KR102227155B1 - Feeder cells expressing OX40L and natural killer cell culture method using the same - Google Patents

Feeder cells expressing OX40L and natural killer cell culture method using the same Download PDF

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KR102227155B1
KR102227155B1 KR1020190044625A KR20190044625A KR102227155B1 KR 102227155 B1 KR102227155 B1 KR 102227155B1 KR 1020190044625 A KR1020190044625 A KR 1020190044625A KR 20190044625 A KR20190044625 A KR 20190044625A KR 102227155 B1 KR102227155 B1 KR 102227155B1
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조덕
천세종
김석호
이재민
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주식회사 박셀바이오
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Abstract

본 발명은 OX40L을 발현하는 배양보조세포 및 이를 이용한 자연살해세포 배양 방법에 관한 것으로서, 본 발명의 따른 OX40L을 발현하는 배양보조세포를 말초혈액단핵구와 함께 공배양시킬 경우, 일반적인 K562 세포주를 배양보조세포로 이용하는 방법과 비교하여 높은 수준의 순도 및 증폭률로 자연살해세포를 증식시킬 수 있는바, 자연살해세포를 이용한 다양한 면역 세포 치료 분야에서 치료 효율 및 치료 효능을 획기적으로 증진시킬 수 있을 것으로 기대된다.The present invention relates to a culture aid cell expressing OX40L and a method for culturing natural killer cells using the same.When the culture aid cell expressing OX40L according to the present invention is co-cultured with peripheral blood mononuclear cells, a general K562 cell line is culture aided. Compared to the method used as a cell, natural killer cells can be proliferated with a high level of purity and amplification, so it is expected that the treatment efficiency and therapeutic efficacy can be dramatically improved in various fields of immune cell therapy using natural killer cells. .

Description

OX40L을 발현하는 배양보조세포 및 이를 이용한 자연살해세포 배양 방법{Feeder cells expressing OX40L and natural killer cell culture method using the same}Culture auxiliary cells expressing OX40L and natural killer cell culture method using the same {Feeder cells expressing OX40L and natural killer cell culture method using the same}

본 발명은 OX40L(CD134 ligand)을 발현하는 배양보조세포(feeder cell) 및 이를 이용한 자연살해세포 배양 방법에 관한 것으로서, 보다 구체적으로는 OX40L의 발현이 증가되도록 형질 전환시킨 배양보조세포를 포함하는 NK 세포 배양용 조성물, 배양보조세포주 및 이를 이용한 NK 세포 배양 방법에 관한 것이다.The present invention relates to a feeder cell expressing OX40L (CD134 ligand) and a method for culturing natural killer cells using the same, and more specifically, NK comprising a transformed culture auxiliary cell to increase the expression of OX40L. It relates to a composition for cell culture, a culture auxiliary cell line, and a NK cell culture method using the same.

자연살해세포(이하 NK 세포)는 혈액 내 백혈구로서, 세포독성 및 면역기능을 조절하는 역할을 하는 것으로 알려져 있으며, 특히, 세포독성 기능과 관련하여 암세포 및 바이러스에 감염된 세포 등을 직접 파괴하는 면역 세포이며, 체내의 암세포, 병원균, 감염된 세포, 이종 세포 등을 제거하는 선천성 면역 기능을 가지는 것으로 알려져 있어, NK 세포는 세포 면역 치료제로서 가능성이 높게 평가되고 있다. Natural killer cells (hereinafter referred to as NK cells) are leukocytes in the blood and are known to play a role in regulating cytotoxicity and immune functions.In particular, immune cells that directly destroy cancer cells and cells infected with viruses in relation to cytotoxic functions. It is known to have an innate immune function to remove cancer cells, pathogens, infected cells, and xenogeneic cells in the body, and NK cells are highly likely to be evaluated as cellular immunotherapy.

NK 세포의 기능에 대한 결함은 다수의 질환에서 보고되었으며, 특히 암 환자의 경우 대다수 NK 세포의 불활성화가 있는 것으로 보고되었다. 일부 연구자들은 정상혈액을 가진 자 또는 NK 세포의 기능이 감소된 환자를 대상으로 NK 세포를 체외에서 증식시키고 활성화시키는 방법에 대한 연구를 진행하였고, 상기 연구 결과 체외에서 증폭된 NK 세포를 다양한 암종에 대하여 반응시킨 결과, 우수한 세포독성(cytotoxicity)을 보인다는 점도 증명되었다. 특히 일부 혈액암에 대하여는 임상시험에서 긍정적인 치료효과를 보여주었다.Defects in the function of NK cells have been reported in a number of diseases, especially in cancer patients, it has been reported that there is inactivation of the majority of NK cells. Some researchers have conducted research on a method of proliferating and activating NK cells in vitro in subjects with normal blood or patients with reduced NK cell function.As a result of this study, NK cells amplified in vitro were converted to various carcinomas. As a result of reacting against it, it was also proved that it exhibits excellent cytotoxicity. In particular, for some hematologic cancers, it has shown positive therapeutic effects in clinical trials.

한편, 배양보조세포를 활용하여 NK 세포를 증폭시키는 방법의 명확한 기전은 정확히 알려져 있지 않으나, 대상자의 혈액으로부터 추출된 말초혈액단핵구와 배양보조세포를 같이 배양할 경우 NK 세포를 선택적으로 증폭시키는 점에서 공통된다. 그러나, 다수의 연구에서는 NK 세포 외에도 T 세포 등 다른 림프구의 증폭이 동반되어, 상기 NK 세포를 활용한 면역 세포 치료의 부적합한 면을 보이기도 한다.On the other hand, the precise mechanism of the method of amplifying NK cells using culture aid cells is not known exactly, but when culturing peripheral blood mononuclear cells extracted from the subject's blood and culture aid cells together, NK cells are selectively amplified. Are common. However, in a number of studies, amplification of other lymphocytes such as T cells in addition to NK cells is accompanied, which also shows an inadequate aspect of immune cell therapy using the NK cells.

한편, 종래 보고된 NK 세포의 증식 방법은 다수의 고가 사이토카인을 고농도로 사용하여야 하는바, 경제력이 있는 일부 환자만이 해당 치료의 적용을 받을 수 있는 실정이다.On the other hand, the conventionally reported NK cell proliferation method requires the use of a large number of expensive cytokines at high concentrations, so only some patients with economic power can be applied to the treatment.

따라서, NK 세포의 선택적 증폭을 위한 효율적인 방법에 대한 개발 연구가 주목받고 있으며, 이에 대한 연구가 이루어지고 있으나(한국등록특허 제10-1525199호), 아직은 미비한 실정이다.Therefore, research on the development of an efficient method for selective amplification of NK cells is attracting attention, and research on this is being made (Korean Patent No. 10-1525199), but it is still insufficient.

본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로서, 본 발명자들은 NK 세포를 선택적으로 증폭시키는 방법에 대한 연구를 진행하던 중, 표면 항원 중 수지상 세포나 T 세포의 분화에 관여하는 것으로 알려진 OX40L의 발현이 증가되도록 형질 전환시킨 배양보조세포를 말초혈액단핵구와 공배양하면, NK 세포를 높은 순도 및 증폭률(Fold expansion)로 얻을 수 있음을 확인하였는바, 이에 기초하여 본 발명을 완성하게 되었다.The present invention was conceived to solve the above problems, and the present inventors were conducting research on a method of selectively amplifying NK cells, OX40L known to be involved in the differentiation of dendritic cells or T cells among surface antigens. It was confirmed that NK cells can be obtained with high purity and fold expansion by co-culturing the transformed culture aid cells with peripheral blood mononuclear cells so that the expression of is increased.Based on this, the present invention was completed.

본 발명의 목적은 OX40L을 발현하는 배양보조세포(feeder cells)를 유효성분으로 포함하는, NK 세포 배양용 조성물을 제공하는 것이다.It is an object of the present invention to provide a composition for culturing NK cells, including feeder cells expressing OX40L as an active ingredient.

본 발명의 다른 목적은 하기 단계를 포함하는 자연살해세포 대량 증식 배양 방법을 제공하는 것이다.Another object of the present invention is to provide a method for mass proliferation of natural killer cells comprising the following steps.

(a) 말초혈액으로부터 말초혈액단핵구(Peripheral blood mononuclear cell, PBMC)를 수득하는 단계; 및(a) obtaining peripheral blood mononuclear cells (PBMC) from peripheral blood; And

(b) 상기 수득한 말초혈액단핵구와 OX40L을 발현하는 배양보조세포(feeder cells)를 사이토카인을 함유하는 배지에서 공배양하는 단계.(b) co-culturing the obtained peripheral blood monocytes and feeder cells expressing OX40L in a medium containing cytokines.

본 발명의 또 다른 목적은 OX40L 유전자로 형질 전환된 NK 세포의 배양보조세포주를 제공하는 것이다.Another object of the present invention is to provide an auxiliary cell line for NK cells transformed with the OX40L gene.

그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

상기 목적을 달성하기 위하여, 본 발명은 In order to achieve the above object, the present invention

OX40L을 발현하는 배양보조세포(feeder cells)를 유효성분으로 포함하는, NK 세포 배양용 조성물을 제공한다.It provides a composition for culturing NK cells, comprising as an active ingredient feeder cells expressing OX40L.

본 발명의 일 구현예로, 상기 배양보조세포는 K562 세포일 수 있다.In one embodiment of the present invention, the culture auxiliary cells may be K562 cells.

본 발명의 다른 구현예로, 상기 OX40L을 발현하는 배양보조세포는 OX40L 유전자가 삽입된 발현 벡터로 형질 전환될 수 있다.In another embodiment of the present invention, the auxiliary culture cells expressing the OX40L may be transformed with an expression vector into which the OX40L gene is inserted.

