KR20120100207A - A method for differentiation and expansion of nk cell from cd14 positive monocytes - Google Patents
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- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/461—Cellular immunotherapy characterised by the cell type used
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- C12N2501/20—Cytokines; Chemokines
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Abstract
Description
본 발명은 자연살해세포(natural killer cell; NK 세포)의 분화 및 증식에 관한 것으로, 보다 상세하게는 제대혈의 단핵세포로부터 유래한 CD14 양성세포를 NK 세포로의 분화 및 증식을 효율적으로 촉진시키는 방법에 관한 것이다.
The present invention relates to the differentiation and proliferation of natural killer cells (NK cells), and more particularly, to efficiently promote the differentiation and proliferation of CD14 positive cells derived from mononuclear cells of cord blood into NK cells. It is about.
면역체계를 구성하는 세포들 중 자연살해세포(natural killer cell, 이하 "NK 세포"라 약칭함)는 비특이적으로 암을 살상할 수 있는 능력이 있는 세포로 알려져 있다. 이러한 NK 세포의 살해능은 림포카인 활성세포(lymphokine activated killer cell, LAK) 및 종양침윤림프구(tumor infiltration lymphocytes, TIL)을 이용하여 고형암(solid tumor) 치료에 이용하거나, 공여자 임파구 주입(donor lymphocyte infusion)을 통한 면역치료법(Tilden. A. B. et al., J. Immunol ., 136: 3910-3915, 1986; Bordignon C, et al., Hematologia 84: 1110-1149, 1999)을 수행함으로써, 골수이식이나 장기 이식시 발생하는 거부반응을 방지하기 위한 새로운 세포치료 요법으로 응용이 시도되고 있다. 또한, NK 세포의 분화와 활성의 결함은 유방암(Konjevic G, et al., Breast Cancer Res . Treat ., 66: 255-263, 2001), 흑색종암(Ryuke Y, et al., Melanoma Res ., 13: 349-356, 2003), 폐암(Villegas FR, et al., Lung Cancer , 35: 23-28, 2002) 등 다양한 암 질환과 관련되어 있음이 보고되어 이러한 질환들을 치료하기 위해 NK 세포 치료법이 대두되고 있다.
Among the cells constituting the immune system, natural killer cells (abbreviated as "NK cells") are known to be cells that are capable of killing cancer nonspecifically. The killing ability of these NK cells can be used for the treatment of solid tumors using lymphokine activated killer cells (LAK) and tumor infiltration lymphocytes (TIL), or donor lymphocyte injection (donor lymphocytes). bone marrow transplantation by performing immunotherapy (Tilden. AB et al., J. Immunol . , 136: 3910-3915, 1986; Bordignon C, et al., Hematologia 84: 1110-1149, 1999). Applications have been attempted as novel cell therapy to prevent rejection in organ transplantation. In addition, defects in the differentiation and activity of NK cells are associated with breast cancer (Konjevic G, et al., Breast Cancer Res . Treat . , 66: 255-263, 2001), Melanoma cancer (Ryuke Y, et al., Melanoma) Res ., 13: 349-356, 2003), lung cancer (Villegas FR , et al., Lung Cancer , 35: 23-28, 2002) has been reported to be associated with a variety of cancer diseases, NK cell therapy has emerged to treat these diseases.
사이토카인(Cytokine) 수용체의 γc의 발현이 결핍된 쥐에서 B세포와 T세포는 발견이 되지만 NK 세포는 발견되지 않는 점에서 γc를 지닌 수용체들이 NK 분화에 중요한 역할을 한다고 알려져 있다(Singer, B et al., Proc . Natl . Acad . Sci . USA 92, 377-381, 1995). 수용체의 γc 형태는 IL-2, IL-4, IL-7, IL-9, IL-15 및 IL-21의 수용체이며, 이 중 IL-2는 성숙된 NK 세포의 증식과 활성화를 증진시키는 기능을 지니고 있음이 보고되고 있다(Shibuya, A. et al., Blood 85, 3538-3546, 1995). IL-2가 결핍된 인간과 마우스에서는 NK 세포의 수가 현저히 감소한다는 보고가 전해지고 있으나(DiSanto, J. P. et al., J. Exp . Med . 171, 1697-1704, 1990), 한편으로는 IL-2 및 IL-2Ra 결핍은 간접적으로 NK 세포의 수와 활성화에 영향을 미친다는 연구 결과도 있다. 게다가, IL-2R 사슬은 IL-15의 수용체를 형성하는데 관여한다고 알려져 있다.
It is known that receptors with γ c play an important role in NK differentiation because B and T cells are found in mice lacking γ c expression of cytokine receptors, but NK cells are not found (Singer , B et al., Proc . Natl . Acad . Sci . USA 92, 377-381, 1995). The γ c form of the receptor is a receptor of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, of which IL-2 promotes proliferation and activation of mature NK cells. It has been reported to have a function (Shibuya, A. et al., Blood 85, 3538-3546, 1995). There are reports of a significant decrease in the number of NK cells in humans and mice deficient in IL-2 (DiSanto, JP et al., J. Exp . Med . 171, 1697-1704, 1990). And IL-2Ra deficiency indirectly affects the number and activation of NK cells. In addition, the IL-2R chain is known to be involved in forming receptors for IL-15.
IL-15는 NK 세포 분화에 관여하고, 이것은 IL-15 생성에 요구되는 전사인자 인터페론(transcription factor interferon, IFN)-조절 인자 1이 결핍된 쥐에서는 NK 세포가 결핍되며(Kouetsu et al., Nature 391, 700-703, 1998), IL-15 또는 IL-15Ra가 결핍된 쥐에서는 NK 세포가 발견되지 않는다는 것에 의해 알게 되었다. 이로써 IL-15는 NK 세포에서 발현되는 IL-15 수용체를 통해서 NK 세포의 성장과 분화를 직접적으로 증진시킨다는 것이 보고되었다(MrozekE et al., Blood 87, 2632-2640,1996).
