Composition comprising GV1001 peptide for reducing CD47 expression in cancer cells and increasing phagocytosis by macrophages, and use thereof

Abstract

The present invention relates to a composition comprising GV1001 peptide for reducing CD47 expression in cancer cells and increasing phagocytosis by macrophages, and a use thereof. Specifically, the present invention relates to: a composition to be used in preventing, treating, or alleviating cancers associated with the expression of CD47; and medical, health functional food and feed uses thereof. The composition of the present invention exhibits excellent effects in preventing, treating or alleviating cancers in which CD47 is expressed, particularly head and neck cancer, gastric cancer and colorectal cancer and / or related symptoms.

Description

Composition for reducing CD47 expression in cancer cells and increasing phagocytosis in macrophages containing GV1001 peptide and uses thereof

[0001] Cross-reference of related applications

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0132419, provisionally filed on September 30, 2024, and Korean Patent Application No. 10-2025-0141023, filed on September 29, 2025, the full text of which is incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a composition for use in preventing, treating, or improving cancer associated with the expression of CD47 (Cluster of Differentiation 47). More specifically, the present invention relates to a pharmaceutical composition, a health functional food, a feed composition, and a kit for use in preventing, treating, or improving cancer and / or related symptoms in subjects who have developed or are at risk of developing cancer associated with the expression of CD47.

[0005]

[0006] CD47 (Cluster of Differentiation 47) is a transmembrane protein and is known to have significantly high expression levels in various tumor types, including head and neck cancers, non-small cell lung carcinoma (NSCLC) (including advanced NSCLC), gastric cancer, colorectal adenocarcinoma, and pancreatic neuroendocrine tumors.

[0007] CD47, belonging to the immunoglobulin superfamily, interacts with several proteins such as integrin, thrombospondin-1, and signal regulatory protein α (SIRPα). In macrophages, the interaction between CD47 and SIRPα activates the "Don't eat me" signal, which inhibits macrophages from eliminating tumor cells through phagocytosis.

[0008] Targeting CD47-SIRPα interactions is emerging as an innovative strategy to disrupt immune checkpoints in various cancers. Nevertheless, blocking CD47-SIRPα interactions alone is insufficient to stimulate macrophage-mediated antitumor activity.

[0009] In addition, the efficacy of anti-CD47 monoclonal antibodies in monotherapy and combination with chemotherapy has been demonstrated in various nonclinical animal models. However, in the case of anti-CD47 antibodies, issues have emerged such as uncertainty regarding therapeutic efficacy, the potential for red blood cell-related side effects (such as hemolysis), and the possibility of attacking normal cells due to a lack of cancer specificity. In particular, clinical development was halted in Gilead's 5F9 (magrolimab) anticancer clinical trial due to unexpected results showing an increase in mortality.

[0010] Therefore, there is a need to develop new substances capable of effectively treating cancers associated with CD47 expression without side effects, as well as anticancer drugs utilizing these substances.

[0011] Meanwhile, the GV1001 peptide, composed of 16 amino acids selected from human telomerase reverse transcriptase (hTERT), has been reported to exhibit anti-inflammatory activity, antioxidant activity, and anticancer activity against some types of cancer, and to be effective in alleviating neurodegenerative diseases, including Alzheimer's disease, and related symptoms. In addition, the toxicity of the GV1001 peptide has been evaluated through numerous clinical trials targeting various diseases, and its safety regarding side effects has been verified.

[0012] However, it has not been studied whether the GV1001 peptide exhibits effective anticancer activity without side effects against cancers associated with CD47 expression.

[0013] Surprisingly, the inventors completed the present invention by confirming that the GV1001 peptide exhibits a significant therapeutic effect without specific side effects on cancers associated with CD47 expression, specifically head and neck cancer, gastric cancer, and colorectal cancer.

[0014] The present invention aims to provide a pharmaceutical composition having excellent preventive or therapeutic effects against cancer associated with CD47 expression, comprising a peptide having an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient.

[0015] In addition, the present invention aims to provide a health functional food having excellent effects in preventing, delaying, or inhibiting the onset of cancer associated with the expression of CD47, comprising a peptide having an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient.

[0016] In addition, the present invention aims to provide a feed composition having excellent effects in preventing, delaying, or inhibiting the onset of cancer associated with CD47 expression, comprising a peptide having an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient.

[0017] In addition, the present invention aims to provide a kit for use in preventing, treating, or improving cancer associated with CD47 expression, comprising a composition including a peptide having an amino acid sequence represented by SEQ ID NO. 1 and instructions for use.

[0018]

[0019] To achieve the aforementioned objective, the present invention provides a pharmaceutical composition for use in preventing or treating cancer associated with CD47 expression, comprising as an active ingredient a peptide having an amino acid sequence represented by SEQ ID NO. 1 (abbreviated herein as 'GV1001 peptide').

[0020] In addition, to achieve the aforementioned objective, the present invention provides a health functional food for use in preventing, delaying, or inhibiting the onset of cancer associated with the expression of CD47, comprising as an active ingredient a peptide having an amino acid sequence represented by SEQ ID NO. 1.

[0021] In addition, to achieve the aforementioned objective, the present invention provides a feed composition for use in preventing, delaying, or inhibiting the onset of cancer associated with the expression of CD47, comprising as an active ingredient a peptide having an amino acid sequence represented by SEQ ID NO. 1.

[0022] In addition, to achieve the aforementioned objective, the present invention provides a kit for use in preventing, treating, or improving cancer associated with the expression of CD47, comprising: a composition comprising a peptide having an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient; and instructions for use.

[0023] In addition, to achieve the aforementioned purpose, the present invention provides a use of a peptide having the amino acid sequence represented by SEQ ID NO. 1 in the manufacture of a composition, such as a pharmaceutical composition or a feed composition or a health functional food, for use in preventing, treating, or improving cancer associated with the expression of CD47.

[0024] In addition, to achieve the aforementioned objective, the present invention provides a use of a peptide having an amino acid sequence represented by SEQ ID NO. 1 in the manufacture of a kit for use in preventing, treating, or improving cancer associated with the expression of CD47.

[0025] In addition, to achieve the aforementioned objective, the present invention provides a method for preventing, treating, or improving cancer associated with the expression of CD47 in a subject, comprising the step of administering a composition containing a peptide having an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient to a subject in need thereof.

[0026]

[0027] The peptide having the amino acid sequence represented by SEQ ID NO. 1 of the present invention is expected to be useful as a monotherapy or in combination with existing anticancer drugs or anticancer therapies, as it can effectively inhibit the development of cancers associated with CD47 expression, particularly head and neck cancer, gastric cancer, and colorectal cancer, and / or improve or treat cancers that have already developed and / or related symptoms.

[0028]

[0029] Figure 1 shows the inhibitory effect of the GV1001 peptide on CD47 gene promoter activity. (a) A graph showing that GV1001 peptide (25, 50, 100 μg / mL) exhibits a concentration-dependent inhibitory effect on CD47 promoter activity in human head and neck cancer cells (SCC17B). (b) A graph showing the relative percentage (%) of the effect on CD47 promoter activity when GV1001 peptide (100 μg / mL), IL-1β (20 ng / mL), and TNF-α (100 ng / mL) were treated alone or in combination. Statistical analysis was performed using one-way analysis of variance (ANOVA). ns = no significant difference; * P<0.05; ** P<0.01; **** P<0.0001.

[0030] Figure 2 shows the inhibitory effect of the GV1001 peptide on CD47 expression in atherosclerotic plaques induced by Pg (Porphyromonas gingivalis). (a) Representative image confirming CD47 protein in mouse aorta by immunofluorescence staining. Red: CD47, Green: α-SMA as a smooth muscle cell marker, Blue: Nucleus stained with DAPI, L: Lumen, Scale bar: 100 μm. (b) Graph quantifying the CD47 staining area via ImageJ analysis. ns: No significant difference; * P <0.05; *** P <0.001; ****P <0.0001.

[0031] Figure 3 shows the inhibitory effect of the GV1001 peptide on CD47 expression. (a) Representative image of immunofluorescence staining of CD47 expression with and without GV1001 peptide treatment in Human coronary artery smooth muscle cells (HCASMCs) exposed to TNF-α and PgLPS. Red: CD47, Blue: Nuclei stained with DAPI, Scale bar: 50 μm. (b) Graph quantifying the degree of CD47 staining. (c) Western blot analysis results of CD47 expression with and without GV1001 peptide treatment in HCASMCs treated with TNF-α or PgLPS. (d) Graph quantifying CD47 protein levels based on Western blot analysis results. (e) RT-qPCR analysis results of CD47 mRNA expression levels with and without GV1001 peptide treatment in HCASMCs treated with TNF-α or PgLPS. ns: No significant difference; *P<0.05, **P<0.01, ***P<0.001.

