CA2516172A1

CA2516172A1 - Anticancer agent comprising lk8 protein as an active ingredient

Info

Publication number: CA2516172A1
Application number: CA002516172A
Authority: CA
Inventors: Hyunkyung Yu; Jang-Seong Kim; Jin-Hyung Ahn; Ho-Jeong Lee; Kwan-Yub Kang; Hyung-Kwon Lim; In-Hwan Lim; Sung-Geun Kim; Hak Kyu Joo; Jihyun Lee; Yeup Yoon; Doo-Hong Park; Jung Hwan Park
Original assignee: Individual
Current assignee: Mogam Biotechnology Research Institute
Priority date: 2003-02-20
Filing date: 2004-02-20
Publication date: 2004-09-02
Also published as: EP1608395A1; WO2004073730A1; AU2004212856B2; CN1753685A; CN100546646C; KR20040075270A; RU2005129273A; RU2306147C2; JP2006518342A; AU2004212856A1; BRPI0407611A; EP1608395A4; KR100595364B1

Abstract

The present invention relates to an anticancer agent comprising LK8 protein as an active ingredient. The anticancer agent of the present invention is effective to repress growth and metastasis of malignant tumor. Thus, the agent can be effectively used not only for repressor of cancer metastasis, but also for therapeutic agent of primary malignant tumor.

Description

AN ACTIVE INGREDIENT
FIEZD OF THE INVENTION
The present invention relates to an anticancer agent comprising a certain protein as an active ingredient, more precisely, to an anticancer agent comprising a protein corresponding to kringle KV3~ of apolipoprotein(a) as an active ingredient.
BAOKGROUND
A tumor is developed by uncontrollable disordered abnormal cell proliferation. If this tumor shows a destructive growth, invasiveness and metastasis, it is regarded as a malignant tumor.
Invasiveness is a character to infiltrate or destroy surrounding tissues, and in particular, a basal layer forming a boundary of tissues is ?0 destroyed by the character, resulting in the local spread and sometimes inflow of a tumor through circulatory system. Metastasis means the spread of tumor cells from the original birthplace to other areas through lymphatic or blood vessels.

