KR102256272B1 - Method for Mass Proliferation of NK cells isolated from whole blood - Google Patents
Method for Mass Proliferation of NK cells isolated from whole blood Download PDFInfo
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- KR102256272B1 KR102256272B1 KR1020190070143A KR20190070143A KR102256272B1 KR 102256272 B1 KR102256272 B1 KR 102256272B1 KR 1020190070143 A KR1020190070143 A KR 1020190070143A KR 20190070143 A KR20190070143 A KR 20190070143A KR 102256272 B1 KR102256272 B1 KR 102256272B1
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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Abstract
본 발명은 전혈로부터 자연살상세포(NK cell)의 직접 분리 방법 및 자연살상세포의 고효율 대량증식 방법에 관한 것으로서, 구체적으로 전혈로부터 말초혈액단핵구(PBMC)의 분리 없이 항체 칵테일, 마그네틱 미세구슬 및 자석만을 이용하여 자연살상세포를 직접 분리하는 방법과 상기 분리된 자연살상세포를 활성화 지지세포 (activated feeder cells)를 이용하여 고효율로 대량 증식하는 방법에 관한 것이다.
본 발명의 자연살상세포 분리방법에 따르면, 기존 말초혈액단핵세포로부터 분리하는 방법에 비하여 소량의 전혈로부터 NK 세포의 회수율을 높이고 그 분리 시간을 획기적으로 줄일 수 있다. 또한, 본 발명의 활성화 지지세포 (activated feeder cells) 제조 방법에 따르면 T 세포의 불활화 및 NK 세포에 대한 민감성을 증가시키고 분리된 NK 세포를 10,000배 이상 증식시킬 수 있다. 또한, 본 발명의 자연살상세포 대량증식 방법에 따르면, 다양한 활성화 수용체의 발현이 증가되고 표적종양세포에 대한 항종양 세포독성을 강하게 증가시킬 수 있다. 본 발명에 따라 대량으로 증식된 세포독성이 높은 NK 세포는 암면역 세포치료제 조성물의 유효성분으로 이용될 수 있다.The present invention relates to a method for direct separation of natural killer cells (NK cells) from whole blood and a method for high-efficiency mass proliferation of natural killer cells, and specifically, antibody cocktails, magnetic microbeads and magnets without separation of peripheral blood mononuclear cells (PBMC) from whole blood. The present invention relates to a method of directly separating natural killer cells using a gulf, and a method of mass-proliferating the isolated natural killer cells with high efficiency using activated feeder cells.
According to the method of isolating natural killer cells of the present invention, it is possible to increase the recovery rate of NK cells from a small amount of whole blood and significantly reduce the separation time compared to the conventional method of separating from peripheral blood mononuclear cells. In addition, according to the method for producing activated feeder cells of the present invention, inactivation of T cells and sensitivity to NK cells can be increased, and isolated NK cells can be proliferated 10,000 times or more. In addition, according to the method for mass proliferation of natural killer cells of the present invention, expression of various activating receptors can be increased and antitumor cytotoxicity to target tumor cells can be strongly increased. High cytotoxic NK cells proliferated in a large amount according to the present invention can be used as an active ingredient of a cancer immune cell therapy composition.
Description
본 발명은 전혈로부터 자연살상세포(NK cell)의 직접 분리 방법 및 자연살상세포의 고효율 대량증식 방법에 관한 것으로서, 구체적으로 전혈로부터 말초혈액단핵구(PBMC)의 분리 없이 항체 칵테일, 마그네틱 미세구슬 및 자석만을 이용하여 자연살상세포를 직접 분리하는 방법과 상기 분리된 자연살상세포를 활성화 지지세포 (activated feeder cells)를 이용하여 고효율로 대량 증식하는 방법에 관한 것이다.The present invention relates to a method for direct separation of natural killer cells (NK cells) from whole blood and a method for high-efficiency mass proliferation of natural killer cells, and specifically, antibody cocktails, magnetic microbeads and magnets without separation of peripheral blood mononuclear cells (PBMC) from whole blood. The present invention relates to a method of directly separating natural killer cells using a gulf, and a method of mass-proliferating the isolated natural killer cells with high efficiency using activated feeder cells.
자연살상세포 (Natural Killer cell; NK cell)는 선천성면역을 담당하는 인체 면역 세포 중의 하나로 바이러스나 암세포에 대한 선택적인 살해능력을 보이는 강력한 세포독성림프구로 알려져 있다. NK 세포는 다양한 면역수용체를 통해 암세포와 정상세포를 구분할 수 있으며 다른 면역세포와는 달리 추가적인 항원인식 없이 표적세포만을 선택적으로 제거할 수 있다 [1, 2]. Natural killer cells (NK cells) are one of the human immune cells responsible for innate immunity and are known as powerful cytotoxic lymphocytes that show selective killing ability against viruses or cancer cells. NK cells can differentiate between cancer cells and normal cells through various immunoreceptors, and unlike other immune cells, only target cells can be selectively removed without additional antigen recognition [1, 2].
NK 세포의 기능은 표적세포 (암세포)에 반응하는 다양한 활성화 수용체 (activating receptor) 및 억제성 수용체 (inhibitory receptor)의 힘의 균형에 의해 조절된다 [1, 3]. NK 세포는 주조직복합성적합체 (major histocompatibility complex; MHC) class I의 발현이 낮거나 결여된 표적세포 (암세포)에 매우 민감하게 반응한다. 하지만 강력한 억제 수용체인 MHC class I의 발현이 높을 경우 효과적으로 표적세포 (암세포)를 제거하지 못하게 된다. 이런 단점을 해결하기 위해서는 NK 세포의 활성화 수용체의 발현을 증가시키는 방법이 필요하다 [1, 4]. The function of NK cells is regulated by the balance of the forces of various activating and inhibitory receptors that respond to target cells (cancer cells) [1, 3]. NK cells respond very sensitively to target cells (cancer cells) with low or no expression of the major histocompatibility complex (MHC) class I. However, when the expression of MHC class I, a potent inhibitory receptor, is high, it is not possible to effectively remove target cells (cancer cells). In order to overcome these shortcomings, a method of increasing the expression of NK cell activation receptors is needed [1, 4].
NK 세포의 활성은 CD16 (Fcγ-receptor), natural killer group 2D (NKG2D), 2B4, 그리고 natural cytotoxicity receptors (NCRs; NKp30, NKp44, NKp46, and NKp80) 등과 같은 다양한 활성화 수용체에 의해 매개되며 [1, 5] NK 세포의 더욱 강력한 활성을 유도하기 위해서는 이들 수용체들의 결합이 요구된다 [6-9]. NK cell activity is mediated by various activating receptors such as CD16 (Fcγ-receptor), natural killer group 2D (NKG2D), 2B4, and natural cytotoxicity receptors (NCRs; NKp30, NKp44, NKp46, and NKp80) [1, 5] In order to induce more potent activity of NK cells, binding of these receptors is required [6-9].
암환자의 경우 정상인에 비해 NK 세포의 수가 감소되어 있거나 기능적인 측면에서 결함이 발견되고 있으며 이러한 부분은 다양한 질병 발생의 원인이 되고 있으며 특히 암 발생과 밀접한 관련성이 있는 것으로 알려지고 있다. 이런 이유로 최근에는 인체내 NK 세포의 활성이 암 발생 억제의 중요한 지표로 간주되고 있으며 이를 측정하기 위한 다양한 방법들이 제시되고 있다. 또한 강력한 세포독성을 가진 NK 세포를 체외에서 대량으로 증식시켜 임상에 적용하고자 다양한 방법들이 고안되고 있다. In the case of cancer patients, the number of NK cells is reduced compared to normal people, or defects in functional aspects are found, and these parts are known to be the cause of various diseases and are particularly closely related to cancer incidence. For this reason, in recent years, the activity of NK cells in the human body is regarded as an important indicator of cancer incidence, and various methods for measuring this have been proposed. In addition, various methods have been devised to proliferate large amounts of NK cells with strong cytotoxicity in vitro and apply them to clinical practice.
NK 세포는 제대혈, 골수, 배아줄기세포, 말초혈액 으로부터 얻을 수 있으며 각기 다양한 방법으로 대량으로 증식하기 위한 방법들이 개발되고 있다. 최근 연구에서는 NK 세포를 대량으로 배양하기 위해 방사선이 조사된 종양세포기반 지지세포 (feeder cells; 종양셀라인, 유전적으로 조작된 K562 세포 및 Epstein-Bar virus를 이식 시킨 lymphoblastoid 세포 등)를 많이 이용하고 있다 [10-14]. 하지만 이런 방법들이 NK 세포를 대량으로 증식시키기는 하지만 위험한 종양세포를 배양과정 중에 사용하기 때문에 임상적용을 위해서는 첨가된 종양세포가 완전히 불활화 및 제거되었다는 것을 증명하여야 한다. 이런 과정은 매우 복잡하고 완전히 제거되지 않을 경우 치명적인 결과를 초래할 수 있다. NK cells can be obtained from umbilical cord blood, bone marrow, embryonic stem cells, and peripheral blood, and methods for mass proliferation in various ways are being developed. In recent studies, radiation-irradiated tumor cell-based feeder cells (tumor cell lines, genetically engineered K562 cells, and lymphoblastoid cells transplanted with Epstein-Bar virus, etc.) have been widely used to cultivate NK cells in large quantities. There is [10-14]. However, although these methods proliferate NK cells in large quantities, since dangerous tumor cells are used during the culture process, it must be proved that the added tumor cells are completely inactivated and removed for clinical application. This process is very complex and can have fatal consequences if not completely eliminated.
다른 방법으로는 종양세포기반 지지세포 대신에 말초혈액단핵구를 지지세포로 이용하여 NK 세포를 증식시키는 방법이 개발되었으나 증식율이 다소 낮다 (1,000배 미만)는 문제점이 있었다. 이런 문제점을 해결하기 위해 선행 발명에서는 5,000배 이상으로 NK 세포를 증식시키는 방법을 개발하였다 [9], 하지만 이 방법은 초기 전처리 시간이 다소 길고 NK 세포 분리 효율이 낮다는 단점이 있었다. As another method, a method of proliferating NK cells by using peripheral blood mononuclear cells as support cells instead of tumor cell-based support cells has been developed, but there is a problem that the proliferation rate is somewhat low (less than 1,000 times). In order to solve this problem, the prior invention has developed a method of proliferating NK cells by more than 5,000 times [9], but this method has a disadvantage in that the initial pretreatment time is somewhat longer and the efficiency of NK cell separation is low.
