KR20210031110A - Compositions for treating sorafenib-resistant liver cancer and Methods for providing information on treating sorafenib-resistant liver cancer - Google Patents

Abstract

The present invention relates to a composition for treating sorafenib-resistant liver cancer and a method for providing information about treatment of sorafenib-resistant liver cancer, and more specifically, to a composition for treating sorafenib-resistant liver cancer and a method for providing information about treatment of sorafenib-resistant liver cancer, which comprises a step of providing information about an effect of administering NK cells to a patient with sorafenib-resistant liver cancer, confirmed to have decreased CEACAM1 expression. In the present invention, it was confirmed that CEACAM1 expression was decreased in SR-Huh7 cells having sorafenib resistance, and it was confirmed that when NK cells, particularly, NK cells treated with the anti-CEACAM1 antibody and SR-Huh7 cells were co-cultured, the cytotoxic effect of SR-Huh7 by NK cells was increased, and thus, the administration regimen of NK cells can be effectively applied to the treatment of sorafenib-resistant liver cancer.

Description

Compositions for treating sorafenib-resistant liver cancer and Methods for providing information on treating sorafenib-resistant liver cancer}

The present invention relates to a composition for the treatment of sorafenib-resistant liver cancer and a method for providing information on the treatment of sorafenib-resistant liver cancer, and more particularly, a composition for treatment of sorafenib-resistant liver cancer including NK cells and a conch that was confirmed to have reduced CEACAM1 expression It relates to a method of providing information on treatment of liver cancer comprising providing information on the effect of administration of NK cells to a patient with phenib-resistant liver cancer.

Liver cancer is a cancer with the second highest mortality rate among all cancers in Korea. Liver cancer with a size of less than 3 cm (small liver cancer) has a 90% chance of survival for one year without special treatment.If surgery is performed, the 5-year survival rate is 40~ It is known that the prognosis is good enough to reach 50%, and early diagnosis of bovine liver cancer is important. However, at the time of discovery, cancer is often quite advanced, and there are many patients who recur even if the liver is deteriorated due to cirrhosis, etc. Therefore, it is necessary to develop a therapeutic agent with high drug efficacy against fundamental cancer recurrence and advanced cancer.

In particular, hepatocellular carcinoma (HCC) is one of the most common cancer types worldwide, especially in Asia. For such liver cancer, Sorafenib is known as the only standard treatment for liver cancer.

Sorafenib is Raf, a serine/threonine kinase in the signaling pathway along with VEGFR-2 and platelet-derived growth factor receptor (PDGFR)-βc-kit, receptor tyrosine kinases that are expected to be overexpressed in tumor cells or tumor vessels. It is known as an oral target anticancer drug (Oral Multikinase Inhibitor) that simultaneously inhibits kinase and attacks only vascular endothelial cells that supply nutrients to cancer cells and cancer cells while leaving normal cells intact. Sorafenib (Nexavar) generated 373 million paid sales in the first half of 2013, and has now obtained approval as a treatment for liver and kidney cancer, and recently approved Nexavar's treatment for thyroid cancer by the US FDA.

However, since many patients show no response to the administration and treatment of sorafenib, or show resistance to sorafenib due to continuous treatment, there is a need to develop a treatment strategy that can improve the effect of sorafenib.

Accordingly, in the present invention, as a result of earnest efforts to develop a method for effectively treating sorafenib-resistant liver cancer, CEACAM1 expression was decreased in SR-Huh7, a sorafenib-resistant cell line, and NK cells, in particular, NK treated with an anti-CEACAM1 antibody. When the cells and SR-Huh7 cells were co-cultured, it was confirmed that the SR-Huh7 cytotoxic effect by NK cells was increased, and the present invention was completed.

It is an object of the present invention to provide a composition for the treatment of sorafenib-resistant liver cancer.

Another object of the present invention is to provide a method of providing information on liver cancer treatment to patients with sorafenib-resistant liver cancer.

To achieve the above object,

The present invention provides a composition for the treatment of sorafenib-resistant liver cancer comprising natural killer cells as an active ingredient.

