206 domain of the human FlnA protein; b) A sequence comprising a fragment of the sequence SEQ ID NO: 1 or a sequence consisting of fragments of the sequence SEQ ID NO: 1, said sequence or said fragment being capable of binding the sequence VRGPGSGGGRGPVPSLQPRAR (SEQ ID NO: 2 or UT-330-350), which defines the 330-350 domain of the UT receptor, or a fragment being capable of binding the sequence SEQ ID NO: 2, particularly a fragment of the sequence GGGRGPVPSLQPRAR (SEQ ID NO: 3 or UT-336-350) which forms the 336-350 domain of the UT receptor, or a fragment of the fragment RGPVPSLQPRAR (SEQ ID NO: 4 or UT-339-350) which defines the 339-350 domain of the UT receptor; c) A sequence containing a fragment of the SEQ ID NO: 2 sequence or a sequence consisting of fragments of the SEQ ID NO: 2 sequence, particularly a fragment forming the 330-350 domain of the UT receptor (SEQ ID NO: 2), a fragment forming the 336-350 domain of the UT receptor (SEQ ID NO: 3), or a fragment forming the 339-350 domain of the UT receptor (SEQ ID NO: 4), and a fragment capable of binding the SEQ ID NO: 1 sequence; d) A sequence that has at least 90% identity with the sequence defined in items a) to c), preferably at least 95% identity.

2. The construct according to claim 1, characterized in that, The at least first binding molecule is an amino acid, optionally modified, particularly β-alanine.

3. The construct according to claim 1 or 2, characterized in that, The interfering amino acid sequence is selected from the amino acid sequences defined in section b), and in particular from the following interfering sequences: - MTAQVTSPSGKTHEAEIVE (SEQ ID NO: 5 or FlnA-D20-CD), GNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 6 or FlnA-D19-FG), THEAEIVEGENHTYCIRF (SEQ ID NO: 7 or FlnA-D20-DE); - A peptide sequence having at least 90% and preferably at least 95% identity with the sequence, and a substantially homologous sequence, preferably a peptide sequence derived from the sequence SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 by one or more conserved substitutions; - RGPVPSLQPRAR (SEQ ID NO: 4 or UT-339-350) and GGGRGPVPSLQPRAR (SEQ ID NO: 3 or UT-336-350), and peptide sequences having at least 90% and preferably at least 95% identity with the sequences of said SEQ ID NO: 3 and SEQ ID NO:

4.

4. The construct according to any one of the preceding claims, characterized in that, The cell penetration sequence is selected from the following sequences: - TCTWLKYH (SEQ ID NO: 8 or Glio) and a peptide sequence having at least 90% and preferably at least 95% identity with the SEQ ID NO: 8 sequence, and substantially homologous sequences, preferably sequences derived from the SEQ ID NO: 8 sequence by one or more conserved substitutions; - A peptide sequence GRRRQRRKKRKCCKRKKRRQRRRG (SEQ ID NO: 9 or CycloGlio), wherein residues 12 and 13 (cysteine) are linked by a disulfide bond bridge, and residues 2 to 10 and 15 to 23 are dextrorotatory (D-type) amino acids, and a peptide sequence having at least 90% and preferably at least 95% identity with the sequence of SEQ ID NO: 9, wherein each end of the cell-penetrating sequence is connected to a first binding molecule via a peptide bond, the first binding molecule being an optionally modified amino acid, the binding molecule itself being connected to an interfering sequence via a peptide bond, the two interfering sequences being identical or different.

5. The construct according to claim 4, characterized in that, The cell-penetrating sequence is selected from the peptide sequence GRRRQRRKKRKCCKRKKRRQRRRG (SEQ ID NO: 9 or CycloGlio), wherein residues 12 and 13 (cysteine) are linked by disulfide bonds, and wherein residues 2 to 10 and 15 to 23 are dextrorotatory (D-type) amino acids, and a peptide sequence having at least 90% and preferably at least 95% identity with the sequence of SEQ ID NO: 9, characterized in that the two first binding molecules are identical, particularly β-alanine molecules; And its characteristics - One of the interfering sequences is selected from the sequences defined in claims a) and b) of claim 1, particularly from the following interfering sequences: MTAQVTSPSGKTHEAEIVE (SEQ ID NO: 5 or FlnA-D20-CD), GNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 6 or FlnA-D19-FG), THEAEIVEGENHTYCIRF (SEQ ID NO: 7 or FlnA-D20-DE), and peptide sequences having at least 90% and preferably at least 95% identity with said sequence; and - Another interfering sequence is selected from the sequence defined in claim 1(c), particularly from the following interfering sequences: RGPVPSLQPRAR (SEQ ID NO: 4 or UT-339-350) and GGGRGPVPSLQPRAR (SEQ ID NO: 3 or UT-336-350) and peptide sequences having at least 90% and preferably at least 95% identity with the sequences said in SEQ ID NO: 3 and SEQ ID NO:

4.

