CN1753685A - Anticancer agent comprising lk8 protein as an active ingredient - Google Patents

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

Anticancer agent containing LK8 protein as an active ingredient

field of invention

The present invention relates to an anticancer agent containing a certain protein as an active ingredient, more specifically, an anticancer agent containing a cysteine kringle (kringle) KV38 with apolipoprotein (a) The corresponding protein is used as the active ingredient.

background

Tumors develop from uncontrolled, chaotic, abnormal cell proliferation. Tumors are considered malignant if they show destructive growth, invasion and metastasis. Invasiveness is the property of infiltrating or destroying surrounding tissue, in particular, the basal layer forming tissue boundaries is disrupted by this property, leading to local spread and sometimes tumor influx through the circulatory system. Metastasis refers to the spread of tumor cells from the primary tumor to other regions through lymphatic or blood vessels. Broadly speaking, metastatic also refers to the direct spread of tumor cells through the peritoneal cavity or other spaces.

So far, surgery, radiation therapy, and chemotherapy have been used alone or in combination for cancer treatment. Surgery is one way to remove diseased tissue. In this way, tumors in specific areas, such as the breast, colon, and skin, can be effectively removed surgically. However, tumors within the spine or scattered tumors such as leukemia cannot be properly treated surgically.

Chemotherapy blocks cell replication or metabolism and has been used in the treatment of breast, lung and testicular cancers. However, cancer patients receiving chemotherapy severely suffer from the side effects of systemic chemotherapy. Common but serious examples of these are motion sickness and vomiting. The side effects of chemotherapy can even affect the patient's life, because these side effects can quickly reduce the patient's fitness. In addition, dose-limiting toxicity (DLT) is also a major side effect of chemotherapy, which calls for careful attention to drug administration. Mucositis is an example of a DLT against anticancer drugs such as the antimetabolite cytotoxic agents 5-fluorouracil, methotrexate, and anticancer antibiotics such as doxorubicin. If a patient is severely affected by the side effects of chemotherapy, he or she should be hospitalized and given pain relievers to relieve suffering. Therefore, the side effects of chemotherapy or radiotherapy are the biggest problem in the treatment of cancer patients.

Therefore, there is an urgent need to develop an anticancer agent derived from the body to alleviate the side effects of chemotherapy. In particular, among substances produced in a living body, a promising substance was found that does not directly attack cancer cells but prevents the growth of cancer cells by acting on numerous endothelial cells that assist the growth of cancer cells. Therefore, anticancer agents containing this substance can not only treat cancer but also prevent metastasis.

Kringle is a protein structure comprising 80 amino acids and three intramolecular disulfide bonds. The Kringle structure is found in many proteins, such as prothrombin (Walz, D.A., etc., Proc.Natl.Acad.Sci., 74:1069-1073, 1977), urokinase (Pennica, D., etc., Nature, 301 : 579-582, 1983), hepatocyte growth factor (Lukker, N.A. et al., Protein Eng., 7: 895-903, 1994) and apolipoprotein (a) (hereinafter referred to as 'apo (a)') ( McLean, J.W. et al., Nature, 330:132-137, 1987). Kringle consists of a single folding unit. The function of Kringle has not yet been fully elucidated.

Apo(a) contains two kringle domains, KIV and KV, and an inactive protease-like domain. Kringle domain KIV is divided into 10 subtypes (KIV-1～KIV-10) according to amino acid homology, and 15-40 copy numbers of this domain are found in many alleles of human apo(a) gene. Apo(a) forms a lipoprotein(a) (hereinafter referred to as 'Lp(a)') through covalent association with apo B-100, a major form of low-density lipoprotein (LDL) Protein composition (Fless, G.M., J, Biol. Chem., 261:8712-8717, 1986). Increased Lp(a) in the cytoplasm is itself a major risk factor for atherosclerosis (Armstrong, V.W. et al., Artherosclerosis, 62: 249-257, 1986; Assmann, G, Am. J. Cardiol., 77: 1179 -1184, 1996).

