KR102669705B1 - Method for Expansion of NK Cell from Umbrical Cord Blood - Google Patents
Method for Expansion of NK Cell from Umbrical Cord Blood Download PDFInfo
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Classifications
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Abstract
본 발명은 단핵세포 집단 또는 NK 세포를 유전적으로 조작된 피더 세포와 접촉시키며 공배양하는 단계를 포함하는 NK 세포의 확장 배양방법에 관한 것으로, 본 발명에 따르면, NK 세포의 세포 증식을 기존 방법보다 10배 이상 증가시킬 수 있어, 암에 대한 세포치료제로 사용되는 NK 세포 치료제를 고효율 제조할 수 있다. The present invention relates to a method for expanding NK cells, which includes the step of co-culturing a population of monocytes or NK cells with genetically engineered feeder cells. According to the present invention, the cell proliferation of NK cells is more efficient than existing methods. It can be increased by more than 10 times, making it possible to manufacture NK cell therapy used as a cell therapy for cancer with high efficiency.
Description
본 발명은 NK 세포의 확장 배양방법에 관한 것으로, 더욱 자세하게는 말초혈액 또는 제대혈 유래 단핵세포 집단 또는 NK 세포를 유전적으로 조작된 피더 세포와 접촉시키며 공배양하는 단계를 포함하는 NK 세포의 확장 배양방법에 관한 것이다.The present invention relates to a method for expanding and culturing NK cells, and more specifically, to a method for expanding and culturing NK cells, which includes the step of co-culturing a mononuclear cell population or NK cells derived from peripheral blood or umbilical cord blood by contacting them with genetically engineered feeder cells. It's about.
면역체계를 구성하는 세포들 중 자연살해세포(natural killer cell, 이하 "NK 세포"라 약칭함)는 특정 항원에 감작되지 않고도 활성 및 억제 수용체의 종합적인 신호전달 균형에 의해 바이러스 감염세포나 암세포를 살상할 수 있는 능력이 있는 면역세포로 알려져 있다. 이러한 NK 세포의 살해능력은 NK 세포 단독 혹은 다른 치료법과 병합하여 각종 혈액암(hematologic maligancy) 및 고형암(solid tumor) 치료에 이용되고 있다. 환자 몸 속에 사이토카인과 같은 약제를 주입하여 체내 NK 세포를 활성화시켜 항암 효과를 보는 방법도 있지만, 체외에서 NK 세포 혹은 유전자 조작 NK 세포를 활성 및 증폭하여 환자 몸에 주입하는 방법이 있다. Among the cells that make up the immune system, natural killer cells (hereinafter abbreviated as “NK cells”) kill virus-infected cells or cancer cells through a comprehensive signaling balance of activating and inhibitory receptors without being sensitized to specific antigens. It is known to be an immune cell with the ability to kill. The killing ability of NK cells is used alone or in combination with other treatments to treat various hematologic malignancies and solid tumors. There is a method of injecting drugs such as cytokines into the patient's body to activate NK cells in the body to achieve anti-cancer effects, but there is also a method of activating and amplifying NK cells or genetically modified NK cells outside the body and injecting them into the patient's body.
NK 세포를 항암 면역 세포치료로 효과적으로 이용하기 위해서는 많은 수의 NK 세포 확보가 필요하다. 그러나 NK 세포는 혈액 내 림프구의 10 ~ 15%를 차지하고 있고 암 환자에서는 종종 NK 세포의 수, 분화 및 기능이 저하되어있어 충분한 세포수의 확보가 어려운 실정이다. 그러므로 NK 세포의 증식 또는 분화를 통한 NK 세포의 다량 확보가 절실히 요구되고 있다. In order to effectively use NK cells as anti-cancer immune cell therapy, it is necessary to secure a large number of NK cells. However, NK cells account for 10 to 15% of lymphocytes in the blood, and in cancer patients, the number, differentiation, and function of NK cells are often reduced, making it difficult to secure a sufficient number of cells. Therefore, there is an urgent need to secure large quantities of NK cells through proliferation or differentiation of NK cells.
NK 세포는 통상적으로 골수의 조혈줄기세포(hematopoietic stem cell, HSC)로부터 유래되어 각종 조직과 혈액에 분포되어 있다고 알려져 있다. 가장 대표적인 것은 지라, 간, 골수, 말초혈액, 제대혈 등이다. 시험관내(in vitro)에서는 제대혈로부터 조혈줄기세포를 분리하여 적당한 사이토카인들을 처리하여 배양함으로서 NK 세포로 분화시키는 방법들이 보고되었다.It is known that NK cells are generally derived from hematopoietic stem cells (HSC) in the bone marrow and are distributed in various tissues and blood. The most representative examples are spleen, liver, bone marrow, peripheral blood, and umbilical cord blood. In vitro methods have been reported to differentiate hematopoietic stem cells into NK cells by isolating hematopoietic stem cells from umbilical cord blood, treating them with appropriate cytokines, and culturing them.
과거에 보고된 NK 세포의 증식 방법은 고가의 다양한 사이토카인을 고농도로 이용하거나 말초혈액단핵구나 암세포주를 함께 사용하는 방법들이었고, 이는 고 비용이 소요될 뿐 아니라 증폭율도 그리 높지 않았다. 그런데, 최근에는 유전자 조작된 암세포주(Imai C et al., Blood. 2005;106:376-83; Denman CJ et al., PLoS One. 2012;7:e30264; Thangaraj JL et al., Cancer Immunol Immunother. 2022;71:613-625)를 활용한 방법들이 소개되어 이런 제한점을 많이 극복하였다. 하지만, NK 세포 증폭율을 개선하기 위해서는 사이토카인 조성과 영양세포 특성 뿐 아니라 배지 조성, 영양세포와 단핵세포(말초혈액, 제대혈)의 비율 등의 여러 배양조건이 최적화되어야 최상의 증폭을 할 수 있다. 또한, 다수의 연구에서는 NK 세포 외에도 T 세포 등 다른 림프구의 증폭이 동반되어 나타나, NK 세포 기반의 면역 세포 치료에 부적합한 면을 보이기도 한다. 따라서, NK 세포만을 선택적으로 증폭하기 위한 효율적인 방법에 대한 개발이 주요한 과제의 대상이 되고 있고, 이에 대한 연구가 이루어지고 있으나 (한국등록특허 10-1525199), 아직도 개선이 필요한 실정이다.The NK cell proliferation methods reported in the past were methods that used various expensive cytokines at high concentrations or a combination of peripheral blood mononuclear cells or cancer cell lines, which were not only expensive but also did not have a very high amplification rate. However, recently, genetically engineered cancer cell lines (Imai C et al., Blood. 2005;106:376-83; Denman CJ et al., PLoS One. 2012;7:e30264; Thangaraj JL et al., Cancer Immunol Immunother 2022;71:613-625) were introduced to overcome many of these limitations. However, in order to improve the NK cell amplification rate, various culture conditions, such as cytokine composition and feeder cell characteristics, as well as medium composition and ratio of feeder cells and mononuclear cells (peripheral blood, cord blood), must be optimized to achieve the best amplification. In addition, many studies show that in addition to NK cells, amplification of other lymphocytes such as T cells is accompanied, which makes them unsuitable for NK cell-based immune cell therapy. Therefore, the development of an efficient method to selectively amplify only NK cells has become a major task, and research is being conducted on this (Korean Patent Registration No. 10-1525199), but improvement is still needed.
이에, 본 발명자들은 NK 세포를 대량 확보하는 방법을 개발하고자 예의 노력한 결과, 제대혈 유래 단핵세포를 막결합성 인터루킨을 발현하도록 유전적으로 조작된 피더세포와 적절한 비율로 배양하고, 적절한 배지를 사용한 경우, 동일 기간에 기존 방법보다 현저히 높은 증식율로 NK 세포를 확보할 수 있다는 것을 확인하고, 본 발명을 완성하게 되었다. Accordingly, the present inventors made diligent efforts to develop a method for securing large quantities of NK cells. As a result, when umbilical cord blood-derived mononuclear cells were cultured in an appropriate ratio with feeder cells genetically engineered to express membrane-bound interleukin and an appropriate medium was used, After confirming that NK cells could be secured at a significantly higher proliferation rate than existing methods in the same period of time, the present invention was completed.
본 발명은 높은 증식율로 NK 세포를 확보할 수 있는 NK 세포의 확장 배양방법을 제공하는데 있다. The purpose of the present invention is to provide a method for expanding NK cells and cultivating them, which can secure NK cells with a high proliferation rate.
상기 목적을 달성하기 위하여, 본 발명은 단핵세포 집단 또는 NK 세포를 막 결합성 인터루킨-18(membrane bound interleukin-18: mbIL-18) 및 막 결합성 인터루킨-21(membrane bound interleukin-21: mbIL-21)을 발현하도록 유전적으로 조작된 피더 세포와 접촉시키며 공배양하는 단계를 포함하는 NK 세포의 확장 배양방법을 제공한다.In order to achieve the above object, the present invention is a mononuclear cell population or NK cells with membrane bound interleukin-18 (mbIL-18) and membrane bound interleukin-21 (mbIL-21). 21) provides a method for expanding and culturing NK cells, which includes the step of contacting and co-culturing with feeder cells genetically engineered to express.
본 발명에 따르면, NK 세포의 세포 증식을 기존 방법 (한국등록특허 10-1525199) 대비 10배 이상 증가시킬 수 있어, 암에 대한 세포치료제로 사용되는 NK 세포 치료제를 고효율 제조할 수 있다. According to the present invention, cell proliferation of NK cells can be increased by more than 10 times compared to the existing method (Korean Patent No. 10-1525199), making it possible to manufacture NK cell therapy used as a cell therapy for cancer with high efficiency.
도 1은 제대혈 유래 단핵세포에서 NK 세포 확장 배양 수율을 여러 조건에서 확인한 결과를 나타낸 것이다.
도 2는 말초혈액유래 단핵세포 및 제대혈 유래 단핵세포에서 NK 세포 확장 배양 수율을 확인한 결과를 나타낸 것이다.
도 3은 말초혈액 유래 단핵세포 및 제대혈 유래 단핵세포에서 NK 세포 확장 후 암세포 살상 효능을 확인한 결과를 나타낸 것이다.
도 4는 확장된 NK 세포의 CD107a, IFn-g 및 TNF-a 분비량을 확인한 결과를 나타낸 것이다.
