RU2306147C2 - Anticancer agent containing lk8 protein as active ingredient - Google Patents

Abstract

FIELD: medicine, anticancer agents.

SUBSTANCE: invention relates to anticancer agent containing LK8 protein SEQ ID NO 1 as active ingredient which effectively suppresses malignant tumor growth and metastasis. Agent of present invention may be used both in cancer metastasis suppression and in therapy of primary malignant tumor.

EFFECT: agent with increased antitumor activity.

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Description

Field of Invention

The present invention relates to an anticancer agent containing a specific protein as an active ingredient, more specifically, to an anticancer agent containing a protein corresponding to kringle KV38 of apolipoprotein (a) as an active ingredient.

State of the art

A tumor develops through uncontrolled, random, abnormal cell proliferation. If the specified tumor shows destructive growth, invasiveness and metastasis, it is considered a malignant tumor. Invasiveness is the property of infiltrating and destroying surrounding tissues, and, in particular, the basal layer that forms the tissue border is destroyed as a result of this property, which leads to local spread and, sometimes, the tumor enters the circulatory system. Metastasis means the spread of tumor cells from the primary focus to other areas through the lymphatic or blood vessels. In a broad sense, metastasis also means the direct spread of tumor cells through the peritoneal cavity or other spaces.

Currently, for the treatment of malignant tumors using surgery, radiation therapy and chemotherapy, individually or together. Surgery is a way to remove pathological tissues. So tumors in specific areas, such as the mammary gland, colon and skin, can be effectively removed with surgery. However, spinal tumors or scattered tumors, such as leukemia, cannot be adequately treated with surgery.

Chemotherapy blocks the replication or metabolism of cells, and it is used to treat breast cancer, lung cancer, and testicular cancer. However, patients with malignant tumors treated with chemotherapy suffer severely from the side effects of systemic chemotherapy. Nausea and vomiting are among the most common but severe effects. Side effects of chemotherapy can affect the patient's life, as they can quickly reduce his adaptability. In addition, dose limiting toxicity (DLT) is also one of the main side effects of chemotherapy, which requires careful attention when administering the drug. Mucositis is an example of DLT against anti-cancer agents such as 5-fluorouracil, which is an antimetabolic cytotoxic agent, and methotrexate, as well as anti-cancer antibiotics such as doxorubicin. If the patient is seriously suffering from these side effects of chemotherapy, he or she should be hospitalized and anodine administered to reduce pain. Thus, the side effects of chemotherapy and radiation therapy are the biggest problem in the treatment of patients with malignant tumors.

Thus, there is an urgent need to develop an anticancer agent derived from a living creature in order to reduce the side effects of chemotherapy. In particular, among the substances produced in a living creature, a promising subject was found that does not attack cancer cells directly, but prevents the growth of cancer cells, acting against various endothelial cells that contribute to the growth of cancer cells. Thus, an anticancer agent containing the specified subject can not only treat malignant tumors, but also prevent metastasis.

Kringle is a type of protein structure that consists of 80 amino acids and three intramolecular disulfide bonds. The kringle structure is found in many proteins, such as throthrombin (Walz, DA et al., Proc. Natl. Acad. Sci., 74: 1069-1073, 1977), urokinase (Pennica, D. et al., Nature, 301: 579-582, 1983), hepatocyte growth factor (Lukker, NA et al., Protein Eng., 7: 895-903, 1994) and apolipoprotein (a) (hereinafter referred to as "apo (a)") ( McLean, JW et al., Nature, 330: 132-137, 1987). Kringle consists of a single collapsed unit. The kringle functions still do not have a clear explanation.