Based on the fact that atherosclerosis and cancer cell growth depend on angiogenesis, the present inventors investigated the anticancer activity of KV38, which is a kind of human apo(a)kringle structure. As a result, the present inventors have completed the present invention and confirmed that this protein is effective as an anticancer agent because it inhibits angiogenesis, which is a key factor in the growth of cancer cells, by a bFGF-like endogenous growth factor. in is required.

Summary of the invention

An object of the present invention is to provide an anticancer agent comprising human apo(a)kringle KV38 (hereinafter referred to as LK8 protein) as an active ingredient.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In order to achieve the above objects of the present invention, the present invention provides an anticancer agent comprising LK8 protein as an active ingredient.

Hereinafter, the present invention is described in detail.

The anticancer agent of the present invention is characterized by containing LK8 protein having the amino acid sequence shown in SEQ.ID.No.1 as an active ingredient. Preferably this agent is used as a metastasis inhibitor, more preferably it is applied to inhibit colon or rectal cancer metastasis to the liver.

In addition, the anticancer agent is preferably used for the treatment of primary tumors. More preferably, the medicament is for the treatment of a cancer selected from the group consisting of prostate cancer, lung cancer, colon cancer and rectal cancer.

The anticancer agent in the present invention preferably contains 0.1-100 mg/kg LK8 protein, more preferably 1-50 mg/kg LK8 protein, and the administration frequency is 1-4 times a day. However, the composition is not limited to the above, and may vary depending on the condition of the patient, the kind and progress speed of the disease.

In the present invention, "KV38" refers to a kind of apo(a) kringle, and "LK8" refers to the recombinant protein of KV38. However, unless otherwise stated, both KV38 and LK8 proteins are collectively referred to as LK8 proteins.

The LK8 protein in the present invention is a domain corresponding to the KV38 kringle of many apo(a) kringle domains, and has an inhibitory effect on cancer cell proliferation, differentiation and metastasis by inhibiting the activity of endothelial cells in vitro and in vivo. As described in the preferred embodiment of the present invention, systemic administration of LK8 protein results in inhibition of primary tumors and their metastasis (see Figures 2-6). Therefore, due to its function of suppressing tumor growth and metastasis, the LK8 protein of the present invention can be effectively used as an anticancer agent against primary tumors and a metastasis suppressor.

If LK8 protein in the present invention is used together with conventional chemotherapy or radiotherapy, its therapeutic effect will be enhanced. Radiation therapy preferably destroys the primary tumor, and if LK8 protein is administered during radiation therapy, metastasis can be more effectively prevented. For chemotherapy, the cytotoxicity of large doses of chemical anticancer agents is the biggest problem. If the LK8 protein of the present invention is administered in chemotherapy, reduced doses of chemical anticancer agents will bring about the same or even improved anticancer effects and lessen cytotoxicity.

In conclusion, if LK8 is administered concurrently with surgery, radiotherapy, chemotherapy or immunotherapy, the therapeutic effect will be maximized. Furthermore, continuous administration of LK8 protein prolongs the dormancy of micrometastases, inhibits primary tumor growth and stabilizes the disease. Many conventional anticancer agents are designed for long-term administration, causing problems such as continuous production of proteins and high prices of products. Their replacement is gene therapy. If used in gene therapy, we still expect the LK8 protein to maximize the effect of anticancer agents or metastasis suppressors.

The anticancer agent containing LK8 protein of the present invention can be administered orally or parenterally and used in general forms of pharmaceutical preparations.