도 5는 확장된 NK 세포의 표면 수용체 발현을 확인한 결과를 나타낸 것이다.Figure 1 shows the results of confirming the NK cell expansion culture yield in umbilical cord blood-derived mononuclear cells under various conditions.
Figure 2 shows the results of confirming the NK cell expansion culture yield in peripheral blood-derived mononuclear cells and umbilical cord blood-derived mononuclear cells.
Figure 3 shows the results of confirming the cancer cell killing efficacy after NK cell expansion in peripheral blood-derived mononuclear cells and umbilical cord blood-derived mononuclear cells.
Figure 4 shows the results of confirming the secretion amounts of CD107a, IFn-g, and TNF-a by expanded NK cells.
Figure 5 shows the results of confirming surface receptor expression of expanded NK cells.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명에서는 제대혈 유래 단핵세포에서 다량의 NK 세포를 증식시키는 방법을 개발하였으며, 보다 구체적으로는 제대혈 유래 단핵세포 집단에서 증폭하여 분리된 NK 세포를 막 결합성 인터루킨-18(membrane bound interleukin-18: mbIL-18) 및 막 결합성 인터루킨-21(membrane bound interleukin-21: mbIL-21)을 발현하도록 유전적으로 조작된 피더 세포와 접촉시키며 공배양하는 방법에서, NK 세포와 피더세포의 비율을 특정비율로 높이는 경우, 단기간(21일) 배양으로 약 100,000배의 NK 세포 증식율을 확인하였으며, 기존 배양방법(약 1,800배)에 비하여, 50 배 이상의 NK 세포 확보가 가능한 것을 확인하였다.In the present invention, we developed a method to proliferate large amounts of NK cells from umbilical cord blood-derived mononuclear cells. More specifically, NK cells amplified and isolated from umbilical cord blood-derived mononuclear cell populations were incubated with membrane bound interleukin-18 (membrane bound interleukin-18: In the method of co-culturing by contacting feeder cells genetically engineered to express mbIL-18) and membrane bound interleukin-21 (mbIL-21), the ratio of NK cells and feeder cells is maintained at a specific ratio. When increased to , a NK cell proliferation rate of approximately 100,000 times was confirmed in short-term (21 days) culture, and it was confirmed that it was possible to secure more than 50 times more NK cells than the existing culture method (about 1,800 times).
따라서, 본 발명은 단핵세포 집단 또는 NK 세포를 막 결합성 인터루킨-18(membrane bound interleukin-18: mbIL-18) 및 막 결합성 인터루킨-21(membrane bound interleukin-21: mbIL-21)을 발현하도록 유전적으로 조작된 피더 세포와 접촉시키며 공배양하는 단계를 포함하는 NK 세포의 확장 배양방법에 관한 것이다.Accordingly, the present invention provides a method for activating monocyte populations or NK cells to express membrane bound interleukin-18 (mbIL-18) and membrane bound interleukin-21 (mbIL-21). It relates to a method for expanding and culturing NK cells, including the step of contacting and co-culturing with genetically engineered feeder cells.
본 발명에 있어서, NK 세포의 확장 배양방법은 다음을 포함할 수 있다:In the present invention, the method for expanding NK cells may include the following:
(a) 단핵세포 또는 NK 세포 집단을 막 결합성 인터루킨-18(membrane bound interleukin-18: mbIL-18) 및 막 결합성 인터루킨-21(membrane bound interleukin-21: mbIL-21) 을 발현하도록 유전적으로 조작된 피더 세포와 공배양하는 단계; 및(a) Monocyte or NK cell populations are genetically modified to express membrane bound interleukin-18 (mbIL-18) and membrane bound interleukin-21 (mbIL-21). co-culturing with the engineered feeder cells; and
(b) 상기 유전적으로 조작된 피더 세포를 첨가하여 재자극한 후 NK 세포를 배양하는 단계.(b) cultivating NK cells after restimulation by adding the genetically engineered feeder cells.
본 발명에서 상기 단핵세포 집단은 말초혈액 유래이거나 제대혈 유래일 수 있다. 단핵세포 내의 NK 세포 수의 측정은 세포 계수기 및 유세포분석 등의 방법을 사용하여 측정할 수 있다. 구체적으로 LUNA-FX7 자동 세포 계수기(Aligned Genetics, Korea)를 이용하여 세포 상등액의 단핵세포 수를 측정하고, 항-인간(Anti-human) CD3-FITC, 항-인간 CD56-APC (BD Biosciences, San Jose, CA)로 염색한 후 유세포 분석을 통해 단핵세포에서의 NK 세포 비율과 이에 따른 실제 세포 수를 산출할 수 있다.In the present invention, the mononuclear cell population may be derived from peripheral blood or cord blood. The number of NK cells within mononuclear cells can be measured using methods such as cell counter and flow cytometry. Specifically, the number of monocytes in the cell supernatant was measured using a LUNA-FX7 automatic cell counter (Aligned Genetics, Korea), and anti-human CD3-FITC and anti-human CD56-APC (BD Biosciences, San Francisco) were used. After staining with (Joseph, CA), the ratio of NK cells in monocytes and the actual number of cells can be calculated through flow cytometry.
본 발명에서 상기 단핵세포 집단은 밀도 구배 원심분리 방법(Density gradient centrifugation)을 이용하여 수득할 수 있다. 구체적으로 건강인의 말초 혈액 또는 제대혈에 2% (v/v) FBS가 포함된 DPBS를 1:1 비율로 희석하여 Lymphoprep상에서 오버레이한 후 상온에서 30 분 동안 400 g 조건으로 브레이크 없이 원심 분리를 진행(가속 1, 감속 0)하여 연막층 (buffy coat layer)에서 세포를 수득할 수 있다. 상기 세포는 LUNA-FX7 자동 세포 계수기를 이용하여 세포 수를 측정하고, 항-인간 CD3-FITC, 항-인간 CD56-APC로 염색한 후 유세포 분석을 통해 NK 세포 비율 및 실제 세포 수를 산출할 수 있다.In the present invention, the mononuclear cell population can be obtained using density gradient centrifugation. Specifically, DPBS containing 2% (v/v) FBS was diluted in the peripheral blood or cord blood of a healthy person at a 1:1 ratio, overlaid on Lymphoprep, and then centrifuged without brake at 400 g for 30 minutes at room temperature. (Acceleration 1, Deceleration 0) to obtain cells from the buffy coat layer. The cell number was measured using a LUNA-FX7 automatic cell counter, stained with anti-human CD3-FITC and anti-human CD56-APC, and then the NK cell ratio and actual cell number could be calculated through flow cytometry. there is.
본 발명에서 상기 NK 세포의 확장 배양방법은 단핵세포를 배양하여 NK 세포를 확장하거나, 단핵세포 집단으로부터 NK 세포를 분리하여, 분리된 NK 세포를 이용하여 수행할 수 있다. In the present invention, the method for expanding and culturing NK cells can be performed by culturing monocytes to expand NK cells, or by isolating NK cells from a mononuclear cell population and using the isolated NK cells.
NK 세포의 분리는 마그네틱 비드가 결합된 항체를 세포에 표지한 후 자석을 이용하여 양성 분리(positive selection) 또는 음성 분리(negative selection) 등의 방법으로 수행할 수 있다. 본 발명의 일 양태에서는, 구체적으로 EasySep™ Human NK Cell Isolation Kit (StemCell Technologies, Cambridge, MA)를 사용하여 단핵세포 집단에 항체가 결합된 마이크로비드를 부착하고, 이를 EasySep™ Magnet에 장착한 후 음성 선택을 통해 NK 세포를 선택적으로 분리할 수 있다.Isolation of NK cells can be performed by labeling the cells with antibodies bound to magnetic beads and then using a magnet to perform positive selection or negative selection. In one aspect of the present invention, antibody-coupled microbeads are attached to a population of mononuclear cells using the EasySep™ Human NK Cell Isolation Kit (StemCell Technologies, Cambridge, MA), mounted on an EasySep™ Magnet, and negative NK cells can be selectively isolated through selection.
본 발명에 있어서, 상기 (a) 단계는 상기 단핵세포 내 자연살해세포: 유전적으로 조작된 피더 세포 수의 비율이 1 : 2~10의 비율로 수행할 수 있으며, 바람직하게는 1: 4~8, 더욱 바람직하게는 1:4~6, 가장 바람직하게는 1 : 4.5~5.5의 비율로 수행할 수 있다. In the present invention, step (a) can be performed at a ratio of natural killer cells in the mononuclear cells to the number of genetically engineered feeder cells of 1:2 to 10, preferably 1:4 to 8. , more preferably at a ratio of 1:4 to 6, and most preferably at a ratio of 1:4.5 to 5.5.
본 발명의 일 양태에서는, 본 발명의 방법으로 증폭하여 수득된 NK 세포의 암 치료제로서의 효능을 확인하였으며, 본 발명에 따른 방법으로 증폭한 NK 세포가 암세포에 대하여, 우수한 세포 독성을 나타내는 것을 확인하였다(도 3 참조).In one aspect of the present invention, the efficacy of NK cells obtained by amplification by the method of the present invention as a cancer treatment agent was confirmed, and it was confirmed that the NK cells amplified by the method of the present invention exhibited excellent cytotoxicity against cancer cells. (See Figure 3).
본 발명의 다른 양태에서는, 본 발명의 방법으로 증폭하여 수득된 NK 세포의 활성도를 확인하기 위해 NK 세포 표면에서의 CD107a 발현과 세포내 염색을 통한 IFN-g 및 TNF-a의 분비량을 측정한 결과, 본 발명에 따른 방법으로 증폭한 NK 세포가 기존 방법으로 증폭한 NK 세포와 유사한 CD107a 및 IFN-g 발현특성을 나타내는 것을 확인하여, 본 발명에 따른 생산력이 높아진 NK 세포 배양방법으로 대량 생산된 NK 세포가 정상적으로 암세포에 대한 살상효능을 가지고 있는 것을 확인하였다(도 4 참조).In another aspect of the present invention, in order to confirm the activity of NK cells obtained by amplification by the method of the present invention, the expression of CD107a on the surface of NK cells and the secretion amount of IFN-g and TNF-a through intracellular staining were measured. , it was confirmed that NK cells amplified by the method according to the present invention exhibited CD107a and IFN-g expression characteristics similar to NK cells amplified by the existing method, and NK mass-produced by the NK cell culture method with increased productivity according to the present invention. It was confirmed that the cells normally have a killing effect on cancer cells (see Figure 4).
