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 membrane-exposed adhesion protein belonging to the immunoglobulin superfamily (IgLON family). Negr1 contains three N-glycosylated C2-type Ig-like (immunoglobulin-like) domains and has an N-terminal signal sequence. A putative GPI attachment site is located near the C-terminus of the protein.

[0004] Negr1 is highly expressed in the brain, and its expression gradually increases during postnatal brain development, reaching 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 regulates the formation of upper layers in cortical regions and the establishment of appropriate 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), first identified as a fusion kinase in a subtype of non-Hodgkin's lymphoma (NPM-ALK) and lung adenocarcinoma (EML4-ALK), is frequently targeted for 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), and ALK is a direct transcriptional target of MYCN (Hasan et al. 2013). 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 predicts the likelihood of a neuroblastoma patient being cured and helps determine 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. Higher-risk patients, on the other hand, 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 it drops to 40% for high-risk patients (Bhoopathi et al. 2021).

[0010] Surgical intervention, alone or in combination with minimal chemotherapy, can increase survival in low-risk cases. However, advanced-stage neuroblastoma remains one of the most intractable childhood cancers, despite recent advances in treatment. Patients with stage III and IV disease (classified according to the International Neuroblastoma Staging System (INSS)) often experience early relapse after chemotherapy, but treatment strategies involving multimodality therapy may improve the prognosis of high-risk patients. Such tumor recurrence 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 tumor cells. Surgical complication rates for patients with neuroblastoma range from 5 to 25%, depending on the stage of the tumor. 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 malignancies, and psychological effects.

[0012] Thus, there is a need to identify novel therapeutic agents effective in the treatment of neuroblastoma. However, this need is not limited to neuroblastoma but extends to virtually all tumor types. Because tumors are known to be able to acquire resistance to conventional therapies, and the increasing prevalence of drug-resistant tumors necessitates continued research to provide new treatments. It is also known that responses to anticancer therapies vary from patient to patient, depending on many factors, including the biology of a particular patient's tumor. This also necessitates 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) showed that overexpression of Negr1 in the human ovarian cancer cell line SKOV-3 resulted in 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 specific 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] To overcome the shortcomings and drawbacks of the prior art, the present inventors first studied the effects of ectopic treatment of various cancer cell lines with a soluble full-length Negr1 protein and found that Negr1 can significantly reduce cancer cell proliferation in vitro and tumor volume ex vivo. This finding is unexpected, because the prior art has neither taught nor suggested that treatment with a soluble Negr1 protein is actually feasible and effective in inhibiting cancer growth (proliferation).

[0017] The present inventors then analyzed the antitumor activity of the detected full-length Ngr1 protein and identified the responsible domain, i.e., the I / PepA (aa1-127) domain of human Negr1. This allowed the present inventors to identify an amino acid stretch within the full-length Negr1 protein that is the minimum active sequence required for antitumor activity. This result is highly advantageous because it allows the provision of various active fragments of Negr1 (Negr1 active fragments) that retain the antitumor activity of the full-length protein. Therefore, the phrase "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 levels.

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

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

[0020] Mutant 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 (cancer-causing) 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 tumor (IMT), and other solid tumors (Villa et al., 2021).

[0021] It is also known that ALK promotes the phosphorylation and aggregation of tau (Park et al., 2021), and pathological tau aggregation occurs in Alzheimer's disease and several other tauopathies, 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] Therefore, 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 mice.

[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) MDMMLLVQGACCSNQWLAAVLLSLCCLLPSCLPAGQSVDFPWAAVDNMMVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTVNEGTNVTLTCLATGKPEPSISWRHISPSAKPFENGQYLDIYGITRDQAGEYECSAENDVSFPDVRKVKVVVNFAPTIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKKLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNPPSTAQYGITGSADVLFSCWYLVLTLSSFTSIFYLKNAILQ (SEQ ID NO: 1)

[0027] UniProtKB-Q80Z24(NEGR1_MOUSE) MVLLAQGACCSNQWLAAVLLSLCSCLPAGQSVDFPWAAVDNMLVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTINEGTNVTLTCLATGKPEPVISWRHISPSAKPFENGQYLDIYGITRDQAGEYECSAENDVSFPDVKKVRVIVNFAPTIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKRLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNPPSTAQYGITGSACDLFSCWSLALTLSSVISIFYLKNAILQ (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) MVLLAQGACCSNQWLAAVLLSLCSCLPAGQSVDFPWAAVDNMLVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTINEGTNVTLTCLATGKPEPVISWRHISPSAKPFENGQYLDIYGITRDQAGEYECSAENDVSFPDVKKVRVIVNFAPTIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKRLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNQSSIPWQVFFMLKVSFLLVCIL (SEQ ID NO: 3)

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

[0031] UniProtKB-D3Z4T6(D3Z4T6_MOUSE) MVLLAQGACCSNQWLAAVLLSLCSCLPAGQSVDFPWAAVDNMLVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTINEGTNVTLTCLATGKPEPVISWRHISPSAKPFENGQYLDIYGITRDQAGEYECSAENDVSFPDVKKVRVIVNFAPTIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKRLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNLWCHLQMCWAHSWQCCLDILQA (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 in which one or more amino acid residues have been 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 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 to 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 can be accomplished using a mathematical algorithm, such as the blastp (blastp BLASTP 2.9.0+) program using the blosum62 matrix.

