Use of Negr1 protein and biologically active fragments thereof in the therapeutic treatment of ALK-associated diseases

Abstract

The present invention relates to novel Negr1 protein fragments and nucleic acids encoding same having ALK-inhibiting or ALK-reducing activity, and to the use of Negr1 protein, its fragments and nucleic acids encoding same in the therapeutic treatment of ALK-associated diseases such as cancer and tauopathy.

Description

[Technical field]

[0001] The present invention is in the field of biopharmaceuticals.

[0002] In particular, the present invention relates to biopharmaceuticals based on the Negr1 protein and their use in the treatment of ALK-associated diseases. [Background technology]

[0003] Negr1 (Neuronal growth regulator 1) is a member of the immunoglobulin superfamily (IgLON family) of membrane exposed adhesion proteins. Negr1 contains three N-glycosylated C2-type Ig-like domains and has an N-terminal signal sequence. A putative GPI attachment site is present near the C-terminus of the protein.

[0004] Negr1 is expressed at high levels in the brain, and its expression gradually increases during postnatal brain development and reaches a steady-state level in adulthood (Miyata et al. 2003). Negr1 expression regulates synaptogenesis, neurite outgrowth, and dendritic spine plasticity in hippocampal neurons (Pischedda et al. 2014; Pischedda and Piccoli 2015). Negr1 controls the formation of upper layers in cortical regions and the establishment of proper cortical-hippocampal connectivity through functional and physical interactions with FGFR2 (Pischedda et al., 2014; Pischedda and Piccoli, 2015; Szczurkowska et al., 2018).

[0005] Negr1 has been identified as a commonly downregulated gene in many types of human cancer tissues (Bobyn et al. 2020 (Non-Patent Document 5); Kim et al. 2014 (Non-Patent Document 6)). For example, expression of the IgLON family has been observed to alter sporadic epithelial ovarian tumors and metastatic neuroblastomas (Takita et al. 2011 (Non-Patent Document 7) and McKie et al. 2012 (Non-Patent Document 8)), suggesting that these genes may play a role as tumor suppressors.

[0006] Neuroblastoma (ORPHA:635) represents approximately 10% of solid tumors in infants and children under the age of 15, with an annual incidence in this age group of approximately 1 / 70,000.

[0007] Neuroblastoma can affect sympathetic nervous system tissues, such as the paravertebral ganglia or adrenal medulla, producing prominent masses in the chest, neck, pelvis, and / or abdomen (Mahapatra and Challagundla 2021 (Non-Patent Document 9)).

[0008] Neuroblastoma has various genetic alterations, including frequent MYCN amplification, loss of heterozygosity at 1p36 and 11q, and gain of genetic material from 17q. Anaplastic lymphoma kinase (ALK) was first identified as a fusion kinase in a subtype of non-Hodgkin's lymphoma (NPM-ALK) and adenocarcinoma of the lung (EML4-ALK), and is frequently targeted in genetic alterations in advanced neuroblastoma and other cancers. The ALK and MYCN genes are located near chromosome 2p. Therefore, amplification of the MYCN locus may also affect the ALK gene. Furthermore, ALK promotes MYCN transcription (Schoenherr et al. 2012 (Non-Patent Document 10)), and ALK is a direct transcriptional target of MYCN (Hasan et al. 2013 (Non-Patent Document 11)). Such a positive feedback loop supports sustained tumor growth (proliferation) (Janoueix-Lerosey et al. 2008).

[0009] Clinically, neuroblastoma is classified as low-risk or high-risk. Risk classification helps predict the probability of cure for neuroblastoma patients and how intensive their treatment should be. For example, patients who are expected to be cured with limited treatment, such as surgery alone, are classified as low-risk. Instead, higher-risk patients are those who are more likely to require more intensive treatment. Risk classification is based on the stage of the cancer, as well as other factors that may affect prognosis, primarily age. The 5-year survival rate for low-risk patients is over 95%, while for high-risk patients it drops to 40% (Bhoopathi et al. 2021 (Non-Patent Document 13)).

[0010] Surgical intervention, alone or in combination with minimal chemotherapy treatment, can increase survival rates in low-risk cases. However, advanced-stage neuroblastoma remains one of the most intractable childhood cancers, despite recent advances in treatment. Stage III and IV patients (classified according to the International Neuroblastoma Staging Classification (INSS)) often experience early relapse after chemotherapy, but treatment strategies involving multimodality therapy may improve the prognosis of high-risk patients. Such recurrence of tumors is often associated with more aggressive growth, resistance to chemotherapy, and metastasis (Speleman et al. 2016).

[0011] Long-term survival rates for patients with metastatic neuroblastoma are low, due in part to the presence of a large number of non-proliferating tumour cells. Surgical complication rates for patients with neuroblastoma range from 5 to 25%, depending on the stage of the tumour. Surgical complications can be varied and severe and should be considered. Chemotherapy regimens used to treat neuroblastoma can result in long-term toxicities, including cardiopulmonary toxicity (anthracyclines), ototoxicity (cisplatin), renal failure (ifosfamide and cisplatin), infertility and impotence (alkylating agents and radiation therapy), secondary cancers and psychological effects.

[0012] Thus, there is a need to find new therapeutic agents that are effective in the treatment of neuroblastoma. However, this need is not limited to neuroblastoma, but extends to virtually all types of tumors, since tumors are known to be able to acquire resistance to conventional therapies, and the increasing prevalence of drug-resistant tumors requires continued research to provide new treatments. It is also known that different patients respond differently to anticancer therapy, depending on many factors, including the biological characteristics of the tumor of a particular patient. This also leads to the need for continued research to develop new and differentiated antitumor agents.

[0013] The prior art teaches that transient expression of Negr1 (by in vitro cell transfection of a Negr1 expression vector) inhibits cell growth (proliferation) in various neuroblastoma lines (Takita et al. 2011 (Non-Patent Document 7)). Kim et al. 2014 (Non-Patent Document 6) show that overexpression of Negr1 in the human ovarian cancer cell line SKOV-3 leads to attenuation of the oncogenic phenotype.

[0014] However, the aforementioned publications are research papers on gene expression profiles in tumor tissues and do not address the issue of providing specifically feasible and effective treatments for cancer patients. [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Miyata S., Matsumoto N., Taguchi K., Akagi A., Iino T., Funatsu N., Maekawa S. (2003) Biochemical and ultrastructural analyzes of IgLON cell adhesion molecules, Kilon and OBCAM in the rat brain. Neuroscience 117, 645–658.

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[0016] In order to overcome the shortcomings and drawbacks of the prior art, the present inventors first study the effect of ectopic treatment of various cancer cell lines with soluble full-length Negr1 protein, and find that Negr1 can significantly reduce cancer cell proliferation in vitro and tumor volume ex vivo.This finding is unexpected, because the prior art does not teach or suggest that treatment with soluble Negr1 protein is actually feasible and effective in blocking cancer growth (proliferation).

[0017] The present inventors then analyzed the antitumor activity of the detected full-length Ngr1 protein and identified the responsible domain, namely, the I / PepA(aa1-127) domain of human Negr1. This allowed the present inventors to identify an amino acid stretch in the full-length Negr1 protein that is the minimum active sequence required for antitumor activity. This result is highly advantageous, since it allows the provision of various active fragments of Negr1 (Negr1 active fragments) that retain the antitumor activity of the full-length protein. Thus, the expression "active fragment(s) of Negr1" or more simply "active fragment(s)" refers to isolated peptide or polypeptide fragment(s) of Negr1 protein that can reduce ALK protein expression level.

[0018] In some respects, peptide or polypeptide fragments may be more advantageous than full-length proteins, because the preparation of full-length proteins may require complex and expensive biological procedures, whereas smaller fragments can be prepared by peptide synthesis. Moreover, considering that the molecular weight of full-length human Negr1 protein is 38,719 Da in the native form and exceeds 50 kDa due to glycosylation, the lower molecular weight of peptide or polypeptide fragments may improve activity and biodistribution.

[0019] The present inventors also found that the anti-tumor activity of Negr1 and its active fragments correlated with their ability to reduce ALK protein expression levels in treated cells.

[0020] Mutated forms of ALK are known to be involved not only in neuroblastoma but also in a wide range of other human tumor types, including thyroid and renal cancer, among others. Furthermore, oncogenic (oncogenic) ALK gene translocations or inversions have been found in rare cases of non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), inflammatory myofibroblastic tumors (IMT), and other solid tumors (Villa et al., 2021).