[0032] Figure 4 shows the concentration- and time-dependent (specific) inhibition of CD47 expression by GV1001 peptide in various cancer cells. (a) Results of Western blot analysis of CD47 expression after treatment of the human head and neck cancer cell line FaDu with GV1001 peptide (5, 10, 50, or 100 μg / mL). (b) Results of Western blot analysis of CD47 expression after treatment of the human head and neck cancer cell line SCC1 with GV1001 peptide (10, 20, 50, or 100 μg / mL). (c) Results of Western blot analysis of CD47 expression after treatment of the human head and neck cancer cell line SCC9 with GV1001 peptide (10, 20, or 50 μg / mL). (d) Results of Western blot analysis of CD47 expression in human head and neck cancer cell line SCC17B treated with GV1001 peptide (10, 20, 50, or 100 μg / mL). (e) Results of Western blot analysis of CD47 expression in mouse head and neck cancer cell line MOC1 treated with GV1001 peptide (5, 10, 20, 30, 40, 50, or 100 μg / mL). (f) Results of Western blot analysis of CD47 expression in human head and neck cancer cell line FaDu treated with GV1001 peptide (100 μg / mL) at 0, 1, 3, 6, 12, 24, and 48 hours. (g) Results of Western blot analysis of CD47 expression after treating human head and neck cancer cell line SCC1 with GV1001 peptide (100 μg / mL) at 0, 1, 3, 6, 12, 24, and 48 hours. (h) Results of Western blot analysis of CD47 expression after treating human head and neck cancer cell line SCC9 with GV1001 peptide (100 μg / mL) at 0, 3, 6, 12, 24, and 48 hours.

[0033] Figure 5 shows the effect of the GV1001 peptide on the phagocytosis of macrophages in normal cells not expressing CD47 and cancer cells expressing CD47. (a) Results of Western blot analysis of CD47 expression in normal human oral keratinocytes (NHOK) treated with GV1001 peptide at concentrations of 0, 50, 100, and 200 μg / mL. (b) Graph showing the number of cells eliminated by macrophage phagocytosis. (c) Results of Western blot analysis of CD47 expression in mouse head and neck cancer cell lines (SCC4) treated with GV1001 peptide at concentrations of 0, 50, 100, and 200 μg / mL. (d) Graph showing the number of cells eliminated by macrophage phagocytosis.

[0034] Figure 6 shows that the phagocytosis of macrophages against human head and neck cancer cell line SCC1 increases in a time-dependent (specific) manner with treatment with GV1001 peptide.

[0035] Figure 7 shows that the phagocytosis of macrophages against mouse head and neck cancer cell line MOC1 increases in a time-dependent (specific) manner with treatment with GV1001 peptide.

[0036] Figure 8 shows that the phagocytosis of macrophages against the human head and neck cancer cell line SCC17B increases in a time-dependent (specific) manner with treatment with GV1001 peptide.

[0037] Figure 9 shows that the phagocytosis of macrophages against the human head and neck cancer cell line FaDu increases in a time-dependent (specific) manner with treatment with GV1001 peptide.

[0038] Figure 10 shows that the GV1001 peptide increases the phagocytosis of macrophages against SCC17B cells to a similar level compared to CD47 mAb.

[0039] Figure 11 shows the results of comparing the degree of phagocytosis after knocking down the CD47 gene by transfecting SCC17B cells, which are cancer cells with high CD47 expression levels, with siCD47.

[0040] Figure 12 shows the results of comparing the degree of phagocytosis after knocking down the CD47 gene by transfecting FaDu cells, which are cancer cells with high CD47 expression levels, with siCD47.

[0041] Figure 13 shows that the GV1001 peptide reduces the expression of inflammatory cytokines and tumor growth factors, as well as CD47, in cancer cells. (ad) Graph showing the effect of GV1001 peptide (100 μg / mL) on the expression of CD47, TNF-α, IL-1β, and TGF-β1 in normal mouse fibroblasts (NMF) and mouse head and neck cancer cells (MOC1). (eg) Graph showing the effect of GV1001 peptide (100 μg / mL) on the expression of CD47, TNF-α, and IL-1β in normal human fibroblasts (NHF) and human head and neck cancer cells (SCC17B). Statistical analysis was performed by one-way analysis of variance (ANOVA). ns = no significant difference; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

[0042] Figure 14 shows the inhibitory effect of GV1001 peptide on c-Myc and Kif4 expression in MOC1 cells. (ac) Results of RT-qPCR analysis confirming the effects of GV1001 peptide (100 μg / mL) on the gene expression of CD47, c-Myc, and Kif4 in mouse head and neck cancer cells (MOC1) treated for 12 and 48 hours, respectively. Statistical analysis was performed using one-way analysis of variance (ANOVA). ns = no significant difference; * P<0.05; ** P<0.01; **** P<0.0001.

[0043] Figure 15 shows that the GV1001 peptide enhances anticancer activity by inhibiting the expression of TNF-α, p-ERK1 / 2, p-p65, p-p38, and ENO-1 proteins, as well as CD47, in cancer cells. (a) Western blot analysis was performed to confirm the effects of GV1001 peptide (100 μg / mL) on the protein levels of CD47, TNF-α, p-ERK1 / 2, p-p65, p-p38, and ENO-1 after treating normal mouse fibroblasts (NMF) and mouse head and neck cancer cells (MOC1) for 12 or 48 hours. (b) Western blot analysis of the effects of treating normal human fibroblasts (NHF) and human head and neck cancer cells (SCC17B) with GV1001 peptide (100 μg / mL) for 12 or 48 hours on the protein levels of CD47, TNF-α, p-ERK1 / 2, p-p65, p-p38, and ENO-1.

[0044] Figure 16 shows the inhibitory effect of GV1001 peptide on HIF-1α and HIF-1β expression. (ab) Results of RT-qPCR analysis confirming the effect of GV1001 peptide (100 μg / mL) on the gene expression of HIF-1α and HIF-1β in mouse head and neck cancer cells (MOC1) treated for 12 and 48 hours, respectively. Statistical analysis was performed using one-way analysis of variance (ANOVA). ns = no significant difference; * P<0.05; *** P<0.001; **** P<0.0001.

[0045]

[0046] The present invention will be described in detail below.

[0047] In one embodiment, the present invention relates to a pharmaceutical composition for use in preventing or treating cancer and / or related symptoms associated with the expression of CD47, comprising as an active ingredient a peptide having the amino acid sequence represented by SEQ ID NO. 1.

[0048] The peptide having the amino acid sequence represented by SEQ ID NO. 1 of the present invention basically refers to a peptide composed of 16 amino acids (EARPALLTSRLRFIPK; SEQ ID NO. 1; 'GV1001 peptide'), but also includes structural or functional equivalents thereof.

[0049] As used herein, the term "structural or functional equivalent" means a peptide that is completely identical to or has a certain sequence identity with respect to the amino acid sequence of the peptide represented by SEQ ID NO. 1 and exhibits substantially the same physiological activity. As used herein, the term "certain sequence identity" means having an identity of about 80.0% to about 99.9%, preferably about 85.0% to about 99.8%, more preferably about 90.0% to about 99.7%, and most preferably about 95.0% to about 99.6% with respect to the amino acid sequence represented by SEQ ID NO. 1. As used herein, the term "similar physiological activity" means exhibiting at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, or more of the physiological activity based on the physiological activity of the GV1001 peptide. Additionally, "structural or functional equivalents" include amino acid sequence variants in which some of the amino acids constituting the natural (wild) peptide are substituted, or some amino acids are deleted or added. The substitution of amino acids is preferably a conservative substitution. Examples of naturally occurring conservative substitutions of amino acids are as follows: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn), and sulfur-containing amino acids (Cys, Met).

[0050] In one embodiment, a structural or functional equivalent of the GV1001 peptide of the present invention may consist of or include the amino acid sequence represented by SEQ ID NO. 1.

[0051] In one embodiment, 1 to 20, preferably 1 to 10, more preferably 1 to 5 amino acids may be additionally added to the N-terminus and / or C-terminus of the GV1001 peptide of the present invention, or an acyl group, an amine group, etc. may be introduced to the side chains of some amino acids constituting the GV1001 peptide, the N-terminus and / or C-terminus of the peptide.

[0052] The GV1001 peptide of the present invention or its structural or functional equivalents can be prepared by methods known to those skilled in the art. Such peptides can be produced in prokaryotic or eukaryotic cells by expressing a polynucleotide encoding the peptide sequence of the present invention as part of a larger polypeptide. Alternatively, the GV1001 peptide of the present invention or its structural or functional equivalents can be synthesized by chemical methods. Methods for the expression of heterologous proteins in a recombinant host, chemical synthesis of polypeptides, and in vitro transcription are well known in the field and are described in detail in the literature (Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed, Cold Sprin Harbor, NY; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc, San Diego, Calif; Merrifield, J (1969) J Am Chem Soc 91:501; Chaiken IM (1981) CRC Crit Rev Biochem 11: 255; Kaiser et al (1989) Ann Rev Biochem 57:957; and Offord, RE (1980) Semisynthetic Proteins, Wiley Publishing).

