KR101145391B1 - A method for expansion of NK cells from Peripheral Blood - Google Patents
A method for expansion of NK cells from Peripheral Blood Download PDFInfo
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Abstract
본 발명은 말초혈액으로부터 자연 살해세포의 증식 방법에 관한 것으로, 더욱 구체적으로 말초혈액으로부터 단핵구를 분리하는 단계 및 상기 단핵구와 방사선 조사된 Jurkat 세포를 사이토카인이 첨가된 배양배지에서 배양하는 단계를 포함하는 NK 세포 증식 방법에 관한 것이다.
본 발명에 따르면, Jurkat 세포를 방사선 조사한 후 사이토카인과 함께 말초혈액 단핵구를 처리함으로써 종래 사이토카인들을 이용하여 NK 세포를 분화 및 증식시키는 방법에 비해 높은 증식능 및 분포도를 나타낼 뿐만 아니라, 현저히 증가된 암세포 살상능력을 나타낸다. 또한 종래 보고된 영양세포들과 비교하여 수율, 순도 및 암세포 살상능도 현저히 우수하다. 따라서 암세포 살상능이 증가된 NK 세포를 이용하여 암세포 치료에 유용하게 이용될 수 있다.The present invention relates to a method for propagating natural killer cells from peripheral blood, and more particularly, to isolate monocytes from peripheral blood and culturing the monocytes and irradiated Jurkat cells in a culture medium to which cytokines have been added. It relates to a NK cell proliferation method.
According to the present invention, by irradiating Jurkat cells and then treating peripheral blood monocytes with cytokines, not only shows a high proliferative capacity and distribution compared to the method of differentiating and proliferating NK cells using conventional cytokines, but also significantly increased cancer cells. Indicate killing ability. In addition, the yield, purity and cancer cell killing ability is significantly superior to the previously reported feeder cells. Therefore, NK cells with increased cancer cell killing ability may be usefully used for cancer cell therapy.
Description
본 발명은 말초혈액으로부터 자연 살해세포의 증식 방법에 관한 것으로, 더욱 구체적으로 말초혈액으로부터 단핵구를 분리하는 단계 및 상기 단핵구와 방사선 조사된 Jurkat 세포를 사이토카인이 첨가된 배양배지에서 배양하는 단계를 포함하는 NK 세포 증식 방법에 관한 것이다.
The present invention relates to a method for propagating natural killer cells from peripheral blood, and more particularly, to isolate monocytes from peripheral blood and culturing the monocytes and irradiated Jurkat cells in a culture medium to which cytokines have been added. It relates to a NK cell proliferation method.
면역계는 복잡한 기전에 의해서 조절이 되며 면역계의 이상이 생길 경우 면역시스템에 불균형이 초래되어 각종 난치성 질병이 발생한다. 면역치료제는 면역계에서 발생하는 불균형을 해소하여 정상상태로 회복, 강화시켜 면역관련 질환을 치료하는 방법이다. 최근 유전체 등에 대한 연구가 활성화되면서 암, 감염성 질환, 자가 면역 질환 등 다양한 병에 대한 면역관련 내용이 새로이 밝혀지고 이를 기반으로 한 치료기술의 개발 가능성이 확대되고 있기 때문에, 면역치료제의 개발이 필수적이다.The immune system is regulated by a complex mechanism, and when an abnormality occurs in the immune system, imbalance in the immune system causes various refractory diseases. Immunotherapy is a method of treating immune-related diseases by relieving imbalances in the immune system and restoring and strengthening them to their normal state. As research on genomes is recently activated, immunizations related to various diseases such as cancer, infectious diseases, and autoimmune diseases are newly revealed, and the development of therapeutic technology based on them is expanding, and therefore, development of immunotherapy is essential. .
종양은 정상세포가 환경적, 유전적 요인에 의해 돌연변이를 일으켜 생기는 자가 세포로 구성된 조직으로서, 오랜 기간을 두고 진행되면서 종양환자의 면역세포는 정상인에 비하여 많은 부분에서 그 기능이 크게 저하되어 있다. 우선 항원을 인식할 수 있는 MHC molecule의 세포 표면 발현이 현저하게 저하되어 있어서 종양항원이 노출되지 않고 있을 뿐만 아니라, B7.1, B7.2, CD40 등의 다양한 공동자극(co-stimulatory) 분자의 발현도 저하되어 있어서 MHC에 의한 항원 인식이 되더라도 보조인자의 자극을 받지 못하여 아네르기(anergy)나 내성(tolerance)으로 진행한다.Tumors are tissues composed of autologous cells in which normal cells are mutated by environmental and genetic factors. As the tumor progresses over a long period, immune cells of tumor patients are significantly degraded in many parts compared to normal individuals. First of all, the surface expression of MHC molecules capable of recognizing antigens is markedly lowered, thereby not exposing tumor antigens, and inducing various co-stimulatory molecules such as B7.1, B7.2, and CD40. Since expression is also reduced, antigen recognition by MHC is not stimulated by the cofactor and proceeds to anergy or tolerance.
종양환자의 경우 이러한 세포독성 T 림프구(cytotoxic T lymphocyte, CTL)나 자연 살해세포(Natural Killer cells, NK 세포)와 같은 효력세포(effector cell)에서 많은 활성화 수용체의 발현은 크게 저하된 반면, 억제 수용체가 다량 확인이 되고 있고 효력세포의 수도 매우 감소되어 있다. 보고에 의하면 위암 환자의 경우 TGF-beta와 같은 면역억제 사이토카인을 많이 분비하는 반면, IFN-gamma, IL-2, IL-15와 같은 면역 활성 사이토카인의 분비는 저하되어 있다 (Lee et al., J. Immunol., 172(12), 7335-7340, 2004). 기존에 사용되고 있는 암의 치료는 수술을 통한 제거, 항암제 치료요법 및 방사선요법으로 이루어지나, 이러한 요법은 세포분열을 억제하는데 그 기전을 두고 있기 때문에 정상세포에의 독성이 커다란 문제점이 되고 있어 정상세포에 독성을 끼치지 않는 세포 치료제의 개발이 필수적이라 할 수 있다.In tumor patients, the expression of many activating receptors in effector cells, such as cytotoxic T lymphocytes (CTLs) or natural killer cells (NK cells), is greatly reduced, whereas inhibitory receptors. Is confirmed in large quantities, and the number of effector cells is greatly reduced. According to the report, gastric cancer patients secrete a lot of immunosuppressive cytokines such as TGF-beta, while the secretion of immunologically active cytokines such as IFN-gamma, IL-2, and IL-15 is reduced (Lee et al. , J. Immunol., 172 (12), 7335-7340, 2004). Conventionally used cancer treatment consists of surgical removal, chemotherapy and radiotherapy, but since these therapies have a mechanism for inhibiting cell division, toxicity to normal cells is a major problem. It is essential to develop a cell therapy that is not toxic.
