KR100941678B1 - Polypeptides or fusion proteins thereof having an inhibitory activity against metastasis of cancer cells - Google Patents

Abstract

The present invention provides a polypeptide or a fusion protein thereof having metastasis inhibitory activity of cancer cells derived from paired immunoglobulin-like receptor α or β (PILRα or PILRβ), which is a kind of transmembrane protein. The present invention also provides a polynucleotide encoding the polypeptide, a vector comprising the same, and a transformant transformed with the vector. In addition, the present invention provides a pharmaceutical composition for inhibiting metastasis of cancer cells comprising the polypeptide or a fusion protein thereof. The polypeptide of the present invention or the fusion protein thereof can inhibit cancer cell metastasis by inhibiting migration and extravasation of cancer cells in connective tissue.

Cancer metastasis, cancer cell migration

Description

Polypeptides or fusion proteins thereof having an inhibitory activity against metastasis of cancer cells

The present invention relates to a polypeptide having a metastasis inhibitory activity of cancer cells derived from PILRα or PILRβ, which is a kind of a transmembrane protein, or a fusion protein thereof. The present invention also relates to a polynucleotide encoding the polypeptide, a vector comprising the same, and a transformant transformed with the vector. The present invention also relates to a pharmaceutical composition for inhibiting metastasis of cancer cells comprising the polypeptide or a fusion protein thereof.

Cancer cells changed by carcinogens proliferate faster than normal cells, form tumor masses, penetrate surrounding tissues, and inhibit normal body function. By inducing angiogenesis, cancer cells are supplied with nutrients and oxygen, and cancer cells are metastasized by angiogenesis. Cancer cells not only grow indefinitely at specific sites, but also move away from existing growth sites and move to new sites to grow. This process is called metastasis. The metastasis process involves the steps of cancer cells turning into highly mobile mesenchymal cells, leaving the existing tissues, invading and moving surrounding connective tissues and capillaries, and moving out of blood vessels and out of blood vessels, within connective tissues. Migration and growth at new sites.

Regulation of cell surface expression and activation of adhesion molecules plays a very important role in the metastasis of tumor cells (Zetter, B. R. (1993). Adhesion molecules in tumor metastasis. Semin Cancer Biol. 4: 219). The process of metastasis of tumor cells is achieved through the regulation of cell surface expression and activity of cell adhesion materials, and understanding of cell adhesion molecules as well as substances that control their expression and function is essential for the full understanding of metastasis. to be. (Bailly, M., Yan, L., Whitesides, GM, Condeelis, JS, and Segall, JE (1998) .regulation of Protusion Shape and Adhesion to the sustratum during chemoacic esponses of mammalian carcinoma cells.Exp Cell Res. 241: 285; Frisch, SM, Vuori, K., Ruoslahti, E., and Chan-Hui., P. (1996) .Control of adhesion-dependent cell survival by focal adhesion kinase.J Cell Biol 134: 793; and Hannigan, GE, Leung-Hagesteijn, C., Fitz-Gibbon, L., Coppolino, MG, Radeva, G., Filmus, J., Bell, JC, and Dedhar, S. (1996) .Regulation of cell adhesion and anchorage- dependent growth by a new β1-integrin-linked protein kinase.Nature 379: 91).

By the present inventors changed the function of when the CD99 molecule is active, β ₁ integrin (β ₁ integrin), by inhibiting the cancer cells are attached to the extracellular matrix (extracellular matrix, ECM), discloses that the cancer metastasis may be involved in the process Suh JS., 2001. Control of invasiveness of human breast carcinoma cell line MCF-7 by CD99 molecule. Kangwon National University. In addition, the present inventors have disclosed that peptide fragments having a specific sequence derived from CD99 can effectively inhibit CD99, thereby inhibiting the growth and / or metastasis of cancer cells and also have angiogenesis and inflammation inhibitory activity. (Korean Patent Registration No. 10-0758006; Korean Patent Application No. 10-2006-0060890; Korean Patent Application No. 10-2006-0067440; International Patent Publication No. WO 2007/037601).

