METHOD FOR MASS PRODUCING NATURAL KILLER CELL AND USE OF NATURAL KILLER CELL OBTAINED BY THE METHOD AS ANTI-CANCER AGENT

Abstract

The present invention relates to a method for producing a large amount of natural killer cells and the use of natural killer cells obtained by the method as an anticancer agent. The use of the method of the present invention can produce fresh NK cells with high purity within a short time compared to conventional method, and can also produce cold-preserved NK cells and thawed cryopreserved NK cells, which have efficacy comparable with that of the fresh NK cells. Furthermore, it can produce NK cells, which have efficacy comparable with that of the fresh NK cells, from cryopreserved CD3-negative cells. The fresh NK cells, cold-preserved NK cells and cryopreserved NK cells produced by the methods of the present invention can exhibit therapeutic effects against various cancers, including colorectal cancer, lung cancer, liver cancer, pancreatic cancer and leukemia, indicating that these NK cells can be effectively used as cellular therapeutic agents. In addition, the present inventors have established doses and methods of administration, which show excellent effects when the fresh NK cells, cold-preserved NK cells and cryopreserved NK cells of the present invention are used as pharmaceutical compositions for cellular therapy.

Claims

1. A method for producing fresh natural killer (NK) cells, the method comprising the steps of: 1) obtaining CD3-negative cells to remove CD3-positive T cells from monocytes; and 2) culturing the CD3-negative cells to treat the CD3-negative cells of step 1) with IL-15 and IL-21, wherein step 1) is performed by allowing the CD3-positive T cells to crosslink to erythrocytes and then isolating the CD3-negative cells by density-gradient centrifugation.

2. A pharmaceutical composition for preventing or treating cancer comprising fresh NK cells produced by the method of claim 1, as an active ingredient.

3. The composition of claim 2, wherein the composition comprises 110.sup.5 to 110.sup.10 NK cells produced by the method of claim 1.

4. The composition of claim 2, wherein the composition is administered once a week for 4 weeks or administered twice a week for 2 weeks.

5. The composition of claim 2, wherein the composition comprises 310.sup.6 fresh NK cells produced by the method of claim 1, and is administered once a week for 4 weeks or administered twice a week for 2 weeks.

6. The composition of claim 2, wherein the cancer is any one cancer selected from a group consisting of colorectal cancer, lung cancer, pancreatic cancer and leukemia.

7. (canceled)

8. (canceled)

9. (canceled)

10. A method for producing cryopreserved NK cells, the method comprising the steps of: 1) obtaining CD3-negative cells to remove CD3-positive T cells from monocytes; 2) culturing the CD3-negative cells to treat the CD3-negative cells of step 1) with IL-15 and IL-21; and 3) freezing the cultured CD3-negative cells of step 2) in a cryopreservation medium containing 10% DMSO (dimethyl sulfoxide) under serum-free, protein-free and animal component-free conditions for 2 months or less, wherein the freezing is performed by stepwise cooling from 70 C. to 200 C.

11. A method for producing thawed cryopreserved NK cells, the method comprising the steps of: 1) obtaining CD3-negative cells to remove CD3-positive T cells from monocytes; 2) culturing the CD3-negative cells to treat the CD3-negative cells of step 1) with IL-15 and IL-21; 3) freezing the cultured CD3-negative cells of step 2) in a cryopreservation medium containing 10% DMSO (dimethyl sulfoxide) under serum-free, protein-free and animal component-free conditions for 2 months or less, wherein the freezing is performed by stepwise cooling from 70 C. to 200 C.; and 4) quick thawing the cryopreserved NK cells at 37 C., and washing out the cryopreservation medium.

12. A pharmaceutical composition for preventing or treating cancer comprising thawed cryopreserved NK cells produced by the method of claim 11, as an active ingredient.

13. The composition of claim 12, wherein the composition comprises 110.sup.5 to 110.sup.10 thawed cryopreserved NK cells produced by the method of claim 11.

14. The composition of claim 12, wherein the composition is administered once a week for 4 weeks or administered twice a week for 2 weeks.

15. The composition of claim 12, wherein the composition comprises 310.sup.6 thawed cryopreserved NK cells produced by the method of claim 11, and is administered once a week for 4 weeks or administered twice a week for 2 weeks.

16. A method of producing NK cells from frozen CD3-negative cells, the method comprising the steps of: 1) obtaining CD3-negative cells to remove CD3-positive T cells from monocytes; 2) freezing the obtained CD3-negative cells of step 1) in a cryopreservation medium containing 10% DMSO (dimethyl sulfoxide) under serum-free, protein-free and animal component-free conditions for 2 months or less, wherein the freezing is performed by stepwise cooling from 70 C. to 200 C.; 3) thawing the frozen CD3-negative cells of step 2); and 4) culturing the thawed CD3-negative cells to treat the thawed CD3-negative cells of step 3) with IL-15 and IL-21.

17. A pharmaceutical composition for preventing or treating cancer comprising fresh NK cells produced by the method of claim 1 and thawed cryopreserved NK cells produced by the method of claim 11, as an active ingredient.

18. The composition of claim 17, wherein the fresh NK cells are administered once a week for 1 week, and the thawed cryopreserved NK cells are administered twice a week for 3 weeks.

Description

DESCRIPTION OF DRAWINGS

[0112] FIG. 1 shows FACS results indicating that differentiation of NK cells from CD3-negative cells isolated either from umbilical cord blood (the top of FIG. 1) or from peripheral blood (the bottom of FIG. 1) was induced after 11-21 days of culture.

[0113] FIG. 2a shows the viability of thawed NK cells obtained by cryopreserving fresh NK cells on 10 days of culture and thawing the cryopreserved NK cells; FIG. 2b shows the degree of differentiation; FIG. 2c shows NK cell receptors; and FIG. 2d show NK cell cytotoxicities.

[0114] FIG. 2e shows the cell recovery rate immediately after thawing of NK cells obtained by differentiation of CD3-negative cells thawed after freezing; FIG. 2f shows fold increase in cell number upon culture after thawing; FIG. 2g shows cell viability; and FIG. 2h shows the degree of differentiation and NK cell receptors.

[0115] FIG. 3a shows the results of detecting NK cells in mice to measure the concentration-dependent detection limit of NK (natural killer) cells.

[0116] V.C (5% HSA): vehicle control group.