Apo (a) includes two types of kringle domains, KIV and KV, and an inactive proteinase-like site. The kringle KIV domain is divided into 10 subtypes (KIV-1 ~ KIV-10), according to amino acid homology, and 15 ~ 40 copies of this domain are found in different alleles of the human apo (a) gene. Apo (a) forms lipoprotein (a) (hereinafter referred to as “Lp (a)”) by covalent bonding with apo B-100, the main protein component of low density lipoprotein (LDL) (Fless, GM, J. Biol. Chem., 261: 8712-8717, 1986). An increase in Lp (a) in the cytoplasm itself is a major risk factor for atherosclerosis (Armstrong, VW et al., Artherosclerosis, 62: 249-257, 1986; Assmann, G., Am. J. Cardiol., 77: 1179-1184 , 1996).

Based on the fact that arteriosclerosis and cancer cell growth are dependent on angiogenesis, the applicants studied the anticancer activity of KV38, one of the human aro (a) kringle. As a result, the applicants completed the present invention, confirming that the protein can be effectively used as an anticancer agent, since it inhibits angiogenesis by an endogenous growth factor, such as bFGF, which is necessary for the growth of cancer cells.

SUMMARY OF THE INVENTION

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

Detailed Description of Preferred Embodiments of the Present Invention

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

Further, the present invention is described in detail.

The anticancer agent of the present invention is characterized by an LK8 protein having the amino acid sequence represented by SEQ. ID No. 1, as an active ingredient. It is preferable to use the agent as an inhibitor of metastasis, more preferably it is used to inhibit metastasis of colon cancer or colon cancer in the liver.

In addition, an anticancer agent is used to treat primary tumors. More preferably, the agent is used to treat cancer selected from the group consisting of prostate cancer, lung cancer, colon cancer, and colon cancer.

The anticancer agent of the present invention preferably contains LK8 protein in an amount of 0.1 ~ 100 mg / kg, more preferably contains LK8 protein in an amount of 1 ~ 50 mg / kg, and the frequency of administration is 1 ~ 4 times per day. However, the composition is not limited to these parameters, and changes are possible in accordance with the condition of the patient and the type and rate of disease progression.

In the present invention, “KV38” means apo (a) kringle, and LK8 means a recombinant KV38 protein. However, both the KV38 protein and the LK8 protein are commonly referred to as the LK8 protein, unless specifically indicated otherwise.

The LK8 protein of the present invention is a domain corresponding to the KV38 kringle of many apo (a) kringle domains and has an inhibitory effect on the proliferation and differentiation of cancer cells and metastasis by suppressing endothelial cell activity in vitro as well as in vivo. As explained in a preferred embodiment of the present invention, systemic administration of the LK8 protein leads to inhibition of the primary tumor and its metastasis (see FIG. 2 ~ FIG. 6). Thus, the LK8 protein of the present invention can be effectively used as an anticancer agent, especially for primary tumors, as well as an inhibitor of metastasis, due to its properties of suppressing tumor growth and metastasis.

The effect of the treatment will be enhanced if the LK8 protein of the present invention is used in conjunction with usually chemotherapy or radiation therapy. Radiation therapy is carried out in order to destroy the primary tumor, and if the LK8 protein is administered during radiation therapy, metastasis can be more effectively prevented. As for chemotherapy, cytotoxicity caused by a huge dose of chemical anticancer agents is the biggest problem. If the LK8 protein of the present invention is administered during chemotherapy, a reduced amount of chemical anticancer agents will have an equivalent or even improved anticancer effect and also reduce cytotoxicity.

In conclusion, we can say that if the introduction of LK8 protein is carried out in conjunction with surgery, radiation therapy, chemotherapy or immunotherapy, the effect of treatment will be maximum. And in addition, continuous administration of LK8 protein lengthens the resting state of micrometastases, inhibits the growth of the primary tumor and stabilizes the condition. Many common anticancer agents are designed for long-term administration, causing problems such as long-term protein production and the high cost of the product. An alternative for them is gene therapy. It is also expected that the LK8 protein maximizes the effect of an anticancer agent or metastasis inhibitor if it is used for gene therapy.

The anti-cancer agent containing the LK8 protein of the present invention can be administered orally or parenterally and used in conventional forms of pharmaceutical compositions.