In a preferred embodiment of the present invention, the sorafenib-resistant liver cancer may have a reduced CEACAM1 expression compared to general liver cancer.

In another preferred embodiment of the present invention, the liver cancer may be hepatocellular carcinoma.

In another preferred embodiment of the present invention, the composition may further include an inhibitor of CEACAM1 expression.

In another preferred embodiment of the present invention, the natural killer cells may be natural killer cells treated with an anti-CEACAM1 antibody.

In order to achieve another object of the present invention,

The present invention provides a method of providing information on liver cancer treatment comprising the step of providing information on the administration effect of natural killer cells to a patient with sorafenib-resistant liver cancer, which has been confirmed to have decreased CEACAM1 expression.

In a preferred embodiment of the present invention, the method is

(a) confirming the expression level of CEACAM1 from a biological sample of a liver cancer patient showing resistance to sorafenib;

(b) providing information on predicting the effect of administration of natural killer cells in patients with sorafenib-resistant liver cancer whose CEACAM1 expression has been reduced compared to general liver cancer patients.

In another preferred embodiment of the present invention, the natural killer cells may be natural killer cells treated with an anti-CEACAM1 antibody.

In another preferred embodiment of the present invention, the liver cancer may be hepatocellular carcinoma.

In the present invention, it was confirmed that CEACAM1 expression was decreased in SR-Huh7 cells having sorafenib resistance, and when co-cultured with NK cells treated with anti-CEACAM1 antibody and SR-Huh7 cells, NK cells Since it was confirmed that the SR-Huh7 cytotoxic effect was increased, the administration regimen of NK cells can be effectively applied to the treatment of sorafenib-resistant liver cancer.

1 is a photograph (A) of observing the cell shape of a Huh7 cell line and a Huh7 (SR-Huh7) cell line having resistance to sorafenib (A) and data comparing the degree of cytotoxicity according to the concentration of the sorafenib concentration (B).
2 is data confirming the expression level of CEACAM1 in Huh7 and SR-Huh7 cells using FACS.
3 is data confirming the expression level of CEACAM1 in Huh7 and SR-Huh7 cells using Western blot.
Figure 4 is data confirming the increase in cytotoxicity of NK cells in Huh7 cells inhibiting the expression of CEACAM1. (A) is data confirming the expression level of CEACAM1 when CEACAM1 is knocked down using shRNA, and (B) is data confirming the degree of cytotoxicity when co-cultured with Huh7 cells and NK cells knocked down CEACAM1 to be.
5 is data confirming the degree of cytotoxicity when NK cells treated with an anti-CEACAM1 antibody were co-cultured with Huh7 cells.
6 is data confirming the degree of cytotoxicity when NK cells are co-cultured with Huh7 cells or SR-Huh7 cells.
7 is data confirming the degree of cytotoxicity when co-cultured SR-Huh7 cells and NK cells treated with anti-CEACAM1 antibody.

Hereinafter, the present invention will be described in more detail.

In one aspect, the present invention relates to a composition for the treatment of sorafenib-resistant liver cancer comprising natural killer cells as an active ingredient.

The sorafenib-resistant liver cancer is characterized in that the expression of CEACAM1 is reduced compared to general liver cancer that responds to sorafenib, and the liver cancer may preferably be hepatocellular carcinoma.

The composition may further include a CEACAM1 expression inhibitor, and the CEACAM1 expression inhibitor may be one or more of the group consisting of antisense oligonucleotides, siRNA, shRNA, aptamers, and antibodies specific for CEACAM1 gene.

The natural killer cells of the present invention may preferably be natural killer cells previously treated with an anti-CEACAM1 antibody.

In another aspect, the present invention relates to a method of providing information on liver cancer treatment comprising the step of providing information on the administration effect of natural killer cells to a patient with sorafenib-resistant liver cancer, which is confirmed to have decreased CEACAM1 expression.

Specifically, the method of providing the above information

(a) confirming the expression level of CEACAM1 from a biological sample of a liver cancer patient showing resistance to sorafenib;

(b) providing information on predicting the effect of administration of natural killer cells in patients with sorafenib-resistant liver cancer whose CEACAM1 expression has been reduced compared to general liver cancer patients.