[0053] The GV1001 peptide of the present invention inhibits the activity of the CD47 promoter in a concentration-dependent manner, and effectively inhibits the activity of the CD47 promoter, particularly in a cancer cell environment (Example and Experimental Example 1: FIG. 1a-1b).

[0054] The GV1001 peptide of the present invention reduces CD47, which is present at high levels in atherosclerotic plaques (benign cancerous lesions) induced by Pg (Porphyromonas gingivalis) (Example and Experimental Example 2; Fig. 2a-b).

[0055] The GV1001 peptide of the present invention inhibits the expression of CD47 in human coronary smooth muscle cells (HCASMC) (Example and Experimental Example 3; Figs. 3a-e).

[0056] The GV1001 peptide of the present invention reduces CD47 levels in human and mouse head and neck cancer cells in a dose-dependent and time-dependent / specific manner (Examples and Experimental Examples 4; Figs. 4a-h).

[0057] The GV1001 peptide of the present invention does not alter the phagocytosis of macrophages against cells that do not express CD47 (Examples and Experimental Examples 5; NHOK cells: Figs. 5a-b; SCC-4 cells: Figs. 5c-d).

[0058] The GV1001 peptide of the present invention increases the phagocytosis of macrophages against SSC1 head and neck cancer cell lines in a time-dependent and specific manner (Example and Experimental Example 6; Fig. 6).

[0059] The GV1001 peptide of the present invention increases the phagocytosis of macrophages against MOC1 head and neck cancer cell lines in a time-dependent and specific manner (Example and Experimental Example 6; Fig. 7).

[0060] The GV1001 peptide of the present invention increases the phagocytosis of macrophages against SCC17B head and neck cancer cell lines in a time-dependent and specific manner (Example and Experimental Example 6; Fig. 8).

[0061] The GV1001 peptide of the present invention increases the phagocytosis of macrophages against FaDu head and neck cancer cell lines in a time-dependent and specific manner (Example and Experimental Example 6; Fig. 9).

[0062] The GV1001 peptide of the present invention increases the phagocytosis of macrophages against SCC17B cells to a level similar to that of CD47 mAb (Example and Experimental Example 7; Fig. 10).

[0063] The GV1001 peptide of the present invention increases the phagocytosis of macrophages by inhibiting the expression of CD47 (Examples and Experimental Examples 8; SSC17B: Fig. 11a-b; FaDu: Fig. 12).

[0064] The GV1001 peptide of the present invention significantly reduces the expression of CD47 as well as inflammatory cytokines (TNF-α, IL-1β) and tumor growth factor (TGF-β1) in cancer cells (Examples and Experimental Examples 9; Figs. 13a-g).

[0065] The GV1001 peptide of the present invention inhibits the expression of factors (c-Myc and Kif4) associated with the transcriptional regulation of the CD47 gene in cancer cells (Example and Experimental Example 10; FIG. 14a-c).

[0066] The GV1001 peptide of the present invention inhibits the expression of CD47 as well as TNF-α, p-ERK1 / 2, p-p65, p-p38, and ENO-1 proteins in cancer cells (Example and Experimental Example 11; Fig. 15a-b).

[0067] The GV1001 peptide of the present invention blocks the transcription of the CD47 gene by inhibiting the expression of hypoxia-inducible factors (HIF-1α and HIF-1β) in cancer cells in a hypoxic environment (Example and Experimental Example 12: Fig. 16a-b).

[0068] The GV1001 peptide of the present invention is composed of a total of 16 amino acids and has a relatively short sequence, so it can easily cross tissue barriers in the body and has excellent cell permeability. It has a small molecular weight, so it can be easily synthesized, making mass production easy and relatively low production costs.

[0069] The GV1001 peptide of the present invention does not act as an immunogen itself in the body and does not substantially exhibit toxicity, so it can be safely administered to or ingested by subjects for a long period of time.

[0070] As used herein, the term “CD47 is (highly) expressed” means that the expression level of CD47 in cancer cells is relatively higher than the expression level of CD47 in normal cells, and means that due to the (high) expression of CD47, the phagocytosis of macrophages, which are immune cells, is suppressed and immune evasion is induced, thereby promoting the activation, growth, and metastasis of cancer (cells). In this specification, “CD47 is (highly) expressed” may be used interchangeably with “CD47 is (overexpressed).”

[0071] In general, representative examples of “cancers associated with CD47 expression” include blood cancers such as non-Hodgkin’s lymphoma (NHL), lymphoblastic lymphoma, lymphoblastic leukemia, acute lymphoblastic leukemia (AML), and multiple myeloma (MM); solid tumors such as liver cancer, small cell lung cancer (SCLC), breast cancer, and colorectal cancer; and other cancers such as kidney cancer and head and neck cancer.

[0072] In one embodiment, the cancer associated with the expression of CD47 according to the present invention is any one selected from the group consisting of head and neck cancer, gastric cancer, and colorectal cancer. Preferably, the cancer associated with the expression of CD47 according to the present invention is a head and neck cancer including oral cancer, nasopharyngeal cancer, laryngeal cancer, salivary gland cancer, etc.

[0073] As used herein, the term “cancer and / or related symptoms associated with CD47 expression” refers to various symptoms and side effects that occur during the course of cancer treatment (surgery, chemotherapy, radiation therapy) or the cancer itself in which CD47 is (highly) expressed. “Cancer and / or related symptoms associated with CD47 expression” refers to various symptoms such as lumps or masses, pain (e.g., localized pain such as chest pain, generalized pain, pain of unknown cause), persistent cough, voice changes, bleeding (including hematuria and bloody stools), weight loss, changes in urine (including volume (polyuria, oliguria), color (hematuria, proteinuria), frequency and number of times (polyuria, nocturia), odor, sensation before / after urination (urgency, residual urine sensation)), changes in bowel movements (including frequency and number of times, shape and texture, and color), chronic fatigue, heartburn, loss of appetite, nausea, vomiting, stomatitis, etc.

[0074] As used herein, the term "prevention" refers to any act of inhibiting, suppressing, delaying, or preventing the occurrence, spread, exacerbation, and recurrence of cancer and / or related symptoms associated with CD47 expression by administering a pharmaceutical composition according to the present invention.

[0075] As used herein, the term "treatment" means any act of slowing (similarly expressed as delaying; retarding), reducing (similarly expressed as decreasing; mitigating), suppressing (similarly expressed as inhibiting), blocking (similarly expressed as preventing), ameliorating (similarly expressed as alleviating), improving, reversing, or curing the cancer and / or related symptoms associated with CD47 expression and their exacerbation (development) in a subject who has developed or is at risk of developing such cancer and / or related symptoms by the administration of the pharmaceutical composition, or any act of altering the subject's overall health status, including the cancer and / or related symptoms, in a manner favorable to the subject's survival.

[0076] In this specification, with respect to active ingredients and / or pharmaceutical compositions, the terms “use” or “administer” mean any act of introducing the GV1001 peptide of the present invention or a pharmaceutical composition containing it as an active ingredient into the body of a subject by administering (taking), injecting (injecting), or inhaling through appropriate means (e.g., syringe, inhaler).

[0077] The pharmaceutical composition of the present invention may be provided in a formulation comprising GV1001 peptide or an equivalent thereof as an active ingredient, optionally comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0078] The pharmaceutical composition of the present invention may contain GV1001 peptide or an equivalent thereof in an amount of about 30 μg / mg to about 10 mg / mg, about 40 μg / mg to about 9 mg / mg, about 50 μg / mg to about 8 mg / mg, about 60 μg / mg to about 7 mg / mg, or about 70 μg / mg to about 6 mg / mg, about 80 μg / mg to about 5 mg / mg, about 90 μg / mg to about 4 mg / mg, or about 100 μg / mg to about 3 mg / mg, based on the total weight of the pharmaceutical composition. The pharmaceutical composition of the present invention may contain GV1001 peptide or an equivalent thereof in an amount of about 10 µg / ml to about 100 mg / ml, about 20 µg / ml to about 95 mg / ml, about 30 µg / ml to about 90 mg / ml, about 40 µg / ml to about 80 mg / ml, or about 50 µg / ml to about 70 mg / ml, based on the total volume of the pharmaceutical composition. When included within the above ranges, it is not only appropriate to produce the intended effect, but also satisfies both the stability and safety of the composition, and it may also be appropriate to include it within the above ranges in terms of cost-effectiveness.

[0079] As used herein, the term "pharmaceuticalally acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. Pharmaceutically acceptable carriers, excipients, or diluents may be used without particular limitation if they are known and commonly used in the pharmaceutical field as buffers, preservatives, analgesics, solubilizers, isotonics, stabilizers, excipients, lubricants, etc.

[0080] Examples of carriers, excipients, or diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc.

[0081] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects, and may be used interchangeably with the terms "effective dose" or "effective dosage." The effective dose or effective dosage may be determined based on factors well known in the medical field, including the subject's health status, the type and / or severity of the disease, the activity of the drug, sensitivity to the drug, the method of administration, the time of administration, the route of administration, the elimination rate, the duration of treatment, and drugs used in combination or concomitantly.

