KR20190118788A - Natural killer cells for treating biliary tract cancer - Google Patents

Abstract

The present invention relates to natural killer cells capable of effectively preventing or treating biliary tract cancer, a method of inducing the same, and a pharmaceutical composition for preventing or treating biliary tract cancer comprising the natural killer cells. Natural killer cells derived from the present invention have excellent killing ability against biliary tract cancer cells, especially biliary tract cancer cells in the liver, and thus can effectively prevent or treat biliary tract cancer in the liver.

Description

NATURAL KILLER CELLS FOR TREATING BILIARY TRACT CANCER}

The present invention relates to natural killer cells capable of effectively preventing or treating biliary tract cancer, a method of inducing the same, and a pharmaceutical composition for preventing or treating biliary tract cancer comprising the natural killer cells.

Biliary cancer is a cancer of the bile ducts of the liver and is thought to occur in stem cells such as liver cancer (Sell and Dunsford Am J. Pathol. 134: 1347-1363, 1989). Globally, the incidence of biliary tract cancers is much lower than that of liver cancer, but in Southeast Asia is much higher than in Europe or North America. Biliary cancers are characterized by high recurrence rates, in which surgical resections are ineffective and inadequate chemotherapy or radiation therapy (Pederson et al Cancer Res. 4325-4332, 1997). In addition, the diagnosis of biliary tract cancer is not easy, and chronic bile duct inflammation resulting from chronic inflammation caused by bacterial or parasitic infection of the biliary tract tends to form biliary tract cancer (Roberts et al., Gastroenterology 112: 269). -279, 1997).

Nevertheless, the mechanism of the cause of the development of biliary tract cancer is not well known, and the target molecule for the treatment of biliary tract cancer is not known. In addition, several cytogenetic studies of biliary tract cancer have been published and very few cell lines have been established (Yamaguchi et al., J. Natl Cancer Inst 75: 29-35, 1985; Ding et al., Br J Cancer 67: 1007-1010, 1993), there is no known method for producing antibodies that bind biliary tract cancer using this cell line.

Natural killer cells (NK cells) are involved in innate immunity to eliminate pathogens and cancer cells, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and macrophages It is known to secrete inflammatory protein-1β (MIP-1β) and other mediators of adaptive immunity. When NK cells meet other cells, the MHC of the NK cells through molecular action sends a signal to the inside of NK cells when there is no MHC Class 1 like cancer cells or when the MHC Class 1 is abnormal, such as cells infected with viruses. I have a system that attacks. However, in several types of cancers, these NK cells have been reported to be defective in function and differentiation ability, and it is known that the survival of cancer cells and the activity of NK cells are closely related. Therefore, studies to increase the number or activity of NK cells for cancer immunotherapy have been conducted in various forms, but no satisfactory results have been obtained so far.

An object of the present invention is to provide a method for inducing natural killer cells that can be used for the prevention or treatment of biliary tract cancer, and natural killer cells induced by the method.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of biliary tract cancer comprising natural killer cells induced in the present invention.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in practice for the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following definitions will be used. The terminology used herein is not intended to be limiting, but rather will be understood to be used herein for the purpose of describing particular embodiments. The articles "a" and "an" are used herein to mean one or more than one (ie, at least one or more than one) of the objects in the grammar of the article.

In the past, attempts have been made to treat immune-related diseases using natural killer cells, but have not yet been satisfactory. In particular, biliary tract cancer is known to be difficult to treat among cancers, and there have been no reports of successful treatment of biliary tract cancer using natural killer cells.

The inventors of the present invention, as a result of earnest research efforts to develop a biliary tract cancer treatment, when using natural killer cells induced by the following method according to the present invention, found that the effect of treating biliary tract cancer is very excellent and completed the present invention.

The present invention relates to a method for inducing natural killer cells, which may first perform a step of obtaining blood, for example whole blood, umbilical cord blood, bone marrow, or peripheral blood, preferably peripheral blood, from a healthy person.