The natural killer cells are characterized in that they are natural killer cells treated with an anti-CEACAM1 antibody, and a CEACAM1 expression inhibitor may be additionally administered in addition to the natural killer cells.

In the present invention, the sample in step (a) may be tissue or blood collected from a patient, and may be derived from a specific tissue or organ containing DNA.

In a specific embodiment of the present invention, a cell model having resistance was constructed by continuously treating Sorafenib, which is a human liver cancer cell line, Huh7, which was designated as "SR-Huh7". As shown in FIG. 1, when sorafenib was treated by concentration, it was confirmed that apoptosis by sorafenib was reduced in SR-Huh7 cells compared to Huh7, which means that SR-Huh7 cells have sorafenib resistance. do.

In the present invention, as a result of confirming that sorafenib resistance is related to CEACAM1, which generally exhibits high expression in cancer cells, as shown in FIGS. 2 and 3, in SR-Huh7 cells having sorafenib resistance, CEACAM1 than human liver cancer cell line Huh7 It was confirmed that the expression was significantly reduced.

The expression of CEACAM1 is an inhibitory ligand of natural killer cells (hereinafter referred to as “NK cells”), and is known to be expressed in target cells to inhibit cytotoxic effects caused by natural killer cells.

As shown in FIG. 4, as a result of knocking down CEACAM1 in Huh7 cells using shRNA, it was confirmed that cytotoxicity of NK cells increased when CEACAM1 expression was suppressed compared to the control, and as shown in FIG. 5, When Huh7 cells and NK cells treated with an anti-CEACAM1 antibody were co-cultured, it was confirmed that the cytotoxicity of NK cells increased and the death rate of Huh7 cells, a liver cancer cell, was increased.

That is, when the expression of CEACAM1 is decreased, it was confirmed that cytotoxicity by NK cells is increased. Accordingly, in the present invention, it was attempted to confirm whether sorafenib-resistant liver cancer cells with reduced CEACAM1 expression are effectively killed by NK cells.

First, in another specific embodiment of the present invention, as shown in FIG. 6, Huh7 and SR-Huh7 cells were co-cultured with NK cells to confirm the cytotoxicity of NK cells, through which the expression of CEACAM1 was low, SR- Huh7 cells were confirmed to be applied advantageously to kill by NK cells.

In addition, as shown in FIG. 7, NK cells administered with CEACMA1 blocking antibody to SR-Huh7 cells were co-cultured to confirm cytotoxicity, and as a result, it was confirmed that the cytotoxic effect by NK cells was remarkably increased.

Therefore, the pharmaceutical composition for the treatment of sorafenib-resistant liver cancer cells comprising NK cells, in particular anti-CEACMA1 antibody-treated NK cells of the present invention, has an effective anti-tumor effect on sorafenib-resistant liver cancer patients with reduced CEACAM1. Can be indicated.

Each of the pharmaceutical compositions of the present invention can be formulated and used in various forms according to conventional methods. It may further include a pharmaceutically acceptable carrier, excipient, and diluent according to each formulation. In addition, it may be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions according to a conventional method.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like. When formulating or formulating the pharmaceutical composition, it is prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The term'administration' as used in the present invention means providing the pharmaceutical composition of the present invention to an individual by any suitable method. The pharmaceutical composition of the present invention is an amount of an active ingredient or a pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human considered by a researcher, veterinarian, doctor or other clinician, that is, alleviation of symptoms of the disease or disorder being treated It can be administered in a therapeutically effective amount, which is an amount that induces. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, the patient's age, weight, and general health condition. , Sex and diet, administration time, administration route and composition ratio, treatment period, and various factors including drugs used simultaneously. The pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, and may be administered once a day or divided into several times.

Hereinafter, the present invention will be described in more detail through examples.

These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Construction of Sorafenib Resistant Cell Line

In the present invention, it was attempted to construct a Sorafenib-resistant cell line.

First, Huh-7 cells, a liver cancer cell line, were cultured in DMEM (Dulbesco's Modified Eagle Medium) medium mixed with sorafenib. The concentration of sorafenib was increased by 0.25uM to finally construct a Huh7 cell model resistant to the concentration of 6uM, which was named "SR-Huh7".