[0082] Furthermore, the pharmaceutical composition of the present invention may be administered as a single agent or in combination with other anticancer agents / therapeutic agents, administered sequentially or simultaneously with conventional anticancer agents / therapeutic agents, and administered as a single or multiple doses. A person skilled in the art can determine, without particular difficulty, an dosage that obtains maximum effect with a minimum amount without side effects by considering all of the above-mentioned factors.

[0083] In one embodiment, the effective dosage of the pharmaceutical composition of the present invention may be, for example, 30 μg / kg / day to 5 mg / kg / day, 35 μg / kg / day to 4.5 mg / kg / day, 40 μg / kg / day to 4.0 mg / kg / day, 45 μg / kg / day to 3.5 mg / kg / day, or 50 μg / kg / day to 3.0 mg / kg / day, based on the amount of active ingredient to be administered to a subject, but is not limited thereto, and the effective dosage may vary depending on the patient's weight, age, disease state, etc. In one embodiment, the pharmaceutical composition of the present invention may be administered once to three times a day, but is not limited thereto, and the number of administrations may vary depending on the patient's weight, age, disease state, etc.

[0084] As used herein, the term "time-specific" means that the desired anticancer efficacy is exhibited at a certain level or higher or at a maximum level at a specific time after administration of the GV1001 peptide of the present invention. The term "exhibits anticancer efficacy at a certain level or higher" means that the expression level of CD47 in cancer cells in which CD47 is (highly) expressed is suppressed by about 10% to a maximum of about 100%, thereby returning it to the expression level of normal cells and / or inactivating the cancer cells, increasing the phagocytosis of macrophages against the cancer cells, and ultimately killing the cancer cells.

[0085] In one embodiment, the GV1001 peptide of the present invention can significantly reduce the expression of CD47 in cancer cells within about 50 hours, about 48 hours, about 46 hours, about 44 hours, about 42 hours, about 40 hours, about 38 hours, about 36 hours, about 34 hours, about 32 hours, about 30 hours, about 28 hours, about 26 hours, about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, and about 2 hours after administration to a subject. Specifically, the GV1001 peptide of the present invention significantly reduces the expression of CD47 in cancer cells within 8 to 48 hours after administration to a subject. More specifically, the GV1001 peptide of the present invention significantly reduces the expression of CD47 in cancer cells within 10 to 40 hours after administration to a subject. Even more specifically, the GV1001 peptide of the present invention significantly reduces the expression of CD47 in cancer cells within 12 to 36 hours after administration to a subject.

[0086] In one embodiment, considering the point in time when the anticancer activity of the GV1001 peptide of the present invention is maximized or maximized, it may be preferable to have a drug-free period of at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, at least about 32 hours, at least about 34 hours, at least about 36 hours, at least about 38 hours, at least about 40 hours, at least about 42 hours, at least about 44 hours, at least about 46 hours, at least about 48 hours, at least about 0.5 days, at least about 1 day, at least about 2 days after receiving an effective dose, but is not limited thereto, and the drug-free period may vary depending on the patient's weight, age, disease state, compliance, etc.

[0087] As used herein, the term “subject” means all animals including primates such as humans, monkeys, orangutans, etc., for whom prevention or treatment of cancer and / or related symptoms associated with CD47 expression is required, or for whom such cancer and / or related symptoms have occurred or are at risk of occurring; experimental animals such as mice, rats, hamsters, gerbils, guinea pigs, rabbits, dogs, pigs, or monkeys, etc.; pets such as cats, dogs, etc.; and domesticated animals such as cattle, horses, camels, deer, goats, sheep, pigs, chickens, turkeys, quail, etc.

[0088] In one embodiment, the subject may be a human patient suffering from cancer and / or related symptoms associated with the expression of CD47, or at risk of developing cancer.

[0089] In one embodiment, the subject may be a human patient who is currently receiving, has received, or is scheduled to receive anticancer chemotherapy(agent) due to cancer and / or related symptoms associated with CD47 expression.

[0090] The route of administration of the pharmaceutical composition of the present invention may be oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal, but the route of administration may be appropriately selected as needed.

[0091] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0092] When the pharmaceutical composition of the present invention is administered parenterally, it may be administered by external application to the skin or by injection (infusion) methods such as intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or thoracic injection, but the method of administration may be appropriately selected as needed.

[0093] The pharmaceutical composition of the present invention may be formulated into a solid dosage form for oral administration, such as a tablet, pill, powder, granule, or capsule.

[0094] The pharmaceutical composition of the present invention may be formulated into a liquid preparation for oral administration, such as a suspension, liquid formulation, emulsion, or syrup.

[0095] The pharmaceutical composition of the present invention may be formulated as a preparation for parenteral administration, such as a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository.

[0096] In one embodiment, the present invention relates to a health functional food for use in preventing or improving cancer and / or related symptoms associated with the expression of CD47, comprising as an active ingredient a peptide having an amino acid sequence represented by SEQ ID NO. 1.

[0097] As used in this specification, the terms “improvement,” and similar terms such as “relief,” “alleviation,” “improvement,” etc. refer to any action in which cancer and / or related symptoms associated with CD47 expression are improved or the health condition of the subject is beneficially altered by the intake of the GV1001 peptide or equivalent thereof according to the present invention, or a health functional food containing it.

[0098] The health functional food of the present invention refers to a food manufactured and / or processed in various forms to prevent or improve cancer and / or related symptoms, which have functional benefits useful to a subject.

[0099] The health functional food of the present invention encompasses not only food in the ordinary sense but also functional food. Accordingly, the term "health functional food" as used herein may be used interchangeably with health functional food composition or functional food composition.

[0100] There are no specific restrictions on the types of food that may include the health functional food of the present invention, but for example, the health functional food of the present invention may be included in meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc.

[0101] The health functional food of the present invention includes all forms such as functional food, nutritional supplement, health food, and food additives, and these types of food can be manufactured in various forms according to conventional methods known in the art. For example, as a health food, it may be manufactured in the form of a liquid drink for consumption, or consumed by granulation, encapsulation, spherical tablet (pill, etc.), and powdering, or may be manufactured in the form of powder, capsule, soft capsule, tablet, gum, or adhesive type liquid composition for consumption. In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruit, jarred fruit, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g., ham, sausage, corned beef, etc.), bread, noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, malt syrup, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable protein, retort foods, frozen foods, health-boosting snacks (nutritious snack, tonic snack, energy-replenishing snack, etc.), and various seasonings (e.g., soybean paste, soy sauce, sauces, etc.).

[0102] The health functional food of the present invention may further include ingredients that are ordinarily added during food manufacturing, provided that such addition does not fall outside the scope of the ultimate purpose of the present invention, for example, may further include proteins, carbohydrates, fats, other nutrients, seasonings, and flavorings.

[0103] The health functional food of the present invention may additionally contain various nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The health functional food of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination.

[0104] The content of the active ingredient in the health functional food of the present invention may be 0.01 to 50 w / w%, 0.1 to 45 w / w%, or 0.5 to 35 w / w% based on the total weight of the health functional food finally manufactured, although this is not limited thereto.

[0105] The content of the active ingredient in the health functional food of the present invention may be 0.01 to 50 w / v%, 0.1 to 45 w / v%, or 0.5 to 35 w / v% based on the total volume of the health functional food finally produced, although this is not limited thereto.

[0106] The effective intake amount of the health functional food of the present invention may be, for example, 1 μg / kg / day to 10 mg / kg / day, 10 μg / kg / day to 1 mg / kg / day, or 50 μg / kg / day to 100 μg / kg / day, but is not limited thereto and may vary depending on various factors such as the age, health condition, and complications of the subject.

[0107] In another aspect, the invention relates to a feed composition for use in preventing or improving cancer and / or related symptoms associated with the expression of CD47, comprising as an active ingredient a peptide having the amino acid sequence represented by SEQ ID NO. 1 or an equivalent thereof.

[0108] The feed composition may be prepared in a liquid form having a concentration of 10 to 90 w / v% of a peptide having the amino acid sequence represented by SEQ ID NO. 1 or an equivalent thereof; or in a powder or granule form having a weight of 10 to 90 w / w% of a peptide having the amino acid sequence represented by SEQ ID NO. 1 or an equivalent thereof. The feed composition may further include additives. Examples of additives include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acidic pyrophosphate, and polyphosphate (polymerized phosphate); and natural antioxidants such as polyphenols, catechins, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid, but are not limited thereto.

[0109] The feed composition may be formulated in the form of a conventional feed and may further include commonly used feed ingredients. The feed composition may further include grains, e.g., ground or crushed wheat, oats, barley, corn, rice, or mixtures thereof; plant proteins, e.g., proteins extracted from soybeans, sunflowers, etc.; animal proteins, e.g., blood meal, meat meal, bone meal, fish meal, or mixtures thereof; sugars; dairy products, etc. In addition, it may further include nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

[0110] The feed composition of the present invention may be fed to target animals alone or mixed with other feed additives using an edible carrier. Additionally, the feed composition of the present invention may be fed by spraying as a top dressing, mixed with animal feed, or administered separately from feed.