IL-15 is involved in NK cell differentiation, which is deficient in NK cells in mice lacking the transcription factor interferon (IFN) -
IL-21은 활성화된 CD4+T 세포에 의해 분비되는 사이토카인이며(Nature, 5:688-697, 2005), IL-21의 수용체(IL-21R)는 수지상세포, NK 세포, T 세포 및 B 세포와 같은 림프구에서 발현되어 있다(Rayna Takaki, et al., J. Immonol 175: 2167- 2173, 2005). IL-21은 구조적으로 IL-2 및 IL-15과 매우 유사하며, IL-21R는 IL-2R, IL-15, IL-7R 및 IL-4R 등과 사슬을 공유하고 있다(Asao et al., J. Immunol, 167: 1-5, 2001). IL-21은 골수로부터의 NK 세포 전구체의 성숙을 유도하는 것으로 보고되었고(Parrish-Novak, et al., Nature , 408: 57-63, 2000), 특히 NK 세포의 사이토카인 생성능 및 세포사멸능과 같은 효과기 기능(effector functions)을 증가시키는 것으로 보고되었으며(M. Strengell, et al., J Immunol , 170: 5464-5469, 2003; J. Brady, et al., J Immunol, 172: 2048-2058, 2004), CD8+T 세포의 효과기 기능도 증가시킴으로써 내재, 적응면역계의 항암반응을 촉진시키는 것으로 보고되었다(Rayna Takaki, et al., J Immunol 175: 2167-2173, 2005; A. Moroz, et al., J Immunol , 173: 900-909, 2004). 또한, 인간의 말초혈액에서 분리한 NK 세포를 활성화 시키며(Parrish-Novak, et al., Nature, 408: 57, 2000), 제대혈에서 분리한 조혈줄기세포로부터 성숙한 NK 세포를 유도하는데 중요한 역할을 하는 것이 보고되었다(J. Brady, et al., J Immunol, 172: 2048, 2004).
IL-21 is a cytokine secreted by activated CD4 + T cells (Nature, 5: 688-697, 2005), and receptors of IL-21 (IL-21R) are dendritic cells, NK cells, T cells and B It is expressed in lymphocytes such as cells (Rayna Takaki, et al., J. Immonol 175: 2167-2173, 2005). IL-21 is structurally very similar to IL-2 and IL-15, and IL-21R shares a chain with IL-2R, IL-15, IL-7R and IL-4R (Asao et al., J.) . Immunol , 167: 1-5, 2001). IL-21 has been reported to induce maturation of NK cell precursors from the bone marrow (Parrish-Novak, et al., Nature , 408: 57-63, 2000), and in particular, cytokine production and apoptosis of NK cells Same effector functions have been reported (M. Strengell, et al., J Immunol , 170: 5464-5469, 2003; J. Brady, et al., J Immunol , 172: 2048-2058, 2004), it has also been reported to promote anticancer responses of the intrinsic, adaptive immune system by increasing the effector function of CD8 + T cells (Rayna Takaki, et al., J Immunol 175: 2167-2173, 2005; A. Moroz, et al. , J Immunol , 173: 900-909, 2004). It also activates NK cells isolated from human peripheral blood (Parrish-Novak, et al., Nature , 408: 57, 2000) and plays an important role in inducing mature NK cells from hematopoietic stem cells isolated from umbilical cord blood. (J. Brady, et al., J Immunol , 172: 2048, 2004).
NK 세포를 항암 면역 세포치료로 효과적으로 이용하기 위해서는 많은 수의 NK세포 확보가 필요하다. 그러나 NK세포는 혈액 내 림프구의 10-15%를 차지하고 있고 암 환자에서는 종종 NK 세포의 수, 분화 및 기능이 저하되어있어 사실상 충분한 세포수의 확보가 어려운 실정이다. 그러므로 NK 세포치료제로 적용하기 위해서는 NK세포의 증식이나 분화를 통한 NK세포의 대량 생산이 요구되고 있다.In order to effectively use NK cells as an anticancer immune cell therapy, it is necessary to secure a large number of NK cells. However, NK cells account for 10-15% of the lymphocytes in the blood, and in cancer patients, the number, differentiation, and function of NK cells are often reduced, making it difficult to obtain sufficient cell numbers. Therefore, the mass production of NK cells through the proliferation or differentiation of NK cells is required to apply to NK cell therapy.
NK 세포는 골수의 조혈줄기세포(hematopoietic stem cell)로부터 유래 된다고 알려져 있다. 시험관내(in vitro)에서는 제대혈로부터 조혈줄기세포를 분리하여 적당한 사이토카인들을 처리하여 배양함으로써 NK세포로 분화시키는 방법들이 보고되었다(Immunity 3: 459-473, 1995; Blood 87:2632-2640, 1996; Eur J Immunol. 33:3439-3447, 2003; Blood 108: 3824-3833, 2006). 즉, CD34+ HSC에 Flt-3L, IL-7, SCF, IL-15을 첨가하여 4주 배양 후 CD3-CD56+의 NK 세포로 분화시킬 수 있다. 그러나 이런 분화 방법은 실제 임상 적용에는 어려움이 있는데 치료에 충분한 양의 세포를 얻기 힘들고 분화하는데 시간과 비용이 많이 요구되는 등의 실제 임상 적용에 대한 많은 어려움이 있다.
NK cells are known to be derived from hematopoietic stem cells of bone marrow. In vitro, methods for separating hematopoietic stem cells from cord blood and treating them with appropriate cytokines and incubating them with NK cells have been reported (Immunity 3: 459-473, 1995; Blood 87: 2632-2640, 1996). Eur J Immunol. 33: 3439-3447, 2003; Blood 108: 3824-3833, 2006). That is, Flt-3L, IL-7, SCF, IL-15 can be added to CD34 + HSC to differentiate into CD3 - CD56 + NK cells after 4 weeks of culture. However, this differentiation method has difficulty in actual clinical application, and there are many difficulties in actual clinical application such as it is difficult to obtain a sufficient amount of cells for treatment and requires time and cost to differentiate.
이에 본 발명자들은 보다 효율적이고 경제적으로 NK 세포를 얻는 방법을 개발하던 중, 제대혈로부터 분리한 단핵세포로부터 CD3, CD34, CD56 및 CD19 양성세포를 제거하여 CD3-CD34-CD56-CD19-세포를 제조한 후, 상기 CD3-CD34-CD56-CD19-세포로부터 CD14 음성세포를 제거하여 CD3-CD34-CD56-CD19-CD14+세포를 제조하였다. 그런 다음, 상기 CD3-CD34-CD56-CD19-CD14+세포에 IL-15 및 IL-21을 혼합처리한 후 배양하는 방법이 NK세포의 분화 및 증식을 현저히 촉진시키고, 우수한 세포 살상능 활성을 가진 NK 세포를 단시간에 유도할 수 있음을 확인함으로써 본 발명을 완성하였다.