In a broad sense, metastasis also means the direct extension of tumor cells through peritoneal cavity or other space.
As of today, surgical operation, radiotherapy and chemotherapy have been used for the treatment of a cancer singly or jointly. The surgical operation is a way to remove diseased tissues.
Thus, tumors in specific regions such as breast, colon and skin can be effectively removed by the surgical operation. However, a tumor in vertebra or dispersive tumor like leuh.emia cannot be properly treated by the surgical operation.
Chemotherapy blocles cell replication or metabolism, and has been used for the treatment of breast cancer, lung cancer and testis cancer.
Though, patients with cancers who have been treated by chemotherapy have seriously suffered from the side effects of systemic chemotherapy.
Motion sickness and vomiting, are common but serious examples of all. The side effects of chemotherapy can even affect the life of a patient since they might drop the adaptability of a patient rapidly. Besides, Dose Limiting Toxicity (DLT) is also one of major side effects of chemotherapy, which draws a careful attention in the administration of a medicine. Mucositis is an example of DZT against anticancer agents such as 5-fluoruracil which is an antimetabolic cytotoxic agent, and methotrexate, and anticancer antibiotics like doxorubicin. If a patient suffers seriously from such side effects of chemotherapy, he or she should be hospitalized and given an anodyne for reducing pain. So, side effects of chemotherapy and radiotherapy are the biggest problem for the treatment of cancer patients.
Therefore, it is an urgent need to develop an anticancer agent originated from a living creature in order to reduce side effects of~ chemotherapy.
In particularo among substances generated in a living creature, a promising subject has been found which does not attack cancer cells directly but prevents cancer cells from growing by working toward various endothelial cells helping cancer cell growth. So, an anticancer agent containing the subject can not only treat cancers but also prevent metastasis.
Kringle is a kind of a protein structure which is composed of 80 amino acids and three intramolecular disulfide bonds. Kringle structure is found in many proteins such as prothrombin (Walt, D.A. et al., Proc. Natl. Acad. Sci., 74:1069-1073, 1977), urokinase (Pennica, D. et al., Nature, 301:579-582, 1983), hepatocyte growth factor (Lukker, N. A. et a1'., Protein Eng., 7:895-903, 1994) and apolipoprotein(a) (refered as 'apo(a)' hereinafter) (McLean, J.W. et al., Nature, 330:132-137, 1987). Kringle is composed of an individual folding unit. The functions of kringle have not been clearly explained, yet.
Apo(a) includes two types of kringle domains, KIV and KV, and an inactive Zarotease-like region.
The kringle domain KIV is divided into 10 subtypes (KIV-1 ~ KIV-10) according t~ the homology of amino acids, aild 15 ~ 40 copy numbers of the domain are found in various human alleles of the apo(a) gene. Apo(a) forms a lipoprotein(a) (referred as 'Lp(a)' hereinafter) by covalent bond with apo B-100, a major protein component of low-density lipoprotein (LDL)(Fless, G.M., J. Biol.
Chem., 261:8712-8717, 1986). The increase of Lp (a) content in cytoplasm itself is a major risk factor of artherosclerosis (Armstrong, V.W. et al., Artherosclerosis, 62:249-257, 1986 Assmann, G., Am. J. Cardiol., 77:1179-1184, 1996).
Based on the fact that artherosclerosis and cancer cell growth depend on angiogenesis, the present inventors have studied on an anticancer activity of KV38, one of human apo(a) kringles.
As a result, the present inventors have completed this invention by confirming that the protein can be effectively used as an anticancer agent because it inhibits angiogenesis by an endogenous growth factor like bFGF which is necessary for cancer cell growth.
SUMMARY OF THE INVENTION
It is an obj ect of this invention to provide an anticancer agent comprising a human apo(a) kringle KV38 (referred as 'LK8 protein' hereinafter) as an active ingredient.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In order to achieve the above object of the present invention, the present invention provides an anticancer agent comprising a LK8 protein as an active ingredient.
Hereinafter, the present invention is described in detail.
The anticancer agent of the present invention is characterized by including LK8 protein having an amino acid sequence represented by SEA. ID. No 1 as an active ingredient. It is preferred to use the agent as a metastasis inhibitor, more preferably, it is used for the suppression of metastasis of colon carcinoma or rectal cancer to liver.
In addition, the anticancer agent is 1~ preferably used for the treatment of primary tumors. More preferablyo the agent is used for the treatment of a cancer selected from. a group consisting of prostate cancer, lung cancer, colon cancer and rectal cancer.
It is preferred for the anticancer agent of the present invention to contain LK8 protein by 0.1 ~ 100 mg/kg, more LK8 preferred to contain protein by 1 ~ 50 mg/kg, and administration times are 1 ~ 4 per day. But the composition is not limited thereto, and is possibly changed according to the conditions of a patient, and types and progressing speed of a disease.
In this invention, "KV38" means an apo(a) kringle, and "LK8" means a recombinant protein of KV38. However, both KV38 and LK8~ protein are called as LK8 protein in general unless mentioned specifically.
LK8 protein of the present invention is a domain corresponding to KV38 kringle of many apo(a) kringle domains and has an inhibiting effect on proliferation and differentiation of cancer cells and metastasis by suppressing the activity of endothelial cells in vitro and in vivo as well. As explained in the preferred embodiment of the present 111ZIe11t1~I'1, the systemic administration of LK8 protein results in the inhibition of a primary tumor and its metastasis (see FIG. 2 ~ FIG. 6) . Therefore, LK8 protein of the present invention can be effectively used as an anticancer agent especially for primary tumors and as a metastasis inhibitor owing to its functions of suppressing tumor growth and metastasis.