[0111] In another aspect, the present invention relates to a kit for use in preventing or treating cancer associated with the expression of CD47, comprising a composition including a peptide having an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient and instructions for use.

[0112] GV1001 peptide, composition (pharmaceutical composition, health functional food, feed composition), cancer associated with CD47 expression, etc. are identical to the definitions described above, so the description is omitted in the scope of overlap.

[0113] Instruction manual may be a means of providing detailed descriptions of one or more of the dosage, administration route, frequency of administration, and precautions of the composition according to the present invention, as well as information on the proper use and storage method of the kit.

[0114] In one embodiment, the instruction manual may be provided in a printed form using text, pictures, photographs, diagrams, illustrations, etc., or in an electronic (digitized) form using barcodes or holograms, etc.

[0115] Specific embodiments of the present invention are as follows:

[0116] Example 1. A pharmaceutical composition for use in preventing or treating cancer caused by abnormal expression of CD47 (Cluster of Differentiation 47), comprising as an active ingredient a peptide having the amino acid sequence represented by SEQ ID NO. 1.

[0117] Embodiment 2. A pharmaceutical composition according to Embodiment 1, wherein the cancer associated with the expression of CD47 is selected from the group consisting of head and neck cancer, gastric cancer, and colorectal cancer.

[0118] Embodiment 3. The pharmaceutical composition of Embodiment 1, wherein the pharmaceutical composition further comprises one or more selected from the group consisting of pharmaceutically acceptable carriers, excipients, and diluents.

[0119] Example 4. A pharmaceutical composition according to Example 1, wherein the peptide exhibits anticancer activity by inhibiting the expression of CD47.

[0120] Embodiment 5. In Embodiment 4, the peptide is c-Myc, Kif4(Kr A pharmaceutical composition exhibiting anticancer activity by inhibiting the expression of one or more proteins selected from the group consisting of uppel-like factor 4), phosphorylated(p)-ERK (Extracellular signal-regulated kinase) 1 / 2, phosphorylated(p)-p65, phosphorylated(p)-p38, and ENO (alpha-enolase)-1.

[0121] Example 6. A pharmaceutical composition according to Example 4, wherein the peptide additionally exhibits anticancer activity through increased phagocytosis of macrophages.

[0122] Example 7. A pharmaceutical composition according to Example 4, wherein the peptide further exhibits anticancer activity by inhibiting the expression of inflammatory cytokines and tumor growth factors.

[0123] Example 8. A pharmaceutical composition according to Example 7, wherein the inflammatory cytokine is one or more selected from the group consisting of TNF-α and IL-1β; and the tumor growth factor is TGF-β1.

[0124] Example 9. A health functional food for use in preventing, delaying, or inhibiting the onset of cancer associated with CD47 expression, comprising as an active ingredient a peptide having the amino acid sequence represented by SEQ ID NO. 1.

[0125] Embodiment 10. A health functional food according to Embodiment 9, wherein the cancer associated with the expression of CD47 is selected from the group consisting of head and neck cancer, gastric cancer, and colorectal cancer.

[0126] Example 11. A feed composition for use in preventing, delaying, or inhibiting the onset of cancer associated with CD47 expression, comprising as an active ingredient a peptide having the amino acid sequence represented by SEQ ID NO. 1.

[0127] Example 12. A feed composition according to Example 11, wherein the cancer associated with the expression of CD47 is selected from the group consisting of head and neck cancer, gastric cancer, and colorectal cancer.

[0128] Embodiment 13. A kit for use in preventing or treating cancer associated with the expression of CD47, comprising: a composition according to any one of Embodiments 1 to 8; and instructions for use.

[0129] Example 14. Use of a peptide having the amino acid sequence represented by SEQ ID NO. 1 in the preparation of a composition, e.g., a pharmaceutical composition, for use in preventing or treating cancer associated with the expression of CD47; or a composition, e.g., a health functional food or feed composition, for use in preventing, delaying, or inhibiting the onset of cancer associated with the expression of CD47.

[0130] Example 15. A method for preventing, treating, or improving cancer associated with CD47 expression in a subject, comprising the step of administering a composition containing a peptide having the amino acid sequence represented by SEQ ID NO. 1 as an active ingredient to a subject in need thereof.

[0131] The terms used in this specification are intended solely for the purpose of describing specific embodiments and are not intended to limit the invention. Terms in which a number is omitted before a noun are not intended to limit the quantity, but rather to indicate that there is one or more objects referred to by the mentioned noun. The terms "comprising," "having," and "containing" are interpreted as open terms.

[0132] In this specification, numeric ranges are merely a convenient alternative to individually referring to each number within the range, and unless otherwise specified, each number should be considered as if it were individually referred to. The end values ​​of all numeric ranges are included within the range and can be combined independently.

[0133] In this specification, the term "approximately" used to describe length, area, volume, time (period), concentration, content, etc., means that there is a tolerance of up to ±20%, and generally ±10%, for the corresponding numerical value or numerical range.

[0134] All methods mentioned herein may be performed in an appropriate order unless otherwise specified or clearly contradictory by the context. The use of any one or all embodiments or exemplary language (e.g., “like”) is merely to better describe the invention, unless otherwise included in the claims, and is not intended to limit the scope of the invention. No language in the specification shall be interpreted as implying that any unclaimed component is essential to the practice of the invention. Unless otherwise defined, technical and scientific terms used herein have the same meaning as generally understood by a person of ordinary skill in the art to which the invention pertains (i.e., a person skilled in the art).

[0135] Preferred embodiment(s) of the present invention include the most optimal mode known to the inventor for carrying out the present invention. Modifications or variations of the preferred embodiment(s) may become apparent to a person skilled in the art upon reading the preceding description. The inventors expect that a person skilled in the art will make appropriate use of such modifications or variations, and expect the present invention to be practiced in a manner different from that described herein. Accordingly, the present invention includes all modifications and / or variations, including equivalents of the essence of the invention mentioned in the appended claims, as permitted by patent law. Furthermore, any combination of the aforementioned components within all possible modifications and / or variations is included within the scope of the present invention unless otherwise specified herein or is clearly contradictory in the context. Although the present invention has been specifically shown and described with reference to exemplary embodiment(s), a person skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the following claims.

[0136]

[0137] Hereinafter, the present invention will be described in detail based on examples and experimental examples to specifically explain the invention. However, the following examples and experimental examples are provided merely to facilitate a better understanding of the invention, and the content of the invention is not limited by the following examples and experimental examples.

[0138] [Preparation Example]

[0139] 1. Preparation of GV1001 Peptide

[0140] A peptide composed of 16 amino acids having the structural formula of Chemical Formula 1 below, represented by SEQ ID NO. 1 (GV1001) selected from human telomerase, was prepared.

[0141] <Chemical Formula 1>

[0142] Peptide GV1001 of SEQ ID NO. 1 was prepared according to a conventionally known solid-phase peptide synthesis method. Specifically, the peptides were synthesized by coupling amino acids one by one starting from the C-terminus via Fmoc solid-phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, South Korea). As follows, peptides with the first amino acid of the C-terminus attached to a resin were used. For example, as follows:

[0143] NH2-Lys(Boc)-2-chloro-Trityl Resin

[0144] NH2-Ala-2-chloro-Trityl Resin

[0145] NH2-Arg(Pbf)-2-chloro-Trityl Resin

[0146] All amino acid raw materials used for peptide synthesis were protected at the N-terminus with Fmoc, and the residues were protected with Trt, Boc, t-Bu(t-butylester), Pbf(2,2,4,6,7-pentamethyl dihydro-benzofuran-5-sulfonyl), etc., which are removed in acid. For example, as follows:

[0147] Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ahx-OH, Trt-Mercaptoacetic acid.

[0148] HBTU[2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminium hexafluorophosphate] / HOBt[N-Hydroxybenzotriazole] / NMM[4-Methylmorpholine] was used as the coupling reagent. Piperidine in 20% DMF was used for Fmoc removal. A cleavage cocktail [TFA(trifluoroacetic acid) / TIS(triisopropylsilane) / EDT(ethanedithiol) / H2O=92.5 / 2.5 / 2.5 / 2.5] was used to separate the synthesized peptide from the resin and remove the protecting group from the residue.

[0149] Each peptide was synthesized by utilizing a state in which starting amino acids with protecting groups were bound to a solid support, reacting the corresponding amino acids with the support, washing with a solvent, and repeating the deprotection process. The synthesized peptides were cleaved from the resin, purified by HPLC, confirmed to be synthesized by MS, and freeze-dried.

[0150] The specific manufacturing process of the GV1001 peptide is described as follows.