[0117] FIG. 3b shows the results of detecting NK cells in the major intra-abdominal organs of mice to mesure the concentration-dependent detection limit of NK cells.

[0118] V.C (5% HSA): vehicle control group.

[0119] FIG. 3c shows the results of analyzing the distribution of NK cells in the major intra-abdominal organs of mice.

[0120] V.C: vehicle control group.

[0121] FIG. 3d shows the results of analyzing the in vivo distribution of NK cells in mice at varying time points.

[0122] FIG. 4a shows an administration schedule for examining the anticancer effect of NK cells against colorectal cancer.

[0123] FIG. 4b shows the results of measuring the tumor size inhibitory effect of NK cells alone or in combination with IL-2 against colorectal cancer when varying number of the NK cells are used.

[0124] V.C: vehicle control group, and

[0125] ADR: adriamycin-treated group.

[0126] FIG. 4c shows the results of measuring the tumor weight reducing effect of NK cells alone or in combination with IL-2 against colorectal cancer when varying number of the NK cells are used.

[0127] V.C: vehicle control group, and

[0128] ADR: adriamycin-treated group.

[0129] FIG. 5a shows administration schedules prepared to examine anticancer effects according to the culture conditions, preservation conditions and administration schedule of NK cells.

[0130] FIG. 5b shows the results of analyzing the tumor volume inhibitory effects of NK cells against colorectal cancer according to the culture conditions, preservation conditions and administration schedule of NK cells.

[0131] V.C: vehicle control group;

[0132] NK-F: fresh NK cell-treated group;

[0133] NK-W/oR-F: group treated with fresh NK cells (Rosettesep-free, that is, w/o Rosettesep);

[0134] NK-4 C.: group treated with NK cells cold-preserved at 4 C.;

[0135] NK-W/oR-4 C.: group treated with fresh NK cells (Rosettesep-free) cold-preserved at 4 C.;

[0136] ADR: adriamycin-treated group.

[0137] FIG. 5c shows the results of analyzing the tumor weight-reducing effects of NK cells against colorectal cancer according to the culture conditions, preservation conditions and administration schedule of NK cells.

[0138] V.C: vehicle control group;

[0139] NK-F: group treated with fresh NK cells;

[0140] NK-W/oR,F: group treated with fresh NK cells (Rosettesep-free);

[0141] NK-4 C. preserved: group treated with NK cells cold-preserved at 4 C.;

[0142] NK-W/oR, 4 C. preserved: group treated with fresh NK cells (Rosettesep-free) cold-preserved at 4 C.;

[0143] ADR: adriamycin-treated group.

[0144] FIG. 6a shows an administration schedule prepared to examine the anticancer effect of NK cells according to freezing or not of NK cells.

[0145] FIG. 6b shows the tumor volume inhibitory effect of NK cells against colorectal cancer according to freezing or not of NK cells.

[0146] V.C: vehicle control group;

[0147] NK cell-Live (4): group administered four times with fresh NK cells;

[0148] NK cell-Live (1)+(3) frozen: group administered once with fresh NK cells and three times with thawed cryopreserved NK cells;

[0149] V.C (serum-free medium): serum-free medium group; NK cell-frozen (4): group administered four times with thawed cryopreserved NK cells;

[0150] NK cell-frozen (8): group administered eight times with thawed cryopreserved NK cells; and

[0151] ADR: adriamycin-treated group.

[0152] FIG. 6c shows the tumor weight-reducing effect of NK cells against colorectal cancer according to freezing or not of NK cells.

[0153] V.C: vehicle control group;

[0154] NK cell-Live (4): group administered four times with fresh NK cells;

[0155] NK cell-Live (1)+(3) frozen: group administered once with fresh NK cells and three times with thawed cryopreserved NK cells;

[0156] V.C (serum-free medium): serum-free medium group;

[0157] NK cell-frozen (4): group administered four times with thawed cryopreserved NK cells;

[0158] NK cell-frozen (8): group administered eight times with thawed cryopreserved NK cells; and

[0159] ADR 2 mpk: adriamycin-treated group.

[0160] FIG. 7a shows administration schedules prepared to examine the anticancer effects of NK cells according to freezing or not of NK cells, the number of the NK cells and the number of administrations of the NK cells.

[0161] FIG. 7b shows the tumor volume effects of NK cells against colorectal cancer according to freezing or not of NK cells, the number of the NK cells and the number of administrations of the NK cells.

[0162] V.C (5% HSA): vehicle control group;

[0163] NK fresh (4) cells: group administered four times with fresh NK cells for 4 weeks;

[0164] NK fresh (2) cells: group administered twice with fresh NK cells for 4 weeks;

[0165] NK fresh (1)+(6) frozen cells: group administered once with fresh NK cells and then administered six times with thawed cryopreserved cells;

[0166] Doxorubicin HCL;

[0167] V.C (serum-free medium): serum-free control group;

[0168] NK frozen (8) cells (serum-free medium): group administered eight times with thawed cryopreserved NK cells in serum-free medium;

[0169] V.C (distilled water): sterile distilled water control group; NK frozen (8) cells (distilled water): group administered eight times with thawed cryopreserved NK cells in sterile distilled water.

[0170] FIG. 7c shows the tumor weight-reducing effects of NK cells against colorectal cancer according to freezing or not of NK cells, the number of the NK cells and the number of administrations of the NK cells.

[0171] V.C (5% HSA): vehicle control group;

[0172] NK fresh (4) cells: group administered four times with fresh NK cells for 4 weeks;

[0173] NK fresh (2) cells: group administered twice with fresh NK cells for 4 weeks;

[0174] NK fresh (1)+(6) frozen cells: group administered once with fresh NK cells and then administered six times with thawed cryopreserved cells;

[0175] Doxorubicin HCL;

[0176] V.C (serum-free medium): serum-free control group;

[0177] NK frozen (8) cells (serum-free medium): group administered eight times with thawed cryopreserved NK cells in serum-free medium;

[0178] V.C (distilled water): sterile distilled water control group;

[0179] NK frozen (8) cells (distilled water): group administered eight times with thawed cryopreserved NK cells in sterile distilled water.

[0180] FIG. 8a shows an administration schedule prepared to examine the anticancer effect of NK cells against lung cancer according to the number of NK cells.

[0181] FIG. 8b shows the tumor volume inhibitory effect of NK cells against lung cancer according to the number of NK cells.

[0182] V.C: vehicle control group; and

[0183] Dox.hcl: Doxorubicin HCL.