본 발명의 또 다른 양태에서는, 본 발명의 방법으로 증폭하여 수득된 NK 세포의 세포 표면 수용체의 표현형 특성을 확인하였으며, 본 발명에 따른 방법으로 증폭한 NK 세포에서 CD16, NKG2A, NKG2C, NKG2D, DNAM-1, NKp30, NKp46 및 CD8a와 같은 표면 마커가 발현되는 것을 확인하여, 본 발명에 따른 방법으로 증폭한 NK 세포가 정상적으로 완전한 기능적 특성을 가지는 것을 확인하였다(도 5 참조).In another aspect of the present invention, the phenotypic characteristics of cell surface receptors of NK cells obtained by amplification by the method of the present invention were confirmed, and CD16, NKG2A, NKG2C, NKG2D, and DNAM in NK cells amplified by the method of the present invention. Surface markers such as -1, NKp30, NKp46, and CD8a were confirmed to be expressed, confirming that NK cells amplified by the method according to the present invention normally have complete functional characteristics (see Figure 5).
본 명세서에서 용어 "자연살해세포(Natural Killer cells)" 또는 "NK 세포"는 선천성 면역계의 주요 성분을 구성하는 세포독성 림프구로서, 대형과립림프구(large granular lymphocyte, LGL)로 정의된다. 상기 "자연살해 세포" 또는 "NK 세포"는 임의의 조직 공급원(source)으로부터 유래된 추가적인 변형이 없는 자연살해세포를 포함하고, 성숙한 자연살해세포 뿐 아니라, 자연살해 전구세포를 포함할 수 있다. 상기 자연살해세포는 인터페론 또는 대식세포-유래 사이토카인에 대한 반응으로 활성화되고, 자연살해 세포는 "활성화 수용체" 및 "억제성 수용체"로 표지되는, 세포의 세포 독성 활성을 제어하는 2가지 유형의 표면 수용체를 포함한다. 자연살해세포는 임의의 공급원, 예를 들어 태반 조직, 태반 관류액, 제대혈, 태반혈, 말초혈, 지라, 간 등으로부터의 조혈세포, 예를 들어 조혈 줄기 또는 전구체로부터 생성될 수 있다.As used herein, the term "Natural Killer cells" or "NK cells" are cytotoxic lymphocytes that constitute a major component of the innate immune system and are defined as large granular lymphocytes (LGL). The “natural killer cells” or “NK cells” include natural killer cells without further modification derived from any tissue source, and may include natural killer progenitor cells as well as mature natural killer cells. The natural killer cells are activated in response to interferon or macrophage-derived cytokines, and natural killer cells are labeled with “activating receptors” and “inhibitory receptors,” which control the cytotoxic activity of the cells. Contains surface receptors. Natural killer cells can be produced from hematopoietic cells, such as hematopoietic stems or precursors, from any source, such as placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc.
일 구체예에 있어서, 상기 자연살해세포는 활성화된 자연살해세포 일 수 있다. 상기 활성화된 자연살해세포는 모세포, 예를 들면, 조혈 세포, 또는 자연살해전구세포에 비해, 세포 독성, 또는 자연살해세포의 본연의 면역조절능이 활성화된 세포를 의미할 수 있다.In one embodiment, the natural killer cells may be activated natural killer cells. The activated natural killer cells may refer to cells in which cytotoxicity or the natural immune regulatory ability of natural killer cells is activated compared to progenitor cells, for example, hematopoietic cells, or natural killer progenitor cells.
일 구체예에 있어서, 상기 NK 세포의 비-제한적 예에는 대식세포, B 림프구, T 림프구, 비만 세포, 단핵구, 수지상 세포, 호산구, 자연 살해 세포, 호염기구, 호중구가 포함된다. 따라서, 특정 구체예들에서, 상기 NK 세포는, 대식세포, B 림프구, T 림프구(CD8+ CTL), 비만 세포, 단핵구, 수지상 세포, 호산구, 자연살해세포, 호염기구, 또는 호중구로 이루어진 군으로부터 선택된 어느 하나인 것일 수 있다. 특정 구체예에서, NK 세포는 자연살해세포, NKT 세포 또는 T 림프구가 혼합된 형태로 증식될 수 있으며, 상기 혼합된 NK 세포는 대식세포, B 림프구, T 림프구, 비만 세포, 단핵구, 수지상 세포, 호산구, 자연살해세포, 호염기구, 및 호중구로 이루어진 군으로부터 선택된 하나 이상을 포함하는 것일 수 있다.In one embodiment, non-limiting examples of NK cells include macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils. Accordingly, in certain embodiments, the NK cells are selected from the group consisting of macrophages, B lymphocytes, T lymphocytes (CD8+ CTL), mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, or neutrophils. It could be any one. In certain embodiments, NK cells may be proliferated as a mixture of natural killer cells, NKT cells, or T lymphocytes, and the mixed NK cells include macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, It may include one or more selected from the group consisting of eosinophils, natural killer cells, basophils, and neutrophils.
구체예에 있어서, 활성화된 자연살해세포 또는 활성화된 자연살해세포가 풍부한(enriched) 집단은 1종 이상의 기능적으로 관련된 마커, 예를 들어 CD16, CD57, CD69, CD94, CD161, CD158a, CD158b, NKp30, NKp44, NKp46, DNAM-1, 2B4, NKp46, CD94, KIR(예를 들어, KIR2DL1, KIR2DL2/3, KIR3DL1), 및 활성화 수용체의 NKG2 패밀리(예를 들어, NKG2A, NKG2C, NKG2D)를 검출함으로써 평가될 수 있다.In an embodiment, activated natural killer cells or a population enriched in activated natural killer cells are selected from one or more functionally relevant markers, such as CD16, CD57, CD69, CD94, CD161, CD158a, CD158b, NKp30, Assessed by detecting NKp44, NKp46, DNAM-1, 2B4, NKp46, CD94, KIRs (e.g., KIR2DL1, KIR2DL2/3, KIR3DL1), and NKG2 family of activating receptors (e.g., NKG2A, NKG2C, NKG2D) It can be.
본 명세서에서 용어 "NK 세포의 자극"은 시험관 내 또는 생체 내에서 자연살해세포의 활성, 예를 들면, 세포독성 활성을 증가시키거나, 활성화된 자연살해세포가 생성, 증가, 증폭, 또는 증식되는 것을 의미할 수 있다.As used herein, the term "stimulation of NK cells" refers to increasing the activity of natural killer cells, for example, cytotoxic activity, in vitro or in vivo, or generating, increasing, amplifying, or proliferating activated natural killer cells. It can mean something.
본 발명의 일 양태에서, 일 양상은 막 결합성 인터루킨-18(membrane bound interleukin-18: mbIL-18) 및 막 결합성 인터루킨-21(membrane bound interleukin-21: mbIL-21)을 발현하도록 유전적으로 조작된 세포주를 피더 세포로 사용한다. In one aspect of the invention, one aspect is genetically modified to express membrane bound interleukin-18 (mbIL-18) and membrane bound interleukin-21 (mbIL-21). Engineered cell lines are used as feeder cells.
본 명세서에서 사용되는 용어, "피더 세포(feeder cells, 지지세포, 배양보조세포 또는 먹이세포라고도 한다)"는 방사선 조사에 의해 분열 증식하는 능력은 없지만 대사 활성이 있기 때문에 여러 가지 대사물질을 생산하여 목적 NK 세포의 증식을 돕는 세포를 의미할 수 있다. 본 명세서에서 사용될 수 있는 피더세포로는 유전자가 도입된 동물 세포주(cell line)로서, 인간만성골수백혈병세포주(예를 들어, K562 세포), RPMI8866, EBV_LCL, 721221, HFWT 등을 사용할 수 있으며, 본 발명의 일 양태에서는 K562 세포(ATCC CCL-243)를 사용하였다.As used herein, the term "feeder cells" (also called support cells, support cells, or feed cells) does not have the ability to divide and proliferate by irradiation, but is metabolically active and produces various metabolites. It may refer to cells that help the proliferation of target NK cells. Feeder cells that can be used in this specification include animal cell lines into which genes have been introduced, such as human chronic myeloid leukemia cell lines (e.g., K562 cells), RPMI8866, EBV_LCL, 721221, and HFWT. In one aspect of the invention, K562 cells (ATCC CCL-243) were used.
본 명세서에서 용어 "유전적 조작 (genetic engineering)" 또는 "유전적으로 조작된 (genetically engineered)"은 세포에 대하여 하나 이상의 유전적 변형 (genetic modification)을 도입하는 행위 또는 그에 의하여 만들어진 세포를 의미한다.As used herein, the term “genetic engineering” or “genetically engineered” refers to the act of introducing one or more genetic modifications into a cell or a cell created thereby.
상세하게는 상기 유전적으로 조작된 세포는 상기 언급된 유전자를 코딩하는 외인성 유전자 (exogenous gene)를 포함하는 것일 수 있다. 용어 "외인성 (exogenous)"은 언급된 분자 (referenced molecule) 또는 언급된 활성(referenced activity)이 숙주 세포로 도입된 것을 의미한다. 분자는 예를 들면, 숙주 염색체 내로의 삽입에 의하는 것과 같은 코딩 핵산 (encoding nucleic acid)의 숙주 유전 물질 내로의 도입 또는 플라스미드와 같은 비염색체 유전물질로서 도입될 수 있다. 코딩 핵산의 발현과 관련하여, 상기 용어 "외인성"은 상기 코딩 핵산이 개체 내로 발현 가능한 형태로 도입된 것을 나타낸다. 생합성 활성과 관련하여, 상기 용어 "외인성"은 숙주 모세포에 도입된 활성을 나타낸다. 그 기원 (source)은 예를 들면, 숙주 모세포에 도입된 후 언급된 활성을 발현하는 동질성 (homologous) 또는 이질성 (heterologous) 코딩 핵산일 수 있다. 그러므로, 용어 "내인성(endogenous)"은 상기 숙주 세포에 존재하는 언급된 분자 또는 활성을 나타낸다. 비슷하게, 코딩 핵산의 발현과 관련하여, 상기 용어 "내인성"은 개체 내에 포함된 코딩 핵산의 발현을 나타낸다. 용어 "이질성(heterologous)"은 언급된 종 외의 다른 기원으로부터의 분자 또는 활성을 나타내고 용어 "동질성(homologous)"은 숙주 모세포로부터의 분자 또는 활성을 나타낸다. 따라서, 코딩 핵산의 외인성 발현은 이질성(heterologous) 또는 동질성 (homologous) 코딩 핵산 중 어느 하나 또는 둘 다를 이용할 수 있다.In detail, the genetically engineered cell may contain an exogenous gene encoding the above-mentioned gene. The term “exogenous” means that the referenced molecule or referenced activity has been introduced into the host cell. The molecule may be introduced, for example, by introduction of an encoding nucleic acid into host genetic material, such as by insertion into a host chromosome, or as non-chromosomal genetic material, such as a plasmid. With regard to expression of a coding nucleic acid, the term “exogenous” refers to the introduction of the coding nucleic acid into an individual in an expressible form. With regard to biosynthetic activity, the term “exogenous” refers to an activity that has been introduced into the host mother cell. The source may be, for example, a homologous or heterologous encoding nucleic acid that, after introduction into the host mother cell, expresses the stated activity. Therefore, the term “endogenous” refers to the referenced molecule or activity present in the host cell. Similarly, with respect to expression of a coding nucleic acid, the term “endogenous” refers to expression of the coding nucleic acid contained within the individual. The term “heterologous” refers to a molecule or activity from an origin other than the species referenced and the term “homologous” refers to a molecule or activity from the host parent cell. Accordingly, exogenous expression of coding nucleic acids can utilize either heterologous or homologous coding nucleic acids, or both.