인체는 면역 반응에 의해 병원체로부터 보호되고 있고, 면역 시스템은 많은 면역관련 세포와 사이토카인 등에 의해 구성되어 있다. 이러한 면역시스템에서 중요적인 역할을 하고 있는 것이 백혈구, 특히 림프구이다. 이 림프구를 구성하는 세포에는 대표적으로 선천성 면역계와 후천성 면역계로 나뉜다. 이러한 면역계 중 자연 살해세포는 대표적인 선천면역세포 중 하나로, 비 특이적으로 암을 살상할 수 있는 능력이 있고 바이러스, 박테리아 등을 인식하여 살상하는 능력을 보유한 세포로 알려져 있으며, 퍼포린(perforin) 및 그랜자임(granzyme) 등의 효소나 Fas-FasL 상호작용으로 병원체를 살상하는 세포로 알려져 있다. 그러나 암환자에서 이러한 NK 세포의 암세포 살상능의 감소로 인해 폐암(Carrega P, et al., Cancer, 112, 863-875, 2008), 간암(Jinushi M, et al., J Hepatol., 43, 1013-1020, 2005), 유방암(Bauernhofer T, et al., Eur J Immunol., 33, 119-124, 2003), 자궁암(Mocchegiani E., et al., Br j Cancer., 79, 244-250, 1999), 혈액암(Tajima F., et al, Lekemia 10, 478-482, 1996) 등의 질환의 발생과 깊은 연관이 있는 것으로 보고되고 있다. 따라서 이런 암의 치료를 위해서 암환자에게서 자연 살해세포의 암세포 살상능 및 활성증가가 필수적이며, 현재 이러한 자연 살해세포의 살상능을 이용하여 고형암이나 혈액 암의 치료에 이용하고 있는 실정이다.The human body is protected from pathogens by an immune response, and the immune system is composed of many immune-related cells, cytokines and the like. White blood cells, especially lymphocytes, play an important role in this immune system. The cells constituting these lymphocytes are typically divided into the innate immune system and the acquired immune system. Natural killer cells of the immune system are one of the representative innate immune cells, and are known as cells that have the ability to kill cancer in a specific manner and have the ability to recognize and kill viruses, bacteria, and the like. It is known as a cell that kills pathogens by enzymes such as granzyme and Fas-FasL interaction. However, in cancer patients, lung cancer (Carrega P, et al., Cancer, 112, 863-875, 2008), liver cancer (Jinushi M, et al., J Hepatol., 43, 1013-1020, 2005), breast cancer (Bauernhofer T, et al., Eur J Immunol., 33, 119-124, 2003), uterine cancer (Mocchegiani E., et al., Br j Cancer., 79, 244-250 , 1999), hematologic cancer (Tajima F., et al, Lekemia 10, 478-482, 1996) has been reported to be closely associated with the development of diseases. Therefore, the cancer cell killing ability and the activity of natural killer cells is essential for the treatment of such cancers, and the situation is currently used in the treatment of solid cancer or blood cancer using the killing ability of natural killer cells.
1980년대 초 미국 NIH의 Rosenberg 그룹에서 고형암, 전이암환자를 대상으로 IL-2 혹은 IL-2 활성화 NK 세포 치료법을 선두로 임상시도가 진행되었고, 그 결과로서 흑색종 환자 혹은 신장암 환자 중 15-30% 정도가 긍정적인 치유효과를 보여주었다. 그러나 IL-2 치료법의 부작용으로는 크게 두 가지가 있다. 하나는 모세혈관 누수 증후군(capillary leak syndrome)에 의해 저혈압을 유도하여 생명을 위협하는 독성을 가지고 있으며, IL-2 활성화된 NK 세포는 혈관내피에서 세포간의 접촉에 의한 예정 세포사에 대한 민감성이 높아서 암부위로의 NK 세포 접근에 제약이 있다. 최근 보고에 의하면, IL-2 보다 IL-15가 NK 세포의 ex vivo expansion, survival, protection from AICD (Activation-Induced Cell death) 등에 효과가 있다고 하지만 고농도의 IL-15를 사용해야 하고 고가인 것이 문제점으로 대두 되고 있다. 최근에는 이들 사이토카인 이외에도 Flt3-L, SCF, IL-7 등과 같은 사이토카인들(early acting cytokines)이 NK 세포수를 증가시키는 데 효과가 있다는 보고가 있다.In the early 1980s, the Rosenberg group of the NIH in the United States led clinical trials in the treatment of IL-2 or IL-2 activated NK cells in patients with solid and metastatic cancer, resulting in 15- of 18 patients with melanoma or kidney cancer. About 30% showed a positive healing effect. However, there are two major side effects of IL-2 therapy. One is life-threatening toxicity by inducing hypotension by capillary leak syndrome, and IL-2 activated NK cells are highly sensitive to the expected cell death by intercellular contact in the vascular endothelium. There is a limit to NK cell access up. According to recent reports, IL-15 is more effective than IL-2 in ex vivo expansion, survival, and protection from AICD (Activation-Induced Cell Death). However, high concentrations of IL-15 are required and expensive. It is emerging. Recently, in addition to these cytokines, there are reports that early acting cytokines such as Flt3-L, SCF, IL-7 and the like are effective in increasing the number of NK cells.
한편, 항암면역치료에 사용하기 위해서는 대량의 활성화된 자연 살해세포를 필요로 하는데, 암환자의 혈액을 대량으로 확보하기 어렵고 말초혈액의 단핵구에서 자연 살해세포를 분리하는데 고비용이 필요하기 때문에 매우 한정된 환자만을 대상으로 선정하는 실정이다. 현재 전 세계적으로 여러 연구팀이 이러한 자연 살해세포를 이용한 항암면역치료법을 개발하고 있고 일부는 그 효력을 테스트하기 위하여 임상시험 중에 있다. 대표적으로 MACS (Magnetic Activated Cell Sorting), cliniMACS, FACs Sorter 등의 장비를 사용하여 혈액에 있는 단핵구나 골수로부터 자연 살해세포를 분리 또는 유도하고 있다. 이러한 방법에는 다음의 단계가 선행되고 있다. 1) 단핵구로부터 초기에 NK 세포를 분리하여 사이토카인을 사용하여 확장배양, 2) 단핵구에 공존하는 T 세포를 제거한 후 사이토카인을 사용하여 NK 세포를 확장배양, 3) 골수에 있는 줄기세포(stem cell)로부터 자연 살해세포로 유도하는 방법 등이 있다. 그러나 기존에 보고되어 있는 모든 방법이 고가의 장비를 사용하고, 고농도 및 고가의 여러 종류의 사이토카인을 이용하고 있어 치료 대상 환자가 경제적으로 안정된 일부환자에만 국한되어 치료가 가능한 실정이다.On the other hand, a large amount of activated natural killer cells are required for use in anti-cancer immunotherapy, which is very limited because it is difficult to obtain a large amount of blood from cancer patients and a high cost is required to separate natural killer cells from monocytes of peripheral blood. The situation is to select only the target. Currently, several research teams around the world are developing anticancer immunotherapy using natural killer cells, and some are in clinical trials to test their efficacy. Typically, MACS (Magnetic Activated Cell Sorting), cliniMACS and FACs Sorter are used to isolate or induce natural killer cells from monocytes or bone marrow. This method is preceded by the following steps. 1) Isolate NK cells from monocytes early to expand culture using cytokines, 2) Remove T cells coexisting with monocytes, and expand cultures of NK cells using cytokines, 3) Stem cells in the bone marrow cell) to natural killer cells. However, all the previously reported methods use expensive equipment, and high concentrations and expensive types of cytokines are used, and thus the treatment target is limited to only some patients who are economically stable and can be treated.
또한 영양세포(feeder cell)를 이용하여 말초혈액 내 단핵구(PBMC)를 자연 살해세포로 유도하는 방법도 소수의 그룹에서 제안이 되었다. 대표적으로 이탈리아 Robin Fo그룹은 B세포 leukemia인 RPMI8866 세포주를 이용하여 NK 세포를 확장시키는 방법을 고안하였고 (Torelli GF, et al., Br J Haematol. 116, 299307, 2002) 일본의 Ohno 그룹에서는 Wilms 종양세포인 HFWT 세포주를 이용하여 NK 세포를 증폭시키는 방법을 보고하였다 (Ishikawa E, et.al., Anti cancer Res. 24, 18611871, 2004). 그러나 이들 영양세포를 사용하여 NK 세포를 확장 및 배양 시 증폭율 이 기대하는 만큼 높지 못하다고 보고되고 있다. 따라서 본 발명에서는 방법이 간단하고 그 비용이 저가인 자연 살해세포 증식법을 고안하여 대상 환자군의 범위를 확대하고 강력한 항암작용을 가지는 항암면역세포로 세포수를 증폭시켜 보다 많은 대상 환자군에게 응용할 수 있는 신규 방법을 개발하고자 한다.