[0151] 1) Coupling step: An amino acid (8 equivalents) protected on NH2-Lys(Boc)-2-chloro-Trityl Resin and coupling reagents HBTU (8 equivalents) / HOBt (8 equivalents) / NMM (16 equivalents) were dissolved in DMF and added, reacted at room temperature for 2 hours, and washed in the order of DMF, MeOH, and DMF.

[0152] 2) Fmoc deprotection step: Piperidine in 20% DMF was added and reacted twice for 5 minutes at room temperature, and washed in the order of DMF, MeOH, and DMF.

[0153] 3) Basic backbone formation step: The reactions of steps 1) and 2) were repeated to form a peptide backbone.

[0154] 4) Cleavage / separation step: A cleavage cocktail was added to the synthesized peptide resin to separate the peptide from the resin.

[0155] 5) Precipitation step: Cooling diethyl ether was added to the obtained mixture, and the peptide obtained by centrifugation was precipitated.

[0156] 6) Purification / Verification / Drying Step: After purification by Prep-HPLC and confirmation of molecular weight by LC / MS, the peptide was finally obtained as a powder by freeze-drying.

[0157] 2. Preparation of a lentivirus vector expressing EGFP (Enhanced Green Fluorescent Protein)-Firefly luciferase

[0158] 293T cells (Human kidney cells, ATCC) were cultured in DMEM medium (containing 10% FBS) at 80–90% density, and then transfected with Lipofectamine using pLentipuro-EGFP-Firefly Luciferase plasmid (Addgene, Watertown, MA), lentiviral packaging plasmid psPAX2 (Watertown, MA), and pMD2.GVSV-G (VSV glycoprotein expression plasmid, Watertown, MA). Six hours after transfection, the medium was changed, and the cells were cultured for an additional 48 or 72 hours.

[0159] Cell residues were removed from the culture supernatant through centrifugation (1,000g, 10 min) and filtration (0.45 μm filter), and then the lentivirus was recovered through centrifugation (10,000g, 30 min).

[0160] 3. Preparation of cells infected with the manufactured lentivirus

[0161] Human head and neck cancer cell lines FaDu, SCC1, SCC9, and SCC17B, and mouse head and neck cancer cell line MOC1 were cultured in a 24-well plate at 60% density. Poloxamer 407 (100 μg / mL, Sigma Aldrich, Burlington, MA) and PGE2 (10 μM, Cayman Chemical, Ann Arbor MI) were added for 30 minutes to increase infection efficiency. Subsequently, 20 μL of lentivirus was added to infect the cells. Starting 48 hours after infection, non-infected cells were removed by adding puromycin (4 μg / mL), and subsequent experiments were conducted using cells that survived puromycin approximately one week later.

[0162] [method]

[0163] 1. Experimental Animals and Administration Method

[0164] 4-week-old male ApoE - / - C57BL / 6 mice (body weight 30g±2) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and acclimatized to the laboratory environment for one week. The experiment was conducted for four weeks in an animal housing room with an automatic setting of a temperature of 22 ± 2℃, humidity of 50 ± 5%, and a 12-hour light-dark cycle (light cycle: 07:00–19:00), during which food and water were provided freely. During the experiment, the health and behavior of the mice were monitored three times a week. The mice were divided into four groups.

[0165] · Group 1 (n=10): PBS (Phosphate-Buffered Saline) intraperitoneally (IP), administered 3 times a week for 10 weeks.

[0166] · Group 2 (n=10): GV1001 peptide (2.0 mg / kg; dissolved in PBS) administered intraperitoneally, 3 times a week for 10 weeks.

[0167] Group 3 (n=10): Pg intraperitoneally, administered 3 times a week for 10 weeks, and PBS, administered 3 times a week for 10 weeks.

[0168] · Group 4 (n=10): GV1001 peptide (2.0 mg / kg; dissolved in PBS) intraperitoneally, 3 times a week for 1 week, 10 weeks and Pg intraperitoneally, 3 times a week for 1 week, 10 weeks.

[0169] 2. Sample and Tissue Collection

[0170] Mice were sacrificed after completing PBS or GV1001 peptide injection under general anesthesia, and whole blood and whole arterial blood were collected for analysis as previously reported (Kim, SY; Kim, YJ; Kim, S.; Momeni, M.; Lee, A.; Treanor, A.; Kim, S.; Kim, RH; Park, NH GV1001 Inhibits the Severity of the Ligature-Induced Periodontitis and the Vascular Lipid Deposition Associated with the Periodontitis in Mice. Int. J. Mol. Sci. 2023, 24, 12566.). Whole blood was collected from mice via cardiac puncture under isoflurane anesthesia (VetOne, Boise, ID, USA). Then, mice were perfused through the left ventricle for 5 minutes with 4% paraformaldehyde (Millipore Sigma, Burlington, MA, USA) dissolved in phosphate-buffered saline (PBS). After perfusion, the entire aorta, dilated to the iliac bifurcation, was exposed and carefully incised from the surrounding tissue and preserved in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA).

[0171] 3. Frozen slices

[0172] Tissue samples (aorta) were embedded in freezing molds using Tissue-Tek OCT complex (Sakura Finetek, Torrence, CA, USA) and stored at -80°C until frozen tissue sections were prepared. The frozen tissue blocks were sectioned to a thickness of 10 μm at -20°C using a Cryostar NX70 cryo-system (Thermo Fisher Scientific, Waltham, MA, USA) according to the previously described procedure (Kim, S.; Bando, Y.; Chang, C.; Kwon, J.; Tarverti, B.; Kim, D.; Lee, SH; Ton-That, H.; Kim, R.; Nara, PL; et al. Topical application of Porphyromonas gingivalis into the gingival pocket in mice leads to chronic-active infection, periodontitis and systemic inflammation. Int. J. Mol. Med. 2022, 50, 5159.).

[0173] 4. Histology and Immunofluorescence (IF) Analysis

[0174] For immunofluorescence staining, frozen sections of cells or tissues (aortic roots) were fluorescence detected using primary antibodies, such as CD47 (Abcam, Cambridge, UK), TNF-α (Abcam, Cambridge, UK), p65 (Santa Cruz Biotechnology, Dallas, TX, USA), p-p65 (Cell Signaling, Danvers, MA, USA), p-ERK1 / 2 (Cell Signaling, Denver, MA, USA), and ENO-1 (Abcam, Cambridge, UK), followed by Alexa Fluor 488 or 546-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA). Sequentially, sections were mounted on slides using VECTASHIELD anti-fade mounting medium and DAPI (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence images were captured using a confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany). All experiments were performed according to the manufacturer's instructions.

[0175] 5. Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)

[0176] Total RNA was extracted from mouse tissue samples or cells (normal cells, cancer cells) and reverse transcribed, after which qPCR was performed. Total RNA was extracted from tissue (mouse aortic root) or cancer cells using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA), and reverse transcribed using the SuperScript® III Reverse Transcriptionase Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the following steps: 5 minutes at 65°C, 2 minutes at 25°C, and 1 hour at 45°C. Then, quantitative PCR (qPCR) was performed using PowerUp™ SYBR-Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as a loading control. Fold induction was calculated using the comparative ΔCq method and relative transcription levels (2 -△△Cq It was represented as ). The primer sequences used for RT-qPCR are shown in Table 1.

[0177] GeneForward primer 5'-3'Reverse primer 5'-3'mIL-1βCACAGCAGCACATCAACAAGGTGCTCATGTCCTCATCCTGmTNF-αTCAGGTTGCCTCTGTCTCAGGCTCTGTGAGGAAGGCTGTGmCD47GGTGGGAAACTACACTTGCGAAGCTCCTCGTAAGAACAGGCTGATCmTGF-β1AAGTGGATCCACCAGCCCAATGTGCGCTCCTGCAAGTGCAGmc-MycTCGCTGCTGTCCTCCGAGTCCGGTTTGCCTCTTCTCCACAGACmKif4TCTGTTTCAGGCTGCTTTCAGCCCTGAAATATTTGATTGGAGmHIF-1 αTCAAGTCAGCAACGTGGAAGTATCGAGGCTGTGTCGACTGm-HIF-1βCTTCCTTCGCTGACCATCAGTGCTGTTGGTGATGTTGTTGmGapdhAGCTTGTCATCAACGGGAAGTTTGATGTTAGTGGGGTCTCGhCD47TATCCTCGCTGTGGTTGGACTGTAGTCCAAGTAATTGTGCTAGAGChTNF-αCCTCTCTCTAATCAGCCCTCTGGAGGACCTGGGAGTAGATGAGhIL-1αTGTGACTGCCCAAGATGAAGCGTGAGTTTCCCAGAAGAAGAGhGapdhAGCCACATCGCTCAGACACGCCCAATACGACCAAATCC

[0178] 6. Measurement of CD47 Promoter Activity To measure CD47 promoter activity, a CD47-luciferase vector was constructed by cloning the corresponding promoter region from LightSwitch Promoter Reporter GoClone (RenSP, S710450; SwitchGear Genomics, Menlo Park, CA), which contains the CD47 promoter sequence, into a pGL3-based vector (Promega, Madison, WI). A reference vector (Promega) containing Renilla luciferase was used in combination for transduction efficiency and quantification.