An anticancer agent can be formulated for oral or parenteral administration by mixing with conventional excipients, binders, wetting agents, disintegrants, diluents such as surfactants or excipients. Solid compositions for oral administration are tablets, pills, dusting powders, granules and capsules. These solid compositions are prepared by mixing with one or more suitable excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple fillers, lubricants such as magnesium stearate, talc, etc. can be used. Liquid compositions for oral administration are suspensions, solutions, emulsions and syrups, and the above compositions may contain various excipients, such as moisturizing agents, sweeteners, flavorings and preservatives, in addition to commonly used simple diluents, such as water or liquid paraffin. Compositions for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions and suppositories. Water-insoluble excipients and suspensions may contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil, such as olive oil, an ester for injection, such as ethyl oleate and the like. Suppositories may contain, in addition to the active compound or compounds, vitepsol, macrogol, tween 61, cocoa butter, lauric oil, glycerin gelatin, and the like.

The LD ₅₀ of LK8 protein is approximately 1000 mg / kg, which indicates the very high safety of the anti-cancer agent of the present invention (see table 2).

Brief Description of the Drawings

The implementation of the preferred variants of the present invention can be better understood using the accompanying drawings.

FIG. 1 is a schematic representation of the pKBRI-LK8 (8.25 kb) expression vector of the LK8 gene, in which the LK8 cDNA (261 bp) is inserted between the AOX1 promoter and the AOX1 terminator.

FIG. 2 is a series of photographs and a graph showing that lung metastasis of the mouse melanoma cell line, B16F10, which was injected into the tail vein of mice (C57BL / 6), is inhibited by treatment with LK8 protein,

(a) the lung of a mouse treated only with PBS,

(b) lung of a mouse treated with 1 mg / kg LK8 protein,

(c) a graph showing that metastasis of B16F10 cells to mouse lung is inhibited by treatment with LK8 protein.

FIG. 3 is a series of photographs and a graph showing that liver metastasis of the CT-26 mouse colorectal cancer cell line transplanted into mouse spleen is inhibited by treatment with LK8 protein,

(a) the liver of a mouse treated with PBS (control) and LK8 protein (10 mg / kg per day),

(b) a graph showing the number of colonies of CT-26 cells metastasized to the liver of a mouse treated with PBS (control) and LK8 protein (10 mg / kg per day),

(c) photographs showing the distribution of CT-26 cancer cells metastasized to the liver of a mouse treated with PBS (control) and LK8 protein (10 mg / kg per day), which was observed with hematoxylin-eosin staining.

FIG. 4 is a series of graphs showing changes in tumor size in a mouse transplanted with PC-3 human prostate cancer cells in accordance with the administration of the LK8 protein,

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

(b) a graph showing changes in tumor size after treatment with LK8 protein of 50 mg / kg per day.

FIG. 5 is a graph showing changes in tumor size in a mouse transplanted with A549 human lung cancer cells in accordance with the administration of the LK8 protein.

FIG.6 is a series of graphs showing changes in tumor size (a) and tumor mass (b) in mice transplanted with colon cancer cells and human colon cancer LS 174T, in accordance with the introduction of protein LK8.

Examples

Practical and currently preferred embodiments of the present invention are illustrative, as shown in the following examples.

However, one should appreciate the fact that specialists, taking into account the present description, can make modifications and improvements within the scope of the idea and scope of the present invention.

Example 1: Production of LK8 Protein

<1-1> Construction of the expressing vector LK8 pMBRI-LK8

In order to efficiently produce the LK8 protein, applicants first constructed the LK8 expression vector.

For expression of the LK8 gene, the pPIC9 vector (8.0 kb, Invitrogen, Netherlands) was used as the base vector. As can be seen on the representative map, the pPIC9 expression vector includes, in order, the AOX1 promoter, which provides high methanol expression, the α-factor secretion signal, which includes the secretion of the expressed protein, 3'AOX1 (TT), the AOX1 polyadenylation signal, which provides an effective termination of transcription and polyadenylation, and a DNA fragment encoding the wild-type histidinol dehydrogenase Pichia pastoris, which is used as a selectable marker for transforming the above strain.