In the present invention, the step of separating and obtaining peripheral blood mononuclear cells (PBMC) from the peripheral blood obtained as described above can be performed.

In the present invention, the peripheral blood mononuclear cells (PBMC) are mammalian cells, preferably mononuclear cells isolated from the peripheral blood of human, mainly immune cells such as B cells, T cells, natural killer cells, And granulocytes such as basophil, eosinophil, neutrophil, and the like. The PBMC can be prepared by a conventional manufacturing method from peripheral blood collected in vivo. Preferably, the PBMC can be separated from peripheral blood using specific gravity centrifugation using Ficoll.

In the present invention, the step of removing the CD3 ⁺ T cells from the isolated peripheral blood mononuclear cells can be performed. The method for removing the CD3 ⁺ T cells in the present invention is not particularly limited, but may be performed using a commercially available immunomagnetic separation product, for example, MACSxpress (Milteyi Biotec, Germany). .

In the present invention, the step of culturing the peripheral blood mononuclear cells from which CD3 ⁺ T cells are removed in a culture medium containing cytokines (cytokine) in an amount of 500 to 1500 IU / ml.

In the present invention, the culture medium is 0.5 to 0.6% by weight of sodium chloride (NaCl), 0.4 to 0.5% by weight of D-glucose (D-glucose), 0.2 to 0.3% by weight of amino acids, 0.15 to 0.25% by weight of NaHCO 3 and HEPES (4- ( 2-hydroxyethyl) -1-piperazineethanesulfonic acid) may include 0.15 to 0.25% by weight, but is not limited thereto. Preferably Cell Science & Technology Institute inc. AlyS505NK-EX of (CSTI) may be used, but is not limited thereto.

In the present invention, the cytokine (cytokine) means an immune activated cytokine that can be used to induce peripheral blood monocytes into natural killer cells. In one embodiment of the invention, such cytokines include, for example, IL-2, IL-15, IL-21, Flt3-L, SCF, IL-7, IL-12, IL18 or a mixture of two or more thereof. Can be used. In particular, since IL-2, IL-15 or IL-21 is known as a cytokine having an excellent effect on differentiation and proliferation into natural killer cells, it is preferable to use these cytokines, most preferably IL-2. have.

For the purpose of the present invention, the cytokine is preferably included in an amount of 500 to 1500 IU / ml, more preferably may be included in an amount of 800 to 1200 IU / ml.

In addition, in the present invention, the culturing may be performed for 10 to 25 days, preferably 5 to 7 in T75 flasks containing peripheral blood mononuclear cells from which CD3 ⁺ T cells have been removed, and culture medium containing 10 to 30 ml. After incubation for one day can be transferred to the T175 flask may be carried out with an additional incubation for 7 to 14 days.

In the present invention, the culture may use a stationary culture or a suspension culture method, and the culture may mean culturing in a state in which the culture is left without agitating or shaking in the incubator. Suspension culture means that the cells are cultured in a suspended state without attaching to the lower or side portion of the reactor through aeration or agitation. In addition, the reactor for stationary culture and the reactor for suspension culture may be the same, or may be different. For example, when the reactor for stationary culture and the reactor for suspension culture are the same, after the stationary culture is completed in the same reactor, the culture medium is additionally supplied by supplying a medium containing necessary nutrients such as cytokines. In the case of using different kinds of incubators, the culture may be transferred to a reactor for suspension culture after the completion of the stationary culture.

In the present invention, natural killer cells induced by the above method have a CD3 ⁻ phenotype, and may have a phenotype of CD56 ^bright or CD56 ^dim .

In addition, the natural killer cells induced as described above in the present invention are particularly highly cytotoxic to intrahepatic bile duct cancer cells among the biliary cancer cells, thereby effectively preventing or treating biliary tract cancer in the liver.

According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating biliary tract cancer comprising natural killer cells obtained by the method of the present invention as an active ingredient.