Therefore, while the inventors developed a method for obtaining NK cells more efficiently and economically, CD3 - CD34 - CD56 - CD19 - cells were prepared by removing CD3, CD34, CD56 and CD19 positive cells from mononuclear cells isolated from umbilical cord blood. then, the CD3 - removal of the CD14 negative cells from the cell CD3 - - CD34 - CD56 - CD19 was produced CD14 + cells - CD34 - CD56 - CD19. Then, the method of culturing after mixing and mixing IL-15 and IL-21 to the CD3 - CD34 - CD56 - CD19 - CD14 + cells significantly promotes the differentiation and proliferation of NK cells, and has excellent cell killing activity The present invention was completed by confirming that NK cells can be induced in a short time.
본 발명의 목적은 제대혈 유래 CD14 양성 단핵세포에 IL-15 및 IL-21을 혼합처리하여 세포 살상능이 우수한 NK 세포의 분화 및 증식을 촉진시키는 방법을 제공하는 것이다.An object of the present invention is to provide a method of promoting the differentiation and proliferation of NK cells having excellent cell killing ability by mixing IL-15 and IL-21 with cord blood-derived CD14 positive mononuclear cells.
상기 목적을 달성하기 위하여, 본 발명은 제대혈 유래 CD14 양성 단핵세포에 IL-15 및 IL-21을 혼합처리하는 단계를 포함하는 CD14 양성 단핵세포로부터 NK 세포를 분화시키는 방법을 제공한다.In order to achieve the above object, the present invention provides a method for differentiating NK cells from CD14 positive mononuclear cells, comprising mixing IL-15 and IL-21 with cord blood-derived CD14 positive mononuclear cells.
또한, 본 발명은 제대혈 유래 CD14 양성 단핵세포에 IL-15 및 IL-21을 혼합처리하는 단계를 포함하는 CD14 양성 단핵세포로부터 NK 세포를 증식시키는 방법을 제공한다. The present invention also provides a method of propagating NK cells from CD14 positive monocytes, comprising mixing IL-15 and IL-21 with cord blood derived CD14 positive monocytes.
또한, 본 발명은 본 발명에 따른 방법에 의하여 제조된 NK 세포를 유효성분으로 함유하는 암 예방 및 치료용 약학적 조성물을 제공한다.
The present invention also provides a pharmaceutical composition for preventing and treating cancer, which contains NK cells prepared by the method according to the present invention as an active ingredient.
본 발명의 CD14 양성세포로부터 NK 세포로의 분화 및 증식 방법은 제대혈로부터 분리한 단핵세포로부터 CD3, CD34, CD56 및 CD19 양성인 T세포, B세포 및 NK세포를 제거하고 얻은 CD14 양성인 세포에 IL-15 및 IL-21을 혼합처리함으로써 NK 세포의 분화 및 증식을 현저하게 촉진시킬 수 있고, 또한 이로부터 우수한 세포 살상능 활성을 가진 NK 세포를 단시간에 유도할 수 있으므로 항암세포 치료에 유용하게 이용될 수 있다.
The method of differentiation and proliferation from CD14 positive cells to NK cells of the present invention is to remove CD3, CD34, CD56 and CD19 positive T cells, B cells and NK cells from mononuclear cells isolated from umbilical cord blood. And IL-21 mixed treatment can significantly promote the differentiation and proliferation of NK cells, and can also be useful for anti-cancer cell treatment because it can induce NK cells having excellent cell killing activity in a short time therefrom. have.
도 1은 사람의 제대혈로부터 단핵세포(mononuclear cell, MNC)를 분리하여 CD3 양성세포를 제거한 다음, CD34, CD19 및 CD56 양성세포를 차례로 제거하고 CD14 양성세포를 얻은 결과, CD14 양성세포의 회수율을 나타낸 도이다.
도 2는 CD34 양성세포의 순도와 CD14 양성세포의 순도를 two-color flow cytometric 분석에 의해 각각 결정한 것으로 우측에 있는 숫자가 각각의 사분면(quadrant)에 해당하는 세포들의 백분율을 나타낸 도이다.
도 3은 제대혈로부터 단핵세포를 분리하여 CD3 양성세포를 제거한 다음, CD34, CD19 및 CD56 양성세포를 차례로 제거하고 얻은 CD14 양성세포를 CD15, CD19, CD33, CD117 및 CD122 마커를 사용하여 FACS로 분석하여 사분면에 해당하는 세포들의 백분율을 나타낸 도이다.
도 4는 제대혈로부터 NK 세포의 근원이 되는 CD34 양성 조혈줄기세포와 CD14 양성 단핵세포를 분리한 후, 각각 IL-15 및 IL-21을 포함하는 NK 분화배지에 12일간 배양하면서 7 일째 NK 분화도를 CD3, CD56 및 CD122 마커를 이용하여 FACS로 확인하여 사분면에 해당하는 세포들의 백분율을 나타낸 도이다.
도 5는 제대혈로부터 NK 세포의 근원이 되는 CD34 양성 조혈줄기세포와 CD14 양성 단핵세포를 분리한 후, 각각 IL-15 및 IL-21을 포함하는 NK 분화배지에 12일간 배양하면서 12 일째 NK 분화도를 CD3, CD56 및 CD122 마커를 이용하여 FACS로 확인하여 사분면에 해당하는 세포들의 백분율을 나타낸 도이다.
도 6은 CD34 양성 조혈줄기세포와 CD14 양성 단핵세포를 IL-15 및 IL-21을 포함하는 NK 분화 배지에 배양하면서 세포를 개수하여 증식률(fold induction)을 나타낸 도이다.
도 7은 CD34 양성 조혈줄기세포로부터 28일 분화시킨 NK 세포와 CD14 양성 단핵세포로부터 12일간 분화시켜 90% 이상의 분화도를 가진 NK 세포들의 51Cr release assay를 수행한 도이다.
E:T ratio는 effector와 target cell의 비율을 나타낸다. Figure 1 shows the isolation of mononuclear cells (MNC) from human umbilical cord blood to remove CD3 positive cells, and then sequentially remove CD34, CD19 and CD56 positive cells to obtain CD14 positive cells, showing the recovery rate of CD14 positive cells. It is also.
FIG. 2 shows the purity of CD34 positive cells and the purity of CD14 positive cells, respectively, by two-color flow cytometric analysis. The figure on the right shows the percentage of cells corresponding to each quadrant.
Figure 3 is to remove the mononuclear cells from the umbilical cord blood to remove the CD3 positive cells, then remove the CD34, CD19 and CD56 positive cells in turn and analyzed the CD14 positive cells obtained by FACS using the CD15, CD19, CD33, CD117 and CD122 markers Figure shows the percentage of cells in quadrant.
