Natural killer cells exhibiting increased anticancer activity and immunotherapeutic use thereof
12319936 ยท 2025-06-03
Assignee
Inventors
Cpc classification
A61K40/15
HUMAN NECESSITIES
A61K2239/59
HUMAN NECESSITIES
A61K2239/38
HUMAN NECESSITIES
International classification
Abstract
Provided are a natural killer cell exhibiting increased anticancer activity and immunotherapeutic use thereof. According to the natural killer cell according to an aspect, a relative mean fluorescence intensity (MFI) of a specific receptor significant for cancers including glioblastoma is increased, and thus effective anticancer immunotherapy is possible.
Claims
1. A method of treating cancer, the method comprising administering an effective amount of an isolated natural killer cell, wherein the natural killer cell has: (1) an expression level of a KIR2DS4 gene on day 14 of culture which is 10 times to 50 times higher compared with that on day 0 of peripheral blood mononuclear cell (PBMC) culture; and (2) one or more characteristics selected from the following (a) to (e) or a population thereof to an individual in need thereof: (a) a relative mean fluorescence intensity (MFI) value of NKG2D exhibits a 1.2-fold to 12-fold increase, as compared with that on day 0 of PBMC culture; (b) a relative MFI value of NKp30 exhibits a 1.5-fold to 15-fold increase, as compared with that on day 0 of PBMC culture; (c) a relative MFI value of NKp44 exhibits a 12-fold to 22-fold increase, as compared with that on day 0 of PBMC culture; (d) a relative MFI value of integrin subunit alpha 1 (ITGA1) exhibits a 1.8-fold to 25-fold increase, as compared with that on day 0 of PBMC culture; and (e) a relative MFI value of integrin subunit alpha 2 (ITGA2) exhibits a 1.4-fold to 6-fold increase, as compared with that on day 0 of PBMC culture; wherein the relative MFI is defined by the following Equation 1:
Relative MFI=Receptor MFI/Isotype MFI.[Equation 1]
2. The method of claim 1, wherein the natural killer cell (i) promotes immune activation, blood-brain barrier penetration, or cell migration; or (ii) inhibits self-tolerance.
3. The method of claim 1, wherein the cancer is one or more selected from the group consisting of lung cancer, laryngeal cancer, stomach cancer, colorectal cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, fat cancer, fibrous cell carcinoma, blood cancer, leukemia, lymphoma, multiple myeloma, and glioma.
4. The method of claim 3, wherein the glioma is astrocytic tumor, oligodendroglial tumor, mixed glioma, or ependymal tumor.
5. The method of claim 4, wherein the astrocytic tumor is glioblastoma, anticancer drug-resistant glioblastoma, or recurrent glioblastoma.
6. The method of claim 1, wherein the cancer secretes or expresses MHC class I polypeptide-related sequence A (MICA) or platelet-derived growth factor-DD (PDGF-DD).
7. The method of claim 1, wherein the relative MFI value of (a) exhibits a 3-fold to 6-fold increase; the relative MFI value of (b) exhibits a 3-fold to 6-fold increase; the relative MFI value of (c) exhibits a 12-fold to 18-fold increase; the relative MFI value of (d) exhibits a 6-fold to 10-fold increase; and the relative MFI value of (e) exhibits a 2-fold to 4-fold increase.
8. The method of claim 1, wherein the natural killer cell further has one or more characteristics selected from CD16 having an MFI value of 10 to 140, LFA-1 having an MFI value of 20 to 160, NKG2D having an MFI value of 5 to 25, NKp30 having an MFI value of 5 to 20, NKp44 having an MFI value of 12 to 25, ITGA1 having an MFI value of 4 to 25, and ITGA2 having an MFI value of 2 to 10.
9. The method of claim 1, wherein the natural killer cell further has one or more characteristics selected from CD2 having an MFI value of 20 to 180, CD27 having an MFI value of 0.1 to 1.5, CD69 having an MFI value of 1 to 10, CD226 having an MFI value of 2 to 12, NKp46 having an MFI value of 2 to 8, CD160 having an MFI value of 0.1 to 4, KIR2DL1 having an MFI value of 0.1 to 4, KIR2DL3 having an MFI value of 0.1 to 5, KIR3DL1 having an MFI value of 0.1 to 4, NKG2A having an MFI value of 0.4 to 16, CD161 having an MFI value of 0.2 to 12, CCR3 having an MFI value of 0.3 to 3, CCR5 having an MFI value of 0.5 to 4, CCR6 having an MFI value of 0.8 to 6, CXCR3 having an MFI value of 0.4 to 5, CXCR1 having an MFI value of 0.4 to 5, CXCR2 having an MFI value of 0.1 to 3, and ITGB7 having an MFI value of 1 to 16.
10. The method of claim 1, wherein the natural killer cell further has any one characteristic selected from KIR2DS1+, KIR2DS2+, KIR2DS3+, CXCR1+, CXCR2+, CXCR3+, CCR3+, CCR5+, CCR6+, PSA-NCAM+, nestin+, tyrosine hydroxylase+, CD147+, CD127+, CD15+, CD31+, CD146+, CD49c+, CD107a+, NKG2A+, CD45+, CD44, CD140a+, CD87, CD11b+, CD10, and CD80 .