The treatment effect will be enhanced if LK8 protein of the present invention is used together with conventional chemotherapy or radiotherapy.
Radiotherapy is performed to destroy a primary tumor, and if LK8 protein is administered during radiotherapy, metastasis can be prevented more effectively. As for chemotherapy, cytotoxicity caused by huge dosage of chemical anticancer agents is the biggest problem. If LK8 protein of the invention is administered during chemotherapy, the decreased amount of chemical anticancer agents will bring equivalent or even improved anticancer effects as well as lessen the cytotoxicity.
In conclusion, if the administration of LK8 protein is performed together with surgical operation, radiotherapy, chemotherapy or immunotherapy, the treatment effect will be maximized. And further, the continuous administration of LK8 protein extends dormancy of micrometastasis, suppresses the growth of a primary tumor and stabilizes conditions. Many conventional anticancer agents are designed for long term administration, causing troubles such as continuous production of a protein and high price of the product. The alternative to them is gene therapy. And LK8 protein is also expected to maximize the effect of an anticancer agent or a metastasis~inhibitor if it is used in gene therapy.
The anticancer agent comprising LK8 protein of the present invention can be administered orally or parenterally and be used in general forms of pharmaceutical formulation.
The anticancer agent can be prepared for oral or parenteral administration by mixing with generally used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactants, or excipients. Solid formulations for oral administration are tablets, pills, dusting powders, granules and capsules.
These solid formulations are preppared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin.
Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, and suppositories. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc.
Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerinated gelatin, etc.
LDSO of the LIFO larotein is about 1, 000 m~/l~g, suggesting that the anticancer agent of the present invention is very much safe (see Table 2).
ERIEF DESCRIPTION OF THE DRAWINGS
The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

FIG. 1 is a schematic representation of the expression vector 'pMBRI-LK8 (8.25 kb)' of LK8 gene, in which LK8 cDNA (261 bp) is inserted between AOX1 promoter and AOX1 terminator, FIG. 2 is a set of photographs and a graph showing that the pulmonary metastasis of a murine melanoma cell line, B16F10, which was injected into mouse (C57BL/6) tail vein, is inhibited by the treatment of LK8 protein, (a) Lung of the mouse treated with PBS only, (b) Lung of the mouse treated with LK8 protein by 1 mg/kg, (c) A graph showing that metastasis of B16F10 cells to lung of the mouse is inhibited by the treatment of LK8 protein, FIG. 3 is a set of photographs and a graph.
showing that the metastasis of liver by a mouse colorectal cancer cell line CT-26 transplanted ,into mouse spleen is inhibited by the treatment of LK8 protein, (a) Liver of the mouse treated with PBS
(control) and LK8 protein (10 mg/kg/day) , (b) A graph showing the colony number of CT-26 cells metastasized into liver of the mouse treated with PBS (control) and LK8 protein (10 mg/
kg/day) , (c) photographs showing the distribution of CT-26 cancer cells metastasized into liver of the mouse treated with PBS (control) and LK8 protein ( 10 mg/kg/day) , which is observed by hematoxylin &
eosin staining, FIG. 4 is a set of graphs showing the changes of the size of a tumor in a mouse transplanted with human prostate carcinoma PC-3 cells, according to the administration of LK8 protein, (a) A graph showing the changes of the size of a tumor after treating LK8 protein by 100 mg/kg /day, (b) A graph showing the changes of the si~.e of a tumor after treating LK8 protein by 50 mg/kg /day, FIG. 5 is a graph showing the changes of the size of a tumor in a mouse transplanted with human lung carcinoma A549 cells, according to the administration of LK8 protein, FIG. 6 is a set of graphs showing the changes of the size of a tumor (a) and the weight of a tumor (b) in mice transplanted with human rectal and colon carcinoma LS 174T cells, according to the administration of LK8 protein.
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example l: Preparation of LK8 protein <1-1> Construction of LK8 expression vector pMERI-In order to prepare LK8 protein effectively, the present inventors constructed LK8 expression vector first.

For the expression of LK8 gene, pPIC9 vector (8.0 kb, Invitrogen, Netherland) was used as a basic vector. As seen in the representative map, pPIC9 expression vector includes promoter AOX1 which provides high expression by methanol, a -factor secretion signal which enables the secretion of an expressed protein, 3'AOX1(TT), AOX1 polyadenylation signal, which enables effective termination of transcription and polyadenylation, and. a DNA fragment coding histidinol dehydrogenase of wild type Pichia pastoris used as a selectable marker for a transformant of the above strain, in order.
At first, LK8 gene was amplified by PCR with primers 'LKBN-Xhol' represented by SEQ. ID. No 2 and °LKBC-EcoRI' represented ~~~r SEQ. ID. No 3~
using pETl5b/LK8 (see PCT/KR99I00554) as a template. The PCR product was digested with restriction enzymes X.h~I and EcoRI, followed by subcloning lay inserting the product into pPIC9 vector which was digested with the same restriction enzymes. Finally, the LK8 gene expression vector 'pMBRI-LK8 (8.25 kb) was constructed (FIG. 1).