본 발명의 또 다른 구현예로, 상기 OX40L 유전자는 서열번호 1로 표시되는 염기서열로 이루어질 수 있다.In another embodiment of the present invention, the OX40L gene may consist of a nucleotide sequence represented by SEQ ID NO: 1.

본 발명의 또 다른 구현예로, 상기 발현 벡터는 플라스미드 벡터, 코스미드 벡터, 박테리오파지 벡터 및 바이러스 벡터로 이루어진 군으로부터 선택될 수 있다.In another embodiment of the present invention, the expression vector may be selected from the group consisting of a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector.

본 발명의 또 다른 구현예로, 상기 바이러스 벡터는 렌티바이러스(lentivirus) 또는 레트로바이러스(retrovirus) 벡터일 수 있다.In another embodiment of the present invention, the viral vector may be a lentivirus or retrovirus vector.

또한, 본 발명은 하기 단계를 포함하는 자연살해세포 배양 방법을 제공한다.In addition, the present invention provides a method for culturing natural killer cells comprising the following steps.

(a) 말초혈액으로부터 말초혈액단핵구(Peripheral blood mononuclear cell, PBMC)를 수득하는 단계; 및(a) obtaining peripheral blood mononuclear cells (PBMC) from peripheral blood; And

(b) 상기 수득한 말초혈액단핵구와 OX40L을 발현하는 배양보조세포(feeder cells)를 사이토카인을 함유하는 배지에서 공배양하는 단계.(b) co-culturing the obtained peripheral blood monocytes and feeder cells expressing OX40L in a medium containing cytokines.

본 발명의 일 구현예로, 상기 사이토카인은 IL-2, IL-15, IL-21, Flt3-L, IL-7, IL-12 및 IL-18로 이루어진 군으로부터 선택되는 1 종 이상일 수 있다.In one embodiment of the present invention, the cytokine may be at least one selected from the group consisting of IL-2, IL-15, IL-21, Flt3-L, IL-7, IL-12, and IL-18. .

본 발명의 다른 구현예로, 상기 OX40L을 발현하는 배양보조세포(feeder cells)는 50 Gy 내지 300 Gy 방사선을 처리하여 수득될 수 있다.In another embodiment of the present invention, the feeder cells expressing OX40L may be obtained by treatment with 50 Gy to 300 Gy radiation.

본 발명의 또 다른 구현예로, 상기 공배양은 2일 내지 30일 동안 수행될 수 있다.In another embodiment of the present invention, the co-culture may be performed for 2 to 30 days.

또한, 본 발명은 OX40L 유전자로 형질 전환된 NK 세포의 배양보조세포주를 제공한다.In addition, the present invention provides a culture aid cell line for NK cells transformed with the OX40L gene.

본 발명에 따른 배양보조세포, 보다 구체적으로 OX40L의 발현이 증가된 K562 세포를 말초혈액단핵구와 함께 공배양시킬 경우, 일반적인 K562 세포를 배양보조세포로 이용하는 방법과 비교하여 높은 수준의 순도 및 증폭률로 NK 세포를 증식시킬 수 있는바, NK 세포를 이용한 다양한 면역 세포 치료 분야에서 치료 효율 및 치료 효능을 획기적으로 증진시킬 수 있을 것으로 기대된다.When the culture auxiliary cells according to the present invention, more specifically, K562 cells with increased expression of OX40L, are co-cultured with peripheral blood mononuclear cells, they have a high level of purity and amplification compared to the method using general K562 cells as culture auxiliary cells. Since NK cells can be proliferated, it is expected that therapeutic efficiency and therapeutic efficacy can be dramatically improved in various fields of immune cell therapy using NK cells.

도 1은 OX40L을 발현하는 배양보조세포를 이용한 NK 세포 증폭 방법을 나타내는 전체 개요도이다.
도 2는 K562-OX40L 세포주 및 K562 세포주의 mRNA 발현에 관한 것으로, (a)는 OX40L mRNA의 발현 여부를 확인한 결과를 나타낸 것이고, (b)는 OX40L mRNA의 발현량을 확인한 결과를 나타낸 것이다.
도 3은 NK 세포의 순도 및 증폭률 측정에 관한 것으로, (a)는 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 순도를 대조군과 비교하여 나타낸 것이고, (b)는 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 증폭률을 대조군과 비교하여 나타낸 것이다.
도 4는 NK 세포의 순도 및 증폭률 측정에 관한 것으로, (a)는 K562 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 순도와 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 순도를 비교한 결과를 나타낸 것이고, (b)는 K562 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 순도와 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 증폭률을 비교한 결과를 나타낸 것이다.
도 5는 NK 세포의 기능 측정에 관한 것으로, (a)는 K562 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 세포독성과 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 세포독성을 비교한 결과를 나타낸 것이고, (b)는 K562 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 ADCC와 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 ADCC를 비교한 결과를 나타낸 것이다.
도 6은 K562 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 세포 표면 수용체와 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 세포 표면 수용체의 발현 결과를 비교하여 나타낸 것이다.
도 7은 NK 세포의 증폭률 측정에 관한 것으로, (a)는 K562-OX40L 세포주 및 IL-21(5 ng/㎖)에 의해 얻어진 NK 세포의 증폭률과 K562 세포주 및 IL-21(5 ng/㎖)에 의해 얻어진 NK 세포의 증폭률을 비교한 결과를 나타낸 것이고, (b)는 상기 IL-21(50 ng/㎖)인 조건 하의 증폭률을 비교한 결과를 나타낸 것이고, (c)는 상기 IL-21(100 ng/㎖)인 조건 하의 증폭률을 비교한 결과를 나타낸 것이고, (d)는 상기 (a) 내지 (c)의 NK 세포의 평균 증폭률을 나타낸 것이다.
도 8은 다양한 농도의 IL-21(0, 50, 100 ng/㎖)과 E:T 비율(2:1, 1:1, 0.5:1)에서, NK 세포의 기능 측정에 관한 것으로, (a)는 K562 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 세포독성 및 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 세포독성을 비교한 결과를 나타낸 것이고, (b)는 상기 각 NK 세포의 ADCC를 비교한 결과를 나타낸 것이다.
도 9는 OX40L-K562 세포주 및 IL-21(0, 5, 50 ng/㎖)에 의해 얻어진 NK 세포의 기능 측정에 관한 것으로, (a)는 탈과립 마커인 CD107a의 발현비를 나타낸 것이고, (b)는 탈과립 마커인 TNF-α의 발현비를 나타낸 것이고, (c)는 탈과립 마커인 IFN-γ의 발현비를 나타낸 것이고, (d)는 텔로미어 길이 증가비를 나타낸 것이다.1 is an overall schematic diagram showing a method for amplifying NK cells using culture aids expressing OX40L.
Figure 2 relates to the mRNA expression of the K562-OX40L cell line and the K562 cell line, (a) shows the result of confirming the expression of OX40L mRNA, (b) shows the result of confirming the expression level of OX40L mRNA.
Figure 3 relates to the measurement of the purity and amplification rate of NK cells, (a) shows the purity of NK cells obtained by co-culturing K562-OX40L cell line and peripheral blood mononuclear cells compared to the control, (b) is K562-OX40L It shows the amplification rate of NK cells obtained by co-culturing the cell line and peripheral blood monocytes compared to the control.
Figure 4 relates to the measurement of the purity and amplification rate of NK cells. The results of comparing the purity are shown, and (b) is the result of comparing the purity of NK cells obtained by co-culture with K562 cell line and peripheral blood monocytes and the amplification rate of NK cells obtained by co-culture with K562-OX40L cell line and peripheral blood monocytes. Is shown.
Figure 5 relates to the measurement of the function of NK cells, (a) is the cytotoxicity of NK cells obtained by co-culture with K562 cell line and peripheral blood mononuclear cells and cells of NK cells obtained by co-culture with K562-OX40L cell line and peripheral blood mononuclear cells. The results of comparing toxicity are shown, and (b) is the result of comparing ADCC of NK cells obtained by co-culturing K562 cell line and peripheral blood monocytes and ADCC of NK cells obtained by co-culturing K562-OX40L cell line and peripheral blood monocytes. Is shown.
Figure 6 shows the comparison of the expression results of the cell surface receptor of NK cells obtained by co-culturing K562 cell line and peripheral blood monocytes and the cell surface receptor of NK cells obtained by co-culturing K562-OX40L cell line and peripheral blood monocytes.
Figure 7 relates to the measurement of the amplification rate of NK cells, (a) is the amplification rate of NK cells obtained by K562-OX40L cell line and IL-21 (5 ng/ml) and K562 cell line and IL-21 (5 ng/ml) It shows the result of comparing the amplification rate of NK cells obtained by, (b) shows the result of comparing the amplification rate under the condition of IL-21 (50 ng/ml), and (c) shows the result of comparing the amplification rate of IL-21 ( 100 ng/ml) shows the results of comparing the amplification rates under the condition, and (d) shows the average amplification rates of the NK cells of the above (a) to (c).
Figure 8 relates to the measurement of the function of NK cells at various concentrations of IL-21 (0, 50, 100 ng/ml) and E:T ratio (2:1, 1:1, 0.5:1), (a ) Shows the results of comparing the cytotoxicity of NK cells obtained by co-culture with K562 cell line and peripheral blood monocytes and the cytotoxicity of NK cells obtained by co-culture with K562-OX40L cell line and peripheral blood monocytes. It shows the result of comparing the ADCC of each NK cell.
Figure 9 relates to the measurement of the function of NK cells obtained by the OX40L-K562 cell line and IL-21 (0, 5, 50 ng/ml), (a) shows the expression ratio of the degranulation marker CD107a, (b ) Represents the expression ratio of the degranulation marker TNF-α, (c) represents the expression ratio of the degranulation marker IFN-γ, and (d) represents the increase ratio of telomere length.