First, the LK8 gene was amplified by PCR using the primers "LK8N-Xhol" represented by SEQ. ID No. 2 and "LK8C-EcoRI" represented by SEQ. ID No. 3, using pET15b / LK8 (see PCT / KR99 / 00554) as a matrix. The PCR product was enzymatically digested using restriction enzymes XhoI and EcoRI, followed by subcloning by insertion of the product into the pPIC9 vector, which was enzymatically digested using the same restriction enzymes. Ultimately, an expression vector of the LK8 gene, “pMBRI-LK8” (8.25 kb), was constructed.

<1-2> Obtaining a transformant comprising pMBRI-LK8

Pichia pastoris, a methylotropic yeast fungus, was used as a host to produce a recombinant transformant.

In particular, the LK8 gene expression vector, "pMBRI-LK8", was treated with the SacI restriction enzyme, which provided linearization. The vector was inserted into the AOXI gene of the chromosome of the above host strain using homologous recombination. At the same time, electroporation was performed for transformation. Recombinant yeast transformant was selected from histidine-deficient media by studying colony formation. PCR was performed to confirm the insertion of LK8 cDNA into the AOX1 region of the chromosome of the selected recombinant transformant. Then, the recombinant transformant was cultured, and LK8 gene expression was induced by methanol. As a result, the expression of the LK8 gene was confirmed, indicating mass secretion of the protein in the culture medium.

The secreted LK8 protein of the present invention was composed of the amino acid sequence represented by SEQ. ID Number 1.

<1-3> Cultivation of the recombinant strain

<1-3-1> Sowing culture

In the present invention, a recombinant strain was obtained by insertion of the LK8 gene in Pichia pastoris. The established strain was cultured after seeding for 24 hours to obtain the appropriate biomass and activity (when diluted 20 times, the OD ₆₀₀ was 0.8-1.2).

A seed culture was obtained in YDP medium (1% yeast extract, 2% peptone, 2% dextrose) for 24 hours with shaking of the culture. A 75 liter fermenter was used. The volume of the initial medium was 20 L, and the final volume of the culture solution was adjusted to 40 L by the fed culture method.

<1-3-2> Main culture

After cultivation of the seed culture in YPD medium, the main culture was obtained using a seed culture solution in an amount of 30% of the primary medium. The main culture was obtained in a fermenter containing the ingredients listed in table 1 below. When the enzyme was saturated with methanol, the enzyme was reduced to more than 10% to produce LK8 protein. Meanwhile, methanol was continuously fed to induce continuous expression of the protein. The procedure was repeated to obtain LK8 protein. The rate of consumption of the carbon source was proportional to the number of cells. Thus, when a certain amount of the enzyme was restored, the feed rate of methanol was automatically adjusted so that it did not go beyond ± 20%. As a result of the repetition of the above cultivation process, during which fermentation lasted more than 200 hours, secreted LK8 protein was obtained in an amount of 250 mg per 1 liter of culture solution.

Table 1
The composition of the medium for the main culture Types of environment Component Concentration The environment for the main culture Methanol
Metal solution
trace elements 500 g / l
8 ml / l Solution composition
metal
trace elements CuSO ₄ · 5H ₂ O
Ki
MnSO ₄ H ₂ O
NaMo · H ₂ O
H ₃ BO ₄
CoCl ₂
ZnCl ₂
FeSO ₄ · H ₂ O
Biotin
H ₂ SO ₄ 4 g / l
0.3 g / l
4 g / l
0.1 g / l
0.01 g / l
0.1 g / l
3 g / l
10 g / l
0.1 g / l
5 ml / l

Example 2: Experimental lung metastasis using intravenous administration of murine melanoma B16F10 cells

Murine melanoma B16F10 (1.8 × 10 ⁵ ) cells (hereinafter referred to as “melanoma cells”) (American Type Culture Collection) were introduced into the tail vein of a C57BL / 6 mouse (Charles River Japan, Inc.). Starting from the next day, the LK8 protein obtained in the above <Example 1> was injected subcutaneously twice (1 mg / kg per day, 0.2 mg / kg per day) per day for 14 days. The control group was injected with PBS instead of LK8 protein. On the 13th day after cell transplantation, the mouse was opened and the lung was removed to count the number of colonies of metastatic malignant cells (melanoma cells).

