KR20130084465A - Method for proliferating natural killer cell from canine peripheral blood - Google Patents
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Abstract
Description
본 발명은 개 유래의 자연살해세포의 대량 증식방법에 관한 것으로, 보다 상세하게는 개로부터 유래된 단핵구세포로부터 자연살해세포를 시험관 내에서 대량 증폭할 수 있는 자연살해세포 증식 및 분화용 배지 조성물 및 이를 이용한 분화 및 증폭방법에 관한 것이다.The present invention relates to a method for mass proliferation of natural killer cells derived from dogs, and more particularly, to a composition for natural killer cell proliferation and differentiation capable of amplifying large numbers of natural killer cells in vitro from mononuclear cells derived from dogs, and It relates to a differentiation and amplification method using the same.
성체 줄기세포 중 하나인 조혈줄기세포(hematopoietic stem cell)는 혈액을 구성하는 모든 세포(적혈구, 백혈구, 혈소판 및 림프구)로 분화할 수 있는 세포로서, 주로 골수에 있는 조혈줄기세포로부터 생체의 면역체계를 구성하는 세포가 지속적으로 자가 재생된다. 이들 면역체계를 구성하는 세포들 중 특히, 자연살해세포(natural killer cell, 이하 'NK 세포'라 약칭함)는 비특이적으로 암세포 및 바이러스 감염세포를 살상할 수 있는 능력이 밝혀지면서 많은 연구가 진행되었으며, NK 세포의 분화와 활성의 결함은 유방암(Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), 흑색종암(Ryuke Y, et al., Melanoma Res., 13: 349-356, 2003), 폐암(Villegas FR, et al., Lung Cancer, 35: 23-28, 2002) 등 다양한 질병들과 관련 되어 있음이 밝혀졌다. 이러한 연구들을 바탕으로 암과 같은 난치성질환 치료에 NK 세포를 이용하는 NK 세포 치료법이 대두되고 있다. NK 세포를 이용한 세포 치료를 위해서는 생체 외(ex vivo) 상에서 NK 세포를 배양하는 것이 필요하고, NK 세포의 생체외 분화 조건을 최적화하는 것은 필수적이며, 생체 외 상에서 조혈줄기세포로부터 성숙한 NK 세포를 분화시키는 방법은 이미 보고되어 있다.One of the adult stem cells, hematopoietic stem cell, is a cell that can differentiate into all the cells constituting the blood (erythrocytes, white blood cells, platelets, and lymphocytes). The cells that make up are constantly self-renewing. Among the cells constituting these immune systems, in particular, natural killer cells (abbreviated as 'NK cells') has been studied a lot as the ability to kill cancer cells and virus infected cells unspecifically Defects in the differentiation and activity of NK cells are found in breast cancer (Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), melanoma cancer (Ryuke Y, et al., Melanoma Res., 13). : 349-356, 2003) and lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002). Based on these studies, NK cell therapy using NK cells has emerged for the treatment of intractable diseases such as cancer. In order to treat cells with NK cells, it is necessary to culture NK cells on ex vivo, and to optimize the in vitro differentiation conditions of NK cells, it is necessary to differentiate mature NK cells from hematopoietic stem cells in vitro. How to do this has already been reported.
그동안 사용되어온 공통 유전형의 마우스 모델(syngenic mouse model)이나 제노그래프트 마우스 모델(xenograft mouse model) 등 실험동물에서의 실험은 개나 영장류를 이용한 실험에 비해 실제 임상적 효능을 예측하기 어려운 것으로 잘 알려져 있고, 실험용 쥐에서 NK 세포의 체외 증폭기술이 확립되지 않아 대부분 NK 세포를 순수 분리한 다음 사이토카인으로 활성화 시켜 사용하여 왔다.It is well known that experiments in experimental animals such as the common genotype mouse model or the xenograft mouse model have been difficult to predict the actual clinical efficacy compared to experiments using dogs or primates. Since the in vitro amplification of NK cells in laboratory mice has not been established, most of them have been isolated from pure NK cells and activated with cytokines.
한편, 개는 개의 게놈(genome) 판독 결과 인간과 개의 게놈이 매우 유사하다고 입증되었으며, 특히 개에서 자연적으로 발생된 암의 유전적, 분자 생물학적, 병리조직학적 양상 및 일반적으로 사용되고 있는 암치료법에 대한 반응이 사람에서 발생되는 암과 거의 비슷하다는 사실이 밝혀졌으며(Lindblad-Toh K et al, 2005; Hoffman MM & Birney E, 2007), 인간의 면역질환 및 감영성질환과 유사한 질병이 자연발생적으로 유발되기 때문에 아주 흥미 있는 실험모델이다. 아울러 개에서 암이나 바이러스 질환 등과 같은 난치성 질환의 발생이 현저히 증가하고 있어 이들 난치성 질환에 대한 새로운 치료제 개발 역시 매우 필요하다.On the other hand, dog genome readouts have shown that the human and dog genomes are very similar, particularly for the genetic, molecular biological, pathologic aspects of cancers naturally occurring in dogs, and for commonly used cancer therapies. It has been shown that the response is almost similar to that in humans (Lindblad-Toh K et al, 2005; Hoffman MM & Birney E, 2007), and spontaneous induction of diseases similar to human immune and emotional diseases It is a very interesting experimental model. In addition, the incidence of intractable diseases, such as cancer or viral diseases in dogs is increasing significantly, it is also very necessary to develop new treatments for these intractable diseases.
이와 같은 생물학적 유사성 때문에 개에서 암에 관한 연구는 사람 또는 실험동물만을 대상으로 한 암 연구와 차별화되는 귀중한 정보를 제공하며, 이와 같은 가치에 대한 인식은 암-관련 유전자 확인 연구를 비롯하여 환경성 위험요소, 암의 생물학적 특성 이해, 그리고 특히 새로운 암치료법 개발 및 효능 평가 등의 연구 분야에서 현저히 높아지고 있다(Paoloni M & Khanna C, 2008). 그러나 아직까지 개에서 세계적으로 NK 세포 증폭 및 이를 이용한 암치료에 관련된 보고가 없다.Because of this biological similarity, research on cancer in dogs provides valuable information that differentiates cancer research from humans or experimental animals only, and recognition of this value can be attributed to environmental risks, including cancer-related genetic identification studies, Significant advances have been made in the areas of research, including understanding the biological properties of cancer and in particular developing new cancer therapies and evaluating efficacy (Paoloni M & Khanna C, 2008). However, there are no reports on amplifying NK cells and treating cancer using them worldwide.