4 is isolated from CD34 positive hematopoietic stem cells and CD14 positive mononuclear cells, which are the source of NK cells from umbilical cord blood, and cultured for 12 days in NK differentiation medium containing IL-15 and IL-21. Fig. 3 shows the percentage of cells corresponding to quadrants identified by FACS using CD3, CD56 and CD122 markers.
5 is isolated from CD34 positive hematopoietic stem cells and CD14 positive monocytes, which are the source of NK cells from cord blood, and cultured for 12 days in NK differentiation medium containing IL-15 and IL-21. Fig. 3 shows the percentage of cells corresponding to quadrants identified by FACS using CD3, CD56 and CD122 markers.
6 is a diagram showing the fold induction by counting cells while culturing CD34 positive hematopoietic stem cells and CD14 positive monocytes in NK differentiation medium containing IL-15 and IL-21.
FIG. 7 is a diagram illustrating 51 Cr release assay of NK cells differentiated from CD34 positive hematopoietic stem cells for 28 days and CD14 positive monocytes for 12 days.
E: T ratio represents the ratio of the effector to the target cell.
이하, 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 제대혈 유래 CD14 양성 단핵세포에 IL-15 및 IL-21을 혼합처리하는 단계를 포함하는 CD14 양성 단핵세포로부터 NK 세포를 분화시키는 방법을 제공한다.The present invention provides a method for differentiating NK cells from CD14 positive mononuclear cells, comprising mixing IL-15 and IL-21 with cord blood-derived CD14 positive mononuclear cells.
상기 제대혈 유래 CD14 양성 단핵세포는 하기의 단계로 제조되는 것이 바람직하나 이에 한정하지 않는다.The cord blood-derived CD14 positive mononuclear cells are preferably prepared by the following steps, but are not limited thereto.
1) 제대혈 유래 단핵세포로부터 CD3, CD34, CD56 및 CD19 양성세포를 제거하여 CD3-CD34-CD56-CD19-세포를 제조하는 단계; 및1) preparing CD3 - CD34 - CD56 - CD19 - cells by removing CD3, CD34, CD56 and CD19 positive cells from cord blood-derived mononuclear cells; And
2) 단계 1)의 CD3-CD34-CD56-CD19-세포로부터 CD14 음성세포를 제거하여 CD3-CD34-CD56-CD19-CD14+세포를 제조하는 단계인 것이 바람직하나 이에 한정하지 않는다.2) It is preferable to remove CD14 negative cells from the CD3 - CD34 - CD56 - CD19 - cells of step 1) to prepare CD3 - CD34 - CD56 - CD19 - CD14 + cells, but not always limited thereto.
본 발명의 한가지 측면에서, CD3-CD34-CD56-CD19-CD14+세포를 제조하기 위하여, 제대혈로부터 단핵세포층(mononuclear cell layer, MNC layer)을 분리한 후, 적혈구를 제거하여 단핵세포를 수득한 다음, CD3 마이크로비드(microbeads)(Miltenyi Biotech)를 첨가하여 CD3 양성 세포에 자성을 가지게 한 후, 이를 MACS 컬럼에 통과시켜 CD3 음성세포를 분리하였다. 그런 다음, 상기 CD3 음성 단핵세포에서 CD34, CD 56 및 CD19 양성세포를 제거한 후, 단핵세포 마커(marker)인 CD14 마이크로비드를 첨가하여 반응시키고 CD14 양성 단핵세포를 수득하였다. 그 결과, 도 1 및 도 2에 나타낸 바와 같이 CD14 양성세포의 회수율은 4.4%였고, 순도는 84.6%인 것을 확인하였다(도 1 및 도 2 참조).In one aspect of the present invention, in order to prepare CD3 - CD34 - CD56 - CD19 - CD14 + cells, after separating the mononuclear cell layer (MNC layer) from the cord blood, erythrocytes are removed to obtain mononuclear cells. CD3 microbeads (Miltenyi Biotech) were added to give CD3 positive cells a magnetism, and then passed through a MACS column to separate CD3 negative cells. Then, CD34,
본 발명의 한가지 측면에서, CD14 양성세포의 발현을 확인하기 위하여, CD14 양성세포를 각각의 CD15, CD19, CD33, CD56, CD117 및 CD122 마커를 사용하여 FACS로 분석한 결과, CD14 양성세포는 CD19와 CD56 음성세포이었고, 또한, CD14 양성세포는 전부 CD33 양성인 표현형을 나타냈으며, 일부는 CD15, CD117 및 CD122와 부분 양성을 나타내는 것을 확인하였다(도 3 참조).In one aspect of the invention, to confirm the expression of CD14 positive cells, CD14 positive cells were analyzed by FACS using the respective CD15, CD19, CD33, CD56, CD117 and CD122 markers. CD56 negative cells were also found, and all of the CD14 positive cells showed a phenotype that was CD33 positive, and some were partially positive with CD15, CD117, and CD122 (see FIG. 3).
본 발명의 한가지 측면에서, CD14 양성세포로부터 NK 세포로의 분화를 비교하기 위하여, CD34 양성조혈줄기세포와 CD14 양성 단핵세포를 사람 IL-15, IL-21 및 하이드로코르티손(hydrocortisone) 배지에서 각각 배양하여, 세포수를 확인하고 NK 분화도를 FACS로 분석한 결과, 단기간에 CD14 양성세포의 NK 분화도가 CD34 양성 세포에 비해 현저히 증가하는 것을 확인하였다(도 4 및 도 5 참조).In one aspect of the invention, in order to compare the differentiation of CD14 positive cells to NK cells, CD34 positive hematopoietic stem cells and CD14 positive monocytes were cultured in human IL-15, IL-21 and hydrocortisone medium, respectively. As a result, cell number was confirmed and NK differentiation was analyzed by FACS. As a result, it was confirmed that NK differentiation of CD14 positive cells was significantly increased in comparison with CD34 positive cells in a short time (see FIGS. 4 and 5).
따라서, CD14 양성세포로부터 NK 세포를 분화시키는 방법이 조혈줄기세포로부터 NK 세포를 분화시키는 방법에 비해, 단기간에 많은 수의 NK 세포를 수득할 수 있음을 확인하였다.
Therefore, it was confirmed that the method of differentiating NK cells from CD14 positive cells can obtain a large number of NK cells in a short time, compared to the method of differentiating NK cells from hematopoietic stem cells.