이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명자들은 표면 항원 중 수지상 세포나 T 세포의 분화에 관여하는 것으로 알려진 OX40L의 발현이 증가되도록 형질 전환시킨 배양보조세포를 말초혈액단핵구와 공배양하여 높은 순도와 증폭률을 가지는 NK 세포를 수득할 수 있음을 확인함으로써, 본 발명을 완성하였다.The present inventors obtained NK cells having high purity and amplification rate by co-culture with peripheral blood mononuclear cells transformed to increase the expression of OX40L, which is known to be involved in the differentiation of dendritic cells or T cells among surface antigens. By confirming that there is, the present invention has been completed.

이에, 본 발명은 OX40L을 발현하는 배양보조세포(feeder cells)를 유효성분으로 포함하는, NK 세포 배양용 조성물을 제공한다.Accordingly, the present invention provides a composition for culturing NK cells, including feeder cells expressing OX40L as an active ingredient.

본 발명에서 사용되는 용어, “OX40L”은 OX40(CD134)의 리간드로서, TNF 패밀리의 구성원이고, B 세포, 대식세포, 내피 세포 및 수지상 세포(DC)를 비롯한 활성화된 항원제시세포(APC)에서 발현되는 것으로 알려져 있다. 또한, OX40L은 활성화 T 세포에 발현하는 OX40을 매개하여 T 세포 수용체(TCR)를 자극하여 T 세포의 증식에 관여하고, IL-2, IL-4, IFN-γ등의 사이토카인 생산의 공(共)자극으로 작용하는 것으로 알려져 있으며, 이외에도 활성화된 혈관내피세포에서 발현되는 OX40L은 활성화된 T 세포가 염증 부위로 침윤하는 것에 관여하는 것으로 알려져 있다. The term "OX40L" used in the present invention is a ligand of OX40 (CD134), a member of the TNF family, and in activated antigen presenting cells (APCs) including B cells, macrophages, endothelial cells and dendritic cells (DC). It is known to be expressed. In addition, OX40L is involved in the proliferation of T cells by mediating OX40 expressed on activated T cells by stimulating T cell receptors (TCRs), and is responsible for the production of cytokines such as IL-2, IL-4, and IFN-γ.共) It is known to act as an stimulus, and in addition, OX40L expressed in activated vascular endothelial cells is known to be involved in the invasion of activated T cells into the inflammatory site.

본 발명에서 사용되는 용어, “배양보조세포(feeder cells, 지지세포라고도 한다)”는 분열 증식하는 능력은 없지만 대사 활성이 있기 때문에 여러 가지 대사물질을 생산하여 목적 NK 세포의 증식을 돕는 세포이다. 본 발명에서 사용될 수 있는 배양보조세포로는 유전자가 도입된 동물 세포주(cell line)나 각종 사이토카인이나 화합물이 처리된 말초혈 백혈구 세포(PBL), 자기 또는 타인의 말초혈 백혈구 세포, T 세포, B 세포 또는 다단핵구(monocyte) 등이 있으며, 바람직하게는 유전자가 도입된 동물 세포주가 사용될 수 있으며, 보다 바람직하게는 인간만성골수백혈병세포주의 하나인 K562 세포일 수 있으나, 이에 제한되는 것은 아니며, 본 발명이 속하는 기술 분야에서 통상적으로 사용 가능한 것으로 알려진 다른 배양보조세포들도 본 발명의 목적에 부합한다면, 제한 없이 사용 가능함은 당연한 것이다.The term “feeder cells” used in the present invention is a cell that does not have the ability to divide and proliferate, but because it has metabolic activity, it produces various metabolites to help the proliferation of target NK cells. The culture auxiliary cells that can be used in the present invention include an animal cell line into which a gene has been introduced, peripheral blood leukocyte cells (PBL) treated with various cytokines or compounds, peripheral blood leukocyte cells of oneself or others, T cells, B cells or mononuclear cells (monocyte), and the like, preferably an animal cell line into which a gene has been introduced may be used, and more preferably, it may be a K562 cell, which is one of the human chronic myelogenous leukemia cell lines, but is not limited thereto, It is natural that other culture aid cells, which are known to be commonly used in the technical field to which the present invention pertains, can also be used without limitation, provided that they meet the object of the present invention.

한편, 본 발명에 있어서, OX40L을 발현하는 배양보조세포는 OX40L 유전자가 삽입된 발현 벡터로 형질 전환된 것을 특징으로 한다.On the other hand, in the present invention, the culture aid cells expressing OX40L are characterized in that they are transformed with an expression vector into which the OX40L gene is inserted.

본 발명에 따른 OX40L 유전자(NCBI accession number : NM_003326)는 서열번호 1의 염기서열로 이루어진 것일 수 있으며, 상기 염기서열의 상동체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 유전자는 서열번호 1의 염기서열과 각각 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다.The OX40L gene (NCBI accession number: NM_003326) according to the present invention may be composed of the nucleotide sequence of SEQ ID NO: 1, and homologs of the nucleotide sequence are included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more, respectively, with the nucleotide sequence of SEQ ID NO: 1. I can.

본 발명에서 사용되는 용어, “발현 벡터”는 적당한 숙주세포에서 목적 단백질 또는 목적 RNA를 발현할 수 있는 벡터로서, 유전자 삽입물이 발현되도록 작동 가능하게 연결된(operably linked) 필수적인 조절요소를 포함하는 유전자 제작물을 말한다.The term “expression vector” used in the present invention is a vector capable of expressing a target protein or target RNA in a suitable host cell, and a gene product comprising essential regulatory elements operably linked to express a gene insert Say.

본 발명에서 용어, “작동 가능하게 연결된(operably linked)”은 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질 또는 RNA를 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 것을 말한다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동 가능하게 연결되어 코딩하는 핵산 서열의 발현에 영향을 미칠 수 있다. 재조합 발현 벡터와의 작동적 연결은 당해 기술 분야에 잘 알려진 유전자 재조합 기술을 이용하여 달성할 수 있으며, 부위-특이적 DNA 절단 및 연결은 해당 기술 분야에 일반적으로 알려진 효소 등을 사용한다.In the present invention, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect the expression of the encoding nucleic acid sequence. The operative linkage with the recombinant expression vector can be achieved using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage use enzymes generally known in the art.

본 발명에 사용 가능한 발현 벡터는 플라스미드 벡터, 코스미드(cosmid) 벡터, 박테리오파아지 벡터, 바이러스 벡터 등을 포함하나 이에 제한되지는 않으며, 바람직하게는 바이러스 벡터일 수 있으며, 보다 바람직하게는 렌티바이러스(lentivirus) 또는 레트로바이러스(retrovirus) 벡터일 수 있으나, 이에 제한되는 것은 아니며, 적합한 발현 벡터는 프로모터(promoter), 오퍼레이터(operator), 개시 코돈(initiation codon), 종결 코돈(termination codon), 폴리아데닐화 신호(polyadenylation signal), 인핸서(enhancer)와 같은 발현 조절 서열 외에도, 막 표적화 또는 분비를 위한 신호서열(signal sequence) 또는 리더서열(leader sequence)을 포함하여 목적에 따라 다양하게 제조될 수 있다. 발현 벡터의 프로모터는 구성적(constitutive) 또는 유도성(inducible)일 수 있다. 또한, 발현 벡터는 벡터를 함유하는 숙주세포를 선택하기 위한 선택 마커를 포함하고, 복제가 가능한 발현 벡터인 경우 복제 기원을 포함한다.Expression vectors usable in the present invention include, but are not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like, and may preferably be a viral vector, more preferably a lentivirus ( lentivirus) or retrovirus vector, but is not limited thereto, and suitable expression vectors include promoter, operator, initiation codon, termination codon, polyadenylation In addition to expression control sequences such as a polyadenylation signal and an enhancer, it may be prepared in various ways according to the purpose, including a signal sequence or a leader sequence for membrane targeting or secretion. The promoter of the expression vector may be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector, and, in the case of an expression vector capable of replication, includes an origin of replication.

본 발명의 다른 양태로서, 본 발명은 하기 단계를 포함하는 자연살해세포 배양 방법을 제공한다.As another aspect of the present invention, the present invention provides a method for culturing natural killer cells comprising the following steps.

(a) 말초혈액으로부터 말초혈액단핵구(Peripheral blood mononuclear cell, PBMC)를 수득하는 단계; 및(a) obtaining peripheral blood mononuclear cells (PBMC) from peripheral blood; And

(b) 상기 수득한 말초혈액단핵구와 OX40L을 발현하는 배양보조세포(feeder cells)를 사이토카인을 함유하는 배지에서 공배양하는 단계.(b) co-culturing the obtained peripheral blood monocytes and feeder cells expressing OX40L in a medium containing cytokines.

본 발명에서 말초혈액단핵구(peripheral blood mononuclear cell, PBMC)는 혈액 내 존재하는 구형 핵을 가진 세포를 의미하며, 이러한 말초혈액단핵구에는 B 세포, T 세포, 대식세포(macrophage), 수지상 세포(dendritic cell), 자연살해세포(NK cell, natural killer cell) 등의 면역세포들이 포함되어 있다.In the present invention, peripheral blood mononuclear cells (PBMCs) refer to cells with spherical nuclei present in blood, and such peripheral blood mononuclear cells include B cells, T cells, macrophages, and dendritic cells. ), natural killer cells (NK cells, natural killer cells) and other immune cells.