[0036] Thus, one 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 comprising, consisting essentially of, or consisting 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 is capable of reducing ALK protein expression levels;

[0037] The ranges of percent identity mentioned 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%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% 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 with 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 with 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 ALK protein expression levels. The present inventors have also surprisingly found that a variant of the above-mentioned minimum biologically active sequence (SEQ ID NO: 7), in which W (tryptophan) is substituted with 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 comprising, consisting essentially of, or consisting of an amino acid sequence 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 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 including the minimal biologically active sequence of SEQ ID NO:6 or SEQ ID NO:7.

[0040] To the best of the inventors' knowledge, no isolated peptide or polypeptide that 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, orthologs, and variant proteins as defined above. Further examples of such fragments include 6 to 35 amino acids, 6 to 70 amino acids, or 35 to 70 amino acids from the amino acid sequence of the Negr1 protein as defined above. Specific examples are fragments containing 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] The 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 biologically active minimum sequence; SEQ ID NO: 12, which is the "PepA-1 variant"; SEQ ID NO: 13, which is the "PepA-2 variant"; SEQ ID NO: 14, which is the "PepA-3 variant"; and SEQ ID NO: 7, which is the minimum variant biologically active sequence.

[0045] Compared with the full-length Negr1 protein, the peptides or polypeptides of the present invention have the advantage of shorter amino acid sequences and less steric hindrance, which may result in easier synthesis and easier penetration into cell membranes. Typically, 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 typically do not accumulate in specific organs (e.g., the kidney or liver), thereby minimizing toxic side effects. Typically, the peptides or polypeptides are also less immunogenic than recombinant antibodies or proteins.

[0046] These characteristics make the peptides or polypeptides of the present invention 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 isolated nucleic acids (DNA or RNA) comprising a nucleotide sequence encoding the Negr1 peptide or polypeptide defined above, and their use in the therapeutic treatment of tumor diseases or tauopathies.

[0048] Also included within the scope of the present invention are expression vectors (preferably viral expression vectors) containing the above-mentioned nucleotide sequences encoding the Negr1 peptides or polypeptides defined above, as well as host cells transformed with the expression vectors.

[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: SEQ ID NO: 24, which encodes PepA-1, and SEQ ID NO: 25, which is optimized for expression in human cells. 2 Optimized peptide 5.

[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 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).

[0062] Preferred tauopathies to be treated are selected from the group consisting of Alzheimer's disease, post-encephalitic parkinsonism, chronic traumatic encephalopathy (CTE), Guam Parkinsonism-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 containing an active ingredient and one or more pharmaceutically acceptable excipients.

[0064] Those skilled in the art will be able to 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 with antitumor activity is 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 in the therapeutic treatment of the tauopathy.

[0068] That is, the present invention includes the following aspects.

[0069] Aspect [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.

[0070] Aspect [2]: The isolated Negr1 protein or a biologically active fragment thereof for use according to Aspect [1], wherein the isolated Negr1 protein is selected from the group consisting of the following (i) to (iii): (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 (in particular, an amino acid sequence comprising 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 comprising, consisting essentially of, or consisting 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 capable of reducing ALK protein expression levels (particularly, an amino acid sequence comprising 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 capable of reducing ALK protein expression levels); and (iii) an amino acid sequence comprising an amino acid sequence 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 consisting essentially of an amino acid sequence 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 consisting of an amino acid sequence 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 comprising a minimum biologically active sequence of SEQ ID NO:6 or SEQ ID NO:7 (particularly, an amino acid sequence comprising an amino acid sequence 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 comprising a minimum biologically active sequence of SEQ ID NO:6 or SEQ ID NO:7);

[0071] Embodiment [3]: An isolated Negr1 protein or a biologically active fragment thereof for use according to embodiment [1] or [2], wherein the biologically active fragment comprises the minimal biologically active sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

[0072] Aspect [4]: ​​An isolated Negr1 protein or a biologically active fragment thereof for use according to any of Aspects [1] to [3], 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.

[0073] Embodiment [5]: An isolated Negr1 protein or a biologically active fragment thereof for use according to any one of embodiments [1] to [4], comprising a stability-increasing modification.

[0074] Aspect [6]: An isolated Negr1 protein or a biologically active fragment thereof for use according to aspect [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.

[0075] Aspect [7]: An isolated Negr1 protein or a biologically active fragment thereof for use according to aspect [5] or [6], wherein the stability-increasing modification is selected from the group consisting of PEGylation, cyclization, N-methylation, substitution of at least two amino acid residues with the corresponding D-stereoisomers, substitution of at least two amides in the backbone with sulfonamides, and any combination thereof.

[0076] Aspect [8]: An isolated Negr1 protein or a biologically active fragment thereof for use according to any of aspects [1] to [7], wherein the disease is a tumor disease and the use is simultaneous, separate or sequential combined use with a compound or substance having antitumor activity or a radiotherapeutic agent.

[0077] Aspect [9]: The isolated Negr1 protein or a biologically active fragment thereof for use according to aspect [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.

[0078] Aspect

[10] : An isolated Negr1 protein or a biologically active fragment thereof for use according to any one of Aspects [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).

[0079] Aspect

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

[0080] Aspect

[12] : An isolated Negr1 protein or a biologically active fragment thereof for use according to any one of Aspects [1] to [7] and Aspect

[11] , wherein the tauopathy is selected from the group consisting of Alzheimer's disease, post-encephalitic 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).