또한, 본 발명은 제대혈 유래 CD14 양성 단핵세포에 IL-15 및 IL-21을 혼합처리하는 단계를 포함하는 CD14 양성 단핵세포로부터 NK 세포를 증식시키는 방법을 제공한다.The present invention also provides a method of propagating NK cells from CD14 positive monocytes, comprising mixing IL-15 and IL-21 with cord blood derived CD14 positive monocytes.
본 발명의 한가지 측면에서, 제대혈로부터 분리한 단핵세포로부터 CD3, CD34, CD56 및 CD19 양성인 T세포, B세포 및 NK세포를 제거하여 CD14 양성 세포를 수득한 결과, 회수율은 4.4%였고, 순도는 84.6%인 것을 확인하였다(도 1 및 도 2 참조).In one aspect of the present invention, CD14 positive cells were obtained by removing CD3, CD34, CD56 and CD19 positive T cells, B cells and NK cells from mononuclear cells isolated from umbilical cord blood, with a recovery of 4.4% and purity of 84.6. It was confirmed to be% (see FIGS. 1 and 2).
본 발명의 한가지 측면에서, CD14 양성세포의 발현을 FACS로 분석한 결과, CD14 양성세포는 CD19와 CD56 음성세포이었고, 또한, CD14 양성세포는 전부 CD33 양성인 표현형을 나타냈으며, 일부는 CD15, CD117 및 CD122와 부분 양성을 나타내는 것을 확인하였다(도 3 참조).In one aspect of the invention, the expression of CD14 positive cells was analyzed by FACS, and the CD14 positive cells were CD19 and CD56 negative cells, and the CD14 positive cells all showed a CD33 positive phenotype, with some CD15, CD117 and It was confirmed to show partial positive with CD122 (see FIG. 3).
본 발명의 한가지 측면에서, CD14 양성세포로부터 NK 세포의 증식을 확인하기 위하여, CD34 양성조혈줄기세포와 CD14 양성 단핵세포를 NK 분화를 촉진시키고 활성을 돕는 것으로 알려진 IL-15 및 IL-21을 포함하는 NK 분화 배지에서 각각 배양하면서 세포 증식률(fold induction)을 조사한 결과, CD34 양성세포는 NK 분화배지에서 증식률이 좋지 않은 반면에, CD14 양성세포는 증식률이 2배 이상 증가하는 것을 확인하였다(도 6 참조).In one aspect of the invention, in order to confirm the proliferation of NK cells from CD14 positive cells, CD34 positive hematopoietic stem cells and CD14 positive monocytes include IL-15 and IL-21, which are known to promote NK differentiation and help activity. As a result of fold induction of each cell culture in NK differentiation medium, CD34 positive cells showed poor proliferation rate in NK differentiation medium, whereas CD14 positive cells increased more than two times (Fig. 6). Reference).
따라서, CD14 양성세포로부터 NK 세포를 증식시키는 방법이 조혈줄기세포로부터 NK 세포를 증식시키는 방법에 비해, 단기간에 많은 수의 NK 세포를 수득할 수 있음을 확인하였다.Therefore, it was confirmed that the method of propagating NK cells from CD14 positive cells can obtain a large number of NK cells in a short period of time as compared to the method of propagating NK cells from hematopoietic stem cells.
또한, 본 발명은 본 발명의 따른 방법에 의하여 제조된 NK 세포를 유효성분으로 함유하는 암 예방 및 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing and treating cancer containing NK cells produced by the method according to the present invention as an active ingredient.
상기 암은 유방암, 흑색종암, 위암, 간암, 대장암 및 폐암 등으로 구성된 군으로부터 선택되는 어느 하나인 것이 바람직하나 이에 한정되지 않는다.The cancer is preferably any one selected from the group consisting of breast cancer, melanoma cancer, gastric cancer, liver cancer, colon cancer, lung cancer and the like, but is not limited thereto.
본 발명의 한가지 측면에서, 제대혈로부터 분리한 단핵세포로부터 CD3, CD34, CD56 및 CD19 양성인 T세포, B세포 및 NK세포를 제거하여 CD14 양성 세포를 수득한 결과, 회수율은 4.4%였고, 순도는 84.6%인 것을 확인하였다(도 1 및 도 2 참조).In one aspect of the present invention, CD14 positive cells were obtained by removing CD3, CD34, CD56 and CD19 positive T cells, B cells and NK cells from mononuclear cells isolated from umbilical cord blood, with a recovery of 4.4% and purity of 84.6. It was confirmed to be% (see FIGS. 1 and 2).
본 발명의 한가지 측면에서, CD14 양성세포의 발현을 FACS로 분석한 결과, CD14 양성세포는 CD19와 CD56 음성세포이었고, 또한, CD14 양성세포는 전부 CD33 양성인 표현형을 나타냈으며, 일부는 CD15, CD117 및 CD122와 부분 양성을 나타내는 것을 확인하였다(도 3 참조).In one aspect of the invention, the expression of CD14 positive cells was analyzed by FACS, and the CD14 positive cells were CD19 and CD56 negative cells, and the CD14 positive cells all showed a CD33 positive phenotype, with some CD15, CD117 and It was confirmed to show partial positive with CD122 (see FIG. 3).
본 발명의 한가지 측면에서, CD14 양성세포로부터 NK 세포의 활성을 비교하기 위하여, CD34 양성 조혈줄기세포로부터 28일간 분화시킨 NK 세포(분화도 92%)와 12일간 CD14 양성 단핵세포로부터 분화시킨 NK 세포(분화도 90%)로 51Cr release assay를 수행하여 세포용해 활성(cytolytic activity)을 비교한 결과, 28일간 분화시킨 CD34-derieved NK 세포 와 14일간 분화시킨 CD14-derived NK 세포의 NK 살상능 활성은 유사한 것을 확인됨에 따라, CD34-derieved NK 세포에 비해 CD14 양성세포로부터 유래된 NK 세포는 단기간의 높은 세포용해 활성이 있는 것을 확인하였다(도 7 참조).In one aspect of the present invention, NK cells (92% differentiation) differentiated from CD34-positive hematopoietic stem cells for 28 days and NK cells differentiated from CD14-positive mononuclear cells for 12 days The NK killing activity of CD34-derived NK cells differentiated for 28 days and that of CD14-derived NK cells differentiated for 14 days were similar to each other by the 51 Cr release assay with differentiation degree of 90% It was confirmed that NK cells derived from CD14-positive cells had a short-term high cytolytic activity as compared to CD34-skinned NK cells (see FIG. 7).