따라서, 본 발명의 주된 목적은 소량의 전혈로부터 NK 세포의 회수율을 높이고 분리 시간을 줄일 수 있는 NK 세포의 분리 방법을 제공하는 데 있다.Accordingly, the main object of the present invention is to provide a method for isolating NK cells capable of increasing the recovery rate of NK cells from a small amount of whole blood and reducing the separation time.
본 발명의 다른 목적은 T 세포의 불활화 및 NK 세포에 대한 민감성을 증가시킨 활성화 지지세포 (activated feeder cells)의 제조방법을 제공하는데 있다.Another object of the present invention is to provide a method for producing activated feeder cells, which has increased sensitivity to NK cells and inactivation of T cells.
본 발명의 다른 목적은 다양한 종양세포주에 대해 강력한 세포독성을 나타내는 활성화 NK 세포의 대량 증식방법을 제공하는데 있다.Another object of the present invention is to provide a method for mass proliferation of activated NK cells exhibiting strong cytotoxicity against various tumor cell lines.
본 발명의 다른 목적은 전혈에서 NK 세포 수의 측정하는 방법 및 분리된 NK 세포의 수율을 측정하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method of measuring the number of NK cells in whole blood and a method of measuring the yield of isolated NK cells.
본 발명에서는 안전성과 세포독성이 높은 NK 세포를 대량으로 배양하기 방법으로 첫째, 전혈로부터 NK 세포의 회수율을 높이는 방법과 그 분리 시간을 줄이는 방법을 개발하였다. 둘째, 분리된 NK 세포를 10,000배 이상 증식시킬 수 있는 활성화 지지세포 (activated feeder cells)의 제조 방법을 개발하였다. 본 발명에서는 NK 세포의 강력한 증식을 유도하기 위한 방법으로 말초혈액단핵세포 (PBMCs)에 CD3 항체, IFN-r, IL-2을 이용하여 활성화 시키고 이 후 방사선을 조사하여 T 세포의 불활화 및 NK 세포에 대한 민감성을 증가시킨 활성화 지지세포 (activated feeder cells)를 제조하였다.In the present invention, as a method for culturing NK cells having high safety and cytotoxicity in large quantities, first, a method of increasing the recovery rate of NK cells from whole blood and a method of reducing the separation time was developed. Second, a method for producing activated feeder cells capable of proliferating isolated NK cells 10,000 times or more was developed. In the present invention, as a method for inducing strong proliferation of NK cells, peripheral blood mononuclear cells (PBMCs) are activated using CD3 antibodies, IFN-r, and IL-2, and then irradiated with radiation to inactivate T cells and NK cells. Activated feeder cells with increased sensitivity to cells were prepared.
본 발명에 따라 대량으로 증식된 NK 세포는 다양한 종양세포주에 대해 강력한 세포독성을 나타내었고 췌장암 및 대장암을 대상으로 한 동물실험에서 종양성장을 효과적으로 억제하였다. 이 방법은 소량의 혈액 (전혈)으로부터 NK 세포의 분리 효율을 획기적으로 높이고 시간 절약적이며 강력한 활성화 지지세포 (activated feeder cells) 제조를 통한 항암면역세포치료를 위한 세포독성이 높은 NK 세포를 대량으로 증식시킬 수 있는 방법을 제공해준다. NK cells proliferated in large quantities according to the present invention exhibited strong cytotoxicity against various tumor cell lines and effectively inhibited tumor growth in animal experiments targeting pancreatic cancer and colon cancer. This method dramatically increases the efficiency of separation of NK cells from a small amount of blood (whole blood), saves time, and produces high-cytotoxic NK cells for anticancer immune cell therapy in large quantities through the production of powerful activated feeder cells. It provides a way to multiply.
본 발명의 한 양태에 따르면, 본 발명은 다음 단계들을 포함하는 전혈로부터 말초혈액단핵구(PBMC)의 분리 없이 직접 자연살상세포(NK cell)를 분리하는 방법을 제공한다:According to one aspect of the present invention, the present invention provides a method of directly separating natural killer cells (NK cells) without separation of peripheral blood mononuclear cells (PBMC) from whole blood comprising the following steps:
a) 인간의 말초혈액이 분주된 튜브에 항체 칵테일(CD3, CD4, CD19, CD36, CD66b, CD123, glycophorin A)과 마그네틱 미세구슬(magnetic microbead)을 넣고 혼합한 후 실온에서 3-6분간 반응시키는 단계;a) Add an antibody cocktail (CD3, CD4, CD19, CD36, CD66b, CD123, glycophorin A) and magnetic microbeads to a tube into which human peripheral blood was dispensed, and then react at room temperature for 3-6 minutes. step;
b) 상기 반응이 끝난 후 튜브를 5,000 가우스 이상의 자력을 가진 자석에 넣어 실온에서 3-6분간 방치하는 단계b) After the reaction is over, the tube is placed in a magnet having a magnetic force of 5,000 Gauss or more and left at room temperature for 3-6 minutes.
c) 새로운 튜브에 자석에 붙지 않은 혈액을 천천히 따라 붓고 마그네틱 미세구슬을 첨가하여 혼합 후 실온에서 3-6분간 반응시키는 단계;c) slowly pouring non-magnetized blood into a new tube, adding magnetic microbeads, mixing, and reacting at room temperature for 3-6 minutes;
d) 상기 반응이 끝난 후 튜브를 5,000 가우스 이상의 자력을 가진 자석에 넣어 실온에서 3-6분간 방치하는 단계;d) after the reaction is over, the tube is placed in a magnet having a magnetic force of 5,000 gauss or more and allowed to stand at room temperature for 3-6 minutes;
e) 새로운 튜브에 자석에 붙지 않은 혈액을 천천히 따라 붓고 항체 칵테일과 마그네틱 미세구슬을 a)단계의 1/5 ~ 1/15의 양으로 넣고 혼합한 후 실온에서 3-6분간 반응시키는 단계;e) slowly pouring non-magnetized blood into a new tube, adding the antibody cocktail and magnetic microbeads in the amount of 1/5 to 1/15 of step a), mixing, and reacting at room temperature for 3-6 minutes;
f) 상기 반응이 끝난 후 튜브를 5,000 가우스 이상의 자력을 가진 자석에 넣어 실온에서 3-6분간 방치하는 단계; 및f) after the reaction is over, the tube is placed in a magnet having a magnetic force of 5,000 Gauss or more and allowed to stand at room temperature for 3-6 minutes; And
g) 새로운 튜브에 자석에 붙지 않은 혈액을 천천히 옮기고 원심분리하여 자연살상세포(NK cell)를 분리하는 단계.g) Separating natural killer cells (NK cells) by slowly transferring the non-magnetized blood to a new tube and centrifuging.
본 발명에 있어서, 상기 자연살상세포(NK cell) 분리 방법은 면역자기 네가티브 선택(immunomagnetic negative selection) 방법을 이용하고 있으므로, a) 단계에서 항체 칵테일은 NK 세포 이외의 세포들의 마커들, 예컨대 CD3, CD4, CD19, CD36, CD66b, CD123, glycophorin A 등에 대한 항체들이 포함될 수 있으며, 이들 항체는 biotin이 결합되어 있어서 monoclonal anti-biotin antibody, streptavidin 또는 dextran이 부착된 마그네틱 미세구슬(magnetic microbead)과 결합할 수 있다.In the present invention, the method for isolating natural killer cells (NK cells) uses an immunomagnetic negative selection method, so the antibody cocktail in step a) includes markers of cells other than NK cells, such as CD3, Antibodies against CD4, CD19, CD36, CD66b, CD123, glycophorin A, etc. may be included, and these antibodies are biotin-bound and thus can bind to monoclonal anti-biotin antibodies, streptavidin, or dextran-attached magnetic microbeads. I can.
본 발명에 있어서, 상기 b), d), f) 단계에서 자석은 NK 세포이외의 혈액 세포들이 결합된 마그네틱 미세구슬(magnetic microbead)을 붙여서 제거할 수 있는 어떠한 자력도 가질 수 있으며, 바람직하게는 5,000 가우스 이상, 더욱 바람직하게는 5,000 내지 10,000 가우스의 자력을 가질 수 있다. 또한, 본 발명에서는, MACS (Magnetic-activated cell sorting) 칼럼을 사용하지 않고 튜브와 자석만으로 NK 세포 이외의 세포들을 손쉽게 제거할 수 있다. In the present invention, in the steps b), d), and f), the magnet may have any magnetic force that can be removed by attaching magnetic microbeads to which blood cells other than NK cells are bound, and preferably It may have a magnetic force of 5,000 Gauss or more, more preferably 5,000 to 10,000 Gauss. In addition, in the present invention, cells other than NK cells can be easily removed with only a tube and a magnet without using a MACS (Magnetic-activated cell sorting) column.
본 발명에 있어서, 상기 c) 단계에서, 항체 카테일 없이 마그네틱 미세구슬만을 첨가하는 이유는 이미 a) 단계에서 넣어준 항체 칵테일이 남아 있어서 다시 넣어줄 필요가 없기 때문이다. 또한, 상기 e) 단계에서, 항체 칵테일과 마그네틱 미세구슬을 1/5 ~ 1/15의 양으로 넣어준 이유는 마지막으로 남아 있는 NK 세포이외의 혈액세포들을 완전히 제거하기 위한 것이다. 본 발명자들은 상기 a), c), e) 단계를 중첩적으로 수행함으로써 소량의 전혈로부터 NK 세포의 회수율을 높이고 그 분리 시간을 획기적으로 줄일 수 있음을 확인하였다.In the present invention, in step c), only magnetic microbeads are added without an antibody category because the antibody cocktail previously added in step a) remains, so there is no need to add it again. In addition, in step e), the reason for adding the antibody cocktail and magnetic microbeads in an amount of 1/5 to 1/15 is to completely remove the blood cells other than the remaining NK cells. The present inventors have confirmed that by performing the steps a), c), and e) overlapping, the recovery rate of NK cells from a small amount of whole blood can be increased and the separation time can be drastically reduced.