6. The interference construct according to any one of claims 4 and 5, characterized in that, The cell-penetrating sequence is selected from the peptide sequence GRRRQRRKKRKCCKRKKRRQRRRG (SEQ ID NO: 9 or CycloGlio), wherein residues 12 and 13 (cysteine) are linked by a disulfide bond bridge, and residues 2 to 10 and 15 to 23 are dextrorotatory (D-type) amino acids, and a peptide sequence having at least 90% and preferably at least 95% identity with the sequence of SEQ ID NO: 9, wherein lysine residues at positions 11 and 14 of the cell-penetrating sequence are acetylated.

7. The interference construct according to any one of the preceding claims, characterized in that, It is selected from the following sequences of constructs: TVKVSFPSGPVHQDAFKINIIYNGXGrrrqrrkkrKCCKrkkrrqrrrGXGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 10, also known as FlnA-CPPD19-FG-CycloGlio), wherein residues 37 and 38 are connected by disulfide bonds, X is an optional modified amino acid at both positions, preferably β-alanine at both positions, and residues 27 to 35 and 40 to 48 are dextrorotatory (D-type) amino acids, wherein lysines at positions 36 and 39 are acetylated; EVIEAEHTKGSPSTVQATMXGrrrqrrkkrKCCKrkkrrqrrrGXMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 11; also known as FlnA-CPPD20-CD-CycloGlio), wherein residues at positions 32 and 33 are connected by a disulfide bond bridge, X is an optional modified amino acid and preferably β-alanine at both positions, and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids, wherein lysines at positions 31 and 34 are acetylated; FRISYTHNEGEVIEAEHTXGrrrqrrkkrKCCKrkkrrqrrrGXTHEAEIVEGENHTYSIRF (SEQ ID NO: 12; also known as FlnA-CPPD20-DE-CycloGlio), wherein residues 31 and 32 are connected by a disulfide bond bridge, X is an optional modified amino acid, preferably β-alanine, and residues 21 to 29 and 34 to 42 are dextrorotatory (D-type) amino acids, and lysines at positions 30 and 33 are acetylated; TCTWLKYHXGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 13; also known as FlnA-CPPD19-FG-Glio), where X is an optional modified amino acid, preferably β-alanine; TCTWLKYHXMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 14; also known as FlnA-CPPD20-CD-Glio), wherein X is an optional modified amino acid, preferably β-alanine; TCTWLKYHXTHEAEIVEGENHTYCIRF (SEQ ID NO: 15; also known as FlnA-CPPD20-DE-Glio), wherein X is an optional modified amino acid, preferably β-alanine; RARPQLSPVPGRGGGXGrrrqrrkkrKCCKrkkrrqrrrGXGGGRGPVPSLQPRAR (SEQ ID NO: 16, also known as UT-CPP336-350-CycloGlio), wherein residues 28 and 29 are connected by a disulfide bond bridge, X is an optional modified amino acid and preferably β-alanine at both positions, and residues 18 to 26 and 31 to 39 are dextrorotatory (D-type) amino acids, and lysines at positions 27 and 30 are acetylated; RARPQLSPVPGRXGrrrqrrkkrKCCKrkkrrqrrrGXRGPVPSLQPRAR SEQ ID NO: 17; also known as UT-CPP339-350-CycloGlio), wherein residues 25 and 26 are linked by a disulfide bond, X is an optional modified amino acid, preferably β-alanine at both positions, and residues 15 to 23 and 28 to 36 are dextrorotatory (D-type) amino acids, wherein lysines at positions 24 and 27 are acetylated. TCTWLKYHXGGGRGPVPSLQPRAR (SEQ ID NO: 18; also known as UT-CPP336-350-Glio), wherein X is an optional modified amino acid, preferably β-alanine; TCTWLKYHXRGPVPSLQPRAR (SEQ ID NO: 19; also known as UT-CPP339-350-Glio), wherein X is an optional modified amino acid, preferably β-alanine; - A construct that has at least 90% identity with the above-mentioned construct, and preferably at least 95% identity.