본 발명에서 배지에 포함될 수 있는 사이토카인(cytokine)은 인터루킨(interleukin) 류에서 선택된 하나 이상인 것이 바람직하며, 인터루킨은 림프구나 단구 및 대식세포 등 면역담당 세포가 생산하는 단백질성 생물활성물질의 총칭으로 본 발명에서 사용가능한 인터루킨으로는 IL-2, IL-15, IL-21, Flt3-L, IL-7, IL-12 및 IL-18로 이루어진 군에서 선택되는 하나 이상인 것이 바람직하며, 특히, IL-2 및 IL-15 또는 IL-2, IL-15 및 IL-21을 사용하는데, IL-21은 지속적 사용이 아닌 단기(배양시작일에 배양액에 한번 투여 후 중단)로 사용함이 바람직하지만, 이에 한정되는 것은 아니며, 본 발명에 속하는 기술분야에서 통상의 지식을 가진 자에게 다른 사이토카인도 본 발명의 목적에 부합하는 한 제한없이 이용 가능함은 자명한 것이다.In the present invention, the cytokine that may be included in the medium is preferably at least one selected from interleukin, and interleukin is a generic term for proteinaceous biologically active substances produced by immune cells such as lymphocytes, monocytes, and macrophages. Interleukin usable in the present invention is preferably one or more selected from the group consisting of IL-2, IL-15, IL-21, Flt3-L, IL-7, IL-12 and IL-18, and in particular, IL -2 and IL-15 or IL-2, IL-15 and IL-21 are used, but it is preferable to use IL-21 for a short term (stop after administration once in the culture medium at the start of culture), but not for continuous use. It is obvious that other cytokines can be used without limitation, as long as they meet the object of the present invention, to those of ordinary skill in the art.

상기 사이토카인들이 NK 세포 분화에 관여한다는 사실은 여러 문헌에서 찾을 수 있다. 사이토카인 수용체의 γ의 발현이 결핍된 쥐에서 B 세포와 T 세포는 발견이 되지만 NK 세포는 발견되지 않는 점에서 γ를 지닌 수용체들이 NK 분화에 중요한 역할을 한다고 알려져 있으며, 수용체의 γ형태는 IL-2, IL-4, IL-7, IL-9, IL-15 또는 IL-21의 수용체이다.The fact that these cytokines are involved in NK cell differentiation can be found in several literatures. In mice deficient in the expression of γ of the cytokine receptor, B cells and T cells are found, but NK cells are not found. Therefore, it is known that receptors with γ play an important role in NK differentiation, and the γ form of the receptor is IL. -2, IL-4, IL-7, IL-9, IL-15 or IL-21 receptor.

상기 IL-2는 성숙된 NK 세포의 증식과 활성화를 증진시키는 기능을 지니고 있음이 보고되고 있다. IL-2가 결핍된 인간과 마우스에서는 NK 세포의 수가 현저히 감소한다는 보고가 있으나, 한편으로는 IL-2 및 IL-2Ra 결핍은 간접적으로 NK 세포의 수와 활성화에 영향을 미친다는 연구 결과도 있으며, IL-2R 사슬은 IL-15의 수용체를 형성하는데 관여한다고 알려져 있다.It has been reported that IL-2 has a function of enhancing the proliferation and activation of mature NK cells. It has been reported that the number of NK cells is significantly reduced in humans and mice deficient in IL-2, but there are also studies showing that IL-2 and IL-2Ra deficiency indirectly affects the number and activation of NK cells. , IL-2R chain is known to be involved in the formation of receptors for IL-15.

상기 IL-15에 관하여, IL-15 또는 IL-15Rα가 결핍된 쥐에서는 NK 세포가 발견되지 않는다는 사실과 IL-15 생성에 요구되는 전사인자 인터페론(transcription factor interferon, IFN)-조절 인자 1이 결핍된 쥐에서는 NK 세포가 결핍됨이 밝혀졌다. 이로써, IL-15가 NK 세포 분화에 관여한다는 것과 IL-15는 NK 세포에서 발현되는 IL-15 수용체를 통해서 NK 세포의 성장과 분화를 직접적으로 증진시킨다는 것이 알려져 있다.Regarding the above IL-15, the fact that NK cells are not found in mice deficient in IL-15 or IL-15Rα, and a lack of transcription factor interferon (IFN)-regulator 1 required for IL-15 production It was found that NK cells were deficient in old mice. Thus, it is known that IL-15 is involved in NK cell differentiation and that IL-15 directly enhances the growth and differentiation of NK cells through the IL-15 receptor expressed in NK cells.

상기 IL-21은 transcription-3(STAT-3)을 자극시킴으로써 NK 세포의 텔로미어 길이(telomere length)를 증가시키고 NK 세포의 활성, 성장, 증폭에 중요한 역할을 하는 것으로 알려져 있다. 특히, 인간의 CD34+ 조혈모세포에서 IL-15와의 조합을 통해 NK 세포의 성장을 돕는다는 것이 보고되었고, 멤브레인에 결합된(membrane-bound, mb) IL-21을 통해 NK 세포를 증폭시킬 시, 인터페론 감마(IFN-γ의 발현이나 세포독성이 증가한다는 것이 확인되었다. 또한, 배양액에 IL-21의 주입을 지속하는 것보다 처음 한 번만 주입하는 것이 NK 세포 증폭에 효과적인 것으로 보고되었다. The IL-21 is known to increase the telomere length of NK cells by stimulating transcription-3 (STAT-3) and play an important role in the activity, growth, and amplification of NK cells. In particular, it has been reported that human CD34+ hematopoietic stem cells help the growth of NK cells through combination with IL-15, and when NK cells are amplified through membrane-bound (mb) IL-21, interferon It was confirmed that the expression of gamma (IFN-γ and cytotoxicity) was increased. In addition, it was reported that the first injection of IL-21 into the culture medium was more effective in amplifying NK cells than to continue.

본 발명은 말초혈액단핵구(Peripheral Blood Mononuclear Cell, PBMC)와 방사선을 조사한 OX40L이 발현된 배양보조세포(feeder cells)를 이용하여 높은 순도 및 증폭률로 NK 세포를 유도하고 증식하는 방법으로서, 이때, 본 발명에 따른 방사선은 감마선일 수 있고, 바람직한 방사선 조사량은 50 Gy 내지 300 Gy일 수 있으며, 보다 바람직하게는 70 Gy 내지 200 Gy일 수 있으며, 가장 바람직하게는 100 Gy일 수 있으나, 이에 제한되는 것은 아니다. The present invention is a method for inducing and proliferating NK cells with high purity and amplification rate using peripheral blood mononuclear cells (PBMC) and feeder cells expressing OX40L irradiated with radiation. The radiation according to the invention may be gamma rays, and a preferred radiation dose may be 50 Gy to 300 Gy, more preferably 70 Gy to 200 Gy, and most preferably 100 Gy, but is limited thereto. no.

NK 세포 증식 방법은 대부분 MACs(magnetic activated cell sorting)를 이용하여 T 세포를 제거하거나, NK 세포만을 분리하여 증식시키는 방법을 사용하였다. 그러나, NK 세포를 분리 시 cliniMACs나 FACs sorter 등 고가 장비의 사용이 필요하고, 별도로 단핵구에 다량으로 존재하는 T 세포를 키트를 사용하여 제거해야하므로 비용이 많이 들고 증식 과정이 복잡한 문제점이 있었다.Most of the NK cell proliferation methods used a method of removing T cells using magnetic activated cell sorting (MACs) or separating and proliferating only NK cells. However, when separating NK cells, expensive equipment such as cliniMACs or FACs sorter is required, and T cells present in large amounts in monocytes must be removed using a kit, which is expensive and the proliferation process is complicated.

상기와 같은 문제를 해결하기 위해서, 본 발명자들이 개발한 방법은 별도의 분리 과정없이 상기 OX40L을 발현하는 배양보조세포(feeder cells)에 방사선을 조사한 후, 말초혈액단핵구와 공동 배양함으로써 높은 순도와 증폭률로 NK 세포를 유도하고 증식시킬 수 있다.In order to solve the above problem, the method developed by the present inventors is a high purity and amplification rate by irradiating the feeder cells expressing the OX40L without a separate separation process and then co-culturing with peripheral blood monocytes. NK cells can be induced and proliferated.

보다 구체적으로, 본 발명의 일실시예에서는 OX40L을 발현하는 배양보조세포주로서, OX40L를 발현하는 K562(K562-OX40L) 및 정상인 유래 말초혈액단핵구를 준비하여(실시예 1 참조), 상기 준비된 배양보조세포주 및 말초혈액단핵구를 사이토카인인 IL-2 및 IL-15 존재 하에서 공배양시켰으며, trypan blue exclusion method 및 FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체를 사용하여 NK 세포 수 및 증폭된 NK 세포의 순도를 확인한 결과, 높은 증폭률 및 순도를 나타내는 NK 세포를 수득할 수 있음을 확인하였다(실시예 2 참조).More specifically, in one embodiment of the present invention, as a culture aid cell line expressing OX40L, K562 (K562-OX40L) expressing OX40L and peripheral blood mononuclear cells derived from normal people were prepared (see Example 1), and the prepared culture aid Cell lines and peripheral blood monocytes were co-cultured in the presence of cytokines IL-2 and IL-15, and NK using trypan blue exclusion method and FITC-anti-human CD3 and PE-Cy5 anti-human CD56 monoclonal antibodies. As a result of confirming the number of cells and the purity of the amplified NK cells, it was confirmed that NK cells exhibiting a high amplification rate and purity can be obtained (see Example 2).