본 발명에 있어서, 기존의 전혈로부터 말초혈액단핵구(PBMC) 분리 후 간접적으로 자연살상세포(NK cell)를 분리하는 방법에 비하여 소량의 혈액으로부터 NK 세포의 분리 효율을 높이고 분리 시간을 줄이는 것을 특징으로 하는 자연살상세포(NK cell)를 분리하는 방법을 제공한다. 종래에는 말초혈액으로부터 먼저 원심분리 및 밀도구배 등을 통해 말초혈액단핵구(PBMC)을 분리한 후, MACS 칼럼을 이용하여 magnetic microbead가 부착된 항체(antibody)와 반응시켜서 자연살상세포(NK cell)를 분리하였는데, 이 경우 비교적 많은 양의 혈액이 필요하고 NK 세포의 분리시간이 오래 걸리며, NK 세포의 분리 효율이 높지 않은 문제점이 있었으나, 본 발명은 이들 문제점을 해결하였다.In the present invention, compared to the method of indirectly separating natural killer cells (NK cells) after separating peripheral blood mononuclear cells (PBMC) from whole blood, it is characterized in that the separation efficiency of NK cells from a small amount of blood is increased and the separation time is reduced. It provides a method for isolating natural killer cells (NK cells). Conventionally, peripheral blood mononuclear cells (PBMC) are first separated from peripheral blood through centrifugation and density gradient, and then natural killer cells (NK cells) are reacted with an antibody to which magnetic microbeads are attached using a MACS column. However, in this case, a relatively large amount of blood is required, the separation time of NK cells takes a long time, and there is a problem that the separation efficiency of NK cells is not high, but the present invention solves these problems.
본 발명의 다른 양태에 따르면, 본 발명은 다음 단계들을 포함하는 자연살상세포(NK cell) 증식을 위한 활성화 지지세포 (activated feeder cells)의 제조방법을 제공한다:According to another aspect of the present invention, the present invention provides a method for producing activated feeder cells for proliferation of NK cells, comprising the following steps:
a) 인체에서 분리된 말초혈액단핵세포 (PBMCs)에 CD3 항체, IFN-r, IL-2을 이용하여 활성화 시키는 단계; 및a) activating peripheral blood mononuclear cells (PBMCs) isolated from the human body using CD3 antibodies, IFN-r, and IL-2; And
b) 상기 활성화된 말초혈액단핵세포 (PBMCs)에 방사선을 조사하는 단계.b) irradiating the activated peripheral blood mononuclear cells (PBMCs).
본 발명에 있어서, 상기 활성화 지지세포 (activated feeder cells)는 T 세포의 불활화 및 NK 세포에 대한 민감성을 증가시키고, 분리된 NK 세포를 10,000배 이상 증식시킬 수 있는 것을 특징으로 하는 활성화 지지세포 (activated feeder cells)의 제조 방법을 제공한다.In the present invention, the activated feeder cells are activated feeder cells, characterized in that the T cells are inactivated and the sensitivity to NK cells is increased, and the isolated NK cells can proliferate 10,000 times or more. activated feeder cells).
본 발명의 다른 양태에 따르면, 본 발명은 다음 단계들을 포함하는 활성화된 자연살상세포(NK cell)의 대량 증식방법을 제공한다:According to another aspect of the present invention, the present invention provides a method for mass proliferation of activated natural killer cells (NK cells) comprising the following steps:
a) 상기 본 발명의 방법에 따라 전혈로부터 말초혈액단핵구(PBMC)의 분리 없이 직접 자연살상세포(NK cell)를 분리하는 단계;a) directly separating natural killer cells (NK cells) without separating peripheral blood mononuclear cells (PBMC) from whole blood according to the method of the present invention;
b) 상기 본 발명의 방법에 따라 활성화 지지세포 (activated feeder cells)를 준비하는 단계; 및b) preparing activated feeder cells according to the method of the present invention; And
c) 상기 분리된 자연살상세포(NK cell)와 준비된 활성화 지지세포(activated feeder cells)를 항-CD16 항체가 고형화된 배양용기에 넣고 배양하는 단계.c) culturing the isolated natural killer cells (NK cells) and prepared activated feeder cells into a culture vessel in which an anti-CD16 antibody is solidified.
본 발명에 있어서, 상기 증식된 NK세포는 다양한 활성화 수용체의 발현이 증가되고 표적종양세포에 대한 항종양 세포독성을 강하게 증가시키는 것을 특징으로 하는 활성화된 자연살상세포(NK cell)의 대량 증식방법을 제공한다.In the present invention, the proliferated NK cells increase the expression of various activating receptors and strongly increase anti-tumor cytotoxicity against target tumor cells. to provide.
본 발명의 다른 양태에 따르면, 본 발명은 다음 단계들을 포함하는 전혈에서 면역세포 수의 측정방법을 제공한다:According to another aspect of the present invention, the present invention provides a method for measuring the number of immune cells in whole blood comprising the following steps:
a) 혈액분석기를 이용하여 전혈 ㎕당 림프구의 수를 측정하는 단계;a) measuring the number of lymphocytes per µl of whole blood using a blood analyzer;
b) 유세포분석기를 이용하여 전혈의 림프구 내 면역세포의 비율을 측정하는 단계; 및b) measuring the percentage of immune cells in the lymphocytes of whole blood using a flow cytometer; And
c) 하기 식 1과 같은 방법으로 전혈에서 분석을 원하는 면역세포의 수를 계산하는 단계.c) calculating the number of immune cells desired to be analyzed in whole blood by a method as shown in
[식 1][Equation 1]
전혈에서 면역세포의 수 = a) 단계에서 구한 전혈 ㎕당 림프구 세포 × b) 단계에서 구한 림프구 내 면역세포의 비율 × 사용한 혈액량 (㎕)Number of immune cells in whole blood = Lymphocyte cells per µl of whole blood obtained in step a) × Ratio of immune cells in lymphocytes obtained in step b) × Blood volume used (µl)
본 발명의 다른 양태에 따르면, 본 발명은 다음 단계들을 포함하는 분리된 면역세포의 수율의 측정방법을 제공한다:According to another aspect of the present invention, the present invention provides a method for measuring the yield of isolated immune cells comprising the following steps:
a) 제 7항에 따라 전혈에서 면역세포 수를 측정하는 단계; a) measuring the number of immune cells in whole blood according to claim 7;
b) 아래 식 2와 같은 방법으로 분리된 순수 면역세포 수를 측정하는 단계; 및b) measuring the number of pure immune cells isolated by the same method as in
[식 2][Equation 2]
분리된 순수 면역세포의 수 = 분리된 면역세포의 수 × 분리된 면역세포의 비율Number of isolated pure immune cells = number of isolated immune cells × ratio of isolated immune cells
c) 아래 식 3과 같은 방법으로 분리된 면역세포의 수율을 계산하는 단계c) Calculating the yield of isolated immune cells by the method shown in Equation 3 below.
[식 3][Equation 3]
분리된 면역세포의 수율 = b) 단계에서 구한 분리된 순수 면역세포의 수 / a) 단계에서 구한 전혈에서 면역세포의 수 × 100Yield of isolated immune cells = Number of isolated pure immune cells obtained in step b) / Number of immune cells in whole blood obtained in step a) × 100
본 발명에 있어서, 상기 면역세포는 NK 세포, T 세포, 또는 B 세포 일 수 있으나, 바람직하게는 자연살상세포(NK cell)인 것을 특징으로 하는 측정방법을 제공한다. 전혈 상태의 면역 세포와 분리된 면역세포의 비율 측정은 유세포 분석기를 통해서 할 수 있으며, 예컨대 NK 세포인 경우 CD3, CD56형광이 부착된 단클론항체를 이용하여 유세포분석기로 NK 세포 (CD3-CD56+ 세포)의 비율을 측정할 수 있다.In the present invention, the immune cells may be NK cells, T cells, or B cells, but preferably provides a measurement method characterized in that they are natural killer cells (NK cells). The ratio of whole-blooded immune cells and isolated immune cells can be measured through a flow cytometer.For example, for NK cells, use a CD3, CD56 fluorescently attached monoclonal antibody, and use a flow cytometer to measure NK cells (CD3 - CD56 + cells). ) Can be measured.
본 발명의 자연살상세포 분리방법에 따르면, 기존 말초혈액단핵세포로부터 분리하는 방법에 비하여 소량의 전혈로부터 NK 세포의 회수율을 높이고 그 분리 시간을 획기적으로 줄일 수 있다. 또한, 본 발명의 활성화 지지세포 (activated feeder cells) 제조 방법에 따르면 T 세포의 불활화 및 NK 세포에 대한 민감성을 증가시키고 분리된 NK 세포를 10,000배 이상 증식시킬 수 있다. 또한, 본 발명의 자연살상세포 대량증식 방법에 따르면, 다양한 활성화 수용체의 발현이 증가되고 표적종양세포에 대한 항종양 세포독성을 강하게 증가시킬 수 있다. 본 발명에 따라 대량으로 증식된 세포독성이 높은 NK 세포는 암면역 세포치료제 조성물의 유효성분으로 이용될 수 있다.According to the method of isolating natural killer cells of the present invention, it is possible to increase the recovery rate of NK cells from a small amount of whole blood and significantly reduce the separation time compared to the conventional method of separating from peripheral blood mononuclear cells. In addition, according to the method for producing activated feeder cells of the present invention, inactivation of T cells and sensitivity to NK cells can be increased, and isolated NK cells can be proliferated 10,000 times or more. In addition, according to the method for mass proliferation of natural killer cells of the present invention, expression of various activating receptors can be increased and antitumor cytotoxicity to target tumor cells can be strongly increased. High cytotoxic NK cells proliferated in a large amount according to the present invention can be used as an active ingredient of a cancer immune cell therapy composition.
도 1은 본 발명에 따라 제조된 활성화 지지세포의 다양한 활성화 및 억제 인자의 발현을 나타낸 그래프이다.
도 2는 본 발명에 따라 증식된 NK 세포의 증식율을 나타낸 그래프이다.