8. The construct according to any one of the preceding claims, characterized in that, It is selected from the following sequences of constructs: TVKVSFPSGPVHQDAFKINIIYNGAGrrrqrrkkrKCCKrkkrrqrrrGAGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 28, also known as FlnA-CPPD19-FG-CycloGlio-syn), wherein residues 37 and 38 are connected by disulfide bonds, alanine at positions 25 and 50 is β-alanine, and residues 27 to 35 and 40 to 48 are dextrorotatory (D-type) amino acids, and lysine at positions 36 and 39 is acetylated; EVIEAEHTKGSPSTVQATMAGrrrqrrkkrKCCKrkkrrqrrrGAMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 29; also known as FlnA-CPPD20-CD-CycloGlio-syn), wherein residues 32 and 33 are connected by a disulfide bond, alanine at positions 20 and 45 is β-alanine, and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids, and lysine at positions 31 and 34 is acetylated; FRISYTHNEGEVIEAEHTAGrrrqrrkkrKCCKrkkrrqrrrGATHEAEIVEGENHTYSIRF (SEQ ID NO: 30; also known as FlnA-CPPD20-DE-CycloGlio-syn), wherein residues 31 and 32 are connected by disulfide bonds, alanine at positions 19 and 44 is β-alanine, and residues 21 to 29 and 34 to 42 are dextrorotatory (D-type) amino acids, and lysine at positions 30 and 33 is acetylated; TCTWLKYHAGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 31; also known as FlnA-CPPD19-FG-Glio-syn), where the alanine at position 9 is β-alanine; TCTWLKYHAMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 32; also known as FlnA-CPPD20-CD-Glio-syn), where the alanine at position 9 is β-alanine; TCTWLKYHATHEAEIVEGENHTYCIRF (SEQ ID NO: 33; also known as FlnA-CPPD20-DE-Glio-syn), where the alanine at position 9 is β-alanine; RARPQLSPVPGRGGGAGrrrqrrkkrKCCKrkkrrqrrrGAGGGRGPVPSLQPRAR (SEQ ID NO: 34; also known as UT-CPP336-350-CycloGlio-syn), wherein residues 28 and 29 are connected by a disulfide bond, alanine at positions 16 and 41 is β-alanine, residues 18 to 26 and 31 to 39 are dextrorotatory (D-type) amino acids, and lysine at positions 27 and 30 is acetylated; RARPQLSPVPGRAGrrrqrrkkrKCCKrkkrrqrrrGARGPVPSLQPRAR SEQ ID NO: 35; also known as UT-CPP339-350-CycloGlio-syn), wherein residues 25 and 26 are connected by a disulfide bond, alanine at positions 13 and 38 is β-alanine, and residues 15 to 23 and 28 to 36 are dextrorotatory (D-type) amino acids, and lysine at positions 24 and 27 is acetylated; TCTWLKYHAGGGRGPVPSLQPRAR (SEQ ID NO: 36; also known as UT-CPP336-350-Glio-syn), where the alanine at position 9 is β-alanine; TCTWLKYHARGPVPSLQPRAR (SEQ ID NO: 37; also known as UT-CPP339-350-Glio-syn), where the alanine at position 9 is β-alanine; A sequence that has at least 90% identity with the above sequence, and preferably at least 95% identity.

9. The construct according to any one of claims 1 to 5, characterized in that, It also contains markers that can penetrate into cells.