On the other hand, the PILR family, which is identified as a natural ligand of CD99, is composed of PILRα and PILRβ. (Mousseau, DD, D. Banville, D. L'Abbe, P. Bouchard, and S.-H. Shen. 2000. PILRα, a novel immunoreceptor tyrosine-based inhibitory motif-bearing protein, recruits SHP-1 upon tyrosine phosphorylation . and is paired with the truncated counterpart PILRβ J. Biol Chem 275:.. 4467; Fournier, N., L. Chalus, I. Durand, E. Garcia, JJ Pin, T. Churakova, S. Patel, C. Zlot ., D. Gorman, S. Zurawski, et al 2000. FDF03, a novel inhibitory receptor of the immunoglobulin superfamily, is expressed by human dendritic and myeloid cells J. Immunol 165:.. 1197). PILRα and PILRβ are type 1 transmembrane proteins that are composed of glycated extracellular, transmembrane and intracellular sites, whereas the extracellular sites of PILRα and PILRβ have very similar amino acid sequences, whereas the cytoplasmic sites are very Different. The cytoplasmic region of PILRα contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which are known to inhibit cell activity. In contrast, PILRβ, which is expressed in natural killer cells or dendritic cells, lacks ITIM, and when ligand is bound, it activates cells unlike PILRα (Shiratori I., Ogasawara K., Saito T., Lanier LL., Arase H. (2004) .Activation of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor.J Exp Med 199: 525).

The present inventors have conducted various studies to develop a ligand capable of inhibiting cancer metastasis by activating a CD99 molecule. As a result, a polypeptide having a specific sequence derived from CDLR, a natural ligand of PILRα or PILRβ, effectively activates CD99, thereby inducing cancer cells. It was found that the metastasis of can be suppressed.

Accordingly, an object of the present invention is to provide a polypeptide derived from PILRα or PILRβ, which has a metastasis inhibiting activity of cancer cells, or a fusion protein thereof.

In addition, an object of the present invention is to provide a polynucleotide encoding the polypeptide and a vector comprising the same.

In addition, an object of the present invention is to provide a transformant transformed host cells with the vector.

In addition, an object of the present invention is to provide a pharmaceutical composition for inhibiting metastasis of cancer cells comprising the polypeptide or a fusion protein thereof as an active ingredient, and comprising a pharmaceutically acceptable carrier.

According to one aspect of the invention, a polypeptide consisting of 7 to 200 amino acids derived from a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, comprising a peptide of 123-129 of SEQ ID NO: 1 or 2, cancer cells A polypeptide having a transfer inhibitory activity of is provided. The polypeptide may be selected from the group consisting of the polypeptides of SEQ ID NOs: 3 to 5 and 7 to 11.

According to another aspect of the present invention, there is provided a fusion protein of the polypeptide with a poly-His region or Fc region, wherein the polyhistidine region and the Fc region have amino acid sequences of SEQ ID NOs: 13 and 14, respectively. Can be.

According to another aspect of the invention, there is provided a polynucleotide encoding said polypeptide, said polynucleotide may be selected from the group consisting of the polynucleotides of SEQ ID NOs: 15-17 and 19-23.

According to another aspect of the invention, a vector comprising a polynucleotide encoding said polypeptide and a host cell with said vector, for example Escherichia genus) or Pichia genus strain is provided.

According to another aspect of the present invention, there is provided a pharmaceutical composition for inhibiting metastasis of cancer cells, comprising the polypeptide or fusion protein as an active ingredient and comprising a pharmaceutically acceptable carrier.

The polypeptide of the present invention or the fusion protein thereof can inhibit cancer cell metastasis by inhibiting migration and extravasation of cancer cells in connective tissue. Therefore, the polypeptide or a fusion protein thereof may be usefully used as a therapeutic agent for metastatic cancer.

We conducted various searches for ligands that can activate CD99 molecules. Considering that PILRα or PILRβ, which is a type of transmembrane protein, can act as a natural ligand of CD99 molecules, ligands of various lengths were prepared and searched from PILRα or PILRβ. Surprisingly, it has been found that polypeptides comprising specific sequences derived from PILRα or PILRβ have high affinity to the surface of cancer cells and inhibit their migration, thereby having activity as a metastatic cancer therapeutic agent.

The present invention is derived from a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, comprising 7 to 200, preferably 7 to 160, more preferably comprising the peptide of 123 to 129 of SEQ ID NO: 1 or 2 Polypeptides consisting of 7 to 151 amino acids include polypeptides having metastatic inhibitory activity of cancer cells. The polypeptide according to the present invention may be selected from the group consisting of the polypeptides of SEQ ID NOs: 3 to 5 and 7 to 11.