In the present invention, the pharmaceutical composition means "cellular therapeutic agent". As used herein, the term "cellular therapeutic agent" refers to a medicinal product (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special manipulation from an individual. Or drugs used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating and screening live autologous, allogeneic, or heterologous cells in vitro or by altering the biological characteristics of the cells in order to restore tissue function. Means.

Natural killer cells included in the pharmaceutical composition of the present invention have excellent selective cytotoxicity to biliary cancer cells, especially in the liver, and can effectively prevent or treat biliary tract cancer.

The pharmaceutical composition of the present invention comprises at least one natural killer cell, at least 1Х10 ^4, at least 1Х10 ^5, at least 3Х10 ^5, at least 1Х10 ^6, at least 3Х10 ^6, at least 6Х10 ⁶ , or at least 1Х10 ⁷ , and at least 1Х10 ¹⁰ Or less than.

As used herein, the term "prevention" refers to any action that inhibits or delays biliary tract cancer by administration of the pharmaceutical composition.

In addition, the term "treatment" used in the present invention means all the actions that improve or cure symptoms of biliary tract cancer by administration of the pharmaceutical composition.

In the present invention, the pharmaceutical composition may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, the pharmaceutical composition may be characterized in that it is intended for humans.

The pharmaceutical compositions of the present invention are not limited to these, but each may be formulated in the form of oral dosage forms, such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions according to conventional methods. Can be. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, suspending agents, disintegrating agents, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, and the like in the case of oral administration, and in the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms. have. And others, solutions, suspensions, tablets, capsules, sustained release preparations and the like.

Examples of suitable carriers, excipients, and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.

Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , Sublingual or rectal. Oral or parenteral release is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention is dependent on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed. The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, extent of disease, drug form, route of administration and duration, and may be appropriately selected by those skilled in the art, and may be 0.0001-50 mg / day. It may be administered at kg or 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

Natural killer cells derived from the present invention have excellent killing ability against biliary tract cancer cells, in particular, biliary tract cancer cells in the liver, and can effectively prevent or treat biliary tract cancer in the liver.