이에, 본 발명자들은 사람에서 암을 극복하기 위한 연구에 귀중한 실질적 자료를 제공할 수 있는 개와 같은 효과적인 동물 실험 모델이 필요한 시점에서, 개 자연살해세포(natural killer cell, NK cell)에 발현하는 특이 표지인자 조차도 밝혀져 있지 않는 개 유래의 자연살해세포의 연구를 진행하던 중, 시험관 내에서 강력한 항암 및 항바이러스 효과를 갖는 개 유래의 고순도의 자연살해세포 대량 증폭방법을 발견하고, 본 발명을 완성하였다.Therefore, the inventors of the present invention have specific markers for expressing natural killer cells (NK cells) in humans when an effective animal experimental model such as a dog that can provide valuable practical data for research to overcome cancer in humans is required. During the study of natural killer cells derived from dogs, whose factors are not known, a method for mass amplification of high-purity natural killer cells derived from dogs with potent anti-cancer and antiviral effects was found in vitro, and the present invention was completed.
본 발명의 목적은 개 유래의 자연살해세포를 시험관 내에서 대량 증폭할 수 있는, 자연살해세포 분화 및 증폭용 배지 조성물을 제공하는 것이다.An object of the present invention is to provide a natural killer cell differentiation and amplification medium composition capable of amplifying a large number of natural killer cells derived from dogs in vitro.
본 발명의 또 다른 목적은 상기 조성물을 이용하여 개로부터 유래된 단핵구세포로부터 고효율로 강력한 항암 및 항바이러스 효과를 갖는 개 유래의 자연살해세포의 대량 증폭방법을 제공하는 것이다.Still another object of the present invention is to provide a method for mass amplification of natural killer cells derived from dogs having a powerful anti-cancer and antiviral effect at high efficiency from monocytes derived from dogs using the composition.
상기의 목적을 달성하기 위하여 본 발명은 IL-2, IL-15 또는 이들의 혼합물을 유효성분으로 함유하는, 개로부터 유래된 자연살해세포(natural killer cell, NK cell) 분화 및 증폭용 배지 조성물을 제공한다.In order to achieve the above object, the present invention provides a medium composition for differentiating and amplifying natural killer cells (NK cells) derived from dogs, which contains IL-2, IL-15 or a mixture thereof as an active ingredient. to provide.
본 발명에 따른 조성물은 유효성분으로 IL-2 10 내지 500 U/㎖ 및 IL-15 0.1 내지 100 U/㎖를 함유하는 것을 특징으로 한다.The composition according to the present invention is characterized in that it contains IL-2 10 to 500 U / ml and IL-15 0.1 to 100 U / ml as the active ingredient.
본 발명은 정상개의 말초혈액에서 밀도 구배 원심방법(density gradient centrifugation)을 이용하여 분리한 단핵구세포(PBMCs)를 상기 조성물이 첨가된 배지에서 피더 세포(Feeder cell)와 함께 공조배양 하는, 개로부터 유래된 자연살해세포(natural killer cell, NK cell)의 분화 및 증폭방법을 제공한다.The present invention is derived from a dog co-cultured with the feeder cells (PBMCs) in the medium to which the composition is added to the mononuclear cells (PBMCs) isolated by using a density gradient centrifugation in the peripheral blood of normal dogs It provides a method for differentiation and amplification of natural killer cells (NK cells).
본 발명에 따른 분화 및 증폭방법은 개 유래의 단핵구세포를 75%(비중 1.057) 내지 80%(비중 1.063)농도의 Lymphoprep, 100%(비중 1.077) 농도의 Lymphoprep 및 100% Histopaque(비중 1.119)의 불연속 밀도구배(discontinuous density gradients) 원심분리를 이용하여 정제하는 것을 특징으로 한다.The differentiation and amplification method according to the present invention is a method of treating the cells derived from dogs from 75% (specific gravity 1.057) to 80% (specific gravity 1.063) Lymphoprep, 100% (specific gravity 1.077) Lymphoprep and 100% Histopaque (specific gravity 1.119) Discontinuous density gradients (discontinuous density) characterized in that the purification using centrifugation.
보다 상세하게는 본 발명에 따른 분화 및 증폭방법은 개 유래의 단핵구세포를 76%(비중 1.058) 내지 77%(비중 1.059) 농도의 Lymphoprep, 100%(비중 1.077) 농도의 Lymphoprep 및 100% Histopaque(비중 1.119)의 불연속 밀도구배(discontinuous density gradients) 원심분리를 이용하여 정제하는 것을 특징으로 한다.More specifically, the method of differentiation and amplification according to the present invention is directed to Lymphoprep at a concentration of 76% (specific gravity 1.058) to 77% (specific gravity 1.059), Lymphoprep at a concentration of 100% (specific gravity 1.077) and 100% Histopaque ( It is characterized in that the purification by using a discontinuous density gradient (discontinuous density gradients) of specific gravity 1.119).
본 발명에 따른 분화 및 증폭방법은 개로부터 유래된 단핵구세포로부터 순도가 95% 이상의 개 유래의 자연살해세포만을 최대 200배 이상 대량 증폭시킬 수 있는 것을 특징으로 한다.The differentiation and amplification method according to the present invention is characterized in that amplification can be performed up to 200 times or more only from natural killer cells derived from dogs having a purity of 95% or more from monocytes derived from dogs.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에 있어서, “Feeder cell(피더 세포)”란, 분열증식하지 못하게 하였으나 대사활성이 있기 때문에 여러 가지 대사물질을 생산하여 목적 세포의 증식을 돕는 세포로서, 최초에 이식한 세포를 ‘피더 세포’라고 한다. In the present invention, "Feeder cell" is a cell that prevents proliferation but has metabolic activity and thus produces various metabolites to help the growth of the target cell. '
본 발명에 있어서, 상기‘Lymphoprep’나 ‘Histopaque’이라는 이름은 실험실에서 널리 사용되는 이름으로서, 그 물건을 용이하게 입수할 수 있고 그로써 물건 자체가 명확히 특정되기도 한다.In the present invention, the name 'Lymphoprep' or 'Histopaque' is a name widely used in a laboratory, and the object can be easily obtained, and thus the object itself is clearly specified.
본 발명은 IL-2, IL-15 또는 이들의 혼합물을 유효성분으로 함유하는, 개로부터 유래된 자연살해세포(natural killer cell, NK cell) 분화 및 증폭용 배지 조성물을 제공한다.The present invention provides a medium composition for differentiating and amplifying natural killer cells (NK cells) derived from dogs, which contains IL-2, IL-15 or a mixture thereof as an active ingredient.