[0021] It is also known that ALK promotes tau phosphorylation and aggregation (Park et al., 2021) and that pathological tau aggregation occurs in Alzheimer's disease and several other taupathies, including, inter alia, postencephalitic parkinsonism, chronic traumatic encephalopathy, Parkinson-dementia complex of Guam, and FTDP-17 caused by MAPT mutations.

[0022] Therefore, the Negr1 protein and biologically active fragments thereof are predicted to be effective in treating tauopathies, such as neurodegenerative disorders characterized by the accumulation of abnormal tau protein in the brain.

[0023] Thus, one aspect of the present invention is an isolated Negr1 protein or a biologically active fragment thereof of at least 6 amino acids in length for use in the therapeutic treatment (therapeutic therapy) of a disease selected from the group consisting of tumor diseases and tauopathies, wherein the biologically active fragment of Negr1 has the ability to reduce ALK protein expression levels.

[0024] As used herein, the expression "Negr1 protein" includes inter alia the human Negr1 protein as well as its orthologues from other animal species, such as mouse.

[0025] The amino acid sequences of the major isoforms of human and mouse Negr1 are available from the UniProt Database under accession numbers Q7Z3B1 and Q80Z24, respectively.

[0026] UniProtKB-Q7Z3B1(NEGR1_HUMAN) MDMMLLVQGACCSNQWLAAVLLSLCCLLPSCLPAGQSVDFPWAAVDNMMVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTVNEGTNVTLTCLATGKPEPSISWRHISPSAKPFENGQYLDYGITRDQAGEYECSAENDVSFPDVRKVKVVVNFAPTIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKKLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNPPSTAQYGITGSADVLFSCWYLVLTLSSFTSIFYLKNAILQ (SEQ ID NO: 1)

[0027] UniProtKB-Q80Z24(NEGR1_MOUSE) MVLLAQGACCSNQWLAAVLLSLCSCLPAGQSVDFPWAAVDNMLVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTINEGTNVTLTCLATGKPEPVISWRHISPSAKPFENGQYLDIIYGITRDQAGEYECSAENDVSFPDVKKVRVIVNFAPTIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKRLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNPPSTAQYGITGSACDLFSCWSLALTLSSVISIFYLKNAILQ (SEQ ID NO: 2)

[0028] Further isoforms of the mouse Negr1 protein are available from the UniProt Database under the accession numbers A0A4W9, H3BKU7 and D3Z4T6.

[0029] UniProtKB-A0A4W9(A0A4W9_MOUSE) MVLLAQGACCSNQWLAAVLLSLCSCLPAGQSVDFPWAAVDNMLVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTINEGTNVTLTCLATGKPEPVISWRHISPSAKPFENGQYLDIIYGITRDQAGEYECSAENDVSFPDVKKVRVIVNFAPTIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKRLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNQSSIPWQVFFMLKVSFLLVCIL (SEQ ID NO: 3)

[0030] UniProtKB-H3BKU7(H3BKU7_MOUSE) MVLLAQGACCSNQWLAAVLLSLCSCLPAGQSVDFPWAAVDNMLVRKGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTINEGTNVT (SEQ ID NO: 4)

[0031] UniProtKB-D3Z4T6(D3Z4T6_MOUSE) MVLLAQGACCSNQWLAAVLLSLCSCLPAGQSVDFPWAAVDNMLVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTINEGTNVTLTCLATGKPEPVISWRHISPSAKPFENGQYLDIIYGITRDQAGEYECSAENDVSFPDVKKVRVIVNFAPTIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKRLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNLWCHLQMCWAHSWQCCLDILQA (SEQ ID NO: 5)

[0032] The expression "Negr1 protein" further includes variant proteins derived from any one of the human and mouse Negr1 proteins identified above and in which one or more amino acid residues are substituted, and the variant proteins are at least 90% identical to any one of the human and mouse Negr1 proteins identified above.

[0033] When making amino acid substitutions, generally the substituted amino acid residue may be a conservative amino acid substitution, e.g., a polar residue is substituted for a polar residue, a hydrophilic residue for a hydrophilic residue, a hydrophobic residue for a hydrophobic residue, a positively charged residue for a positively charged residue, or a negatively charged residue for a negatively charged residue.

[0034] Since all amino acid sequences in the UniProt Database that show at least 90% identity with human Negr1 are neuron growth regulator 1 (Negr1) proteins, variant proteins that are at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 are predicted to retain the ability to reduce ALK protein expression levels.

[0035] The comparison of sequences and determination of percent (%) identity between two sequences is accomplished using a mathematical algorithm, such as the blastp (blastp BLASTP 2.9.0+) program using the blosum62 matrix.

[0036] Thus, a preferred embodiment of the present invention is an isolated Negr1 protein or a biologically active fragment thereof of at least 6 amino acids in length for use in the therapeutic treatment of a tumor disease or a tauopathy, wherein the isolated Negr1 protein is selected from the group consisting of: (i) an amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; (ii) an amino acid sequence that comprises, consists essentially of, or consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and is capable of reducing ALK protein expression levels;

[0037] The ranges of percent identity above are as follows: at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% with SEQ ID NO:1; at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% with SEQ ID NO:2; at least 91%, at least 92%, at least 93%, at least 94% with SEQ ID NO:3 %, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%; SEQ ID NO:4 and at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%; SEQ ID NO:5 and at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%.

[0038] As described in the experimental section below, the present inventors have found that SEQ ID NO:6 is a minimal active sequence of the Negr1 amino acid sequence that maintains the biological activity of reducing the expression level of ALK protein. The present inventors have also surprisingly found that a variant (SEQ ID NO:7) of the above-mentioned minimum biologically active sequence, in which W (tryptophan) is replaced by I (isoleucine), retains the ALK expression reducing activity of SEQ ID NO:6.

[0039] Therefore, another preferred embodiment of the present invention is an isolated Negr1 protein or a biologically active fragment thereof for use in the therapeutic treatment of a tumor disease or a tauopathy, wherein the isolated Negr1 protein is selected from the group consisting of: (iii) an amino acid sequence that comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or that essentially consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or that consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and that includes a biologically active minimal sequence, SEQ ID NO:6 or SEQ ID NO:7.

[0040] To the best of the inventors' knowledge, no isolated peptide or polypeptide, which is a biologically active fragment of Negr1 protein capable of reducing the expression level of ALK protein, has been disclosed in the prior art.

[0041] Therefore, the scope of the present invention also includes isolated peptides or polypeptides that are fragments of Negr1 protein having a length of at least 6 amino acids and capable of reducing ALK protein expression levels, wherein the expression "Negr1 protein" includes isoforms, orthologs and variant proteins as defined above.

[0042] Examples of such fragments include 6 to 96 amino acids from the amino acid sequence of the Negr1 protein, where the expression "Negr1 protein" includes isoforms, orthologues and variant proteins as defined above. Further examples of such fragments include 6 to 35 amino acids or 6 to 70 amino acids or 35 to 70 amino acids from the amino acid sequence of the Negr1 protein as defined above. Particular examples are fragments comprising 6, 33, 70 or 96 amino acids from the amino acid sequence of the Negr1 protein as defined above.

[0043] Preferred embodiments of such fragments include the minimal biologically active sequences SEQ ID NO:6 or SEQ ID NO:7.

[0044] Most preferred embodiments of such fragments are SEQ ID NO:8, which is "PepA"; SEQ ID NO:9, which is "PepA-1"; SEQ ID NO:10, which is "PepA-2"; SEQ ID NO:11, which is "PepA-3"; SEQ ID NO:6, which is the minimal biologically active sequence; SEQ ID NO:12, which is a "PepA variant"; SEQ ID NO:13, which is a "PepA-1 variant"; SEQ ID NO:14, which is a "PepA-2 variant"; SEQ ID NO:15, which is a "PepA-3 variant"; and SEQ ID NO:7, which is a minimum variant biologically active sequence.

[0045] The peptide or polypeptide of the present invention has the advantage of a shorter amino acid sequence and less steric hindrance compared to full-length Negr1 protein, which may facilitate synthesis and penetration into cell membranes. Usually, peptide or polypeptide fragments have high activity, specificity and affinity, minimal drug-drug interactions, and biological and chemical diversity. Another advantage of peptides or polypeptides as drugs is that they usually do not accumulate in specific organs (e.g., kidney or liver), which can minimize toxic side effects. Usually, said peptides or polypeptides are also less immunogenic than recombinant antibodies or proteins.