1 is a schematic view showing a design of an in-vivo experiment using a nude mouse in one embodiment of the present invention. FIG. 1 (a) shows the concentration-dependent stability of SMT01 natural killer cells obtained in the present invention. Corresponds to experimental design for toxicity assessment. In addition, Figure 1 (b) corresponds to the experimental design for evaluating the concentration-dependent efficacy of SMT01 natural killer cells obtained in the present invention.
Figure 2 shows the results of flow cytometry analysis of ex-vivo amplified SMT01 in one embodiment of the present invention, and cytotoxicity for biliary tract cancer cell line. Figure 2 (a) is a result of analyzing the phenotype of the cell group of SMT01, anti-CD56 / CD16 antibody was used to analyze the phenotype of NK cells, anti-CD3 antibody was used for T cell population analysis, DC And anti-CD40 antibodies were used for B cell analysis. FIG. 2 (b) shows HuCCT-1 (hepatic biliary cancer cell line), SNU1196 (hepatic biliary tract cell line), and SNU478 (Barter bulge adenocarcinoma cell line) corresponding to intrahepatic biliary cancer cell line and extrahepatic biliary cancer cell line. The results of analysis of cell lysis of SMT01. Here, the K562 cell line corresponds to the positive control.
Figure 3 shows the tumor growth inhibitory effect of SMT01. Figures 3 (a), 3 (b) and 3 (c) show the change in photographs, tumor volume and tumor weight of nude mice subjected to G1 to G5 treatment and tumors isolated therefrom.
4 shows in-vivo nude mouse experiments of SMT01. Figure 4 (a) shows the change in body weight, Figure 4 (b) shows the change in tumor volume. Here, the tumor volume was measured using a caliper ((CD-15CX, Mitutoyo, Japan) according to the following formula: tumor volume (mm ³ ) = L (mm) x W ² (mm ² ) x 1/2. Figure 4 (c) measures the change in tumor weight The average tumor weight measured in each group was as follows: G1, 2.904 g (IR: 0.0%); G2, 1.892 g (IR: 34.8%) G3, 2.292 g (IR: 21.1%); G4, 2.252 g (IR: 22.4%); G5, 1.385 g (IR: 52.3%), wherein the IR was measured using the following formula: IR ( %) = (1-T / C) ×× 100 (T: mean tumor weight in experimental and positive controls, C: mean tumor weight in negative controls).
Figure 5 shows the results of H & E staining and IHC experiment of nude mouse tumors. The overall morphology and CD56 IHC results of the representative tumors in each group were observed at low magnification (12.5 X) and magnified pictures are shown in the right column (100X). CD56-positive NK cells in the G2 to G4 groups were found to be distributed along the cleavage between tumor nodules (red arrow).
Figure 6 shows the histological observations of apoptotic tumor cells and infiltrating natural killer cells. Figure 6 (a) shows the results of TUNEL staining of the tumor sections of all groups, TUNEL positive cells were observed more frequently in the G2 to G5 group compared to the G1 group. Figure 6 (b) shows the results of the IHC experiment, where a and b show consecutive sections of the G2 group showing NCR1-positive NK cell infiltration (red arrows) along loose tissue between tumor nodules, and c and d are Serial sections of the G3 group showing CD56 and NCR1-positive killer cells are shown. e to h show serial sections of the G3 group, and apoptosis signals of tumor cells detected by IHC using anti-human caspase 3a antibodies were mainly observed at the site of infiltration of natural killer cells. i and j show serial sections of the G4 group. Caspase 3a positive cells (black arrows) in tumors were observed at sites where infiltrated natural killer cells were observed (red arrows). k and 1 show serial sections of the G4 group, and caspase 3a positive cells (black arrows) were observed to be present at sites of infiltration of natural killer cells (red arrows) along the loose tissue between tumor nodules. m to p show serial sections showing multiple caspase 3a positive cells (black arrows) distributed in tumors unrelated to infiltration of natural killer cells in the G1 and G5 groups.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example

1. Preparation of natural killer cells

Blood was obtained from two healthy donors and PBMCs isolated. CDS ⁺ T cells were removed using MACSxpress (Milteyi Biotec, Germany). PBMCs from which T cells were removed were washed twice using DPBS buffer, and then cultured in a T75 flask containing 20 ml of NK amplification medium (ALyS505NK-IL2 1000 IU / ml, Cell Science & Technology Inst). Fresh medium was added to IL-2 activated natural killer cells every 3 days and transferred to T175 flasks after 5-7 days of culture. Natural killer cell amplification was further performed for 7-14 days with continued addition of fresh medium until the target cell number was reached. Survival and number of natural killer cells (SMT01 cells) amplified by trypan blue counting were measured using an automatic cell counter.

2. Design of in-vivo experiments in nude mice

NCr athymic nude mice (BALB / cSlc-nu / nu) (Charles River Laboratories, Japan SLC, Inc) were used as OECD Good Laboratory Practice regulations (ENV / MC / CHEM (98) 17 as revised in 1997 ) And in a microisolator cage according to Good Laboratory Practice (21 CFR Part 58, Food and Drug Administration, United States of America (Apr. 1, 2015)) for nonclinical laboratory studies. Manipulation and breeding.

In order to evaluate dose-dependent safety and toxicity, the natural killer cells obtained as described above were prepared in three different doses (FIG. 1 (a)). For low dose infusions, 4X10 ⁴ cells / animals corresponding to doses of 2X10 ⁶ cells / kg in humans were designated as 2X10 ⁵ cells / animals and 1X10 ⁶ cells / animals for medium and high doses, respectively. Subsequently, 50 male and female mice, including the recovery group, amplified spontaneous killer cells ex-vivo (weights of male and female, respectively, at 18 weeks corresponded to 18.7-22.5g and 16.1-18.8g). Intravenously, twice weekly, three weeks apart for 27 weeks. A total of 18 SMT01 cells were injected intravenously.