도 3은 본 발명에 따라 증식된 NK 세포의 다양한 활성화 수용체의 발현을 나타낸 그래프이다. (도 3a. NK 세포의 다양한 활성화 수용체의 발현변화를 나타낸 그래프, 도3b.resting NK의 대표적인 histogram, 도3c. expanded NK의 대표적인 histogram)
도 4는 본 발명에 따라 증식된 NK 세포는 표적종양세포에 대한 항종양 세포독성을 나타낸 그래프이다. (도 4a. K562 세포주, 도 4b. PANC-1 세포주, 도 4c. SW480 세포주)
도 5는 본 발명에 따라 증식된 NK 세포의 췌장암 및 대장암 마우스 모델에서 항종양효과을 나타낸 그래프이다. (도 5a. 췌장암 모델, 도 5b-c. 대장암 모델)1 is a graph showing the expression of various activation and inhibitory factors of activated support cells prepared according to the present invention.
2 is a graph showing the proliferation rate of NK cells proliferated according to the present invention.
3 is a graph showing the expression of various activating receptors of NK cells proliferated according to the present invention. (Fig. 3a. Graph showing the expression change of various activating receptors in NK cells, Fig. 3b. Representative histogram of resting NK, Fig. 3c. Representative histogram of expanded NK)
Figure 4 is a graph showing the anti-tumor cytotoxicity of NK cells proliferated according to the present invention to target tumor cells. (Fig. 4a. K562 cell line, Fig. 4b. PANC-1 cell line, Fig. 4c. SW480 cell line)
5 is a graph showing the anti-tumor effect of NK cells proliferated according to the present invention in a mouse model of pancreatic cancer and colon cancer. (Fig. 5a. pancreatic cancer model, Fig. 5b-c. colon cancer model)
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 through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 종양세포주 배양Example 1. Tumor cell line culture
본 발명에 사용된 백혈명 유래 세포주 (K562), 대장암 유래 세포주 (SW480), 췌장암 유래 세포주 (MIA PaCa-2, PANC-1)는 100 U/㎖ penicillin과 100 ㎍/㎖ streptomycin, 10 % fetal bovine serum (FBS)이 첨가된 RPMI 1640 (K562, SW480)과 Dulbecco’s Modified Eagle’s medium (MIA PaCa-2, PANC-1) 배지로 5% CO2가 유지되는 37˚C 배양기에서 배양하였다.The leukemia-derived cell line (K562), colon cancer-derived cell line (SW480), and pancreatic cancer-derived cell line (MIA PaCa-2, PANC-1) used in the present invention were 100 U/ml penicillin and 100 µg/ml streptomycin, 10% fetal. RPMI 1640 (K562, SW480) added with bovine serum (FBS) and Dulbecco's Modified Eagle's medium (MIA PaCa-2, PANC-1) were cultured in a 37°C incubator maintaining 5% CO 2.
실시예 2. 활성화 지지세포 (activated feeder cells) 제조Example 2. Preparation of activated feeder cells
사람의 말초혈액 10 ~ 15 cc을 채혈한다. 10 ~ 15 cc of human peripheral blood is collected.
1) 채혈된 일부의 혈액(4cc 미만)은 NK cell 분리에 사용한다. 1) Some of the collected blood (less than 4cc) is used for NK cell separation.
2) 일반혈액검사(CBC) 및 세포검사를 위해 소량의 혈액 2 cc 미만을 사용한다.2) A small amount of less than 2 cc of blood is used for general blood test (CBC) and cytology.
3) 활성화 지지세포 제조를 위해 나머지 혈액은 생리식염수 (normal saline)을 첨가하여 잘 혼합하고 밀도구배 용액인 Histopaque-1077 위에 올린 후 400 × g에서 30분간 실온에서 원심분리하여 말초혈액단핵세포 (PBMC; peripheral blood mononuclear cell)를 얻었다. 분리된 말초혈액단핵세포 (PBMCs)를 생리식염수 또는 NK cell 배양 배지로 잘 혼합하여 U형 또는 V형 반응용기에 넣는다. 이후 CD3 항체 (500 ng/ml), IFN-r (1000U/ml), IL-2 (1000U/ml)을 첨가한 후, 37°C에 30분 이상 반응하여 말초혈액단핵세포의 활성화를 유도한다. 반응 후 남은 항체와 사이토카인을 제거하기 위해 생리식염수로 원심세척하였다. 이후 차가운 NK cell 배양 배지를 첨가하여 잘 혼합한 후 25 Gy 이상의 방사선을 조사하였다. 3) For the preparation of activated support cells, the remaining blood is mixed well with normal saline, placed on the density gradient solution Histopaque-1077, and centrifuged at 400 × g for 30 minutes at room temperature to produce peripheral blood mononuclear cells (PBMC). ; peripheral blood mononuclear cell) was obtained. The separated peripheral blood mononuclear cells (PBMCs) are mixed well with physiological saline or NK cell culture medium and placed in a U-type or V-type reaction vessel. After that, CD3 antibody (500 ng/ml), IFN-r (1000U/ml), IL-2 (1000U/ml) are added, and then reacted at 37°C for 30 minutes or more to induce activation of peripheral blood mononuclear cells. . Centrifugal washing was performed with physiological saline to remove remaining antibodies and cytokines after the reaction. After that, the cold NK cell culture medium was added, mixed well, and irradiated with radiation of 25 Gy or more.
실시예 3. 전혈상태에서 짧은 시간 (30 ~ 40분)에 회수율 (80% 이상)이 높은 NK 세포 분리 Example 3. Isolation of NK cells with high recovery rate (80% or more) in a short time (30 to 40 minutes) in whole blood state
혈액 4 cc를 15 cc U형 튜브에 천천히 분주한다. NK cell을 negative selection으로 분리하기 위한 항체 칵테일 (CD3, CD4, CD19, CD36, CD66b, CD123, glycophorin A)과 magnetic microbead를 각각 잘 섞어서 준비해 둔다. 이후 항체 칵테일 200 ㎕와 magnetic microbead 200㎕를 혈액이 분주된 U형 튜브에 넣어주고 잘 혼합한다. 실온에서 5분간 반응시킨다. 가능한 5분을 넘지 않도록 주의한다. 반응시간이 길어질수록 회수되는 NK cell이 적어진다. 반응이 끝난 후 튜브를 7,000 가우스 이상의 자력을 가진 자석에 넣어 실온에서 5분간 방치한다. 5분 후 새로운 U형 튜브에 자석에 붙지 않은 혈액을 천천히 따라 붓는다. 이후 magnetic microbead 200㎕을 첨가하여 잘 혼합 후 실온에서 5분간 반응시킨다. 가능한 5분을 넘지 않도록 주의한다. 반응이 끝난 후 튜브를 7,000 가우스 이상의 자력을 가진 자석에 넣어 실온에서 5분간 방치한다. 5분 후 새로운 U형 튜브에 자석에 붙지 않은 혈액을 천천히 따라 붓는다. 마지막으로 항체 칵테일 200 ㎕와 magnetic microbead를 처음 넣는 량의 1/5 ~ 1/15을 넣어주고 잘 혼합한 후 실온에서 5분간 반응한다. 반응이 끝난 후 튜브를 7,000 가우스 이상의 자력을 가진 자석에 붙여 실온에서 5분간 방치한다. 5분 후 새로운 U형 튜브에 자석에 붙지 않은 혈액을 천천히 옮기고 400×g에서 5분간 원심분리 한다. 생리식염수를 첨가하여 원심세척 후 분리한 세포수를 확인한다. 대조군으로는 기존의 분리 방법으로써 전혈상태가 아닌 원심구배법으로 분리된 말초혈액단핵세포에 CD56 또는 CD3, CD14, CD19 등과 같은 magnetic bead가 부착된 항체와 반응시켜 칼럼을 통해 자연살상세포를 분리하는 방법을 사용하였다.Slowly dispense 4 cc of blood into a 15 cc U-shaped tube. Prepare an antibody cocktail (CD3, CD4, CD19, CD36, CD66b, CD123, glycophorin A) and magnetic microbeads to separate NK cells by negative selection. After that, put 200 µl of the antibody cocktail and 200 µl of magnetic microbeads into a U-shaped tube into which blood has been dispensed, and mix well. It was reacted for 5 minutes at room temperature. Be careful not to exceed 5 minutes. The longer the reaction time is, the fewer NK cells are recovered. After the reaction is over, the tube is placed in a magnet with a magnetic force of 7,000 gauss or more and left at room temperature for 5 minutes. After 5 minutes, slowly pour the non-magnetized blood into a new U-shaped tube. Then, 200 µl of magnetic microbeads were added, mixed well, and reacted at room temperature for 5 minutes. Be careful not to exceed 5 minutes. After the reaction is over, the tube is placed in a magnet with a magnetic force of 7,000 gauss or more and left at room temperature for 5 minutes. After 5 minutes, slowly pour the non-magnetized blood into a new U-shaped tube. Finally, add 200 µl of the antibody cocktail and 1/5 to 1/15 of the amount of the magnetic microbead first, mix well, and react at room temperature for 5 minutes. After the reaction is over, the tube is attached to a magnet with a magnetic force of 7,000 gauss or more and left at room temperature for 5 minutes. After 5 minutes, slowly transfer the non-magnetized blood to a new U-shaped tube and centrifuge at 400×g for 5 minutes. After centrifugal washing by adding physiological saline, check the number of separated cells. As a control, the conventional separation method is to separate natural killer cells through a column by reacting with antibodies with magnetic beads such as CD56 or CD3, CD14, CD19, etc. to peripheral blood mononuclear cells separated by centrifugal gradient method rather than whole blood. Method was used.
실시예 4. 면역세포의 이론적인 수치 분석법과 분리된 면역세포의 수율 분석법Example 4. Theoretical Numerical Analysis of Immune Cells and Yield Analysis of Isolated Immune Cells
1) 전혈에 포함되어 있는 림프구의 수를 측정하기 위해 혈액분석기의 흡입관을 통해 전혈을 흡입시켜 분석한다. 혈액분석기로부터 나온 결과는 전혈 1㎕에 포함되어 있는 세포수로 나타내어진다(예 : 1.40 × 103 cells/μL)1) In order to measure the number of lymphocytes contained in whole blood, the whole blood is sucked through the suction tube of the blood analyzer and analyzed. The result from the blood analyzer is expressed as the number of cells contained in 1 µl of whole blood (e.g., 1.40 × 10 3 cells/µL).