[0081] Embodiment

[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 the expression level of ALK protein.

[0082] Embodiment

[14] : The isolated peptide or polypeptide according to embodiment

[13] , which is a fragment of a Negr1 protein selected from the group consisting of (i) and (ii) below: (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 (in particular, an amino acid sequence comprising 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 comprising, consisting essentially of, or consisting 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 (particularly, an amino acid sequence comprising 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);

[0083] Embodiment

[15] : An isolated peptide or polypeptide according to embodiment

[13] or

[14] , comprising the minimal biologically active sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

[0084] Aspect

[16] : An isolated peptide or polypeptide according to any one of aspects

[13] to

[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.

[0085] Embodiment

[17] : An isolated peptide or polypeptide according to any one of embodiments

[13] to

[16] , which comprises a modification that increases stability.

[0086] Embodiment

[18] : The isolated peptide or polypeptide according to embodiment

[17] , 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.

[0087] Embodiment

[19] : The isolated peptide or polypeptide of embodiment

[17] or

[18] , 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 peptide backbone with sulfonamides, and any combination thereof.

[0088] Aspect

[20] : An isolated nucleic acid comprising a nucleotide sequence encoding the peptide or polypeptide according to any one of aspects

[13] to

[19] .

[0089] Aspect

[21] : An isolated nucleic acid comprising a nucleotide sequence encoding a Negr1 protein or a peptide or polypeptide described in any of aspects

[13] to

[19] , for use in the therapeutic treatment of a disease selected from tumor diseases and tauopathies.

[0090] Aspect

[22] : An expression vector comprising a nucleotide sequence encoding the peptide or polypeptide according to any one of aspects

[13] to

[19] .

[0091] Embodiment

[23] : A host cell comprising the expression vector according to embodiment

[22] .

[0092] Aspect

[24] : A pharmaceutical composition comprising an isolated peptide or polypeptide according to any one of aspects

[13] to

[19] or an isolated nucleic acid according to aspect

[21] , and a pharmaceutically acceptable excipient.

[0093] That is, the aspect

[24] is an isolated peptide or polypeptide according to any one of aspects

[13] to

[19] , or The present invention may also be a pharmaceutical composition comprising a Negr1 protein or an isolated nucleic acid comprising a nucleotide sequence encoding said peptide or polypeptide, and a pharmaceutically acceptable excipient, for use in the therapeutic treatment of a disease selected from tumor diseases and tauopathies.

[0094] Aspect

[25] : An isolated peptide or polypeptide (Negr1 protein or a biologically active fragment thereof) according to any one of aspects

[13] to

[19] for use in reducing the expression level of ALK protein in a subject suffering from a tumor disease or tauopathy.

[0095] 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 explanation of the drawings]