The invention also encompasses fusion proteins of the polypeptide with a poly-His region or Fc region. The polyhistidine (poly-His) region is one of the label peptides, can be used to separate and purify the polypeptide of the present invention by binding to histidine binding resin. Preferably, in the fusion protein of the present invention, the polyhistidine (poly-His) region may have an amino acid sequence of SEQ ID NO: 13. The Fc region can be used to increase the stability of the polypeptide in the blood. Preferably, in the fusion protein of the present invention, the Fc region may have an amino acid sequence of SEQ ID NO: 14.

The present invention includes a polynucleotide encoding the polypeptide. The polynucleotide can be prepared according to a conventional method from known nucleic acid sequences encoding PILRα or PILRβ. Preferably, the polynucleotide may have a nucleotide sequence of SEQ ID NOs: 15 to 17 and 19 to 23.

The invention also includes a vector comprising a polynucleotide encoding the polypeptide. As a vector usable as a cloning vector of the vector, various known vectors can be used. For example, pPICZα A, B, C (Invitrogen, USA) and the like can be used. In addition, the cloning vector may be a vector into which a DNA encoding a polyhistidine (poly-His) region is inserted, for example, a pET28a (+) vector (Novagene, USA) or a DNA encoding an Fc region (for example, CDNA) consisting of the nucleotide sequence of SEQ ID NO: 25 can be used in a conventional vector, for example, a pET28a (+) vector (Novagene, USA). The vector may be prepared by inserting the gene encoding the polypeptide into a cloning vector digested with an appropriate restriction enzyme by a conventional method. The vector according to the present invention may be directly contained in a gene therapy composition for the purpose of gene therapy, or may be used for the preparation of a transformant.

The present invention also includes a transformant transformed into a host cell with the vector. The host cell is not particularly limited as long as the polypeptide can be effectively expressed, and Escherichia genus strain (e.g., Escherichia coli )), Pichia genus strains (eg, X-33 Pichia; Invitrogen, USA) and the like can be used as preferred host cells.

The present invention includes a pharmaceutical composition for inhibiting metastasis of cancer cells, comprising the polypeptide or a fusion protein thereof as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention may include excipients such as lactose, corn starch, lubricants such as magnesium stearate, emulsifiers, suspending agents, buffers, tonicity agents, and the like which are well known, and may be in oral or parenteral dosage forms. Preferably in a parenteral dosage form. In the case of intramuscular, intraperitoneal, subcutaneous and intravenous dosage forms, a sterile solution of the active ingredient is usually prepared and may comprise a buffer which can suitably adjust the pH of the solution, and for intravenous administration isotonic in the formulation. It may include isotonic agents so that it is endowed. In addition, the pharmaceutical composition of the present invention may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier such as saline having a pH of 7.4, and may be locally introduced into the intramuscular blood flow of the patient in the form of a solution. have.

By effectively inhibiting the metastasis of cancer cells, the pharmaceutical composition of the present invention can be administered to cancer patients such as breast cancer, gastric cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, and various solid cancers or myeloid leukemia, and the dosage is usually administered to each patient. It may be administered at a dose that can generally vary depending on the age, body weight and symptoms of the patient, eg, from about 0.01 to 10 mg / kg per day.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the present invention is not limited to these examples.

Example  One. Of polypeptide  synthesis

The cDNA fragments of SEQ ID NOs: 15 to 24 encoding the polypeptides of SEQ ID NOs: 3 to 12, respectively, were inserted into the pET28a (+) vector to prepare six types of pET28a-hPILRα or pET28a-hPILRβ vectors. That is, after obtaining cDNA fragments of SEQ ID NOs: 15 to 24 through PCR, cutting the terminal portions with EcoRI, and then binding the cDNA fragments to the pET28a (+) vector digested with the same restriction enzyme by using a binding enzyme, pET28a-hPILRα and pET28a-hPILRβ vectors were prepared, respectively. Also, in the case of SEQ ID NO: 8, a cDNA encoding a human immunoglobulin Fc region (ie, a cDNA consisting of the nucleotide sequence of SEQ ID NO: 25) was inserted into a pET28a (+) vector prepared by inserting it into a pET28a (+) vector. , pET28a-hPILRα VI-Fc (or pET28a-hPILRβ VI-Fc) vector was prepared.