2) 혈액분석기로는 림프구에 포함되어 있는 다양한 세포를 확인할 수 없기 때문에 다음과 같이 유세포분석기를 이용하여 분석하였다. 전혈에 포함되어 있는 다양한 면역세포의 비율을 확인하기 위해 소량의 혈액 (100~500㎕)에 CD3, CD4, CD8, CD19, CD25, CD45, CD56 형광이 부착된 단클론항체와 RBC 제거 용액을 첨가하여 10 ~ 20분간 반응을 시킨다. Foxp3를 분석하기 위해 세포내 염색 (intracellular staining)을 실시한다. 이후 유세포분석기를 이용하여 백혈구세포 (CD45+ 세포)를 찾아내고 백혈구 세포 중에 림프구 영역을 확인 후 각 면역세포(예컨대, NK 세포, T 세포, B 세포)의 비율을 확인하였다(림프구내의 다양한 세포의 비율을 확인). 2) Since various cells contained in lymphocytes cannot be identified with a blood analyzer, the analysis was performed using a flow cytometer as follows. In order to check the ratio of various immune cells contained in whole blood, CD3, CD4, CD8, CD19, CD25, CD45, CD56 fluorescently attached monoclonal antibody and RBC removal solution were added to a small amount of blood (100~500µl). Let it react for 10 to 20 minutes. Intracellular staining is performed to analyze Foxp3. Subsequently, leukocytes (CD45 + cells) were identified using a flow cytometer, and the lymphocyte region among the leukocytes was identified, and the ratio of each immune cell (e.g., NK cells, T cells, B cells) was confirmed (of various cells in lymphocytes). Check the ratio).
3) 전혈에서 분석하고자 하는 면역세포의 수를 확인하기 위해서는 1)에서 분석된 림프구수에 2)에서 측정된 림프구내의 면역세포의 비율과 전혈의 량을 곱하면 확인할 수 있다. 분석의 예로, 혈액분석기를 통해 분석한 림프구 수가 1.40 × 103 cells/μL이고 2)에서 측정한 림프구내의 면역세포 중 하나인 NK cell (CD3-CD56+ 세포)의 비율 9.8%이다. 전혈의 량이 4cc의 경우 4cc에 포함되어 있는 NK 세포의 수는 다음과 같이 계산된다.3) To check the number of immune cells to be analyzed in whole blood, it can be confirmed by multiplying the number of lymphocytes analyzed in 1) by the ratio of immune cells in the lymphocytes measured in 2) and the amount of whole blood. As an example of the analysis, the number of lymphocytes analyzed through a blood analyzer is 1.40 × 10 3 cells/μL, and the ratio of NK cells (CD3- CD56 + cells), one of the immune cells in the lymphocytes measured in 2), is 9.8%. In the case of 4cc of whole blood, the number of NK cells contained in 4cc is calculated as follows.
림프구 수(1.40 × 103 cells/μL) × NK 세포의 비율(0.098) × 혈액량(4,000μL) = 5.49 × 105 cellsLymphocyte count (1.40 × 10 3 cells/μL) × NK cells ratio (0.098) × blood volume (4,000 μL) = 5.49 × 10 5 cells
4) 실시예 3.에서 분리된 NK 세포의 수율을 계산하기 위해서는 위의 3)에서 분석된 전혈에서의 NK 세포의 수를 기준으로 설정하여 계산한다. 3)에서 전혈 4cc에는 NK 세포가 5.49 × 105 cells가 포함되어 있다. 실시예 3.에서 분리된 NK 세포의 수(혈구계수기로 측정된 세포의 수)가 5.21 × 105 cells 이고 유세포분석기를 통해 얻은 NK 세포(CD3-CD56+ 세포)의 비율이 98.31% 였다면 순수한 NK 세포의 수는 5.12 × 105 cells이 된다(5.21 × 105 cells × 0.9831). 최종적으로 분리된 NK 세포의 수율은 전혈에 포함되어 있는 NK 세포를 이론적인 수치로 설정하고 분리된 NK 세포와의 비율을 수율로 한다. 예시의 분리된 NK 세포의 수율은 순수한 NK 세포의 수(5.12 × 105 cells)/전혈에서의 NK 세포의 수(5.49 × 105 cells) × 100 = 93.26% 이다.4) In order to calculate the yield of NK cells isolated in Example 3, it is calculated based on the number of NK cells in whole blood analyzed in 3) above. In 3), 4cc of whole blood contains 5.49 × 10 5 cells of NK cells. If the number of NK cells isolated in Example 3 (the number of cells measured by a hemocytometer) was 5.21 × 10 5 cells and the ratio of NK cells (CD3 - CD56 + cells) obtained through flow cytometry was 98.31%, then pure NK was obtained. The number of cells becomes 5.12 × 10 5 cells (5.21 × 10 5 cells × 0.9831). As for the yield of finally isolated NK cells, the NK cells contained in whole blood are set to a theoretical value, and the ratio with the isolated NK cells is used as the yield. An exemplary yield of isolated NK cells is the number of pure NK cells (5.12 × 10 5 cells)/number of NK cells in whole blood (5.49 × 10 5 cells) × 100 = 93.26%.
위와 같은 면역세포의 이론적인 수치 분석법과 분리된 면역세포의 수율 분석법을 일반적인 계산식으로 나타내면 다음과 같다.The theoretical numerical analysis method of immune cells as described above and the yield analysis method of isolated immune cells are expressed in general calculation formulas as follows.
① 전혈에서 분석하고자 하는 면역세포의 수 : 혈액분석기로부터 전혈 ㎕당 림프구 세포 × 유세포분석기로부터 전혈의 림프구에서 분석하고자 하는 면역세포의 비율 × 사용한 혈액량 (㎕) ① Number of immune cells to be analyzed from whole blood: Lymphocyte cells per µl of whole blood from a blood analyzer × The ratio of immune cells to be analyzed from lymphocytes of whole blood from a flow cytometer × Amount of blood used (µl)
② 분리된 순수 면역세포의 수 : 분리된 면역세포의 수 × 유세포분석기로부터 분리된 면역세포의 비율② Number of separated pure immune cells: Number of separated immune cells × Ratio of separated immune cells from flow cytometry
③ 분리된 면역세포의 수율 : ②/① × 100③ Yield of isolated immune cells: ②/① × 100
본 분석법은 임상진단에 위해 사용 가능하도록 calibration 시약을 사용하여 검증한 후 분석하였다.This assay was analyzed after being verified using a calibration reagent so that it can be used for clinical diagnosis.
분석의 예로, 분리된 NK cell (CD3-CD56+ 세포)의 비율은 CD3, CD56형광이 부착된 단클론항체를 이용하여 유세포분석기로 분석하였다. 또한 전혈에서 ㎕당 림프구 세포의 수는 혈액분석기를 이용하여 확인한다. NK cell의 이론적인 수치와 분리된 NK cell의 수율은 아래의 계산식으로부터 얻을 수 있다. As an example of the analysis, the ratio of isolated NK cells (CD3- CD56 + cells) was analyzed by flow cytometry using CD3 and CD56 fluorescently attached monoclonal antibodies. In addition, the number of lymphocyte cells per µl in whole blood is checked using a blood analyzer. The theoretical value of NK cell and the yield of separated NK cell can be obtained from the following calculation formula.
① NK cell의 이론적인 수치 : 혈액분석기로부터 ㎕당 림프구 세포 × 유세포분석기로부터 전혈의 림프구에서 NK cell 비율 × 분리시 사용한 혈액량 (㎕) ① Theoretical value of NK cells: Lymphocyte cells per µl from blood analyzer × NK cell ratio from lymphocytes of whole blood from flow cytometer × blood volume used for separation (µl)
② 분리된 순수 NK cell 수 : 분리된 NK cell 수 × 유세포분석기로부터 분리된 NK cell의 비율 (CD3-CD56+ 세포)② Number of separated pure NK cells: Number of separated NK cells × Ratio of NK cells separated from flow cytometer (CD3 - CD56 + cells)
③ NK cell 수율 : ②/① × 100③ NK cell yield: ②/① × 100
실시예 5. NK 세포 배양 Example 5. NK cell culture
1) 항체 (antibody) 코팅 배양용기 조제1) Preparation of antibody-coated culture vessel
생리식염수로 배양용기를 2 ~ 3회 세척한다. 항 CD16 항체 (1 ㎍/ml)를 배양용기에 첨가하여 냉장상태 (2~8℃)에서 18 ~ 24시간 동안 반응시키거나 37°C 배양기에서 4시간 이상 반응시킨다. 이후 배양용기에 부탁되지 않고 남아 있는 항 CD16 항체 용액을 제거한다. Wash the
2) NK cell 배양2) NK cell culture
항체 고형화 배양용기에 전혈로부터 분리된 NK cell와 말초혈액단핵구를 이용한 활성화 지지세포를 배지와 잘 혼합 (NK 세포 : 활성화 유도체세포 = 1 : 20)하여 넣고 5% HS (human serum) 및 500 U/㎖ 인터류킨-2 (이하 ‘배양배지’ 라고 한다)를 첨가하여 37℃, 5 % CO2가 유지되는 배양기에서 배양한다. 이후 6 ~ 8일 동안 2일 간격으로 2배수로 배양용기를 널려주며 배양배지의 양은 초기양의 2배로 유지할 수 있도록 배지를 첨가해준다. 이 후 더 넓은 배양용기에 세포를 옮기고 배양배지를 2 ~ 2.5배를 추가하였다. NK cell의 증식 정도에 따라 배양배지는 1 ~ 3일 마다 첨가하여 18 ~ 21동안 배양하였다. NK cell의 증식율 및 표면항원을 확인하기 배양 7일, 14일 및 18 ~ 21일에 수거하여 증식율을 확인하였다.In an antibody solidification culture vessel, NK cells isolated from whole blood and activated support cells using peripheral blood mononuclear cells were mixed well with a medium (NK cells: activated derivative cells = 1: 20), and 5% HS (human serum) and 500 U/ Add ㎖ interleukin-2 (hereinafter referred to as'culture medium') and incubate in an incubator maintained at 37°C and 5% CO 2. After that, for 6 to 8 days, the culture vessel is spread out at twice the interval of 2 days, and the medium is added so that the amount of the culture medium is maintained at twice the initial amount. After that, the cells were transferred to a wider culture vessel and 2 to 2.5 times the culture medium was added. Depending on the degree of proliferation of NK cells, the culture medium was added every 1 to 3 days and cultured for 18 to 21. To confirm the proliferation rate and surface antigen of NK cells, the proliferation rate was confirmed by harvesting on the 7th, 14th and 18th to 21st days of culture.