따라서 본 발명에서 유전적으로 조작된 피더 세포는 mbIL-18 및 mbIL-21을 암호화하는 핵산을 포함하는 것일 수 있다. 더욱 상세하게는 상기 세포는 mbIL-18 및 mbIL-21을 암호화하는 핵산을 포함하는 벡터로 형질 전환된 것일 수 있다.Therefore, the genetically engineered feeder cells in the present invention may contain nucleic acids encoding mbIL-18 and mbIL-21. More specifically, the cells may have been transformed with a vector containing nucleic acids encoding mbIL-18 and mbIL-21.
본 명세서에서 사용되는 용어, "벡터"는 적당한 숙주세포에서 목적 단백질을 발현할 수 있는 벡터로서, 유전자 삽입물이 발현되도록 작동 가능하게 연결된 조절 요소를 포함하는 유전자 작제물을 지칭한다. 일 실시예에 따른 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널, 및/또는 인핸서와 같은 발현 조절 요소를 포함할 수 있으며, 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 또한, 상기 벡터는, 숙주 세포 내에서 안정적으로 상기 융합 단백질을 발현시킬 수 있는, 발현용 벡터일 수 있다. 상기 발현용 벡터는 당업계에서 식물, 동물 또는 미생물에서 외래의 단백질을 발현하는 데 사용되는 통상의 것을 사용할 수 있다. 상기 재조합 벡터는 당업계에 공지된 다양한 방법을 통해 구축될 수 있다. 예를 들어, 상기 벡터는 벡터를 함유 하는 숙주세포를 선택하기 위한 선택성 마커를 포함하고, 복제 가능한 벡터인 경우, 복제 기원을 포함할 수 있다. 또한, 벡터는 자가 복제하거나 숙주 DNA에 도입될 수 있으며, 상기 벡터는 플라스미드, 렌티바이러스, 아데노바이러스, 아데노-관련 바이러스, 레트로바이러스, 헤르페스 심플렉스 바이러스, 및 배시니아 바이러스로 구성되는 군으로 부터 선택되는 것일 수 있다.As used herein, the term “vector” refers to a vector capable of expressing a protein of interest in a suitable host cell, and refers to a genetic construct containing regulatory elements operably linked to express the gene insert. A vector according to one embodiment may include expression control elements such as a promoter, operator, start codon, stop codon, polyadenylation signal, and/or enhancer, and the promoter of the vector may be constitutive or inducible. Additionally, the vector may be an expression vector that can stably express the fusion protein in a host cell. The expression vector may be a conventional vector used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector can be constructed through various methods known in the art. For example, the vector may include a selectable marker for selecting host cells containing the vector, and if the vector is replicable, it may include an origin of replication. Additionally, the vector may self-replicate or be introduced into host DNA, and the vector may be selected from the group consisting of plasmids, lentiviruses, adenoviruses, adeno-associated viruses, retroviruses, herpes simplex viruses, and vaccinia viruses. It may be possible.
또한, 상기 벡터에서, 전술한 융합 단백질을 암호화하는 폴리뉴클레오티드 서열은 프로모터에 작동 가능하게 연결되어 있을 수 있다. 본 명세서에서 사용된 용어, "작동 가능하게 연결된"은 핵산 발현 조절 서열(예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 번역을 조절하게 된다.Additionally, in the vector, the polynucleotide sequence encoding the above-described fusion protein may be operably linked to a promoter. As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional factor binding sites) and another nucleic acid sequence, thereby The regulatory sequences regulate transcription and/or translation of the other nucleic acid sequences.
본 명세서에서 "막 결합성 인터루킨(membrane bound interleukin)"은 세포막에 결합된 인터루킨을 의미하는 것으로, 세포 외로 인터루킨을 분비하는 것과는 구분되는 의미일 수 있다.As used herein, “membrane bound interleukin” refers to interleukin bound to the cell membrane, and may have a distinct meaning from secreting interleukin outside the cell.
mbIL-18 염기서열은 NheI-Kozak-GM-CSF signal + IL-18 + Modified IgG4 Hinge + IgG4 Fc + CD4 TM + T2A 순으로 구성되어 있고 구체적인 염기 서열은 서열번호 1에 나타내었다. The mbIL-18 base sequence consists of the following order: NheI-Kozak-GM-CSF signal + IL-18 + Modified IgG4 Hinge + IgG4 Fc + CD4 TM + T2A, and the specific base sequence is shown in SEQ ID NO: 1.
mbIL-18 염기서열은 상기 서열번호 1의 핵산 서열 또는 그의 아미노산 서열과 약 70% 이상, 약 75% 이상, 약 80% 이상, 약 85%이상, 약 90% 이상, 약 92% 이상, 약 95% 이상, 약 97% 이상, 약 98% 이상, 또는 약 99% 이상의 서열 상동성을 갖는 것일 수 있다.The mbIL-18 base sequence is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more of the nucleic acid sequence of SEQ ID NO: 1 or its amino acid sequence. It may have sequence homology of at least %, at least about 97%, at least about 98%, or at least about 99%.
mbIL-21의 서열은 GM-CSF signal + IL-21 + Modified IgG4 Hinge + IgG4 Fc + CD4 TM + NotI 구성되어 있고 구체적인 염기 서열은 서열번호 2에 나타내었다. The sequence of mbIL-21 consists of GM-CSF signal + IL-21 + Modified IgG4 Hinge + IgG4 Fc + CD4 TM + NotI, and the specific base sequence is shown in SEQ ID NO: 2.
상기 서열번호 2의 핵산 서열 또는 그의 아미노산 서열과 약 70% 이상, 약 75% 이상, 약 80% 이상, 약 85%이상, 약 90% 이상, 약 92% 이상, 약 95% 이상, 약 97% 이상, 약 98% 이상, 또는 약 99% 이상의 서열 상동성을 갖는 것일 수 있다.The nucleic acid sequence of SEQ ID NO: 2 or its amino acid sequence and about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more It may have sequence homology of at least about 98%, or at least about 99%.
유전자 조작된 배양보조세포 k562-mbIL18/IL21의 최종 구조와 mbIL-18 염기서열 및 mbIL-21 염기서열의 구성은 다음과 같으며, 서열번호 3에 나타내었다: NheI-Kozak-GM-CSF signal + IL-18 + Modified IgG4 Hinge + IgG4 Fc + CD4 TM + T2A (No STOP) + GM-CSF signal + IL-21 + Modified IgG4 Hinge + IgG4 Fc + CD4 TM + NotI The final structure of the genetically engineered culture feeder cells k562-mbIL18/IL21 and the composition of the mbIL-18 base sequence and mbIL-21 base sequence are as follows and are shown in SEQ ID NO: 3: NheI-Kozak-GM-CSF signal + IL-18 + Modified IgG4 Hinge + IgG4 Fc + CD4 TM + T2A (No STOP) + GM-CSF signal + IL-21 + Modified IgG4 Hinge + IgG4 Fc + CD4 TM + NotI
일 구체예에 있어서, 상기 공배양은 사이토카인의 존재 하에서 이루어지는 것일 수 있다.In one embodiment, the co-culture may be performed in the presence of cytokines.
일 구체예에 있어서, 상기 사이토카인은 인터루킨 또는 이의 돌연변이이다. 다수의 인터루킨들은 보조 CD4 T 림프구들, 뿐만 아니라 단핵구, 대식세포, 및 내피 세포에 의해 합성된다. 인터루킨들은 T 및 B 림프구들 및 조혈세포들의 발달 및 분화를 촉진시킬 수 있다. 인터루킨들의 비-제한적 예들에는 IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, 또는 IL36이 포함된다. 그러므로, 특정 구체예들에서, 상기 사이토카인은 IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11,IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28,IL29, IL30, IL31, IL32, IL33, IL35, 또는 IL36의 야생형 및 돌연변이 형태들을 비롯한 (그러나 이에 제한되는 것은 아니다) 인터루킨 또는 이의 돌연변이이다.In one embodiment, the cytokine is interleukin or a mutant thereof. Many interleukins are synthesized by helper CD4 T lymphocytes, as well as monocytes, macrophages, and endothelial cells. Interleukins can promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Non-limiting examples of interleukins include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL36. Therefore, in certain embodiments, the cytokines include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18 , including but not limited to wild-type and mutant forms of IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL36. Interleukin or its mutation.
다른 구체예에 있어서, 상기 공배양에 첨가되는 사이토카인은 IL-2일 수 있다. 더욱 상세하게는, 상기 IL-2는 1 ~ 200 U/㎖, 바람직하게는 1 ~ 100 U/㎖, 더욱 바람직하게는 1 ~ 50 U/㎖, 가장 바람직하게는 1 ~ 20 U/㎖의 농도로 사용될 수 있다. In another embodiment, the cytokine added to the co-culture may be IL-2. More specifically, the concentration of IL-2 is 1 to 200 U/ml, preferably 1 to 100 U/ml, more preferably 1 to 50 U/ml, and most preferably 1 to 20 U/ml. It can be used as
다른 구체예에 있어서, 상기 재자극에 첨가되는 사이토카인은 IL-2일 수 있다. 더욱 상세하게는, 상기 IL-2는 10 ~ 500 U/㎖ , 바람직하게는 10 ~ 300 U/㎖, 더욱 바람직하게는 10 ~ 250 U/㎖의 농도로 사용될 수 있다. In another embodiment, the cytokine added for restimulation may be IL-2. More specifically, the IL-2 can be used at a concentration of 10 to 500 U/ml, preferably 10 to 300 U/ml, and more preferably 10 to 250 U/ml.