Colonies obtained by transforming the resulting expression vectors into BL21 (DE3) cells were incubated in LB medium for about 4 to 6 hours, and when the absorbance (A600) reached 0.4-0.6 for 7 to 9 hours, isopropyl β-D- Expression was induced by the addition of 1-thiogalactopyranoside (isopropyl β-D-1-thiogalactopyranoside (IPTG) (1.4 mM). The cells were precipitated by centrifugation, washed with phosphate buffered saline (PBS), and then precipitated again to remove impurities from the medium. It was confirmed whether expression was induced through SDS-PAGE gel.

8M urea buffer (8M urea, 0.01M Tris-Cl, 0.1M NaH ₂ PO ₄ ) was used for separation of the expressed protein, and pH concentrations of 8.0, 6.3, 4.5, etc. were used according to the separation step. After lysing the cells with pH 8.0 buffer to which protease inhibitors (1 mM PMSF, 10 μg / ml leupeptin, 1 μg / ml, pepstatin, 1 μg / ml aprotinin) were added, centrifuged at 4 ° C. and 13000 rpm for 20 minutes. Separated. The obtained supernatant was mixed with Ni-NTA His Bind Resin (Novagen, USA), a histidine binding resin, on a 1 ml Eppendorf tube, and reacted at 4 ° C. for 16 hours to react with the histidine of the expressed protein. Bonding of resins was induced. The resulting reaction mixture was centrifuged, the supernatant was discarded and washed with pH 6.3 buffer. After dialysis with PBS, quantitatively stored in refrigeration.

A polypeptide prepared as described above and a cDNA sequence encoding the same are summarized in Table 1 and FIG. 1. As can be seen in SEQ ID NOS: 1 and 2, PILRα V and VI have the same amino acid sequence and nucleotide sequence as the sequences of PILRβ V and VI.

snippet Polypeptide cDNA PILR α I 1-151 SEQ ID NO: 3 SEQ ID NO: 15 PILR α II 67-151 SEQ ID NO: 4 SEQ ID NO: 16 PILR α III 120-151 SEQ ID NO: 5 SEQ ID NO: 17 PILR α IV 67-119 SEQ ID NO: 6 SEQ ID NO: 18 PILR α V 120-129 SEQ ID NO: 7 SEQ ID NO: 19 PILR α VI 123-129 SEQ ID NO: 8 SEQ ID NO: 20 PILR β I 1-151 SEQ ID NO: 9 SEQ ID NO: 21 PILR β II 67-151 SEQ ID NO: 10 SEQ ID NO: 22 PILR β III 120-151 SEQ ID NO: 11 SEQ ID NO: 23 PILR β IV 67-119 SEQ ID NO: 12 SEQ ID NO: 24 PILR β V 120-129 SEQ ID NO: 7 SEQ ID NO: 19 PILR β VI 123-129 SEQ ID NO: 8 SEQ ID NO: 20

Example  2. Polypeptides  Preparation of the composition comprising

Polypeptides of SEQ ID NOS: 3-12 were dissolved in PBS to prepare a concentration of 3 μg in 100 μl volume. The obtained protein solution was used in the following test example.

Test Example  1. Human breast cancer cells Extracellular matrix  Hepatic adhesion inhibition test

The effects of the polypeptides of SEQ ID NOS: 3-6 and 9-12 on the attachment to fibronectin of human breast cancer cells (MCF-7) were measured.

Fibronectin, one of the extracellular matrix components, was plated in 96 well cell culture plates and dried under ultraviolet light. MCF-7 cells were prepared to be 5 × 10 ^{4 per} well, and the peptide solutions of SEQ ID NOs. 3 to 6 or 9 to 12 prepared in Example 2 were treated with the cells at a concentration of 3 μg, and then cultured for 1 hour. After washing three times with PBS, cells adhered to the extracellular matrix were removed using trypsin-EDTA, and then stained with Trypan-blue solution, and hemacytometer. The number of adherent cells was measured using. The results are shown in Figures 2a and 2b.

As can be seen in Figures 2a and 2b, when treated with the polypeptide of the present invention, the number of endothelial cells adhered to fibronectin is reduced by about 10 to 40% compared to the control. However, the polypeptide of SEQ ID NO: 6 or 12, which did not include the 123rd to 129th amino acids of SEQ ID NOs: 1 and 2, showed a similar degree of adhesion to the control.

