WO2010013947A2 - 자연살해세포의 증식방법 - Google Patents
자연살해세포의 증식방법 Download PDFInfo
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- WO2010013947A2 WO2010013947A2 PCT/KR2009/004228 KR2009004228W WO2010013947A2 WO 2010013947 A2 WO2010013947 A2 WO 2010013947A2 KR 2009004228 W KR2009004228 W KR 2009004228W WO 2010013947 A2 WO2010013947 A2 WO 2010013947A2
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- natural killer
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0602—Vertebrate cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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Definitions
- the present invention relates to a method for propagating natural killer cells (NK cells) in high yield, and more specifically, natural killer cells are peripheral blood leukocyte cells in a medium containing anti-CD3 antibody and interleukin protein. It relates to a method for propagating natural killer cells, including the step of culturing in the presence of.
- NK cells natural killer cells
- NK cells Natural killer cells
- NK cells Natural killer cells
- NK cells are lymphocytic cells that play a role in the immune response. Although these cells have many functions, in particular, they have a strong activity of killing tumor cells, and thus are thought to be important members of the immune monitoring apparatus for removing cells with tumorous or tumorigenic abnormalities in the body. Therefore, research has been conducted for a long time to effectively use these cells for tumor treatment and removal of virus-infected cells that are supposed to be the source of tumors.
- NK cells in the normal body are in an inactive state. Many researchers have studied the activation of NK cells from normal blood or from inactivated patient blood.
- NK cells activated in vitro opens up the possibility of immunocytotherapy and has been confirmed in vitro or in animal experiments on various carcinomas.
- tumor cell lines were used to identify in-vitro cytotoxicity and showed significant cytotoxicity against various cancers including hematologic cancer, liver cancer, lung cancer, kidney cancer, pediatric nerve cancer and skin cancer.
- hemologic and pediatric neurocarcinomas showed clinical and non-clinical effects.
- NK cells present in the body is not very high, and the number of effective NK cells required for the therapeutic effect is very high. Therefore, leukapheresis is performed to collect a large amount of leukocytes. Even if a single blood collection can only be one or two treatments.
- NK cells do not proliferate due to insufficient growth of NK cells
- the biggest problem applied to clinical practice is In order to overcome this problem, many studies on NK cell proliferation have been made, but they have not reached the level applicable to the clinical practice. In general, the study of NK cell proliferation using IL-2 or other cytokines and chemicals has not reached an increase of 3 to 10 times the number of initially isolated NK cells.
- the present inventors secured safety by using inactivated autologous peripheral blood cells as feeder cells, and simultaneously treated with OKT-3 antibody and IL-2 to maintain natural killer cell killing ability while proliferating rate significantly. The increase was confirmed, and the present invention was completed.
- the present invention comprises a step of culturing natural killer cells in the presence of peripheral blood lymphocytes (PBL) in a medium containing anti-CD3 antibody and interleukin protein, high purity natural Provided is a method for explosively proliferating killer cells.
- PBL peripheral blood lymphocytes
- the present invention simultaneously treats OKT-3 antibody and IL-2 while using natural PBL (peripheral blood lymphocyte) as a feeder cell in culture of natural killer cells.
- PBL peripheral blood lymphocyte
- the method includes removing the anti-CD3 antibody from the culture and adding the culture medium from which the anti-CD3 antibody has been removed to the medium containing the interleukin protein, wherein further culturing is performed. It is preferable that the culture medium from which the anti-CD3 antibody to be added is removed contains natural killer cells at a concentration of 1 x 10 5 to 3 x 10 6 cells / well.
- 1 is a result of analyzing the surface type of the initial NK cells isolated from the peripheral blood of normal people.
- Figure 2 shows the results of analyzing the surface type of NK-cell obtained by culturing according to the method of the present invention.
- Figure 3 is the result confirming the tumor killing ability of NK-cell obtained by culturing according to the method of the present invention.
- NK-cells obtained by culturing in each culture condition according to the presence or absence of support cells PBMC, anti-CD3 antibody OKT-3 and IL-2.
- Figure 6 is a result of comparing the proliferation capacity of NK-cell obtained by culturing by varying the seeding (seeding) concentration of natural killer cells.
- the present invention includes a step of culturing a natural killer cell (NK cell) in the presence of peripheral blood lymphocytes (PBL) in a medium containing anti-CD3 antibody and interleukin protein. It relates to a method for propagating natural killer cells.