Experiments were performed to evaluate the dose-dependent efficacy of SMT01 on the same nude mice (FIG. 1 (b)). Specifically, implantation and engraftment were performed by subcutaneously injecting HuCCT1 cells (5 × 10 ⁶ cells / 0.2 mL) into each group of 10 nude mice (G1 to G5): G1, saline (negative control); G2-G4, SMT01 injection; G5, gemcitabine + cisplatin (Gem + CDDP) (positive control). In each group, eight of the well-grafted mice with tumor volumes of 84-119 mm ³ (19.3-20.5 g body weight range) were selected and treatment was performed.

More specifically, nude mice containing HuCCT-1 tumors were injected intravenously with SMT01 five times at 10 day intervals. As a positive control, gemcitabine (Gem) and cis-diamineplatinum (II) dichloride (CDDP) were administered at doses of 120 mg / kg and 3 mg / kg, respectively. Three groups of mice were administered at different doses upon infusion of SMT01. G2-G4: G2, low dose (4X10 ⁴ cell / animal); G3, medium dose (2X10 ⁵ cells / animal); G4, high dose (1 × 10 ⁶ cells / animal). G1 and G5 correspond to negative control (physiological saline) and positive control (CDDP + Gem), respectively. Disposable syringes (26G, 1 ml) were used for cell injection. The injection volume was 0.2 ml / head, and after 14 days after the last injection, the mice containing the tumor were euthanized and evaluated (FIG. 1 (b)).

3. Human Cell Line

Human cholangiocarcinoma cell line used in this experiment, HuCCT-1 (hepatic cholangiocarcinoma cell line) was purchased from Health Science Research Resources Bank (Osaka, Japan), SNU1196 (extrahepatic cholangiocarcinoma cell line) and SNU478 (barter bulge adenocarcinoma cell line) ) Was purchased from Korea Cell Line Bank (Seoul, Korea). Cell lines were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO), 100 U / ml penicillin, and 100 mg / ml streptomycin under a humidified environment containing 5% CO ₂ .

4. Phenotypic flow cytometric analysis

To analyze the SMT01 cell population, cells were stained with PE conjugated anti-human CD56 and anti-human CD40 antibodies, FITC conjugated anti-human CD3 and anti-human CD16 monoclonal antibodies (BD Biosciences). For staining, SMT01 was washed twice in phosphate buffer saline (PBS) and resuspended in FACS staining buffer (PBS with 0.5% bovine serum albumin). Cells were then stained for 30 minutes at 4 ° C. using appropriate antibodies. After washing three times with PBS buffer, cells were analyzed on a FACS flow cytometer (Beckton Dickinson AriaIII). Natural killer cells were defined as CD56 ⁺ / CD3 ⁻ and CD56 ⁺ / CD16 ⁺ to analyze NK cell populations.

5. Cytotoxicity Assay

The cytotoxic activity of natural killer cells was measured using Cell Counting Kit-8 (CCK-8) (Dojindo Mol. Tech., USA). K562 cells were used as a positive control to evaluate the cytotoxic activity of natural killer cells against human cholangiocarcinoma cell line. 96-well plates were inoculated with SMT01 effector cells at 1 × 10 ⁴ cells per well and incubated for 24 hours. The CCK8 kit was used to measure the cell viability of the target cell lines at three different effector: target (E: T) cell ratios (1: 5, 1: 1, and 5: 1). Absorbance was measured at 450 nm using a microplate reader. Cytotoxicity results were measured using the following formula.

Cytotoxicity (%) = 100%-((ODe + t -ODb)-(ODe-ODb)) / (ODt-ODb) X 100% (OD, absorbance; e, effector; t, target; b, blank) .