[0046] Due to these characteristics, the peptides or polypeptides of the present invention are particularly suitable for use as therapeutic agents, although as mentioned above, the full-length Negr1 protein is also effective.

[0047] The scope of the present invention also includes an isolated nucleic acid (DNA or RNA) comprising a nucleotide sequence encoding a Negr1 peptide or polypeptide as defined above, and its use in the therapeutic treatment of a tumor disease or a tauopathy.

[0048] Also included within the scope of the present invention is an expression vector (preferably a viral expression vector) containing the above-mentioned nucleotide sequence encoding the Negr1 peptide or polypeptide defined above, as well as a host cell transformed with said expression vector.

[0049] As a non-limiting example, the nucleotide sequence of the 33 aa peptide "PepA-1" or "PepA-1 variant" optimized for expression in human cells is shown below:

[0050] GACGGCGCCAGCAAGGGCGCTTGGCTGAACCGGAGCAGCATCATCTTCGCCGGCGGAGATAAGTGGTCCGTGGACCCTAGAGTGTCTATCAGCACCCTG (PepA-1 nt sequence) (SEQ ID NO: 21)

[0051] GACGGCGCCAGCAAGGGCGCTTGGCTGAACCGGAGCAGCATCATCTTCGCCGGCGGAGATAAGATCTCTGTGGACCCTAGAGTGTCCATCAGCACCCTG (PepA-1 variant nt sequence) (SEQ ID NO: 22)

[0052] Upstream of these two sequences, the leader sequence of serum albumin preproprotein NP_000468 may be inserted:

[0053] ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTTCTGTTCAGCTCCGCCTACTCT (leader sequence of serum albumin preproprotein NP_000468) (SEQ ID NO: 23)

[0054] Based on the above nucleic acids, two expression vectors can be constructed, each containing one of the following exogenous nucleic acids:

[0055] ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTTCTGTTCAGCTCCGCCTACTCTGACGGCGCCAGCAAGGGCGCTTGGCTGAACCGGAGCAGCATCATCTTCGCCGGCGGAGATAAGTGGTCCGTGGACCCTAGAGTGTCTATCAGCACCCTG (leader sequence + PepA-1) (SEQ ID NO: 24)

[0056] ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTTCTGTTCAGCTCCGCCTACTCTGACGGCGCCAGCAAGGGCGCTTGGCTGAACCGGAGCAGCATCATCTTCGCCGGCGGAGATAAGATCTCTGTGGACCCTAGAGTGTCCATCAGCACCCTG (leader sequence + PepA-1 variant) (SEQ ID NO: 25)

[0057] These nucleic acids encode the following peptides: peptide optimized SEQ ID NO:24, which encodes PepA-1, and peptide optimized SEQ ID NO:5 for expression in human cells.

[0058] MKWVTFISLLFLFSSAYSDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTL (leader sequence + PepA-1) (SEQ ID NO: 26)

[0059] MKWVTFISLLFLFSSAYSDGASKGAWLNRSSIIFAGGDKISVDPRVSISTL (leader sequence + PepA-1 variant) (SEQ ID NO: 27).

[0060] Based on the known nucleotide sequence encoding Negr1, those skilled in the art can, without undue burden, use common general knowledge available in the field of recombinant DNA technology to design and produce further embodiments of the nucleic acids, expression vectors and host cells of the present invention.

[0061] As far as therapeutic applications are concerned, preferred tumor diseases to be treated are selected from the group consisting of neuroblastoma, ganglioglioma, gangliocytoma, adenocarcinoma of the lung, renal cell carcinoma, esophageal squamous cell carcinoma, breast carcinoma, thyroid carcinoma, colon adenocarcinoma, glioblastoma, esophageal squamous cell carcinoma, malignant melanoma, ovarian carcinoma, non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL) and inflammatory myofibroblastic tumor (IMT).

[0062] Preferred tauopathies to be treated are selected from the group consisting of Alzheimer's disease, post-encephalitic parkinsonism, chronic traumatic encephalopathy (CTE), Guam Parkinson-Dementia Complex, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), primary age-related tauopathy (PART) dementia, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), vacuolar tauopathy, meningioangiomatosis, and subacute sclerosing panencephalitis (SSPE).

[0063] The Negr1 protein and biologically active fragments thereof may be formulated into pharmaceutical compositions comprising an active ingredient and one or more pharma- ceutically acceptable excipients.

[0064] One skilled in the art can select the route of administration, pharmaceutical dosage form and dose of the active ingredient to be administered depending on several factors, including, inter alia, the particular disease being treated and the characteristics of the patient.

[0065] The Negr1 protein and its biologically active fragments for use in the present invention may be administered to a patient suffering from a tumor as a combination therapy, i.e., as simultaneous, separate or sequential combined administration with a compound or substance having antitumor activity or a radiotherapeutic agent.

[0066] According to a preferred embodiment, the compound or substance having antitumor activity is selected from the group consisting of cyclophosphamide, cisplatin, carboplatin, vincristine, doxorubicin, etoposide, topotecan, melphalan, busulfan, thiotepa, dinutuximab, dinutuximab beta, crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, ensartinib, entrectinib, and any combination thereof.

[0067] Similarly, the Negr1 protein and biologically active fragments thereof for use in the present invention may be administered to patients suffering from a tauopathy as a combination therapy, i.e., by simultaneous, separate or sequential co-administration with compounds or substances effective for the therapeutic treatment of the tauopathy.