따라서, CD14 양성세포로부터 활성이 있는 NK세포로의 분화가 CD34 조혈줄기세포보다 단기간 내에 이루어지고 더 많은 수를 얻을 수 있어서 보다 효율적인 것을 확인하였다.
Therefore, it was confirmed that the differentiation of CD14-positive cells into NK cells which are active is more efficient than that of CD34 hematopoietic stem cells, since more numbers can be obtained.
본 발명의 조성물은 상기 NK 세포에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 투여를 위해서는 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 산제, 정제, 캡슐제, 환, 과립 또는 주사액제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar function in addition to the NK cells. For administration, it may be prepared further comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, as necessary, including antioxidants, buffers, Other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, rings, granules or injection solutions. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.
본 발명의 조성물은 비경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 조성물의 일일 투여량은 0.01 ~ 5000 ㎎/㎏이며, 바람직하게는 0.01 ~ 10 ㎎/㎏ 이며, 하루 일 회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다.
The composition of the present invention may be administered parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically), and the dosage may be weight, age, sex, health condition, diet, time of administration, method of administration, The range varies depending on the rate of excretion and the severity of the disease. The daily dose of the composition according to the present invention is 0.01 to 5000 mg / kg, preferably 0.01 to 10 mg / kg, and more preferably administered once to several times a day.
또한, 본 발명은 약학적으로 유효한 양의 본 발명의 따른 조성물을 암에 걸린 개체에 투여하는 단계를 포함하는 암 치료 방법을 제공한다.The present invention also provides a method of treating cancer comprising administering a pharmaceutically effective amount of a composition according to the present invention to a subject with cancer.
아울러, 본 발명은 약학적으로 유효한 양의 본 발명의 따른 조성물을 개체에 투여하는 단계를 포함하는 암 예방 방법을 제공한다.In addition, the present invention provides a method for preventing cancer, comprising administering to a subject a pharmaceutically effective amount of a composition according to the present invention.
상기 암은 유방암, 흑색종암, 위암, 간암, 대장암 및 폐암 등으로 구성된 군으로부터 선택되는 어느 하나인 것이 바람직하나 이에 한정되지 않는다.The cancer is preferably any one selected from the group consisting of breast cancer, melanoma cancer, gastric cancer, liver cancer, colon cancer, lung cancer and the like, but is not limited thereto.
본 발명의 조성물은 상기 NK 세포에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 투여를 위해서는 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar function in addition to the NK cells. For administration, it may be prepared further comprising one or more pharmaceutically acceptable carriers.
본 발명의 조성물은 비경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 조성물의 일일 투여량은 0.01 ~ 5000 ㎎/㎏이며, 바람직하게는 0.01 ~ 10 ㎎/㎏ 이며, 하루 일 회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다. The composition of the present invention may be administered parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically), and the dosage may be weight, age, sex, health condition, diet, time of administration, method of administration, The range varies depending on the rate of excretion and the severity of the disease. The daily dose of the composition according to the present invention is 0.01 to 5000 mg / kg, preferably 0.01 to 10 mg / kg, and more preferably administered once to several times a day.
이하, 본 발명을 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 한정되는 것은 아니다.
However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.
<< 실시예Example 1> 제대혈로부터 1> From cord blood 조혈줄기세포의Of hematopoietic stem cells 분리 및 배양 Isolation and Cultivation
병원으로부터 연구용으로 제공(건양대학병원 산부인과)받은 제대혈을 RPMI 1640을 이용하여 2:1로 희석하여 준비한 다음, Ficoll-Paque 상층부에 준비된 제대혈을 조심스레 얹은 후, 20,000 rpm, 30분간 원심분리하여 단핵세포층(mononuclear cell layer; MNC layer)을 수득한 후, 조심스럽게 취한 세포에서 적혈구를 제거하여 단핵세포를 수득하였다. 조혈줄기세포의 마커(marker) CD34 마이크로비드를 첨가하여 표지한 후, 이를 MS/RS 컬럼과 MACS를 이용하여 CD34+ 세포를 분리하였다.Umbilical cord blood received from the hospital for research (Konyang University Hospital Obstetrics and Gynecology) was diluted 2: 1 using RPMI 1640, and then carefully placed on the cord blood prepared above Ficoll-Paque, followed by centrifugation at 20,000 rpm for 30 minutes. After obtaining a cell layer (mononuclear cell layer (MNC layer)), cells were carefully taken off the red blood cells to obtain mononuclear cells. After labeling by adding marker CD34 microbeads of hematopoietic stem cells, CD34 + cells were isolated using an MS / RS column and MACS.
그 결과, 도 1 및 도 2에 나타낸 바와 같이 CD34 양성세포의 회수율은 0.6% 이였으며 순도는 81.1%이였다(도 1 및 도 2).
As a result, the recovery of CD34 positive cells was 0.6% and the purity was 81.1%, as shown in Figs. 1 and 2 (Figs. 1 and 2).
<< 실시예Example 2> 제대혈의 2> cord blood 단핵세포로부터From monocytes CD14CD14 양성세포의 분리 Positive Cell Isolation
병원으로부터 연구용으로 제공(건양대학병원 산부인과)받은 제대혈을 RPMI 1640을 이용하여 2:1로 희석하여 준비한 다음, Ficoll-Paque 상층부에 준비된 제대혈을 조심스레 얹은 후, 20,000 rpm, 30분간 원심분리하여 단핵세포층(mononuclear cell layer; MNC layer)을 수득한 후, 조심스럽게 취한 세포에서 적혈구를 제거하여 단핵세포를 수득하였다. 상기 단핵세포에 T 세포 표지자로 CD3 마이크로비드(microbead)(Miltenyi Biotech)를 첨가하여 30 분 4℃로 반응시킨 후, 세척하여 MACS 버퍼(buffer)에 현탁시켜 Vario MACS를 이용하여 CD 컬럼(column)을 통과시켜 CD3 음성 세포를 얻었다. 그런 다음, 상기 CD3 음성 단핵세포에 CD34, CD56 및 CD19가 표지 된 각각의 마이크로비드를 첨가하여 CD34, CD 56 및 CD19 양성세포를 제거한 후, 단핵세포 마커(marker)인 CD14 마이크로비드를 첨가하여 반응시키고 MS/RS 컬럼과 MACS를 이용하여 CD14 양성 단핵세포를 수득하였다. 이렇게 수득한 CD14 양성세포 순도는 유세포 분석기(flow cytometry, FACS)로 확인하였다. Umbilical cord blood received from the hospital for research (Konyang University Hospital Obstetrics and Gynecology) was diluted 2: 1 using RPMI 1640, and then carefully placed on the cord blood prepared above Ficoll-Paque, followed by centrifugation at 20,000 rpm for 30 minutes. After obtaining a cell layer (mononuclear cell layer (MNC layer)), cells were carefully taken off the red blood cells to obtain mononuclear cells. After adding CD3 microbead (Miltenyi Biotech) as a T cell marker to the monocytes and reacting at 4 ° C. for 30 minutes, washing and suspending it in a MACS buffer was performed using a Vario MACS CD column. Passed through to obtain CD3 negative cells. Then, CD34, CD56 and CD19-labeled microbeads were added to the CD3 negative monocytes to remove CD34, CD56 and CD19 positive cells, and then CD14 microbeads, which are monocyte markers, were added to react. And CD14 positive monocytes were obtained using an MS / RS column and MACS. CD14 positive cell purity thus obtained was confirmed by flow cytometry (FACS).