6. Histology, immunohistochemistry (IHC) and TUNEL analysis

For histological analysis, HuCCT-1 tumors were isolated from nude mice and fixed in paraffin. Tissue sections were prepared to a size of 4 μm and then stained with hematoxylin and eosin (H & E) and used for IHC experiments. Antibodies used in IHC experiments are anti-human CD56 (BD Sciences), natural cytotoxicity triggering receptor 1 (NCR1) (BD Sciences), and anti-human Caspase 3a antibody (Abcam). Tumor slice slides were used to perform in situ analysis of apoptotic cells using TUNEL staining. TUNEL analysis was performed using the TumorTACS in situ apoptosis kit (R & D Systems, Minneapolis, MN, USA). Olympus MVX10 was used for microscopic observation, and slide sections were photographed using Olympus BX51.

7. Statistical Analysis

Body weight, tumor volume, and tumor weight were evaluated using SAS (Version 9.3, SAS Institute Inc., USA). Each value is expressed as mean ± SD. Bartlett test was used for equal variance and Dunnett t-test was used for statistical significance. Statistical significance was determined at P <0.05.

8. Amplification of SMT01, Cytotoxic Natural Killer Cells from PBMC

In order to produce SMT01, natural killer cells were isolated from human peripheral blood using MACSexpress and then CD3-positive T cells were removed. The isolated natural killer cells were then incubated for 14 days in the presence of IL-2. To determine the SMT01 cell population, natural killer cells amplified in vitro using antibodies that recognize natural killer cells (CD56 / CD3), T cells (CD3), and lymphocyte cells (CD40) such as dendritic cells and B cells were used. Stained. CD56 / CD16 antibodies were used to determine natural killer cell subtypes. Many of the cells amplified based on the CD56 and CD3 phenotypes were expressed as natural killer cells. CD3 ⁺ T cell population was found to be distributed at a very low level of 0.2% (Fig. 2 (a)). 99.2% of SMT01 cell populations were identified as CD56 ⁺ / CD3 ⁻ natural killer cells. Among the killer cells, the CD56 ^dim and CD56 ^bright phenotypes were also observed, and SMT01 was found to contain a lot of CD56 ^dim killer cells.

 The cell lytic activity of SMT01 was evaluated (FIG. 2 (b)). To evaluate the cytotoxicity of natural killer cells, the number of viable target cells was measured using the cell counting kit-8 (CCK-8). K562 cells were used as a positive control to compare the survival rate of human intrahepatic and extrahepatic cholangiocarcinoma (CC) cell lines. SMT01 showed increased cytolytic activity against K562 cells. In the bile duct cancer cell line, it was confirmed that SMT01 more effectively kills HuCCT1, which is an intrahepatic biliary cancer cell line, than the SNU1196 and SNU478 cell lines. Through this, natural killer cells induced in accordance with the present invention was found to be remarkably excellent cell lysis effect on hepatic biliary cancer cell line.

9. In Vitro Study of SMT01 in Nude Mice

In order to evaluate the dose dependent safety, toxicity and efficacy of SMT01, in vivo experiments were performed on three doses of natural killer cells (Tables 1 and 2). SMT01 was injected into the tail vein of nude mice. At this time, the low dose of SMT01 was set to 4X10 ⁴ cells / animals corresponding to 2X10 ⁶ cells / kg in humans, and the medium dose and the high dose were set to 2X10 ⁵ cells / animal and 1x10 ⁶ cells / animal, respectively. It was. For dose dependent safety and toxicity assessment, SMT01 was injected twice weekly at three week intervals, for a total of 18 injections, with three different doses (Table 1, FIG. 1). As a result, even when injecting the maximum dose of SMT01 was confirmed that the safety without a separate lethal response. In addition, although not included in the drawings, the internal organs were observed in all SMT01 treatment groups, and no toxicity was observed in relation to the administration of natural killer cells. In addition, no special changes in T and B lymphocytes were observed, and it was found that SMT01 according to the present invention did not cause immune-related side effects (Table 1).