[0096] [Figure 1] Figure 1 shows experimental results obtained by the present inventors, demonstrating 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 rates of wild-type N2a cells and three different clones stably expressing Negr1 (colonies 3, 7, and 11) were analyzed. N2a clones were seeded at a constant aliquot (20,000 cells) on day 0. Cell numbers were counted on day 5; n = 12, **p < 0.01 vs. wild-type. (C) 20,000 N2a wild-type or stably expressing Negr1 (col3 and 11) cells were seeded in soft agar and cultured for 14 days. The 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. [Figure 2]Figure 2 shows experimental results obtained by the present inventors, demonstrating that PepA, an embodiment of the present invention, inhibits neuroblastoma growth 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 fixed dose (20,000 cells) on day 0 and cultured for 5 days. Culture viability was measured by MTT assay. A graph showing cell viability expressed as fold-over of untreated cultures; n = 10, **p < 0.01 vs. GFP. (B) A 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 volume (20,000 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 volume (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 increase over vehicle; n=8, **p<0.01 vs. GFP. [Figure 3]Figure 3 shows the experimental results obtained by the present inventors, demonstrating that PepA inhibits neuroblastoma growth in vivo. (A) One million N2a cells were subcutaneously injected into CD1 immunodeficient nude mice. When tumors reached a volume of 100 mm, 2 μg of GFP or PepA was injected in situ. This treatment was repeated every other day. Tumor growth was monitored 1 to 9 days after injection. A graph shows tumor volume growth expressed as fold increase relative to day 1; n = 9, **p < 0.01 vs. GFP. (B) The presence of GFP and PepA in tumors, liver, and kidneys 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 (red, to stain immature blood vessels) and DAPI (blue, to visualize cell nuclei). (D-E) Graphs showing the number of blood vessels (total area) (D) and the average area (arbitrary units) (E); n = 4, *p < 0.05 vs. GFP. [Figure 4]Figure 4 shows the experimental results obtained by the inventors, demonstrating that soluble Negr1-derived peptides (i.e., PepA-1, PepA-2, and PepA-3) further inhibit neuroblastoma growth in vitro and in vivo. (A) Diagram showing the boundaries of the peptides. (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 rate (20,000 cells) on day 0, cultured for 5 days, and finally measured by MTT assay. A graph showing cell vitality at day 5, normalized to GFP values; 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 fixed dose (20,000 cells) on day 0, cultured for 5 days, and finally measured by MTT assay. Cell vitality at day 5, normalized to values ​​measured during vehicle (for lorlatinib) or GFP (for rNegr1 and PepA-1) treatment, is shown; n = 10; ***p < 0.01 vs. vehicle, ***p < 0.01 vs. GFP. (D) Primary cortical neurons were treated daily with 40, 80, 160, or 200 ng / ml of PepA-1 or GFP starting on day 7. Five days later, cytotoxicity was assessed by MTT. Cell vitality normalized to GFP values ​​is shown; n = 6. (E) One million N2a cells were subcutaneously injected into CD1 immunodeficient nude mice. When tumors reached a volume of 100 mm3, 2 μg of GFP or PepA-1 was 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 increase relative to day 1; n=3, *p<0.05 vs. GFP. [Figure 5]Figure 5 shows the experimental results obtained by the inventors, demonstrating that synthetic PEG-PepA-1 inhibits neuroblastoma growth in vitro and in vivo. (A) One million N2a cells were subcutaneously injected into CD1 immunodeficient nude mice. When tumors reached a volume of 100 mm, 10 μg of PEG-scrambled peptide or PEG-PepA-1 was injected in situ. This treatment was repeated every other day. After injection, tumor growth was monitored daily. A graph showing tumor volume growth expressed as fold increase over day 1; n = 6, *p < 0.05, **p < 0.01 vs. PEG-scrambled. (B) One million N2a cells were subcutaneously injected into CD1 immunodeficient nude mice. When tumors reached a volume of 100 mm, 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. A graph showing tumor volume growth expressed as fold increase over day 1; n = 6-8, *p < 0.05 vs. PEG-scrambled. [Figure 6]Figure 6 shows the experimental results obtained by the inventors, demonstrating that the peptide PepA regulates ALK signaling. (A) Diagram showing the boundaries of PepA, PepB, and PepC. (B) 50,000 N2a cells were treated with 300 ng / ml of GFP, rNegr1, or PepA daily for 5 days. On DIV5, cultures were processed for Western blotting to assess 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 differences over GFP-treated cells; n = 6, *p < 0.05 vs. GFP. (E) 50,000 N2a cells were treated with 3 μM of GFP, lorlatinib, or PepA-1 daily for 5 days. On day 5 (DIV), cultures were processed for Western blotting to assess protein expression levels for ALK and MYCN, as well as actin and S6 ribosomal protein. (F-G) Graphs show ALK (F) and MYC (G) levels normalized to actin and S6RP amounts and expressed as fold changes relative to GFP-treated cells; n = 6, *p < 0.05, ***p < 0.001 vs. GFP. (H) 150,000 N2a cells were treated with 3 μM GFP, lorlatinib, or PepA-1 for 10 minutes. Cultures were then processed for Western blotting to assess protein expression levels for AKT, phosphorylated AKT (phospho-AKT), ERK1 / 2, and phosphorylated ERK1 / 2 (phospho-ERK1 / 2), as well as S6 ribosomal protein. (I-J) Graphs showing the pAKT / AKT ratio (I) and pERK / ERK ratio (J) expressed as fold over vehicle-treated cells; n=6, **p<0.01, ***p<0.001 vs. vehicle. [Figure 7]Figure 7 shows the experimental results obtained by the inventors, demonstrating that a synthetic version of PepA-1 and a scrambled peptide containing a biologically active minimal sequence variant regulate ALK signaling. (A) Schematic showing the location of the biologically active minimal sequence GDKWSV (SEQ ID NO: 8) (Source: PDB: 6DLD). (B) Sequence of the synthetic peptide. (C) 150,000 N2a cells were treated with vehicle or the following synthetic peptides (10 μM, 10 μM for 10 min): scrambled peptide, scr-GDKISV, a PepA-1-derived peptide 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 the phosphorylated ERK / ERK (phospho-ERK / ERK) ratio as fold over vehicle; n=8, *p<0.05, ***p<0.01 vs. vehicle. (E) 150,000 N2a cells were treated with 3 μM or 30 μM vehicle, lorlatinib, or GDKWSV peptide for 10 minutes. Cultures were then processed for dot blotting to assess the expression levels of ERK1 / 2 and phosphorylated ERK1 / 2. (F) Graph showing the phosphorylated ERK / ERK ratio as fold over vehicle; n=5, **p<0.01 vs. vehicle. (G) 150,000 N2a cells were treated with the following synthetic peptides (10 μM for 10 min): scrambled peptide, PepA-1, and PepA-1 6aa scramble, a PepA-1-derived peptide 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 the phosphorylated ERK / ERK ratio as fold over vehicle; n = 4, ***p < 0.01 vs. other conditions. [Figure 8]Figure 8 shows experimental results obtained by the inventors regarding the efficacy of a pegylated form of PepA-1, designated PEG-PepA-1. (A) 150,000 N2a cells were treated with 3 μM or 30 μM vehicle, lorlatinib, or PEG-PepA-1 for 10 minutes. Cultures were then processed for Western blotting to assess protein expression levels for ERK1 / 2 and phosphorylated ERK1 / 2. (B) Graph showing the phosphorylated ERK / ERK ratio as fold increase over vehicle upon treatment with 30 μM; n = 8, **p < 0.01 vs. vehicle. (C) 150,000 N2a cells were treated with 5 μM vehicle, lorlatinib, scrambled peptide, or PEG-PepA-1 for 24 or 48 hours. Cultures were then processed for Western blotting to assess protein expression levels for ALK. Graphs show 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. Graphs show normalized ALK optical density as fold over vehicle; n=4, **p<0.01 vs. scrambled peptide. DETAILED DESCRIPTION OF THE INVENTION

[0097] The results obtained by the present inventors are described in detail below.