그 결과, 도 1 및 도 2에 나타낸 바와 같이 CD14 양성세포의 회수율은 4.4%였고, 순도는 84.6%이였다(도 1 및 도 2).
As a result, the recovery of CD14 positive cells was 4.4% and the purity was 84.6%, as shown in Figs. 1 and 2 (Figs. 1 and 2).
<< 실험예Experimental Example 1> 1> CD14CD14 양성세포의 발현 분석 Positive Cell Expression Analysis
상기 <실시예 2>에서 분리한, 제대혈 단핵세포(mononuclear cell, MNC)로부터 분리한 CD14 양성세포를 각각의 CD15, CD19, CD33, CD56, CD117 및 CD122 마커를 사용하여 FACS로 분석하였다.CD14 positive cells isolated from cord blood mononuclear cells (MNC) isolated in Example 2 were analyzed by FACS using respective CD15, CD19, CD33, CD56, CD117 and CD122 markers.
그 결과, 도 3에 나타낸 바와 같이 CD14 양성세포는 CD19와 CD56 음성세포이였고, 또한, CD14 양성세포는 전부 CD33 양성인 표현형을 나타냈으며, 일부는 CD15, CD117 및 CD122와 부분 양성을 나타내는 것을 확인하였다(도 3).
As a result, as shown in FIG. 3, the CD14 positive cells were CD19 and CD56 negative cells, and all of the CD14 positive cells showed a CD33 positive phenotype, and some were partially positive with CD15, CD117 and CD122 ( 3).
<< 실험예Experimental Example 2> 2> CD34CD34 양성세포 및 Positive cells and CD14CD14 양성세포로부터 From positive cells NKNK 세포로의 분화 비교 Comparison of Differentiation into Cells
상기 <실시예 1> 및 <실시예 2>에서 각각 분리한 CD34 양성조혈줄기세포와 CD14 양성 단핵세포를 CD3 및 CD56 항체로 염색하여 NK 분화도(CD3-CD56+인 NK 세포군의 비율)를 FACS로 분석하였다. 구체적으로 CD34 양성조혈줄기세포와 CD14 양성 단핵세포를 각각 12-웰 플레이트(Falcon)에 1×106 세포/ml의 농도로 사람 IL-15(10 ng/ml, PeproTech)와 IL-21(10 ng/ml, PeproTech) 및 하이드로코르티손(hydrocortisone)(10-6 M, stem cell Tech.)이 첨가된 Myelocult(Stem cell Technology) 완전 배지를 사용하여 37℃, 5% CO2에서 14일 또는 12일 동안 배양하였고, 배양하는 도중에 세포의 농도가 2×106 세포/웰이 넘게 되면 초기 사용한 조건의 배지를 사용하여 세포를 나누어 주었다. 4일, 8일, 14일, 18일 및 21일 별로 세포수를 각각 확인하였고, 7일째 및 12일째 CD3, CD122 및 CD56 항체로 염색하여 NK 분화도를 FACS 분석하였다.CD34 positive hematopoietic stem cells and CD14 positive mononuclear cells isolated in <Example 1> and <Example 2>, respectively, were stained with CD3 and CD56 antibodies, and the degree of NK differentiation (proportion of NK cell group of CD3 - CD56 + ) was determined by FACS. Analyzed. Specifically, CD34-positive hematopoietic stem cells and CD14-positive mononuclear cells were collected in human IL-15 (10 ng / ml, PeproTech) and IL-21 (10) at concentrations of 1 × 10 6 cells / ml in 12-well plates (Falcon), respectively. ng / ml, PeproTech) and Myelocult (Stem cell Technology) complete medium with hydrocortisone ( 10-6 M, stem cell Tech.) for 14 or 12 days at 37 ° C., 5% CO 2 When the concentration of the cells exceeded 2 × 10 6 cells / well during the culture, the cells were divided using medium under the conditions of initial use. Cell numbers were determined at 4, 8, 14, 18 and 21 days, respectively, and stained with CD3, CD122 and CD56 antibodies at 7 and 12 days, and NK differentiation was analyzed by FACS.
그 결과, 도 4 및 도 5에 나타낸 바와 같이 7일째 CD34 양성세포의 NK 분화도(CD3-CD56+)는 13.5%이고, CD14 양성세포의 NK 분화도는 78.5%인 것을 확인하였으며, 12일째 CD34 양성세포의 NK 분화도(CD3-CD56+)는 41.3%이고, CD14 양성세포의 NK 분화도는 90.2% 로인 것을 확인하였다. 따라서, 단기간에 CD14 양성세포의 NK 분화도가 CD34 양성 세포에 비해 현저히 증가하는 것을 확인하였다(도 4 및 도 5).
As a result, as shown in FIGS. 4 and 5, it was confirmed that the NK differentiation (CD3 - CD56 + ) of CD34 positive cells on day 7 was 13.5%, and the NK differentiation of CD14 positive cells was 78.5%, on day 12 CD34 positive cells. The degree of NK differentiation (CD3 - CD56 + ) was 41.3%, and the level of NK differentiation of CD14 positive cells was 90.2%. Therefore, it was confirmed that NK differentiation of CD14 positive cells increased significantly compared to CD34 positive cells in a short period of time (Figs. 4 and 5).