As a result, huge colonies formed in the lungs of the mice of the control group, which were injected with PBS, which indicates the transfer of melanoma cells (FIG. 2A). In contrast, the number and size of the colonies formed by the transfer of melanoma cells were significantly smaller in the experimental group of mice injected with LK8 protein (FIG. 2b). In conclusion, we can say that in the group treated with LK8 protein 1 mg / kg, there was a 53% inhibition of metastasis compared with the control group (FIG. 2c).

Example 3: Experimental liver metastasis using intravenous administration of CT-26 mouse colorectal cancer cells

CT-26 cells (American Type Culture Collection), mouse colorectal cancer cells, were injected into the spleen to induce liver metastasis. Then studied the effect of inhibiting the formation of metastases of the protein LK8. In particular, CT-26 cells, which were 80% grown on a plate, were washed with PBS, followed by singularization of 0.02% EDTA. Single cells were washed again with PBS, then carefully resuspended in PBS. The suspension was stained with trypan blue to count the number of cells. The cell density was adjusted to 5 · 10 ⁵ / ml, which were then injected into each mouse in 100 μl. Used 6 ~ 8-week-old Balb / c mice (Charles River Japan, Inc.). After a surgical incision in the right abdomen, a suspension of cancer cells was carefully injected into the spleen with a 30 gauge needle. LK8 protein was subcutaneously injected to the experimental group twice a day at 10 mg / kg, and physiological saline was injected in the same way to the control group. After 14 days, the mice were sacrificed for liver examination (FIG. 3a) and the number of colonies visible on the surface of the liver, which was fixed in a 10% formalin solution, was counted. There was not much difference in weight between the groups. However, the number of colonies that were transferred to the liver was significantly less in the experimental group, which was injected with LK8 protein in the amount of 10 mg / kg per day than in the control group. The decrease in the number of colonies in the experimental group, which was injected with LK8 protein in the amount of 10 mg / kg per day, was approximately 60%, compared with the control group (FIG. 3b). In addition, a section of liver tissue that was fixed in a formalin solution was stained with hematoxylin-eosin and studied. As a result, the tumor region was significantly limited in the experimental group treated with LK8 protein, compared with the control group (FIG. 3c).

Example 4: Inhibition of Primary Tumor Growth

In order to study the effect of LK8 protein on in vivo antiangiogenesis, a xenograft tumor model was used. Each relevant experiment was performed using 4-week-old, blindly selected female Balb / c nu / nu nude mice (Charles River Japan, Inc.) that were grown under sterile conditions.

<Human Prostate Cancer (RS-3)>

Human prostate cancer cells PC-3 (American Type Culture Collection) were cultured in RPMI 1640 medium (GIBCO ™, Invitrogen Corporation) supplemented with 10% FBS and approximately 5 · 10 ⁶ PC-3 cells were injected subcutaneously into the central muscle region of the athymic mouse . Exactly 10 days after transplantation, LK8 protein was injected in an amount of 100 mg / kg per day. At the same time, only PBS was injected into the control group instead of LK8 protein. The treatment was continued for 30 days, then the tumor size was measured every 3-4 days. As a result, tumor growth was suppressed by treatment with LK8 protein, which was approximately 60% inhibition compared to the control group (FIG. 4a). In the case when the LK8 protein was injected in an amount of 50 mg / kg per day, tumor growth was similarly suppressed by more than 60% compared with the control group (FIG. 4b).