본 발명에 있어서, 상기 유효성분은 보다 바람직하게는 IL-2 및 IL-15의 혼합물인 것을 특징으로 한다.In the present invention, the active ingredient is more preferably a mixture of IL-2 and IL-15.
본 발명에 있어서, 상기 유효성분은 IL-2 10 내지 500 U/㎖ 및 IL-15 0.1 내지 100 U/㎖를 함유하며, 바람직하게는 IL-2 50 내지 200 U/㎖ 및 IL-15 10 내지 100 U/㎖, 더욱 바람직하게는 IL-2 70 내지 150 U/㎖ 및 IL-15 50 내지 100 U/㎖를 함유한다.In the present invention, the active ingredient contains IL-2 10 to 500 U / ml and IL-15 0.1 to 100 U / ml, preferably IL-2 50 to 200 U / ml and IL-15 10 to 100 U / ml, more preferably IL-2 70-150 U / ml and IL-15 50-100 U / ml.
본 발명은The present invention
1) 개로부터 유래된 단핵구세포를 피콜-하이파크(Ficoll-Hypaque)에 중첩하고 원심분리하는 단계;1) superimposing monocytes derived from dogs on Ficoll-Hypaque and centrifuging;
2) 상기 분리된 단핵구세포를 피콜(Ficoll)로 정제하는 단계; 및2) purifying the isolated monocytes with Ficoll; And
3) 피더 세포(Feeder cell) 존재 하에, 상기 IL-2, IL-15 또는 이들의 혼합물을 유효성분으로 함유하는, 개로부터 유래된 자연살해세포(natural killer cell, NK cell) 증식 및 분화용 배지 조성물을 상기 2)에서 정제된 단핵구세포(mononuclear cells)와 공조 배양하는 단계:3) A medium for proliferation and differentiation of natural killer cells (NK cells) derived from dogs containing the IL-2, IL-15 or a mixture thereof as an active ingredient in the presence of a feeder cell. Co-culturing the composition with mononuclear cells purified from 2):
를 포함하는 것을 특징으로 하는, 개로부터 유래된 자연살해세포(natural killer cell, NK cell)의 분화 및 증폭방법을 제공한다.Characterized in that, it provides a method for differentiation and amplification of natural killer cells (NK cells) derived from dogs.
보다 상세하게는 본 발명은 More specifically, the present invention
1) 개로부터 유래된 단핵구세포를 1.077 내지 1.119 g/㎖ 비중의 피콜-하이파크(Ficoll-Hypaque)에 중첩하고 원심분리하는 단계;1) superimposing monocytes derived from dogs on Ficoll-Hypaque at 1.077-1.119 g / ml specific gravity and centrifuging;
2) 상기 분리된 단핵구세포를 75%(비중 1.057) 내지 80%(비중 1.063)농도의 Lymphoprep, 100%(비중1.077) 농도의 Lymphoprep 및 100% Histopaque(비중 1.119)의 불연속 밀도구배(discontinuous density gradients) 원심분리를 이용하여 정제하는 단계; 및2) Discontinuous density gradients of the isolated monocytes from 75% (specific gravity 1.057) to 80% (specific gravity 1.063) Lymphoprep, 100% (specific gravity 1.077) Lymphoprep and 100% Histopaque (specific gravity 1.119) Purification using centrifugation; And
3) K562 피더 세포(Feeder cell) 존재 하에, 상기 2)의 정제된 단핵구세포를 IL-2 10 내지 500 U/㎖ 및 IL-15 0.1 내지 100 U/㎖를 포함하는 배지에서 10 내지 25일 동안 공조 배양하는 단계:3) Purified mononuclear cells of 2) in the presence of K562 feeder cells for 10-25 days in a medium comprising IL-2 10-500 U / ml and IL-15 0.1-100 U / ml Air Conditioning Steps:
를 포함하는 것을 특징으로 하는, 개로부터 유래된 자연살해세포(natural killer cell, NK cell)의 분화 및 증폭방법을 제공한다.Characterized in that, it provides a method for differentiation and amplification of natural killer cells (NK cells) derived from dogs.
또한, 본 발명은In addition,
1) 개로부터 유래된 단핵구세포를 1.077 내지 1.119 g/㎖ 비중의 피콜-하이파크(Ficoll-Hypaque)에 중첩하고 원심분리하는 단계;1) superimposing monocytes derived from dogs on Ficoll-Hypaque at 1.077-1.119 g / ml specific gravity and centrifuging;
2) 상기 분리된 단핵구세포를 76%(비중 1.058) 내지 77%(비중 1.059) 농도의 Lymphoprep, 100%(비중 1.077) 농도의 Lymphoprep 및 100% Histopaque(비중 1.119)의 불연속 밀도구배(discontinuous density gradients) 원심분리를 이용하여 정제하는 단계; 및2) Discontinuous density gradients of the isolated monocytes from 76% (1.058 specific gravity) to 77% (1.059 specific gravity) Lymphoprep, 100% (1.077 specific gravity) Lymphoprep and 100% Histopaque (specific gravity 1.119) Purification using centrifugation; And
3) K562 피더 세포(Feeder cell) 존재 하에, 상기 2)의 정제된 단핵구세포를 IL-2 10 내지 500 U/㎖ 및 IL-15 0.1 내지 100 U/㎖를 포함하는 배지에서 10 내지 25일 동안 공조 배양하는 단계:3) Purified mononuclear cells of 2) in the presence of K562 feeder cells for 10-25 days in a medium comprising IL-2 10-500 U / ml and IL-15 0.1-100 U / ml Air Conditioning Steps:
를 포함하는 것을 특징으로 하는, 개로부터 유래된 자연살해세포(natural killer cell, NK cell)의 분화 및 증폭방법을 제공한다.Characterized in that, it provides a method for differentiation and amplification of natural killer cells (NK cells) derived from dogs.
본 발명에 따른 분화 및 증폭방법은 상기 1) 단계의 1.077 내지 1.119 g/㎖ 비중으로 조절된 피콜-하이파크(Ficoll-Hypaque) 용액에서의 부유밀도구배 원심분리 방법을 이용함으로써 단핵구세포를 과립구(granulocytes)의 오염 없는 고농도의 전체 살아있는 단핵구세포군을 수득할 수 있다.Differentiation and amplification method according to the present invention by using a floating density gradient centrifugation method in a Ficoll-Hypaque solution adjusted to the specific gravity of 1.077 to 1.119 g / ㎖ of the step 1) granulocytes ( High concentrations of whole living monocytes without contamination of granulocytes) can be obtained.