Test Example  2. Infiltration of cancer cells Inhibitory ability  exam

Fibronectin, an integrin ligand, was previously plated into transwells, and then 5 x 10 ⁵ human breast cancer cell lines MCF-7 were placed on top of the transwells and cultured. After 24 hours, when the cells grow more than 80%, the peptides of SEQ ID NOS: 3 to 8 were treated at a concentration of 3 μg and incubated for 1 hour in a 37 ° C., 5% CO ₂ incubator. 0.1% BSA was added to the lower part of the transwell, and the infiltration-inducing medium (0.005% vitamin C, 1% BSA, culture supernatant obtained by culturing for 24 hours in DMEM serum-free culture medium) was added to the transwell every 24 hours. The number of cells dropped to the bottom of the wells was measured three times. Each test was performed three times and the results were statistically processed. Fc peptide was treated in place of the peptide as a control. The result is shown in FIG. 3.

As can be seen in Figure 3, when treated with the peptides of SEQ ID NOs: 3 to 5 and 7 to 8 of the present invention, the degree of infiltration of extracellular material of human breast cancer cells compared to the control group treated with the peptide of Fc is about 20 Suppressed to the% level. Considering that cancer cells that have escaped from blood vessels are transferred to new sites through invasion of the bottom membrane or surrounding connective tissue, the polypeptide of the present invention has an effective cancer cell metastasis inhibiting activity.

Test Example  3. In vitro cancer cells in in vitro ) Extravascular  outflow Inhibitory ability  exam

Supernatant obtained by incubating in a transwell with a pore diameter of 8 µm for 16 hours incubation in DMEM serum-free medium containing 0.005% vitamin C and 0.1% bovine albumin, infiltrating induction medium (mouse fibroblast NIH / 3T3) ), Divided into three groups. The first group treated 5 × 10 ⁵ human breast cancer cell line MCF-7 with 3 μg of control peptide consisting of Fc (control), the second group treated equal numbers of MCF-7 cells with 3 μg of peptide of SEQ ID NO: 7 In the third group, the same number of MCF-7 cells were treated with 3 μg of the peptide of SEQ ID NO: 8. Each well was incubated for 1 hour in a 37 ° C., 5% CO ₂ incubator, after which 0.1% BSA was added and 6 hours later, the number of cells escaping to the bottom of the transwell containing the infiltration medium was measured. The result is shown in FIG. 4.

As can be seen in Figure 4, when treated with the polypeptide of the present invention, extravasation of human breast cancer cells is suppressed to about 80% level. Given that cancer cells infiltrating into blood vessels are flowed out of blood vessels in order to move along blood vessels and metastasize to organs, the polypeptide of the present invention is expected to have an effective cancer cell metastasis inhibiting activity.

1 shows the structure of the polypeptides of SEQ ID NOs: 3-12.

2a and 2b show the results of measuring the effect of the polypeptide of the present invention on the attachment to fibronectin of human breast cancer cell line MCF-7.

Figure 3 shows the results of measuring the number of human breast cancer cells infiltrated with fibronectin when the peptide of the present invention is treated.

Figure 4 shows the results of measuring the number of human breast cancer cells leaked out of blood vessels when the human breast cancer cell line MCF-7 was treated with the polypeptide of the present invention.