[0184] FIG. 8c shows the tumor weight-reducing effect of NK cells against lung cancer according to the number of NK cells.

[0185] V.C: vehicle control group; and

[0186] Dox.hcl: Doxorubicin HCL.

[0187] FIG. 8d shows the results of H-E staining performed to confirm that NK cells infiltrate into tumor tissue.

[0188] V.O: serum-free control group;

[0189] arrow: dead cancer cells; and

[0190] CA: cancer cells.

[0191] FIG. 8e shows the results of CD56 analysis perfoLmed to confirm that NK cells infiltrate into tumor tissue.

[0192] V.O: serum-free medium control group,

[0193] arrow: CD56-positive cells, and

[0194] CA: cancer cells.

[0195] FIG. 9a shows an administration schedule prepared to examine the anticancer effects of NK cells against lung cancer, liver cancer and pancreatic cancer.

[0196] CB NK cells: umbilical cord blood-derived NK cells; and

[0197] PBL NK cells: peripheral cell-derived NK cells.

[0198] FIG. 9b shows the tumor volume inhibitory effects of NK cells against lung cancer (A549), liver cancer (SNU-709) and pancreatic cancer (MIA-PaCa-2).

[0199] V.C: vehicle control group,

[0200] CB NK cells: umbilical cord blood-derived NK cells; and

[0201] PBL NK cells: peripheral cell-derived NK cells.

[0202] FIG. 9c shows the tumor weight-reducing effects of NK cells against lung cancer (A549), liver cancer (SNU-709) and pancreatic cancer (MIA-PaCa-2).

[0203] V.C: vehicle control group,

[0204] CB NK cells: umbilical cord blood-derived NK cells; and

[0205] PBL NK cells: peripheral cell-derived NK cells.

BEST MODE

[0206] Hereinafter, the present invention describes in detail with reference to examples and experimental examples. It is to be understood, however, that these examples and experimental examples are for illustrative purposes and are not intended to limit the scope of the present invention.

EXAMPLE 1

Production of NK Cells

[0207] Umbilical cord blood and peripheral blood, provided for research purposes from the Department of Obstetrics and Gynecology, Konyang University Hospital (Korea) and the Department of Obstetrics and Gynecology, Chungnam National University Hospital (Korea) (approved by the IRB of each hospital), were diluted at 2:1 with RPMI 1640 to prepare a blood dilution. The prepared blood dilution was placed carefully in the upper layer of Ficoll-Paque, and then centrifuged at 2,000 rpm for 30 minutes to obtain a mononuclear cell layer (MNC layer). Cells were carefully collected from the mononuclear cell layer, and erythrocytes were removed from the collected cells to obtain monocytes. CD3 microbeads (Miltenyi Biotech) were added to the obtained monocytes to label with CD3, and then CD3-positive cells were removed using CS column and Vario MACS to obtain CD3-negative cells. Specifically, the CD3 microbeads (Miltenyi Biotech) recognized CD3 chains and capture CD3-positive cells from the monocytes so as to be magnetized. Then, among the monocytes, CD3-positive cells to which the microbeads were attached were passed through a MACS column reacting with a magnet, and thus CD3-positive cells remained in the column, and only CD3-negative cells were separated from the column.

[0208] Blood was diluted with saline and treated with a suitable amount of RosetteSep capable of cross-linking to CD3-positive cells, depending on the counted number of cells, after which the blood was agitated at room temperature for 20 minutes. After agitation, the blood were diluted 2-fold and placed onto Ficoll-Paque solution in such a manner that layers would not be mixed, after which the resulting solution was centrifuged at 2,000 rpm at room temperature for 20-30 minutes. After removal of the supernatant, the separated monocyte layer was collected and washed to obtain CD3-negative cells. The RosetteSep component is a tetrameric complex comprising mouse- and rat-derived monoclonal antibodies, glycoporin A antibody, and P9 antibody or P9 F(ab) antibody serving as a support. In the process of isolating the CD3-negative cells, the tetrameric complex of RosetteSep added to the blood crosslinks to CD3-positive cells in the blood to form immunorosettes, and the immunorosettes having a density higher than that of Ficoll is located below Ficoll by Ficoll-based density gradient centrifugation, and CD3-negative cells that did not bind to the tetrameric complex are located above Ficoll and isolated. The isolated CD3-negative cells were seeded into a T75 flask at a concentration of 110.sup.6 cells/ml, and cultured with IL-15 and IL-21 in alpha-MEM complete medium under the conditions of 37 C. and 5% CO.sub.2 for 10-21 days. During culture, the concentration of the cells did not exceed 210.sup.6 cells/ml, and was adjusted to a concentration of 110.sup.6 cells/ml by use of a medium having the same conditions as those of the original medium. On 4, 8, 14, 18 and 21 days, the cell number was counted, and on 4, 8, 14 and 21, the cells were stained with CD3 and CD56 antibodies, and the proportion of CD3.sup.CD56.sup.+ NK cells was analyzed by FACS according to a known method.

[0209] As a result, as shown in FIG. 1, differentiation of NK cells from the CD3-negative cells isolated from umbilical cord blood (the top of FIG. 1) and peripheral blood (the bottom of FIG. 1) was induced after 11-21 days of culture.

EXAMPLE 2

Production of Cryopreserved NK Cells

[0210] 2-1: Production of Cryopreserved NK Cells from Differentiated NK Cells

[0211] The NK cells (after 10 days of culture) produced by the method of Example 1 was cryopreserved to produced cryopreserved NK cells. The frozen storage was performed using a cryopreservation medium (Cryostor) containing 10% DMSO (dimethyl sulfoxide) under serum-free, protein-free and animal component-free conditions, and the differentiated NK cells were frozen at a concentration of 2.2510.sup.7 cells/1.5 ml (1.510.sup.7 cells/ml). The freezing was performed using a cryopreservation box containing isopropyl alcohol, and the cells were cooled stepwise from 70 C. (deep freezer) and finally preserved at 200 C. (LN2).

[0212] The cryopreservation was performed for 1 month. Immediately before use, the cryopreserved cells were thawed rapidly at 37 C. by washing the cells with saline to remove the cryopreservation medium. The thawed cryopreserved NK cells were analyzed by FACS to determine the proportion of CD3.sup.CD56.sup.+ NK cells, the proportion of NK receptors, the cell viability, and their cytotoxicity to CML (chronic myelogenous leukemia) cells, and the characteristics thereof were compared with fresh NK cells (fresh NK cells, not cryopreserved) of the same origin.