본 발명에 있어서, 2) 단계는 단핵구세포를 75%(비중 1.057) 내지 80%(비중 1.063)농도 농도의 Lymphoprep, 100%(비중 1.077) 농도의 Lymphoprep 및 100% Histopaque(비중 1.119)의 불연속 밀도구배(discontinuous density gradients) 원심분리를 이용하여 정제할 수 있으며, 보다 바람직하게는 분리된 단핵구세포를 76%(비중 1.058) 내지 77%(비중 1.059) 농도의 Lymphoprep, 100%(비중 1.077) 농도의 Lymphoprep 및 100% Histopaque(비중 1.119)의 불연속 밀도구배(discontinuous density gradients) 원심분리를 이용할 수 있다.In the present invention, step 2) is a mononuclear cell at a concentration of 75% (specific gravity 1.057) to 80% (specific gravity 1.063) concentration of Lymphoprep, 100% (specific gravity 1.077) concentration of Lymphoprep and 100% Histopaque (specific gravity 1.119) Gradient (discontinuous density gradients) can be purified using centrifugation, and more preferably the isolated monocytes are concentrated at a concentration of 76% (specific gravity 1.058) to 77% (specific gravity 1.059) Lymphoprep, 100% (specific gravity 1.077) Discontinuous density gradients of Lymphoprep and 100% Histopaque (specific gravity 1.119) can be used.
본 발명에 있어서, 상기 불연속 밀도구배에 사용되는 Lymphoprep의 농도는 본 발명의 목적을 달성에 중요한 의미를 가진다. 즉, 사람에서와는 달리 개의 과립구 특히, 호산구(eosinophil)의 밀도는 단핵구세포의 밀도와 매우 유사하여 일반적인 단핵구세포의 분리방법으로 호산구의 오염을 줄이기 매우 어렵다. 뿐만 아니라 개체의 차이는 있지만 개의 말초혈액 내에는 사람에 비해 매우 적은 수의 자연살해세포가 존재하기 때문에 과립구의 오염이 많을수록 분리된 단핵구세포 내에 존재하는 자연살해세포의 비율이 작아짐으로써 증폭의 효과를 저하시키는 원인이 된다.In the present invention, the concentration of Lymphoprep used in the discontinuous density gradient has an important meaning in achieving the object of the present invention. In other words, unlike in humans, the density of dog granulocytes, in particular, eosinophil, is very similar to that of monocytes, so it is very difficult to reduce the contamination of eosinophils by the general method of separating monocytes. In addition, although there are differences among individuals, there are very few natural killer cells in the peripheral blood of dogs compared to humans, so the greater the contamination of granulocytes, the smaller the percentage of natural killer cells in isolated monocytes. It causes the deterioration.
이에, 상기 본 발명에 따른 75%(비중 1.057) 내지 80%(비중 1.063)농도의 Lymphoprep, 보다 바람직하게는 76%(비중 1.058) 내지 77%(비중 1.059) 농도의 Lymphoprep은 적은 양의 채혈로 호산구의 오염을 현저히 줄이고, 많은 수의 자연살해세포를 수득에 중요한 의미를 가진다. 만약 Lymphoprep을 75%(비중 1.058) 농도 미만으로 사용하여 단핵구세포를 정제할 경우, 호산구의 오염은 현저히 줄일 수 있었지만 회수할 수 있는 단핵구세포의 총 개수가 매우 적을 뿐만 아니라 회수된 단핵구세포 내 림프구의 수가 현저히 감소하고, 단핵세포(monocytes)의 수가 많아 자연살해세포의 증폭 효과는 급격히 감소하는 문제점이 있으며, Lymphoprep을 80%(비중 1.063) 농도를 초과하여 사용할 경우에는 호산구의 오염을 줄이기 어려운 문제점이 있다.Thus, the Lymphoprep concentration of 75% (specific gravity 1.057) to 80% (specific gravity 1.063), more preferably 76% (specific gravity 1.058) to 77% (specific gravity 1.059) concentration according to the present invention is a small amount of blood collection Significantly reduces the contamination of eosinophils, and is important for obtaining a large number of natural killer cells. If lymphocytes were purified using Lymphoprep below 75% (specific gravity 1.058), eosinophil contamination could be reduced significantly, but the total number of monocytes that could be recovered was very small, as well as the lymphocytes in the recovered monocytes. The number of remarkable decreases and the number of monocytes are so large that the amplification effect of natural killer cells rapidly decreases. When Lymphoprep is used in excess of 80% (specific gravity 1.063), it is difficult to reduce eosinophil contamination. have.
본 발명에 있어서, 상기 2) 단계 후 정제된 단핵구세포는 1×104 내지 1×108 cells/well의 농도로 접종되는 것이 좋고, 더욱 바람직하게는 1×105 내지 3×107 cells/well의 농도가 좋다. 특히, 3×106 cells/well의 농도로 접종한 경우, 배양 후 14일에 약 200배의 증식율을 보이는 것을 실험을 통해 확인하였다.In the present invention, the purified monocytes after step 2) is preferably inoculated at a concentration of 1 × 10 4 to 1 × 10 8 cells / well, more preferably 1 × 10 5 to 3 × 10 7 cells / Well concentration is good. In particular, when inoculated at a concentration of 3 × 10 6 cells / well, it was confirmed through experiments that the growth rate of about 200 times at 14 days after incubation.
본 발명에 따른 분화 및 증폭방법은 상기 1) 및 2) 단계로부터 분리 정제된 단핵구세포와 함께 IL-2 10 내지 500 U/㎖ 및 IL-15 0.1 내지 100 U/㎖을 동시에 처리 배양함으로써 강력한 항암 효과를 갖는 고순도의 자연살해세포를 단기간에 더욱 폭발적으로 증식시킬 수 있는 것을 특징으로 한다.Differentiation and amplification method according to the present invention is a powerful anti-cancer by treating and incubating IL-2 10 to 500 U / ㎖ and IL-15 0.1 to 100 U / ㎖ together with the monocytes separated and purified from the steps 1) and 2) It is characterized in that the high-purity natural killer cells having an effect can be expanded more explosively in a short time.