11. The method of claim 1, wherein 50% to 90% of the population expresses NKp44; 50% to 90% of the population expresses KIR2DS2; or 60% to 100% of the population expresses NKG2D.
12. The method of claim 1, wherein the natural killer cell is treated with PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, or PDGF-AB, when cultured from PBMC.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
DETAILED DESCRIPTION
(14) Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to these exemplary embodiments.
Example. Preparation of Natural Killer Cells Exhibiting Increased Immune Receptor Activity
(15) To prepare natural killer cells expressing immune receptors significant for glioblastoma, natural killer cells were cultured as follows.
(16) 1. Preparation of Peripheral Blood Mononuclear Cells
(17) 1.1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) and Plasma from Blood
(18) Blood was prepared by collecting blood from the vein of a normal person. At this time, a blood collection tube containing heparin was used as a blood collection container. Each 30 ml of the blood collected from a patient was carefully transferred to two tubes (#352070, BD, or equivalent or higher) containing Ficoll (#17-1440-02, GE Healthcare, or equivalent or higher). The tube containing the blood was centrifuged at 2,500 rpm in a break-off state for 10 minutes, and then the plasma portion of the upper layer was transferred to a new tube. The transferred plasma was inactivated in a heat block for 30 minutes, and then centrifuged at 4,000 rpm for 5 minutes. The supernatant from the centrifuged tube was transferred to a new tube, labeled as plasma, and stored at 2 C. to 8 C.
(19) Plasma was collected from the tube in which the blood and Ficoll were put and centrifuged, and a remaining pale yellow layer in the lower layer was transferred to a new tube while taking care not to mix with a red blood cell layer, and then Ca/Mg-free Dulbecco's phosphate-buffered saline (DPBS) (#14190, Gibco) was added thereto. Then, after centrifugation at 1,500 rpm for 5 minutes, the supernatant was discarded. Precipitated cells remaining after discarding the supernatant were suspended in 5 ml of red blood cell (RBC) lysis buffer (#158904, Qiagen). Thereafter, the cell suspension was centrifuged at 1,500 rpm for 5 minutes to discard the supernatant, and Ca/Mg free DPBS was added to the tube from which the supernatant was discarded, and centrifuged again at 1,500 rpm for 5 minutes. Precipitated cells remaining after discarding the supernatant were suspended in 1 ml of Alys505NK-EX (#01410P10, CSTI) medium.
(20) A small amount of the cell suspension suspended in Alys505NK-EX was taken and diluted 100 times with Ca/Mg free DPBS. Then, a small amount of the diluted solution was taken, and mixed with an equal volume of trypan blue. The mixture was put on a hemocytometer, and the number of cells and viability were determined.
(21) 1.2. Freezing of PBMCs
(22) All cell suspensions obtained in Example 1.1 were centrifuged at 1,500 rpm for 5 minutes, and then the supernatant was discarded. Cells were suspended in Cryostor CS10 or a mixture of ALyS505NK-EX+Albumin+DMSO stored at 2 C. to 8 C. such that the number of cells was 110.sup.6 cells/ml to 10010.sup.6 cells/ml. 1 mL of the cell suspension was aliquoted into 2 mL cryovials, which were then subjected to a first stage of freezing using a controlled rate freezer (CRF) at 0 C. for 10 minutes to 15 minutes, at 12 C. for 5 minutes to 10 minutes, and 42 C. for 0.5 minutes to 1 minute. After the first stage of freezing, the cryovials were subjected to a second stage of freezing at 25 C. for 1 minute to 3 minutes, and 15 C. for 1 minute to 3 minutes. After the second stage of freezing, the cryovials were subjected to freezing at 42 C. for 20 minutes to 40 minutes, and 120 C. for 20 minutes to 50 minutes. Alternatively, after the first stage of freezing in the range of 4 C. to 40 C. at 3 C./m, the second stage of freezing was performed in the range of 40 C. to 90 C. at 5 C./m, followed by freezing in the range of 90 C. to 120 C. at 5 C./m. The frozen cells were transferred to an LN.sub.2 tank and stored (below 130 C.).
(23) 1.3. Thawing of Frozen PBMCs
(24) A heat block was set at 37 C., and a culture medium supplemented with 10% plasma was put in a T flask. Depending on the cell density, the volume of the culture medium may be variously adjusted to, for example, 4 ml, 6 ml, 8 ml, or 10 ml. The frozen PBMCs were thawed by putting the cryovials frozen in Example 2.2 into the heat block. When the frozen PBMCs were partially thawed, they were transferred to a T flask containing the culture medium. Subsequently, the T flask was placed in a 5% CO.sub.2 incubator at 37 C., and incubated for one day. After collecting the cultured PBMCs in a tube, Ca/Mg free DPBS was added thereto, followed by centrifugation at 1,500 rpm for 5 minutes. Then, the supernatant was discarded. The cells isolated by centrifugation were suspended in a small amount of culture medium, and then the number of cells was counted. Table 1 shows viability after thawing of the cryopreserved cells. As shown in Table 1, 93% or more of PMBCs thawed after cryopreserving according to the present disclosure survived, indicating that high viability was maintained.