In addition, a small number of groups have proposed a method for inducing peripheral blood mononuclear cells (PBMC) into natural killer cells using feeder cells. Representatively, Robin Fo group in Italy devised a method of expanding NK cells using RPMI8866 cell line, a B-cell leukemia (Torelli GF, et al., Br J Haematol. 116, 299307, 2002). A method for amplifying NK cells using HFWT cell line, which is a cell, has been reported (Ishikawa E, et.al., Anti cancer Res. 24, 18611871, 2004). However, it is reported that amplification rate is not as high as expected when NK cells are expanded and cultured using these feeder cells. Therefore, in the present invention, by devising a natural killer cell proliferation method which is simple and inexpensive, it expands the range of the target patient group and amplifies the cell number into anti-cancer immune cells with potent anticancer activity, which can be applied to more target patient groups. We want to develop a new method.
따라서, 본 발명의 주된 목적은 말초혈액 단핵구와 방사선 조사된 Jurkat 세포를 사이토카인 존재 하에 배양함으로써 자연 살해세포로 효율적으로 분화 및 증식시킬 수 있는 말초혈액으로부터 자연 살해세포의 증식 방법을 제공하는 데 있다.Accordingly, a main object of the present invention is to provide a method for proliferating natural killer cells from peripheral blood which can efficiently differentiate and proliferate into natural killer cells by culturing peripheral blood monocytes and irradiated Jurkat cells in the presence of cytokines. .
본 발명의 다른 목적은 말초혈액 단핵구와 방사선 조사된 Jurkat 세포를 사이토카인 존재하에 배양함으로써 강력한 암세포 살상능력을 갖는 자연 살해세포의 제조방법을 제공하는 데 있다.Another object of the present invention is to provide a method for producing natural killer cells having potent cancer cell killing ability by culturing peripheral blood monocytes and irradiated Jurkat cells in the presence of cytokines.
본 발명의 다른 목적은 상기 방법으로 제조된 자연 살해세포를 포함하는 암 예방 및 치료용 조성물을 제공하는데 있다.
Another object of the present invention to provide a composition for preventing and treating cancer comprising natural killer cells prepared by the above method.
본 발명의 한 양태에 따르면, 본 발명은 말초혈액으로부터 단핵구를 분리하는 단계, 및 상기 단핵구와 방사선 조사된 Jurkat 세포를 사이토카인이 첨가된 배양배지에서 배양하는 단계를 포함하는 NK 세포 증식 방법을 제공한다.According to an aspect of the present invention, the present invention provides a NK cell proliferation method comprising the step of isolating monocytes from peripheral blood, and culturing the monocytes and irradiated Jurkat cells in a cytokine-added culture medium. do.
본 발명은 항암면역치료를 위해 말초혈액단핵구(Peripheral Blood Mononuclear Cell, PBMC)와 방사선을 조사한 영양세포(feeder cell)를 이용하여 항암면역기능이 증가된 자연 살해세포(NK 세포)를 유도하고 증식하는 방법이다. 기존의 NK 세포 증식 방법은 대부분 MACs(magnetic activated cell sorting)를 이용하여 T 세포를 제거하거나, NK 세포만을 분리하여 자연 살해세포를 증식시키는 방법을 사용하였다. 그러나 cliniMACs나 FACs sorter 등 고가 장비를 사용하여 자연 살해 세포를 분리하여야 하고 또한 별도로 단핵구에 다량으로 존재하는 T 세포를 키트를 사용하여 제거해야 하므로, 이에 따른 많은 비용이 발생하며 복잡한 과정이 필요하다. 그러나 본 발명자들이 개발한 방법은 별도의 분리과정이 없으며 영양세포로 사용된 Jurkat 세포에 방사선을 조사한 후 공동 배양함으로써 높은 비율과 암세포 살상능을 지닌 자연 살해세포를 유도 및 증식시킬 수 있다.The present invention utilizes peripheral blood mononuclear cells (PBMC) and irradiated feeder cells to induce and proliferate natural killer cells (NK cells) with increased anticancer immune function for chemotherapy. It is a way. Conventional NK cell proliferation methods have mostly used T cells to remove T cells using MACs (magnetic activated cell sorting), or to separate natural NK cells to propagate natural killer cells. However, natural killer cells must be separated using expensive equipment such as cliniMACs or FACs sorter, and a large amount of T cells present in monocytes must be removed using a kit. However, the method developed by the present inventors does not have a separate separation process and can be induced and proliferated in natural killer cells having high ratio and cancer cell killing ability by irradiating and co-culturing Jurkat cells used as feeder cells.
본 발명에 사용된 “Jurkat 세포주”는 혈액암(immortalized acute T cell leukemia) 세포주로 University of California at San Francisco의 Dr. Arthur Weiss가 개발한 세포주이다. 다양한 케모카인 수용체가 발현하고 있으며 IL-2를 생산할 수 있는 세포로서, 본 발명자들이 말초혈액 단핵구로부터 NK 세포로의 분화 및 증식을 위해 다수의 혈액암 세포주를 스크리닝하여 찾아낸 영양세포주이다. 세포 표면에 자연 살해세포 활성억제 인자인 MHC class I을 높이 발현하기 때문에 항암면역치료를 위한 영양세포(feeder cells)의 후보로 가능성이 전혀 대두되지 못했던 세포주이다. 따라서 자연 살해세포의 확장배양에 영양세포로 사용될 수 있음이 본 발명자들에 의해 처음으로 밝혀진 것이다. 본 발명에 사용되는 Jurkat 세포는 ATCC로부터 입수할 수 있으며, 100 내지 500 Gy 방사선을 조사함으로써 영양세포로서 사용할 수 있다."Jurkat cell line" used in the present invention is a blood cancer cell line (immortalized acute T cell leukemia), Dr. University of California at San Francisco. It is a cell line developed by Arthur Weiss. As a cell expressing various chemokine receptors and capable of producing IL-2, it is a feeder cell line which the present inventors have found by screening a number of blood cancer cell lines for differentiation and proliferation from peripheral blood monocytes to NK cells. Since MHC class I, which is a natural killer cell inhibitor, is highly expressed on the cell surface, it is a cell line that has never emerged as a candidate for feeder cells for anticancer immunotherapy. Therefore, the present inventors have been found for the first time that it can be used as a feeder cell in the expansion culture of natural killer cells. Jurkat cells used in the present invention can be obtained from ATCC and can be used as feeder cells by irradiation with 100 to 500 Gy radiation.
본 발명은 방사선 조사된 Jurkat 세포를 이용하여 말초혈액의 단핵구를 고 순도의 암세포 살상능을 가진 자연 살해세포로 유도 및 증식시킬 수 있는 방법을 제공하며, 최종적으로 획득한 자연 살해세포의 수율과 순도는 기존에 보고된 다른 영양세포들에 비하여 수율, 순도, 및 암세포 살상능이 월등하게 우월하다.The present invention provides a method for inducing and propagating mononuclear cells of peripheral blood into natural killer cells having high purity of cancer cell killing ability using irradiated Jurkat cells, and finally yielding and purity of natural killer cells obtained Is far superior in yield, purity, and cancer cell killing capacity to other previously reported vegetative cells.