또한, 본 발명에 따른 분화 및 증폭방법은 순차적으로 상기 1) 단계 후, 상기 2) 단계의 불연속 밀도구배 원심분리에 의해 정제된 단핵구세포를 상기 3) 단계의 조성물 및 배양조건을 이용하여 자연살해세포로 분화 유도시킴으로써 형태, 표현형, 기능적인 면에서 전형적인 개 유래의 자연살해세포로 분화됨을 확인할 수 있었다.In addition, the differentiation and amplification method according to the present invention is a natural killing of the mononuclear cells purified by the discontinuous density gradient centrifugation of step 1) after the step 1) using the composition and culture conditions of step 3) By inducing differentiation into cells, it was confirmed that they differentiate into typical killer cells derived from typical dogs in terms of morphology, phenotype, and function.
본 발명에서 사용되는 피더 세포는 유전자가 도입된 동물 세포주(cell line)나 각종 사이토카인이나 화합물이 처리된 말초혈 백혈구 세포(PBL), T-cell, B-cell, 또는 단핵구 등을 사용할 수 있으나, 가장 바람직하게는 K562 세포로 자연살해세포를 순수 자연살해세포만 선택적으로 증식하게 할 수 있는 장점이 있다.The feeder cells used in the present invention may be animal cell lines into which genes are introduced, or peripheral blood leukocyte cells (PBLs) treated with various cytokines or compounds, T-cells, B-cells, or monocytes. Most preferably, K562 cells have the advantage of selectively proliferating natural killer cells.
본 발명에 따른 분화 및 증폭방법은 개의 단핵구세포로부터 자연살해 세포를 선택적으로 증폭시킬 수 있으며, 적은 양의 채혈로 많은 수의 자연살해 세포를 수득할 수 있는 장점이 있다.The differentiation and amplification method according to the present invention can selectively amplify natural killer cells from dog monocytes, and has a merit of obtaining a large number of natural killer cells with a small amount of blood collection.
보다 상세하게 상기 방법으로 분화된 개 유래의 자연살해세포에 대하여, 전형적인 자연살해세포로의 분화가 이루어 졌음을 확인하기 위해, 세포 표면의 자연살해세포 관련 유전자(NK cell-related gene)의 발현 변화를 확인하기 위해 RT-PCR을 수행하였다.In more detail, the expression of NK cell-related genes on the cell surface was changed in order to confirm that the NK cell-derived natural killer cells were differentiated into typical natural killer cells. RT-PCR was performed to confirm.
그 결과, 상기 분화 및 증폭방법에 따른 개 유래의 자연살해세포는 CD16, NKG2D, NKp30, NKp44, 및 Ly49로 이루어진 활성 수용체의 발현이 증가가 되었고, 상기 활성 수용체가 증가된 자연살해세포를 비교적 단기간에 200배 이상 증식시킬 수 있으며, 궁극적으로 95% 이상의 고순도의 강력한 항암 효과를 갖는 개 유래의 자연살해세포를 다량 얻을 수 있는 것을 확인할 수 있었다.As a result, the expression of an active receptor consisting of CD16, NKG2D, NKp30, NKp44, and Ly49 in the dog-derived natural killer cells according to the differentiation and amplification method was increased, and the natural killer cells in which the active receptor was increased were relatively short-term. It can be confirmed that it can multiply more than 200 times and ultimately obtain a large amount of natural killer cells derived from dogs having a strong anticancer effect of 95% or higher high purity.
보다 상세하게는 정상개의 말초혈액에서 밀도 구배 원심방법(density gradient centrifugation)을 이용하여 단핵구세포(PBMCs)를 분리한 다음, 10% FBS와 IL-2(100 U/ml)와 IL-15(10 U/ml)가 첨가된 RPMI-1640 media에서 단핵구세포를 피더 세포(Feeder cell)와 함께 14일간 공조배양하여 개로부터 유래된 자연살해세포의 증폭률을 확인한 결과, 최대 200배 이상 증폭됨을 확인하였으며, 14일 후 자연살해세포의 순도는 최대 95% 이상인 것을 확인할 수 있었다. 도 1을 참조한다.More specifically, mononuclear cells (PBMCs) were isolated from normal dog peripheral blood using density gradient centrifugation, followed by 10% FBS, IL-2 (100 U / ml) and IL-15 (10). U / ml) was added to RPMI-1640 media added with monocytes and co-cultured with feeder cells (Feeder cells) for 14 days to confirm the amplification rate of natural killer cells derived from dogs, it was confirmed that the amplification more than 200 times, After 14 days, natural killer cells were found to have a purity of 95% or more. Please refer to Fig.
상기 분화 및 증폭방법에 따른 개 유래의 자연살해세포는 임상 적용이 가능한, 다량의 활성화된 자연살해세포를 이용하여 종양치료나 종양의 발생원이 되는 것으로 상정되고 있는 바이러스 감염세포의 제거에 유효한 세포치료제를 제조할 수 있다.Natural killer cells derived from dogs according to the differentiation and amplification methods are effective cell therapy agents for the treatment of virus-infected cells that are supposed to be a tumor treatment or tumor source using a large amount of activated natural killer cells that can be clinically applied. Can be prepared.
본 발명에 따른 개로부터 유래된 자연살해세포 증식 및 분화용 배지 조성물은 개 유래의 단핵구세포 및 피더 세포와 함께 배지에 첨가하여 추가 배양함으로써 강력한 항암 효과를 갖는 고순도의 자연살해세포를 단기간에 폭발적으로 증식시킬 수 있는 장점이 있다.The natural killer cell proliferation and differentiation medium composition derived from the dog according to the present invention is added to the medium with the monocytes and the feeder cells derived from the dog and further cultured to explode the high purity natural killer cells having a strong anticancer effect in a short time. There is an advantage to multiply.
본 발명에 따른 자연살해세포 분화 및 증폭방법은 개 유래의 자연살해세포의 살해능을 강력히 증가시키면서 최대 증식이 가능한 획기적인 방법이므로, 적은 양의 채혈로 많은 수의 강력한 자연살해세포를 수득할 수 있게 됨으로써 세포치료제로의 상용화에 크게 기여할 것이다.Natural killer cell differentiation and amplification method according to the present invention is a breakthrough method capable of maximum proliferation while strongly increasing the killing ability of natural killer cells derived from dogs, so that a large number of powerful natural killer cells can be obtained with a small amount of blood collection. This will greatly contribute to the commercialization of cell therapy products.