또한, 본 발명의 다른 일실시예에서는 상기 준비된 배양보조세포주 및 말초혈액단핵구를 사이토카인인 IL-2, IL-15 및 IL-21(한번만 노출) 존재 하에서 공배양시켰으며, trypan blue exclusion method 및 FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체를 사용하여 NK 세포 수 및 증폭된 NK 세포의 순도를 확인한 결과, 높은 증폭률을 나타내는 NK 세포를 수득할 수 있음을 확인하였다(실시예 3 참조).Further, in another embodiment of the present invention, the prepared culture auxiliary cell line and peripheral blood monocytes were co-cultured in the presence of cytokines IL-2, IL-15 and IL-21 (exposed only once), and the trypan blue exclusion method and As a result of confirming the number of NK cells and the purity of amplified NK cells using FITC-anti-human CD3 and PE-Cy5 anti-human CD56 monoclonal antibodies, it was confirmed that NK cells exhibiting a high amplification rate can be obtained ( See Example 3).

본 발명의 또 다른 양태로서, 본 발명은 OX40L 유전자로 형질 전환된, NK 세포 배양보조세포주를 제공한다.In another aspect of the present invention, the present invention provides an NK cell culture auxiliary cell line transformed with the OX40L gene.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[실시예][Example]

실시예 1. 실험준비 및 실험방법Example 1. Experimental Preparation and Experimental Method

1-1. OX40L 발현 렌티바이러스의 생산1-1. Production of OX40L expressing lentivirus

본 발명에 따른 OX40L을 발현하는 배양보조세포를 생산하기 위해서, 인간 유래 OX40L 유전자를 렌티바이러스 벡터인 pLVX-IRES-ZsGreen에 클로닝하여 재조합 렌티바이러스 생산용 벡터를 제작하였다. 이후, 바이러스 생산을 위해 상기로부터 제작된 재조합 유전자(OX40L-pLVX-IRES-ZsGreen)를 293FT 세포에 패키징 벡터와 함께 lipofectamin3000(Invitrogen)을 이용하여 형질 주입시킨 다음, 24시간이 지나면, 새로운 배지로 교환하여 48시간 동안 세포를 배양한 후, 바이러스가 함유된 배지를 회수하였다. 회수된 배지는 500 × g 에서 10분간 원심분리하고, 0.45 ㎛ 필터를 이용하여 바이러스가 함유된 순수한 배지만을 분리하여 OX40L 발현 렌티바이러스를 생산하였다. 마지막으로, Centricon을 이용하여 렌티바이러스를 농축하고 멸균된 1.5 ㎖ 튜브에 1 ㎖씩 분주한 후 -70 ℃에 보관하였다.In order to produce a culture aid cell expressing OX40L according to the present invention, a human-derived OX40L gene was cloned into a lentiviral vector, pLVX-IRES-ZsGreen, to produce a vector for producing a recombinant lentivirus. Thereafter, for virus production, the recombinant gene (OX40L-pLVX-IRES-ZsGreen) produced from the above was transfected into 293FT cells using lipofectamin3000 (Invitrogen) with a packaging vector, and after 24 hours, exchanged with a new medium. After culturing the cells for 48 hours, the medium containing the virus was recovered. The recovered medium was centrifuged at 500 × g for 10 minutes, and only pure medium containing virus was separated using a 0.45 μm filter to produce OX40L-expressing lentivirus. Finally, the lentivirus was concentrated using Centricon, 1 ml each was dispensed into a sterilized 1.5 ml tube, and stored at -70°C.

1-2. OX40L 발현 K562(K562-OX40L) 세포주 생산1-2. OX40L expression K562 (K562-OX40L) cell line production

상기 실시예 1-1의 OX40L 발현 렌티바이러스의 생산 하루 전에 배양보조세포인 K562를 미리 준비하고, 준비된 K562 세포 배지 9 ㎖에 냉동 보관한 OX40L 발현 렌티바이러스 1 ㎖를 녹여 polybrene(8 ㎍/㎖)을 함께 첨가한 후, 미리 준비된 K562 세포 배지와 교환하고, 48시간 동안 세포 배양을 진행하였다. 이후, 초고속 유세포 자동분리기를 이용한 녹색 형광 발현 세포의 선별과정을 통하여 감염된 세포만을 선별하였다. 또한, 역전사 중합효소 연쇄반응(RT-PCR)을 통해 감염된 세포의 OX40L mRNA 발현 여부를 확인하고, 유세포분석기(FACS)를 통해 감염된 세포 표면에서 OX40L 단백질의 발현 여부를 확인하여, OX40L이 발현된 K562 세포주를 생산하였다.One day before production of the OX40L-expressing lentivirus of Example 1-1, K562 was prepared in advance, and 1 ml of OX40L-expressing lentivirus stored frozen in 9 ml of prepared K562 cell medium was dissolved and polybrene (8 µg/ml). After adding together, it was exchanged with a K562 cell medium prepared in advance, and cell culture was performed for 48 hours. Thereafter, only the infected cells were selected through the selection process of green fluorescent-expressing cells using an ultra-high-speed flow cell automatic separator. In addition, by checking the expression of OX40L mRNA in infected cells through reverse transcription polymerase chain reaction (RT-PCR), and by checking the expression of OX40L protein on the surface of infected cells through flow cytometry (FACS), K562 expressing OX40L The cell line was produced.

1-3. OX40L 발현 K562(K562-OX40L)의 배양1-3. Culture of OX40L-expressing K562 (K562-OX40L)

상기 실시예 1-2로부터 생산한 OX40L을 발현하는 K562(K562-OX40L) 세포주를 5 % CO2가 공급된 배양기를 이용하여, 37 ℃의 25 ㎖의 완전 RPMI 1640 배지가 들어있는 T-75 플라스크에서 배양시켰다. 이후, 400 × g 조건 하에서 3분 동안 원심 분리를 진행하였으며, 5 ㎖의 완전 RPMI 1640 배지에서 세포 펠릿을 재부유시켜 배양보조세포를 수확한 뒤, 상기 배양보조세포의 과도한 성장을 방지하기 위해서, 상기 배양보조세포에 Gammacell 3000 Elan 방사기를 이용하여 100 Gy로 감마선을 조사하였다.The K562 (K562-OX40L) cell line expressing OX40L produced from Example 1-2 was used in an incubator supplied with 5% CO 2 in a T-75 flask containing 25 ml of complete RPMI 1640 medium at 37°C. Incubated in. Thereafter, centrifugation was performed for 3 minutes under 400 × g conditions, and the cell pellet was resuspended in 5 ml of complete RPMI 1640 medium to harvest the culture supplemental cells, and in order to prevent excessive growth of the culture supplemental cells, The culture auxiliary cells were irradiated with gamma rays at 100 Gy using a Gammacell 3000 Elan radiator.

1-4. 말초혈액단핵구(Peripheral Blood Mononuclear Cells, PBMCs)의 분리1-4. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

말초혈액단핵구(PBMCs)의 분리를 위해서, 정상인 공여자의 전혈을 PBS와 1:2 비율(10 ㎖ 전혈 : 20 ㎖ PBS)로 희석하여, 15 ㎖ Lymphoprep 상에서 오버레이하고, 상온에서 1200 × g 조건으로 25분 동안 중지없이 원심 분리를 진행하고(가속 1, 감속 0), 연막층(buffy coat layer)에서 세포를 수확하고, 7분 동안 400 × g 에서 PBS로 세 번 세척하였다. 이후, 세포 펠릿을 탭핑(tapping)하여 재부유시킨 다음, NK 세포 배양 배지를 추가하여 trypan blue exclusion method를 통해 세포 수를 카운트하였다. For the isolation of peripheral blood mononuclear cells (PBMCs), whole blood from a normal donor was diluted with PBS in a ratio of 1:2 (10 mL whole blood: 20 mL PBS), and then overlaid on 15 mL Lymphoprep, and at room temperature at 1200 × g at 25 Centrifugation was performed without stopping for a minute (acceleration 1, deceleration 0), cells were harvested from a buffery coat layer, and washed three times with PBS at 400 × g for 7 minutes. Thereafter, the cell pellet was tapped to resuspend, and then NK cell culture medium was added, and the number of cells was counted through the trypan blue exclusion method.