10. The construct according to claim 9, characterized in that, It is selected from the following sequences of constructs: TVKVSFPSGPVHQDAFKINIIYNGAGrrrqrrkkrKCCKrkkrrqrrrGAGNYIINIKFADQHVPGSPFSVKVT (SEQ ID: 38, also known as Mq-FlnA-CPPD19-FG-CycloGlio-syn), wherein residues 37 and 38 are connected by disulfide bonds, alanine at positions 25 and 50 is β-alanine, and residues at positions 27 to 35 and 40 to 48 are dextrorotatory (D-type) amino acids, and residues at positions 36 and 39 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bonds; EVIEAEHTKGSPSTVQATMAGrrrqrrkkrKCCKrkkrrqrrrGAMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 39; also known as Mq-FlnA-CPPD20-CD-CycloGlio-syn), wherein residues 32 and 33 are connected by disulfide bonds, alanine at positions 20 and 45 is β-alanine, and residues at positions 22 to 30 and 35 to 43 are dextrorotatory (D-type) amino acids, and residues at positions 31 and 34 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bonds; FRISYTHNEGEVIEAEHTAGrrrqrrkkrKCCKrkkrrqrrrGATHEAEIVEGENHTYSIRF (SEQ ID NO: 40; also known as Mq-FlnA-CPPD20-DE-CycloGlio-syn), wherein residues 31 and 32 are connected by disulfide bonds, alanine at positions 19 and 44 is β-alanine, and residues at positions 21 to 29 and 34 to 42 are dextrorotatory (D-type) amino acids, and residues at positions 30 and 33 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bonds; ATCTWLKYHAGNYIINIKFADQHVPGSPFSVKVT (SEQ ID NO: 41; also known as Mq-FlnA-CPPD19-FG-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond; ATCTWLKYHAMTAQVTSPSGKTHEAEIVE (SEQ ID NO: 42; also known as Mq-FlnA-CPPD20-CD-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond; ATCTWLKYHATHEAEIVEGENHTYCIRF (SEQ ID NO: 43; also known as Mq-FlnA-CPPD20-DE-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond; RARPQLSPVPGRGGGAGrrrqrrkkrKCCKrkkrrqrrrGAGGGRGPVPSLQPRAR (SEQ ID NO: 44; also known as Mq-UT-CPP336-350-CycloGlio-syn), wherein residues 28 and 29 are connected by disulfide bonds, alanine at positions 16 and 41 is β-alanine, and residues at positions 18 to 26 and 31 to 39 are dextrorotatory (D-type) amino acids, and residues at positions 27 and 30 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bonds; RARPQLSPVPGRAGrrrqrrkkrKCCKrkkrrqrrrGARGPVPSLQPRAR SEQ ID NO: 45; also known as Mq-UT-CPP339-350-CycloGlio-syn), wherein residues 25 and 26 are connected by a disulfide bond, the alanine at positions 13 and 38 is β-alanine, and residues 15 to 23 and 28 to 36 are dextrorotatory (D-type) amino acids, and residues 24 and 27 are each connected to the carboxylic acid group of 5-carboxytetramethylrhodamine via peptide bonds. ATCTWLKYHAGGGRGPVPSLQPRAR (SEQ ID NO: 46; also known as Mq-UT-CPP336-350-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond; ATCTWLKYHARGPVPSLQPRAR (SEQ ID NO: 47; also known as Mq-UT-CPP339-350-Glio-syn), wherein the alanine at position 1 and the alanine at position 10 are β-alanine, and the alanine at position 1 is linked to the carboxylic acid group of 7-diethylaminocoumarin-3-carboxylic acid via a peptide bond. A sequence that has at least 90% identity with the above sequence, and preferably at least 95% identity.

11. The construct according to any one of claims 1 to 10, for use as a medicine or diagnostic / prognostic agent, particularly for the treatment of cancer, especially solid carcinomas, including breast cancer, lung cancer (particularly for the treatment of lung adenocarcinoma), melanoma, prostate cancer, colon cancer (particularly colorectal adenocarcinoma), gastric cancer (particularly gastric adenocarcinoma), renal cancer (particularly renal cell carcinoma), nasopharyngeal carcinoma (particularly nasopharyngeal squamous cell carcinoma), pancreatic cancer, diffuse glioma, and glioblastoma, for use in subjects in need, particularly humans.

12. A pharmaceutical composition comprising, as an active substance, at least one construct according to any one of claims 1 to 10, and a pharmaceutically acceptable solvent, excipient, and / or carrier.

13. The composition according to claim 12, characterized in that, It is in solid form, particularly powder, gel or injectable solution, optionally in colloidal form.

14. A nucleic acid encoding any one of the sequences according to any one of claims 1 to 10, particularly a nucleic acid encoding a construct selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:

7.

15. A viral vector comprising the nucleic acid according to claim 14, preferably an adenovirus vector or a lentivirus vector.

16. A combination product comprising at least one construct according to the invention, particularly constructs selected from the following sequence constructs: SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 11, SEQ ID NO: 29, SEQ ID NO: 12, SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 36, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 15, SEQ ID NO: 33, and an anticancer agent, said anticancer agent being particularly selected from tyrosine kinase inhibitors, particularly gefitinib, and alkylating agents that induce DNA double-strand breaks and cell death, particularly temozolomide TMZ.

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