[0179] CD47-luciferase vector (45 ng) and reference vector (5 ng) were transfected into cells (SCC17B) using Lipofectamine 2000 (Invitrogen), a positively charged lipid complex that acts to transport DNA, which is difficult to pass through the cell membrane, into the cell. After 48 hours had passed since transfection, the cells were treated with GV1001 peptide (100 μg / mL), IL-1β (20 ng / mL), IL-1β (20 ng / mL) + GV1001 peptide (100 μg / mL), TNF-α (100 ng / mL), or TNF-α (100 ng / mL) + GV1001 peptide (100 μg / mL) for 48 hours. After treatment with each substance, the luciferase activity of the cell lysates was measured using the Dual-Luciferase Reporter Assay System (Promega). CD47 promoter activity was quantified by calculating the Firefly / Renilla luciferase ratio.

[0180] 7. Analysis of in vitro phagocytosis

[0181] THP-1 cells, a human mononuclear cell line derived from patients with acute mononuclear leukemia, were used in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA) containing PMA (200 ng / mL, Sigma Aldrich) in a 24-well plate (5 x 10 4 After culturing for 24 hours in a cell / well, they were differentiated into macrophages. After differentiation induction, cancer cells (2 x 10⁻¹⁰) infected with lentivirus were cultured into the macrophages. 5 cell) was added.

[0182] Subsequently, APC-conjugated F4 / 80 antibody (Novus Biologicals, Carlsbad, CA) was added for macrophage labeling, and DAPI (Vector Laboratories, Burlingame, CA) was added for nuclear staining.

[0183] After taking images using a confocal microscope (LSM 700, Carl Zeiss, Oberkochen, Germany), quantitative analysis was performed using Image Pro Plus software.

[0184] 8. Western Blotting

[0185] Western blot was performed on whole-cell extracts (WCE) of cultured cells (normal or cancer cells). The WCEs were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). After fractionating the WCEs by SDS-PAGE, they were transferred to Immobilon®-P membranes (Millipore, Billerica, MA, USA). The membranes were sequentially incubated with primary and secondary antibodies, and then exposed to a chemiluminescent reagent (Bio-Rad, Hercules, CA, USA) for signal detection.

[0186] 9. Statistical Analysis

[0187] All graphs were created using GraphPad Prism Software, and statistical analysis results were calculated using GraphPad Prism 9 (GraphPad Software, Boston, MA, USA). One-way analysis of variance (ANOVA) with the Newman-Keuls test was used for multiple comparisons. A p-value less than 0.05 was considered significant. All in vitro results were verified through at least three independent experiments. Error bars represent the mean ± standard deviation.

[0188]

[0189] [Examples and Experimental Examples]

[0190] 1. The GV1001 peptide inhibits the activity of the CD47 promoter in a concentration-dependent manner and effectively inhibits the activity of the CD47 promoter, particularly in a cancer cell environment.

[0191] We evaluated whether the GV1001 peptide directly affects the promoter activity of the CD47 gene and further analyzed whether it controls CD47 expression induced by inflammatory cytokines.

[0192] To this end, a reporter vector containing a cloned CD47 promoter region was prepared and transduced into a human head and neck cancer cell line (SCC17B). Subsequently, after administering the GV1001 peptide alone or in combination with the inflammatory cytokines IL-1β (20 ng / mL) and TNF-α (100 ng / mL), CD47 promoter activity was measured using the Dual-Luciferase assay.

[0193] GV1001 peptide (25, 50, 100 μg / mL) not only inhibited CD47 promoter activity in SCC17B cells in a concentration-dependent manner but also significantly reduced the increase in CD47 promoter activity induced by IL-1β or TNF-α (Fig. 1a-b).

[0194] These results strongly suggest that some of the mechanisms by which the GV1001 peptide inhibits CD47 expression occur at the transcriptional stage of the CD47 gene. Furthermore, this implies that the GV1001 peptide can act through various pathways, such as directly binding to the CD47 promoter to inhibit its activity, or indirectly reducing promoter activity by inhibiting the expression of TNF-α and IL-1β.

[0195] 2. GV1001 peptide reduces CD47, which is present at high levels in atherosclerotic plaques (benign cancer) induced by Pg (Porphyromonas gingivalis).

[0196] CD47 is a representative anti-phagocytic protein that enables evasion of phagocytosis by macrophages and plays an important role in cell survival in the environment of atherosclerosis (a dangerous benign vascular cancer) and tumors.

[0197] In this study, we aimed to investigate the expression pattern of CD47 in mouse aortic root tissues with Pg-induced atherosclerotic plaques. Figure 2a shows the fluorescence staining results, in which CD47 levels were observed to be negligible in the control group (PBS+PBS) and the group treated with GV1001 peptide alone (2 mg / kg body weight). In the Pg-treated group, CD47 levels were significantly increased, whereas they were inhibited by GV1001 peptide treatment.

[0198] These results suggest that the GV1001 peptide effectively inhibits CD47 expression, thereby exhibiting a therapeutic effect by inhibiting the anti-phagocyte activity of cancers in which CD47 is expressed.

[0199] 3. GV1001 peptide inhibits the expression of CD47 induced by inflammatory mediators, TNF-α and PgLPS.

[0200] When human coronary artery smooth muscle cells (HCASMCs) were treated with TNF-α (100 ng / mL) or PgLPS (20 μg / mL), CD47 protein levels (Figs. 3a-d) and mRNA levels (Fig. 3e) significantly increased. On the other hand, when GV1001 peptide (100 μg / mL) was simultaneously treated under the same conditions, CD47 levels were significantly inhibited.

[0201] These results suggest that the GV1001 peptide effectively inhibits CD47 expression, which is increased due to inflammatory mediators abundant in cancer cells and atherosclerotic plaques of blood vessels, and inhibits the anti-phagocyte activity caused by CD47 expression.

[0202] 4. GV1001 peptide reduces CD47 levels in human and mouse head and neck cancer cells in a dose-dependent and time-dependent / specific manner.

[0203] CD47 is a "don't eat me" signaling protein that inhibits phagocytosis by the innate immune system and is overexpressed in cancer cells, participating in immune evasion. In this study, we aimed to investigate the effects of the GV1001 peptide on CD47 expression in human head and neck cancer cell lines FaDu, SCC1, SCC9, and SCC17B, and the mouse head and neck cancer cell line MOC1.

[0204] When human head and neck cancer cell lines FaDu, SCC1, SCC9, and SCC17B and mouse head and neck cancer cell line MOC1 were treated with various concentrations of GV1001 peptide (5, 10, 20, 30, 40, 50, or 100 μg / mL) for about 0.5 hours for 2 days, CD47 expression decreased in a concentration-dependent manner (Figs. 4a-4e).

[0205] Based on the concentration (100 μg / mL) of the GV1001 peptide that significantly inhibited CD47 expression in human head and neck cancer cell lines FaDu, SCC1, and SCC9, treatment was performed at 0, 1, 3, 6, 12, 24, and 48 hours. As a result, CD47 expression was generally reduced at all times, but was found to be significantly reduced particularly after 12 hours (Fig. 4f-4h).

[0206] These results suggest that the GV1001 peptide inhibits the expression of CD47 in various cancer cells in a concentration- and time-dependent, or more strictly time-specific, manner.

[0207] 5. GV1001 peptide does not alter the phagocytosis of macrophages against normal cells not expressing CD47 and cancer cells expressing CD47.

[0208] The effect of the GV1001 peptide on the phagocytosis of macrophages against cancer cells in normal human oral keratinocytes (NHOK) and mouse head and neck cancer cell lines (SCC4) that do not express CD47 was confirmed through an 'in vitro phagocytosis analysis'.

[0209] After treating with GV1001 peptide at concentrations of 0, 50, 100, and 200 μg / mL and checking CD47 expression after about 0.5 hours, it was found that GV1001 peptide did not have a significant effect on CD47 expression in both normal cells and cancer cells.

[0210] These results suggest that it does not alter the phagocytosis of macrophages against normal cells that do not express CD47 and cancer cells that express CD47.

[0211] 6. GV1001 peptide increases the phagocytosis of macrophages against various head and neck cancer cell lines in a time-dependent (specific) manner.

[0212] To verify whether the GV1001 peptide induces and promotes phagocytosis of macrophages in human and mouse-derived head and neck cancer cells, the results were analyzed at different time points of GV1001 treatment.

[0213] When GV1001 peptide (100 μg / mL) was treated to various head and neck cancer cell lines SCC1 (Fig. 6), MOC1 (Fig. 7), SCC17B (Fig. 8), and FaDu (Fig. 9), phagocytosis by macrophages increased in a time-dependent (specific) manner.