In vivo cell lysis activity of SMT01 was evaluated in biliary cancer cell lines. To this end, xenograft cancer cells were xenografted in nude mice. More specifically, HuCCT-1 cells were implanted subcutaneously in the back of 10 nude mice of each group in an amount of ⁵ × 10 ⁶ cells / ml. Eight days after bile duct cancer cell transplantation, nude mice well implanted with similar tumor volumes were selected and used for in vivo experiments. Eight mice selected from each treatment group, including the negative control (G1, saline infusion), were treated for each treatment by the method shown in Table 2 and FIG. 1 below. Chemo-administration was performed on the same number of mice as positive controls (G5, CDDP + Gem). Five doses of SMT01 were divided into three different concentrations of doses (G2-G4). Tumors were collected from nude mice and tumor volume and weight were measured (FIG. 3).

The mean tumor volume observed after 55 days of administration in each of the G1, G2, G3, G4, and G5 groups corresponded to 5324, 2769, 3491, 3193, and 2167 mm ³ (FIG. 3 (b)). Inhibition of tumor growth was observed in all treatment groups except negative control (G1). It can be seen that the mean weight difference of the isolated tumors in the treatment group and the negative control group, respectively, is very large (FIG. 3 (a)). Through these results, the SMT01 natural killer cells according to the present invention was confirmed that the growth inhibitory effect of biliary tract cancer is remarkably excellent. Although maximal growth inhibition was observed in the chemotherapy group, there was no statistical significance. Furthermore, as a result of measuring the weight change in all treatment groups, it was confirmed that the weight of mice was significantly reduced in the chemotherapy group (FIG. 4 (a)), which was not related to the diet (FIG. 4 (b). )). By the way, according to the present invention it was confirmed that the body weight of the mouse is maintained constant in the SMT01 natural killer cells treated group. Through this, natural killer cell therapy is related to the quality of life, and it can be seen that it has an effective effect on weight maintenance which is an important parameter in cancer treatment.

Through the above in vivo studies, it was found that SMT01 natural killer cells according to the present invention have excellent cytolytic activity against hepatic biliary cancer cells and do not cause any special side effects in vivo.

group
(Number of animals) Number of cells injected
(Cell count / animal) Injection volume
(mL / animal) Mean proliferation of lymphocytes (%) T cell
(Male / female) B cell
(Male / female) G1 Negative control
20 0 0.2 100/100 100/100 G2 Low capacity (20) 4X10 ⁴ 0.2 97.5 / 99.1 99.8 / 102.2 G3 Medium capacity (20) 2X10 ⁵ 0.2 98.8 / 103.3 104.2 / 102.2 G4 High capacity (20) 1 X 10 ⁶ 0.2 97 / 104.2 104.2 / 97.6

group/
Volume N Apoptosis Necrosis Rate (%) ± + ++ ± + ++ +++ ++++ G1 /
0 cells / two 8 8.17 ± 1.26 8 0 0 0 5 3 0 0 G2 / 4X10 ⁴ cells / two 8 12.42 ± 2.59 2 6 0 0 One 5 One One G3 / 2X10 ⁵ cells / two 8 11.71 ± 3.73 3 5 0 One 3 2 2 0 G4 / 1X10 ⁶ cells / two 8 10.88 ± 2.25 3 5 0 0 4 3 One 0 G5 / 3 + 120mg / kg 8 23.00 ± 3.35 0 2 6 0 4 2 2 0 G1: negative control (physiological saline)
G2: SMT01, low dose (4X10 ⁴ cells / two)
G3: SMT01, medium dose (2X10 ⁵ cells / two)
G4: SMT01, high dose (1 × 10 ⁶ cells / two)
G5: cis-diamineplatinum (II) dichloride (CDDP) + gemcitabine (Gem)
N: number of animals
Degree: ±: Very little, +: Less, ++: Normal, +++: High, ++++: Serious

10. Assessment of Histological Effects of SMT01 Activity in Nude Mouse Tumors

Tumor tissue sections were used to assess the cytolytic activity of SMT01 (FIGS. 5 and 6). As shown in FIG. 3, tumors isolated from nude mice were fixed in paraffin to prepare tissue sections. Anti-human CD56 antibodies were used for IHC experiments to determine whether natural killer cells penetrated the HuCCT-1 tumors of nude mice injected with SMT01. The same tumor tissue sections were used for TUNEL analysis.