[0098] In a first series of experiments, we observed that Negr1 protein was downregulated in the brain cancer cell lines SH5Y (human neuroblastoma), U87 (glioblastoma), and N2a (mouse neuroblastoma). We also found that ectopic expression of Negr1 in N2a cells significantly reduced cell proliferation, and that the ability of Negr1-expressing N2a cell clones to form colonies in soft agar was significantly reduced. Furthermore, in in vivo studies using a xenograft mouse model, an established tool for investigating cancer progression in vivo, we observed that the size of tumors derived from subcutaneously injected Negr1-expressing N2a cells was significantly smaller than that of tumors derived from wild-type N2a cells.

[0099] These findings demonstrate that overexpression of Negr1 can inhibit cancer growth (proliferation) both in vitro and in vivo.

[0100] 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).

[0101] The present inventors also conducted a series of experiments aimed at identifying the minimal amino acid sequence capable of exerting antineoplastic (anti-tumor) activity. The Negr1 protein contains three IgG-like domains. The amino acid sequence of the human full-length Negr1 protein is available from UniProtKB under accession number Q7Z3B1 (NEGR1_HUMAN) and is designated as SEQ ID NO: 1 in the attached sequence listing. Therefore, the present inventors cloned and characterized each of the IgG-like domains. 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). We treated naive N2a cells 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 cell proliferation, whereas PepB and PepC did not. We also tested the ability of Negr1 and PepA to inhibit 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 cancer lines analyzed.

[0102] The antitumor activity of PepA was also confirmed in in vivo experiments using 6-week-old CD1 nude mice. In these experiments, the inventors observed that orthotopic PepA treatment robustly reduced tumor growth. Furthermore, the inventors found that, upon in situ administration, PepA was localized in tumors, liver, and kidneys, whereas the control GFP protein was primarily localized in tumors. This suggests that PepA can reach the bloodstream and be distributed throughout the body. Furthermore, the inventors analyzed tumor angiogenesis by detecting CD34 protein and observed that angiogenesis was significantly reduced in PepA-treated tumors.

[0103] To further narrow down the biologically active minimal sequence of PepA, we performed 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 observed that all tested Negr1-derived peptides significantly reduced N2a growth (proliferation), with PepA-1 and PepA-3 exerting a stronger inhibitory effect. Furthermore, in MTT assays, we demonstrated that rNegr1, PepA-1, and lorlatinib (an FDA-approved ALK inhibitor) significantly reduced N2a growth (proliferation). a We observed that treatment with increasing concentrations of PepA-1 reduced the growth (proliferation) of primary cortical neurons to a similar extent. Remarkably, we further observed that treatment with increasing concentrations of PepA-1 did not induce greater toxicity in primary cortical neurons than treatment with GFP. The significant antineoplastic activity of PepA-1 was also confirmed in vivo in 6-week-old nude mice.

[0104] 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 had antitumor activity both in vitro against N2a cells and in vivo in 6-week-old CD1 nude mice.

[0105] The present inventors also clarified 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, as well as the phosphorylation of ERK kinase and AKT kinase.

[0106] Importantly, we also identified the 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.

[0107] Based on the above experimental results, not only the Negr1 peptide of SEQ ID NO: 8 (PepA) but also the minimal biologically active sequence located between positions 45 and 50 of SEQ ID NO: 8, SEQ ID NO: No. 6 It can be concluded that Negr1 peptide fragments having a length of at least 6 amino acids, including the following, also have antitumor activity.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] Furthermore, the Negr1 protein and its biologically active fragments, peptides, or polypeptides may all be provided in modified forms (modified forms) to improve their pharmacokinetic properties, for example, to increase potency, prolong activity, and / or prolong 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, and the like. Any of a 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; containing at least two D-amino acids as disclosed in Vlieghe et al. 2010; Contains at least two β-amino acids as disclosed in Hook et al. 2005; N-methylation as disclosed in 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.

[0113] 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.

[0114] Experimental section Materials and Methods Constructs and peptides

[0115] The 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.

[0116] scramble: DGGSKGGFADLSSIIWARWNKARVDPLVSISTS (SEQ ID NO: 18) Scramble-GDKISV: NRGDKISVSVSSSSGGRAWDWDIAKPGLAILTF (SEQ ID NO: 19) PepA-1-6aa scramble: DGASKGAWLNRSSIIFAGVWGSKDDPRVSISTL (SEQ ID NO: 20)

[0117] Cell culture and transfection

[0118] 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% CO. Cortical neuron cultures were prepared from mouse embryos (E17.5-18.5; C57BL / 6 strain).

[0119] 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 ).

[0120] 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).