In order to confirm whether the SR-Huh7 cells actually exhibit resistance to sorafenib, Huh7 cells and SR-Huh7 cells were cultured in DMEM medium added with 10% FBS, and then Sorafenib was added to each cell line at 0, 1.25, 2.5. , 5, 7.5, 10, 15, 20uM concentration was treated and reacted for 48 hours, and then the cell viability was measured by the MTT assay method.

As a result, as shown in FIG. 1, when sorafenib was treated by concentration, it was confirmed that SR-Huh7 cells decreased apoptosis due to sorafenib compared to Huh7, which indicates that SR-Huh7 cells exhibited resistance to sorafenib. Means to have.

Confirmation of CEACAM1 expression level in sorafenib-resistant cells

2-1: Confirmation of changes in the expression of ligands capable of binding to NK cells

In the present invention, it was attempted to confirm whether a change in the expression of a ligand capable of binding to NK cells occurs in sorafenib-resistant cells.

Specifically, when NK (Natural Killer) cells and cancer cells bind, well-known ligands ( MHC-1, ICAM1, CEACAM1, ULBP-1, ULBP-2, ULBP-3, TRAIL-R1, HLA-G, MIC A/B) were fluorescently stained and confirmed using a FACS instrument.

As a result, as shown in FIG. 2, it was confirmed that the expression of CEACAM1 ligand, which inhibits the activity of NK cells in SR-Huh7 cells, was less expressed in Huh7 cells.

2-2: Confirmation of CEACAM1 expression level through Western blot

The degree of expression of the CEACAM1 protein was confirmed by Western blot.

First, 200 µl of RIPA buffer was added to each of SR-Huh7 cells and Huh7 cells to lyse the cells. Then, the protein was isolated, and CEACAM1 protein expression was confirmed in each cell through Western blot.

As a result, as shown in FIG. 3, it was confirmed that the expression of CEACAM1 was reduced in SR-Huh7 cells compared to Huh7 cells, similar to the results obtained through FACS.

Confirmation of increase in NK cell activity by inhibition of CEACAM1 expression

3-1: Construction of Huh7 cell line with knockdown of CEACAM1 expression

To confirm the correlation between the expression of CEACAM1 and the cytotoxicity of NK cells, shCEACAM1 (SIGMA-ALDRICH, Lentiviral Transduction Particles, Clone ID: NM_001712.3-2050s21c1; SEQ ID NO: 1: CCG GCT ATC ACT CTA ATT CGG ATT TCT CGA GAA ATC CGA ATT AGA GTG ATA GTT TTT G) was treated on Huh-7 cells to obtain a stable cell line in which CEACAM1 expression was knocked down.

3-2: Confirmation of cytotoxicity of NK cells by inhibition of CEACAM1 expression

The CEACAM1 knockdown Huh7 cells prepared in Example 3-1 were tested for resistance to NK cells (separated from healthy humans using magnetic-activated cell sorting (MACS) after blood collection).

First, Huh7 cells are ^{dispensed into a 24-well plate so that 1 x 10 5} cells/well, and then NK cells are each 1 (1 x 10 ⁵ cells/well) with Huh7: 1 or 5 (5 x 10 ⁵ cells/well). It was co-cultured by adding it so that it might be in a ):1 ratio.

During cultivation, 2 ml of RPMI1640 medium was added to each well, and 10 ng/ml of IL-12 was added together to help the activity of NK cells by co-culture for 6 hours. After 6 hours, the Huh7 cells were recovered from the plate using trypsin-EDTA, and then the cells were precipitated by centrifugation. The cells were resuspended by adding 1 ml of 1X PBS to the precipitated cells, and then the cells were transferred to a FACS tube. After adding 3 ml of 1X PBS, centrifugation to precipitate the cells, 200 µl of 1X PBS was added to the precipitated cells to resuspend the cells. 1 µl of a reagent for staining dead cells (To-pro-3 iodide) was added to the tube containing the cells, and then Huh7 apoptosis by NK cells was measured using a FACS instrument.