<110> KNU-Industry Cooperation Foundation <120> Polypeptides or fusion proteins according to an inhibitory          activity against metastasis of cancer cells <130> PN0154 <160> 25 <170> KopatentIn 1.71 <210> 1 <211> 303 <212> PRT <213> Homo sapiens <400> 1 Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Pro Leu Leu Leu Pro Pro   1 5 10 15 Ala Phe Leu Gln Pro Ser Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu              20 25 30 Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser          35 40 45 Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Thr Ala      50 55 60 Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Arg Gln Ser  65 70 75 80 Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg                  85 90 95 Leu Phe Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu Arg Ile             100 105 110 Ser Asn Leu Gln Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg Val Glu         115 120 125 Leu Asp Thr Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile Glu Gly     130 135 140 Thr Lys Leu Ser Ile Thr Gln Ala Val Thr Thr Thr Thr Thr Gln Arg Pro 145 150 155 160 Ser Ser Met Thr Thr Thr Trp Arg Leu Ser Ser Thr Thr Thr Thr Thr                 165 170 175 Gly Leu Arg Val Thr Gln Gly Lys Arg Arg Ser Asp Ser Trp His Ile             180 185 190 Ser Leu Glu Thr Ala Val Gly Val Ala Val Ala Val Thr Val Leu Gly         195 200 205 Ile Met Ile Leu Gly Leu Ile Cys Leu Leu Arg Trp Arg Arg Arg Lys     210 215 220 Gly Gln Gln Arg Thr Lys Ala Thr Thr Pro Ala Arg Glu Pro Phe Gln 225 230 235 240 Asn Thr Glu Glu Pro Tyr Glu Asn Ile Arg Asn Glu Gly Gln Asn Thr                 245 250 255 Asp Pro Lys Leu Asn Pro Lys Asp Asp Gly Ile Val Tyr Ala Ser Leu             260 265 270 Ala Leu Ser Ser Ser Thr Ser Pro Arg Ala Pro Pro Ser His Arg Pro         275 280 285 Leu Lys Ser Pro Gln Asn Glu Thr Leu Tyr Ser Val Leu Lys Ala     290 295 300 <210> 2 <211> 227 <212> PRT <213> Homo sapiens <400> 2 Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Leu Leu Leu Gln Pro Pro   1 5 10 15 Ala Phe Leu Gln Pro Gly Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu              20 25 30 Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser          35 40 45 Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Ile Val      50 55 60 Pro Asn Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser  65 70 75 80 Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg                  85 90 95 Leu Phe Leu Asn Trp Thr Glu Gly Gln Glu Ser Gly Phe Leu Arg Ile             100 105 110 Ser Asn Leu Arg Lys Glu Asp Gln Ser Val Tyr Phe Cys Arg Val Glu         115 120 125 Leu Asp Thr Arg Arg Ser Gly Arg Gln Gln Leu Gln Ser Ile Lys Gly     130 135 140 Thr Lys Leu Thr Ile Thr Gln Ala Val Thr Thr Thr Thr Thr Thr Trp Arg 145 150 155 160 Pro Ser Ser Thr Thr Thr Ile Ala Gly Leu Arg Val Thr Glu Ser Lys                 165 170 175 Gly His Ser Glu Ser Trp His Leu Ser Leu Asp Thr Ala Ile Arg Val             180 185 190 Ala Leu Ala Val Ala Val Leu Lys Thr Val Ile Leu Gly Leu Leu Cys         195 200 205 Leu Leu Leu Leu Trp Trp Arg Arg Arg Lys Gly Ser Arg Ala Pro Ser     210 215 220 Ser Asp Phe 225 <210> 3 <211> 151 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 3 Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Pro Leu Leu Leu Pro Pro   1 5 10 15 Ala Phe Leu Gln Pro Ser Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu              20 25 30 Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser          35 40 45 Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Thr Ala      50 55 60 Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser  65 70 75 80 Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg                  85 90 95 Leu Phe Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu Arg Ile             100 105 110 Ser Asn Leu Gln Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg Val Glu         115 120 125 Leu Asp Thr Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile Glu Gly     130 135 140 Thr Lys Leu Ser Ile Thr Gln 145 150 <210> 4 <211> 85 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 4 Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser Phe Tyr   1 5 10 15 Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg Leu Phe              20 25 30 Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu Arg Ile Ser Asn          35 40 45 Leu Gln Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg Val Glu Leu Asp      50 55 60 Thr Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile Glu Gly Thr Lys  65 70 75 80 Leu Ser Ile Thr Gln                  85 <210> 5 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 5 Gln Ser Val Tyr Phe Cys Arg Val Glu Leu Asp Thr Arg Ser Ser Gly   1 5 10 15 Arg Gln Gln Trp Gln Ser Ile Glu Gly Thr Lys Leu Ser Ile Thr Gln              20 25 30 <210> 6 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 6 Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser Phe Tyr   1 5 10 15 Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg Leu Phe              20 25 30 Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu Arg Ile Ser Asn          35 40 45 Leu Gln Lys Gln Asp      50 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 7 Gln Ser Val Tyr Phe Cys Arg Val Glu Leu   1 5 10 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 8 Tyr Phe Cys Arg Val Glu Leu   1 5 <210> 9 <211> 151 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 9 Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Leu Leu Leu Gln Pro Pro   1 5 10 15 Ala Phe Leu Gln Pro Gly Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu              20 25 30 Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser          35 40 45 Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Ile Val      50 55 60 Pro Asn Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser  65 70 75 80 Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg                  85 90 95 Leu Phe Leu Asn Trp Thr Glu Gly Gln Glu Ser Gly Phe Leu Arg Ile             100 105 110 Ser Asn Leu Arg Lys Glu Asp Gln Ser Val Tyr Phe Cys Arg Val Glu         115 120 125 Leu Asp Thr Arg Arg Ser Gly Arg Gln Gln Leu Gln Ser Ile Lys Gly     130 135 140 Thr Lys Leu Thr Ile Thr Gln 145 150 <210> 10 <211> 85 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 10 Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser Phe Tyr   1 5 10 15 Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg Leu Phe              20 25 30 Leu Asn Trp Thr Glu Gly Gln Glu Ser Gly Phe Leu Arg Ile Ser Asn          35 40 45 Leu Arg Lys Glu Asp Gln Ser Val Tyr Phe Cys Arg Val Glu Leu Asp      50 55 60 Thr Arg Arg Ser Gly Arg Gln Gln Leu Gln Ser Ile Lys Gly Thr Lys  65 70 75 80 Leu Thr Ile Thr Gln                  85 <210> 11 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 11 Gln Ser Val Tyr Phe Cys Arg Val Glu Leu Asp Thr Arg Arg Ser Gly   1 5 10 15 Arg Gln Gln Leu Gln Ser Ile Lys Gly Thr Lys Leu Thr Ile Thr Gln              20 25 30 <210> 12 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 12 Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser Phe Tyr   1 5 10 15 Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg Leu Phe              20 25 30 Leu Asn Trp Thr Glu Gly Gln Glu Ser Gly Phe Leu Arg Ile Ser Asn          35 40 45 Leu Arg Lys Glu Asp      50 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> poly-His fragment <400> 13 His His His His His His   1 5 <210> 14 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Fc fragment <400> 14 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys   1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr              20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser          35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser      50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr  65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                  85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 15 <211> 453 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 15 atgggtcggc ccctgctgct gcccctactg cccttgctgc tgccgccagc atttctgcag 60 cctagtggct ccacaggatc tggtccaagc tacctttatg gggtcactca accaaaacac 120 ctctcagcct ccatgggtgg ctctgtggaa atccccttct ccttctatta cccctgggag 180 ttagccacag ctcccgacgt gagaatatcc tggagacggg gccacttcca caggcagtcc 240 ttctacagca caaggccgcc ttccattcac aaggattatg tgaaccggct ctttctgaac 300 tggacagagg gtcagaagag cggcttcctc aggatctcca acctgcagaa gcaggaccag 360 tctgtgtatt tctgccgagt tgagctggac acacggagct cagggaggca gcagtggcag 420 tccatcgagg ggaccaaact ctccatcacc cag 453 <210> 16 <211> 255 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 16 gtgagaatat cctggagacg gggccacttc cacaggcagt ccttctacag cacaaggccg 60 ccttccattc acaaggatta tgtgaaccgg ctctttctga actggacaga gggtcagaag 120 agcggcttcc tcaggatctc caacctgcag aagcaggacc agtctgtgta tttctgccga 180 gttgagctgg acacacggag ctcagggagg cagcagtggc agtccatcga ggggaccaaa 240 ctctccatca cccag 255 <210> 17 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 17 cagtctgtgt atttctgccg agttgagctg gacacacgga gctcagggag gcagcagtgg 60 cagtccatcg aggggaccaa actctccatc acccag 96 <210> 18 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 18 gtgagaatat cctggagacg gggccacttc cacaggcagt ccttctacag cacaaggccg 60 ccttccattc acaaggatta tgtgaaccgg ctctttctga actggacaga gggtcagaag 120 agcggcttcc tcaggatctc caacctgcag aagcaggac 159 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 19 cagtctgtgt atttctgccg agttgagctg 30 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 20 tatttctgcc gagttgagct g 21 <210> 21 <211> 453 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 21 atgggtcggc ccctgctgct gcccctgctg ctcctgctgc agccgccagc atttctgcag 60 cctggtggct ccacaggatc tggtccaagc tacctttatg gggtcactca accaaaacac 120 ctctcagcct ccatgggtgg ctctgtggaa atccccttct ccttctatta cccctgggag 180 ttagccatag ttcccaacgt gagaatatcc tggagacggg gccacttcca cgggcagtcc 240 ttctacagca caaggccgcc ttccattcac aaggattatg tgaaccggct ctttctgaac 300 tggacagagg gtcaggagag cggcttcctc aggatctcaa acctgcggaa ggaggaccag 360 tctgtgtatt tctgccgagt cgagctggac acccggagat cagggaggca gcagttgcag 420 tccatcaagg ggaccaaact caccatcacc cag 453 <210> 22 <211> 255 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 22 gtgagaatat cctggagacg gggccacttc cacgggcagt ccttctacag cacaaggccg 60 ccttccattc acaaggatta tgtgaaccgg ctctttctga actggacaga gggtcaggag 120 agcggcttcc tcaggatctc aaacctgcgg aaggaggacc agtctgtgta tttctgccga 180 gtcgagctgg acacccggag atcagggagg cagcagttgc agtccatcaa ggggaccaaa 240 ctcaccatca cccag 255 <210> 23 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 23 cagtctgtgt atttctgccg agtcgagctg gacacccgga gatcagggag gcagcagttg 60 cagtccatca aggggaccaa actcaccatc acccag 96 <210> 24 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> nucleotide fragment <400> 24 gtgagaatat cctggagacg gggccacttc cacgggcagt ccttctacag cacaaggccg 60 ccttccattc acaaggatta tgtgaaccgg ctctttctga actggacaga gggtcaggag 120 agcggcttcc tcaggatctc aaacctgcgg aaggaggac 159 <210> 25 <211> 993 <212> DNA <213> Artificial Sequence <220> <223> Fc fragment sequence <400> 25 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc cccgggtaaa tga 993  