<< 실험예Experimental Example 3> 3> CD34CD34 양성세포 및 Positive cells and CD14CD14 양성세포로부터 From positive cells NKNK 세포의 증식 비교 Comparison of Cell Proliferation
상기 <실시예 1> 및 <실시예 2>에서 제조한 CD34 양성조혈줄기세포와 CD14 양성 단핵세포를 NK 분화를 촉진시키고 활성을 돕는 것으로 알려진 IL-15 및 IL-21을 포함하는 NK 분화 배지에서 각각 배양하면서, 1, 4, 6, 7 및 12일 후 세포의 수를 개수하여 세포 증식률(fold induction)을 조사하였다. CD34 positive hematopoietic stem cells and CD14 positive mononuclear cells prepared in <Example 1> and <Example 2> in an NK differentiation medium containing IL-15 and IL-21 known to promote NK differentiation and help activity While culturing, respectively, the number of cells after 1, 4, 6, 7 and 12 days was counted to investigate the cell fold induction.
그 결과, 도 6에 나타낸 바와 같이, CD34 양성세포는 NK 분화배지에서 증식률이 좋지 않은 반면에, CD14 양성 세포는 증식률이 2배 이상 증가하는 것을 확인하였다(도 6).
As a result, as shown in Figure 6, CD34 positive cells were not good growth rate in NK differentiation medium, while CD14 positive cells were confirmed that the growth rate increased more than two times (Fig. 6).
<< 실험예Experimental Example 4> 4> CD34CD34 양성세포 및 Positive cells and CD14CD14 양성세포로부터 From positive cells NKNK 세포의 활성 비교 Cell activity comparison
CD34 양성 조혈줄기세포로부터 28일간 분화시킨 NK 세포(분화도 92%)와 14일간 CD14 양성 단핵세포로부터 분화시킨 NK 세포(분화도 90%)로 51Cr release assay를 수행하여 세포용해 활성(cytolytic activity)를 비교하였다. 구체적으로 NK-민감성 타켓(sentive target)인 K562 세포를 방사선 동위원소 51Cr으로 37℃, 1시간 표지시킨 후 세척하여 준비하였다. 시험관내(In vitro)에서 분화시킨 NK 세포를 세척 후 effector:target ratio에 따라 타겟 세포인 51Cr-labelled K562 세포(104/웰)와 함께 96 웰 둥근 바닥 플레이트(well round bottom plate)에서 4시간 동안 배양하였다. 4시간 후, 배양 상층액 100 ㎕를 취하여 방사능을 γ-counter로 측정하였고, lysis(%)는 (sample release-spontaneous/maximum release-spontaneous release)×100으로 계산하였다.Cytolytic activity was performed by 51 Cr release assay with NK cells differentiated from CD34 positive hematopoietic stem cells for 28 days (92% differentiation) and NK cells differentiated from CD14 positive monocytes (90% differentiation) for 14 days. Compared. Specifically, K562 cells, which are NK-sensitive targets (sentive targets), were prepared by labeling with radioactive isotope 51 Cr at 37 ° C. for 1 hour and washing. NK cells differentiated in vitro were washed in 4 wells in 96 well round bottom plates with 51 Cr-labelled K562 cells (10 4 / well), the target cells, according to the effector: target ratio. Incubated for hours. After 4 hours, 100 μl of the culture supernatant was taken to measure radioactivity by γ-counter, and lysis (%) was calculated as (sample release-spontaneous / maximum release-spontaneous release) × 100.
그 결과, 도 7에 나타낸 바와 같이, E/T 비율 10:1에서 lysis(%)가 70% 정도로 28일간 분화시킨 CD34-derieved NK 세포와 14일간 분화시킨 CD14-derived NK 세포의 NK 살상능 활성은 유사한 것을 확인됨에 따라, CD34-derieved NK 세포에 비해 CD14 양성세포로부터 유래된 NK 세포는 단기간의 높은 세포용해 활성이 있는 것을 확인하였다. 따라서, CD14 양성세포로부터 활성이 있는 NK세포로의 분화가 CD34 조혈줄기세포보다 단기간 내에 이루어지고 더 많은 수를 얻을 수 있어서 보다 효율적인 것을 확인하였다(도 7).
As a result, as shown in FIG. 7, the NK killing activity of CD34-derived NK cells differentiated for 28 days and CD14-derived NK cells differentiated for 14 days at an E / T ratio of 10: , It was confirmed that NK cells derived from CD14-positive cells had a short-term high cytolytic activity compared to CD34-skinned NK cells. Therefore, it was confirmed that differentiation from CD14 positive cells to active NK cells is more efficient since CD34 hematopoietic stem cells can be obtained in a shorter time and more numbers are obtained (FIG. 7).
Claims (5)
A method of differentiating NK cells from CD14 positive mononuclear cells, comprising mixing IL-15 and IL-21 with cord blood-derived CD14 positive mononuclear cells.
A method for propagating NK cells from CD14 positive monocytes, comprising mixing IL-15 and IL-21 with cord blood derived CD14 positive monocytes.
1) 제대혈 유래 단핵세포로부터 CD3, CD34, CD56 및 CD19 양성세포를 제거하여 CD3-CD34-CD56-CD19-세포를 제조하는 단계; 및
2) 단계 1)의 CD3-CD34-CD56-CD19-세포로부터 CD14 음성세포를 제거하여 CD3-CD34-CD56-CD19-CD14+세포를 제조하는 단계를 포함하는 방법에 의해 제조되는 것을 특징으로 하는 방법.
The method of claim 1 or 2, wherein the C14 positive monocytes are
1) preparing CD3 - CD34 - CD56 - CD19 - cells by removing CD3, CD34, CD56 and CD19 positive cells from cord blood-derived mononuclear cells; And
2) removing the CD14 negative cells from the CD3 - CD34 - CD56 - CD19 - cells of step 1) to prepare the CD3 - CD34 - CD56 - CD19 - CD14 + cells. .
A pharmaceutical composition for preventing and treating cancer, comprising NK cells prepared by the method of claim 1 or 2 as an active ingredient.
[Claim 5] The pharmaceutical composition for preventing and treating cancer according to claim 4, wherein the cancer is any one selected from the group consisting of breast cancer, melanoma cancer, gastric cancer, liver cancer, colon cancer, lung cancer and the like.
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| CN114058584B (en) * | 2022-01-07 | 2022-07-01 | 山东省齐鲁干细胞工程有限公司 | Preparation method of clinical natural killer cells |
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| JP2020114248A (en) * | 2014-03-07 | 2020-07-30 | エメルセル エスエーエス | Pooled nk cell derived from umbilical cord, and application of the same for therapy of cancer and chronic infectious disease |
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