[0068] Further features and advantages of the present invention will become apparent from the following detailed description of experiments conducted by the inventors, taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0069] [Figure 1]Figure 1 shows the experimental results obtained by the inventors, showing that overexpression of Negr1 inhibits neuroblastoma growth in vitro and in vivo. (A) Western blot analysis of human neuroblastoma (SH5Y), glioblastoma (U87), mouse neuroblastoma (N2a) and mouse primary cortical neurons. Samples were stained with anti-Negr1 and anti-actin antibodies. (B) The proliferation rate of wild-type N2a cells and three different clones stably expressing Negr1 (colonies 3, 7 and 11) was analyzed. N2a clones were seeded at a constant amount (20000 cells) on day 0. Cell numbers were counted on DIV5; n=12, **p<0.01 vs. wild-type. (C) 20000 N2a wild-type or stably expressing Negr1 (col3 and 11) cells were seeded in soft agar and cultured for 14 days. Cells were then stained with crystal violet. (D) Graph showing the number of colonies (clusters containing more than 50 cells); n=6, **p<0.01 vs. wild type. (E) One million N2a cells (wild type, clone 3, and clone 11) were injected subcutaneously into CD1 immunodeficient nude mice. Tumor growth was monitored 3, 5, 7, 9, 13, and 15 days after injection. Images show two representative tumor masses, scale bar=1 cm. (F) Graph showing tumor volume; n=6, **p<0.01 vs. wild type. [Diagram 2]FIG. 2 shows the experimental results obtained by the present inventors, showing that PepA, an embodiment of the present invention, inhibits the growth of neuroblastoma in vitro. (A) The proliferation rate of wild-type N2a cells treated with recombinant GFP or Negr1 (200 ng, daily) was analyzed. N2a clones were seeded at a constant amount (20,000 cells) on day 0 and cultured for 5 days. Culture viability was measured by MTT assay. Graph showing cell viability expressed as fold-over of untreated culture; n=10, **p<0.01 vs. GFP. (B) Cartoon showing the boundaries of the Negr1 IgG-like domain reported in NCBI (NP_776169.2.). The proliferation rate of wild-type N2a cells treated with recombinant GFP, Negr1, or Negr1-derived PepA, PepB, and PepC (200 ng, daily) was analyzed. N2a cells were seeded at a constant amount (20000 cells) on day 0 and cultured for 5 days. Graph showing cell number at day 5; n=12, **p<0.01 vs. GFP. (C) Proliferation rates of U87, SK-MEL5, MCF7, A549 and SH-SY5Y cells treated with recombinant GFP, Negr1 or PepA (200 ng daily) were analyzed. Cells were seeded at a constant amount (20000 cells) on day 0 and cultured for 5 days. Culture viability was measured by MTT assay. Graph showing cell viability expressed as fold over vehicle; n=8, **p<0.01 vs. GFP. [Diagram 3]Figure 3 shows the experimental results obtained by the inventors, showing that PepA inhibits neuroblastoma growth in vivo. (A) One million N2a cells were injected subcutaneously into CD1 immunodeficient nude mice. When the tumors reached a volume of 100 mm3, 2 μg of GFP or PepA were injected in situ. This treatment was repeated every other day. Tumor growth was monitored 1-9 days after injection. Graph showing tumor volume growth expressed as fold over day 1; n=9, **p<0.01 vs. GFP. (B) The presence of GFP and PepA in tumors, liver and kidney was investigated by STREP-pulldown. (C) 20 μm thick slices taken from tumors treated with GFP or PepA were analyzed by immunofluorescence. Samples were stained with anti-CD34 antibody (to stain immature blood vessels, red) and DAPI (to visualize cell nuclei, blue). (D-E) Graphs showing the number of blood vessels (E) and the average area (arbitrary units) (E); n=4, *p<0.05 vs. GFP. [Figure 4]FIG. 4 shows the experimental results obtained by the inventors, showing that soluble Negr1-derived peptides (i.e., PepA-1, PepA-2 and PepA-3) further inhibit neuroblastoma growth in vitro and in vivo. (A) The boundaries of the peptides are shown. (B) The proliferation rate of wild-type N2a cells treated with recombinant GFP, PepA, PepA-1, PepA-2 or PepA-3 (500 ng, daily) was analyzed. N2a cells were seeded at a constant amount (20,000 cells) on day 0, cultured for 5 days and finally measured by MTT assay. Graph showing cell vitality at day 5, normalized to GFP value; n=10, **p<0.01 vs. GFP. (C) The proliferation rate of wild-type N2a cells treated with vehicle, lorlatinib, GFP, rNegr1 and PepA-1 (3 μM, daily) was analyzed. N2a cells were seeded at a constant amount (20000 cells) on day 0, cultured for 5 days, and finally measured by MTT assay. Graph showing cell vitality at day 5, normalized to values ​​measured upon vehicle (for lorlatinib) or GFP (for rNegr1 and PepA-1) treatment; n=10, ***p<0.01 vs. vehicle, °°°p<0.01 vs. GFP. (D) Primary cortical neurons were treated daily from DIV7 with 40-80-160-200 ng / ml PepA-1 or GFP. After 5 days, cytotoxicity was assessed by MTT. Graph showing cell vitality normalized to GFP values; n=6. (E) One million N2a cells were injected subcutaneously into CD1 immunodeficient nude mice. When tumors reached a volume of 100 mm3, 2 μg of GFP or PepA-1 were injected in situ. This treatment was repeated every other day. Tumor growth was monitored 3, 5, 7 and 10 days after injection. Graph showing tumor volume growth expressed as fold over day 1; n=3, *p<0.05 vs. GFP. [Diagram 5]FIG. 5 shows the experimental results obtained by the inventors, showing that synthetic PEG-PepA-1 inhibits the growth of neuroblastoma in vitro and in vivo. (A) One million N2a cells were injected subcutaneously into CD1 immunodeficient nude mice. When the tumor reached a volume of 100 mm3, 10 μg of PEG-scramble peptide or PEG-PepA-1 was injected in situ. This treatment was repeated every other day. After the injection, the growth of the tumor was monitored daily. Graph showing the growth of tumor volume expressed as fold over day 1; n=6, *p<0.05, **p<0.01 vs. PEG-scramble. (B) One million N2a cells were injected subcutaneously into CD1 immunodeficient nude mice. When tumors reached a volume of 100 mm3, 150 μg of PEG-scrambled peptide or PEG-PepA-1 was injected intraperitoneally. This treatment was repeated every other day. Tumor growth was monitored daily after injection. Graph showing tumor volume growth expressed as fold over day 1; n=6-8, *p<0.05 vs. PEG-scrambled. [Figure 6]FIG. 6 shows the experimental results obtained by the inventors, showing that peptide PepA regulates ALK signaling. (A) Schematic showing the boundaries of PepA, PepB and PepC. (B) 50000 N2a cells were treated with 300ng / ml GFP, rNegr1 or PepA daily for 5 days. At DIV5, cultures were processed for Western blotting to evaluate protein expression levels for ALK and MYCN as well as actin and S6 ribosomal protein. (C-D) Graphs showing ALK levels (C) and MYC levels (D) normalized to actin and S6RP amounts and expressed as fold over GFP-treated cells; n=6, *p<0.05 vs. GFP. (E) 50000 N2a cells were treated with 3μM GFP, lorlatinib or PepA-1 daily for 5 days. At DIV5, cultures were processed for Western blotting to assess protein expression levels for ALK and MYCN, as well as actin and S6 ribosomal proteins. (F-G) Graphs showing ALK (F) and MYC (G) levels normalized to actin and S6RP amounts and expressed as fold over GFP-treated cells; n=6, *p<0.05, ***p<0.001 vs. GFP. (H) 150000 N2a cells were treated with 3 μM GFP, lorlatinib or PepA-1 for 10 min. Cultures were then processed for Western blotting to assess protein expression levels for AKT, phospho-AKT, ERK1 / 2 and phospho-ERK1 / 2, as well as S6 ribosomal proteins. (I-J) Graphs showing pAKT / AKT ratios (I) and pERK / ERK ratios (J) expressed as fold relative to vehicle-treated cells; n=6, **p<0.01, ***p<0.001 vs. vehicle. [Figure 7]FIG. 7 shows the experimental results obtained by the inventors, showing that a synthetic version of PepA-1 and a scrambled peptide containing a biologically active minimal sequence variant modulate ALK signaling. (A) A diagram showing the location of the biologically active minimal sequence GDKWSV (SEQ ID NO: 8) (source PDB: 6DLD). (B) Sequence of the synthetic peptide. (C) 150000 N2a cells were treated with vehicle or the following synthetic peptides (10 μM, 10 min 10 μM): scrambled peptide, scr-GDKISV, a peptide derived from PepA-1 but containing the biologically active minimal sequence variant GDKISV (SEQ ID NO: 9), and PepA-1. Cultures were then processed for Western blotting to assess the expression levels of ERK1 / 2 and phosphorylated ERK1 / 2. (D) Graph showing phospho-ERK / ERK ratio as fold over vehicle; n=8, *p<0.05, ***p<0.01 vs. vehicle. (E) 150000 N2a cells were treated with vehicle, lorlatinib or GDKWSV peptide at 3 μM or 30 μM for 10 min. Cultures were then processed for dot blotting to assess expression levels of ERK1 / 2 and phosphorylated ERK1 / 2. (F) Graph showing phospho-ERK / ERK ratio as fold over vehicle; n=5, **p<0.01 vs. vehicle. (G) 150000 N2a cells were treated with the following synthetic peptides (10 μM, 10 μM for 10 min): scrambled peptide, PepA-1, and PepA-1 6aa scramble, a PepA-1 derived peptide but with a scrambled sequence instead of the GDKISV hexapeptide. Cultures were then processed for Western blotting to assess the expression levels of ERK1 / 2 and phosphorylated ERK1 / 2. (H) Graph showing phosphorylated ERK / ERK ratio as fold over vehicle; n=4, ***p<0.01 vs other conditions. [Figure 8]Figure 8 shows the experimental results obtained by the inventors regarding the efficacy of the pegylated form of PepA-1, called PEG-PepA-1. (A) 150000 N2a cells were treated with vehicle, lorlatinib or PEG-PepA-1 at 3 μM or 30 μM for 10 min. Cultures were then processed for Western blotting to assess protein expression levels for ERK1 / 2 and phosphorylated ERK1 / 2. (B) Graph showing phosphorylated ERK / ERK ratio as fold over vehicle upon treatment with 30 μM; n=8, **p<0.01 vs. vehicle. (C) 150000 N2a cells were treated with vehicle, lorlatinib, scrambled peptide or PEG-PepA-1 at 5 μM for 24 or 48 h. Cultures were then processed for Western blotting to assess protein expression levels for ALK. Graph showing normalized ALK optical density as fold over control; n=4, **p<0.01 vs. PEG-PepA-1, °p<0.01 vs. scrambled peptide. (D) 150,000 N2a cells were treated with vehicle, lorlatinib + scrambled peptide, or lorlatinib + PEG-PepA-1 at 5 μM for 24 hours. Cultures were then processed for Western blotting to assess protein expression levels for ALK. Graph showing normalized ALK optical density as fold over vehicle; n=4, **p<0.01 vs. scrambled peptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] The results obtained by the inventors are described in detail below.