(25) TABLE-US-00001 TABLE 1 PBMC viability after thawing Origin Viability (%) Donor 1 98.07 Donor 2 93 Donor 3 97.4
2. 2.1. Culture of Natural Killer Cells Preparation of Culture Flask Coated with Fibronectin and Gamma Globulin (Primary)
(26) In a 15 ml tube, 0.01 ml of fibronectin (#FC-010, Millipore) and 0.121 ml of a gamma globulin (#020A1004, Green Cross) solution were put, and then 9.859 ml of Ca/Mg free DPBS was added. The prepared coating solution was put into a T75 flask (#156499, Nunc) using a pipette, and allowed to react at 2 C. to 8 C. for 16 hours or more. Before cell culture, the remaining coating solution was washed with Ca/Mg free DPBS and then removed.
(27) 2.2. Primary Culture of Natural Killer Cells
(28) The cell suspension prepared in Example 1 was taken and put in the coating flask prepared in Example 2.1, and 1.5 ml of autologous plasma, 0.075 ml of IL-18 (#B003-2, R&D), 0.075 ml of PDGF-DD (1159-SB, R&D), 0.03 ml of anti-NKp46 (#MAB1850, R&D), and 13.4625 ml of Alys505NK-EX (#01410P10, CSTI) were added thereto, followed by incubation in a CO.sub.2 incubator for two days to three days. Thereafter, 1.5 ml of autologous plasma and 13.5 ml of Alys505NK-EX were added to the flask, followed by incubation in a CO.sub.2 incubator for one day to two days.
(29) 2.3. Preparation of Culture Flask Coated with Fibronectin and Gamma Globulin (Secondary)
(30) In a 50 ml tube, 0.025 ml of fibronectin (#FC-010, Millipore) and 0.303 ml of a gamma globulin (#020A1004, Green Cross) solution were put, and then 24.647 ml of Ca/Mg free DPBS was added. The prepared coating solution was put into a T175 flask (#159910, Nunc) using a pipette, and allowed to react at 2 C. to 8 C. for 16 hours or more. Before cell culture, the remaining coating solution was washed with Ca/Mg free DPBS and then removed.
(31) 2.4. Secondary Culture of Natural Killer Cells and Treatment with New Function Enhancement Substances
(32) After the primary culture in Example 2.2, the T75 flask, in which the cells were cultured, was taken out from the incubator, and the cells were collected and transferred to a T175 flask (#159910, Nunc). 3 ml of plasma, 0.06 ml of anti-NKp46 (#MAB1850, R&D), and 27 ml of Alys505NK-EX (#01410P10, CSTI) were added to the T175 flask, followed by incubation in a CO.sub.2 incubator for 1 day to 2 days. Thereafter, the remaining plasma and 0.12 ml of anti-NKp46 solution, 0.03 ml of IL-18, 0.03 ml of PDGF-DD (1159-SB, R&D), 53.85 ml of Alys505NK-EX (#01410P10, CSTI), and one or two or more of 50 ng/ml of PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD (1159-SB, R&D), and PDGF-AB as new function enhancement substances were added, followed by incubation in a CO.sub.2 incubator for 1 day to 2 days.
(33) 2.5. Tertiary Culture of Natural Killer Cells
(34) The cells and plasma of the T175 flask incubated in Example 2.4 were put in a culture medium containing 2000 IU/ml of IL-2, followed by incubation in a CO.sub.2 incubator. After 2 days to 3 days, an equal volume of fresh culture medium (a culture medium containing 2000 IU/ml of IL-2) was mixed with the cell suspension in which the cells were cultured, followed by incubation in a CO.sub.2 incubator.
(35) In the above cultures (all of primary culture, secondary culture, and tertiary culture), instead of using the culture medium supplemented with IL-2, a predetermined amount of IL-2 was added to an immune cell culture medium to which IL-2 was not added.
(36) 3. Phenotypic Analysis of Natural Killer Cells According to Culture Period
(37) The activated natural killer cells cultured by the culture method according to the above exemplary embodiment were characterized on days 0, 6, 10, and 14 during the culture period of differentiation and expansion of natural killer cells from PBMCs before culture.
(38) For characterization of the cells, CD3, CD56, CD19, CD16, CD14, CD4, and CD8 were identified as major markers. During the culture period, a percentage of CD3.sup.CD56.sup.+ NK cells increased, and on day 14 of culture, NK cells accounted for 85.5% (4.92) as a main component. In addition, CD3.sup.+CD56.sup. T cells decreased to 10% on day 14 of culture, and CD3.sup.+CD56.sup.+ NKT cells were present at a constant ratio of 5%. Monocytes and B cells were not detected as 0%. In addition, a percentage of CD8.sup.+ cells was higher than that of CD4.sup.+ cells among T cells after culture.
(39)
(40)
(41) The results of
(42) 4. Relative MFI Values of NK Cell Receptor Expression Before Culture (D0) and after Culture (D14)
(43) The relative MFI values of activated natural killer cells cultured by the culture method according to the above exemplary embodiments were measured.