- NK cell natural killer cell
- PBL peripheral blood lymphocytes
- the natural killer cell proliferation method of the present invention is not particularly limited and may be performed, for example, including the following steps:
- NK cells Natural killer cells
- NK cells are present in about 10-15% of normal blood and have high killing capacity when reacted with non-self. In cells infected with various viruses, invading bacteria, or generating abnormal cells, NK cells react nonspecifically and immediately remove foreign substances. However, since the number of NK cells present in the body is not very high, and the number of effective NK cells required for showing a therapeutic effect should be very large, there is a need for an effective NK cell proliferation method.
- peripheral blood mononuclear cells or peripheral blood mononuclear cells (PBMC) cell
- PBMC peripheral blood mononuclear cells
- the present invention relates to a method for amplifying an isolated natural killer cell (NK cell) of the former, characterized in that the proliferation method according to the present invention also uses a feeder cell.
- NK cell isolated natural killer cell
- Feeder cell is a cell that prevents division and proliferation but has metabolic activity and produces various metabolites to help the growth of the target cell. do.
- the term 'feeder cell' is also used as a term support cell.
- feeder cells used in the present invention animal cell lines into which genes have been introduced, peripheral blood leukocyte cells (PBL) treated with various cytokines or compounds, or peripheral blood leukocyte cells (PBLs) of self or others ), T-cell, B-cell, or monocytes. Most preferably, magnetic peripheral blood leukocytes (PBLs) can be used.
- PBL peripheral blood leukocyte cells
- PBLs peripheral blood leukocyte cells
- the magnetic peripheral blood leukocyte cells used as the support cells are inactivated to ensure safety.
- a conventional method known in the art may be used, and for example, a method of irradiating gamma-rays may be used.
- Such inactivated feeder cells include isolated T-cells.
- Proliferation method using a support cell as in the present invention is a method of purely separating the natural killer cells and then proliferating, there is an advantage that can continue to grow only pure natural killer cells thereafter.
- the proliferation method of the present invention is characterized in that the natural killer cells are cultured in a medium containing anti-CD3 antibody and interleukin protein.
- a CD3 antibody is a protein that specifically reacts with a CD3 antigen, a group of molecules that associate with a T cell receptor (TCR) to form an antigen recognition complex.
- CD3 molecules have a longer intracellular region and a antigen recognition signal in the cell compared to TCR. It is in charge of conveying.
- OKT-3 As an example of the anti-CD3 antibody which can be used by this invention, OKT-3, UCHT1, HIT3a, etc. are mentioned, Preferably it is an OKT-3 antibody.
- Interleukin (IL) protein is a generic term for proteinaceous biologically active substances produced by immunoreactive cells such as lymphocytes, monocytes, and macrophages, and refers to a group of molecular species in cytokines.
- interleukin protein which can be used by this invention, IL-2, IL-15, IL-12, IL-18, IL-21, etc. are mentioned, Preferably it is an IL-2 protein.
- NK cells and PBLs isolated from human peripheral blood are added to a culture medium for animal cell culture such as AIM-V media, RIMI1640, CellGro SCGM, and X-VIVO20, and the anti-CD3 antibody and Incubate by adding interleukin protein.
- a culture medium for animal cell culture such as AIM-V media, RIMI1640, CellGro SCGM, and X-VIVO20
- the anti-CD3 antibody and Incubate by adding interleukin protein In one embodiment of the present invention was cultured by the addition of the OKT-3 antibody and IL-2.
- the concentration of the OKT-3 antibody to be added is 0.1-100 ng / ml, preferably about 10 ng / ml, and the concentration of IL-2 is 10-2000 U / ml, preferably about 500 U / ml. .
- the kind of serum or plasma added to the medium is not particularly limited, and commercially available ones derived from various animals can be used, but those derived from humans are more preferable as human origin.
- a method known to those skilled in the art can be used, such as a combination of cytokines for propagating lymphocytes from PBMCs, lectins for stimulating lymphocyte proliferation, and the like.
- the present invention provides an optimal culture concentration of NK cells capable of significantly proliferating NK cells.
- the concentration of natural killer cells seeded in the medium significantly affects the growth rate.
- natural killer cells are inoculated at a concentration of 1 ⁇ 10 5 to 1 ⁇ 10 6 cells / well, more preferably 1 ⁇ 10 5 to 3 ⁇ 10 6 cells / well.
- concentration of 1 ⁇ 10 5 to 1 ⁇ 10 6 cells / well more preferably 1 ⁇ 10 5 to 3 ⁇ 10 6 cells / well.
- natural killer cells are treated with anti-CD3 antibody such as OKT-3 antibody and interleukin protein such as IL-2 at the appropriate concentration while using support cells, thereby using only support cells or only OKT-3 antibody stimulation.
- anti-CD3 antibody such as OKT-3 antibody
- interleukin protein such as IL-2
- the present invention relates to natural killer cells obtained by the above method.