[0121] Purification with STREP resin HEK293 cells transfected with the relative constructs were incubated for 1 hour at 4°C in RIPA buffer (150 mM NaCl, 50 mM HEPES, 0 . Proteins were dissolved in 5% NP40, 1% sodium deoxycholate, and then processed for streptavidin immunoprecipitation. Proteins were eluted from the STREP resin with elution buffer (2.5 mM desthiobiotin, 100 mM Tris-HCl) for 1 h at 4°C with gentle agitation. lThe STREP resin was eluted in RIPA buffer (150 mM NaCl, 150 mM NaCl, 1 mM EDTA). Protein concentration was measured by a 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 h at 4°C and then processed for streptavidin immunoprecipitation. Proteins were eluted from the STREP resin in Laemmli buffer.

[0122] 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 4°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 culture 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.

[0123] 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 agitation for 1 hour, the lysate was clarified by centrifugation at 16,000 g for 20 minutes. All experimental procedures were performed at 4°C. Protein concentration was 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 a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Sigma-Aldrich) at 80 V for 120 minutes at 4°C. The 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), and 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), and the optical density of specific bands was quantified with ImageLab software (Bio Rad).

[0124] animal One million N2a cells were suspended in 150 μl of DMEM high glucose and administered subcutaneously to the left rear paw of 8-week-old nu / nu mice (Charles River) by injection using a microsyringe (Hamilton). All animal procedures were approved by Institutional and National Agencies (authorization 559 / 2016-PR).

[0125] Immunofluorescence Tumors were dissected, post-fixed in 4% PFA for 2 hours, then placed in OCT and stored at -80°C until processing. Serial sectioning was performed on a Leica cryostat to obtain 14-μm sections, which were then mounted on Polysine slides (Thermo Fischer Scientific). The sections were immersed in 2.5% BSA, 10% NGS, 0.2% Triton X-100, and PBS 1X for 1 hour at room temperature (RT), and then 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. The 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 stored in the dark at 4°C until images were acquired with a Zeiss Axio Imager M2 equipped with a forty-fold (40X) objective.

[0126] Statistical Analysis and Guidelines All data were presented as boxplots showing minimum, maximum, and median values. Normality of data distribution was determined using the D'Agostino and Pearson omnibus normality test followed by an unpaired Student's t test, analysis of variance (ANOVA) followed by Tukey's post-hoc test, or two-way ANOVA followed by a Bonferroni or Student's t post-hoc test, as appropriate. The number of experiments (n) and significance level (p) are indicated throughout the text. All methods were performed in accordance with relevant guidelines and national regulations.

[0127] result Negr1 overexpression inhibits cancer growth (proliferation) in vitro and in vivo. Protein samples obtained 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 was detectable 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 rate (20,000 cells) on day 0 in vitro (DIV0). Cell proliferation was assessed on DIV5. We found that ectopic Negr1 expression significantly reduced N2a proliferation (Figure 1B).

[0128] Cancer cells are known to be able to grow independently of solid surfaces. Such anchorage-independent growth is a hallmark of carcinogenesis. This characteristic can be reproduced in vitro by monitoring colony growth in a soft agar matrix. Therefore, the soft agar colony formation assay tests whether cells have undergone malignant transformation. Therefore, 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 clone had a significantly reduced ability to form colonies in soft agar (Figure 1C-D).

[0129] Xenograft mouse models are an established tool for investigating cancer progression in vivo. To this end, one million N2a cells (naive, clone 3, and clone 11) were subcutaneously injected into immunodeficient CD1 nude mice. Tumor volume was monitored ex vivo by measuring tumor size with a caliper. Tumor volume was calculated using the ellipsoid formula (length × width × height × 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).

[0130] Soluble Negr1 inhibits cancer growth (proliferation) in vitro and in vivo. At physiological levels, Negr1 exists as both a GPI-anchored membrane-associated protein and a soluble protein 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. To this end, we expressed and purified Strep-FLAG Negr1 and Strep-GFP from HEK293 cells. The latter is a biologically inactive protein used as a control. Next, 10,000 naive N2a cells were seeded and treated daily with 200 ng of rNegr1 or GFP. On DIV5, cell growth was assessed by MTT assay. We observed that treatment with Negr1 significantly reduced N2a growth (Figure 2A).

[0131] Next, we aimed to identify the minimal sequence capable of exerting the anti-neoplastic effect of Negr1. 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 then expressed and purified these three domains (hereafter referred to as PepA, PepB, and PepC) from HEK293 cells.

[0132] 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 observed that treatment with Negr1 and PepA significantly reduced N2a proliferation (Figure 2B).

[0133] Next, we tested the ability of Negr1 and the peptide PepA to inhibit the growth of human cancer cell lines: U87 (glioblastoma), SK-Mel5 (malignant melanoma), MCF7 (breast adenocarcinoma), A549 (adenocarcinoma of the alveolar basal epithelium), and SH-SY5Y (neuroblastoma). To this end, each line was treated daily with 200 ng of GFP, rNegr1, or PepA. On Day 5, 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). These results are in good agreement with experimentally detected expression of the receptor ALK in cancer cell lines of ectodermal origin, including small cell lung cancer, breast cancer, malignant melanoma, neuroblastoma, and glioblastoma (Dirks et al. 2002).

[0134] To further evaluate the antineoplastic activity of PepA, 1 million N2a cells were injected subcutaneously into 6-week-old CD1 nude mice. 3 When tumors reached a volume of 10 μg, they were injected in situ with 2 μg of GFP or PepA. This treatment was repeated every other day, and tumor growth was monitored daily. We found that PepA treatment significantly reduced tumor growth (Figure 3A).