<4-2> Cancer of the lung of a person (A549)

A549 human lung cancer cells (American Type Culture Collection) were cultured in DMEM (GIBCO ™, Invitrogen Corporation) supplemented with 10% FBS. Then, approximately 1 · 10 ⁷ tumor cells were subcutaneously injected into the central muscle region of the back of the athymic mouse. Exactly 5 days after transplantation, LK8 protein was injected in an amount of 50 mg / kg per day. The control group was only injected with PBS instead of LK8 protein. Treatment was continued for 46 days, then the tumor size was measured every 3-4 days. As a result, tumor growth was suppressed by treatment with LK8 protein, which was 61% inhibition compared to control (FIG. 5).

<4-3> Cancer of the colon and rectum of a person (LS 174T)

Human colon and rectal cancer cells LS 174T (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 region of the back of a nude mouse. Exactly 5 days after transplantation, LK8 protein was injected in an amount of 50 mg / kg per day. Only PBS was administered to the control group instead of LK8 protein. Treatment was continued for 34 days, then tumor size was measured every 3-4 days. As a result, tumor growth was suppressed by treatment with LK8 protein, which was 64% inhibition compared to the control (FIG. 6a). Also, the tumor weight decreased during treatment with LK8 protein by approximately 68.7%, as measured on the last day of the experiment (FIG.6b).

Example 5: LK8 Protein Acute Toxicity Test

In the acute toxicity test, 5-week-old SPF SD rats (Sprague Dawley) were used. Rats were divided into 5 groups and 5 rats of each group were injected once with LK8 protein at doses of 260 μg, 364 mg / kg, 510 mg / kg, 714 mg / kg and 1000 mg / kg, respectively, by intravenous injection (table 2). Within 14 days after administration of the test material, LK8 protein, death, clinical symptoms, and changes in body weight were observed. Hematological tests and biochemical blood tests were performed, and any pathological signs in the gastrointestinal tract, organs of the chest and abdomen were examined visually during the autopsy. As a result, mild toxicity was detected in the group that was injected with 1000 mg / kg of LK8 protein, but neither toxicity nor death was detected in other groups in most tests, including changes in body weight, blood tests, hematological tests and biochemical blood tests, autopsy, etc. Thus, the LK8 protein used in this experiment was evaluated as a safe substance because it did not cause any toxic changes in rats up to the level of 1000 mg / kg, and its established LD50 values were significantly greater than 1000 mg / kg for rats (table 2).

table 2
The number of deaths by days after administration of LK8 protein The entered amount (mg / kg) Number of deaths /
total number of rats in the test Days After LK8 Protein Administration 0 one 2 3 four 5 6 7 8 9 10 eleven 12 13 fourteen 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 APPLICABILITY

As explained earlier in this document, the LK8 protein has an inhibitory effect on metastasis, in particular on the growth of human prostate cancer, lung cancer, colon cancer and colon cancer, when administered systemically. Thus, the anticancer agent containing the LK8 protein of the present invention can be effectively used as an agent for treating a primary tumor or an inhibitor of metastasis.

Those skilled in the art will appreciate the fact that the concepts and specific embodiments of the present invention disclosed herein can easily be used as a basis for modifying or developing other embodiments of the same objectives of the present invention. Those skilled in the art will also appreciate the fact that these equivalent embodiments of the present invention do not depart from the idea and scope of the present invention as set forth in the appended claims.

Claims (7)

1. An anti-cancer agent containing LK8 protein represented by SEQ ID NO 1 as an effective ingredient. 2. The anti-cancer agent of claim 1, wherein the effective dose of the LK8 protein is 1 ~ 100 mg / kg. 3. The anti-cancer agent according to claim 2, wherein the effective dose of LK8 protein is 1 ~ 50 mg / kg. 4. The anticancer agent according to claim 1, wherein the agent is used to inhibit cancer metastasis. 5. The anticancer agent according to claim 1, wherein the cancer metastasis is metastasis of colon cancer or colon cancer. 6. The anti-cancer agent according to claim 1, wherein the agent is used to treat a primary tumor. 7. The anti-cancer agent of claim 6, wherein the primary tumor is selected from the group consisting of prostate cancer, colon cancer, colon cancer, and lung cancer.

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