The anticancer agent can be prepared for oral or parenteral administration by mixing with common fillers, bulking agents, binders, wetting agents, disintegrating agents, diluents such as surfactants, or excipients. Solid preparations for oral administration include tablets, pills, powders, granules or capsules. These solid preparations are prepared by mixing one or more suitable excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin and the like. Besides simple excipients, lubricating agents such as magnesium stearate, talc, etc. may also be used. Liquid preparations for oral administration are suspensions, solutions, emulsions and syrups, which may contain various excipients such as wetting agents, sweeteners, fragrances and preservatives in addition to common diluents such as water and liquid paraffin. Preparations for parenteral administration are sterile aqueous solutions, water-insoluble excipients, suspensions, emulsions and suppositories. Water-insoluble excipients and suspensions may contain, in addition to one or more active compounds, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Suppositories may contain, in addition to one or more active compounds, witepsol, polyethylene glycol, Tween 61, cocoa butter, lauryl butter, glycerinated gelatin, and the like.

The LD ₅₀ of LK8 protein is about 1,000 mg/kg, indicating that the anticancer agent of the present invention is very safe (see Table 2).

Brief description of the drawings

Preferred embodiments of the present invention are best understood with reference to the accompanying drawings, in which:

Figure 1 is a schematic diagram of the expression vector 'pMBRI-LK8 (8.25kb)' of the LK8 gene, wherein the LK8 cDNA (261bp) is inserted between the AOX1 promoter and the AOX1 terminator.

Figure 2 is a set of photographs and a graph showing that the lung metastasis of murine melanoma cell line B16F10 injected in the tail vein of mice (C57BL/6) was inhibited by treatment with LK8 protein.

(a) Lungs of mice treated with PBS only.

(b) Lungs of mice treated with 1 mg/kg LK8 protein.

(c) Graph showing that the migration of B16F10 cells to the lungs of mice was inhibited by LK8 protein treatment.

Fig. 3 is a set of photographs and a graph showing that the metastasis of the colorectal cancer cell line CT-26 implanted in the mouse spleen to the liver was inhibited by LK8 protein treatment.

(a) Mouse livers treated with PBS (control) and LK8 protein (10 mg/kg/day).

(b) Graph showing the number of clones of CT-26 cells transferred into livers of mice treated with PBS (control) and LK8 protein (10 mg/kg/day).

(c) Photographs showing the distribution of CT-26 cancer cells metastasized into livers of mice treated with PBS (control) and LK8 protein (10 mg/kg/day), stained with hematoxylin & eosin.

Fig. 4 is a set of graphs showing changes in tumor size in mice implanted with human prostate cancer PC-3 cells following administration of LK8 protein.

(a) Graph showing changes in tumor size after treatment with 100 mg/kg/day LK8 protein.

(b) Graph showing changes in tumor size after treatment with 50 mg/kg/day LK8 protein.

Fig. 5 is a graph showing changes in tumor size in mice implanted with human lung cancer A549 cells following administration of LK8 protein.

Fig. 6 is a set of graphs showing changes in tumor size (a) and tumor weight (b) in mice implanted with human colorectal cancer LS174T cells following the administration of LK8 protein.

Example

Practical and presently preferred embodiments of the invention are illustrated in the following examples.

However, those skilled in the art will appreciate that, in light of this disclosure, modifications and improvements can be made within the spirit and scope of the invention.

Embodiment 1: LK8 protein preparation

<1-1> Construction of LK8 expression vector pMBRI-LK8

In order to efficiently prepare LK8 protein, the inventors first constructed an LK8 expression vector.

For expression of the LK8 gene, pPIC9 vector (8.0 kb, Invitrogen, Netherlands) was used as the base vector. As shown in the schematic diagram, the sequence of the pPIC9 expression vector contains the AOX1 promoter that can provide methanol-induced high expression, the α-factor secretion signal that can make the expressed protein secreted, the 3'AOX1 (TT), the efficient termination of transcription and the polyglandular The glycosylated AOX1 polyadenylation signal, and a DNA fragment encoding wild-type Pichia pastoris histidinol dehydrogenase were used as selectable markers for transformants of the above strains.