실시예 6. 활성화 지지세포 및 NK cell 표면항원 변화 분석 Example 6. Analysis of changes in activated support cells and NK cell surface antigens
활성화 지지세포 및 NK cell 표면항원 변화 분석을 위해 아래의 형광이 부착된 단클론항체 (monoclonal antibodies)를 사용하여 유세포분석기로 분석하였다.For analysis of changes in activated support cells and NK cell surface antigens, the following fluorescence-attached monoclonal antibodies were used and analyzed by flow cytometry.
HLA-ABC-FITC, MICA-PE, MICB-PE, ULBP-1-PE, ULBP-2-PE, ULBP-3-PE, Anti-CD3-PE, CD16-PE, Anti-CD45-FITC, CD48-FITC, CD56-PE-Cy5, CD244 (2B4)-FITC, CD226 (DNAM-1)-FITC, CD314 (NKG2D)-PE, CD335 (NKp46)-PE, CD336 (NKp44)-PE, CD337 (NKp30)-PE. Isotype control을 기준으로 분석 하였다.HLA-ABC-FITC, MICA-PE, MICB-PE, ULBP-1-PE, ULBP-2-PE, ULBP-3-PE, Anti-CD3-PE, CD16-PE, Anti-CD45-FITC, CD48- FITC, CD56-PE-Cy5, CD244 (2B4)-FITC, CD226 (DNAM-1)-FITC, CD314 (NKG2D)-PE, CD335 (NKp46)-PE, CD336 (NKp44)-PE, CD337 (NKp30)- PE. It was analyzed based on isotype control.
실시예 7.Example 7. NK 세포의 세포독성 (cytotoxicity) 검사Cytotoxicity test of NK cells
본 발명에서는 NK 세포의 표적종양세포로 (K562, SW480, PANC-1)을 사용하였다. 각각의 표적종양세포에 5 uM 5-carboxyfluorescein diacetate succinmidyl ester (CFSE)을 첨가하여 37℃, 5 % CO2 존재 하에서 10분 동안 반응시켰다. 이 후 10% HS이 포함된 배지를 이용하여 2~3회 원심세척하였다. NK 세포 (effector cell)와 CFSE-labeled 표적종양세포 (target cell)를 10 : l, 5 : 1, 2.5 : 1, 1 : 1 비율로 반응 튜브 또는 96-well plate에 첨가하여 37℃, 5 % CO2 존재 하에서 4시간 동안 배양하였다. 배양이 완료된 튜브는 즉시 얼음물 (ice water)에 넣고 50 ㎍/ml propidium iodide (PI)을 첨가하여 1시간 이내에 유세포분석기를 이용하여 자연살상세포 (NK cell)의 세포독성 (cytotoxicity)을 확인하였다. *양성대조군 또는 병용약물로 대장암 모델에서는 4Gy 방사선이 조사된 SW480 세포주 (방사선 조사 후 2 ~ 3일 후 사용) 및 cetuximab (10 ㎍/ml)처리된 SW480 세포주를 사용하였고 췌장암 모델에서는 gemcitabne 0.01μM, 0.1μM, 0.5μM이 처리된 PANC-1 세포주 (약물 처리 후 2 ~ 3일 후 사용)를 사용하였다.In the present invention, (K562, SW480, PANC-1) was used as target tumor cells for NK cells. 5 uM 5-carboxyfluorescein diacetate succinmidyl ester (CFSE) was added to each target tumor cell and reacted for 10 minutes at 37°C in the presence of 5% CO2. After that, centrifugal washing was performed 2-3 times using a medium containing 10% HS. Add NK cells (effector cells) and CFSE-labeled target cells (target cells) to a reaction tube or 96-well plate at a ratio of 10: l, 5: 1, 2.5: 1, 1: 1, and then 37℃, 5% Incubated for 4 hours in the presence of CO 2. The tube after the culture was completed was immediately placed in ice water and 50 µg/ml propidium iodide (PI) was added, and the cytotoxicity of NK cells was confirmed using a flow cytometer within 1 hour. *For colon cancer model as a positive control or combination drug, SW480 cell line irradiated with 4Gy radiation (used 2 to 3 days after irradiation) and SW480 cell line treated with cetuximab (10 μg/ml) were used, and gemcitabne 0.01 μM in pancreatic cancer model. , 0.1 μM, 0.5 μM-treated PANC-1 cell line (used 2-3 days after drug treatment) was used.
실시예 8. NK 세포의 동물효능 실험Example 8. Animal efficacy test of NK cells
NK 세포의 동물효능 실험을 위해 5 ~ 6주령의 비비만성 당뇨병 및 중증 복합성 면역 부전증 (nonobese diabetic/severe combined immunodeficiency, NOD/SCID) NOD.CB17-Prkdcscid/ARC 마우스를 이용하였다. 인간 대장암 세포주 SW480 (2~5 × 106세포)는 마우스 오른쪽 다리 (대퇴부)에 피하로 접종하였다. 인간 췌장암 세포주 PANC-1 (2~5 × 106세포)는 마우스 등에 피하로 접종하였다. 종양 이식 7 ~ 8일 후에, 대장암 모델은 선형가속기 (Infinity, ELEKTA)를 이용하여 오른쪽 다리 (대퇴부)에 4 Gy 방사선을 조사하였다. 방사선을 조사한 후, NK 세포 (1 × 107세포)를 마우스 꼬리 정맥으로 주사 하였다. 방사선 조사 및 NK 세포 주사는 주 1회 간격으로 3회 실시하였다. 췌장암 모델은 gemciatbine 투여 후 2 ~ 3일 후에 NK 세포 (1 × 107세포)를 마우스 꼬리 정맥으로 주 1회 간격으로 3회 주사 하였다. Cetuximab (30 mg/kg, SW480 양성 대조군) 및 gemcitabine (120 mg/kg 및 240 mg/kg, PANC-1 양성 대조군)은 NK 세포 주사 2 ~ 3일 전에 매회 투여하였다. 종양 사이즈는 (부피 = 깊이 × 넓이2 × 0.5)는 주 2회 측정하였다. For the animal efficacy test of NK cells, 5 to 6 weeks old nonobese diabetic/severe combined immunodeficiency (NOD/SCID) NOD.CB17-Prkdcscid/ARC mice were used. The human colorectal cancer cell line SW480 (2-5 × 10 6 cells) was inoculated subcutaneously on the right leg (femur) of the mouse. The human pancreatic cancer cell line PANC-1 (2-5 × 10 6 cells) was inoculated subcutaneously in mice or the like. 7 to 8 days after tumor implantation, the colorectal cancer model was irradiated with 4 Gy radiation to the right leg (femur) using a linear accelerator (Infinity, ELEKTA). After irradiation with radiation, NK cells (1 × 10 7 cells) were injected into the mouse tail vein. Irradiation and injection of NK cells were performed three times a week at intervals. In the pancreatic cancer model, NK cells (1 × 10 7 cells) were injected into the tail vein of mice 3 times a
실험 결과Experiment result
결과 1. NK 세포의 분리 효율.
1) NK 세포의 이론적인 수치 분석과 분리된 NK 세포의 수율 분석1) Theoretical numerical analysis of NK cells and yield analysis of isolated NK cells
본 발명에서는 NK 세포의 분리 효율을 향상시켜 공여자로부터 채혈량을 최소화 하고자 하였다. 본 발명에서는 소량의 전혈로부터 NK 세포를 분리하는 방법을 개발하였고 기존의 방법 (MACS법)과 비교하여 분리 수율을 3배 이상 증가시켰다 (기존 24.93%, 본 연구 89.42%). 기존 방법의 혈액량이 적을수록 분리효율이 떨어지며 전치리 과정으로 말초혈액단핵세포 (PBMC)를 분리해야 한다는 단점이 있다. 또한 NK 세포의 분리 시간은 본 발명에서는 30~40분이면 완료되지만 기존의 방법으로는 3시간 이상 소요되어 효율적이지 못하다는 단점이 있다. 본 발명에서는 NK 세포의 정확한 세포 수 및 분리 수율을 계산할 수 있는 방법을 고안하였다. 기존에는 말초혈액에서 PBMC를 분리하고 유세포분석기를 이용하여 NK 세포의 비율과 PBMC 세포 수를 곱하여 간접적으로 계산하였다. 하지만 이 방법은 전혈에 있는 모든 세포를 대표하기에는 무리가 있고 PBMC 분리 과정에서 세포의 소실이 발생 할 수 가 있어 정확한 세포수를 계산하기에는 힘든 부분이 있었다. 본 발명에서는 이런 문제점을 해결하기 위해 소량의 전혈 (100~200㎕)을 이용하여 RBC만 용해시킨 후 백혈구세포 전체에서 NK 세포의 비율을 유세포분석기를 이용하여 분석하였다. 또한 혈액분석기를 이용하여 전혈에서 ㎕당 림프구 세포를 측정하여 아래의 ①과 같은 방법으로 NK 세포의 수를 정확히 계산하였다. 분리된 NK 세포의 수율 (회수율)은 아래의 ②와 ③의 방법을 이용하여 계산하였다. 또한 이 방법은 소량의 전혈 (100~500㎕)을 이용하여 다양한 면역세포 (CD3, CD4, CD8, CD19, CD25, CD45, CD56, Foxp3)을 분석할 수 있다. 본 발명은 기존의 PBMC 분리 후 유세포분석기를 이용하여 간접적으로 계산하는 방법보다 소량의 혈액을 이용하여 정확하고 개관적인 방법으로 면역세포를 분석 가능하기 때문에 인체 면역세포의 모니터링에 유용하게 사용할 수 있다. 대표적인 실시예로 NK 세포에 대해 아래와 같이 계산하였다.In the present invention, it was attempted to minimize the amount of blood collected from a donor by improving the separation efficiency of NK cells. In the present invention, a method for isolating NK cells from a small amount of whole blood was developed and the separation yield was increased by more than 3 times compared to the conventional method (MACS method) (existing 24.93%, this study 89.42%). The smaller the blood volume of the conventional method, the lower the separation efficiency, and there is a disadvantage in that the peripheral blood mononuclear cells (PBMC) must be separated through a pretreatment process. In addition, the separation time of NK cells is completed in 30 to 40 minutes in the present invention, but there is a disadvantage in that it is not efficient because it takes 3 hours or more with the conventional method. In the present invention, a method capable of calculating the exact cell number and separation yield of NK cells was devised. Previously, PBMCs were isolated from peripheral blood and calculated indirectly by multiplying the ratio of NK cells and the number of PBMC cells using a flow cytometer. However, this method is unreasonable to represent all cells in whole blood, and cell loss may occur during the PBMC separation process, making it difficult to accurately calculate the number of cells. In the present invention, in order to solve this problem, only RBC was lysed using a small amount of whole blood (100-200 µl), and then the ratio of NK cells in the total white blood cells was analyzed using a flow cytometer. In addition, lymphocyte cells per µl in whole blood were measured using a blood analyzer, and the number of NK cells was accurately calculated as shown in ① below. The yield (recovery rate) of the isolated NK cells was calculated using the method of ② and ③ below. In addition, this method can analyze various immune cells (CD3, CD4, CD8, CD19, CD25, CD45, CD56, Foxp3) using a small amount of whole blood (100~500µl). The present invention can be usefully used for monitoring human immune cells because it is possible to analyze immune cells in an accurate and overview method using a small amount of blood than a method of indirectly calculating using a flow cytometer after separation of PBMCs. As a representative example, NK cells were calculated as follows.