본 발명에서는 영양세포로서 Jurkat 세포가 NK 세포의 분화 및 증식에 미치는 영향을 알아보기 위해, IL-2만을 처리한 군을 대조군으로 하고 말초혈액 유래 단핵구와 Jurkat 세포를 IL-2 존재하에 공동 배양한 실험군을 이용하여 NK 세포군의 비율을 측정하였다. 그 결과, IL-2만 처리한 대조군은 배양 16일째까지 변화가 없었으나 IL-2 존재 하에 Jurkat 세포와 공동 배양한 실험군은 배양 16일 후 대조군과 비교하여 약 130배 증가함을 확인하였다 (도 1). 또한 NK 세포의 증식 뿐만 아니라 분포(enrichment)의 정도를 확인한 결과, 배양 15일 경과 후 IL-2만 처리한 대조군에 비해 IL-2 존재하에 Jurkat 세포주와 공동배양한 실험군에서 NK 세포의 표현형인 CD56+, CD3- 세포의 비율이 92% 이상까지 증가함을 확인하였다 (도 2a).In the present invention, in order to determine the effect of Jurkat cells on the differentiation and proliferation of NK cells as feeder cells, the control group was treated only with IL-2 and co-cultured peripheral blood-derived monocytes and Jurkat cells in the presence of IL-2. The ratio of the NK cell group was measured using the experimental group. As a result, the control group treated only with IL-2 did not change until day 16, but the experimental group co-cultured with Jurkat cells in the presence of IL-2 increased about 130-fold compared to the control after 16 days of culture (Fig. One). In addition, after confirming the proliferation as well as the degree of enrichment of NK cells, CD56 +, a phenotype of NK cells in the experimental group co-cultured with Jurkat cell line in the presence of IL-2 compared to the control group treated with IL-2 after 15 days of culture. , It was confirmed that the proportion of CD3- cells increased up to 92% or more (Fig. 2a).
또한 본 발명에서는 다른 영양세포와 비교하여 NK 세포의 증식에 미치는 영향을 알아보기 위해, Jurkat 세포 이외에 다른 혈액암 세포주인 CEM, 및 K562 세포를 이용하여 NK 세포군의 enrichment 정도를 측정하였다. 그 결과, CEM 세포주는 IL-2만 처리한 대조군과 유사한 낮은 NK 세포의 분포를 보였고, K562 세포주의 경우 15일 경과 후 약 40%의 NK 세포의 분포를 보였으며, 본 발명의 Jurkat 세포주는 약 80%의 NK 세포의 분포를 보였다. 따라서 본 발명의 방사선 조사된 Jurkat 세포에 의한 NK 세포 증식 방법은 Jurkat 세포에 특이적인 반응에 의해 이루어짐을 확인하였다 (도 3).In addition, in the present invention, in order to determine the effect on the proliferation of NK cells compared to other feeder cells, the enrichment of the NK cell population was measured using CEM, and K562 cells, other blood cancer cell lines in addition to Jurkat cells. As a result, the CEM cell line showed a low NK cell distribution similar to the control group treated with IL-2 only, and the K562 cell line showed a distribution of about 40% NK cells after 15 days, and the Jurkat cell line of the present invention was about It showed distribution of NK cells of 80%. Therefore, the NK cell proliferation method by irradiated Jurkat cells of the present invention was confirmed by the reaction specific to Jurkat cells (Fig. 3).
본 발명의 NK 세포 증식 방법에서, 상기 말초혈액은 정상인 또는 암환자로부터 얻은 것일 수 있다. 사람의 말초혈액으로부터 얻은 단핵구는 본 발명의 NK 세포 증식 방법에 따라 NK 세포로 분화, 증식될 수 있고 이렇게 얻은 NK 세포를 암 예방을 목적으로 정상인, 암의 위험성을 갖는 환자, 또는 암환자에 처치될 수 있다. 또한 암환자의 말초혈액으로부터 얻은 단핵구를 NK 세포로 증식시키고 이렇게 얻은 NK 세포를 암 치료를 목적으로 암의 위험성을 갖는 환자 또는 암환자에게 처치될 수 있다.In the NK cell proliferation method of the present invention, the peripheral blood may be obtained from normal or cancer patients. Monocytes obtained from human peripheral blood can be differentiated and proliferated into NK cells according to the NK cell proliferation method of the present invention, and the NK cells thus obtained are treated to patients with cancer risk or normal patients for cancer prevention purposes. Can be. In addition, the monocytes obtained from the peripheral blood of cancer patients can be propagated to NK cells and the NK cells thus obtained can be treated to patients or cancer patients with cancer risk for the purpose of treating cancer.
본 발명의 NK 세포 증식 방법에서, 상기 사이토카인은 말초혈액 단핵구를 NK 세포로 분화시키는데 사용될 수 있는 면역 활성화 사이토카인으로서, 예를 들어 IL-2, IL-15, IL-21, Flt3-L, SCF, IL-7 또는 이들을 2종 이상 혼합 처리하여 말초혈액 단핵구를 NK 세포로 분화시킬 수 있다. 특히 IL-2, IL-15 및 IL-21이 NK 세포로의 분화 및 증식에 탁월한 효과가 있는 사이토카인으로 알려져 있으므로, 이들 사이토카인을 이용하는 것이 좋으며, 바람직하게는 IL-2이다. In the NK cell proliferation method of the present invention, the cytokine is an immune activating cytokine that can be used to differentiate peripheral blood monocytes into NK cells, for example, IL-2, IL-15, IL-21, Flt3-L, Peripheral blood monocytes can be differentiated into NK cells by treatment of SCF, IL-7 or two or more thereof. In particular, since IL-2, IL-15 and IL-21 are known to be cytokines which have an excellent effect on differentiation and proliferation into NK cells, it is preferable to use these cytokines, preferably IL-2.
상기 사이토카인들이 NK 세포 분화에 관여한다는 사실은 여러 문헌에서 찾을 수 있다. 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.The fact that these cytokines are involved in NK cell differentiation can be found in several literatures. 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). Although there have been reports of a significant decrease in the number of NK cells in humans and mice lacking IL-2 (DiSanto, JP et al., J. Exp. Med. 171, 1697-1704, 1990), on the other hand, IL-2 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.
상기 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-15Rα가 결핍된 쥐에서는 NK 세포가 발견되지 않는 다는 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).The IL-15 is involved in NK cell differentiation, which is deficient in NK cells in mice lacking the transcription factor interferon (IFN) -
상기 IL-21은 활성화된 CD4+ T세포에 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-15와 매우 유사하며, IL-21R은 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 the receptor of IL-21 (IL-21R) is 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 the maturation of NK cell precursors from the bone marrow (Parrish-Novak, et al., Nature, 408: 57-63, 2000), in particular with cytokine production and apoptosis of NK cells The same effector functions have been reported to increase (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).
본 발명의 NK 세포 증식 방법에서, 상기 배양시 1일 내지 15일 후 방사선 조사된 Jurkat 세포와 IL-2를 추가하는 단계를 더 포함할 수 있다. 활성화된 NK 세포는 생존시간이 짧기 때문에 활성화된 NK 세포를 이용한 면역 치료에 있어서 문제점을 가지고 있다. 본 발명에서는 NK 세포 증식 후 배양 1일 내지 15일 후 방사선 조사된 Jurkat 세포와 IL-2를 추가함으로서 활성화된 NK 세포의 생존기간을 연장시키고 배양 15일 이후에도 NK 세포를 증식시킬 수 있음을 확인하였다 (도 6).
In the NK cell proliferation method of the present invention, the culture step may further comprise the step of adding irradiated Jurkat cells and IL-2 after 1 day to 15 days. Since activated NK cells have a short survival time, there is a problem in immunotherapy using activated NK cells. In the present invention, it was confirmed that after 1 to 15 days of NK cell proliferation, by adding irradiated Jurkat cells and IL-2, prolonged survival of activated NK cells and proliferating NK cells even after 15 days of culture. (FIG. 6).
본 발명의 다른 양태에 따르면, 본 발명은 말초혈액으로부터 단핵구를 분리하는 단계, 및 상기 단핵구와 방사선 조사된 Jurkat 세포를 사이토카인이 첨가된 배양배지에서 배양하는 단계를 포함하는 암세포 살상능력이 증가된 NK 세포 제조 방법을 제공한다.According to another aspect of the invention, the present invention is to increase the cancer cell killing capacity comprising the step of separating the monocytes from peripheral blood, and culturing the monocytes and irradiated Jurkat cells in a culture medium added to the cytokine Provided are methods for preparing NK cells.