도 1은 본 발명의 분화 및 증폭방법으로 체외 증폭된 개 유래의 자연살해세포의 형태적 특징을 확인한 결과이고
(A: 0일, B: 18일, C: 13일(배지 내))
도 2는 본 발명의 분화 및 증폭방법으로 체외 증폭된 개 유래의 자연살해세포에 대한 표현형 분석 결과이며,
도 3은 본 발명의 방법분화 및 증폭방법으로 체외 증폭된 개 유래의 자연살해세포에 대한 자연살해세포 관련 유전자의 발현 결과이며,
도 4는 본 발명의 분화 및 증폭방법으로 체외 증폭된 개 유래의 자연살해세포에 대한 iTCRα chain의 발현 결과이고,
도 5는 본 발명의 분화 및 증폭방법으로 체외 증폭된 개 유래의 자연살해세포에 대한 IFN-gamma 생산능을 분석한 결과이며,
도 6은 본 발명의 분화 및 증폭방법으로 체외 증폭된 개 유래의 자연살해세포에 대한 개 갑상샘암 세포 살상능을 분석한 결과이고,
도 7은 본 발명의 분화 및 증폭방법으로 체외 증폭된 개 유래의 자연살해세포로 개 갑상샘암을 공격시킨 후 세포를 염색하여 현미경으로 관찰한 결과이며,
도 8은 본 발명의 방법분화 및 증폭방법으로 체외 증폭된 개 유래의 자연살해세포에 대한 종양 살상능을 확인한 결과이다.
(A: 개 유선종양세포, B: 방광이행상피종)1 is a result of confirming the morphological characteristics of natural killer cells derived from dogs amplified in vitro by the differentiation and amplification method of the present invention
(A: 0 days, B: 18 days, C: 13 days (within the badge))
Figure 2 is a phenotypic analysis of natural killer cells derived from dogs amplified in vitro by the differentiation and amplification method of the present invention,
Figure 3 is a result of the expression of natural killer cell genes for natural killer cells derived from dogs amplified in vitro by the method differentiation and amplification method of the present invention,
Figure 4 is a result of the expression of iTCRα chain on natural killer cells derived from dogs amplified in vitro by the differentiation and amplification method of the present invention,
5 is a result of analyzing the IFN-gamma production capacity for natural killer cells derived from dogs amplified in vitro by the differentiation and amplification method of the present invention,
Figure 6 is a result of analyzing the dog thyroid cancer cell killing ability against natural killer cells derived from dogs amplified in vitro by the differentiation and amplification method of the present invention,
7 is a result of observing the microscope after staining cells after attacking dog thyroid cancer with natural killer cells derived from dogs amplified in vitro by the differentiation and amplification method of the present invention,
8 is a result of confirming the tumor killing ability of natural killer cells derived from dogs amplified in vitro by the method differentiation and amplification method of the present invention.
(A: canine mammary tumor cells, B: bladder metastasis epithelium)
본 발명은 하기 실시예에 의하여 더욱 구체적으로 설명한다. 그러나, 하기 실시예는 본 발명의 이해를 돕기 위한 것일 뿐, 어떤 의미로든 본 발명의 범위가 이러한 실시예에 의하여 한정되는 것은 아니다.The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.
이때, 사용되는 기술 용어 및 과학 용어에 있어서 다른 정의가 없다면, 이 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 통상적으로 이해하고 있는 의미를 가지며, 하기의 설명 및 첨부 도면에서 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능 및 구성에 대한 설명은 생략한다.
Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.
[실시예 1] 개 유래의 말초혈액으로부터 단핵구세포의 정제Example 1 Purification of Monocytes from Peripheral Blood from Dogs
(1) 피콜-하이파크(Ficoll-Hypaque)를 이용한 단핵구세포의 분리(1) Isolation of Monocytes Using Ficoll-Hypaque
개의 말초혈액(perpheral blood)은 공주대학교 특수동물학과의 실험동물사육시설에서 사육하는 개 중에서 혈액검사와 정기적인 건강검진을 통해 건강하다고 인정되는 8세의 성견의 경정맥에서 15 내지 30 ㎖의 말초혈액을 무균적으로 채혈하였으며, 채혈된 말초혈액은 헤파린튜브(heparin tube; BD vacutainer)에 보관하여 2시간 내에 사용하였다. Peripheral blood in dogs is 15 to 30 ml of peripheral blood in the jugular vein of an 8-year-old dog that is considered healthy through blood tests and regular medical examinations among dogs raised in the experimental animal breeding facility of the Department of Special Zoology, Kongju National University. Aseptically, blood was collected and the collected peripheral blood was stored in a heparin tube (BD vacutainer) and used within 2 hours.
(2) 단핵구세포의 정제(2) Purification of Monocytes
상기 채취한 말초혈액을 PBS 버퍼(phosphate-buffered saline buffer)로 1:2 비율로 희석하였다. 또한, 불연속 밀도구배(discontinuous density gradients) 용액은 76% 농도의 Lymphoprep(비중 1.058), 77% 농도의 Lymphoprep(비중 1.059), 용액은 78% 농도의 Lymphoprep(비중 1.060), 용액은 79% 농도의 Lymphoprep(비중 1.062), 용액은 80% 농도의 Lymphoprep(비중 1.063) 10 ㎖ 각각을 100% 농도의 Lymphoprep(비중 1.077) 5 ㎖와 100% 농도의 Histopaque(비중 1.119) 용액 위에 조심스럽게 올린 후, 상온에서 25분 동안 400 × g로 원심분리 하여 단핵구세포를 정제한 후, 과립구의 오염(호산구의 오염) 정도 및 단핵구세포의 수를 조사하여 하기 표 1에 나타내었다.The collected peripheral blood was diluted in a 1: 2 ratio with PBS buffer (phosphate-buffered saline buffer). Discontinuous density gradients solution also contains 76% Lymphoprep (1.058 specific gravity), 77% Lymphoprep (1.059 specific gravity), 78% Lymphoprep (1.060 specific gravity) and 79% Lymphoprep (specific gravity 1.062), the solution is carefully placed 10 ml of 80% concentration of Lymphoprep (1.063 specific gravity) on top of 5 ml of 100% concentration of Lymphoprep (1.077 specific gravity) and 100% Histopaque (specific gravity 1.119) solution, and then room temperature After purifying the monocytes by centrifugation at 400 × g for 25 minutes at 25 minutes, the degree of contamination of granulocytes (contamination of eosinophils) and the number of monocytes were investigated.