[0213] The results are shown in FIG. 2a (1. viability), FIG. 2b (2. degree of differentiation), FIG. 2c (3. receptors), FIG. 2d (4. cytotoxicity). As can be seen in FIGS. 2a to 2d, it was shown that the thawed cryopreserved NK cells had characteristics similar to those of fresh NK cells.

[0214] 2-2: Production of NK Cells from Cryopreserved CD3-Negative Cells

[0215] According to the same method as described in Example 1, CD3-negative cells were obtained from umbilical cord blood, and the CD3-negative cells were cryopreserved using the same cryopreservation medium as described in Example 2-1. The concentration of the CD3-negative cells during cryopreservation was 2.2510.sup.7 cells/1.5 ml (1.510.sup.7 cells/ml), and the cryopreservation was performed for about 1 month.

[0216] To obtain differentiated NK cells from the cryopreserved CD3-negative cells, the frozen cells were thawed and cultured in the same manner as described in Example 1. On 0, 2, 4, 7, 9 and 12 days after thawing, the number and viability of the cells were measured, and on 0, 7 and 9 days after thawing, the proportion of CD3-CD56+ NK cells was analyzed by FACS.

[0217] As a result, it was shown that the NK cells obtained from the cryopreserved CD3-negative cells showed characteristics similar to those of fresh NK cells with respect to all the recovery rate of cells recovered after thawing (FIG. 2e), the number of cells (FIG. 2f), the viability of cells (FIG. 2g) and the degree of differentiation (FIG. 2h).

EXAMPLE 3

Examination of In Vivo Distribution of NK Cells in Mice

[0218] In order to examine the tissue distribution of the fresh NK cells produced in Example 1, the following experiment was performed.

[0219] To examine the distribution of NK cells, the NK cells were labeled with DiR. Specifically, 110.sup.7 cells were suspended in 10 ml of 1PBS (phosphate buffered saline) containing 3.5 g/ml of DiR (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine, Sigma, USA) dye and 0.5% ethanol, and were incubated at 37 C. for 1 hour. After incubation, the cells were washed twice with 1PBS and stained with tryphan blue to examine the in vivo distribution of the NK cells.

[0220] In order to examine the tissue distribution of the DiR-labeled NK cells at varying time points, 5% HSA or DiR-labeled NK cells were injected intravenously into BALB/C female nude mice at cell concentrations of 110.sup.3, 110.sup.4, 510.sup.4, 110.sup.5, 510.sup.5, 110.sup.6 and 510.sup.6 cells, and after a given amount of time, and the distribution of the NK cells in the mouse or the major organs (liver, spleen, heart and kidney) extracted from the mice by autopsy was examined using a live animal imaging system (PHOTONE IMAGER, Biospace) according to the manufacturer's protocol.

[0221] To measure the detection limit of the DiR-labeled NK cells at varying concentrations, the in vivo distribution of the DiR-labeled NK cells was examined 24 hours after injection of the cells. As a result, as shown in FIG. 3a, at cell concentrations equal to or lower than 510.sup.4 cells, the distribution pattern of the DiR-labeled NK cells was not clear, and at cell concentrations equal to or higher than 110.sup.5 cells, the image signal was strongly detected in the abdomen of the mice in a concentration-dependent manner (FIG. 3a). FurtheLmore, the major intra-abdominal organs (liver, spleen, kidney, heart and lung) were extracted and imaged, and as a result, it was shown that, at cell concentrations equal to or lower than 510.sup.4 cells, a weak distribution of the NK cells was observed only in the lung, but at cell concentrations equal to higher than 110.sup.5 cells, the image signal was strongly detected in the liver, the spleen and the lung in a concentration-dependent manner (FIG. 3b).

[0222] Next, in order to examine the distribution of the DiR-labeled NK cells in tissue, 110.sup.7 cells were administered intravenously into the mice, and after 30 minutes and 2 hours, the distribution of the cells was measured. As a result, as shown in FIG. 3c, the distribution of the DiR-labeled NK cells in the abdomen was strongly detected from the start of the measurement, and image signals, which were 4-fold and 3.8-fold higher than the vehicle control group, were detected after 30 minutes and 2 hours, respectively. In addition, the liver, spleen, kidney, heart and lung, which are the major intra-abdominal organs, were extracted and imaged, and as a result, the distribution of the DiR-labeled NK cells in the liver, spleen and lung was strongly detected (FIG. 3c). Particularly, in the liver, image signals, which were about 9.5-fold and 5.1-fold higher than those in the vehicle control group, were detected at 30 minutes and 2 hours, respectively.

[0223] Next, the distribution was measured three times a week during a period ranging from one day after intravenous administration of the DiR-labeled NK cells to the day on which the DiR-labeled NK cells were not detected. As a result, as shown in FIG. 3d, up to days after administration, the DiR-labeled NK cells were strongly detected, and after 14 days, the image signal started to be weakened, and on 42 days, the DiR-labeled NK cells were not detected in the measured image (FIG. 3d). The above results indicate that the NK cells of the present invention, after administered in vivo, survive in vivo for at least 30 days, and are abundantly distributed in lung, liver, spleen and the like.

EXPERIMENTAL EXAMPLE 1

Examination of the Anticancer Effect of Administration of Fresh NK Cells against Colorectal Cancer

[0224] 1-1: Examination of Tumor Volume Inhibition According to the Number of Administrations of NK Cells and the Use of NK Cells in Combination with IL-2

[0225] In order to examine the anticancer effect of the NK cells produced by the method of Example 1, mouse models xenografted with human colorectal cancer SW620 cells (Korea Research Institute of Bioscience and Biotechnology, Korea) were used.