본 발명에 따라 제조된 암세포 살상능력이 증가된 NK 세포는 현저히 높은 암세포 살상능력을 나타낸다. 본 발명에서는 상기 방법으로 제조된 NK 세포의 암세포 살상능력을 확인하기 위하여, 종양세포를 표적세포로 하여 IL-2만 처리한 대조군과 암세포 살상능력을 비교하였다. 그 결과, 상기 방법으로 제조된 NK 세포는 IL-2만 처리한 대조군에 최대 5배의 증가된 암세포 살상능을 나타내었으며, 효력세포와 표적세포의 비율이 0.7:1 인 경우에도 상당한 살상능을 보유하고 있음을 확인하였다 (도 4). 따라서 본 발명에 따라 IL-2 존재하에 Jurkat 세포로 처리한 말초혈액 단핵구의 경우 NK 세포로의 높은 enrichment 뿐만 아니라 높은 암세포 살상능을 보임을 확인할 수 있었다.
NK cells with increased cancer cell killing ability prepared according to the present invention exhibit significantly higher cancer cell killing ability. In the present invention, in order to confirm the cancer cell killing ability of the NK cells prepared by the above method, tumor cells were compared with the control group treated with IL-2 and cancer cell killing ability using the tumor cells as target cells. As a result, NK cells prepared by the above method showed up to 5 times increased cancer cell killing ability to IL-2 only control group, and even when the ratio of effector cells and target cells was 0.7: 1, It was confirmed that it holds (Fig. 4). Therefore, it was confirmed that the peripheral blood mononuclear cells treated with Jurkat cells in the presence of IL-2 showed high cancer cell killing ability as well as high enrichment of NK cells.
본 발명의 다른 양태에 따르면, 본 발명은 상기 방법으로 제조된 암세포 살상능력이 증가된 NK 세포를 함유하는 암 예방 및 치료용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a composition for preventing and treating cancer containing NK cells with increased cancer cell killing ability prepared by the above method.
본 발명에서, 상기 조성물은 비특이적으로 암을 살상할 수 있는 능력이 있는 NK 세포를 함유하고 있고, 암환자에서 이러한 NK 세포가 폐암(Carrega P, et al., Cancer, 112, 863-875, 2008), 간암(Jinushi M, et al., J Hepatol., 43, 1013-1020, 2005), 유방암(Bauernhofer T, et al., Eur J Immunol., 33, 119-124, 2003.), 자궁암(Mocchegiani E., et al., Br j Cancer., 79, 244-250, 1999), 혈액암(Tajima F., et al, Lekemia, 10, 478-482, 1996) 등의 질환의 발생과 깊은 연관이 있는 것으로 보고되고 있으므로, 이러한 암의 치료를 위해서 암환자에게서 NK 세포의 암세포 살상능 및 활성 증가가 필수적이며, 현재 이러한 NK 세포의 살상능을 이용하여 다양한 암의 치료에 이용하고 있다.In the present invention, the composition contains NK cells capable of nonspecifically killing cancer, and in cancer patients, these NK cells are known as lung cancer (Carrega P, et al., Cancer, 112, 863-875, 2008). ), Liver cancer (Jinushi M, et al., J Hepatol., 43, 1013-1020, 2005), breast cancer (Bauernhofer T, et al., Eur J Immunol., 33, 119-124, 2003.), uterine cancer ( Mocchegiani E., et al., Br j Cancer., 79, 244-250, 1999), blood cancer (Tajima F., et al, Lekemia, 10, 478-482, 1996) It is reported that there is a need to increase the cancer cell killing capacity and activity of NK cells in cancer patients for the treatment of such cancers, and is currently used in the treatment of various cancers using the killing ability of these NK cells.
본 발명에서, 상기 조성물은 암 예방 및 치료를 목적으로 환자 자신이나 타인 또는 다른 동물의 말초혈액 단핵구를 채취하여 본 발명에 따른 방법으로 NK 세포로 분화 및 증식시켜 암 예방 및 치료를 필요로 하는 대상에게 투여될 수 있다.In the present invention, the composition is a subject in need of preventing and treating cancer by collecting the peripheral blood mononuclear cells of the patient himself or another person or other animals for the purpose of preventing and treating cancer to differentiate and proliferate into NK cells by the method according to the present invention. It can be administered to.
본 발명의 암 예방 및 치료용 조성물은 당업계에 일반적인 제형, 예컨대 주사제의 형태로 제조될 수 있으며, 외과수술적으로 예상투여 부위에 직접 이식하거나, 정맥에 투여된 후 예상 투여부위로 이동할 수 있다. 본 발명의 조성물은 암의 유형, 투여경로, 환자의 나이 및 성, 및 질병의 정도에 따라 적절히 선택될 수 있으나, 바람직하게는 평균 성인의 경우, 1×106 내지 1011 세포를 투여할 수 있다.The cancer prevention and treatment compositions of the present invention may be prepared in the form of formulations common in the art, such as injections, and may be surgically implanted directly into the expected site of administration, or may be administered intravenously and then moved to the expected site of administration. . The composition of the present invention may be appropriately selected depending on the type of cancer, the route of administration, the age and sex of the patient, and the extent of the disease, but in the case of the average adult, 1 × 10 6 to 10 11 cells may be administered. have.
또 다른 제형으로서, 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 제제화 할 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.As another formulation, 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 main formulations, powders, tablets, capsules, pills, granules or injections, such as aqueous solutions, suspensions, emulsions and the like. 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.
상기 제제화된 본 발명의 조성물은 또한 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 일회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다.
The formulations of the present invention formulated above may also be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) and the dosage may be determined by the patient's weight, age, sex, health condition, diet, time of administration. The range varies depending on the method of administration, the rate of excretion and the severity of the disease. The daily dosage 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.
이상 설명한 바와 같이, 본 발명에 따르면 Jurkat 세포는 NK 세포로의 유도 및 증식을 위해 영양세포로 사용될 수 있음을 본 발명자들이 처음으로 밝혀내었다. 상기 Jurkat 세포를 방사선 조사한 후 사이토카인과 함께 말초혈액 단핵구를 처리하는 경우 종래 사이토카인들을 이용하여 NK 세포를 분화 및 증식시키는 방법에 비해 높은 증식능 및 분포도를 나타낼 뿐만 아니라, 현저히 증가된 암세포 살상능력을 나타낸다. 또한 종래 보고된 영양세포들과 비교하여 수율, 순도 및 암세포 살상능도 현저히 우수하다.
As described above, according to the present invention, the inventors have found for the first time that Jurkat cells can be used as feeder cells for induction and proliferation into NK cells. Treatment of peripheral blood mononuclear cells with cytokines after irradiation of the Jurkat cells shows higher proliferation and distribution than conventional methods for differentiating and proliferating NK cells using cytokines, as well as significantly increasing cancer cell killing ability. Indicates. In addition, the yield, purity and cancer cell killing ability is significantly superior to the previously reported feeder cells.
도 1은 사람에서 분리한 말초혈액 단핵구(PBMC)에 IL-2를 500 U/ml를 처리한 PBMC 그룹(◆)과 IL-2 500U/ml에 Jurkat 세포를 영양세포로 처리한 그룹(□)을 5, 10, 16일째 트립판 블루를 사용하여 세포수를 센 후 유세포분석기를 이용하여 CD56+, CD3- 세포의 분포를 분석한 그래프이다.
도 2a는 말초혈액 단핵구에서 NK 세포의 분포를 유세포 분석기를 이용하여 분석한 결과이다.