- the surface type characteristics of the natural killer cells proliferated and cultured according to the above method are described below.
- CD3 + T cells are relatively reduced and more CD3- / CD56 + NK cells are observed at 7 days, and almost all CD3 + T cells disappear at around 10 days from the day of culture, and all of the cells are 95% or more.
- high-purity natural killer cells having a surface type of CD16 + can be obtained by proliferating in a short time.
- a cell therapy agent can be prepared for tumor treatment or removal of virus-infected cells that are supposed to be the source of tumors.
- Example 1 Preparation of feeder cells and isolation of natural killer cells
- AIM-V media Invitrogen, 12055091
- 5% of the cell culture solution hAB serum (Sigma, H4522)
- 10 ul of the cell solution was transferred to a microtube and trypan blue (trypan).
- trypan trypan blue
- blue (Gibco) 90 ul, mix well with a pipette, stain with trypan blue (Gibco, 15250-061) dye and count the cells using an inverted microscope (Olympus, CK2-TRC-2).
- Cell culture medium was added and diluted to 5 x 10 6 cell / ml.
- Example 1- (1) Initial cells were cultured in 12-well plates (Falcon). 500 ul of the supporting cells prepared in Example 1- (1) was put in the well, and 500 ul of the natural killer cells isolated in Example 1- (2) was further added to the well containing the supporting cells.
- Plates were incubated for 5 days in a 37 degree Celsius wet incubator containing 5% carbon dioxide, at which time no culture or cytokine was added.
- the remaining cells were diluted with 2 ml of cell culture solution, 10 ul of diluted cells were taken into a microtube, mixed well with 90 ul of trypan blue solution, and stained with trypanblue to measure the number of cells.
- Cell culture medium was added and diluted to 2 x 10 5 cells / well.
- IL-2 was added to 500 U / ml, mixed with the cells, and then seeded with 2 x 10 5 cells / ml / well in a 12-well plate. The plates were placed in a 37 ° C. wet incubator containing 5% carbon dioxide and further incubated for 12 days.
- the cells were transferred to a new flak, and 5 ml of the cell culture solution containing IL-2 (500 U / ml) was added every day until the 17th day.
- Example 3 Surface type analysis of the obtained NK-cell
- Cells were harvested before, during or after the culture, and centrifuged at 12000 rpm for 5 minutes, and the culture solution was removed by suction. Cell number was measured by diluting with 1 ml FACS buffer (2.5% FBS + PBS), and diluted with FACS buffer to 5 x 10 6 cells / ml. 100 ul of the diluted cell solution was added to the FACS tube (Falcon), and the antibody was added as follows.
- Tube 2 anti-human CD3 -FITC (BD Pharmingen, 5555339) + anti-human CD56 -APC (BD Pharmingen, 555518) + anti-human CD16-PE (BD Pharmingen, 555407)
- Tube 3 anti-CD16-FITC (Color control) (BD Pharmingen, 555406)
- Tube 4 anti-CD56-PE (Color control) (BD Pharmingen, 555516)
- Tube 5 anti-CD56-APC (Color control)
- the tubes were left to stand at refrigeration temperature for 30 minutes and stained. Then, 2 ml FACS buffer was added to the stained cells and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, and again, 2 ml FACS buffer was added and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed again, and 300 ul FACS buffer was added and vortexed to release the cells. Surface type was analyzed using FACSCalibur (Becton Dickinson).
- Target cell (E: T) ratio was adjusted to 30: 1.
- Target cell (E: T) ratio 10: 1.
- 500 ul of cells were taken and placed in a new tube, and the mixture was added to the culture medium and further mixed with 4.5 ml, and Effector: Target cell (E: T) ratio was adjusted to 3: 1.
- CEM chronic lymphoblastic leukemia cell line
- CML chronic myelogenous leukemia
- 80% confluency were prepared and collected. These cell lines were collected, placed in a 15 ml conical tube and centrifuged at 1200 rpm for 5 minutes. Separated. Supernatant was removed and cells were diluted in 5 ml cell culture. The cell number was measured and transferred to a new 15 ml tube by 1 ⁇ 10 6 cells. Cell culture solution was added to the transferred cells to 10 ml, and then centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, 25 ul of FBS was added to dilute cells, and 100 ul of Cr-51 (Perkin Elmer) was added thereto.
- CEM chronic lymphoblastic leukemia cell line
- CML chronic myelogenous leukemia
- the cells were taken out, filled with the cell culture solution to 10 ml, and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed and washed twice more in the same manner. After washing the cells, 10 ml of the cell culture solution was evenly diluted with a pipette.
- the diluted cell line was added 100 ul per target to the U-bottom 96-well plate (FALCON) containing the previously prepared effector cells.