First, the LK8 gene was amplified by PCR with the primer 'LK8N-Xhol' shown in SEQ.ID.No 2 and the primer 'LK8C-EcoRI' shown in SEQ.ID.No 3, and pET15b/LK8 (see PCT/KR99 /00554) as a template. The PCR product was digested with restriction enzymes XhoI and EcoRI, and then the digested product was inserted into pPIC9 vector digested with the same restriction enzymes. Finally, construct the LK8 gene expression vector 'pMBRI-LK8 (8.25kb) (Figure 1).

<1-2> Preparation of transformants containing pMBRI-LK8

Pichia pastoris, a methylotrophic yeast, was used as a host for recombinant transformants.

Specifically, the LK8 gene expression vector 'pMBRI-LK8' was treated with restriction endonuclease SacI to form linearity. This vector is inserted into the AOXI gene of the above-mentioned host strain chromosome by homologous recombination. At this point, transformation is performed by electroporation. Recombinant yeast transformants were selected from histidine-deficient media by checking for colony formation. Use PCR to verify whether the LK8cDNA has been inserted into the AOX1 region of the selected recombinant transformants. Then, the recombinant transformants were cultured and the expression of LK8 gene was induced by methanol. As a result, the expression of the LK8 gene was verified, indicating that the protein is secreted in a large amount in the culture medium.

The secreted LK8 protein in the present invention consists of the amino acid sequence shown in SEQ.ID.No 1.

<1-3>Cultivation of recombinant strains

<1-3-1> Seed cultivation

In the present invention, a recombinant strain is obtained by inserting the LK8 gene into Pichia pastoris. The constructed strains were seed cultured for 24 hours to obtain appropriate biomass and activity (OD ₆₀₀ was 0.8-1.2 when diluted 20 times).

The seed culture was shaken in YDP medium (1% yeast extract, 2% peptone, 2% glucose) for 24 hours. A 75L fermenter was used. The initial medium volume was 20 L, and the final culture volume was adjusted to 40 L by fed-batch culture.

<1-3-2> Main culture

After completion of seed culture in YPD medium, main culture was carried out with the seed medium as 30% of the initial medium. The main culture was carried out in a fermenter containing the ingredients shown in Table 1 below. When the fermentation was saturated with methanol supply, more than 10% of the ferment was recovered to produce LK8 protein. Simultaneously, methanol was continuously added to induce sustained protein expression. This process was repeated to produce LK8 protein. The rate of carbon source consumption is proportional to the number of cells. Therefore, when a part of the fermented product is recovered, the supply rate of methanol is automatically adjusted within ±20%. By repeating the above-mentioned culture process, wherein the fermentation was carried out continuously for more than 200 hours, the protein culture solution of 250 mg/L LK8 secreted was obtained.

<Table 1>

The composition of the medium for the main culture Medium type Element concentration main medium Methanol Trace Metal Solution 500g/L8ml/L Micro heavy metal solution medium composition CuSO ₄ 5H ₂ OKIMnSO ₄ H ₂ ONaMo H ₂ OH ₃ BO ₄ CoCl ₂ ZnCl ₂ FeSO ₄ H ₂ O biotin H ₂ SO ₄ 4g/L0.3g/L4g/L0.1g/L0.01g/L0.1g/L3g/L10g/L0.1g/L5ml/L

Example 2: Lung metastasis experiment by intravenous injection of B16F10 murine melanoma cells

B16F10 cells (1.8×10 ⁵ ), mouse melanoma (hereinafter referred to as 'melanoma cells') (American Type Culture Collection), were injected into the tail vein of C57BL/6 mice (Charles River Japan, Inc.). From the next day, the LK8 protein prepared by the above <Example 1> was subcutaneously injected twice a day for 14 consecutive days (1 mg/kg/day, 0.2 mg/kg/day). The control group was injected with PBS instead of LK8 protein. On the 13th day of cell implantation, the mice were cut open and the lungs were taken to count the clones of metastatic cancer cells (melanoma cells).

As a result, giant colonies were formed in the lungs of the PBS-injected control mouse group, indicating the metastasis of melanoma cells (Fig. 2). In contrast, the number and size of clones formed by melanoma cell metastasis in the experimental mouse group injected with LK8 protein were much smaller and less (Fig. 2b). Overall, LK8 protein treatment at 1 mg/kg showed 53% inhibition of metastasis compared to the control group (Fig. 2c).