[0135] On the 9th day after injection (or when the tumor was 2.5 cm 3 On the day of tumor mass (or earlier if tumor volume reached 100 μg / day), the animals were euthanized, and mouse tissues were collected and analyzed for the presence of GFP and PepA. Notably, by immobilization on STREP resin, we observed that PepA was present in the tumor, liver, and kidney, while GFP protein was primarily present in the tumor (Figure 3B). This biochemical evidence suggests that PepA can reach the bloodstream and be distributed throughout the body.

[0136] 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).

[0137] Characterization of a 33 amino acid long biological anti-neoplastic peptide To further narrow down the minimal biologically active sequence within PepA, we performed in silico analysis of the PepA structure. 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 from HEK293 cells as strep-FLAG-tagged recombinant peptides. 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. On Day 5, cell growth (proliferation) was assessed by MTT assay. We observed that the minimal peptide PepA-1 significantly reduced the growth (proliferation) of N2a (FIG. 4B).

[0138] To further investigate the efficacy of PepA1, we performed a randomized controlled trial comparing N2 and N3 mice treated with vehicle, lorlatinib (an ALK inhibitor approved by the FDA), GFP, rNegr1, and PepA-1 (all molecules at 3 μM daily). a Cell proliferation was investigated. On DIV5, cell vitality was assessed by MTT assay. rNegr1, PepA-1, and lorlatinib inhibited N2 aInterestingly, treatment with increasing concentrations of PepA-1 (daily from DIV7 to DIV12) did not cause excessive toxicity to primary cortical neurons at DIV12 compared with treatment with GFP (Fig. 4D).

[0139] Next, 1 million N2a cells were injected subcutaneously into 6-week-old nude mice. 3 When tumors reached a volume of 10 μg, they were injected in situ with 2 μg of GFP or PepA-1. This treatment was repeated every other day, and tumor growth was monitored daily. We observed that PepA-1 treatment significantly reduced growth (Figure 4E).

[0140] Characterization of a 33-amino acid long synthetic anti-neoplastic peptide. Biological evidence suggests that PepA1 is glycosylated at the asparagine residue (N) at position +10. To mimic glycosylation, we designed a synthetic peptide with a mini-PEG1 moiety (MW: 152 Da) attached to the N-terminus of the PepA-1 sequence. The resulting peptide (hereafter referred to as PEG-PepA-1) is soluble in water at a concentration of 5 mg / ml.

[0141] To evaluate the antineoplastic activity of PEG-PepA-1, 1 million N2a cells were injected subcutaneously into 6-week-old CD1 nude mice. 3 When 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 observed that PepA treatment significantly reduced tumor growth (Figure 5A).

[0142] Finally, we tested the efficacy of PEG-PepA-1 by systemic administration. To this end, 1 million N2a cells were injected subcutaneously into 6-week-old CD1 nude mice. Tumors were 100 mm 3When tumors reached a volume of 100 μg, 150 μg of PEG-PepA-1 or PEG-scrambled peptide was intraperitoneally injected. This treatment was repeated every other day, and tumor growth was monitored daily. We observed that PEG-PepA-1 treatment significantly reduced tumor growth (Figure 5B).

[0143] Given the significant effects on cancer cell growth (proliferation) observed in vitro and in vivo, we sought 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, along with 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).

[0144] We monitored whether treatment with rNegr1 or PepA affected the ALK-MYCN pathway. To this end, 50,000 N2a cells were treated daily for 5 days with 300 ng / ml of GFP, rNegr1, or PepA. On DIV5, cultures were processed for Western blotting to assess the expression levels of ALK and MYCN proteins. PepA treatment significantly reduced ALK and MYCN protein levels (Figures 6B–D).

[0145] Next, 50,000 N2a cells were treated daily for 5 days with 3 μM GFP, lorlatinib, or PepA-1 (50 ng / ml). On DIV5, cultures were processed for Western blotting to assess ALK and MYCN protein expression. Notably, we observed that chronic treatment with PepA-1 led to a strong downregulation of ALK and MYCN (Figures 6E-G).

[0146] ALK induces a signaling cascade involving the 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. Acute treatment (3 μM, 10 min) with PepA-1 significantly reduced the phosphorylation of ERK and AKT (Figures 6H-J).

[0147] Furthermore, 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 tryptophan (W) was replaced with isoleucine (I)), and a scrambled peptide (10 μM, 10 min). Notably, we found that scr-GDKISV and synPepA-1 reduced ERK1 / 2 phosphorylation (Figure 7C-D).

[0148] 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).

[0149] Finally, we tested the biological activity of the GDKWSV peptide alone. To this end, 150,000 N2a cells were treated with either the synthetic GDKWSV peptide or the ALK inhibitor lorlatinib (30 nM, 10 min). Using dot plots, we observed that treatment with the GDKWSV peptide correlated with a significant decrease 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. Following acute treatment (30 μM, 10 min), PEG-PepA-1 significantly reduced ERK phosphorylation (Figure 8A-B).

[0150] We also investigated the combined effects of PEG-PepA-1 and lorlatinib. We found that with long-term treatment, PEG-PepA-1 significantly reduced ALK protein levels, while lorlatinib induced strong upregulation of the receptor (both at 5 μM for 24 or 48 hours, Figure 8C). Notably, coadministration of PEG-PepA-1 significantly reduced ALK protein levels, which was observed with long-term treatment with lorlatinib (5 μM for 24 hours, Figure 8D).