- FALCON U-bottom 96-well plate
- 100ul of the target cell was placed in three wells without effector cells, and 100ul of the cell culture solution was added.
- 100ul of target cell was put in three wells without effector cells, and 100ul of PBS containing 1% triton X-100 was incubated for 4 hours.
- Example 5 Evaluation of cell proliferation capacity of cultured NK-cell (CFSE-proliferation assay)
- NK cells were isolated from the peripheral blood of 15 ml using Rossetsep, and autologous peripheral blood cells were irradiated to inhibit proliferation, and then used as feeder cells.
- Anti-CD3 antibody (OKT-3) was stimulated with PBL for 5 days at low concentration and then cultured for 17 days in media containing IL-2 to confirm up to 600-fold NK cell proliferation.
- NK cell + IL-2 500 U / ml
- NK cell + IL-2 500 U / ml
- irradiated PBMC OKT-3 (10 ng / ml)
- NK cells were isolated using Rosettesep, as in Example 1, and irradiated PBMCs were seeded at 5 times the number of NK cells. On the fifth day from the culture day, each cell was harvested and the number of cells was measured.
- 1 x 10 6 cells were prepared for each of the above conditions, transferred to a 5 ml tube, the cell culture was filled to a final 500 ul, 5 ⁇ M CFSE solution, and then left for 30 minutes in a cell incubator. After washing three times with PBS, 530 nm wavelength was analyzed using FACS Calibure.
- Example 2 the initial culture and OKT-3 removal were performed in the same manner, and when the number of NK-cells was measured, the cells were diluted at different concentrations as follows, and then seeded in a 12-well plate, followed by 9 days in a cell incubator. Further incubation, the cell number was measured
- Natural killer cell proliferation method is a breakthrough method capable of maximal proliferation while maintaining the killer ability of natural killer cells, compared to the conventional method, it is possible to obtain a large number of natural killer cells with a small amount of blood collection It is useful for commercialization as a therapeutic agent.
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Claims (12)
- 자연살해세포를 항-CD3 항체 및 인터루킨 단백질이 함유된 배지에서 말초혈 백혈구 세포의 존재하에 배양하는 공정을 포함하는, 자연살해세포의 증식방법.
- 제1항에 있어서, 항-CD3 항체는 OKT3, UCHT1 및 HIT3a로 이루어진 군에서 선택되는 것을 특징으로 하는 자연살해세포의 증식방법.
- 제2항에 있어서, 항-CD3 항체는 OKT-3항체인 것을 특징으로 하는 자연살해세포의 증식방법.
- 제1항에 있어서, 상기 인터루킨 단백질은 IL-2, IL-15, IL-12, IL-18 및 IL-21로 이루어진 군으로부터 선택되는 것을 특징으로 하는 자연살해세포의 증식방법.
- 제4항에 있어서, 상기 인터루킨 단백질은 인터루킨-2(IL-2)인 것을 특징으로 하는 자연살해세포의 증식방법.
- 제1항에 있어서, 상기 말초혈 백혈구 세포는 불활성화된 것임을 특징으로 하는 자연살해세포의 증식방법.
- 제6항에 있어서, 상기 불활성화된 말초혈 백혈구 세포는 분리된 T 세포(purified T cell)인 것을 특징으로 하는 자연살해세포의 증식방법.
- 제1항에 있어서, 상기 말초혈 백혈구 세포는 자기 말초혈 백혈구 세포인 것을 특징으로 하는 자연살해세포의 증식방법.
- 제1항에 있어서, 자연살해세포를 항-CD3 항체 및 인터루킨 단백질이 함유된 배지에서 말초혈 백혈구 세포의 존재하에 배양하는 공정,상기 배양물에서 항-CD3 항체를 제거하는 공정, 및상기 항-CD3 항체가 제거된 배양액을 인터루킨 단백질이 함유된 배지에 첨가하여 추가 배양하는 공정을 포함하는 자연살해세포의 증식방법.
- 제9항에 있어서, 상기 항-CD3 항체는 OKT-3항체이고, 상기 인터루킨 단백질은 인터루킨-2(IL-2)인 것을 특징으로 하는 자연살해세포의 증식방법.
- 제9항에 있어서, 상기 항-CD3 항체가 제거된 배양액은 1 x 105 내지 1 x 106 cells/well의 농도로 첨가하는 것을 특징으로 하는 자연살해세포의 증식방법.
- 제11항에 있어서, 상기 항-CD3 항체가 제거된 배양액은 1 x 105 내지 3 x 106 cells/well의 농도로 첨가하는 것을 특징으로 하는 자연살해세포의 증식방법.
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