실시예 2. 분리된 말초혈액단핵구(PBMCs) 및 OX40L 발현 K562(K562-OX40L)를 이용한 NK 세포의 배양을 통한 NK 세포의 순도 및 증폭률 확인Example 2. Confirmation of purity and amplification rate of NK cells through culture of NK cells using isolated peripheral blood monocytes (PBMCs) and OX40L-expressing K562 (K562-OX40L)

본 발명에 따라 상기 실시예 1로부터 준비한 말초혈액단핵구(PBMCs) 및 OX40L 발현 K562(K562-OX40L) 세포주를 이용하여 NK 세포의 증폭을 수행하였으며, 실험디자인은 도 1에 나타낸 바와 같다. 보다 구체적으로, 3 × 106 개의 말초혈액단핵구 및 0.5 × 106 개의 방사선 100 Gy가 조사된 K562-OX40L 세포주를 1 ㎖ NK 세포 배지가 포함된 24 웰 플레이트 상에 접종한 다음, 20 U/㎖ IL-2가 함유된 1 ㎖ NK 세포 배지를 추가하여 총 배지 부피는 2 ㎖/well, IL-2의 최종 농도는 10 U/㎖로 만든 뒤, 부드럽게 피펫팅(pipetting) 하여 혼합시킨 후, 37 ℃, 5 % CO2 조건의 배양기에서 배양하였다. In accordance with the present invention, NK cells were amplified using the peripheral blood monocytes (PBMCs) and OX40L-expressing K562 (K562-OX40L) cell lines prepared from Example 1, and the experimental design is as shown in FIG. More specifically, 3 × 10 6 peripheral blood monocytes and 0.5 × 10 6 radiation 100 Gy-irradiated K562-OX40L cell line was inoculated onto a 24-well plate containing 1 ml NK cell medium, and then 20 U/ml After adding 1 ㎖ NK cell medium containing IL-2, the total medium volume was 2 ㎖/well, and the final concentration of IL-2 was 10 U/ ㎖, and then gently pipetted to mix, 37 It was cultured in an incubator under conditions of ℃, 5% CO 2

상기 방법으로 만들어진 K562-OX40L 세포주가 제대로 OX40L를 발현하는지 확인하기 위해, RT-PCR실험을 수행하였다. 그 결과, 도 2a에서 나타낸 바와 같이 K562-OX40L 세포주에서 OX40L mRNA의 발현을 확인하였다. 또한, 도 2b에서 나타낸 바와 같이 K562-OX40L 세포주에서 발현된 OX40L mRNA의 양을 확인하였다.In order to confirm whether the K562-OX40L cell line made by the above method properly expresses OX40L, an RT-PCR experiment was performed. As a result, as shown in Figure 2a, the expression of OX40L mRNA was confirmed in the K562-OX40L cell line. In addition, as shown in Figure 2b, the amount of OX40L mRNA expressed in the K562-OX40L cell line was confirmed.

① 배양 3일 및 5일 째: 배지의 절반을 제거하고 새로운 IL-2를 함유하는 1 ㎖의 새로운 배지를 추가하여 2 ㎖ 최종 부피에 대해 IL-2가 10 U/㎖가 되도록 만들었다.① Day 3 and Day 5 of cultivation: Half of the medium was removed, and 1 mL of fresh medium containing new IL-2 was added to make IL-2 to 10 U/mL for a final volume of 2 mL.

② 배양 7일 째: 배지에 포함된 세포의 수를 카운트하였다. 이 때, 하나의 웰에 세포를 500 μℓ씩 나누고 여기에 방사선 100 Gy가 조사된 K562-OX40L 0.5 × 106 개를 첨가하여 재접종을 한 후, 총 배지 부피를 2 ㎖/well, IL-2의 농도를 100 U/㎖로 증가시키고, 추가적으로 5 ng/㎖의 IL-15를 배지에 첨가하였다. ② 7th day of culture: The number of cells contained in the medium was counted. At this time, 500 μℓ of cells were divided into one well, and 0.5 × 10 6 K562-OX40L irradiated with 100 Gy of radiation were added thereto, followed by re-inoculation, and then the total medium volume was 2 ml/well, IL-2. The concentration of was increased to 100 U/ml, and an additional 5 ng/ml of IL-15 was added to the medium.

한편, 이 방법에서 NK 세포는 전체 21일 중 0, 7, 14일 째에만 K562-OX40L 세포로 총 세 번 자극시켰으며, 매주 FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체를 사용하여 NK 세포의 순도 및 증폭률(Fold expansion)을 평가하였다. On the other hand, in this method, NK cells were stimulated with K562-OX40L cells three times only on days 0, 7, and 14 of the total 21 days, and weekly FITC-anti-human CD3 and PE-Cy5 anti-human CD56 monoclonal The antibody was used to evaluate the purity and fold expansion of NK cells.

③ 배양 10일 및 12일 째: 배지의 절반을 제거하고, 200 U/㎖의 IL-2 및 10 ng/㎖의 IL-15가 함유된 1 ㎖의 새로운 배지를 추가하여, 총 2 ㎖의 배지 내 농도가 100 U/㎖의 IL-2 및 5 ng/㎖의 IL-15가 되도록 하였다. ③ Day 10 and Day 12 of cultivation: Half of the medium was removed, and 1 mL of fresh medium containing 200 U/mL of IL-2 and 10 ng/mL of IL-15 was added, for a total of 2 mL of medium. The concentration within was made to be 100 U/ml of IL-2 and 5 ng/ml of IL-15.

④ 배양 14일 째: 배양 14일 째에는 2주 동안 배양된 세포의 수를 카운트했으며, 7일 째와 마찬가지로, 다시 한번 세포를 하나의 웰 당 500 μℓ씩 나누고 여기에 0.5 × 106 개의 방사선 100 Gy가 조사된 K562-OX40L 첨가하여 재접종을 한 후, 총 배지 부피를 2 ㎖/well, IL-2의 농도를 100 U/㎖로, 5 ng/㎖의 IL-15를 배지에 첨가하였다. 전술한 바와 같이, FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체를 사용하여 NK 세포의 순도 및 증폭률을 평가하였다.④ On the 14th day of culture: On the 14th day of culture, the number of cells cultured for 2 weeks was counted, and as on the 7th day, the cells were once again divided by 500 μℓ per well and 0.5 × 10 6 radiations 100 After re-inoculation with the addition of K562-OX40L irradiated with Gy, the total volume of the medium was 2 ml/well, the concentration of IL-2 was 100 U/ml, and 5 ng/ml of IL-15 was added to the medium. As described above, FITC-anti-human CD3 and PE-Cy5 anti-human CD56 monoclonal antibodies were used to evaluate the purity and amplification rate of NK cells.

또한, NK 세포의 기능을 평가하기 위해, NK 세포의 독성(cytotoxicity), ADCC(antibody dependent cellular cytotoxicity), 세포 아형 검사를 실시하였다. 보다 구체적으로, 세포독성 및 ADCC의 경우 K562 세포주와 리툭시맙(Retuximab) 항체가 코팅된 Raji 세포를 CFSE를 통해 염색하고 두 번의 세척 과정 후, 증폭된 NK 세포와 2:1, 1:1, 0.5:1의 비율로 4시간 동안 공조 배양하였다. 그 다음, Propidium iodide(PI)로 염색하여 유세포분석법으로 죽은 K562 세포, Raji 세포의 비율을 조사하였다.In addition, in order to evaluate the function of NK cells, NK cell cytotoxicity, ADCC (antibody dependent cellular cytotoxicity), and cell subtype tests were performed. More specifically, in the case of cytotoxicity and ADCC, K562 cell line and Raji cells coated with Rituximab antibody were stained through CFSE, and after two washing procedures, amplified NK cells and 2:1, 1:1, It was air-conditioned for 4 hours at a ratio of 0.5:1. Then, it was stained with Propidium iodide (PI) and the ratio of dead K562 cells and Raji cells was investigated by flow cytometry.

NK 세포의 아형 검사의 경우, 튜브 두 개에 각각 하기 표 1의 단클론성 항체를 넣고 빛이 차단된 공간에서 15분 동안 실온으로 항온한 후 세척 과정을 거쳐 유세포 분석기로 분석하였다. In the case of subtype testing of NK cells, the monoclonal antibodies of Table 1 were added to two tubes, respectively, and incubated at room temperature for 15 minutes in a light-blocked space, followed by washing, and then analyzed with a flow cytometer.

Figure 112019039185538-pat00001
Figure 112019039185538-pat00001

⑤ 배양 17일 및 19일 째: 배지의 절반을 제거하고, 200 U/㎖의 IL-2 및 10 ng/㎖의 IL-15가 함유된 1 ㎖의 새로운 배지를 추가하였다. ⑤ Culture 17 and 19 days: Half of the medium was removed, and 1 mL of fresh medium containing 200 U/mL IL-2 and 10 ng/mL IL-15 was added.

⑥ 배양 21일 째: trypan blue exclusion method을 통해서, NK 세포 수를 카운트하고, FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체를 사용하여 증폭된 NK 세포의 순도를 확인하였으며, 이 때, NK 세포 수는 총 말초혈액단핵구와 NK 세포의 분포의 비율을 곱하여 'NK 세포의 순도(purity)'를 산정하였다. ⑥ Day 21 of culture: The number of NK cells was counted through the trypan blue exclusion method, and the purity of the amplified NK cells was confirmed using FITC-anti-human CD3 and PE-Cy5 anti-human CD56 monoclonal antibodies. In this case, the number of NK cells was calculated by multiplying the ratio of the distribution of total peripheral blood mononuclear cells and NK cells to calculate the'purity of NK cells'.

⑦ 배양 23일 및 24일 째: 배지의 절반을 제거하고, 200 U/㎖의 IL-2 및 10 ng/㎖의 IL-15가 함유된 1 ㎖의 새로운 배지를 추가하였다. ⑦ Culture 23 and 24 days: Half of the medium was removed, and 1 mL of fresh medium containing 200 U/mL IL-2 and 10 ng/mL IL-15 was added.

⑧ 배양 25 및 26 일 째: 배지의 절반을 제거하고, 200 U/㎖의 IL-2 및 10 ng/㎖의 IL-15가 함유된 1 ㎖의 새로운 배지를 추가하였다. 8 Culture days 25 and 26: Half of the medium was removed, and 1 mL of fresh medium containing 200 U/mL of IL-2 and 10 ng/mL of IL-15 was added.

⑨ 배양 28일 째: trypan blue exclusion method를 통해서, NK 세포 수를 카운트하고, FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체를 사용하여 증폭된 NK 세포의 순도를 확인 후 남은 세포는 얼려서 보관해 두었다. ⑨ Day 28 of culture: Count the number of NK cells through the trypan blue exclusion method, and confirm the purity of NK cells amplified using FITC-anti-human CD3 and PE-Cy5 anti-human CD56 monoclonal antibodies. The remaining cells were frozen and stored.