① 전혈에서 NK 세포의 수 : 혈액분석기로부터 전혈 ㎕당 림프구 세포 × 유세포분석기로부터 전혈의 림프구에서 NK 세포의 비율 × 사용한 혈액량 (㎕) ① Number of NK cells in whole blood: Lymphocyte cells per µl of whole blood from a blood analyzer × The ratio of NK cells in lymphocytes of whole blood from a flow cytometer × Amount of blood used (µl)
② 분리된 순수 NK 세포의 수 : 분리된 NK 세포의 수 × 유세포분석기로부터 분리된 NK 세포의 비율② Number of separated pure NK cells: Number of separated NK cells × Ratio of separated NK cells from flow cytometry
③ 분리된 NK 세포의 수율 : ②/① × 100③ Yield of isolated NK cells: ②/① × 100
[표 1][Table 1]
2) 본 발명에서의 NK 세포 분리 방법의 장점2) Advantages of the method for isolating NK cells in the present invention
[표 2][Table 2]
결과 2. CD3 항체, IFN-r 및 IL-2를 전처리 후 방사선을 조사한 활성화 지지세포(activated feeder cells)는 NK 세포의 감수성을 증가시키는 다양한 리간드의 발현을 증가시키고 NK 세포의 증식율을 강화한다.
본 발명에서는 NK 세포의 감수성을 증가시키기 위한 방법으로 강력한 활성화 지지세포 (activated feeder cells) 제조방법을 개발하였다. 기존의 방사선 단독의 방법으로도 NK 세포의 감수성을 증가시킬 수 있었으나 환자의 채혈에 대한 부담을 줄이기 위해서는 NK 세포의 증식율 향상이 필요하였고 이를 해결하기 위해서는 더욱 강력한 활성화 지지세포가 필요하였다. 본 발명에서는 CD3 항체, IFN-r 및 IL-2를 말초혈액단핵구 (PBMCs)에 전처리 후 방사선을 조사하여 강력한 활성화 지지세포를 제조하였다. 방사선 처리 24시간 후 세포를 수거하여 유세포분석기를 이용하여 다양한 활성화 및 억제 인자의 발현을 확인 하였고 결과치는 평균형광광도 (mean fluorescence intensities; MFIs)로 나타내었다 (도 1). 기존의 방사선 단독의 방법과 비교하여 NK 세포의 감수성을 증가시키는 NKG2D 리간드인 MICB, ULBP1, ULBP2, ULBP3의 발현이 현저히 증가하였다. 2B4의 리간드인 CD48은 방사선 단독과 비교하여 큰 차이를 보이지 않았다. 또한 NK 세포의 감수성을 억제시키는 인자인 CD155는 방사선 단독과 비교하여 현저히 억제되었고 CD112와 HLA-ABC는 유의한 차이를 보이지 않았다. 이렇게 개발된 강력한 활성화 지지세포 (activated feeder cells)와 분리된 NK 세포를 21일 동안 공동 배양하였을 때 10,000배 이상의 NK 세포의 증식율을 나타내었다 (도 2). 따라서 이 결과들 CD3 항체, IFN-r, IL-2 및 방사선 병용이 말초혈액단핵구 (PBMCs)를 강력한 활성화 지지세포 (activated feeder cells)로 만들며 NK 세포의 증식율을 확연히 증가시키는 것으로 나타났다.In the present invention, a method for producing powerful activated feeder cells was developed as a method for increasing the sensitivity of NK cells. The sensitivity of NK cells could be increased by the conventional radiation alone method, but in order to reduce the burden on blood collection of the patient, it was necessary to improve the proliferation rate of NK cells, and to solve this problem, more powerful activated support cells were needed. In the present invention, the CD3 antibody, IFN-r, and IL-2 were pretreated to peripheral blood mononuclear cells (PBMCs) and irradiated with radiation to prepare strong activated support cells. Cells were harvested 24 hours after radiation treatment, and expression of various activation and inhibitory factors was confirmed using a flow cytometer, and the results were expressed as mean fluorescence intensities (MFIs) (FIG. 1). Compared with the conventional radiation-only method, the expression of the NKG2D ligands MICB, ULBP1, ULBP2, and ULBP3, which increases the sensitivity of NK cells, was significantly increased. CD48, a ligand of 2B4, showed no significant difference compared to radiation alone. In addition, CD155, a factor that inhibits the sensitivity of NK cells, was significantly suppressed compared to radiation alone, and there was no significant difference between CD112 and HLA-ABC. When the developed powerful activated feeder cells and the isolated NK cells were co-cultured for 21 days, the proliferation rate of NK cells was 10,000 times or more (FIG. 2). Therefore, these results showed that the combination of CD3 antibody, IFN-r, IL-2 and radiation turns peripheral blood mononuclear cells (PBMCs) into powerful activated feeder cells and significantly increases the proliferation rate of NK cells.
도 1에서, 통계적 유의성; # P < 0.05, ### P < 0.0005 (#IR versus [Anti-CD3+IFN-r+IL-2]+IR).1, statistical significance; # P <0.05, ### P <0.0005 ( # IR versus [Anti-CD3+IFN-r+IL-2]+IR).
결과 3. 활성화 지지세포 (activated feeder cells)에 의해 증식된 NK 세포는 다양한 활성화 수용체의 발현이 증가된다.Results 3. NK cells proliferated by activated feeder cells increase the expression of various activated receptors.
활성화 지지세포 (activated feeder cells)에 의해 증식된 NK 세포는 resting NK 세포 (말초혈액에서 바로 분리한 NK 세포)와 비교 하여 CD3, CD56, CD16와 NKG2D의 발현은 유의적인 차이를 보이지 않지만 NK 세포의 활성화 수용체인 DNAM-1, 2B4, NKp30, NKp44와 NKp46에서는 resting NK 세포와 비교하여 확연히 증가되었다. 또한 NK 억제 수용체인 NKB1에서는 resting NK와 비교하여 유의적인 감소를 나타내었다. 다른 억제 수용체인 CD158a (KIR2DL1), CD158b (KIRDL2/DL3)와 CD159a (NKG2A)에서는 유의적인 변화를 보이지 않았 (A). resting NK의 대표적인 histogram (B), expanded NK의 대표적인 histogram (C). 따라서 본 발명에 의해 개발된 활성화 지지세포(activated feeder cells)는 NK 세포의 활성화 수용체의 발현을 강하게 유도하며 억제 수용체의 발현을 억제하는 효과를 보였다.NK cells proliferated by activated feeder cells showed no significant difference in the expression of CD3, CD56, CD16, and NKG2D compared to resting NK cells (NK cells isolated directly from peripheral blood). The activation receptors DNAM-1, 2B4, NKp30, NKp44 and NKp46 were significantly increased compared to resting NK cells. In addition, NKB1, an NK inhibitory receptor, showed a significant decrease compared to resting NK. Other inhibitory receptors, CD158a (KIR2DL1), CD158b (KIRDL2/DL3) and CD159a (NKG2A), did not show significant changes (A). Representative histogram of resting NK (B), representative histogram of expanded NK (C). Therefore, the activated feeder cells developed by the present invention strongly induces the expression of the activation receptor of NK cells, and has an effect of inhibiting the expression of the inhibitory receptor.
도 3에서, 통계적 유의성; # P < 0.05, ## P < 0.005, ### P < 0.0005 (#resting NK versus expanded NK).3, statistical significance; # P <0.05, ## P <0.005, ### P <0.0005 ( # resting NK versus expanded NK).
결과 4. 활성화 지지세포 (activated feeder cells)에 의해 증식된 NK 세포는 표적종양세포에 대한 항종양 세포독성을 강하게 증가시킨다.
본 발명에서는 우선 NK 세포에 가장 민감한 세포주 중에 하나로 알려진 K562 세포를 이용하여 활성화 지지세포 (activated feeder cells)에 의해 증식된 NK 세포와 resting NK 세포 (말초혈액에서 바로 분리한 NK 세포)의 항종양 세포독성을 평가하였다. 이 후 활성화 지지세포 (activated feeder cells)를 이용하여 증식시킨 NK세포의 췌장암 및 대장암 세포주에 대한 세포독성을 확인하기 위해 PANC-1 과 SW480에 대한 세포독성 평가를 실시하였다. 활성화 지지세포 (activated feeder cells)에 의해 유도된 NK 세포는 K562 세포 대한 항종양 세포독성을 매우 강하게 나타내었다. 특히 resting NK 세포와 비교하여 더욱 높은 항종양 세포독성을 보였다. 활성화 지지세포 (activated feeder cells)에 의해 증식된 NK 세포는 췌장암 세포주 PANC-1에 대한 높은 세포독성을 보였으며 gemciatbine과 병용 시 세포독성이 더욱 증가하였다. 또한 gemciatbine 병용 시 농도 의존적으로 세포독성이 증가하는 경향을 보였다. 활성화 지지세포 (activated feeder cells)에 의해 증식된 NK 세포는 대장암 세포주 SW480에 대한 높은 세포독성을 보였으며 방사선 또는 cetuximab 병용 시 세포독성이 더욱 증가하였다. 특히 활성화 지지세포 (activated feeder cells) 의해 증식된 NK 세포는 방사선과 cetuximab을 병용하였을 경우 더욱 강력한 항종양 세포독성을 보였다. 이러한 결과는 활성화 지지세포 (activated feeder cells)에 의해 증식된 NK 세포와 표준치료의 병용처리가 췌장암 및 대장암 세포주에 대한 항종양효과를 더욱 증가시킬 것이라는 것을 나타낸다.In the present invention, first, anti-tumor cells of NK cells proliferated by activated feeder cells and resting NK cells (NK cells directly isolated from peripheral blood) using K562 cells known as one of the most sensitive cell lines to NK cells. Toxicity was evaluated. Thereafter, cytotoxicity evaluation for PANC-1 and SW480 was performed to confirm the cytotoxicity of NK cells proliferated using activated feeder cells against pancreatic and colorectal cancer cell lines. NK cells induced by activated feeder cells showed very strong antitumor cytotoxicity against K562 cells. In particular, it showed higher antitumor cytotoxicity compared to resting NK cells. NK cells proliferated by activated feeder cells showed high cytotoxicity against the pancreatic cancer cell line PANC-1, and when combined with gemciatbine, cytotoxicity was further increased. In addition, when gemciatbine was used in combination, cytotoxicity tended to increase in a concentration-dependent manner. NK cells proliferated by activated feeder cells showed high cytotoxicity against colorectal cancer cell line SW480, and cytotoxicity was further increased when radiation or cetuximab was used in combination. In particular, NK cells proliferated by activated feeder cells showed stronger antitumor cytotoxicity when combined with radiation and cetuximab. These results indicate that the combination treatment of NK cells proliferated by activated feeder cells and standard therapy will further increase the antitumor effect against pancreatic and colon cancer cell lines.