상기 표 1에서도 확인할 수 있듯이, Lymphoprep을 76%(비중 1.058) 농도 미만으로 사용하여 단핵구세포를 정제하였을 때, 호산구의 오염은 현저히 줄일 수 있었지만 회수할 수 있는 단핵구세포의 총 개수가 매우 적을 뿐만 아니라 회수된 단핵구세포 내 림프구의 수가 현저히 감소하고, 단핵세포(monocytes)의 수가 많아 자연살해세포의 증폭 효과는 급격히 감소하는 것을 확인할 수 있었고, Lymphoprep을 80%(비중 1.063) 농도를 초과하여 사용하였을 때, 호산구의 오염도가 높은 것을 확인할 수 있었다.As can be seen from Table 1, when the mononuclear cells were purified using Lymphoprep below 76% (specific gravity 1.058), eosinophil contamination was significantly reduced, but the total number of monocytes that could be recovered was not only very small. The number of lymphocytes recovered in monocytes was significantly reduced, and the number of monocytes was significantly increased, and the amplification effect of natural killer cells was rapidly reduced. When Lymphoprep was used in excess of 80% (specific gravity 1.063), , Eosinophils were confirmed to have a high degree of contamination.
상기의 결과로부터 개의 말초혈액으로부터 분리된 단핵구세포의 정제시에는 Lymphoprep는 76%(비중 1.058) 내지 77%(비중 1.059) 농도를 이용함으로써 적은 양의 채혈로부터 호산구의 오염을 현저히 줄일 수 있음을 확인한 결과이고, 나아가 실험관 내에서 많은 수의 자연살해세포를 증폭할 수 있음을 확인한 결과이기도 하다.
From the above results, it was confirmed that Lymphoprep can significantly reduce the eosinophil contamination from a small amount of blood by using Lymphoprep in the concentration of 76% (1.058) to 77% (1.059). It is also a result confirming that a large number of natural killer cells can be amplified in a test tube.
[실시예 2] 개 유래의 자연살해세포(natural killer cell, NK cell)의 증폭 Example 2 Amplification of Natural Killer Cells (NK Cells) from Dogs
상기 실시예 1의 76% 농도의 Lymphoprep(비중 1.058) 10 ㎖, 100% 농도의 Lymphoprep(비중 1.077) 5 ㎖ 및 100% 농도의 Histopaque(비중 1.119)를 이용한 불연속 밀도구배(discontinuous density gradients) 방법으로 정제한 단핵구세포를 이용하였다. Discontinuous density gradient method using 10 ml of Lymphoprep (specific gravity 1.058), 5 ml of 100 percent concentration of Lymphoprep (1.077 specific gravity), and 100% concentration of Histopaque (specific gravity 1.119) of Example 1 Purified monocytes were used.
24 well plate에 상기 정제한 단핵구세포(1×106 cells/well)를 넣은 후, K562 또는 K562-mb15-41BBL 세포주(kindly provided by Dr. Campana, St. Jude Children’s Research Hospital, USA) 0.5×106 cells/well을 추가하여 37℃, 5% CO2 배양기에서 14일 동안 배양하여, 체외에서 개 유래의 자연살해세포를 증폭시켰다.After the purified mononuclear cells (1 × 10 6 cells / well) were placed in a 24 well plate, K562 or K562-mb15-41BBL cell line (kindly provided by Dr. Campana, St. Jude Children's Research Hospital, USA) 0.5 × 10 In addition, 6 cells / well were incubated for 14 days in an incubator at 37 ° C. and 5% CO 2 to amplify natural killer cells derived from dogs in vitro.
상기 배양은 RPMI1640 배지에 10% fetal bovine serum(FBS)을 첨가하고 100 IU/ml human IL-2(PeproTec, USA) 및 10 IU/ml human IL-15(PeproTec, USA)를 첨가한 배지를 이용하였고. 배양액은 매 2일에 한 번씩 교체하였다.
The culture was performed by adding 10% fetal bovine serum (FBS) to RPMI1640 medium and adding 100 IU / ml human IL-2 (PeproTec, USA) and 10 IU / ml human IL-15 (PeproTec, USA). And Cultures were changed every 2 days.
[실시예 3] 개 유래의 자연살해세포의 표현형 확인Example 3 Confirmation of Phenotype of Natural Killer Cells Derived from Dogs
상기 실시예 2로부터 증폭된 개 유래의 자연살해세포의 형태적 특징을 확인하기 위하여, 체외 증폭된 자연살해세포를 May-Grunwald-Giemsa(MGG)염색하여 현미경으로 관찰하였다. In order to confirm the morphological characteristics of the natural killer cells derived from the dog amplified from Example 2, in vitro amplified natural killer cells were stained with May-Grunwald-Giemsa (MGG) and observed under a microscope.
그 결과 도 1에서도 확인할 수 있듯이, 배양 시작일(0일)에는 다양한 종류의 세포로 구성된 단핵구세포의 분포가 많았으나 배양 8일째 다수 세포의 세포질에 자연살해세포 특이적인 퍼포린(perforin)/그랜자임(granzyme)을 함유한 단일 종류 세포의 증폭을 확인할 수 있었으며, 배양 14일째는 대부분의 세포가 세포질에 perforin/granzyme granule들을 함유한 자연살해세포임을 확인할 수 있었다.As a result, as shown in Figure 1, the start of the culture (day 0) was a large number of monocytes composed of a variety of cells distribution, but perforin / granzyme specific to natural cells in the cytoplasm of a large number of cells on the 8th day of culture Amplification of single cell type containing (granzyme) was confirmed, and on
또한, 상기 증폭된 개 유래의 자연살해세포의 표현형(phenotype)을 확인하기 위하여 세포 표면에 발현된 단백질 인자를 APC, RPE 또는 FITC가 결합된 단클론 항체(mAbs)를 이용하여 유세포 분석 결과를 하기 표 2에 나타내었다.In addition, in order to identify the phenotype (phenotype) of the natural killer cells derived from the amplified dogs, the results of flow cytometry using APC, RPE or FITC-coupled monoclonal antibody (mAbs) expressed on the cell surface. 2 is shown.
그 결과 상기 표 2, 도 2 및 도 3에서도 확인할 수 있듯이, 증폭된 개 유래의 자연살해세포는 대부분 CD5 low positive, CD3 positive, CD4 negative, CD8 positive였으며, 배양 시작일(0일)의 단핵구세포와 비교하여 CD11c와 CD11d의 발현이 증가됨을 확인할 수 있었으며, 배양 14일 후 CD21+ 세포는 거의 포함되어 있지 않음을 확인할 수 있었다.
As a result, as can be seen in Table 2, Figure 2 and Figure 3, the natural killer cells derived from amplified dogs were mostly CD5 low positive, CD3 positive, CD4 negative, CD8 positive, monocytes and the start of culture (day 0) In comparison, it was confirmed that the expression of CD11c and CD11d was increased, and after 14 days of culture, almost no CD21 + cells were included.