도 3a 및 도 3b에서 나타낸 바와 같이, 확보된 NK 세포의 순도 및 증폭률은 대조군에 비해 2.5 배 내지 3 배 정도 높았으며, 높은 순도를 나타내는 것을 확인할 수 있었다.As shown in FIGS. 3A and 3B, the purity and amplification rate of the NK cells obtained were 2.5 to 3 times higher than that of the control group, and it was confirmed that the purity was high.

또한, 상기 배양방법으로 배양한 결과, 도 4a 및 도 4b에서 나타낸 바와 같이 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 순도 및 증폭률은 일관되게 K562 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 순도 및 증폭률보다 유의하게 높음을 확인하였다. In addition, as a result of culturing by the above culture method, the purity and amplification rate of NK cells obtained by co-culturing K562-OX40L cell line and peripheral blood mononuclear cells as shown in FIGS. 4A and 4B were consistently co-cultured with K562 cell line and peripheral blood mononuclear cells. It was confirmed that the purity and amplification rate of the obtained NK cells were significantly higher.

한편, 도 5a 및 도 5b에서 나타낸 바와 같이, K562 세포주와 말초혈액단핵구를 공배양하여 얻은 NK 세포의 세포독성 및 ADCC의 측정값과 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻은 NK 세포의 세포독성 및 ADCC의 측정값은 유의한 차이가 없음을 확인하였다. On the other hand, as shown in Figures 5a and 5b, the cytotoxicity of NK cells and ADCC obtained by co-culture of K562 cell line and peripheral blood mononuclear cells, and of NK cells obtained by co-culture of K562-OX40L cell line and peripheral blood mononuclear cells. It was confirmed that there was no significant difference between the cytotoxicity and ADCC measurements.

또한, 도 6에서 나타낸 바와 같이, K562 세포주와 말초혈액단핵구를 공배양하여 얻은 NK 세포의 세포 표면 수용체의 발현 결과와 K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻은 NK 세포의 세포 표면 수용체의 발현 결과는 유의한 차이가 없음을 확인하였다.In addition, as shown in Figure 6, the expression results of the cell surface receptors of NK cells obtained by co-culturing K562 cell line and peripheral blood monocytes, and the cell surface receptors of NK cells obtained by co-culture of K562-OX40L cell line and peripheral blood monocytes. It was confirmed that there was no significant difference in the expression results.

상기 결과를 종합하여 보면, K562-OX40L 세포주를 배양보조세포로 하고, IL-2 및 IL-15를 첨가하여 말초혈액단핵구 세포와 공배양시킨 경우, NK 세포가 높은 순도로 증폭되는 것을 확인할 수 있었다. 또한, 상기 공배양으로 얻어진 NK 세포는 일반적인 배양보조세포주인 K562에 의해 얻어진 NK 세포와 기능에 있어 차이가 없음을 확인할 수 있었다.In summarizing the above results, when the K562-OX40L cell line was used as a culture auxiliary cell, and IL-2 and IL-15 were added to co-culture with peripheral blood mononuclear cells, it was confirmed that NK cells were amplified with high purity. . In addition, it was confirmed that the NK cells obtained by the co-culture had no difference in function from the NK cells obtained by K562, which is a general culture auxiliary cell line.

실시예 3. 분리된 말초혈액단핵구 (PBMCs) 및 IL-21, K562-OX40L를 이용한 NK 세포의 배양을 통한 NK 세포 증폭률 및 기능 확인Example 3. Confirmation of NK cell amplification rate and function through culture of NK cells using isolated peripheral blood monocytes (PBMCs) and IL-21, K562-OX40L

본 발명에 따라 상기 실시예 1로부터 준비한 말초혈액단핵구 및 K562-OX40L 세포주를 이용하여 NK 세포의 증폭을 수행하였다. 전체적인 실험 방법은 실시예 2와 같지만 처음 말초혈액단핵구와 K562-OX40L 배양 시 다양한 농도의 IL-21을 '한 번만' 첨가한다는 점에서 차이가 있다. 보다 구체적으로, 3 × 106 개의 PBMC 및 방사선 100 Gy가 조사된 K562-OX40L 0.5 × 106 개를 1 ㎖ NK 세포 배지가 포함된 24 웰 플레이트 상에 접종한 다음, 20 U/㎖ IL-2가 함유된 1 ㎖ NK 세포 배지를 추가하여 총 배지 부피는 2 ㎖/well, IL-2의 최종 농도는 10 U/㎖로 한 뒤, 여기에 5, 50, 100 ng/㎖의 IL-21을 각각 다른 다른 웰에 첨가한 후, 부드럽게 피펫팅(pipetting) 하여 혼합한 후 37 ℃5 % CO2 배양기에서 배양하였다. In accordance with the present invention, NK cells were amplified using the peripheral blood mononuclear cells and K562-OX40L cell lines prepared from Example 1 above. The overall experimental method is the same as in Example 2, but there is a difference in that when the peripheral blood mononuclear cells and K562-OX40L are first cultured, various concentrations of IL-21 are added'only once'. More specifically, 3 × 10 6 PBMCs and 0.5 × 10 6 K562-OX40L irradiated with 100 Gy of radiation were inoculated onto a 24-well plate containing 1 ml NK cell medium, and then 20 U/ml IL-2 Add 1 ㎖ NK cell medium containing NK cells to make the total medium volume 2 ㎖/well and the final concentration of IL-2 10 U/ ㎖, and then 5, 50, 100 ng/mL IL-21 After the addition to each of the different wells, gently pipetting (pipetting) to mix, and incubated in a 37 ℃ 5% CO 2 incubator.

배양 7, 14, 21, 28, 35일 째에 NK 세포 수를 카운트하고, NK 세포의 순도를 확인하여 처음 NK세포에 비해 증폭된 정도를 나타내는 증폭률을 산출하였다.On days 7, 14, 21, 28, and 35 of culture, the number of NK cells was counted, and the purity of NK cells was checked to calculate an amplification rate indicating the degree of amplification compared to the first NK cells.

배양 21일 째: trypan blue exclusion method을 통해서, NK 세포 수를 카운트하고, FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체를 사용하여 증폭된 NK 세포의 순도를 확인하였으며, 이 때, NK 세포 수는 총 PBMC와 NK 세포의 분포의 비율을 곱하여 'NK 세포의 순도'를 산정하였다. Day 21 of culture: Through the trypan blue exclusion method, the number of NK cells was counted, and the purity of the amplified NK cells was confirmed using FITC-anti-human CD3 and PE-Cy5 anti-human CD56 monoclonal antibodies, At this time, the number of NK cells was multiplied by the ratio of the distribution of total PBMCs and NK cells to calculate the'purity of NK cells'.

상기 배양방법으로 배양한 결과, 도 7a 내지 도 7d에서 나타낸 바와 같이 말초혈액단핵구를 K562 세포주와 공배양하는 경우에는 IL-20(5, 50, 100 ng/㎖)에 노출되더라도 NK 세포가 크게 증폭되지 않았으나, K562-OX40L 세포주와 공배양하는 경우에는 IL-20(5, 50, 100 ng/㎖)에 노출되면 NK 세포가 크게 증폭되는 것을 확인하였다. As a result of culturing by the above culture method, as shown in Figs. 7A to 7D, when peripheral blood mononuclear cells are co-cultured with the K562 cell line, NK cells are greatly amplified even when exposed to IL-20 (5, 50, 100 ng/ml). However, in the case of co-culture with the K562-OX40L cell line, it was confirmed that NK cells were greatly amplified when exposed to IL-20 (5, 50, 100 ng/ml).

또한, NK 세포의 기능을 평가하기 위해, NK 세포의 세포독성, ADCC, 세포 아형 검사, 탈과립(degranulation) 마커인 CD107a, IFN-g, TNF-α 분비능, 텔로미어 길이(Telemere length)를 분석하였다. 보다 구체적으로, 탈과립 마커인 CD107a, IFN-g, TNF-α 측정은 0.5 × 106 개의 증폭된 NK 세포와 0.5 × 106 개의 K562 세포를 배양 후 1시간 후에 brefeldin A와 monensin을 첨가하여 세포 외로 단백질을 이동을 막은 후, 4시간 후에 PE anti-human CD107a와 FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체를 이용하여 CD107a의 발현 정도를 확인하였으며, IFN-g 와 TNF-α는 FITC-anti-human CD3 및 PE-Cy5 anti-human CD56 모노클로날 항체로 염색한 후 세척, 고정, 투과 과정 후 PE anti-human IFN-g 과 APC mouse anti-human TNF-α를 30분 동안 염색한 후 유세포 분석기로 확인하였다. 또한, 텔로미어 길이(Telemere length)는 21일 동안 증폭된 NK 세포를 대상으로 Telemere PNA kit/FITC를 이용하여 상대적인 텔로미어 길이를 확인하였다. In addition, in order to evaluate the function of NK cells, cytotoxicity of NK cells, ADCC, cell subtyping, degranulation markers CD107a, IFN-g, TNF-α secretion capacity, and telomere length were analyzed. More particularly, the degranulation marker CD107a, IFN-g, TNF-α measurements are outside of 0.5 × 10 6 of the amplified NK cells and 0.5 × 10 6 of K562 cells after 1 hour incubation by the addition of brefeldin A and monensin cells After blocking the protein migration, the expression level of CD107a was confirmed using PE anti-human CD107a and FITC-anti-human CD3 and PE-Cy5 anti-human CD56 monoclonal antibodies 4 hours later. IFN-g and TNF -α was stained with FITC-anti-human CD3 and PE-Cy5 anti-human CD56 monoclonal antibody, followed by washing, fixation, and permeation, followed by PE anti-human IFN-g and APC mouse anti-human TNF-α. After staining for minutes, it was confirmed by flow cytometry. In addition, Telomere length (Telemere length) was confirmed relative telomere length using Telemere PNA kit/FITC targeting NK cells amplified for 21 days.