일 구체예에 있어서, 상기 공배양은 2일 ~ 30일 동안 수행되는 것일 수 있으며, 바람직하게는 3~24일 동안 수행될 수 있으며, 더욱 바람직하게는 5~21일 동안 수행될 수 있다.In one embodiment, the co-culture may be performed for 2 to 30 days, preferably for 3 to 24 days, and more preferably for 5 to 21 days.
본 발명에 있어서, 상기 (a) 단계는 배양 및 (b) 단계의 배양은 상등액의 글루코스 농도가 50~100mg/dL 이하인 경우, 배지를 교환하는 것을 특징으로 할 수 있다.In the present invention, the culture in step (a) and the culture in step (b) may be characterized by changing the medium when the glucose concentration of the supernatant is 50 to 100 mg/dL or less.
본 발명에 있어서, 상기 배양은 NK MACS Supplements 및 인간 AB 혈청이 포함된 NK MACS 배지인 것을 특징으로 할 수 있다. In the present invention, the culture may be characterized as NK MACS medium containing NK MACS Supplements and human AB serum.
본 발명에 있어, 상기 단핵세포 또는 NK 세포는 1 x 104 ~ 3 x 106 세포/cm2 밀도로 포함할 수 있으며, 1 x 105 ~ 1 x 106 세포/cm2 또는 1 x 105 ~ 1.5 x 105 세포/cm2,로 포함할 수 있다. In the present invention, the mononuclear cells or NK cells may be included at a density of 1 x 10 4 to 3 x 10 6 cells/cm 2 , and 1 x 10 5 to 1 x 10 6 cells/cm 2 or 1 x 10 5 It can contain ~1.5 x 10 5 cells/cm 2 .
본 발명에 있어서, 상기 (b) 단계의 배양은 5~7일 간격으로 계대배양을 실시하며, 계대배양은 세포 밀도 5.0 x 104 ~ 4.0 x 107 cells/cm2이 되도록 희석하여 수행할 수 있다.In the present invention, the culture in step (b) is subcultured at intervals of 5 to 7 days, and subculture can be performed by diluting the cells to a cell density of 5.0 x 10 4 to 4.0 x 10 7 cells/cm 2 . there is.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시 예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be obvious to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
실시예 1. 세포의 준비Example 1. Preparation of cells
1-1. 말초혈액 유래 단핵세포(Peripheral blood-derived mononuclear cells, PBMC)의 준비1-1. Preparation of peripheral blood-derived mononuclear cells (PBMC)
말초혈액 유래 단핵세포는 StemCell Technologies (Vancouver, BC, Canada)에서 구매하여 사용하였다. 말초혈액 유래 단핵세포를 해동하여 선행문헌(대한민국 공개특허 10-2021-0152934)에 기술된 10% (v/v) 인간 AB 혈청이 포함된 DS (DMEM + Supplements) 배지에 현탁하였고, LUNA-FX7 자동세포계수기(Aligned Genetics, Korea)를 이용하여 세포 수를 측정하였다.Peripheral blood-derived mononuclear cells were purchased from StemCell Technologies (Vancouver, BC, Canada). Peripheral blood-derived mononuclear cells were thawed and suspended in DS (DMEM + Supplements) medium containing 10% (v/v) human AB serum as described in the prior literature (Korea Patent Publication No. 10-2021-0152934), and LUNA-FX7 Cell numbers were measured using an automatic cell counter (Aligned Genetics, Korea).
1-2. 제대혈 유래 단핵세포(Umbilical cord blood-derived mononuclear cells, UCBMC)의 준비1-2. Preparation of umbilical cord blood-derived mononuclear cells (UCBMC)
건강한 공여자로부터 채취한 제대혈(삼성서울병원)에 2% (v/v) FBS (Fetal bovine serum, ThermoFisher, USA)가 포함된 DPBS(Dulbecco's phosphate buffered saline, WELGENE)를 1:1 비율로 추가하였다. 그리고 Lymphoprep (Serumwerk, Bernburg AG, Oslo, Norway) 밀도구배 원심분리 방법(Density gradient centrifugation)을 이용하여 제대혈 유래 단핵세포(UCBMC)를 분리한 후 배양방법에 따라 DS 배지 또는 1% (v/v) NK MACS Supplements, 10% (v/v) 인간 AB 혈청(Human AB serum, sigma, USA)이 포함된 NK MACS 배지(MiltenyiBiotec, Bergisch Gladbach, FRG)에 현탁하였고, LUNA-FX7 자동세포계수기(Aligned Genetics, Korea)를 이용하여 세포 수를 측정하였다.DPBS (Dulbecco's phosphate buffered saline, WELGENE) containing 2% (v/v) FBS (Fetal bovine serum, ThermoFisher, USA) was added to umbilical cord blood (Samsung Seoul Hospital) collected from a healthy donor at a 1:1 ratio. Then, umbilical cord blood-derived mononuclear cells (UCBMC) were isolated using density gradient centrifugation from Lymphoprep (Serumwerk, Bernburg AG, Oslo, Norway) and then cultured in DS medium or 1% (v/v) depending on the culture method. NK MACS Supplements were suspended in NK MACS medium (MiltenyiBiotec, Bergisch Gladbach, FRG) containing 10% (v/v) human AB serum (Sigma, USA), and analyzed using a LUNA-FX7 automated cell counter (Aligned Genetics). , Korea) was used to measure the number of cells.
자연살해세포(NK cell)와 유전적으로 조작된 피더세포의 1:5 공배양 비율에 따라 분주할 피더세포 수를 결정하고자 제대혈 유래 단핵세포에서의 자연살해세포 세포 비율을 확인하였다. 구체적으로, 제대혈 유래 단핵세포 2.0 x 105개를 회수하여 DPBS로 세척한 후 Fixable Viability Stain 780 (BD Biosciences, San Jose, CA)로 15분 동안 염색하였다. 그 후, 2% (v/v) FBS가 포함된 DPBS 버퍼로 세척한 후 항-인간(Anti-human) CD3-FITC, 항-인간 CD56-APC (BD Biosciences, San Jose, CA)로 4℃에서 30분 동안 추가 염색하였다. 상기 세포를 다시 2% (v/v) FBS가 포함된 DPBS로 세척한 후 CytoFLEX (Beckman Coulter, Brea, CA)를 이용하여 데이터를 획득하고 FlowJo 소프트웨어(Tree Star, Ashland, OR)로 분석하였다.To determine the number of feeder cells to be divided according to the 1:5 co-culture ratio of natural killer cells (NK cells) and genetically engineered feeder cells, the cell ratio of natural killer cells in umbilical cord blood-derived mononuclear cells was confirmed. Specifically, 2.0 Afterwards, washed with DPBS buffer containing 2% (v/v) FBS and incubated with anti-human CD3-FITC and anti-human CD56-APC (BD Biosciences, San Jose, CA) at 4°C. Additional staining was performed for 30 minutes. The cells were again washed with DPBS containing 2% (v/v) FBS, and data were acquired using CytoFLEX (Beckman Coulter, Brea, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR).
1-3. 유전적으로 조작된 피더세포(feeder cell)의 준비1-3. Preparation of genetically engineered feeder cells
막 결합성 인터루킨-18(membrane bound interleukin-18: mbIL-18) 및 막 결합성 인터루킨-21(membrane bound interleukin-21: mbIL-21)을 발현하는 피더세포를 선행문헌(대한민국 공개특허 10-2021-0152934)에 따라 제조하였다. Feeder cells expressing membrane bound interleukin-18 (mbIL-18) and membrane bound interleukin-21 (mbIL-21) were described in prior literature (Korean Patent Publication No. 10-2021). -0152934).
구체적으로, 인간 유래 mbIL18 유전자 및 mbIL21 유전자를 렌티바이러스 벡터인 pCDHCMV-RFP(System Biosciences, Palo Alto, CA, USA)에 클로닝하여 재조합 렌티바이러스 생산용 벡터를 제작하였다. 이후, 바이러스 생산을 위해 상기 제작된 재조합 유전자 (pCDH-CMV-RFP-mbIL-1821)를 293FT 세포(ThermoFisher Scientific, USA)에 패키징 벡터와 함께 lipofectamin3000(Invitrogen)을 이용하여 형질주입시켰다. 24 시간 후, 새로운 배지로 교환하여 48 시간 동안 세포를 배양한 후, 바이러스가 함유된 배지를 회수하였다. 회수된 배지는 500 g에서 10 분간 원심분리하고, 0.45㎛ 필터를 이용하여 바이러스가 함유된 순수한 배지만을 분리하여 mbIL18-mbIL21 발현 렌티바이러스를 생산하였다. Specifically, the human-derived mbIL18 gene and mbIL21 gene were cloned into the lentiviral vector pCDHCMV-RFP (System Biosciences, Palo Alto, CA, USA) to construct a vector for recombinant lentivirus production. Then, for virus production, the constructed recombinant gene (pCDH-CMV-RFP-mbIL-1821) was transfected into 293FT cells (ThermoFisher Scientific, USA) using lipofectamin3000 (Invitrogen) along with a packaging vector. After 24 hours, the medium was replaced with new medium and the cells were cultured for 48 hours, and then the medium containing the virus was recovered. The recovered medium was centrifuged at 500 g for 10 minutes, and only the pure medium containing the virus was separated using a 0.45㎛ filter to produce mbIL18-mbIL21 expressing lentivirus.