[0226] Specifically, SW620 cells were suspended in PBS at a concentration of 210.sup.7 cells/ml, and then injected subcutaneously into the axilla between the right shoulder and the chest wall in an amount of 0.3 ml per mouse. At 2 hours after injection of the cancer cells, the NK cells were injected into the mice at concentrations of 310.sup.5, 110.sup.6, 310.sup.6 and 110.sup.7 cells/mouse. In M addition, IL-2 was diluted in PBS, and the mice were treated with the IL-2 dilution at an IL-2 concentration of 10,000 U/mouse. During the experimental period, 0.2 ml of the NK cells were injected into the tail vein of each mouse once a week, a total of four times (0, 7, 14 and 21 days). Then, to measure the change in the tumor volume, the sizes in the three directions of the tumor were measured using vernier calipers a total of 7 times during a period ranging from the day of start of drug administration to the day of autopsy, and then the tumor cell volume was measured using the following equation 1:

Tumor cell volume=(length+width+height)/2 Equation 1

[0227] As a result, as shown in FIG. 4b, the groups injected with the NK cells at concentrations of 310.sup.5, 1>10.sup.6, 310.sup.6 and 110.sup.7 cells/mouse showed tumor growth inhibitions of 23.8%, 53.4% (p<0.001), 59.4% (p<0.001) and 76.8% (p<0.001), respectively, compared to that in the vehicle control group, and the positive control group administered with adriamycin showed a tumor growth inhibition of 58.3% (p<0.001). In addition, when the mice were treated with the NK cells in combination with IL-2, the groups injected with the NK cells at concentrations of 310.sup.5, 110.sup.6, 310.sup.6 and 110.sup.7 cells/mouse showed tumor growth inhibitions of 17.0%, 33.8% (p<0.01), 49.8% (p<0.01) and 73.5% (p<0.001), respectively (FIG. 4b).

[0228] 1-2: Examination of Tumor Weight Reduction According to the Number of Administrations of NK Cells and the Use of NK Cells in Combination with IL-2

[0229] In order to examine the anticancer effects of the NK cells according to the number of administration of the NK cells and the use of the NK cells in combination with IL-2, the weights of tumors in drug-treated mice were measured.

[0230] According to the same method as described in Experimental Example 1-1, mice were treated with the NK cells alone or in combination with IL-2. On day 23, the mice were sacrificed using CO.sub.2 gas, and tumors were separated from the sacrificed mice and weighed using a chemical balance. The tumors were photographed and fixed in liquid nitrogen. All the measurements were analyzed by t-TEST to determine the statistical significance between the vehicle control group and the administered groups.

[0231] As a result, as shown in FIG. 4c, the groups injected with the NK cells at concentrations of 310.sup.5, 110.sup.6, 310.sup.6 and 110.sup.7 cells/mice showed tumor weight reductions of 23.4%, 54.0%, 59.1% (p<0.05) and 78.8% (p<0.01), respectively, compared to the vehicle control group (FIG. 4c). In addition, when the mice were treated with the NK cells in combination with IL-2, the groups injected with the NK cells at concentrations of 310.sup.5, 110.sup.6, 310.sup.6 and 110.sup.7 cells/mice showed tumor weight reductions of 17.0%, 34.3%, 47.0% and 75.6% (p<0.01), respectively, and the positive control group administered with adriamycin showed a tumor weight reduction of 58.2% (p<0.05) (FIG. 4c).

[0232] 1-3: Examination of Cytotoxicity According to the Number of NK Cells and the Use of NK Cells in Combination with IL-2

[0233] In order to examine the cytotoxicity of the NK cells to the mice of Experimental Example 1-1 according to administration of the NK cells alone or in combination with IL-2, the change in body weight and general symptoms of the mice were observed.

[0234] As a result, in the groups administered with the NK cells alone or in combination with IL-2, a normal increase in the body weight was observed without general symptoms during the experimental period, unlike the vehicle control group. However, the positive control group administered with adriamycin showed a statistically significant weight reduction of 21.8% (p<0.01).

[0235] In conclusion, the NK cells of the present invention, when administered alone or in combination with IL-2, did not show general symptoms and toxic symptoms such as weight loss. Regarding the anticancer effects, the group administered with the NK cells alone showed an excellent tumor growth inhibition of 70% or more, and the group administered with the NK cells in combination with IL-2 did not show increased effects compared to the group administered with the NK cells alone.

EXPERIMENTAL EXAMPLE 2

Examination of Anticancer Effects of NK Cells According to the Incubation, Preservation Conditions and Administration Schedules of NK Cells

[0236] 2-1: Examination of Tumor Size Inhibition According to the Incubation, Preservation Conditions and Administration Schedules of NK Cells

[0237] In order to examine the anticancer effects of the NK cells of the present invention according to the NK cell incubation method (comprising removing CD3-positive T cells by Rosettesep or not comprising the removal process), preservation conditions (fresh cells or cold-preserved cells) and administration schedules (once a week for 4 weeks, or twice a week for 2 weeks).

[0238] Specifically, NK cells were divided according to culture conditions into fresh NK, fresh NK (w/o Rosettesep), 4 C. cold-preserved NK and 4 C. cold-preserved NK (w/o Rosettesep), and the fresh NK cells were produced in the same manner as described in Example 1. The fresh NK (w/o Rosettesep) cells were produced in the same manner as described in Example 1, except that the step of removing CD3-positive cells was omitted. The 4 C. cold-preserved NK cells were obtained by preserving fresh NK cells at 4 C. for 12 hours, and the 4 C. cold-preserved NK (w/o Rosettesep) cells were obtained by producing NK cells without the step of removing CD3-positive cells, and preserving the produced cells at 4 C. for 12 hours. In order to administer the produced NK cells to mouse models, cancer cells were transplanted into mice in the same manner as described in Experimental Example 1-1, and when the average tumor volume reached about 50.0 mm.sup.3, and the NK cells were administered to the mice at a concentration of 310.sup.6 cells/mouse, and 0.2 ml of the NK cells were injected into the tail vein of each mouse. In the administration schedule, the NK cells were administered once a week for 4 weeks or administered twice a week for 2 weeks (FIG. 5a).

[0239] In order to examine the toxicity of the NK cells during the experimental period, the weight change and general symptoms of the mice were observed. As a result, in the groups administered with the NK cells once a week for 4 weeks [fresh NK, fresh NK (w/o Rosettesep)] and twice a week for 2 weeks [fresh NK, fresh NK (w/o Rosettesep), 4 C. cold-preserved NK, or 4 C. cold-preserved NK (w/o Rosettesep)], a normal increase in the weight was observed without general symptoms during the experimental period, compared to the vehicle control group. However, in the positive control group administered with adriamycin, two dead animals and a statistically significant weight reduction of 31.5% (p<0.001) were observed.