(44) The relative MFI means a value of the expression intensity of positive cells, relative to that of isotype, and is defined by Equation 1 below.
Relative MFI=Receptor MFI/Isotype MFI[Equation 1]
(45) The relative MFI is a concept distinct from an expression ratio, which measures an expression ratio of the positive cells relative to the isotype. Even though having the same % of expression ratio, the strength of each receptor function differs depending on the MFI value, and it may be understood that the function is actually increased when the relative MFI value is high.
(46) In this exemplary embodiment, as in 2.5. above, a novel natural killer cell having enhanced function, treated with a novel substance, was prepared, and MFI values of specific receptors thereof were measured to define the characteristics of the novel natural killer cell.
(47) To measure the relative MFI, first, cells before and after culture were collected to prepare 110.sup.7 cells, which were centrifuged at 1500 rpm for 5 minutes. Then, the supernatant was discarded, followed by dilution with 2 ml of FACS buffer (PBS containing 2% FBS). Antibodies containing fluorescent materials against the substances shown in Table 2 below were put in a 5 ml FACS tube according to each condition, and 100 l of the diluted cell solution was dispensed and stained in a refrigerator for 30 minutes. After staining, 500 l of PBS was added, followed by centrifugation at 3200 rpm for 3 minutes. Then, the supernatant was discarded. The pellet of the stained cells was fixed by adding 500 l of 1% PFA, and then expression of immune receptors of the cells was analyzed using a flow cytometer (Bechman Coulter, USA), and the relative MFI values were determined according to Equation 1 above.
(48) The results of the relative MFI values are shown in Table 2 below.
(49) TABLE-US-00002 TABLE 2 Relative MFI (N = 4~5) of natural killer cell receptor D0 D14 Natural killer cell receptor Range Mean SD Range Mean SD Activating Receptor CD2 67-181 117.3 51.4 27-165 115.8 60.7 CD16 156-451 266.7 132.1 19-124 53.5 48.8 CD27 1-1.3 1.2 0.1 0.3-0.9 0.7 0.3 CD69 0.7-1.5 1.2 0.4 1.4-6.8 4.2 2.3 NKG2D 1.9-5 3.4 1.3 8.4-20.3 13.7 6.1 CD226 (DNAM-1) 1.7-7.9 4.1 2.7 4-7.6 5.3 1.5 NKp30 1.1-3.6 2.5 1.1 7.2-14.3 9.6 4.0 NKp44 1-1.2 1.1 0.1 16.3-20.3 18.7 2.1 NKp46 2.4-4.7 3.6 1.2 3.2-5.5 4.2 1.2 LFA-1 263-291 275.7 14.3 34-143 109.3 50.1 CD160 2.2-5.5 3.2 1.6 0.3-1.2 0.9 0.4 Inhibitory CD158a (KIR2DL1) 1.1-2.1 1.4 0.4 0.3-1.0 0.8 0.4 Receptor CD158b 1.1-21.8 6.3 10.3 0.3-1.6 1.1 0.5 (KIR2DL3) NKB1 1.0-1.3 1.1 0.2 0.3-1.1 0.8 0.3 (KIR3DL1) NKG2A 1.2-1.6 1.4 0.2 0.6-8.0 5.7 3.5 (CD159a) CD161 3.9-6.2 4.8 1.0 0.5-5.0 3.2 2.0 (NKRP1A) Chemokine CCR3 0.8-1.6 1.2 0.3 0.6-0.8 0.7 0.1 Receptor CCR5 1.5-2.1 1.8 0.2 1.0-1.8 1.7 0.5 CCR6 0.7-1.1 8.0 5.9 1.5-2.3 1.9 0.4 CXCR3 0.7-1.1 1.0 0.2 0.8-1.4 1.1 0.3 CXCR1 1.7-4.9 2.6 1.3 0.9-1.6- 1.2 0.2 CXCR2 1.3-7.0 3.5 2.1 0.3-0.9 0.5 0.3 Integrin ITGA1 0.9-2.8 1.4 0.9 6.5-21.1 13.4 6.2 Receptor (CD49a) ITGA2 1.1-1.5 1.4 0.2 2.7-5.6 4.1 1.3 (CD49b) ITGB7 2.0-8.5 5.3 2.9 2.7-7.7 5.8 2.5 (CD49d)
(50) As shown in Table 2, the relative MFI values of NKG2D, NKp30, NKp44, ITGA1, and ITGA2, which are factors related to anticancer activity and activation of natural killer cells, were found to increase from 1.5-fold up to 25-fold. The above results indicate that the natural killer cell treated with the novel substance according to one embodiment is a novel natural killer cell having a specific MFI value and having increased anticancer activity and increased activity of the cell itself.
(51) 5. Comparative Analysis of Immune Receptor Expression
(52) Expression of immune receptors was compared and analyzed between activated natural killer cells cultured by the culture method according to the above exemplary embodiments and PBMCs before culture.