이후, K562 세포(American Type Culture Collection, ATCC number; CCL-243)를 포함하는 배지 9 ㎖에 mbIL18-mbIL21 발현 렌티바이러스 1 ㎖를 녹여 폴리브렌(polybrene)(8 ㎍/ml)과 함께 첨가하여, 48 시간 동안 세포 배양을 진행하였다. 다음으로, 감염된 세포만 선별하기 위해서, 초고속 유세포자동분리기를 이용하여 적색 형광을 발현하는 세포만 선별하였고, mbIL18-mbIL21의 발현 여부를 유세포분석기 (FACS)를 통해 분석하여, mbIL18-mbIL21이 발현된 K562 세포주(이하, "K562-mbIL18-mbIL21")를 제조하였다. Afterwards, 1 ml of mbIL18-mbIL21 expressing lentivirus was dissolved in 9 ml of medium containing K562 cells (American Type Culture Collection , ATCC number; CCL-243) and added with polybrene (8 μg/ml). Cell culture was performed for 48 hours. Next, in order to select only infected cells, only cells expressing red fluorescence were selected using an ultra-high-speed flow cytometer, and the expression of mbIL18-mbIL21 was analyzed using flow cytometry (FACS) to determine whether mbIL18-mbIL21 was expressed. K562 cell line (hereinafter referred to as “K562-mbIL18-mbIL21”) was prepared.
K562-mbIL-18/21 세포주는 적절한 크기의 T-플라스크에 10% (v/v) FBS가 포함된 RPMI1640 (Gibco, USA) 배지를 넣고 37℃, 5% CO2 인큐베이터에서 배양하였다. 그리고 배양 플라스크에서 회수하여 400 g에서 3분간 원심분리를 진행하고, 10% (v/v) FBS가 포함된 RPMI1640 배지에 현탁시킨 후 Gammacell 3000 Elan 감마선 조사기에서 100 Gy로 조사하여 불활성화하였다. 상기 세포는 다시 400 g에서 3분간 원심분리하고, 배양방법에 따라 DS 배지 또는 1% (v/v) NK MACS Supplements, 10% (v/v) 인간 AB 혈청이 포함된 NK MACS 배지에 현탁하여 준비하였다.The K562-mbIL-18/21 cell line was cultured in a T-flask of an appropriate size in RPMI1640 (Gibco, USA) medium containing 10% (v/v) FBS in an incubator at 37°C and 5% CO 2 . Then, it was recovered from the culture flask, centrifuged at 400 g for 3 minutes, suspended in RPMI1640 medium containing 10% (v/v) FBS, and then inactivated by irradiation with 100 Gy in a Gammacell 3000 Elan gamma irradiator. The cells were centrifuged again at 400 g for 3 minutes and suspended in DS medium or NK MACS medium containing 1% (v/v) NK MACS Supplements and 10% (v/v) human AB serum, depending on the culture method. Ready.
실시예 2. 자연살해세포의 배양Example 2. Culture of natural killer cells
2-1. 말초혈액 유래 단핵세포, 제대혈 유래 단핵세포 및 피더세포를 이용한 자연살해세포 배양(기존 방법) 2-1. Natural killer cell culture using peripheral blood-derived mononuclear cells, umbilical cord blood-derived mononuclear cells, and feeder cells (existing method)
선행문헌(대한민국 공개특허 10-2021-0152934)에 기술된 바와 같이 진행하였다. It was carried out as described in a prior document (Korean Patent Publication No. 10-2021-0152934).
구체적으로, 배양 0일 차에는 말초혈액 유래 단핵구 또는 제대혈 유래 단핵구 각각 3 x 106 개와 100 Gy로 조사된 피더세포 0.5 x 106 개(단핵구: 피더세포 비율=6:1)를 1 mL DS 배지가 포함된 24 웰 플레이트 상에 접종한 후, 20 U/mL IL-2(Peprotech, Rocky Hill, NJ)가 포함된 1 mL DS 배지를 추가하여 총 배지 부피는 2 mL/well, IL-2의 최종 농도는 10 U/mL로 한 뒤, 37℃, 5% CO2 인큐베이터에서 3일 간 배양하였다. 배양 3일차 및 5일차에 각각 배양 배지를 절반 덜어내고, 20 U/mL IL-2가 포함된 1 mL DS 배지를 넣어 배지 절반 교체를 하였다.Specifically, on day 0 of culture, 3 After inoculation on a 24-well plate containing , 1 mL DS medium containing 20 U/mL IL-2 (Peprotech, Rocky Hill, NJ) was added to bring the total medium volume to 2 mL/well, of IL-2. The final concentration was set to 10 U/mL, and then cultured for 3 days in an incubator at 37°C and 5% CO 2 . On the 3rd and 5th days of culture, half of the culture medium was removed, and half of the medium was replaced by adding 1 mL DS medium containing 20 U/mL IL-2.
배양 7일 차에 1개 웰에 있는 세포를 4개의 웰로 나누어 계대배양 하면서 피더세포 5 Х105 개를 넣어 재자극하였다. 이때 배지는 최종 농도 100 U/mL IL-2, 5 ng/mL IL-15가 되도록 교체하였다.On the 7th day of culture, the cells in one well were divided into 4 wells and subcultured, and 5 Х10 5 feeder cells were added to restimulate them. At this time, the medium was changed to a final concentration of 100 U/mL IL-2 and 5 ng/mL IL-15.
이후 배양 2~3일 간격으로 1개 웰에 있는 세포를 2개의 웰로 나누어 계대배양 하면서 21일간 배양하였다.Afterwards, at intervals of 2 to 3 days, cells in one well were divided into two wells and subcultured for 21 days.
2-2. 제대혈 유래 단핵세포 및 피더세포를 이용한 자연살해세포 배양(본 발명의 방법)2-2. Natural killer cell culture using umbilical cord blood-derived mononuclear cells and feeder cells (method of the present invention)
실시예 1-1과 1-2의 방법으로 각각 준비한 말초혈액 유래 단핵세포 및 제대혈 유래 단핵세포로부터 NK 세포를 수득하기 위해 300 g에서 3분간 원심분리를 진행하고, 2% (v/v) FBS와 1 mM EDTA가 포함된 EasySep buffer에 현탁하였다. LUNA-FX7 자동 세포계수기를 이용하여 세포 수를 측정한 후 5.0 x 107 cells/mL이 되도록 농도를 맞추고, EasySep™ Human NK Cell Isolation Cocktail을 상기 세포 현탁액 1 mL 당 50 ㅅL 농도로 첨가하여 상온에서 5분 간 반응하였다. 다음으로, EasySep™ Dextran RapidSpheres™을 상기 세포 현탁액 1 mL 당 50 ㅅL 농도로 첨가한 후 EasySep buffer를 이용하여 총 2.5 mL의 부피가 되도록 맞추고 EasySep™ Magnet에 장착하고 상온에서 3분 간 반응하였다. 이후 상등액만 새로운 튜브에 흘려주어 NK 세포를 수득하였다.To obtain NK cells from peripheral blood-derived mononuclear cells and umbilical cord blood-derived mononuclear cells prepared by the methods of Examples 1-1 and 1-2, centrifugation was performed at 300 g for 3 minutes, and 2% (v/v) FBS was added. and was suspended in EasySep buffer containing 1mM EDTA. After measuring the number of cells using the LUNA-FX7 automatic cell counter, adjust the concentration to 5.0 reacted for 5 minutes. Next, EasySep™ Dextran RapidSpheres™ was added at a concentration of 50 ㅅL per 1 mL of the cell suspension, then adjusted to a total volume of 2.5 mL using EasySep buffer, mounted on EasySep™ Magnet, and reacted at room temperature for 3 minutes. Afterwards, only the supernatant was flowed into a new tube to obtain NK cells.
분리한 NK 세포 수의 측정은 상기 내용과 동일하게 세포 계수기 및 유세포분석 등의 방법을 사용하여 측정할 수 있다. 구체적으로, LUNA-FX7 자동 세포 계수기를 이용하여 세포 수를 측정하였고, 실시예 1-2 설명에 따라 분리한 NK 세포 2.0 x 105개를 회수하여 최종 NK 세포 순도를 산출하였다.The number of separated NK cells can be measured using methods such as cell counter and flow cytometry in the same manner as described above. Specifically, the number of cells was measured using a LUNA-FX7 automatic cell counter, and 2.0 x 10 5 NK cells separated according to the description in Example 1-2 were recovered to calculate the final NK cell purity.
배양 0일 차, G-Rex24 배양 용기(Wilson Wolf, Saint Paul, MN)에 분리한 NK 세포를 5.0 x 104 cells/cm2이 되도록 분주하였다.On day 0 of culture, the isolated NK cells were dispensed into G-Rex24 culture vessels (Wilson Wolf, Saint Paul, MN) at 5.0 x 10 4 cells/cm 2 .
자연살해세포 수 : 유전적으로 조작된 피더세포의 비율을 각각 1:1, 1:2, 1:5, 1:10 및 1:20의 비율로 분주하여 공배양을 실시하였다. 그리고 200 U/mL IL-2, 10 ng/mL IL-15, 1% (v/v) NK MACS Supplements, 10% (v/v) 인간 AB 혈청이 (Sigma-Aldrich) 포함된 NK MACS 배지(Miltenyi Biotec)를 총 8 mL까지 첨가한 후 37℃, 5% CO2 인큐베이터에서 7일 간 배양하였다.Co-culture was performed by distributing the number of natural killer cells: genetically engineered feeder cells at a ratio of 1:1, 1:2, 1:5, 1:10, and 1:20, respectively. and NK MACS medium (Sigma-Aldrich) containing 200 U/mL IL-2, 10 ng/mL IL-15, 1% (v/v) NK MACS Supplements, and 10% (v/v) human AB serum (Sigma-Aldrich). Miltenyi Biotec) was added up to a total of 8 mL and cultured in an incubator at 37°C and 5% CO 2 for 7 days.
7일 배양 기간동안 3~5일 간격으로 약 20 ㅅL의 배양 상등액을 수득한 후 CareSens N Voice 혈당측정기(i-Sens, Korea)를 이용하여 글루코스(Glucose) 농도를 측정하였다. 글루코스 농도가 50 ~ 100 mg/dL 이하인 경우 배양 상등액 4 mL을 조심스럽게 제거하고 그 위에 200 U/mL IL-2, 10 ng/mL IL-15, 1 v/v% NK MACS Supplements, 10% (v/v) 인간 AB 혈청이 포함된 NK MACS 배지 4 mL을 넣어 총 배지 양의 절반을 교체하였다.During the 7-day culture period, approximately 20 ㅅL of culture supernatant was obtained at intervals of 3 to 5 days, and then the glucose concentration was measured using a CareSens N Voice blood glucose meter (i-Sens, Korea). If the glucose concentration is below 50 to 100 mg/dL, carefully remove 4 mL of culture supernatant and add 200 U/mL IL-2, 10 ng/mL IL-15, 1 v/v% NK MACS Supplements, 10% ( v/v) 4 mL of NK MACS medium containing human AB serum was added to replace half of the total amount of medium.