[0240] In addition, in order to examine the anticancer effects of the NK cells according to the incubation, preservation conditions and administration schedules of the NK cells, the change in tumor volume was examined. As a result, as shown in FIG. 5b, the group, administered with fresh NK and fresh NK (w/o Rosettesep) cells once a week for 4 weeks at a concentration of 310.sup.6 cells/mouse, showed tumor growth inhibitions of 50.8% (p<0.001) and 32.7% (p<0.05), respectively, compared to the vehicle control group, and the groups, administered with fresh NK, fresh NK (w/o Rosettesep) and 4 C. cold-preserved NK cells twice a week for 2 weeks, showed tumor growth inhibitions of 35.4%, 10.8% and 33.0%, respectively. The positive control group administered with adriamycin showed a tumor growth inhibition of 71.7% (p<0.01). However, the group administered with 4 C. cold-preserved NK (w/o Rosettesep) cells showed no tumor growth inhibition. This suggests that, when the process of removing CD3-positive T cells is not performed, the proportion of NK cells is low, and thus the anticancer effect of the NK cells is reduced. Thus, it can be seen that the process of removing CD3-positive T cells is necessary to increase the anticancer effect of the NK cells.

[0241] 2-2: Examination of Tumor Weight Reduction According to the Incubation, Preservation Condition and Administration Schedule of NK Cells

[0242] In order to the anticancer effect of the NK cells of the present invention according to the incubation method, preservation condition and administration schedule of the NK cells, the reduction in tumor weight was examined.

[0243] Specifically, cancer cells was transplanted into mice in the same manner as described in Experimental Example 1-1, and NK cells were administered under the same conditions as described in Experimental Example 2-1. On 27 days after drug administration, the tumor was excised and weighed.

[0244] As a result, as shown in FIG. 5c, the groups, administered with fresh NK and fresh NK (w/o Rosettesep) cells once a week for 4 weeks at a concentration of 310.sup.6 cells/mouse, showed tumor weight reductions of 49.0% (p<0.001) and 33.1% (p0.05), compared to the vehicle control group. Furthermore, the groups, administered with fresh NK, fresh NK (w/o Rosettesep) and 4 C. cold-preserved NK cells twice a week for 2 weeks, showed tumor weight reductions of 30.3%, 7.2% and 29.0%, respectively, and the positive control group administered with adriamycin showed a tumor growth inhibition of 70.2% (p<0.05) (FIG. 5c).

[0245] In conclusion, when the NK cells incubated under fresh NK incubation conditions were administered once a week for 4 weeks at a concentration of 310.sup.6 cells/mouse, these cells showed excellent anticancer effects. In addition, when the NK cells were administered twice a week for 2 weeks, the fresh NK and the 4 C. cold-preserved NK cells showed higher anticancer activities higher than the fresh NK (w/o Rosettesep) and the 4 C. cold-preserved NK (w/o Rosettesep) cells.

EXPERIMENTAL EXAMPLE 3

Examination of Anticancer Effect According to Freezing or Not of NK Cells

Example 3-1

Examination of Tumor Volume Inhibition According to Freezing or Not of NK Cells

[0246] In order to examine the anticancer effect of the NK cells of the present invention according to freezing or not of the NK cells, the reduction in tumor volume was examined.

[0247] Specifically, cancer cells were transplanted into mice in the same manner as described in Experimental Example 1-1, and after 2 hours, the NK cells were administered to the mice at a concentration of 310.sup.6 cells/mouse, and 0.2 ml of the NK cells were injected into the tail vein of each mouse. Herein, the NK cells were administered under the following conditions: fresh NK cells (once a week for 4 weeks, a total of four times), thawed cryopreserved NK cells (once a week for 4 weeks, a total of four times), thawed cryopreserved NK cells (twice a week for 4 weeks, a total of eight times), and fresh NK cells (once a week for one week) plus thawed cryopreserved NK cells (once a week for three weeks, a total of three times). Vehicle control groups were administered with 5% HAS and serum free medium, and a positive control group was administered intraperitoneally with 1 mg/kg of adriamycin at 2-day intervals (FIG. 6a).

[0248] To examine toxicity during the experimental period, the weight change and general symptoms of the mice were observed. As a result, in all the groups administered with the NK cells, a normal increase in the weight was observed without general symptoms during the experimental period, compared to the vehicle control groups. However, in the positive control group administered with adriamycin, two dead animals and a statistically significant weight reduction of 32.1% (p<0.001) appeared.

[0249] In addition, in order to examine the anticancer effect of the NK cells according to freezing or not of the NK cells, the change in tumor volume on day 27 was examined. As a result, as shown in FIG. 6b, the groups, administered with fresh NK cells (a total of four times), thawed cryopreserved cells (a total of eight times), and fresh NK cells (once) plus thawed cryopreserved NK cells (a total of three times), showed tumor growth inhibitions of 58.8% (p<0.001), 45.2% (p<0.001) and 19.2%, respectively, and the positive control group showed a tumor growth inhibition of 60.1% (p<0.01) (FIG. 6b).

[0250] 3-2: Examination of Tumor Weight Reduction According to Freezing or Not of NK Cells

[0251] In order to examine the anticancer effect according to freezing or not of the NK cells of the present invention, the reduction in tumor weight was examined.

[0252] Specifically, cancer cells were transplanted into mice in the same manner as described in Experimental Example 1-1, and then NK cells were administered to the mice under the same conditions as described in Experimental Example 4-1. On 27 days after drug administration, the tumor was excised and weighed.

[0253] As a result, as shown in FIG. 6c, the groups, administered with fresh NK cells (a total of 4 times), thawed cryopreserved NK cells (a total of 8 times), and fresh NK cells (once) plus thawed cryopreserved NK cells (a total of 3 times), showed tumor weight reductions of 58.5% (p<0.001), 46.2% (p<0.01) and 19.5%, respectively, and the positive control group showed a tumor weight reduction of 60.5% (p<0.05) (FIG. 6c).

[0254] In conclusion, the above-described results indicate that, when the fresh NK cells (once a week for 4 weeks, a total of 4 times) or the thawed cryopreserved NK cells (twice a week for 4 weeks, a total of 8 times) are administered at a concentration of 310.sup.6 cells/mouse, they show significant anticancer effects without general symptoms and toxic symptoms such as weight loss.

Experimental Example 4

Examination of Anticancer Effects of NK Cells According to Freezing or Not of NK Cells and the Number of Administrations of NK Cells

[0255] 4-1: Examination of Tumor Volume Inhibition According to Freezing or Not of NK Cells and the Number of Administrations of NK Cells

[0256] In order to examine the anticancer effects of the NK cells of the present invention according to freezing or not of the NK cells and the number of administrations of the NK cells, the reduction in tumor volume was examined.