(53) In detail, in the same manner as in 4, expression of the immune receptors of the cells was analyzed using antibodies containing fluorescent materials against the substances shown in Table 3 below, and the results are shown in
(54) TABLE-US-00003 TABLE 3 FITC PE APC 1 IgG IgG IgG 2 CD3 CD16 CD56 3 CD3 CD69 CD56 4 CD3 NKG2D CD56 5 CD3 NKP30 CD56 6 CD3 NKP44 CD56 7 CD3 NKP46 CD56 8 CD3 CD226 (DNAM-1) CD56 9 CD3 CD158b (KIR2DL3) CD56 10 CD3 NKB1 (KIR3DL1) CD56 11 CD3 NKG2A (CD159a) CD56 12 CD3 CD158a (KIR2DL1) CD56 13 CD3 CD2 CD56
(55)
(56) As shown in
(57) 6. Analysis of Expression of Factors Related to Brain Tissue and Blood-Brain Barrier Permeability or Cell Migration Promotion of Activated Natural Killer Cells
(58) Expression of factors related to brain tissue and blood-brain barrier permeability or cell migration promotion of activated natural killer cells cultured by the culture method according to the above exemplary embodiments was compared and analyzed.
(59) In detail, expression of factors related to brain tissue and blood-brain barrier permeability or cell migration promotion were analyzed in the same manner as in 4, except that antibodies containing fluorescent materials against the substances described in Table 4 below were used, and the results are shown in Table 4.
(60) TABLE-US-00004 TABLE 4 Expression marker Expression (%) 1 PSA-NCAM 99.8 2 Nestin 99.4 3 S100B 96.9 4 Tyrosine Hydroxylase 89.7 5 CD147 100 6 CD29 100 7 CD49c 99.9 8 CD146 55.3 9 CD15 99.1 10 CD31 57.2
7. Analysis of Expression of KIR2DS4 in Activated Natural Killer Cells
(61) The KIR2DS4 mRNA expression pattern of the activated natural killer cells cultured by the culture method according to the above exemplary embodiment was compared with that of PBMCs before culture.
(62) In detail, each RNA was extracted from cells before culture and cells after culture by a trizol isolation method, and total RNA sequencing was performed to identify mRNA expression patterns. The results are shown in Table 5 below.
(63) TABLE-US-00005 TABLE 5 Fold change Gene (P14 NK cells/ ID symbol P0 PBMC) 9513 KIR2DS4 32.562
(64) As shown in Table 5, it was found that KIR2DS4 expression was increased about 32-fold in the natural killer cells according to one embodiment, as compared with PBMCs before culture.
Experimental Example 1. Analysis of Anticancer Material Expression
(65) Expression of anticancer materials (granzyme B, perforin, interferon-gamma, and CD107a) of activated natural killer cells cultured by the culture method according to the above exemplary embodiments and PBMCs before culture was compared and analyzed.
(66) First, granzyme B and perforin were analyzed as follows.
(67) 510.sup.5 cells were prepared for each sample, centrifuged at 1500 rpm for 5 minutes, and then the supernatant was discarded to obtain a cell pellet. The cell pellet was diluted with 100 l of FACS buffer, anti-IgG1 k-FITC (ebioscience, 11-4714-42), anti-IgG1k-APC (ebioscience, 17-4714-42), and anti-CD3-FITC (ebioscience, 11-0038-42), anti-CD56-APC (ebioscience, 17-0567-42) antibodies were added to stain surface antigens at room temperature for 15 minutes. Then, 500 l of PBS was added thereto, followed by centrifugation at 6000 rpm for 3 minutes. For intracellular staining, a fixation/permeabilization solution kit (BD, 554714) was used, and the fixation/permeabilization solution was added and allowed to react in a refrigerator for 20 minutes. A perm/wash buffer was added and centrifuged at 6000 rpm for 3 minutes twice. The cell pellet obtained by discarding the supernatant was diluted in 100 l of perm/wash buffer, respectively, and anti-IgG1 k-PE (ebioscience, 12-4714-42), anti-Perforin-PE (ebioscience, 12-9994-42), anti-GranzymeB-PE (ebioscience, 12-8899-41) antibodies were added, and intracellular staining was performed in a refrigerator for 30 minutes. After staining, 500 l of PBS was added to the cell solution, followed by centrifugation. The cells were fixed with 1% PFA, and analyzed using a flow cytometer.
(68) With regard to interferon gamma, cell pellets were obtained in the same manner as above, and then diluted with a culture medium prepared by adding 10% FBS and 1% penicini to an RPMI medium without phenol red, and 500 l thereof was dispensed in each well of a 24-well plate. Then, cells were treated with 0.5 l of PMA/lonomycin (Biolegend) and 0.5 l of GolgiPlug (BD bioscience, USA) and allowed to react in a 5% CO.sub.2 incubator at 37 C. for 4 hours. 4 hours after reaction, cells were collected, and centrifuged at 6000 rpm for 3 minutes to discard the supernatant. Surface antigens of the cell pellet were stained with anti-IgG1k-FITC (ebioscience, 11-4714-42), anti-IgG1k-APC (ebioscience, 17-4714-42) and anti-CD3-FITC (ebioscience, 11-0038-42), anti-CD56-APC (ebioscience, 17-0567-42) antibodies, and intracellular staining was performed. Intracellular staining was performed by staining with anti-IgG1k-PE (ebioscience, 12-4714-42) and anti-INF--PE (ebioscience, 12-8899-41) antibodies, followed by fixation with 1% PFA and analysis using a flow cytometer.