Example 3: Liver metastasis experiment by intrasplenic administration of mouse colorectal cancer cell CT-26

CT-26 cells (American Type Culture Collection), mouse colorectal cancer cells, were injected into the spleen to induce metastasis to the liver. Then, the metastasis suppressing effect of LK8 protein was investigated. Specifically, CT-26 cells, grown to 80% maturity on plates, were washed with PBS and then dispersed with 0.02% EDTA. Single cells were washed again with PBS and carefully resuspended in PBS. The suspension was stained with trypan blue to count the cells. The cell density was adjusted to 5×10 ⁵ /ml, and each mouse was injected with 100 μl. 6-8 week old BALB/c mice (Charles River Japan, Inc.) were used. After surgical incision on the right side of the abdomen, the cancer cell suspension was carefully injected into the spleen with a 30-gauge needle. LK8 protein was subcutaneously injected into the experimental group twice a day, 10 mg/kg/day, similarly, saline was injected into the control group. After 14 days, the mice were sacrificed to examine their livers (Fig. 3a), and the number of clones seen on the liver surface, which had been fixed with 10% formalin solution, was counted. There were no major differences in the weights of the groups. However, the number of clones transferred to the liver in the experimental group administered with 10 mg/kg/day LK8 protein was much less than that in the control group. Compared with the control group, the number of clones in the experimental group injected with 10 mg/kg/day LK8 protein decreased by about 60% (Fig. 3b). In addition, formalin-fixed liver tissue sections were observed by H&E staining. As a result, the tumor area was much more limited in the experimental group treated with LK8 than in the control group (Fig. 3c).

Example 4: Growth Inhibition of Primary Tumors

In order to study the inhibitory effect of LK8 protein on angiogenesis in vivo, a xenograft tumor model was used. For each related experiment, 4-week-old randomly crossed female Balb/c nu/nu nude mice (Charles River Japan, Inc.) were used and fed under sterile conditions.

<4-1> Human Prostate Cancer Cell (PC-3)

Human prostate cancer PC-3 cells (American Type Culture Collection) were cultured in RPMI 1640 medium (GIBCO ^TM , Invitrogen Corporation) supplemented with 10% FBS, and then approximately 5×10 ⁶ PC-3 cells were subcutaneously injected into the central muscle of the back of nude mice district. LK8 protein was injected at 100 mg/kg/day for exactly 10 days after implantation. Meanwhile, the control group was only injected with PBS instead of LK8 protein. This treatment was continued for 30 days, after which the size of the tumors was measured every 3 or 4 days. The results showed that the growth of tumor was inhibited by LK8 protein, about 60% inhibition compared with the control group (Fig. 4a). When LK8 protein was injected at 50 mg/kg/day, tumor growth was inhibited in a similar manner with more than 60% inhibition compared to control (Fig. 4b).

<4-2> Human lung cancer cells (A549)

Human lung cancer A549 cells (American Type Culture Collection) were cultured in DMEM medium (GIBCO ^™ , Invitrogen Corporation) supplemented with 10% FBS. Then 1×10 ⁷ tumor cells were subcutaneously injected into the central muscle area of the back of the nude mice. Exactly 5 days after implantation, LK8 protein was injected at 50 mg/kg/day. The control group was administered only PBS instead of LK8 protein. Treatment was continued for 46 days, after which tumor size was measured every 3 or 4 days. The results showed that tumor growth was inhibited by the treatment of LK8 protein by 61% compared with the control group ( FIG. 5 ).