도 4A에서, K562 세포주 대상 통계적 유의성; ### P < 0.0005 (#Resting NK versus Expanded NK).In Figure 4A, statistical significance for the K562 cell line; ### P <0.0005 ( # Resting NK versus Expanded NK).
도 4B에서, PANC-1 세포주 대상 통계적 유의성; # P < 0.05, ## P < 0.005, ### P < 0.0005 (#Control versus other group s). * P < 0.05, ** P < 0.005, *** P < 0.0005 (*Gem 0.01 uM group versus other groups). In Fig. 4B, statistical significance for PANC-1 cell line; # P <0.05, ## P <0.005, ### P <0.0005 ( # Control versus other group s). * P <0.05, ** P <0.005, *** P <0.0005 ( * Gem 0.01 uM group versus other groups).
도 4C에서, SW480 세포주 대상 통계적 유의성; $ P < 0.05, $$ P < 0.005, $$$ P < 0.0005 ($NK alone versus other groups). # P < 0.05 (#Cetuximab + NK versus IR + NK or Cetuximab + IR + NK). @ P < 0.05, @@@ P < 0.0005 (@IR + NK versus Cetuximab + IR +NK).In Figure 4C, statistical significance for the SW480 cell line; $ P <0.05, $$ P <0.005, $$$ P <0.0005 ( $ NK alone versus other groups). # P <0.05 ( # Cetuximab + NK versus IR + NK or Cetuximab + IR + NK). @ P <0.05, @@@ P <0.0005 ( @ IR + NK versus Cetuximab + IR + NK).
결과 5. 췌장암 및 대장암 NOD/SCID 마우스 모델에서 활성화 지지세포 (activated feeder cells)에 의해 증식된 NK 세포는 강력한 항종양효과를 나타낸다.
본 발명에서 췌장암 및 대장암 NOD/SCID 마우스 모델을 이용하여 활성화 지지세포 (activated feeder cells)를 이용하여 증식시킨 NK세포의 항종양효과를 확인하였다. PANC-1 (인간 췌장암 세포주) 및 SW480 (인간 대장암 세포주) 세포는 NOD/SCID 마우스의 오른쪽 다리 (SW480) 또는 등 (PANC-1)에 접종하였다. 대장암 모델에서 방사선은 마우스의 종양부위에 4 Gy 용량으로 조사하고 활성화 지지세포 (activated feeder cells)를 이용하여 증식시킨 NK세포를 꼬리 정맥으로 주사하였다. Gemcitabine (췌장암 모델에서 양성대조군)과 cetuximab (대장암 모델에서 양성대조군)은 NK 주사 2~3일전에 주사하였다. 활성화 지지세포 (activated feeder cells)를 이용하여 증식시킨 NK세포는 췌장암 및 대장암및 폐암 NOD/SCID 마우스 모델 모두에서 종양성장을 확연히 억제하였다. 췌장암 xenograft 모델에서 대조군에 비하여 gemcitabine 및 NK세포를 병용 투여한 그룹에서 종양성장이 현저하게 억제되었다. 특히, 240 mg/kg의 gemcitabine과 병용 투여시 종양성장이 더욱 유의적으로 억제됨을 확인하였다. 대장암 xenograft 모델에서 증식된 NK 세포의 항종양효과는 방사선 또는 cetuximab 병용에 의해 더욱 상승하였다. 특히 증식된 NK 세포와 cetuximab 및 방사선 병용은 모든 마우스에서 종양을 완전히 제거하였다. 따라서 본 발명에서 개발한 활성화 지지세포 (activated feeder cells)를 이용하여 증식시킨 NK세포는 in vivo에서 강력한 항종양효과를 나타내는 것으로 확인되었고 표준치료와 병용시 서로간의 시너지 효과로 더욱 강력한 항종앙효과를 보였다.In the present invention, the antitumor effect of NK cells proliferated using activated feeder cells was confirmed using a pancreatic cancer and colon cancer NOD/SCID mouse model. PANC-1 (human pancreatic cancer cell line) and SW480 (human colon cancer cell line) cells were inoculated on the right leg (SW480) or back (PANC-1) of NOD/SCID mice. In the colon cancer model, radiation was irradiated to the tumor site of mice at a dose of 4 Gy, and NK cells proliferated using activated feeder cells were injected into the tail vein. Gemcitabine (positive control in pancreatic cancer model) and cetuximab (positive control in colorectal cancer model) were injected 2-3 days before NK injection. NK cells proliferated using activated feeder cells markedly inhibited tumor growth in both pancreatic and colon cancer and lung cancer NOD/SCID mouse models. In the pancreatic cancer xenograft model, tumor growth was significantly inhibited in the group administered with gemcitabine and NK cells compared to the control group. In particular, it was confirmed that tumor growth was more significantly inhibited when administered in combination with 240 mg/kg of gemcitabine. The antitumor effect of proliferated NK cells in the colon cancer xenograft model was further increased by radiation or cetuximab combination. In particular, the combination of proliferated NK cells with cetuximab and radiation completely removed tumors in all mice. Therefore, NK cells proliferated using activated feeder cells developed in the present invention were found to exhibit strong anti-tumor effects in vivo. Showed.
도 5A에서, 췌장암 모델의 통계적 유의성; ### P < 0.0005 (#Control versus other groups). @ P < 0.05, @@@ P < 0.0005 (@Gem 120 mg/kg group versus other groups). $$ P < 0.005 , $$$ P < 0.0005 ($Gem 240 mg/kg group versus other groups). ※※※ P < 0.0005 (※NK group versus other groups). * P < 5(*NK + Gem 120 mg/kg group versus NK + Gem 240 mg/kg group).In Figure 5A, the statistical significance of the pancreatic cancer model; ### P <0.0005 (#Control versus other groups). @ P <0.05, @@@ P <0.0005 ( @
도 5B-C에서, 대장암 모델의 통계적 유의성; @@ P < 0.005, (@Control versus NK or Cetuximab). ## P < 0.005 (#NK versus IR). $ P < 0.05 ($IR at 4 Gy versus NK +Cetuximab, IR + NK or IR + NK + Cetuximab). □□ P < 0.005 (□NK + Cetuximab versus IR + NK, IR + Cetuximab or IR + NK + Cetuximab). ◆ P < 0.005, (◆ IR + NK versus IR + Cetuximab or IR + NK + Cetuximab). ★★★ P < 0.0005, (★ IR + Cetuximab versus IR + NK + Cetuximab)In Figures 5B-C, the statistical significance of the colon cancer model; @@ P <0.005, ( @ Control versus NK or Cetuximab). ## P <0.005 ( # NK versus IR). $ P <0.05 ( $ IR at 4 Gy versus NK +Cetuximab, IR + NK or IR + NK + Cetuximab). □□ P <0.005 ( □ NK + Cetuximab versus IR + NK, IR + Cetuximab or IR + NK + Cetuximab). ◆ P <0.005, ( ◆ IR + NK versus IR + Cetuximab or IR + NK + Cetuximab). ★★★ P <0.0005, ( ★ IR + Cetuximab versus IR + NK + Cetuximab)
참고문헌references
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Claims (9)
a) 인체에서 분리된 말초혈액단핵세포 (PBMCs)에 CD3 항체, IFN-r 및 IL-2을 이용하여 활성화 시키는 단계; 및
b) 상기 활성화된 말초혈액단핵세포 (PBMCs)에 방사선을 조사하는 단계.
A method for producing activated feeder cells for proliferation of NK cells, comprising the following steps:
a) activating peripheral blood mononuclear cells (PBMCs) isolated from the human body using CD3 antibodies, IFN-r and IL-2; And
b) irradiating the activated peripheral blood mononuclear cells (PBMCs).
The activated feeder cell according to claim 3, wherein the activated feeder cells increase T cell inactivation and sensitivity to NK cells, and can proliferate isolated NK cells 10,000 times or more. (activated feeder cells) manufacturing method.
a) 전혈로부터 자연살상세포(NK cell)를 분리하는 단계;
b) 제 3항의 방법에 따라 활성화 지지세포 (activated feeder cells)를 준비하는 단계; 및
c) 상기 분리된 자연살상세포(NK cell)와 준비된 활성화 지지세포(activated feeder cells)를 항-CD16 항체가 고형화된 배양용기에 넣고 배양하는 단계.
Mass proliferation method of activated natural killer cells (NK cells) comprising the following steps:
a) separating natural killer cells (NK cells) from whole blood;
b) preparing activated feeder cells according to the method of claim 3; And
c) culturing the isolated natural killer cells (NK cells) and prepared activated feeder cells into a culture vessel in which an anti-CD16 antibody is solidified.
The method of claim 5, wherein the proliferated NK cells increase expression of various activating receptors and strongly increase antitumor cytotoxicity to target tumor cells. .
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