As shown in FIG. 4, as a result of knocking down CEACAM1 in Huh7 cells using shRNA, it was confirmed that cytotoxicity of NK cells increased when CEACAM1 expression was suppressed compared to the control.

That is, it was confirmed that when the expression of CEACAM1 is decreased, the death of liver cancer stem cells by NK cells is increased.

Confirmation of increase in cytotoxicity of NK cells using anti-CEACAM1 antibody

In the present invention, when NK cells were treated with an anti-CEACAM1 antibody, it was attempted to determine whether the toxicity of liver cancer cells by NK cells increased. IgG was treated as a control.

First, NK cells isolated from blood obtained from healthy people using magnetic-activated cell sorting (MACS) were divided into two groups, and 20 µg/ml of anti-pan CEACAM1 antibody (abcam, Cat.ab4567) was added to one side, The other was added 20 µg/ml of anti-IgG (Santacruz, sc-3877) as a control, followed by incubation at _{37° C. and 5% CO 2 for 30 minutes.} Then, 1X PBS was added, centrifuged to wash, and repeated 3 times. Huh7 cells after washing were ^{dispensed into a 24-well plate to be 1 x 10 5} cells/well, and then Huh7 cells by NK cells in the same manner as in Example 3-2. Apoptosis was measured.

As a result, as shown in FIG. 5, when the Huh7 cells and NK cells treated with the anti-CEACAM1 antibody were co-cultured, it was confirmed that the cytotoxicity of the NK cells increased and the Huh7 cell death rate was increased.

Confirmation of cytotoxicity by NK cells in Sorafenib-resistant cells

5-1: Confirmation of cytotoxicity according to co-culture of SR-Huh7 cells and NK cells

In the present invention, it was attempted to confirm whether sorafenib-resistant liver cancer cells with reduced CEACAM1 expression are effectively killed by NK cells.

First, NK cells were co-cultured in each of SR-Huh7 cells and Huh7 cells, and it was carried out in the same manner as in Example 3-1.

As a result, as shown in FIG. 6, it was confirmed that SR-Huh7 cells with low expression of CEACAM1 increased cytotoxicity by NK cells compared to Huh7 cells.

5-2: Confirmation of cytotoxicity according to co-culture of SR-Huh7 cells and NK cells treated with anti-CEACAM1 antibody

In the present invention, when NK cells treated with an anti-CEACAM1 antibody were administered to sorafenib-resistant liver cancer cells with reduced CEACAM1 expression, it was attempted to confirm whether the liver cancer cytotoxicity by NK cells increased.

First, SR-Huh7 cells and Huh7 cells were co-cultured with NK cells treated with an anti-CEACAM1 antibody, respectively, and carried out in the same manner as in Example 4.

As a result, as shown in FIG. 7, it was confirmed that the cytotoxicity of NK cells treated with the anti-CEACAM1 antibody was high in SR-Huh7 showing sorafenib resistance.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Compositions for treating sorafenib-resistant liver cancer and Methods for providing information on treating sorafenib-resistant liver cancer <130> 1066016 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 58 <212> RNA <213> Artificial Sequence <220> <223> shCEACAM1 <400> 1 ccggctatca ctctaattcg gatttctcga gaaatccgaa ttagagtgat agtttttg 58

Claims (6)

Sorafenib-resistant liver cancer treatment composition comprising natural killer cells as an active ingredient.
The composition for treating sorafenib-resistant liver cancer according to claim 1, wherein the expression of CEACAM1 in the sorafenib-resistant liver cancer is reduced compared to general liver cancer.
The composition of claim 1, wherein the composition further comprises an inhibitor of CEACAM1 expression.
The composition for treating sorafenib-resistant liver cancer according to claim 1, wherein the natural killer cells are natural killer cells treated with an anti-CEACAM1 antibody.
A method of providing information on treatment of liver cancer, comprising providing information on the effect of administration of natural killer cells to a patient with sorafenib-resistant liver cancer whose CEACAM1 expression is confirmed to be reduced.
The method of claim 5, wherein the natural killer cells are natural killer cells treated with an anti-CEACAM1 antibody.

Images