<4-3> Human colorectal cancer cells (LS 174T)

Human colorectal cancer LS 174T cells (American Type Culture Collection) were cultured in RPMI medium (GIBCO ^™ , Invitrogen Corporation) supplemented with 10% FBS. Then 5×10 ⁶ tumor cells were subcutaneously injected into the central muscle area of the back of the nude mice. Exactly 5 days after implantation, LK8 protein was injected at 50 mg/kg/day. The control group was administered only PBS instead of LK8 protein. Treatment was continued for 34 days, and tumor sizes were then measured every 3 or 4 days. The results showed that tumor growth was inhibited by LK8 protein treatment with 64% inhibition compared with the control group (Fig. 6a). Moreover, measurements on the last day of the experiment showed that LK8 protein treatment reduced tumor weight by 68.7% (Fig. 6b)

Embodiment 5: Acute toxicity test of LK8 protein

Five-week-old SPF SD (Sprague Dawley) rats were used for acute toxicity experiments. The rats were divided into 5 groups, and each of these 5 groups of rats was given LK8 protein 260mg/kg, 364mg/kg, 510mg/kg, 714mg/kg and 1000mg/kg by intravenous injection (Table 2). After 14 days, the experimental material, LK8 protein, medication, death, clinical symptoms and weight changes in the rats were observed. Blood phase and blood biochemical tests are performed, and any abnormalities in the gastrointestinal or thoracic and abdominal organs are checked with the naked eye during autopsy. The results showed that slight toxicity was detected in the group given 1000 mg/kg LK8 protein, but neither toxicity nor death occurred in most of the tests in the other groups, including weight change, blood tests, blood phase tests and blood biochemical tests Examination, autopsy, etc. Therefore, the LK8 protein used in this experiment is considered a safe substance because it does not cause any toxic changes in rats at a level as high as 1,000 mg/kg in rats, and its estimated LD ₅₀ value is in large Much higher than 1,000mg/kg in mice (Table 2).

<Table 2>

Number of deaths as a function of days after LK8 protein administration Application amount (mg/kg) Number of deaths/total number of experimental mice Days after LK8 protein administration 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 260 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 364 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 510 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 714 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1000 3/5 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0

industrial application

As mentioned above, LK8 protein has inhibitory effect on metastasis, especially, growth inhibition on human prostate, lung, colon and rectal cancer when administered systemically. Therefore, the anticancer agent comprising the LK8 protein of the present invention can be effectively used as a therapeutic agent or a metastasis suppressor for primary tumors.

It should be appreciated by those skilled in the art that the conception and specific embodiment 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 Listing

<110> Muyan Institute of Biotechnology

<120>Anticancer agent containing LK8 protein as an active ingredient

<130>2p-11-19

<160>3

<170>KopatentIn 1.71

<210>1

<211>75

<212>PRT

<213> Human

<400>1

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Lys Ala Thr Thr

1 5 10 15

Val Thr Gly Thr Pro Cys Gln Glu Trp Ala Ala Gln Glu Pro His Arg

20 25 30

His Ser Thr Phe Ile Pro Gly Thr Asn Lys Trp Ala Gly Leu Glu Lys

35 40 45

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ile Asn Gly Pro Trp Cys Tyr

50 55 60

Thr Met Asn Pro Arg Lys Leu Phe Asp Tyr Cys

65 70 75

<210>2

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> LK8N-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 (7)

1. an anticarcinogen comprises the LK8 albumen shown in the SEQ.ID.No1 as effective ingredient.

2. the anticarcinogen described in claim 1, wherein the proteic effective dose of LK8 is 0.1～100mg/kg.

3. the anticarcinogen described in claim 2, wherein the proteic effective dose of LK8 is 1～50mg/kg.

4. the anticarcinogen described in claim 1, wherein this medicament is used for the inhibition of cancerometastasis.

5. the anticarcinogen described in claim 1, wherein cancer metastasis is the transfer of colon cancer or rectal cancer.

6. the anticarcinogen described in claim 1, wherein this medicament is used for the treatment of primary tumor.

7. the anticarcinogen described in claim 6, wherein primary tumor is selected from the group of being made up of carcinoma of prostate, colon cancer, rectal cancer and pulmonary carcinoma.

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