[0071] In the first series of experiments, we found (by observation) that Negr1 protein was downregulated in brain cancer-derived cell lines SH5Y (human neuroblastoma), U87 (glioblastoma) and N2a (mouse neuroblastoma). We also found that ectopic Negr1 expression in N2a cells significantly reduced cell proliferation, and that Negr1-expressing N2a cell clones had a significantly reduced ability to generate colonies on soft agar. Furthermore, in an in vivo study using a xenograft mouse model, an established tool for investigating cancer progression in vivo, we found that the size of tumors derived from subcutaneously injected (sc) Negr1-expressing N2a cells was significantly smaller than that of tumors derived from wild-type N2a cells.

[0072] These findings demonstrate that overexpression of Negr1 can inhibit cancer growth (proliferation) both in vitro and in vivo.

[0073] In a second series of experiments, the present inventors observed that treatment of naive N2a cells with soluble recombinant full-length Negr1 protein significantly reduced N2a growth (proliferation).

[0074] The present inventors also performed a series of experiments aimed at identifying the minimal amino acid sequence capable of exerting antineoplastic (antitumor) activity. Negr1 protein contains three IgG-like domains. The amino acid sequence of human full-length Negr1 protein is available from UniProtKB under the accession number Q7Z3B1 (NEGR1_HUMAN) and is designated as SEQ ID NO: 1 in the attached sequence listing. Thus, the present inventors cloned and characterized each IgG-like domain. The three IgG-like domains of Negr1 are hereinafter referred to as PepA (SEQ ID NO: 8), PepB (SEQ ID NO: 16) and PepC (SEQ ID NO: 17). When naive N2a cells were treated with Negr1, PepA, PepB, PepC, or GFP (green fluorescent protein) as a control (GFP is approximately the same size as full-length Negr1), we found that Negr1 and PepA significantly reduced N2a proliferation, whereas PepB and PepC did not. We also tested the ability of Negr1 and PepA to block the growth of other cancer cell lines, namely U87 (glioblastoma), SK-Mel5 (malignant melanoma), MCF7 (breast adenocarcinoma), A549 (adenocarcinomic alveolar basal epithelial cells), and SH-SY5Y (neuroblastoma). Treatment with rNegr1 and PepA significantly reduced the growth of all the cancer lines analyzed.

[0075] The antitumor activity of PepA was also confirmed in an in vivo experiment using 6-week-old CD1 nude mice, where the inventors found that orthotopic PepA treatment robustly reduced tumor growth. Furthermore, the inventors found that, when administered in situ, PepA was present in the tumor, liver, and kidney, whereas the GFP protein used as a control was mainly present in the tumor. This suggests that PepA can reach the bloodstream and be distributed throughout the body. Furthermore, the inventors analyzed tumor neovascularization by detecting CD34 protein and found that angiogenesis was significantly reduced in tumors treated with PepA.

[0076] To further narrow down the biologically active minimal sequence of PepA, we performed an in silico analysis of the PepA structure. We cloned and characterized three partially overlapping peptides spanning the entire amino acid sequence of PepA. These peptides are hereafter referred to as PepA-1 (SEQ ID NO: 9), PepA-2 (SEQ ID NO: 10) and PepA-3 (SEQ ID NO: 11). When N2a cells were treated with GFP (control), PepA, PepA-1, PepA-2 or PepA-3, we found that all tested Negr1-derived peptides significantly reduced the growth of N2a, but PepA-1 and PepA-3 did so more strongly. Furthermore, in the MTT assay, we found that rNegr1, PepA-1 and lorlatinib (an ALK inhibitor approved by the FDA) reduced the growth of N2A to the same extent. Notably, we further observed that treatment with increasing concentrations of PepA-1 did not induce greater toxicity in primary cortical neurons compared with treatment with GFP.The significant antineoplastic activity of PepA-1 was also confirmed in vivo in 6-week-old nude mice.

[0077] Because biological evidence suggests that the human Negr1 protein is glycosylated at the position corresponding to position +10 in the amino acid sequence of PepA-1, we generated a PEGylated version of PepA-1 and confirmed that it has antitumor activity both in vitro against N2a cells and in vivo in 6-week-old CD1 nude mice.

[0078] The present inventors also elucidated the molecular mechanism underlying the antitumor activity of the Negr1-derived peptide of the present invention by identifying it in the ALK-MYCN pathway. The present inventors found that treatment of N2a cells with PepA or PepA-1 reduced the expression levels of ALK receptor and MYCN transcription factor proteins, and also reduced the phosphorylation of ERK kinase and AKT kinase.

[0079] Importantly, we also identified a minimal biologically active sequence of Negr1 required for antitumor activity, i.e., GDKWSV (SEQ ID NO: 6), which is present in all Negr1-derived peptides shown to be biologically active, i.e., PepA, PepA-1, PepA-2, and PepA-3, but absent from PepB and PepC, which were shown to be inactive.

[0080] Based on the above experimental results, it can be concluded that not only the Negr1 peptide of SEQ ID NO: 8 (PepA), but also a Negr1 peptide fragment of at least 6 amino acids in length containing SEQ ID NO: 86, which is the minimal biologically active sequence located between positions 45 and 50 of SEQ ID NO: 8, has anti-tumor activity.

[0081] Illustrative examples of such peptide fragments include, but are not limited to, PepA-1 (SEQ ID NO: 9), PepA-2 (SEQ ID NO: 10) and PepA-3 (SEQ ID NO: 11). A further illustrative example is a hexapeptide consisting of the amino acid sequence of SEQ ID NO: 6.

[0082] The results obtained by the inventors also show that a functional variant of the Negr1 peptide defined above, in which the minimal biologically active sequence SEQ ID NO:6 is replaced by SEQ ID NO:7, also has antitumor activity.

[0083] Illustrative examples of such variant peptide fragments include, but are not limited to, PepA-1v (SEQ ID NO: 13), PepA-2v (SEQ ID NO: 14) and PepA-3v (SEQ ID NO: 15). A further illustrative example is a hexapeptide consisting of the amino acid sequence of SEQ ID NO: 7.

[0084] All Negr1 fragments of the present invention can be provided in a PEGylated form, which mimics the glycosylated form of the naturally occurring human Negr1 protein.

[0085] Furthermore, the Negr1 protein and any of its biologically active fragments, peptides or polypeptides may be provided in modified forms (modified forms) to improve their pharmacokinetic properties, for example to increase potency, sustain activity and / or extend half-life. Such modifications include, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands or other proteins, etc. Any of a large number of chemical modifications may be performed by known techniques. Specific illustrative examples of such modifications known per se to those skilled in the art and may be used in the context of the present invention include the following: · Cyclic peptides as disclosed in Kong and Heinis 2021; Contains at least two D-amino acids as disclosed in Vlieghe et al. 2010; · containing at least two β-amino acids as disclosed in Hook et al. 2005; · N-methylation as disclosed by Linde et al. 2008; containing different PEG groups as disclosed in Daepp et al. 2012; and / or Replacement of at least two amides of the peptide backbone with sulfonamides as disclosed in de Bont et al. 1999.

[0086] The following experimental section is provided for illustrative purposes only and is not intended to limit the scope of the invention, which is defined in the appended claims.

[0087] Experimental section Materials and Methods Constructs and peptides

[0088] mNegr1 cDNA (Addgene clone C3342IRCKp5014P057-rzpdm13-21) was cloned into the strep-FLAG pcDNA3.1 vector. The mNegr1-peptide was subcloned into the strep-FLAG pcDNA3.1 vector.

[0089] scramble: DGGSKGGFADLSSIIWARWNKARVDPLVSISTS (SEQ ID NO: 18) Scramble-GDKISV: NRGDKISVSVSSSSGGRAWDWDIAKPGLAILTF (SEQ ID NO: 19) PepA-1-6aa scramble: DGASKGAWLNRSSIIFAGVWGSKDDPRVSISTL (SEQ ID NO: 20)

[0090] Cell culture and transfection

[0091] N2a (Neuro2a, ATCC CCL-131), HEK293 (ATCC CRL-1573), U87 MG (ATCC® HTB14), SKMEL5 (ATCC® HTB70), MCF7 ATCC® HTB-22, A549 ATCC® CCL-185, and SHSY5Y (ATCC® CRL 2266™) cells were cultured in DMEM high glucose (Gibco) containing 10% FBS, 1% penicillin / streptomycin, and 1% glutamine at 37°C in a humidified atmosphere of 5% CO2. Cortical neuronal cultures were prepared from mouse embryos (E17.5-18.5; C57BL / 6 strain).