[0214] This suggests that the GV1001 peptide can induce and promote phagocytosis by macrophages and enhance tumor removal ability by inhibiting CD47 expression in various head and neck cancer cell lines in a time-dependent (specific) manner.

[0215] 7. GV1001 peptide increases macrophage phagocytosis of SCC17B cells to a level similar to CD47 mAb.

[0216] We determined whether the GV1001 peptide increased the phagocytosis of macrophages compared to CD47 mAb. After exposing SCC17B cells to GV1001 peptide (100 μg / mL) and CD47 mAb (0.2 μM, 1.0 μM) for 2 days, the number of SCC17B cells engulfed by macrophages was quantified through an 'in vitro phagocytosis assay'.

[0217] As a result, the GV1001 peptide was found to increase the phagocytosis of macrophages on SCC17B cells to a level similar to that of CD47 mAb (0.2 μM) (Fig. 10).

[0218] 8. GV1001 peptide increases macrophage phagocytosis by inhibiting CD47 expression.

[0219] We wanted to determine whether the mechanism by which the GV1001 peptide increases the phagocytosis of macrophages is induced through the inhibition of CD47 expression.

[0220] SCC17B cells (Fig. 11a-b) and FaDu cells (Fig. 12), which are cancer cells with high CD47 expression levels, were transfected with siCD47 to knock down the CD47 gene, and then the degree of phagocytosis was compared.

[0221] CD47 expression was significantly reduced in both the GV1001 peptide treatment group and the siCD47 treatment group, and phagocytosis by macrophages was significantly increased in both groups. Even when GV1001 and siCD47 were treated in combination, phagocytosis did not increase further and remained at a level similar to that of the single-treatment group. This suggests that the increase in macrophage phagocytosis induced by the GV1001 peptide is based on a mechanism involving the inhibition of CD47 expression.

[0222] 9. GV1001 peptide significantly reduces the expression of CD47, as well as inflammatory cytokines and tumor growth factors, in cancer cells.

[0223] RT-qPCR was performed to confirm whether the GV1001 peptide affects the expression of not only CD47 but also inflammatory cytokines and tumor necrosis factor in cancer cells.

[0224] MOC1 and SCC17B head and neck cancer cells were treated with GV1001 peptide (100 μg / mL), and the expression of CD47, TNF-α, IL-1β, and TGF-β1 was analyzed by qPCR (Fig. 13a-g).

[0225] As a result, the GV1001 peptide was found to significantly reduce the expression of CD47, TNF-α, IL-1β, and TGF-β1.

[0226] On the other hand, no significant changes were observed in normal mouse-derived fibroblasts (NMF) and human fibroblasts (NHF). This suggests that the action of the GV1001 peptide is specific to cancer cells.

[0227] 10. GV1001 peptide inhibits the expression of c-Myc and Kif4, factors involved in the transcriptional regulation of the CD47 gene in cancer cells.

[0228] c-Myc directly binds to the CD47 promoter to activate transcription, and Kif4 is a gene involved in CD47 expression and cell migration. We investigated the regulatory effects of GV1001 on the expression of c-Myc and Kif4, factors related to CD47 transcriptional regulation.

[0229] When MOC1 cells were treated with the GV1001 peptide, the expression of CD47, c-Myc, and Kif4 was all significantly inhibited in a time-dependent manner (Fig. 14a-c).

[0230] This result implies that the GV1001 peptide can utilize the inhibition mechanisms of c-Myc and Kif4 as additional pathways to indirectly regulate CD47 expression.

[0231] 11. GV1001 peptide enhances anticancer activity by inhibiting the expression of TNF-α, p-ERK1 / 2, p-p65, p-p38, and ENO-1 proteins, as well as CD47, in cancer cells.

[0232] To determine the effects of GV1001 peptide on various inflammatory mechanisms associated with CD47 expression in cancer cells, mouse head and neck cancer cells (MOC1) and human head and neck cancer cells (SCC17B) were treated with GV1001 peptide (100 μg / mL) for 12 or 48 hours, and protein levels were analyzed by Western blot.

[0233] In MOC1 cells, the levels of the inflammatory cytokine TNF-α and phosphorylated proteins p-ERK1 / 2, p-p65, and p-p38, which are involved in cell proliferation and inflammatory responses, were reduced in a time-dependent manner by the GV1001 peptide (Fig. 15a). In particular, the level of ENO-1, which is highly expressed in cancer cells, was significantly reduced by GV1001 treatment. Since ENO-1 is a glycolysis enzyme known to promote CD47 expression as well as cancer cell migration and invasion, this implies that GV1001 inhibits both metabolic and inflammation-related mechanisms.

[0234] In SCC17B cells as well, treatment with GV1001 peptide was shown to significantly inhibit the expression of CD47, as well as TNF-α, p-ERK1 / 2, and p-p65 (Fig. 15b).

[0235] On the other hand, no significant changes were observed in normal mouse fibroblasts (NMF) and normal human fibroblasts (NHF) (Fig. 15a-b). This suggests that the action of the GV1001 peptide is specific to cancer cells.

[0236] Consequently, it can be inferred that the GV1001 peptide acts on complex molecular mechanisms associated with CD47 expression to effectively reduce the immune evasion ability of cancer cells.

[0237] 12. GV1001 peptide blocks the transcription of the CD47 gene by inhibiting the expression of HIF-1α and HIF-1β in cancer cells under a hypoxic environment.

[0238] According to recent studies, HIF-1 has been reported to directly activate the transcription of CD47 in hypoxic breast cancer cells.

[0239] To verify whether the GV1001 peptide can regulate CD47 transcriptional activity induced by HIF-1, changes in HIF-1 mRNA expression induced by the GV1001 peptide were examined.

[0240] When MOC1 cells were treated with the GV1001 peptide, the levels of both HIF-1α and HIF-1β were significantly inhibited (Fig. 16a-b). These results suggest that the GV1001 peptide inhibits CD47 activity and is involved in the HIF-1 inhibition mechanism in addition to the promotion of macrophage phagocytosis and inflammation-related mechanisms.

[0241]

[0242] The peptide having the amino acid sequence of SEQ ID NO. 1 of the present invention has an excellent effect of inhibiting the expression of CD47 and can also inhibit the expression of cancer-related proteins such as c-Myc, Kif4, p-ERK (Extracellular signal-regulated kinase) 1 / 2, p-p65, p-p38, and ENO (alpha-enolase)-1, inflammatory cytokines, and tumor growth factors, as well as significantly increase the phagocytosis of macrophages. Therefore, it can be usefully employed to prevent, treat, or improve cancer and / or related symptoms associated with the expression of CD47, and is expected to have great utility value in anticancer drugs and related fields.

Claims

1. A pharmaceutical composition for use in preventing or treating cancer associated with the expression of CD47 (Cluster of Differentiation 47), comprising as an active ingredient a peptide having the amino acid sequence represented by SEQ ID NO.

1.

2. In Claim 1, A pharmaceutical composition in which the cancer associated with the expression of CD47 is selected from the group consisting of head and neck cancer, gastric cancer, and colorectal cancer.

3. In Claim 1, The above pharmaceutical composition further comprises one or more selected from the group consisting of pharmaceutically acceptable carriers, excipients, and diluents.

4. In Claim 1, A pharmaceutical composition in which the above peptide exhibits anticancer activity by inhibiting the expression of CD47.

5. In Claim 4, A pharmaceutical composition in which the above peptide exhibits anticancer activity by inhibiting the expression of one or more selected from the group consisting of c-Myc, Kif4, phosphorylated(p)-ERK (Extracellular signal-regulated kinase) 1 / 2, phosphorylated(p)-p65, phosphorylated(p)-p38, and ENO (alpha-enolase)-1.

6. In Claim 4, A pharmaceutical composition in which the above peptide additionally exhibits anticancer activity through increased phagocytosis of macrophages.

7. In Claim 4, A pharmaceutical composition in which the above peptide additionally exhibits anticancer activity by inhibiting the expression of inflammatory cytokines and tumor growth factors.

8. In Claim 7, The above inflammatory cytokines are one or more selected from the group consisting of TNF-α and IL-1β, and A pharmaceutical composition in which the tumor growth factor is TGF-β1.

9. A health functional food for use in preventing, delaying, or inhibiting the onset of cancer associated with CD47 expression, comprising as an active ingredient a peptide having the amino acid sequence represented by SEQ ID NO.

1.

10. In Claim 9, A health functional food in which the cancer associated with CD47 expression is selected from the group consisting of head and neck cancer, gastric cancer, and colorectal cancer.

11. A feed composition for use in preventing, delaying, or inhibiting the development of cancer associated with CD47 expression, comprising as an active ingredient a peptide having the amino acid sequence represented by SEQ ID NO.

1.

12. In Claim 11, A feed composition in which the cancer associated with CD47 expression is selected from the group consisting of head and neck cancer, gastric cancer, and colorectal cancer.

13. As a kit for use in preventing or treating cancer associated with CD47 expression, A composition according to any one of claims 1 to 8; and A kit including an instruction manual.

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