배양 7일 차에 유전적으로 조작된 피더세포 재자극을 위하여 G-Rex24 배양 용기의 배양 상등액 절반을 조심스럽게 제거한 후 LUNA-FX7 자동 세포계수기를 이용하여 세포 수를 측정하였고, 실시예 1-2 설명에 따라 배양 세포 2.0 x 105개를 회수하여 자연살해세포 순도를 확인하였다. 총 세포 수를 기준으로 세포 밀도는 5.0 x 104 ~ 4.0 x 107 cells/cm2 이 되도록 유지하고, 그 위에 실시예 1-3 에 따라 준비한 유전적으로 조작된 피더세포를 배양 중인 자연살해세포 수 대비 1:1, 1:2, 1:5, 1:10 및 1:20의 비율로 분주하여 재자극하였다. 그리고 200 U/mL IL-2, 10 ng/mL IL-15, 1% (v/v) NK MACS Supplements, 10% (v/v) 인간 AB 혈청이 포함된 NK MACS 배지를 총 8 mL까지 첨가한 후 배양하였다.To restimulate the genetically engineered feeder cells on the 7th day of culture, half of the culture supernatant from the G-Rex24 culture vessel was carefully removed, and the number of cells was measured using a LUNA-FX7 automatic cell counter. Example 1-2 Description According to this, 2.0 x 10 5 cultured cells were recovered and the purity of natural killer cells was confirmed. Based on the total number of cells , the cell density was maintained at 5.0 Restimulation was performed at ratios of 1:1, 1:2, 1:5, 1:10, and 1:20. Then add NK MACS medium containing 200 U/mL IL-2, 10 ng/mL IL-15, 1% (v/v) NK MACS Supplements, and 10% (v/v) human AB serum up to a total of 8 mL. and then cultured.
21일 차까지의 배양 기간동안 3~5일 간격으로 글루코스 농도를 측정하여 적절한 시기에 배양 배지를 절반씩 교체하였고, 또한, 5~7일 간격으로 세포 수를 측정한 후 계대 배양 시기를 결정하여 총 세포 수를 기준으로 세포 밀도 5.0 x 104 ~ 4.0 x 107 cells/cm2이 되도록 200 U/mL IL-2, 10 ng/mL IL-15, 1% (v/v) NK MACS Supplements, 10% (v/v) 인간 AB 혈청이 포함된 NK MACS 배지로 희석하여 배양하였다.During the culture period until the 21st day, the glucose concentration was measured at intervals of 3 to 5 days, and half of the culture medium was replaced at an appropriate time. Additionally, the number of cells was measured at intervals of 5 to 7 days, and the subculture period was determined. 200 U/ mL IL- 2 , 10 ng/mL IL-15, 1% (v/v) NK MACS Supplements, to a cell density of 5.0 The cells were diluted with NK MACS medium containing 10% (v/v) human AB serum and cultured.
배양 21일 차에, 전술한 바와 같이 LUNA-FX7 자동 세포계수기를 이용하여 배양된 세포 수를 측정하였고, Fixable Viability Stain 780, 항-인간 CD3-FITC, 항-인간 CD56-APC를 이용하여 자연살해세포의 순도 및 증폭률을 평가하였다.On the 21st day of culture, the number of cultured cells was measured using a LUNA-FX7 automatic cell counter as described above, and natural killing was performed using Fixable Viability Stain 780, anti-human CD3-FITC, and anti-human CD56-APC. The purity and amplification rate of cells were evaluated.
그 결과, 도 2에 나타난 바와 같이, 제대혈 유래 자연살해세포 수 : 유전적으로 조작된 피더세포의 비율을 1:5로 하였을 때, 단기간(21일) 배양으로 약 100,000배의 NK 세포 증식율을 확인하였으며, 기존 배양방법(약 1,800배)에 비하여, 50 배 이상의 NK 세포 확보가 가능한 것을 확인하였다. As a result, as shown in Figure 2, when the ratio of the number of natural killer cells derived from umbilical cord blood: genetically engineered feeder cells was 1:5, an NK cell proliferation rate of approximately 100,000 times was confirmed in short-term (21 days) culture. , It was confirmed that it was possible to secure more than 50 times more NK cells than the existing culture method (about 1,800 times).
실시예 3. 증폭된 NK 세포의 암세포 독성 확인Example 3. Confirmation of cancer cell toxicity of amplified NK cells
실시예 2-2의 방법으로 증폭하여 수득된 NK 세포의 암 치료제로서의 효능을 확인하기 위하여 표적 세포에 대한 NK 세포의 14일째 세포독성을 CFSE-기반 분석에 의해 4시간 동안 측정하였다. To confirm the efficacy of NK cells obtained by amplification by the method of Example 2-2 as a cancer treatment agent, the cytotoxicity of NK cells against target cells on day 14 was measured by CFSE-based analysis for 4 hours.
구체적으로, 냉동보관된 14일째까지 증폭시킨 NK 세포를 RPMI 1640 complete 배지(Gibco-BRL)에 해동 후 48시간 동안 싸이토카인 IL-2 (200U), IL-15 (10ng/mL)이 첨가된 RPMI1640 complete 배지에서 회복을 진행하였다. 이후 표적 세포 (K562, Raji)를 FACS 버퍼에서 10분 동안 0.5μM CFSE로 염색하고, RPMI1640 배지로 2회 세척하였다. NK 세포와 표적세포 계수 후 1 Х 104 개의 표적 세포를 96-웰 둥근 바닥 플레이트에 정상인 말초혈액 유래 증폭된 NK세포와 제대혈 유래 증폭된 NK 세포 effector-to-target (E:T)의 비율 1:1 및 2:1이 되도록 혼합하였다. 플레이트를 1,500 rpm에서 2분 동안 원심분리한 후 37℃, 5% CO2 배양기에서 4시간 동안 공배양하였다. 이후, 혼합된 세포를 FACS 튜브로 옮기고 각각의 튜브에 1 ㎕의 1 mg/mL PI(propidium iodide) (Sigma Aldrich, St. Louis, MO, USA)를 첨가하였다. 이후, FACS lyric에서 결과를 획득하고, Kaluza 소프트웨어(Beckman Coulter) 를 사용하여 분석하였다.Specifically, NK cells expanded until the 14th day of frozen storage were thawed in RPMI 1640 complete medium (Gibco-BRL) and then incubated in RPMI1640 complete with the addition of cytokines IL-2 (200U) and IL-15 (10ng/mL) for 48 hours. Recovery was carried out in medium. Afterwards, target cells (K562, Raji) were stained with 0.5 μM CFSE in FACS buffer for 10 minutes and washed twice with RPMI1640 medium. After counting NK cells and target cells, 1 Х 10 4 target cells were placed in a 96-well round bottom plate. Ratio of amplified NK cells derived from normal peripheral blood and amplified NK cells derived from umbilical cord blood effector-to-target (E:T) 1 :1 and 2:1 were mixed. The plate was centrifuged at 1,500 rpm for 2 minutes and then co-cultured at 37°C in a 5% CO 2 incubator for 4 hours. Afterwards, the mixed cells were transferred to a FACS tube, and 1 μl of 1 mg/mL PI (propidium iodide) (Sigma Aldrich, St. Louis, MO, USA) was added to each tube. Afterwards, the results were obtained from FACS lyric and analyzed using Kaluza software (Beckman Coulter).
그 결과, 도 3에 나타낸 바와 같이, 말초혈액 유래 증폭된 NK 세포의 암세포 살상 효능은 기존 방법으로 증폭한 NK 세포(실시예 2-1)와 본 발명에 따른 개선된 방법(실시예 2-2)으로 증폭한 NK 세포에서 유사한 세포 독성을 나타내는 것을 확인할 수 있었고, 제대혈 유래 증폭된 NK 세포의 암세포 살상 효능은 본 발명에 따른 개선된 방법(실시예 2-2)으로 증폭한 NK 세포가 보다 우수한 세포 독성을 나타내는 것을 확인하였다.As a result, as shown in Figure 3, the cancer cell killing efficacy of peripheral blood-derived amplified NK cells was compared to NK cells amplified by the existing method (Example 2-1) and the improved method according to the present invention (Example 2-2 ), it was confirmed that NK cells amplified showed similar cytotoxicity, and the cancer cell-killing efficacy of umbilical cord blood-derived amplified NK cells was superior to that of NK cells amplified by the improved method according to the present invention (Example 2-2). It was confirmed that it exhibits cytotoxicity.
실시예 4. 자연살해세포의 탈과립화(CD107a), IFN-g 및 TNF-a 분비량 확인 Example 4. Confirmation of degranulation of natural killer cells (CD107a) and secretion amounts of IFN-g and TNF-a
실시예 2-2의 방법에 의해 증폭된 NK 세포의 활성도를 확인하기 위해 NK 세포 표면에서의 CD107a 발현과 세포내 염색을 통한 IFN-g 및 TNF-a의 분비량을 측정하였다. In order to confirm the activity of NK cells amplified by the method of Example 2-2, the expression of CD107a on the surface of NK cells and the secretion amount of IFN-g and TNF-a were measured through intracellular staining.
보다 구체적으로, 냉동 후 회복된 NK 세포의 CD107a, IFN-g 및 TNF-α의 측정은 2 × 104 개의 NK 세포와 2 × 104 개의 K562 세포를 1시간 공배양 후 brefeldin A와 monensin을 첨가하여 세포 외로 단백질을 이동을 막은 뒤, 4시간 추가 공배양을 진행하였다. 4시간 후에 PE anti-human CD107a와 FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체로 표면 수용체를 먼저 염색한 후, 세척, 고정 및 투과 과정 후 BV421 IFN-g 모노클로날 항체를 이용하여 30분 동안 염색한 후 IFN-g의 발현정도를 확인하였다. TNF-α는 APC-CY7-anti-human CD3 및 PE-Cy7 anti-human CD56 모노클로날 항체로 표면 수용체 염색한 후 위 과정과 동일하게 세척, 고정 및 투과 과정 진행 뒤 APC anti-human TNF-α를 30분 동안 염색 한 후 FACS lyric에서 세포를 획득하고, Kaluza 소프트 웨어를 사용하여 분석하였다.More specifically, the measurement of CD107a, IFN-g, and TNF-α in NK cells recovered after freezing was performed by co-culturing 2 × 10 4 NK cells and 2 × 10 4 K562 cells for 1 hour, then adding brefeldin A and monensin. After preventing the protein from moving out of the cell, co-culture was performed for an additional 4 hours. After 4 hours, surface receptors were first stained with PE anti-human CD107a, FITC-anti-human CD3, and PE-Cy5 anti-human CD56 monoclonal antibodies, followed by washing, fixation, and permeabilization, followed by BV421 IFN-g monoclonal antibodies. After staining for 30 minutes using an antibody, the expression level of IFN-g was confirmed. TNF-α was stained with surface receptors using APC-CY7-anti-human CD3 and PE-Cy7 anti-human CD56 monoclonal antibodies, followed by washing, fixation, and permeabilization in the same manner as above. APC anti-human TNF-α After staining for 30 minutes, cells were obtained from FACS lyric and analyzed using Kaluza software.