[0257] Specifically, cancer cells were transplanted into mice in the same manner as described in Experimental Example 1-1. After 2 hours, fresh NK cells and thawed cryopreserved NK cells were administered to the mice at concentrations of 310.sup.6 cells/mouse and 610.sup.6 cells/mouse, and 0.2 ml of the NK cells were injected into the tail vein of each mouse. Herein, the NK cells were administered under the following conditions: 310.sup.6 fresh NK cells/mouse (once a week for 4 weeks, a total of 4 times); 610.sup.6 fresh NK cells/mouse (once in two weeks for 4 weeks; a total of two times); and fresh NK cells (310.sup.6 cells/mouse; once a week for 1 week) plus thawed cryopreserved cells (310.sup.6 cells/mouse; twice a week for 3 weeks, a total of 6 times); thawed cryopreserved NK cells (serum free medium; twice a week for 4 weeks, a total of 8 times); and thawed cryopreserved NK cells (distilled water; twice a week for 4 weeks, a total of 8 times). Vehicle control groups were injected intravenously with the same amount of 5% HAS, serum-free medium and distilled water, respectively, and a positive control group was administered intraperitoneally with 2 mg/kg of doxorubicin HCL at 2-day intervals (FIG. 7a).

[0258] In order to examine toxicity during the experimental period, the weight change and general symptoms of the animals were observed. As a result, in all the groups injected with the NK cells, a noLmal increase in the weight was observed without general symptoms, compared to the vehicle control group. However, in the positive control group administered with doxorubicin HCL, two dead animals during the administration period and a statistically significant weight reduction of 25.0% (p<0.01) on the last day appeared.

[0259] In addition, in order to examine the anticancer effects of the NK cells according to freezing or not of the NK cells and the number of administrations of the NK cells, the change in tumor volume on day 26 was examined. As a result, as shown in FIG. 7b, the groups, administered with 310.sup.6 fresh NK cells/mouse (a total of 4 times), 610.sup.6 fresh NK cells/mouse (a total of two times), fresh NK cells (310.sup.6 cells/mouse; once) plus thawed cryopreserved NK cells (310.sup.6 cells/mouse; a total of 6 times), thawed cryopreserved NK cells (serum free medium; a total of 8 times), and thawed cryopreserved NK cells (distilled water; a total of 8 times), showed tumor growth inhibitions of 68.3% (p<0.001), 61.5% (p<0.001), 63.7% (p<0.001), 55.1% (p<0.01) and 38.8%, respectively. The positive control group administered with doxorubicin HCl showed a tumor growth inhibition of 72.9% (p<0.01) on the last day (FIG. 7b).

[0260] 4-2: Examination of Tumor Weight Reduction According to Freezing or Not of Cells, Number of Cells and the Number of Administrations of Cells

[0261] In order to examine the anticancer effects of the NK cells of the present invention according to freezing or not of the NK cells, the number of the NK cells and the number of administrations of the NK cells, the reduction in tumor weight was examined.

[0262] Specifically, cancer cells were transplanted into mice in the same manner as described in Experimental Example 1-1, and then NK cells were administered to the mice under the same conditions as described in Experimental Example 4-1. On 26 days after drug administration, the tumor was excised and weighed. As a result, as shown in FIG. 7c, the groups, administered with 310.sup.6 fresh NK cells/mouse (a total of 4 times), 610.sup.6 fresh NK cells/mouse (a total of two times), fresh NK cells (310 cells/mouse; once) plus thawed cryopreserved NK cells (310.sup.6 cells/mouse; a total of 6 times), thawed cryopreserved NK cells (serum free medium; a total of 8 times), and thawed cryopreserved NK cells (distilled water; a total of 8 times), showed tumor weight reductions of 68.6% (p<0.001), 61.8% (p<0.01), 59.1% (p<0.01), 50.2% (p<0.05) and 40.8% (p<0.05), respectively. The group administered with the positive control Doxorubicin HCl showed a tumor weight reduction of 70.4% (p<0.01) (FIG. 7c).

[0263] In conclusion, the above-described results indicated that, when fresh NK cells (once a week for 4 weeks, a total of 4 times; or once in two weeks for 4 weeks, a total of two times), thawed cryopreserved NK cells (serum free medium; twice a week for 4 weeks, a total of 8 times), and fresh NK cells (once a week for 1 week) plus thawed cryopreserved NK cells (twice a week for 3 weeks, a total of 6 times), were injected into the tail veins of each mouse at the above-described concentrations, these cells showed statistically significant excellent effects on the inhibition of tumor growth without causing general symptoms and toxic symptoms such as weight loss.

EXPERIMENTAL EXAMPLE 5

Examination of the Anticancer Effect of NK Cells Against Lung Cancer According to the Number of NK Cells

[0264] 5-1: Examination of the Effects of NK Cells on Tumor Growth Inhibition and Tumor Weight Reduction According to Number of NK Cells

[0265] Using mouse models xenografted with human lung cancer NCI-H460 cells (Korea Research Institute of Bioscience and Biotechnology, Korea), the anticancer effect of the NK cells of the present invention was examined according to the number of the NK cells by intravenously injecting varying doses of the NK cells in a repeated manner.

[0266] Specifically, cancer cells were transplanted into mice in the same manner as described in Experimental Example 1-1, and when the average tumor volume reached 50.0 mm.sup.3, NK cells were injected into the mice at concentrations of 310.sup.5, 110.sup.6, 310.sup.6 and 110.sup.7 cells/mouse. 0.2 ml of the NK cells were injected into the tail vein of each mouse once a week for 4 weeks (a total of 4 times). A solvent control group was administered with 5% HSA, and a positive control group was administered intraperitoneally with 2 mg/kg of doxorubicin HCL at 2-day intervals (FIG. 8a).

[0267] In order to examine toxicity during the experimental period, the weight change and general symptoms of the mice were observed. As a result, in all the groups administered with varying doses of the NK cells, a normal increase in the weight was observed without general symptoms during the experimental period, compared to the vehicle control group. However, in the positive control group administered with doxorubicin HCL, a statistically significant weight reduction of 43.3% (p<0.001) appeared.