[0151] 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. A. F., Drexler H. G. (2002) Expression and functional analysis of the anaplastic lymphoma kinase (ALK) gene in tumor cell lines. Int J Cancer 100, 49-56. ·Hasan M. K., Nafady A., Takatori A., Kishida S., Ohira M., Suenaga Y., Hossain S., et al. (2013) ALK is a MYCN target gene and regulates cell migration and invasion in neuroblastoma. Sci Rep 3, 3450. ·Hook D. F., Bindschaedler P., Mahajan Y. R., Sebesta R., Kast P., Seebach D. (2005) The Proteolytic Stability of ‘Designed’β-Peptides Containing α-Peptide-Bond Mimics and of Mixed α,β-Peptides: Application to the Construction of MHC-Binding Peptides. Chemistry & Biodiversity 2, 591-632. ·Janoueix-Lerosey I., Lequin D., Brugieres L., Ribeiro A., Pontual L. de, Combaret V., Raynal V., et al. (2008) Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma. Nature 455, 967-970. ·Kim H., Hwang J.-S., Lee B., Hong J., Lee S. (2014) Newly Identified Cancer-Associated Role of Human Neuronal Growth Regulator 1 (NEGR1). J Cancer 5, 598-608.·Kong X.-D., Heinis C. (2021) Towards the Development of Orally Available Peptide Therapeutics. Chimia (Aarau) 75, 514-517. ·Linde Y., Ovadia O., Safrai E., Xiang Z., Portillo F. P., Shalev D.E., Haskell-Luevano C., Hoffman A., Gilon C. (2008) Structure-activity relationship and metabolic stability studies of backbone cyclization and N-methylation of melanocortin peptides. Peptide Science 90, 671-682. ·Mahapatra S., Challagundla K. B. (2021) Neuroblastoma, in StatPearls. StatPearls Publishing, Treasure Island (FL). ·McKie A. B., Vaughan S., Zanini E., Okon I. S., Louis L., Sousa C. de, Greene M. I., et al. (2012) The OPCML tumor suppressor functions as a cell surface repressor-adaptor, negatively regulating receptor tyrosine kinases in epithelial ovarian cancer. Cancer Discov 2, 156-171. ·Miyata S., Matsumoto N., Taguchi K., Akagi A., Iino T., Funatsu N., Maekawa S. (2003) Biochemical and ultrastructural analyses of IgLON cell adhesion molecules, Kilon and OBCAM in the rat brain. Neuroscience 117, 645-658. ·Park J, Choi H, Kim YD, Kim SH, Kim Y, Gwon Y, Lee DY, Park SH, Heo WD, Jung YK. Aberrant role of ALK in tau proteinopathy through autophagosomal dysregulation. Mol Psychiatry. 2021 Jan 15. ·Pischedda F., Montani C., Obergasteiger J., Frapporti G., Corti C., Rosato Siri M., Volta M., Piccoli G. (2018) Cryopreservation of Primary Mouse Neurons: The Benefit of Neurostore Cryoprotective Medium. Front Cell Neurosci 12, 81. ·Pischedda F., Piccoli G. (2015) The IgLON Family Member Negr1 Promotes Neuronal Arborization Acting as Soluble Factor via FGFR2. Front Mol Neurosci 8, 89. ·Pischedda F., Szczurkowska J., Cirnaru M. D., Giesert F., Vezzoli E., Ueffing M., Sala C., et al. (2014) A cell surface biotinylation assay to reveal membrane-associated neuronal cues: Negr1 regulates dendritic arborization. Mol. 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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. 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 the following (i) to (iii): (i) an amino acid sequence comprising 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 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 is capable of reducing ALK protein expression levels; and (iii) 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, substitution of at least two amino acid residues with the corresponding D-stereoisomers, substitution 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 4, wherein the disease is a tumor disease and the use is simultaneous, separate or sequential combined use with a compound or substance having anti-tumor 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 anti-tumor 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. 5. The isolated Negr1 protein or a biologically active fragment thereof for use according to any one of claims 1 to 4, wherein the tumor disease is selected from the group consisting of neuroblastoma, ganglioglioma, ganglioneuroma, 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 4, wherein the disease is tauopathy, and the use is simultaneous, separate or sequential combined use with a compound or substance effective for the therapeutic treatment of tauopathy.

12. 5. The isolated Negr1 protein or a biologically active fragment thereof for use according to any one of claims 1 to 4, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, post-encephalitic 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 the expression level of ALK protein.

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) and (ii) (i) an amino acid sequence comprising 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 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. The isolated peptide or polypeptide of 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. The isolated peptide or polypeptide of 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-stereoisomers, 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. 17. 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 tumor diseases and tauopathies.

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

23. A host cell comprising the expression vector of claim 22.

24. An isolated peptide or polypeptide according to any one of claims 13 to 16, or A pharmaceutical composition comprising a Negr1 protein or an isolated nucleic acid comprising a nucleotide sequence encoding said peptide or polypeptide, and a pharmaceutically acceptable excipient, for use in the therapeutic treatment of a disease selected from tumor diseases and tauopathies.

25. 17. An isolated peptide or polypeptide according to any one of claims 13 to 16 for use in reducing ALK protein expression levels in a subject suffering from a tumor disease or thaupaty.

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