(69) The results for granzyme B, perforin, and interferon gamma are shown in
(70)
(71) As shown in
(72) In addition, to compare expression of CD107a degranulation between natural killer cells according to one specific embodiment and PBMCs before culture, K562, which is a lymphoblastoid cell extracted from the bone marrow of a chronic myelogenous leukemia patient, was reacted as a target cell, and expression levels of CD107a were analyzed.
(73) In detail, the target K562 cell was prepared at a density of 110.sup.5 cells per condition, centrifuged at 1500 rpm for 5 minutes, and then the supernatant was discarded to obtain a pellet. The cell pellet was diluted by adding 250 l of a culture medium prepared by adding 10% FBS and 1% penicillin-streptomycin (10,000 U/mL) (gibco, 15140122) to an RPMI medium without phenol red. The natural killer cells were prepared at a density of 110.sup.5 cells per condition to prepare activated natural killer cells and target cells at a ratio of 5:1. After centrifuging the prepared cells, the supernatant was discarded, and 250 l of the same culture medium as used in dilution of the target cells was added and suspended. Thereafter, the prepared activated natural killer cells and target cells were put in a 24-well plate at a ratio of 5:1, and then anti-IgG1k-PE (ebioscience) and anti-CD107a-PE (ebioscience) antibodies were added, followed by incubation in an incubator under conditions of 37 C. and 5% CO.sub.2 for 4 hours. When the reaction was completed for 4 hours, the cells are harvested, and stained with anti-IgG1 k-APC (ebioscience), anti-CD3-FITC (ebioscience), and anti-CD56-APC (ebioscience) with fluorescent materials to identify only natural killer cells. After staining, 500 l of PBS was added, followed by centrifugation. Then, the cells were washed, and fixed with 1% PFA, and analyzed using a flow cytometer. The results are shown in
(74)
(75) As shown in
Experimental Example 2. Analysis of Interactions Between Ligands of Glioblastoma Cell Lines and Activated Natural Killer Cells
(76) Interactions between ligands of glioblastoma cell lines and the natural killer cells according to a specific embodiment were analyzed.
(77) First, U-87MG and T98G cells, which are glioblastoma cell lines, were cultured in a DMEM medium supplemented with 10% FBS and 1% Penicillin-Streptomycin (10,000 U/mL), respectively, and U-373MG and A172 cells, which are another glioblastoma cell lines, were cultured in an RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin (10,000 U/mL), respectively. Subsequently, staining was performed using antibodies against HLA-ABC, HLA-E, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, PVR, ICAM-1, ICAM-2, ICAM-3, LFA-3, B7-H6, PVR, and Necin-2, which are ligands of T98G, U-87MG, A172, U-373MG glioblastoma cell lines for natural killer cells, and expression levels in the cells were analyzed.
(78) In detail, 0.25% trypsin-EDTA (1) and phenol red were added to each cancer cell line cultured in a T75 flask, and allowed to react in a 5% CO.sub.2 incubator at 37 C. for 3 minutes to 5 minutes, and then the cells were suspended, and harvested by inactivating the enzyme with a culture medium supplemented with 10% FBS. The number of the harvested cells was counted and prepared at a density of 8.510.sup.6 cells, followed by centrifugation at 1500 rpm for 5 minutes. Then, the supernatant was discarded, and the resultant was diluted with 1.7 ml of FACS buffer (PBS containing 2% FBS). Next, the antibodies with fluorescent materials shown in Table 6 below were put into a 5 ml FACS tube according to each condition, and 100 l of the diluted cell solution was dispensed thereto, and stained at room temperature for 15 minutes. The stained cells were fixed by adding 500 l of 1% PFA, and then analyzed using a flow cytometer (Bechman Coulter, USA). The results are shown in
(79) TABLE-US-00006 TABLE 6 FITC PE APC 1 IgG IgG IgG 2 MICA 3 MICB 4 ULBP-1 5 ULBP-2 6 ULBP-3 7 ULBP-4 8 B7-H6 9 PVR (CD155) 10 Necin-2 (CD112) 11 LFA-3 (CD56) 12 ICAM-1 (CD54) 13 ICAM-2 (CD102) 14 ICAM-3 (CD50) 15 HLA-E 16 HLA-ABC
(80)
(81) As shown in
Experimental Example 3. Examination of Cytotoxicity Against Glioblastoma Cell Lines
(82) To examine the direct cytotoxicity of the natural killer cells according to one specific embodiment, a blood cancer cell line K562, and glioblastoma cell lines A172, U-87MG, U-373MG, and T98G cells, which have high sensitivity to natural killer cells, were subjected to a cytotoxicity test.