<1-2> Preparation of a transformant includin pMBRI-LK8 Pichia pastoris, a methylotrophic yeast, was used as a host for the preparation of a recombinant transformant.
Particularly, LK8 gene expression vector 'pMBRI-LK8' was treated with a restriction enzyme SacI, leading to linearization. The vector was inserted into AOXI gene of the above host strain chromosome by homologous recombination. At that time, electroporation was performed for transformation. A recombinant yeast transformant was selected from histidine-deficient medium by examining colony forming. PCR was performed to confirm if LK8 cDNA was inserted into A0~1 region of chromosome of the selected recombinant transformant. And then, the recombinant transformant was cultured and the expression of LK8 gene was induced by methanol. As a result, the expression of LK8 gene was confirmed, suggesting that the protein was mass-secreted in the culture medium.
The secreted LK8 protein of the present invention was composed of amino acid sequence represented by SEQ. ID. No 1.
<1-3> Cultivation of a recombinant strain <1-3-1> Seed culture In this invention, a recombinant strain was obtained by inserting LK8 gene into Pichia pastoris. The established strain was seed cultured for 24 hours to obtain appropriate biomass and activity (when it was diluted by 20 times, OD6oo was 0 . 8 - 1 . 2 ) .
Seed culture was performed in YDP medium (10 yeast extract, 2~ peptone, 2% dextrose) for 24 hours by shaking culture. 75 L fermenter was used.
The volume of a beginning medium was 20 L and the final volume of culture solution was adjusted to 40 L by fed-batch culture method.
<1-3-2> Main culture After completing seed culture in YPD medium, main culture was performed with seed culture solution as much as 300 out of the primary medium.
Main culture was performed in a fermenter containing ingredients seen in the below table 1.
When the ferment was saturated by the supply of methanol, the ferment was recovered more than 100 for the production of LK8 protein. In the meantime, methanol was being supplied continuously to induce the continuous expression of the protein.
The procedure was repeated to produce LK8 protein.
Consumption speed of a carbon source was in proportion to the amount of cells. So, when some of ferment was recovered, the speed of methanol supply was automatically regulated not to be out of ~200. From the repetition of the above culture process, during which fermentation was continued for over 200 hours, the secreted LK8 protein was obtained by 250 mg per 1 L of culture solution.
<Table 1>
Medium composition for main culture Kinds of medium Component Concentration Medium for main Methanol 500 g/L

culture Trace metal 8 m.~/L

solution Medium composition CuSO4 ' 5H20 4 g/L

of trace metal KI 0.3 g/L

solution MnS04 ' H20 4 g/L

0.1 g/L

NaMo ~ HBO
0.01 g/L

H3B04 0.1 g/L

CoCl2 3 g/L

ZnCl2 10 g/L

FeS04 ' H20 0. 1 g/L

Biotin 5 mk/L

Example 2: Experimental lung metastasis b intravenous administration of B16F10 murine melanoma cells B16F10 cells (1.8 ~ 105), mouse melanoma (referred as °melanoma cells° hereinafter) (American Type Culture Collection), were administered in tail vein of a C57BL/6 mouse (Charles River Japan, Inc.). From the next day, LK8 protein, prepared in the above <Example 1>, was injected subcutaneously twice (1 mg/kg/day, 0 . 2 mg/kg/day) a day for 14 days . A control group was injected with PBS instead of LK8 protein. On the 13~'' day from cell transplantation, the mouse was dissected, and lung was taken to count the number of colony of the metastasized cancer cells (melanoma cells).
1~