도 2b와 2c는 NK 세포의 분포 정도를 시간에 따른 증가 정도를 나타낸 그래프이다.
도 3은 사람에게서 분리한 말초혈액 단핵구에 IL-2를 500 U/ml로 처리한 PBMC 그룹(◆)과 IL-2 500 U/ml에 K562(□), CEM(■), Jurkat(△) 세포주를 영양세포로 각각 처리한 후 4일, 10일, 14일째 유세포 분석기를 이용하여 NK 세포의 분포를 분석한 결과이다.
도 4는 IL-2만 처리한 대조군(△)과 IL-2 및 방사선 조사된 Jurkat 세포로 공동배양한 실험군(■)을 15일간 배양한 후 종양세포 살상능을 확인한 그래프이다. 표적세포에 동위원소를 표지한 후 말초혈액의 단핵구를 4시간동안 공동배양한 후 상등액을 취하여 감마카운터에 측정하여 도출된 수치를 퍼센트화 한 것이다.
도 5는 위암 및 대장암 환자의 말초혈액 단핵구를 얻어 IL-2 단독으로 배양하거나 IL-2와 Jurkar 세포를 영양세포로 처리한 그룹 간의 NK 세포의 분포(왼쪽) 및 살상능(오른쪽)을 분석한 결과이다.
도 6a와 6b는 말초혈액 단핵구의 생존률을 증가시키기 위해, IL-2를 처리하고 Jurkat 세포를 영양세포로 사용한 말초혈액 단핵구를 배양 후 10일째 방사선으로 조사한 Jurkat 세포를 한번 더 공동 배양하여 세포수를 나타낸 그래프이다. 도 6a는 말초혈액단핵구의 세포수를 측정한 그래프이며, 도 6b는 NK 세포수를 측정한 그래프이다.1 is a PBMC group (◆) treated with 500 U / ml of IL-2 in peripheral blood mononuclear cells (PBMC) isolated from humans and a group treated with Jurkat cells as feeders for 500 U / ml of IL-2 (□). 5, 10, and 16 days after trypan blue counting the number of cells using a flow cytometer is a graph analyzing the distribution of CD56 +, CD3- cells.
Figure 2a is the result of analyzing the distribution of NK cells in peripheral blood monocytes using a flow cytometer.
2b and 2c are graphs showing the degree of increase in distribution of NK cells over time.
FIG. 3 shows PBMC group (◆) treated with 500 U / ml of IL-2 in peripheral blood monocytes isolated from humans and K562 (□), CEM (■), Jurkat (△) in IL-2 500 U / ml The results of analyzing the distribution of NK cells using flow cytometry on the 4th, 10th and 14th day after the cell lines were treated with feeder cells, respectively.
FIG. 4 is a graph confirming tumor cell killing ability after 15 days of culturing the experimental group (■) co-cultured with IL-2 only control (△) and IL-2 and irradiated Jurkat cells. Isotopes were labeled with target cells, and monocytes in peripheral blood were co-cultured for 4 hours, then the supernatant was taken and measured on the gamma counter to percentage the value derived.
FIG. 5 shows the distribution (left) and killing ability (right) of NK cells between groups treated with IL-2 alone or cultured with IL-2 alone or treated with feeders of IL-2 and Jurkar cells in patients with gastric and colorectal cancer. One result.
Figures 6a and 6b is to increase the survival rate of peripheral blood monocytes, co-cultured Jurkat cells irradiated with radiation twice a
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 말초혈액 단핵구로부터 NK 세포의 제조Example 1 Preparation of NK Cells from Peripheral Blood Monocytes
1) 정상인의 혈액을 채혈한 후, Ficoll(Ficoll-paqueTM PLUS, GE healthcare)을 이용하여 2500 rpm에서 30분간 원심분리한 후 연막층(buffy coat)에서 단핵세포(PBMC)를 분리하였다. 그 후 트립판 블루(Tryphan Blue)로 염색한 후 염색된 세포를 제외하고 염색되지 않은 세포만을 헤마토사이토미터(hematocytometer)를 이용하여 측정한 후 세포수를 계산하였다.1) After collecting blood from normal people, the cells were centrifuged at 2500 rpm for 30 minutes using Ficoll (Ficoll-paque TM PLUS, GE healthcare) and mononuclear cells (PBMC) were separated from the buffy coat. Then, after staining with tryphan blue (Tryphan Blue), except for the stained cells were measured using a hematocytometer (hematocytometer), the cell number was calculated.
2) 영양세포(feeder cell)로 사용되는 Jurkat 세포주(ACTT No. TIB-152)는 RPMI1640 배지에 10% FBS와 1% penicillin/streptomycin을 넣은 human RPMI 배지에 75T 플라스크에 37℃, 5% CO2 의 조건에서 배양 하였다. 또한 다른 혈액 암세포주인 CEM 세포(ACTT No. CCL-119)와 K562 세포(ACTT No. CCL-243)도 human RPMI 배지에 37℃, 5% CO2를 공급받는 상황에서 75 T 플라스크에서 배양하였다. 2-3일 마다 한번씩 IL-2가 500 U/ml로 첨가된 hRPMI 배지를 넣어주었으며, 5일 간격으로 세포를 모두 수확한 후 새로운 IL-2와 hRPMI 배지를 넣어주었다.2) Jurkat cell line (ACTT No. TIB-152) used as feeder cells was 37 ° C., 5% CO 2 in 75T flask in human RPMI medium containing 10% FBS and 1% penicillin / streptomycin in RPMI1640 medium. Incubated under the conditions of. In addition, CEM cells (ACTT No. CCL-119) and K562 cells (ACTT No. CCL-243), which are other blood cancer cell lines, were also cultured in 75 T flasks at 37 ° C. and 5% CO 2 in human RPMI medium. Once every 2-3 days, hRPMI medium added with 500 U / ml of IL-2 was added. After harvesting all cells at 5-day intervals, fresh IL-2 and hRPMI medium were added.
3) 세포수를 헤마토사이토미터를 이용하여 측정한 후 1×106/ml의 농도의 Jurkat, CEM 및 K562 세포주에 2.25 Gy/min의 세기로 300 Gy 방사선을 조사하였다. IL-2가 500 U/ml 농도로 첨가된 hRPMI 배지를 24웰 플레이트에 넣은 후, 방사선 조사된 각각의 Jurkat, CEM 및 K562 세포주와 상기 1)에서 분리한 단핵구를 각각 1:1 비율로 37℃, 5% CO2가 공급되는 배양기에서 배양하였다. 또한 상기와 동일한 방법을 이용하여 영양세포주를 첨가하지 않고 단핵구에 IL-2만 처리한 군을 대조군으로 하였다.
3) The cell number was measured using a hematocytometer, and then 300 Gy radiation was irradiated at the intensity of 2.25 Gy / min to Jurkat, CEM and K562 cell lines at a concentration of 1 × 10 6 / ml. HRPMI medium added with 500 U / ml of IL-2 was added to a 24-well plate, and each of the irradiated Jurkat, CEM, and K562 cell lines and the monocytes isolated from 1) were 37 ° C. in a 1: 1 ratio. , And incubated in an incubator fed with 5% CO 2 . In addition, using the same method as the control group was treated with IL-2 to monocytes without adding the feeder cell line as a control.