(83) Target cancer cells (K562, U-87MG, U-373MG, A172, T98G) were collected, and centrifuged at 1500 rpm for 5 minutes, respectively. Each supernatant was discarded. Then, each cell pellet was diluted with DPBS and washed, and the cell pellet after washing was suspended in a culture medium prepared by adding 10% FBS to an RPMI medium without phenol red. 110.sup.5 cells per condition were prepared, and stained with CFSE (Life technologies) at a concentration of 5 M by incubating for 10 minutes in an incubator under 5% CO.sub.2 conditions. The cells were washed with DPBS twice, and diluted with a culture medium prepared by adding 10% FBS to an RPMI medium without phenol red. Activated natural killer cells were prepared according to E:T ratios to the target cells (1:1, 1.25:1, 2.5:1, 5:1, 10:1, 20:1), and dispensed, together with the target cells, in a 24-well plate, and mixed. The cells were reacted for 4 hours, and treated with 7-aminoactinomycin D (7AAD) 20 minutes before the end of the reaction. After the reaction was completed, the cells were collected in a 5 ml FACS tube, and the cytotoxicity of the cells was analyzed through a flow cytometer. The results are shown in
(84)
(85)
(86) As shown in
(87) These results suggest that the novel natural killer cell according to one specific embodiment not only expresses significant immune receptors for glioblastoma, but also expresses immune receptors capable of overcoming self-tolerance, and thus may be usefully applied to the treatment of blood cancer, glioblastoma, etc.
Experimental Example 4. Blocking Assay of Natural Killer Cells
(88) It was examined whether cytotoxicity against cancer cells is inhibited when expression of specific factors is suppressed in the natural killer cells according to one specific embodiment.
(89) In detail, an antibody against NKp30, an antibody against NKp44, and an antibody against NKG2D were used to prepare natural killer cells in which the activities of these receptors were blocked. Thereafter, in the same manner as in Experimental Example 3, cytotoxicity against U-87MG, U-373MG, A172, and T98G was examined, and the results are shown in
(90)
(91) As shown in
(92) These results indicate that the activity of NKp30, NKp44, and/or NKG2D in the natural killer cells according to one specific embodiment is a major factor for cytotoxicity, and the natural killer cells having a specific MFI value of NKp30, NKp44, and/or NKG2D according to one specific embodiment are cells with remarkably increased anticancer activity.
Experimental Example 5. Analysis of Anticancer Activity of Natural Killer Cells
(93) 5.1. Analysis of Anticancer Activity in Ovarian Cancer Animal Model
(94) Anticancer activity of the natural killer cells according to one specific embodiment was examined in vivo.
(95) First, 110.sup.7 cells/head of OVCAR3, which is an ovarian cancer cell line, was subcutaneously administered to NOD-SCID mice to prepare xenograft animal models. Thereafter, experimental groups were set up as shown in Table 7 below.
(96) TABLE-US-00007 TABLE 7 Administration No. Group Dosage route Note 1 G1 Vehicle 5% albumin: dextran i.v. Negative infusion = 1:1, 100 l control 2 G2 Cisplatin Cisplatin 1.5 mg/kg i.p. Positive control 3 G3 NK Live 1 10.sup.7 cells/head i.v. Fresh NK cells 4 G4 NK 1 10.sup.7 cells/head i.v. Frozen NK Freeze cells
(97) A negative control G1 vehicle group was prepared at a ratio of 5% albumin:dextran infusion=1:1, and 100 l thereof was intravenously administered. A positive control G2 cisplatin group received 1.5 mg/kg of cisplatin. Natural killer cell-administered groups include G3 NK Live group and G4 NK Freeze group, which differ from each other in terms of fresh and frozen cells. With regard to G4 NK Freeze group, frozen NK cells were thawed, and 110.sup.7 cells thereof was administered to each animal. With regard to G3 NK Live group, cells were collected during culture, and 110.sup.7 cells thereof was administered to each animal. The administration group was administered twice a week for a total of 6 times. During the test period, the survival rate, tumor size, and symptoms of the mice were observed. Monitoring was performed for 78 days. After 78 days, the animals were sacrificed, and the weight of the extracted tumor was measured. The results are shown in Table 8 below and
(98) TABLE-US-00008 TABLE 8 Group Tumor weight (mean, g) G1 1.74 0.27 G2 1.15 0.10 G3 0.49 0.07 G4 0.57 0.09
(99)
(100) As shown in Table 8 and
(101) 5.2. Analysis of Anticancer Activity in Stomach Cancer Animal Model
(102) Anticancer activity of the natural killer cells according to one specific embodiment was examined in vivo.
(103) First, 110.sup.6 cells/head of NCI-N87, which is a stomach cancer cell line, was administered to NOD-SCID mice to prepare xenograft animal models. 6 days after tumor transplantation, experimental groups were set up as shown in Table 9 below.