[0092] High density (750~1000 cells / mm 2 ) Neuronal cultures were seeded and grown in 24-well plastic tissue culture plates as previously described (Iwaki; Bibby Sterilin) ​​(Pischedda et al. 2018).

[0093] N2a and HEK293 cells were transfected with different constructs for 48 hours using Lipofectamine 2000 (Invitrogen). Stable N2a clones expressing mNegr1 were isolated by neomycin selection (1 mg / ml).

[0094] Purification with STREP resin HEK293 cells transfected with the relative constructs were lysed in RIPA buffer (150 mM NaCl, 50 mM HEPES, 0.5% NP40, 1% sodium deoxycholate) for 1 h at 4°C and then processed for streptavidin immunoprecipitation. Proteins were eluted from the STREP resin in elution buffer (2.5 mM desthiobiotin, 100 mM Tris-HCL, 150 mM NaCl, 1 mM EDTA) with gentle agitation for 1 h at 4°C. Protein concentration was measured by standard Bradford assay (Bio-Rad) and protein purity was assessed by SDS-PAGE followed by silver staining. Synthetic peptides were purchased from Genscript Nederland. Mouse tissues were lysed in RIPA buffer (150 mM NaCl, 50 mM HEPES, 0.5% NP40, 1% sodium deoxycholate, 1 ml / mg of tissue) for 1 hour at 4° C. and then processed for streptavidin immunoprecipitation. Proteins were eluted from the STREP resin into Laemmli buffer.

[0095] MTT assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess culture vitality. N2a cells were cultured in 96-well plates at a concentration of 5 × 10 3 cells / cm 2 The cells were cultured at 100° C. for 30 min at 37° C. The treatment conditions are shown in the results section. At the end of the treatment, MTT solution was added to the cell medium at a final concentration of 0.25 mg / mL. The incubation was continued at 37° C. for 30 min. The medium was then removed and the formazan precipitate was collected in DMSO (200 μL). As a surrogate for cell viability, the absorbance was measured at 570 nm using a spectrophotometer (Varioskan LUX, Thermofisher). The fold increase in cell viability relative to the control condition is expressed.

[0096] Western blotting Protein levels and relative phosphorylation were assessed by Western blotting. Briefly, after washing with PBS, cells were solubilized in lysis buffer (150 mM NaCl, 50 mM HEPES, 0.5% NP40, 1% sodium deoxycholate). After gentle stirring for 1 h, the lysates were clarified by centrifugation at 16,000 g for 20 min. All experimental procedures were performed at 4°C. Protein concentrations were assessed by Bradford assay (Bio-Rad, USA). For Western blotting experiments, equal amounts of protein were diluted with 0.25% 5x Laemmli buffer. Samples were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Sigma-Aldrich) at 80 V for 120 min at 4°C. Primary antibodies were as follows: β-actin (Santa Cruz Biotechnology, sc-47778), ALK (Santa Cruz Biotechnology, sc-398791), N-myc (Santa Cruz Biotechnology, sc-56729), Phospho-p44 / 42 MAPK(Erk1 / 2)(Thr202 / Tyr204)(Cell Signaling Technology), p44 / 42 MAPK(Erk1 / 2)(137F5) Rabbit mAb (Cell Signaling Technology), Akt(pan)(11E7) Rabbit mAb (Cell Signaling Technology), Phospho-Akt(Ser473)(D9E)XP® Rabbit mAb (Cell Signaling Technology), S6 Ribosomal Protein(5G10) Rabbit mAb (Cell Signaling Technology). Antibodies were applied overnight in blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk). Proteins were detected using the ECL prime detection system (GE Healthcare).ECL signals were acquired using an imaging ChemiDoc Touch system (Bio Rad Laboratory Italy, Segrate, Italy). The optical density of specific bands was quantified with ImageLab software (Bio Rad).

[0097] animal One million N2a cells were suspended in 150 μl DMEM high glucose and administered subcutaneously into the left rear paw of 8-week-old nu / nu mice (Charles River) by injection with a microsyringe (Hamilton). All animal procedures were approved by Institutional and National Agencies (authorization 559 / 2016-PR).

[0098] Immunofluorescence Tumors were dissected and post-fixed in 4% PFA for 2 hours, then placed in OCT and stored at -80°C until processing. 14 μm sections were serially sectioned on a Leica cryostat and mounted on Polysine Slides (Thermo Fischer Scientific). Sections were soaked in 2.5% BSA, 10% NGS, 0.2% Triton X-100, PBS 1X for 1 hour at room temperature (RT) and incubated overnight (O / N) with primary antibody (anti-CD34 antibody [EP373Y], ABCAM, ab81289) at 4°C. After washing three times with PBS 1X-0.2% Triton X-100, sections were incubated with conjugated secondary antibody (AffiniPure Goat Anti-Rabbit IgG (H+L) Alexa Fluor 594, Jackson ImmunoResearch, 111-585-003) for 1 hour at room temperature. Sections were then rinsed three times with PBS 1X-0.2% Triton X-100, once with PBS 1X, and then mounted in aqueous mounting medium. Sections were kept in the dark at 4°C until images were acquired with a Zeiss Axio Imager M2 equipped with a forty-fold (40X) objective.

[0099] Statistical Analysis and Guidelines All data were presented as box plots showing minimum, maximum and median values. Normality of data distribution was determined using D'Agostino and Pearson omnibus normality test followed by unpaired Student's t test, ANOVA followed by Tuckey's post-hoc test or two-way ANOVA followed by Bonferroni or Student's t post-hoc test, as appropriate. Number of experiments (n) and significance levels (p) are indicated throughout the text. All methods were performed in accordance with the relevant guidelines and national regulations.

[0100] result Negr1 overexpression inhibits cancer growth (proliferation) in vitro and in vivo. Protein samples from human neuroblastoma (SH5Y), glioblastoma (U87), and mouse neuroblastoma (N2a) were processed by Western blotting. We found that Negr1 protein was downregulated in brain cancer-derived lines. In contrast, Negr1 could be detected in terminally differentiated mouse cortical neurons (Figure 1A). Next, we evaluated whether Negr1 protein levels correlated with cell proliferation. To this end, we generated and characterized several N2a clones expressing Strep-FLAG Negr1 fusion protein. N2a clones were seeded at a constant amount (20,000 cells) on day 0 in vitro (DIV0). Cell proliferation was evaluated at DIV5. We found that ectopic Negr1 expression significantly reduced N2a proliferation (Figure 1B).

[0101] It is known that cancer cells can grow independently of a solid surface. Such anchorage-independent growth is a hallmark of carcinogenesis. This feature can be reproduced in vitro by monitoring colony growth in a soft agar matrix. Thus, the soft agar colony formation assay tests whether cells have undergone malignant transformation. Thus, a fixed amount (20,000 cells) of wild-type and Negr1-expressing cells was seeded in soft agar and cultured for 14 days. The cells were then visualized by crystal violet staining. By scoring the number of colonies (clusters containing more than 50 cells), we observed that the Negr1-expressing N2a cell clones had a significantly reduced ability to generate colonies on soft agar (Figure 1C-D).

[0102] Xenograft mouse models are an established means to investigate cancer progression in vivo. For this purpose, one million N2a cells (naive, clone 3 and clone 11) were injected subcutaneously into immunodeficient CD1 nude mice. Tumor volumes were monitored ex-vivo by measuring the size of the tumor with a caliper. Tumor volumes were calculated using the formula for an ellipsoid (length x width x height x 0.52). We observed that the size of tumors derived from Negr1-expressing cells was significantly smaller than that of tumors derived from wild-type N2a cells (Figure 1E-F).

[0103] Soluble Negr1 inhibits cancer growth (proliferation) in vitro and in vivo. At the physiological level, Negr1 exists as a GPI-anchored membrane-bound protein and as a soluble protein that is released upon metalloprotease cleavage (Sanz et al. 2015; Pischedda and Piccoli 2015). Therefore, we investigated whether ectopic treatment with soluble recombinant Negr1 (Negr1) could regulate cancer cell growth. For this purpose, we expressed and purified Strep-FLAG Negr1 and Strep-GFP from HEK293 cells. The latter is a biologically inactive protein used as a control. Then, 10000 naive N2a cells were seeded and treated daily with 200 ng of rNegr1 or GFP. At DIV5, cell growth was assessed by MTT assay. We observed that treatment with Negr1 significantly reduced N2a growth (Fig. 2A).