Claims (18)

delete A polypeptide selected from the group consisting of the polypeptides of SEQ ID NOs: 3 to 5 and 7 to 11. A fusion protein of the polypeptide of claim 2 with a poly-Histidine (poly-His) region. 4. The fusion protein of claim 3, wherein said polyhistidine region has the amino acid sequence of SEQ ID NO: 13. A fusion protein of the polypeptide of claim 2 with the Fc region. The fusion protein of claim 5, wherein the Fc region has an amino acid sequence of SEQ ID NO: 14. 7. A polynucleotide encoding the polypeptide of claim 2. 8. The polynucleotide of claim 7, wherein the polynucleotide is selected from the group consisting of polynucleotides of SEQ ID NOs: 15-17 and 19-23. A vector comprising a polynucleotide encoding the polypeptide of claim 2. The vector according to claim 9, wherein the vector has a cDNA inserted therein encoding a poly-His region or an Fc region. The transformant transformed the host cell with the vector of claim 9. The method of claim 11, wherein the host cell Escherichia genus) transformant or Pichia genus strain. A polypeptide consisting of 7 to 200 amino acids derived from a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and comprising peptides 123 to 129 of SEQ ID NO: 1 or 2, and having a metastasis inhibiting activity of cancer cells A pharmaceutical composition for inhibiting metastasis of cancer cells, comprising as an active ingredient, a pharmaceutically acceptable carrier. A polypeptide consisting of 7 to 200 amino acids derived from a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and comprising peptides 123 to 129 of SEQ ID NO: 1 or 2, and having a metastasis inhibiting activity of cancer cells A pharmaceutical composition for inhibiting metastasis of cancer cells comprising a fusion protein with a polyhistidine (poly-His) region as an active ingredient and a pharmaceutically acceptable carrier. A polypeptide consisting of 7 to 200 amino acids derived from a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and comprising peptides 123 to 129 of SEQ ID NO: 1 or 2, and having a metastasis inhibiting activity of cancer cells And a fusion protein of the Fc region as an active ingredient, and a pharmaceutical composition for inhibiting metastasis of cancer cells comprising a pharmaceutically acceptable carrier. A pharmaceutical composition for inhibiting metastasis of cancer cells comprising a polypeptide selected from the group consisting of the polypeptides of SEQ ID NOs: 3 to 5 and 7 to 11 as an active ingredient, and comprising a pharmaceutically acceptable carrier. A cancer cell comprising a fusion protein of a poly-His region with a polypeptide selected from the group consisting of the polypeptides of SEQ ID NOs: 3 to 5 and 7 to 11, and comprising a pharmaceutically acceptable carrier Pharmaceutical composition for inhibiting metastasis. A pharmaceutical composition for inhibiting metastasis of cancer cells comprising a fusion protein of an Fc region and a polypeptide selected from the group consisting of the polypeptides of SEQ ID NOs: 3 to 5 and 7 to 11, and comprising a pharmaceutically acceptable carrier.

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