실시예 2. NK 세포의 증식능 측정Example 2. Measurement of proliferative capacity of NK cells
영양세포가 자연 살해세포의 증식에 미치는 영향을 보기 위하여, 실시예 1에서 사용한 방법으로서 방사선이 조사된 Jurkat 세포와 단핵구(PBMC)를 IL-2로 공동배양한 실험군과, 단핵구만을 IL-2로 배양한 대조군을 각각 37℃, 5% CO2가 공급되는 배양기에서 배양한 후, 0, 5, 10, 16일에 각각 세포수를 측정하고 형광이 부착된 CD56과 CD3에 대한 항체를 이용하여 염색한 후 유세포 분석기(flow cytometry)로 분석하여 NK 세포(CD56+, CD3-)군을 분석하였다. 그 후 NK 세포수를 총 PBMC와 NK 세포의 분포를 이용하여 계산하였다.In order to see the effect of feeder cells on the proliferation of natural killer cells, the method used in Example 1, the experimental group co-cultured with irradiated Jurkat cells and monocytes (PBMC) with IL-2, and only monocytes with IL-2 The cultured controls were incubated in an incubator with 37 ° C. and 5% CO 2 , respectively, and then cell numbers were measured at 0, 5, 10 and 16 days, respectively, and stained using antibodies to CD56 and CD3 with fluorescence. After analysis by flow cytometry, NK cells (CD56 +, CD3-) groups were analyzed. NK cell number was then calculated using the distribution of total PBMC and NK cells.
그 결과 도 1에서 볼 수 있듯이, NK 세포의 증가는 IL-2만을 처리한 대조군(●)과 IL-2와 영양세포를 함께 처리한 실험군(□)에서 배양 5일째까지는 거의 없었으나, 10일째에는 대조군에 비해 실험군에서 약 65배 증가하였고 16일째에는 약 130배 증가함을 확인하였다.
As a result, as shown in Figure 1, the increase of NK cells in the control group treated with IL-2 (●) and the experimental group treated with both IL-2 and feeder cells (□) was almost until the 5th culture, but on the 10th day In comparison with the control group was increased about 65 times in the experimental group was confirmed to increase by about 130 times at 16 days.
실시예 2. NK 세포의 enrichment 정도 측정Example 2 Determination of Enrichment Level of NK Cells
Jurkat 세포와 IL-2로 공동 배양한 실험군에서 NK 세포로의 enrichment 정도를 측정하기 위하여, 실시예 1에서 방사선 조사된 Jurkat 세포와 단핵구를 IL-2로 공동 배양한 실험군과 단핵구만을 IL-2로 배양한 대조군을 각각 37℃, 5% CO2가 공급되는 배양기에서 배양한 후, 0, 5, 10, 15일에 각각 형광이 부착된 CD56, CD3 항체를 이용하여 염색한 후 유세포 분석기로 분석하였다. 또한 NK 세포로 enrichment가 되는 시점을 파악하기 위하여 같은 방법으로 0, 3, 5일에 측정하였고, 6일부터 10일까지 매일 NK 세포의 분포를 유세포 분석기로 측정하였다.In order to measure the enrichment of NK cells in the experimental group co-cultured with Jurkat cells and IL-2, only the experimental group and monocytes co-cultured with IL-2 with Jurkat cells and monocytes irradiated in Example 1 were IL-2. The cultured controls were incubated in an incubator fed with 37 ° C. and 5% CO 2 , respectively, and stained using CD56 and CD3 antibodies with fluorescence at 0, 5, 10 and 15 days, respectively, and analyzed by flow cytometry. . In addition, to determine the time of enrichment with NK cells were measured on the 0, 3, 5 days by the same method, and the distribution of NK cells daily from 6 to 10 days by flow cytometry.
도 2a에서, 15일 경과 후 IL-2만 처리한 대조군에 비해 IL-2에 방사선을 조사한 Jurkat 세포주를 영양세포로 처리한 실험군에서 자연 살해세포의 표현형인 CD56+, CD3- 세포의 비율이 92% 이상까지 증가함을 확인하였다. In FIG. 2A, the ratio of CD56 + and CD3- cells, which are phenotypes of natural killer cells, was 92% in the experimental group treated with Jurkat cell line irradiated with IL-2 as a feeder cell compared to the control group treated with IL-2 after 15 days. It confirmed that the increase to the above.
도 2b와 2c에서, 상기와 같은 자연 살해세포의 enrichment가 어느 시점에서 되는지를 확인하기 위해 시간별로 자연 살해세포의 표현형을 분석한 결과, 5일째까지는 큰 변화가 없었으나, 6일째부터 영양세포를 처리한 실험군에서 자연 살해세포의 표현형인 CD56+, CD3- 세포의 비율이 증가하여 10일 정도에는 PBMC 내에 자연 살해세포의 비율이 80% 이상임을 확인할 수 있었다.
In Figures 2b and 2c, as a result of analyzing the phenotype of natural killer cells by time to confirm at what time the enrichment of the natural killer cells as described above, there was no significant change until day 5, the feeder cells from day 6 In the treated experimental group, the ratio of CD56 + and CD3- cells, which are phenotypes of natural killer cells, increased, indicating that the ratio of natural killer cells in PBMC was about 80% or more in about 10 days.
실시예 3. 다른 혈액암 세포주를 이용한 NK 세포의 enrichment 정도 비교Example 3. Comparison of enrichment levels of NK cells using different hematologic cancer cell lines
본 발명에서 영양세포로 사용된 Jurkat 세포 이외에 다른 혈액암 세포주의 NK enrichment 정도를 파악하기 위하여, CEM 및 K562 세포주를 300 Gy의 방사선으로 조사한 후 실시예 1에서 분리된 단핵구와 IL-2를 넣고 공동배양한 후, 상기 실시예 2와 동일한 방법을 이용하여 0, 4, 10, 14일째에서 NK세포의 분포를 유세포 분석기로 분석하였다. 동일하게 IL-2만 처리한 군을 대조군으로 하였다.In order to determine the degree of NK enrichment of other hematological cancer cell lines in addition to the Jurkat cells used as feeder cells in the present invention, CEM and K562 cell lines were irradiated with 300 Gy of radiation, and the monocytes and IL-2 isolated in Example 1 were added to the cavity. After incubation, the distribution of NK cells was analyzed by flow cytometry at 0, 4, 10 and 14 days using the same method as in Example 2. Similarly, the group treated with IL-2 was used as a control.
도 3에서, CEM 세포주는 IL-2만 처리한 대조군과 유사한 NK 세포의 분포를 보였고, K562 세포주의 경우 15일 경과 후 약 40%의 NK 세포의 분포를 보였으며, 본 발명의 Jurkat 세포주는 약 80%의 NK 세포의 분포를 보였다. 따라서 본 발명의 방사선 조사된 Jurkat 세포에 의한 자연 살해세포 유도법은 Jurkat 세포에 특이적인 반응에 의해 이루어짐을 확인하였다.
In FIG. 3, the CEM cell line showed a distribution of NK cells similar to the control group treated only with IL-2, and the K562 cell line showed a distribution of about 40% of NK cells after 15 days. The Jurkat cell line of the present invention was about It showed distribution of NK cells of 80%. Therefore, natural killer cell induction by irradiated Jurkat cells of the present invention was confirmed to be made by a specific response to Jurkat cells.
실시예 4. NK 세포의 살상능 측정Example 4. Determination of Killing Capacity of NK Cells
본 발명에 따른 Jurkat 세포를 이용하여 생성된 NK 세포의 암세포 살상능을 평가하기 위하여 종양세포(K562, Jurkat)를 표적세포로 하여 Cr51 release assay를 수행하였다. 구체적으로 암세포인 K562와 Jurkat 세포의 세포수를 측정한 후 동위원소인 51Cr을 각각 암세포에 표지하여 1시간 동안 세포배양기에서 반응시켰다. 동위원소로 표지된 암세포를 hRPMI 배지로 동위원소를 2회 씻어준 후 Jurkat 세포와 IL-2로 배양한 단핵구와 IL-2만을 처리한 단핵구의 세포수를 hematocytometer를 이용하여 측정한 후 동위원소가 표지된 암세포와 20:1, 7:1, 2:1, 0.7:1의 비율로 4시간동안 공동 배양하였다. 4시간 후 2500 rpm에서 5분간 원심분리한 후 상등액을 튜브에 넣어 감마 카운터(gamma counter)로 측정하였다.Cr51 release assay was performed using tumor cells (K562, Jurkat) as target cells to evaluate cancer cell killing ability of NK cells generated using Jurkat cells according to the present invention. Specifically, after measuring the cell number of cancer cells K562 and Jurkat cells, the 51 isotope 51 Cr was labeled on the cancer cells and reacted in the cell incubator for 1 hour. Isotope-labeled cancer cells were washed twice with hRPMI medium, and then the cell counts of monocytes treated with Jurkat and IL-2 and monocytes treated with IL-2 were measured using a hematocytometer. The labeled cancer cells were co-cultured for 4 hours at a ratio of 20: 1, 7: 1, 2: 1, 0.7: 1. After 4 hours, centrifugation was performed at 2500 rpm for 5 minutes, and the supernatant was put into a tube and measured by a gamma counter.