(104) TABLE-US-00009 TABLE 9 Administration No. Group Dosage route Note 1 G1 Vehicle 5% albumin: dextran i.v. Negative control infusion = 1:1, 200 l 2 G2 HER2 Herceptin 1 mg/kg i.p. Positive control 3 G3 NK Live 1 10.sup.7 cells/head i.v. Fresh NK cells 4 G4 NK 1 10.sup.7 cells/head i.v. Frozen NK cells Freeze 5 G5 NK 1 10.sup.7 cells/ i.v. Fresh NK cells + L + Herceptin head + Herceptin Herceptin 6 G6 NK 1 10.sup.7 cells/ i.v. Frozen NK F + Herceptin head + Herceptin cells + Herceptin
(105) A negative control G1 vehicle group was prepared at a ratio of 5% albumin:dextran infusion=1:1, and 200 l thereof was intravenously administered. A positive control G2 HER2 group received 1 mg/kg of Herceptin twice a week for a total of 6 times. G3 NK Live group and G4 NK Freeze group, which were groups administered with the natural killer cells alone, were intravenously administered with 110.sup.7 cells/head twice a week for a total of 6 times. With regard to G3 NK Live group, cells obtained by recovering the cells during culture were administered thereto. With regard to G4 NK Freeze group, frozen NK cells were thawed, and 110.sup.7 cells/head thereof was administered thereto. G5 NK L+Herceptin group and G6 F+Herceptin group, which were groups co-administered with NK cells and Herceptin, were intravenously administered with the same cells as in the group administered with NK cells alone, in combination with 1 mg/kg of Herceptin, twice a week for a total of 6 times.
(106) During the test period, the survival rate, tumor size, and symptoms of the mice were observed. Monitoring was performed for 52 days. After 52 days, the animals were sacrificed, and the weight of the extracted tumor was measured. The results are shown in Table 10 below and
(107) TABLE-US-00010 TABLE 10 Group G1 Vehicle G2 HER2 Tumor weight 3.38 0.22 2.99 0.28 (mean, g) Group G3 NK live G4 NK freeze Tumor weight 1.15 0.23 1.28 0.23 (mean, g) Group G5 NK L + HER2 G6 NK F + HER2 Tumor weight 0.64 0.15 0.68 0.23 (mean, g)
(108)
(109) As shown in Table 10 and
(110) It was also confirmed that when Herceptin and the natural killer cells were co-administered, the weight of the tumor was reduced by about 17% to about 20%, as compared with that of the group administered with the natural killer cells alone. This means that antibody-dependent cell cytotoxicity (ADCC) by co-administration of the natural killer cells and Herceptin may provide high tumor growth inhibitory effect and a maintenance period of anticancer efficacy.
(111) 5.3. Analysis of Anticancer Activity in Glioblastoma Animal Model
(112) Anticancer activity of the natural killer cells according to one specific embodiment was examined in vivo.
(113) First, 110.sup.4 U87MG-luci cells obtained by transducing a luciferase gene into a human brain glioblastoma-derived cell line U87MG were transplanted into the right brain cerebral hemisphere skull of 7-week-old NOG female mouse to construct an experimental orthotopic animal model. At a predetermined period of time after cell transplantation, Bioluminescence (BLI) of the tumor was measured using an IVIS bio-imaging equipment (PerkinElmer, USA), and subjects were selected such that the BLI value had an average value on the 7.sup.th day after transplantation. The selected animals were divided according to experimental groups, as shown in Table 11 below.
(114) TABLE-US-00011 TABLE 11 Administration No. Group Dosage route Note 1 G1 5% albumin: i.v. Negative Vehicle dextran infusion = control 1:1, 200 l 2 G2 NK 1 10.sup.6 cells/head i.v. Experimental cells group
(115) A negative control G1 vehicle group was prepared at a ratio of 5% albumin:dextran infusion=1:1, and 200 l thereof was intravenously administered to each animal. An experimental G2 NK cell group was intravenously administered with the natural killer cells at a dose of 110.sup.6 cells/head twice a week for a total of 6 times. During the monitoring period, an IVIS bioimaging system (perkinelmer, U.S.A.) was used to measure the tumor volume twice a week. To measure the tumor volume, 150 mg/kg of luciferin (promega) was intraperitoneally administered at a dose of 10 mL/kg. After 10 minutes, each individual thus administered was sequentially anesthetized using an inhalation anesthetic. For the individuals fully anesthetized, bioluminescence (BLI) of the tumor was sequentially measured using the IVIS system, and the measured bioluminescence value of the region of interest (ROI) was analyzed according to Threshold corresponding to the internal standard of a committed research institute, Woojung Bio (Gyeonggi-do, Korea). The results are shown in Table 12 below and
(116) TABLE-US-00012 TABLE 12 Bioluminescence (p/sec/cm.sup.2/sr) Time after administration (day) Group 1 3 7 9 13 16 G1 Mean 4.28.E+08 1.14.E+09 4.43.E+09 2.98.E+09 6.10.E+09 1.09.E+10 Dosage: S.E. 7.61.E+07 3.41.E+08 1.00.E+09 5.90.E+08 1.21.E+09 2.94.E+09 0 cell/0.2 N 5 5 5 5 5 5 mL/head G2 Mean 3.19.E+08 5.70.E+08 5.19.E+09 2.58.E+09 3.17.E+09 1.78.E+09 Dosage: S. E. 8.28.E+07 1.52.E+08 1.35.E+09 7.16.E+08 1.40.E+09 7.69.E+08 1 10.sup.6 cells/ N 5 5 5 5 5 5 0.2 mL/head
(117) Each number represents mean+S.E. (n=5) S.E.: Standard error N: Number of animals
(118)
(119) As shown in Table 12 and