As a result, huge colonies were formed in lungs of the control group mice injected with PBS, suggesting the transfer of melanoma cells (FIG.
2a) . On the contrary, the number and the size of colonies generated by the transfer of melanoma cells were much less and smaller in the experimental group mice inj acted with LIC8 protein (FIG. 2b). In conclusion, the group treated with LK8 protein by 1 mg/kg showed 53% metastasis inhibition, comparing to the control group (FIG.
2c) .
Exam 1e 3: Experimental liver metastasis by intra-s~lenic administration of marine colorectal cancer cell CT-26 CT-26 cells (American Type Culture Collection), mouse colorectal cancer cells, were injected in spleen to induce metastasis into liver.
Then, metastasis-inhibiting effect of LI~C8 protein was investigated. Particularly, CT-26 cells, which were 80o grown up on a plate, were washed with PBS, followed by singularizing with 0.020 EDTA. The single cells were washed with PBS again, then resuspended in PBS carefully. The suspension was stained with Trypan blue to count the number of cells . Cell density was adjusted to 5 X 105/m.~, which was injected into each mouse by 100 ,cte. 6 8 week old BALB/c mice (Charles River Japan, Inc.) were used. After the right side of abdomen was surgically excised, cancer cell suspension was carefully injected to the spleen using a 30-gauge needle. ZK8 protein was subcutaneously injected to a experimental group twice a day by 10 mg/kg /day, and likewise, saline was injected as a control group. 14 days later, the mice were sacrificed to examine th.e liver (FIG. 3a), and the number of colonies seen on surface of the liver was counted, which was fixed in 10~ formalin solution. There was no big difference in weight among groups. However, the number of colonies which were transferred to liver was much less in an experimental group which was administered with LK8 protein by 10 mg/kg/day than in a control group. The decrease of the number of colonies in an experimental group injected with LK8 protein by 10 mg/kg/day was about 600, comparing to a control group (FIG. 3b). Besides, liver tissue section which was fixed in formalin solution was stained with H&E, and observed. As a result, a tumor region was much limited in an experimental group treated with LK8 protein comparing to a control group (FIG. 3c) .
Example 4: Growth inhibition of a primary tumor In order to investigate the effect of LK8 protein on the anti-angiogenesis .in vivo, a xenografted tumor model was used. Every relevant experiment was performed with 4 week old randomly crossed female Balb/c nu/nu nude mice (Charles River Japan, Inc.), which were raised under sterilised condition.
<4-1> Human ~rostatic carcinoma (PC-3 Human prostatic carcinoma PC-3 cells (American Type Culture Collection) were cultured in RPMI 1640 medium (GIBCOTM, Invitrogen Corporateon) supplemented with 10o FBS, and about 5 ~ 106 PC-3 cells were subcutaneously injected in central muscular region of the back of the nude mouse. Exactly 10 days after the transplantation, LK8 protein was injected by 100 mg/kg/day.

Meanwhile, a control group was injected PBS only instead of LK8 protein. The treatment was continued for 30 days, then, the size of a tumor was measured every 3 or 4 days. As a result, tumor growth was inhibited by the treatment of LK8 protein, which was about 60o inhibition comparing to a control group (FIG. 4a). When the LK8 protein was injected by 50 mg/kg/day, tumor growth was inhibited in similar fashion over 600 comparing to a control group (FIG. 4b).
<4-2~ Human lunct carcinoma (A549 Human lung carcinoma A549 cells (American Type Culture Collection) were cultured in DMEM
medium (GIBCOT~~ Invitrogen Corporation) supplemented with 10o FBS. Then, 1 ~ 107 tumor cells were subcutaneously injected in central muscular region of the back of the nude mouse.
Exactly 5 days after the transplantation, LK8 protein was injected by 50 mg/kg/day. A control group was administered PBS only instead of LK8 protein. The treatment was continued for 46 days, then, the size of a tumor was measured every 3 or 4 days. As a result, tumor growth was inhibited by the treatment of LK8 protein, which was 61%