The results of IHC experiments on tissue sections obtained from G1 (physiological saline input group) and G5 (chemistry-administered mouse group) showed CD56 positive signals at the background level, whereas tumor nodules in the SMT01 injection group (G2-G4). Idu signals of CD45 cells mainly distributed in the cleavage between the nodules. That is, SMT01 natural killer cells were found to infiltrate into HuCCT-1 tumors formed in nude mice (FIG. 5). In addition, IHC signal was observed mainly in the tissue mass and loosely distributed areas inside the tumor for vascular penetration (Fig. 5, G2-G4, arrow)

Infiltration of CD56 positive NK cells infiltrating tumor tissue could be more clearly observed under high resolution microscope (FIG. 6 (b)). As shown in Figure 6 (b), CD56 positive natural killer cells can be observed that the cells in the G2 to G4 mouse group injected with SMT01 densely distributed at the site where the cells penetrated the xenografted tumor tissue there was. Similar results were obtained in an IHC experiment on NCR1, an activator of natural killer cells, indicating that the infiltrating cells were natural killer cells (FIG. 6 (b), NCR1 panel). Apoptosis signals were detected through IHC experiments on caspase 3a, TUNEL staining and apoptosis markers (FIG. 6 (a), (b)). As a result, a large number of tumor cell suicides were observed in tumors injected with SMT01 natural killer cells according to the present invention (FIG. 6 (a)). In addition, apoptotic cells were found to be significantly observed in the natural killer cell population. Through this, the outflowed natural killer cells showed cytolytic activity against tumor cells near tumor vessels. SMT01 natural killer cells were found to be particularly cytotoxic to the hepatobiliary cancer cell line.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations can be made without departing from the technical spirit of the present invention described in the claims. It will be obvious to those of ordinary skill in the field.

Claims (8)

Removing CD3 ⁺ T cells from Peripheral blood mononuclear cells (PBMC); And
Culturing the peripheral blood mononuclear cells from which the CD3 ⁺ T cells have been removed for 10 to 25 days in a culture medium containing interleukin-2 (IL-2) in an amount of 500 to 1500 IU / ml Including, natural killer cells induction method. The method of claim 1,
Peripheral blood mononuclear cells from which the CD3 ⁺ T cells have been removed are cultured in a culture medium containing interleukin-2 in an amount of 800 to 1200 IU / ml. The method of claim 1,
Peripheral blood mononuclear cells from which the CD3 ⁺ T cells have been removed are incubated for 5 to 7 days in a T75 flask containing 10 to 30 ml of culture medium, and then transferred to a T175 flask to further culture for 7 to 14 days. Induction method of killer cells. The method of claim 1,
The culture medium is 0.5 to 0.6 wt% sodium chloride (NaCl), 0.4 to 0.5 wt% D-glucose, 0.2 to 0.3 wt% amino acid, 0.15 to 0.25 wt% NaHCO3 and 4- (2-hydroxyethyl ) -1-piperazineethanesulfonic acid) 0.15 to 0.25% by weight, comprising the method of inducing natural killer cells. The method of claim 1,
The natural killer cells include natural killer cells having a phenotype of CD56 ^bright or CD56 ^dim , natural killer cells. A pharmaceutical composition for preventing or treating biliary tract cancer comprising natural killer cells induced by the method of any one of claims 1 to 5 as an active ingredient. The method of claim 6,
The biliary tract cancer is intrahepatic biliary tract cancer, pharmaceutical composition. The method of claim 6,
The composition comprises a natural killer cells 1Х10 ⁴ to 1Х10 ¹⁰ , the pharmaceutical composition.

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