도 4에서, IL-2와 방사선 조사된 Jurkat 세포로 공동배양한 실험군(■)에서 암세포 살상능이 대조군(△)에 비해 약 3-5배 현저히 증가함을 확인하였다. 또한, E:T(효력세포:표적세포)의 비율이 0.7:1 임에도 불구하고 상당한 살상능을 보유하고 있음을 확인할 수 있었다.In FIG. 4, it was confirmed that cancer cell killing ability in the experimental group co-cultured with IL-2 and irradiated Jurkat cells (■) was significantly increased about 3-5 times compared to the control group (△). In addition, despite the ratio of E: T (effective cell: target cell) of 0.7: 1, it was confirmed that it has significant killing ability.
이러한 결과들을 종합해 볼 때, 본 발명에 따른 IL-2와 방사선 조사된 Jurkat 세포를 처리한 말초혈액 단핵구의 경우 자연 살해세포로의 높은 enrichment 뿐 만 아니라 높은 암세포 살상능을 보임을 확인 할 수 있었다.
Based on these results, it was confirmed that peripheral blood mononuclear cells treated with IL-2 and irradiated Jurkat cells according to the present invention showed high cancer cell killing ability as well as high enrichment of natural killer cells. .
실시예 5. 암환자의 말초혈액 단핵구에서 적용 여부Example 5 Application in Peripheral Blood Monocytes of Cancer Patients
본 발명에 따른 방사선 조사된 Jurkat 세포주를 이용한 NK 세포 생성법이 위암과 대장암 환자군에서도 적용되는지를 파악하기 위하여, 고려대학교 안암병원과 구로병원에서 암환자 검체(말초 정맥혈)를 3-5 ml 정도씩 얻어 PBMC를 분리하여 유세포분석기로 NK 세포의 분포도를 조사하였고, 방사선이 조사된 Jurkat 세포와 IL-2를 단핵구(PBMC)에 넣고 배양하였고, NK 세포의 분포를 15일후 측정하였다. 또한 15일후 NK 세포의 암세포 살상능을 측정하기 위해 Jurkat 세포를 타겟(target) 세포로 하여 실시예 4와 동일한 방법을 이용하여 10:1 비율로 암세포 살상능을 측정하였다.In order to determine whether NK cell generation method using the irradiated Jurkat cell line according to the present invention is applied to gastric and colorectal cancer patients, cancer samples (peripheral venous blood) of 3-5 patients each in Korea University Anam Hospital and Guro Hospital The PBMCs were isolated and examined for distribution of NK cells by flow cytometry. The irradiated Jurkat cells and IL-2 were cultured in monocytes (PBMC), and the distribution of NK cells was measured after 15 days. In addition, in order to measure cancer cell killing ability of NK cells after 15 days, cancer cell killing ability was measured at a ratio of 10: 1 using Jurkat cells as target cells using the same method as in Example 4.
도 5에서, IL-2만 처리한 대조군(PBMC)의 경우 단핵구에서 자연 살해세포의 분포가 10% 정도였던 반면, Jurkat 세포를 함께 배양한 실험군(PBMC+Jurkat)에서는 70%가 넘는 자연 살해세포 분포를 보였으며, 암세포 살상능도 현격히 증가함을 확인하였다.
In FIG. 5, the control group treated only with IL-2 (PBMC) had a distribution of about 10% of natural killer cells in monocytes, whereas more than 70% of natural killer cells in the experimental group (PBMC + Jurkat) cultured with Jurkat cells. It showed distribution, and cancer cell killing ability was also markedly increased.
실시예 6. NK 세포의 증식기간 연장Example 6. Extension of Proliferation Period of NK Cells
본 발명에 따른 방사선 조사된 Jurkat 세포를 이용한 NK 세포가 좀 더 오랜기간 동안 증식능을 유지시켜 주기 위하여, 본 발명자들은 말초혈액에서 분리한 단핵구에 방사선 조사된 Jurkat 세포와 IL-2를 넣어준 후 37℃, 5% CO2가 유지되는 배양기에서 10일간 배양한 후, 10일째 다시 방사선이 조사된 Jurkat 세포와 IL-2를 넣어주었고, 20일까지 NK 세포의 증식을 헤마토사이토미터를 이용하여 세포수를 측정하고 유세포 분석기를 이용하여 NK 세포의 분포를 측정하여 정확한 NK 세포수를 측정하였다.In order to maintain the proliferative capacity of NK cells using irradiated Jurkat cells according to the present invention for a longer period of time, the present inventors put irradiated Jurkat cells and IL-2 in monocytes isolated from peripheral blood 37 After incubation for 10 days in an incubator maintained at 5 ° C. and 5% CO 2 , the irradiated Jurkat cells and IL-2 were added at 10 days, and the proliferation of NK cells by 20 days was performed using a hematocytometer. The number of cells was measured and the distribution of NK cells was measured using a flow cytometer to determine the exact number of NK cells.
도 6a와 6b에서, IL-2만 처리한 대조군(◆) 및 IL-2와 Jurkat 세포를 처리한 실험군(□)과 비교하여 10일째에 다시 방사선이 조사된 Jurkat 세포와 IL-2를 처리한 추가실험군(△)에서 자연 살해세포의 세포수가 15일과 20일째에도 증가함을 확인하였다.6a and 6b, compared with the control group treated with IL-2 (◆) and the experimental group treated with IL-2 and Jurkat cells (□) treated with Jurkat cells and IL-2 irradiated again on
따라서 본 발명에 따른 IL-2와 방사선이 조사된 Jurkat 세포를 이용한 자연 살해세포의 유도방법은 기존의 방법에 비하여 경제적이며 간단하게 강력한 암세포 살상능을 갖는 자연 살해세포유도 방법으로서, 현재 난치병질환이나 암환자의 치료와 생존률을 증가시킬 수 있는 세포치료제로서의 가능성을 확인했다고 할 수 있다.Therefore, the method of inducing natural killer cells using IL-2 and irradiated Jurkat cells according to the present invention is a natural killer cell-derived method having economical and simple powerful cancer cell killing ability compared to the conventional method, It can be said that it has confirmed the possibility as a cell therapy that can increase the treatment and survival rate of cancer patients.
Claims (7)
Isolating monocytes from peripheral blood, and NK cell proliferation method comprising the step of culturing the monocytes and irradiated blood cancer Jurkat cells in a culture medium added to the cytokine.
The method of claim 1, wherein said peripheral blood is obtained from a normal or cancer patient.
The method of claim 1, wherein said cytokine is IL-2.
The method of claim 1, further comprising adding IL-2 and Jurkat cells that were irradiated on days 1 to 15 of the culture.
The method of claim 1, wherein the method has a CD3-CD56 + cell distribution of at least 80% of the cultured cells.
Separation of monocytes from peripheral blood, and NK cell production method of increased cancer cell killing capacity comprising the step of culturing the monocytes and irradiated blood cancer Jurkat cells in a culture medium containing cytokines.
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