[0104] Next, we aimed to identify the minimal sequence that can exert the Negr1 anti-neoplastic effect. The Negr1 protein contains three IgG-like domains. Therefore, we cloned and characterized each IgG-like domain as a strep-FLAG tagged recombinant protein. We expressed and purified these three domains (hereafter referred to as PepA, PepB, and PepC) from HEK293 cells.

[0105] Next, 10,000 naive N2a cells were seeded and treated daily with 200 ng of Negr1, PepA, PepB, PepC or GFP. Cell numbers were assessed on DIV5. We found that treatment with Negr1 and PepA significantly reduced N2a proliferation (Figure 2B).

[0106] Next, we tested the ability of Negr1 and peptide PepA to block the growth of human cancer-derived cell lines, namely U87 (glioblastoma), SK-Mel5 (malignant melanoma), MCF7 (breast adenocarcinoma), A549 (adenocarcinoma alveolar basal epithelial cells) and SH-SY5Y (neuroblastoma). For this purpose, each line was treated daily with 200 ng of GFP, rNegr1 or PepA. On DIV5, cell growth was assessed by MTT assay. We found that treatment with rNegr1 and PepA significantly reduced the growth of all cancer lines tested (Figure 2C). Such results are in full agreement with the expression of the receptor ALK experimentally detected in cancer cell lines of ectodermal origin, including small cell lung cancer, breast cancer, malignant melanoma, neuroblastoma and glioblastoma (Dirks et al. 2002).

[0107] To further evaluate the antineoplastic activity of PepA, one million N2a cells were injected subcutaneously into 6-week-old CD1 nude mice. 3 When the tumors reached a volume of 10 μg, 2 μg of GFP or PepA was injected in situ. This treatment was repeated every other day, and tumor growth was monitored daily. We found that PepA treatment significantly reduced tumor growth (Figure 3A).

[0108] On the 9th day after injection (or when the tumor was 2.5 cm 3 On the first day (or earlier if the tumor volume reached 100 μg / kg / day), the animals were euthanized and mouse tissues were harvested to analyze the presence of GFP and PepA. Notably, by immobilization on STREP resin, we found that PepA was present in the tumor, liver, and kidney, while GFP protein was mainly present in the tumor (Figure 3B). This biochemical evidence suggests that PepA can reach the bloodstream and be distributed throughout the body.

[0109] Cancer growth requires and induces angiogenesis. In particular, cancer tumors are characterized by the presence of immature blood vessels decorated with CD34 protein. Therefore, we analyzed tumor neovascularization by detecting CD34 protein. Tumors treated with PepA showed significantly reduced angiogenesis in terms of total area stained with CD34 and average vascular area (Figures 3C-E).

[0110] Characterization of a 33 amino acid long biological anti-neoplastic peptide To further narrow down the biologically active minimal sequence within PepA, the PepA structure was analyzed in silico. Consistent with the predicted secondary structure, we cloned and characterized three peptides covering the PepA sequence (Figure 4A). The three peptides were expressed and purified as strep-FLAG tagged recombinant peptides from HEK293 cells. These three peptides are hereafter referred to as PepA-1 (33 amino acids), PepA-2 (70 amino acids), and PepA-3 (70 amino acids). To evaluate the in vitro activity of these three peptides, 10,000 naive N2a cells were seeded and treated daily with 500 ng / ml of GFP, PepA, PepA-1, PepA-2, and PepA-3. At DIV5, cell growth (proliferation) was evaluated by MTT assay. We found that the minimal peptide PepA-1 significantly reduced the growth (proliferation) of N2a (FIG. 4B).

[0111] To further investigate the efficacy of PepA1, we investigated the proliferation of N2A cells in response to chronic treatment with vehicle, lorlatinib (an ALK inhibitor approved by the FDA), GFP, rNegr1 and PepA-1 (daily, all molecules at 3 μM). At DIV5, cell vitality was assessed by MTT assay. rNegr1, PepA-1 and lorlatinib reduced N2A growth (proliferation) to a similar extent (Figure 4C). Interestingly, treatment with increasing concentrations of PepA-1 (daily, from DIV7 to 12) did not cause excessive toxicity to primary cortical neurons at DIV12 compared to treatment with GFP (Figure 4D).

[0112] Next, 1 million N2a cells were injected subcutaneously into 6-week-old nude mice. 3 When the tumors reached a volume of 100 μg, 2 μg of GFP or PepA-1 was injected in situ. This treatment was repeated every other day, and tumor growth was monitored daily. We found that PepA-1 treatment significantly reduced growth (Figure 4E).

[0113] Characterization of a 33 amino acid long synthetic antineoplastic peptide. Biological evidence suggests that PepA1 is glycosylated at the asparagine residue (N) at position +10. We designed a synthetic peptide with a mini-PEG1 moiety (MW: 152 Da) attached to the N-terminus of the PepA-1 sequence to mimic glycosylation. The resulting peptide (hereafter referred to as PEG-PepA-1) is soluble in water at a concentration of 5 mg / ml.

[0114] To evaluate the antineoplastic activity of PEG-PepA-1, one million N2a cells were injected subcutaneously into 6-week-old CD1 nude mice. 3 When the tumors reached a volume of 10 μg, 10 μg of PEG-PepA-1 or PEG-scrambled peptide was injected in situ. Treatment was repeated every other day, and tumor growth was monitored daily. We found that PepA treatment significantly reduced tumor growth (Figure 5A).

[0115] Finally, we tested the efficacy of PEG-PepA-1 by systemic administration. For this purpose, one million N2a cells were injected subcutaneously into 6-week-old CD1 nude mice. 3 When the tumor volume reached 100 μg, 150 μg of PEG-PepA-1 or PEG-scrambled peptide was injected intraperitoneally. This treatment was repeated every other day, and tumor growth was monitored daily. We found that PEG-PepA-1 treatment significantly reduced tumor growth (Figure 5B).

[0116] Given the significant effect on cancer cell growth (proliferation) in vitro and in vivo, we aimed to elucidate the molecular mechanisms underlying the anti-neoplastic effects induced by Negr1-derived peptides (Figure 6A). Altered activity and gene amplification of the ALK receptor, and the associated increased expression of the downstream MYCN transcription factor, are important molecular hallmarks of neuroblastoma. Furthermore, the ALK gene can be oncogenically altered in several malignancies, including non-small cell lung cancer (NSCLC) and anaplastic large cell lymphoma (ALCL).

[0117] We monitored whether treatment with rNegr1 or PepA affected the ALK-MYCN pathway. To this end, 50,000 N2a cells were treated with 300 ng / ml of GFP, rNegr1 or PepA daily for 5 days. At DIV5, cultures were processed for Western blotting to evaluate the expression levels of ALK and MYCN proteins. PepA treatment significantly reduced ALK and MYCN protein levels (Figure 6B-D).

[0118] Next, 50,000 N2a cells were treated with 3 μM GFP, lorlatinib or PepA-1 (50 ng / ml) daily for 5 days. On DIV5, cultures were processed for Western blotting to evaluate the expression of ALK and MYCN proteins. Notably, we observed that chronic treatment with PepA-1 caused a strong downregulation of ALK and MYC-N (Figure 6E-G).

[0119] ALK induces a signaling cascade involving phosphorylation of ERK and AKT kinases. Therefore, to complement the above findings, we evaluated the effect of PepA-1 on the phosphorylation of ERK and AKT. In acute treatment (3 μM, 10 min), PepA-1 significantly reduced the phosphorylation of ERK and AKT (Figure 6H-J).

[0120] Additionally, N2a cells were treated with the following synthetic peptides: a synthetic version of PepA-1 (synPepA-1), a scrambled peptide containing the GDKISV sequence (scr-GDKISV, in which the potentially oxidizable W (tryptophan) was replaced by I (isoleucine)), and a scrambled peptide (10 μM, 10 min). Notably, we found that scr-GDKISV and synPepA-1 reduced the phosphorylation of ERK1 / 2 (Figure 7C-D).