[0268] In order to examine the anticancer effect of the NK cells, the tumor volume was measured a total of 11 times according to the method of Experimental Example 1 during a period ranging from the day of start to the day of autopsy. On the last day (day 26), the mice were sacrificed, and the tumor was separated from the sacrificed mice, and then the volume of the tumor was measured to determine the tumor growth inhibitory effect of the NK cells. As a result, as shown in FIG. 8b, the group, administered with NK cells at a concentration of 110.sup.7 cells/mouse, showed a significant tumor growth inhibition of 47.9% (p<0.001), compared to the vehicle control group. The groups, administered with NK cells at concentrations of 310.sup.5, 110.sup.6 and 310.sup.6 cells/mouse, showed tumor growth inhibition up to 10 days after administration of the NK cells, but after 10 days, the tumor growth in these group showed a tendency to increase as the inhibition rate decreased. Furthermore, the positive control group showed a tumor growth inhibition of 53.8% (p<0.001) (FIG. 8b).

[0269] In addition, the effect of the NK cells on tumor weight reduction was examined. As a result, as shown in FIG. 8c, the group, administered with NK cells at a concentration of 110.sup.7 cells/mouse, showed a significant tumor weight reduction of 46.5 (p<0.001), compared to the vehicle control group, and the positive control group showed a tumor weight reduction of 52.4% (p<0.001) (FIG. 8c).

[0270] 5-2: Examination of NK Cell Infiltration into Tumor Tissue

[0271] In order to examine the amount of NK cells that infiltrated into tumor tissue when the NK cells were administered under the conditions described in Experimental Example 5-1, the following experiment was performed.

[0272] Specifically, mouse cancer tissue was fixed in 10% formalin solution at 4 C. for 12 hours. The fixed tissue was sectioned thinly and placed in PBS solution. For immunohistochemical staining of the NK cells, the tissue section was placed in 3% hydrogen peroxide solution for 30 minutes, and then placed in a solution containing 0.1 M PBS (pH 7.4), 0.1% triton X-100, serum bovine albumin and CD56 antibody (1:500, PharMigen, USA), followed by incubation at 4 C. for 12 hours. Thereafter, the section was incubated in a solution containing fluorescence-labeled anti-mouse IgG (1:200, PharMigen, USA) at room temperature for 1 hour, and then observed with a microscope. Alternatively, the fixed tissue was directly stained with hematoxylin and eosin and was observed with a microscope.

[0273] As a result, as shown in FIGS. 8d and 8e, when the NK cells were administered to the mice at concentrations of 310.sup.5, 110.sup.6 and 310.sup.6 cells/mouse, the number of dead cancer cells (arrow) increased in a manner dependent on the number of NK cells injected (FIG. 8d), and the number of the NK cell marker CD56-positive cells (arrow) significantly increased compared to that in the normal control group administered with the vehicle, and also CD56-positive cells mainly infiltrated around cancer tissue (FIG. 8e).

[0274] In conclusion, it was shown that, when the NK cells were injected into the tail veins of the mice once a week for 4 weeks at a concentration of 110.sup.7 cells/mouse, these NK cells showed significant tumor inhibitory effects against human lung cancer without causing general symptoms and toxic symptoms such as weight loss.

EXPERIMENTAL EXAMPLE 6

Examination of Anticancer Effects of NK Cells against Lung Cancer, Liver Cancer and Pancreatic Cancer

[0275] In order to the anticancer effects of the NK cells of the present invention against various kinds of cancer, the anticancer effects of the NK cells were examined by repeated intravenous injections into mouse models xenografted with human lung cancer A549 cells (Korea Research Institute of Bioscience and Biotechnology, Korea), liver cancer SNU-709 cells (Korea Research Institute of Bioscience and Biotechnology, Korea) and pancreatic cancer MIA-Paca-2 cells (Korea Research Institute of Bioscience and Biotechnology, Korea).

[0276] Specifically, cancer cells were transplanted into mice in the same manner as described in Experimental Example 1-1, and when the average tumor volume reached 50.0 mm.sup.3, NK cells were injected into the mice at a concentration of 610.sup.6 cells/mouse. 0.2 ml of the NK cells were injected into the tail vein of each mouse once a week for 4 weeks (a total of 4 times). A solvent control group was administered with 5% HSA (FIG. 9a).

[0277] In order to examine toxicity during the experimental period, the weight change and general symptoms of the mice were observed. As a result, in all the groups administered with varying doses of the NK cells, a normal increase in the weight was observed without general symptoms during the experimental period, compared to the vehicle control group.

[0278] In order to examine the anticancer effect of the NK cells, the tumor volume was measured a total of 11 times according to the method of Experimental Example 1 during a period ranging from the day of start to the day of autopsy. On the last day (day 25), the mice were sacrificed, and the tumor was separated from the sacrificed mice, and then the volume of the tumor was measured to determine the tumor growth inhibitory effect of the NK cells. As a result, as shown in FIG. 9b, in the lung cancer mouse models, the groups administered with umbilical cord blood-derived NK cells and peripheral blood-derived NK cells showed tumor growth inhibitions of 24.7% (p<0.05) and 9.0%, respectively, compared to the vehicle control group. In the liver cancer mouse models, the group administered with umbilical cord blood-derived NK cells showed a significant tumor growth inhibition of 37.7% (p<0.01). In the pancreatic cancer mouse models, the group administered with umbilical cord blood-derived NK cells showed a significant tumor growth inhibition of 28.2% (p<0.01) (FIG. 9b).

[0279] In addition, on 25 days after administration of the NK cells, the effects of the NK cells on tumor weight reduction were examined. As a result, as shown in FIG. 9c, in the lung cancer mouse models, the groups administered with umbilical cord blood-derived NK cells and peripheral blood-derived NK cells showed tumor growth inhibitions of 20.4% (p<0.01) and 10.8%, respectively, compared to the vehicle control group. In the liver cancer mouse models, the group administered with umbilical cord blood-derived NK cells showed a tumor weight reduction of 37.6% (p<0.01), and in the pancreatic cancer mouse models, the group administered with umbilical cord blood-derived NK cells showed a tumor weight reduction of 23.9% (p<0.01) (FIG. 9c).

[0280] In conclusion, it was shown that, when the NK cells of the present invention were injected into the tail veins of the mice once a week for 4 weeks at a concentration of 610.sup.6 cells/mouse, these NK cells showed significant tumor inhibitory effects against human lung cancer, liver cancer and pancreatic cells without causing general symptoms and toxic symptoms such as weight loss.