그 결과, 도 8a 및 도 8b에서 나타낸 바와 같이, 다양한 농도의 IL-21(0, 50, 100 ng/㎖)과 E:T 비율(2:1, 1:1, 0.5:1)에서, K562-OX40L 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 세포독성 및 ADCC의 측정값과 IL-21의 첨가없이 K562 세포주와 말초혈액단핵구를 공배양하여 얻어진 NK 세포의 세포독성 및 ADCC의 측정값은 유의한 차이가 없음을 확인하였다.As a result, as shown in FIGS. 8A and 8B, at various concentrations of IL-21 (0, 50, 100 ng/ml) and E:T ratio (2:1, 1:1, 0.5:1), K562 -Measured values of cytotoxicity and ADCC of NK cells obtained by co-culture of OX40L cell line and peripheral blood monocytes, and cytotoxicity and ADCC of NK cells obtained by co-culture of K562 cell line and peripheral blood monocytes without the addition of IL-21 It was confirmed that there was no significant difference.

또한, 도 9a 내지 도 9d에서 나타낸 바와 같이, 『IL-21의 첨가없이 K562 세포주를 배양보조세포』로 하여 말초혈액단핵구와 공배양하여 얻어진 NK 세포의 CD107a, TNF-α, IFN-γ의 발현비 및 텔로미어 길이 증가비와 『IL-21을 배양 초기에 한번만 첨가하고 OX40L-K562 세포주를 배양보조세포』로 하여 말초혈액단핵구와 공배양하여 얻어진 NK 세포의 CD107a, TNF-α, IFN-γ의 발현비 및 텔로미어 길이 증가비는 유의한 차이가 없음을 확인하였다.In addition, as shown in Figs. 9A to 9D, expression of CD107a, TNF-α, and IFN-γ in NK cells obtained by co-culture with peripheral blood monocytes using "the K562 cell line without the addition of IL-21 as a culture aid cell" CD107a, TNF-α, IFN-γ of NK cells obtained by co-culture with peripheral blood monocytes using the ratio and telomere length increase ratio and 『IL-21 was added only once at the beginning of culture and OX40L-K562 cell line was used as a culture aid cell』. It was confirmed that there was no significant difference between the expression ratio and the telomere length increase ratio.

상기 결과를 종합하여 보면, K562-OX40L을 배양보조세포로 하고 IL-21이 한번 첨가되어 말초혈액단핵구와 공배양하여 얻어진 NK 세포는, K562를 배양보조세포로 하고 IL-21이 한번 첨가되어 말초혈액단핵구와 공배양하여 얻어진 NK 세포에 비해 높은 순도 및 증폭률을 가지는 것을 확인할 수 있었다. 또한, 상기 공배양으로 얻어진 NK 세포는 IL-21의 첨가 없이 K562 세포주와 공배양하여 얻어진 NK 세포와 기능면에서 차이가 없음을 확인하였다.In summarizing the above results, NK cells obtained by co-culture with peripheral blood mononuclear cells by using K562-OX40L as a culture aid cell and IL-21 was added once are peripheral blood mononuclear cells with K562 as culture aid cells, and IL-21 was added once. It was confirmed that it has a higher purity and amplification rate than NK cells obtained by co-culture with blood monocytes. In addition, it was confirmed that the NK cells obtained by the co-culture did not differ in function from the NK cells obtained by co-culture with the K562 cell line without the addition of IL-21.

전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Feeder cells expressing OX40L and natural killer cell culture <130> PD17-112-P1 <150> KR 10-2018-0061385 <151> 2018-05-29 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 552 <212> DNA <213> OX40L <400> 1 atggaaaggg tccaacccct ggaagagaat gtgggaaatg cagccaggcc aagattcgag 60 aggaacaagc tattgctggt ggcctctgta attcagggac tggggctgct cctgtgcttc 120 acctacatct gcctgcactt ctctgctctt caggtatcac atcggtatcc tcgaattcaa 180 agtatcaaag tacaatttac cgaatataag aaggagaaag gtttcatcct cacttcccaa 240 aaggaggatg aaatcatgaa ggtgcagaac aactcagtca tcatcaactg tgatgggttt 300 tatctcatct ccctgaaggg ctacttctcc caggaagtca acattagcct tcattaccag 360 aaggatgagg agcccctctt ccaactgaag aaggtcaggt ctgtcaactc cttgatggtg 420 gcctctctga cttacaaaga caaagtctac ttgaatgtga ccactgacaa tacctccctg 480 gatgacttcc atgtgaatgg cggagaactg attcttatcc atcaaaatcc tggtgaattc 540 tgtgtccttt ga 552 <110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Feeder cells expressing OX40L and natural killer cell culture <130> PD17-112-P1 <150> KR 10-2018-0061385 <151> 2018-05-29 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 552 <212> DNA <213> OX40L <400> 1 atggaaaggg tccaacccct ggaagagaat gtgggaaatg cagccaggcc aagattcgag 60 aggaacaagc tattgctggt ggcctctgta attcagggac tggggctgct cctgtgcttc 120 acctacatct gcctgcactt ctctgctctt caggtatcac atcggtatcc tcgaattcaa 180 agtatcaaag tacaatttac cgaatataag aaggagaaag gtttcatcct cacttcccaa 240 aaggaggatg aaatcatgaa ggtgcagaac aactcagtca tcatcaactg tgatgggttt 300 tatctcatct ccctgaaggg ctacttctcc caggaagtca acattagcct tcattaccag 360 aaggatgagg agcccctctt ccaactgaag aaggtcaggt ctgtcaactc cttgatggtg 420 gcctctctga cttacaaaga caaagtctac ttgaatgtga ccactgacaa tacctccctg 480 gatgacttcc atgtgaatgg cggagaactg attcttatcc atcaaaatcc tggtgaattc 540 tgtgtccttt ga 552

Claims (16)

OX40L(CD134 ligand)을 발현하는 배양보조세포(feeder cells)를 유효성분으로 포함하되, 상기 배양보조세포는 K562 세포인 것을 특징으로 하는, NK 세포 증식용 조성물.
Comprising feeder cells expressing OX40L (CD134 ligand) as an active ingredient, wherein the culture auxiliary cells are K562 cells, characterized in that, NK cell proliferation composition.
 삭제delete 제1항에 있어서,
상기 OX40L을 발현하는 배양보조세포는 OX40L 유전자가 삽입된 발현 벡터로 형질 전환된 것을 특징으로 하는, 조성물.
The method of claim 1,
The OX40L-expressing culture aid cell is characterized in that transformed with an expression vector into which the OX40L gene is inserted, the composition.
 제3항에 있어서,
상기 OX40L 유전자는 서열번호 1로 표시되는 염기서열로 이루어진 것을 특징으로 하는, 조성물.
The method of claim 3,
The OX40L gene is characterized in that consisting of a nucleotide sequence represented by SEQ ID NO: 1, composition.
 제3항에 있어서,
상기 발현 벡터는 플라스미드 벡터, 코스미드 벡터, 박테리오파지 벡터 및 바이러스 벡터로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 조성물.
The method of claim 3,
The expression vector is characterized in that selected from the group consisting of a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector.
 제5항에 있어서,
상기 바이러스 벡터는 렌티바이러스(lentivirus) 또는 레트로바이러스(retrovirus) 벡터인 것을 특징으로 하는, 조성물.
The method of claim 5,
The viral vector is a lentivirus or retrovirus vector, characterized in that, the composition.
 (a) 말초혈액으로부터 말초혈액단핵구(Peripheral blood mononuclear cell, PBMC)를 수득하는 단계; 및
(b) 상기 수득한 말초혈액단핵구와 OX40L을 발현하는 배양보조세포(feeder cells)를 사이토카인을 함유하는 배지에서 공배양하는 단계를 포함하고,
상기 배양보조세포는 K562 세포인 것을 특징으로 하는, 자연살해세포 증식 방법.
(a) obtaining peripheral blood mononuclear cells (PBMC) from peripheral blood; And
(b) co-culturing the obtained peripheral blood monocytes and feeder cells expressing OX40L in a medium containing cytokines,
The culture auxiliary cell is characterized in that the K562 cells, natural killer cell proliferation method.
 제7항에 있어서,
상기 사이토카인은 IL-2, IL-15, IL-21, Flt3-L, IL-7, IL-12 및 IL-18로 이루어진 군으로부터 선택되는 1 종 이상인 것을 특징으로 하는, 방법.
The method of claim 7,
The cytokine is characterized in that at least one member selected from the group consisting of IL-2, IL-15, IL-21, Flt3-L, IL-7, IL-12 and IL-18.
 제7항에 있어서,
상기 OX40L을 발현하는 배양보조세포(feeder cells)는 50 Gy 내지 300 Gy 방사선을 처리하여 수득된 것을 특징으로 하는, 방법.
The method of claim 7,
The method of claim 1, wherein the OX40L-expressing feeder cells are obtained by treatment with 50 Gy to 300 Gy radiation.
 제7항에 있어서,
상기 공배양은 2 일 내지 30 일 동안 수행되는 것을 특징으로 하는, 방법.
The method of claim 7,
The method, characterized in that the co-culture is carried out for 2 to 30 days.
 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete
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