[0121] As a complementary approach, we treated N2a cells with a PepA-1 derived peptide in which the GDKWSV sequence was replaced with a scrambled sequence (PepA-1-6aa scramble, 10 μM, 10 min). We observed that PepA-1-6aa scramble did not alter ERK phosphorylation (Figure 7E-F).

[0122] Finally, we tested the biological activity of the GDKWSV peptide alone. To this end, 150,000 N2a cells were treated with the synthetic GDKWSV peptide or the ALK inhibitor lorlatinib (30 nM, 10 min). By dot plots, we observed that treatment with the GDKWSV peptide correlated with a significant reduction in ERK1 / 2 phosphorylation (Figure 7E-F). This finding suggests that the hexapeptide GDKWSV is essential for maintaining the biological activity of the PepA-1 peptide. Finally, we monitored the pharmacodynamic properties of PEG-PepA1. Upon acute treatment (30 μM, 10 min), PEG-PepA-1 significantly reduced ERK phosphorylation (Figure 8A-B).

[0123] We also investigated the combined effect of PEG-PepA-1 and lorlatinib. We found that with long-term treatment, PEG-PepA-1 significantly reduced the levels of ALK protein, while lorlatinib induced a strong upregulation of the receptor (both at 5 μM, 24 h or 48 h, FIG. 8C). Notably, coadministration of PEG-PepA-1 significantly reduced the levels of ALK protein observed with long-term treatment with lorlatinib (5 μM, 24 h, FIG. 8D).

[0124] References ·Bhoopathi P., Mannangatti P., Emdad L., Das SK, Fisher PB(2021) The quest to develop an effective therapy for neuroblastoma. J Cell Physiol. ·Bobyn A., Zarrei M., Zhu Y., Hoffman M., Brenner D., Resnick AC,Scherer SW, Gallo M. (2020) Ancestry and frequency of genetic variants in the general population are confounders in the characterization of germline variants linked to cancer. BMC Med Genet 21, 92. ·Bont DBA de, Sliedregt-Bol KM, Hofmeyer LJF, Liskamp RMJ (1999) Increased stability of peptidesulfonamide peptidomimetics towards protease catalyzed degradation. Bioorganic & Medicinal Chemistry 7, 1043-1047. ·Daepp S., Mueller C., Garayoa E. G., Blaeuenstein P., Maes V., Brans L., Tourwe D. A., Schibli R. (2012) PEGylation, increasing specific activity and multiple dosing as strategies to improve the risk-benefit profile of targeted radionuclide therapy with 177Lu-DOTA-bombesin analogues. EJNMMI Research 2, 24. ·Dirks W. G., Faehnrich S., Lis Y., Becker E., MacLeod R. 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Claims

1. An isolated Negr1 protein or a biologically active fragment thereof for use in the therapeutic treatment of a disease selected from tumor diseases and taupathies, wherein the isolated Negr1 protein or a biologically active fragment thereof is capable of reducing ALK protein expression levels, and the fragment has a length of at least 6 amino acids.

2. The isolated Negr1 protein or a biologically active fragment thereof for use according to claim 1, wherein the isolated Negr1 protein is selected from the group consisting of: (i) an amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; (ii) an amino acid sequence that comprises, consists essentially of, or consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and is capable of reducing ALK protein expression levels; and (iii) an amino acid sequence that comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or that essentially consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or that consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and that includes a minimum biologically active sequence of SEQ ID NO:6 or SEQ ID NO:

7.

3. The isolated Negr1 protein or a biologically active fragment thereof for use according to claim 2, wherein the biologically active fragment comprises the minimal biologically active sequence of SEQ ID NO: 6 or SEQ ID NO:

7.

4. The isolated Negr1 protein or a biologically active fragment thereof for use according to claim 1, wherein the biologically active fragment is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:

15.

5. 5. An isolated Negr1 protein or a biologically active fragment thereof for use according to any one of claims 1 to 4, comprising a stability-increasing modification.

6. The isolated Negr1 protein or a biologically active fragment thereof for use according to claim 5, wherein the modification that increases stability is selected from the group consisting of an N-terminal modification, a C-terminal modification, a side chain modification, an amino acid modification, a backbone modification and any combination thereof.

7. The isolated Negr1 protein or a biologically active fragment thereof for use according to claim 5, wherein the stability-increasing modification is selected from the group consisting of PEGylation, cyclization, N-methylation, replacement of at least two amino acid residues with the corresponding D-stereoisomers, replacement of at least two amides in the backbone with sulfonamides, and any combination thereof.

8. The isolated Negr1 protein or a biologically active fragment thereof for use according to any one of claims 1 to 7, wherein the disease is a tumor disease and the use is a simultaneous, separate or sequential combined use with a compound or substance having antitumor activity or a radiotherapeutic agent.

9. 9. The isolated Negr1 protein or a biologically active fragment thereof for use according to claim 8, wherein the compound or substance having antitumor activity is selected from the group consisting of cyclophosphamide, cisplatin, carboplatin, vincristine, doxorubicin, etoposide, topotecan, melphalan, busulfan, thiotepa, dinutuximab, dinutuximab beta, crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, ensartinib, entrectinib, and any combination thereof.

10. The isolated Negr1 protein or a biologically active fragment thereof for use according to any one of claims 1 to 9, wherein the tumor disease is selected from the group consisting of neuroblastoma, ganglioglioma, gangliocytoma, lung adenocarcinoma, renal cell carcinoma, esophageal squamous cell carcinoma, breast cancer, thyroid cancer, colon adenocarcinoma, glioblastoma, esophageal squamous cell carcinoma, malignant melanoma, ovarian cancer, non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL) and inflammatory myofibroblastic tumor (IMT).

11. The isolated Negr1 protein or a biologically active fragment thereof for use according to any one of claims 1 to 7, wherein the disease is a tauopathy and the use is a simultaneous, separate or sequential combined use with a compound or substance effective for the therapeutic treatment of a tauopathy.

12. 12. The isolated Negr1 protein or a biologically active fragment thereof for use according to any one of claims 1 to 7 and 11, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, postencephalitic parkinsonism, chronic traumatic encephalopathy (CTE), Parkinson-dementia complex of Guam, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), primary age-related tauopathy (PART) dementia, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), vacuolar tauopathy, meningioangiomatosis, and subacute sclerosing panencephalitis (SSPE).

13. An isolated peptide or polypeptide of at least 6 amino acids in length which is a fragment of Negr1 protein and is capable of reducing ALK protein expression levels.

14. 14. The isolated peptide or polypeptide of claim 13, which is a fragment of a Negr1 protein selected from the group consisting of: (i) an amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; and (ii) an amino acid sequence that comprises, consists essentially of, or consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or an amino acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5,

15. 15. An isolated peptide or polypeptide according to claim 14, comprising the minimal biologically active sequence SEQ ID NO:6 or SEQ ID NO:

7.

16. 16. The isolated peptide or polypeptide of claim 15, selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:

15.

17. 17. An isolated peptide or polypeptide according to any one of claims 13 to 16, which comprises a modification that increases its stability.

18. 18. The isolated peptide or polypeptide of claim 17, wherein the stability-increasing modification is selected from the group consisting of an N-terminal modification, a C-terminal modification, a side chain modification, an amino acid modification, a backbone modification, and any combination thereof.

19. 18. The isolated peptide or polypeptide of claim 17, wherein the stability-enhancing modification is selected from the group consisting of PEGylation, cyclization, N-methylation, replacement of at least two amino acid residues with the corresponding D-stereoisomer, replacement of at least two amides in the peptide backbone with sulfonamides, and any combination thereof.

20. An isolated nucleic acid comprising a nucleotide sequence encoding the peptide or polypeptide of any one of claims 13 to 16.

21. An isolated nucleic acid comprising a nucleotide sequence encoding a Negr1 protein or a peptide or polypeptide according to any one of claims 13 to 16 for use in the therapeutic treatment of a disease selected from a tumor disease and a tauopathy.

22. An expression vector comprising a nucleotide sequence encoding the peptide or polypeptide according to any one of claims 13 to 16.

23. A host cell comprising the expression vector of claim 21.

24. A pharmaceutical composition comprising an isolated peptide or polypeptide according to any one of claims 13 to 19 or an isolated nucleic acid according to claim 21, and a pharma- ceutically acceptable excipient.

25. 20. The isolated Negr1 protein or a biologically active fragment thereof according to any one of claims 13 to 19, for use in reducing ALK protein expression levels in a subject suffering from a tumor disease or thaupaty.

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