상기 사용한 항체 FITC-conjugated anti-human CD3 및 PE-Cy5-conjugated anti-human CD56는 BD Biosciences에서 구입하였고, APC-Cy7-conjugated anti-human CD3 및 PE-Cy7-conjugated anti-human CD56은eBioscience에서 구입하였으며, PE-conjugated anti-human CD107a, BV421-conjugated anti-human IFN-γ 및 APC-conjugated anti-human TNF-α는 BD Biosciences에서 구입하여 사용하였다. The antibodies used above, FITC-conjugated anti-human CD3 and PE-Cy5-conjugated anti-human CD56, were purchased from BD Biosciences, and APC-Cy7-conjugated anti-human CD3 and PE-Cy7-conjugated anti-human CD56 were purchased from eBioscience. PE-conjugated anti-human CD107a, BV421-conjugated anti-human IFN-γ, and APC-conjugated anti-human TNF-α were purchased from BD Biosciences.
그 결과, 도 4에 나타낸 바와 같이, 본 발명에 따른 개선된 방법(실시예 2-2)으로 증폭한 NK 세포는 기존 방법으로 증폭한 NK 세포(실시예 2-1)와 유사한 CD107a 및 IFN-g 발현특성을 나타내는 것을 확인할 수 있었다. 이러한 결과의 의미는 생산력이 높아진 NK 세포 배양방법으로 대량 생산된 NK 세포는 정상적으로 암세포에 대한 살상효능을 가지고 있음을 의미한다.As a result, as shown in Figure 4, NK cells amplified by the improved method according to the present invention (Example 2-2) have CD107a and IFN- similar to NK cells amplified by the existing method (Example 2-1). g expression characteristics were confirmed. These results mean that NK cells mass-produced using an NK cell culture method with increased productivity normally have a killing effect on cancer cells.
실시예 5; 증폭된 NK 세포의 표현형 특성Example 5; Phenotypic characteristics of amplified NK cells
냉동 보관된 증폭된 NK 세포를 RPMI 1640 complete 배지에 해동 후 NK세포 표면 수용체의 표현형 특성을 확인하였다. The frozen and stored amplified NK cells were thawed in RPMI 1640 complete medium, and then the phenotypic characteristics of the NK cell surface receptor were confirmed.
구체적으로, 해동 후 회복된 NK 세포 2 Х 104 개를 FACS 버퍼(FBS 1% 포함 PBS)로 세척한 후, APC-Cyanine7-접합 마우스 항-인간 CD3 및 PE-Cyanine7-접합 항인간 CD56 멤브레인 항체로 15분간 처리하였다. 이후, 각각 다른 형광-접합된 항-인간 CD16, CD8a, NKG2A, NKG2C, NKG2D, DNAM-1, NKp30, NKp46, CD57, 및 CD62L 멤브레인 항체로 30분 동안 추가 염색하였다. 이후, 상기 세포를 FACS 버퍼로 세척 한 뒤, FACS Lyric를 이용하여 데이터를 획득하고 Kaluza로 분석하였다.Specifically, 2 Х 10 4 NK cells recovered after thawing were washed with FACS buffer (PBS containing 1% FBS) and then incubated with APC-Cyanine7-conjugated mouse anti-human CD3 and PE-Cyanine7-conjugated anti-human CD56 membrane antibodies. Treated for 15 minutes. Afterwards, additional staining was performed with different fluorescent-conjugated anti-human CD16, CD8a, NKG2A, NKG2C, NKG2D, DNAM-1, NKp30, NKp46, CD57, and CD62L membrane antibodies for 30 minutes. Afterwards, the cells were washed with FACS buffer, and data were acquired using FACS Lyric and analyzed with Kaluza.
상기 사용한 항체는 APC-Cy7-conjugated anti-human CD3, PE-Cy7-conjugated anti-human CD56, pacific blue-conjugated anti-human CD16, pacific blue-conjugated anti-human NKp46, PerCP-conjugated anti-human NKG2D, PerCP-conjugated anti-human CD8a, FITC-conjugated anti-human CD62L, FITC-conjugated anti-human CD57 및 PE-conjugated anti-human NKp30 는eBioscience에서 구입하였으며, APC-conjugated anti-human DNAM-1는 BD Biosciences에서 구입하였고, APC-conjugated anti-human NKG2A, PE-conjugated anti-human NKG2C는 R&D systems에서 구입하여 사용하였다. The antibodies used were APC-Cy7-conjugated anti-human CD3, PE-Cy7-conjugated anti-human CD56, pacific blue-conjugated anti-human CD16, pacific blue-conjugated anti-human NKp46, PerCP-conjugated anti-human NKG2D, PerCP-conjugated anti-human CD8a, FITC-conjugated anti-human CD62L, FITC-conjugated anti-human CD57, and PE-conjugated anti-human NKp30 were purchased from eBioscience, and APC-conjugated anti-human DNAM-1 was purchased from BD Biosciences. APC-conjugated anti-human NKG2A and PE-conjugated anti-human NKG2C were purchased and used from R&D systems.
그 결과, 도 5에 나타낸 바와 같이, 본 발명에 따른 개선된 방법(실시예 2-2)으로 증폭한 NK 세포에서 CD16, NKG2A, NKG2C, NKG2D, DNAM-1, NKp30, NKp46 및 CD8a와 같은 표면 마커가 발현되는 것을 확인할 수 있었다. 즉, 본 발명에 따른 개선된 방법으로 증폭한 NK 세포는 정상적으로 완전한 기능적 특성을 가지는 것을 알 수 있다. As a result, as shown in Figure 5, surface surfaces such as CD16, NKG2A, NKG2C, NKG2D, DNAM-1, NKp30, NKp46 and CD8a in NK cells amplified by the improved method according to the present invention (Example 2-2) It was confirmed that the marker was expressed. In other words, it can be seen that NK cells amplified by the improved method according to the present invention normally have complete functional characteristics.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (14)
(a) 단핵세포 집단 또는 NK 세포를 막 결합성 인터루킨-18(membrane bound interleukin-18: mbIL-18) 및 막 결합성 인터루킨-21(membrane bound interleukin-21: mbIL-21)을 발현하도록 유전적으로 조작된 피더 세포와 공배양하는 단계; 및
(b) 상기 유전적으로 조작된 피더 세포를 첨가하여 재자극한 후 NK 세포를 배양하는 단계;
여기서, 상기 (a) 단계 및 (b) 단계는 상기 단핵세포 내 자연살해세포: 유전적으로 조작된 피더 세포 수의 비율이 1 : 4~6의 비율로 수행하는 것을 특징으로 함.
상기 유전적으로 조작된 피더 세포는 막-결합된 인터루킨-15 및 막-결합된 4-1BB 리간드를 발현되도록 유전적으로 조작되지 않은 피더 세포인 것을 특징으로 함.
Expansion culture method of NK cells comprising the following steps:
(a) Monocyte populations or NK cells are genetically modified to express membrane bound interleukin-18 (mbIL-18) and membrane bound interleukin-21 (mbIL-21). co-culturing with the engineered feeder cells; and
(b) cultivating NK cells after restimulation by adding the genetically engineered feeder cells;
Here, steps (a) and (b) are characterized in that the ratio of the number of natural killer cells in the mononuclear cells: the number of genetically engineered feeder cells is 1:4 to 6.
The genetically engineered feeder cells are characterized in that they are feeder cells that have not been genetically engineered to express membrane-bound interleukin-15 and membrane-bound 4-1BB ligand.
The method of claim 2, wherein the mononuclear cell population is derived from peripheral blood or umbilical cord blood.
The method of claim 2, wherein step (a) is IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, performed in the presence of one or more cytokines selected from the group consisting of IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, and IL36. How to feature.
The method of claim 5, wherein the cytokine is IL-2.
The method according to claim 2, wherein co-culture is performed for 2 to 20 days.
The method of claim 2, wherein the culture in step (b) is IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, Performed in the presence of one or more cytokines selected from the group consisting of IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, and IL36 A method characterized by:
The method of claim 8, wherein the cytokines are IL-2 and IL-15.
The method according to claim 2, wherein in the culture in step (a) and the culture in step (b), the medium is exchanged when the glucose concentration of the supernatant is 50 to 100 mg/dL or less.
The method of claim 2, wherein the culture is performed in NK MACS medium containing NK MACS Supplements and human AB serum.
The method of claim 2, wherein the mononuclear cells or NK cells are included at a density of 1 x 10 4 to 3 x 10 6 cells/cm 2 .
According to claim 2, the culture in step (b) is subcultured at intervals of 5 to 7 days, and the subculture is performed by diluting the cells to a cell density of 5.0 x 10 4 to 4.0 x 10 7 cells/cm 2 . How to feature.
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| KR20190133732A (en) * | 2017-03-27 | 2019-12-03 | 싱가포르국립대학교 | Stimulating Cell Lines for EX VIVO Expansion and Activation of Natural Killer Cells |
| WO2022051749A1 (en) * | 2020-09-02 | 2022-03-10 | Nkarta, Inc. | Enhanced expansion and cytotoxicity of engineered natural killer cells and uses thereof |
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| KR20190133732A (en) * | 2017-03-27 | 2019-12-03 | 싱가포르국립대학교 | Stimulating Cell Lines for EX VIVO Expansion and Activation of Natural Killer Cells |
| WO2022051749A1 (en) * | 2020-09-02 | 2022-03-10 | Nkarta, Inc. | Enhanced expansion and cytotoxicity of engineered natural killer cells and uses thereof |
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