inhibition comparing to a control (FIG. 5).
<4-3> Human colon and rectal carcinoma (LS 174T
Human colon and rectal carcinoma LS 174T
cells (American Type Culture Collection) were cultured in RPMI medium (GIBC~TM, Invitrogen Corporation) supplemented with 10o FBS. Then, 5 106 tumor cells were subcutaneously injected in central muscular region of the back of the nude mouse. Exactly 5 days after the transplantation, LK8 protein was injected by 50 m~/k~Oday. A
control group was administered PBS only instead of LK8 protein. The treatment was continued for 34 days, then, the sire of a tumor was measured every 3 or 4 days. As a result, tumor growth was inhibited by the treatment of LK8 protein, which was 64o inhibition comparing to a control (FIG.
6a). And, the weight of the tumor was decreased by the treatment of LK8 protein about 68.70, which was measured on the final day of the experiment (FIG. 6b) .
Example 5: Acute toxicity test of LK8 protein 5-week old SPF SD (Sprague Dawley) line rats were used in the test for acute toxicity. Rats were divided into 5 groups, and 5 rats per each group were administered once with LK8 protein by the dosage of 260 mg/kg, 364 mg/kg, 510 mg/kg, 714 mg /kg and 1000 °mg/kg, respectively, by intravenous injection (Table 2). For 14 days after test material, LK8 protein, administration, death, clinical symptoms and weight change in rats were observed. The hematological tests and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were examined visually during autopsy. As a result, a weak toxicity was detected in the group administered with 1000 mg/kg of LK8 protein, but neither toxicity nor death was found in other groups in most tests including weight change s blood test8 hematological tests and biochemical tests of blood, autopsy, etc. Therefore, the LK8 protein used in this experiment was evaluated to be safe substance since it did not cause any toxic change in rats up to the level of 1,000 mg/kg in rats and its estimated LDSO values were much greater than 1, 000 mg/kg in rats (Table 2) .
<Table 2>

The number of deaths according to days after LK8 protein administration Amou Days after LK

protein nt The administration of number 0 1 2 3 4 5 6 7 8 9 1 1 1 1 1 admi of 0 1 2 3 4 nist deaths rati /Total on test (mg/ rats kg) INDUSTRIAL APPLICA~ILIT'.~
As explained hereinbefore, LK8 protein has an inhibiting effect on metastasis, in particular, on the growth of human prostatic cancer, lung cancer, colon cancer and rectal cancer as being systemically administered. Thus, an anticancer agent containing LK8 protein of the present invention can be effectively used as a treatment agent for a primary tumor or a metastasis inhibitor.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

SEQUENCE FISTING
<110> MOGAM BIOTECHNOLOGY INSTITUTE
<120> Anticancer agent comprising LK8 protein as an active ingredient <130> 2p-11-19 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 75 <212> PRT
<213> Homo Sapiens <400> 1 Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Lys Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Glu Trp Ala Ala Gln Glu Pro His Arg His Ser Thr Phe Ile Pro Gly Thr Asn Lys Trp Ala Gly Leu Glu Lys 3 5 40 4.5 Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ile Asn Gly Pro Trp Cys Tyr Thr Met Asn Pro Arg Lys Leu Phe Asp Tyr Cys <210>2 <211>34 <212>DNA

<213>Artificial Sequence <220>

<223>LKBN-XhoI
primer <400> 2 tccgctcgag aaaagagaac aagactgtat gttt 34 <210>3 <211>31 <212>DNA

<213>Artificial Sequence <220>

<223>LK8C-EcoRI
primer <400> 3 cgaattctta agaggatgca cagagaggga t 31

Claims

1. An anticancer agent containing LK8 protein represented by SEQ. ID. No 1 as an effective ingredient.

2. The anticancer agent as set forth in claim 1, wherein the effective dose of LK8 protein is 0.1 ~ 100 mg/kg.

3. The anticancer agent as set forth in claim 2, wherein the effective dose of LK8 protein is 1 ~ 50 mg/kg.

4. The anticancer agent as set forth in claim 1, wherein the agent is used for the inhibition of metastasis of cancer.

5. The anticancer agent as set forth in claim 1, wherein the metastasis of cancer is the metastasis of colon cancer or rectal cancer.

6. The anticancer agent as set forth in claim 1, wherein the agent is used for the treatment of a primary tumor.

7. The anticancer agent as set forth in claim 6, wherein the primary tumor is selected from a group consisting of prostate cancer, colon cancer, rectal cancer and lung cancer.