[실시예 4] 자연살해세포 관련 유전자의 발현 조사Example 4 Expression Survey of Natural Killer Cell-Related Genes
자연살해세포는 분화되면서 세포 표면에 특정 수용체를 발현시키는데, 이들 수용체를 통해서 NK 세포가 분화되는 단계를 확인할 수 있게 된다.As natural killer cells are differentiated, they express specific receptors on the cell surface. Through these receptors, NK cells can be identified.
상기 실시예 2로부터 분리된 배양 시작일(0일)의 단핵구세포와 체외 증폭된 자연살해세포에서 자연살해세포 관련 유전자(NK cell-related gene)의 발현에 대한 RT-PCR을 수행하였다.RT-PCR was performed for the expression of NK cell-related genes in monocytes and in vitro amplified natural killer cells on the start of culture (day 0) isolated from Example 2.
개 유래의 자연살해세포에 대한 특이 표지자가 아직 밝혀져 있지 않기 때문에 공지된 다른 포유동물에서 자연살해세포와 관련된 여러 분자들(CD56, CD16, NKG2D, NKp30, NKp44, Ly49)에 대한 발현 양상을 확인하였다. 상기 분자들에 대한 프라이머는 하기 표 3에 나타내었다. Since no specific marker for natural killer cells derived from dogs has yet been identified, expression patterns of various molecules related to natural killer cells (CD56, CD16, NKG2D, NKp30, NKp44, Ly49) in other known mammals were confirmed. . Primers for the molecules are shown in Table 3 below.
도 3의 A에서도 확인할 수 있듯이, NK세포와 관련된 수용체들에 대한 유전자 즉, CD56, CD16, NKG2D, NKp30, NKp44, Ly49에서의 mRNA발현 양상은 배양 시작일(0일)의 단핵구세포와 비교하였을 경우, 14일간 배양하여 체외 증폭된 자연살해세포에서 CD56을 제외한 모든 유전자들의 발현이 현저히 증가되는 것을 확인할 수 있었으며 특히, Ly49는 배양 전 단핵구세포에서는 전혀 발현되지 않고 증폭된 자연살해세포에서만 발현되는 것을 확인할 수 있어, Ly49가 개 유래의 자연살해세포를 구분하는 특이 표지인자로 사용될 수 있음 역시 확인할 수 있었다.As can be seen from A of Figure 3, the gene expression pattern for the receptors associated with NK cells, namely CD56, CD16, NKG2D, NKp30, NKp44, Ly49 when compared to monocytes at the start of culture (day 0) , 14 days of in vitro amplified natural killer cells were found to significantly increase the expression of all genes except CD56. Especially, Ly49 was found to be expressed only in amplified natural killer cells but not in monocytes before culture. Ly49 could be used as a specific marker to distinguish natural killer cells from dogs.
또한, 자연살해세포의 세포독성 능력과 관련이 깊은 퍼포린(perforin)과 그랜자임(granzyme) B의 발현 양상을 분석한 결과도 자극하기 전 배양 시작일(0일)의 단핵구세포와 비교하여 14일간 시험관에서 증폭된 자연살해세포에서 발현이 현저히 증가됨을 확인할 수 있었다.In addition, the results of analyzing the expression patterns of perforin and granzyme B, which are closely related to the cytotoxic ability of natural killer cells, were also compared with monocytes at the start of culture (day 0) before stimulation. In vitro, amplified natural killer cells were found to significantly increase expression.
반면, 사람의 자연살해세포에 대한 특이 표지인자인 CD56의 경우는 개 자연살해세포에는 전혀 발현되지 않음을 확인하였고, 상기 사용한 프라이머와 RT-PCR의 결과의 정확성을 재확인하기 위하여 개 뇌세포(positive control)와 개의 간세포(negative control)에서 CD56에 대한 발현을 동일한 프라이머로 분석하였다. On the other hand, CD56, a specific marker for human natural killer cells, was not expressed at all in natural killer cells of dogs. In order to reconfirm the accuracy of the primer and RT-PCR results, the dog brain cells (positive) Expression of CD56 in control and negative control of dogs was analyzed with the same primers.
그 결과 도 3의 B에서도 확인할 수 있듯이, 뇌세포에서 CD56 발현이 확인되었고 반면, 간에서는 전혀 발현이 되지 않음을 확인한 바, 상기 분석 결과가 정확하였음을 확인할 수 있었다.As a result, as can be seen in Figure 3B, CD56 expression was confirmed in the brain cells, while it was confirmed that not expressed in the liver at all, it was confirmed that the analysis results were accurate.
상기 결과는 상기 실시예 2의 방법으로 분화된 개 유래의 자연살해세포가 T 세포의 특성을 갖는 것을 확인한 결과로서, 이를 세포가 자연살해 T 세포(NKT cell)인지를 확인하기 위하여, NKT 세포에만 발현되는 iTCRα의 발현 여부를 RT-PCR로 확인하였다. The result is a result of confirming that the natural killer cells derived from dogs differentiated by the method of Example 2 have the characteristics of T cells, to determine whether the cells are natural killer T cells (NKT cells), only NKT cells The expression of iTCRα expressed was confirmed by RT-PCR.
상기 NKT 세포는 내재면역의 한 수행원으로서 비교적 근래에 그 기능들이 밝혀지고 있는 T 세포의 한 종류이다.The NKT cell is a type of T cell whose function has been revealed relatively recently as an agent of endogenous immunity.
그 결과 도 4에서 확인할 수 있듯이, iTCRα를 발현하는 세포는 소수로 대부분의 증폭된 세포는 NKT 세포와 상관이 없는 것을 확인할 수 있었다.
As a result, as can be seen in Figure 4, a small number of cells expressing iTCRα was confirmed that most amplified cells are not correlated with NKT cells.
[실시예 5] 개 유래의 자연살해세포의 IFN-gamma 생산 능력 비교Example 5 Comparison of IFN-gamma Production Capacity of Canine-derived Natural Killer Cells
상기 실시예 2의 체외에서 증폭된 개 유래의 자연살해세포가 효과적으로 IFN-gamma를 생산하는지를 ELISA법(BD OptEIATMSetCanineKit, BD Bioscience)을 통하여 확인하였다.It was confirmed by ELISA method (BD OptEIA ™ SetCanineKit, BD Bioscience) that the natural killer cells derived from the dog amplified in vitro of Example 2 effectively produced IFN-gamma.
그 결과 도 5에서 확인할 수 있듯이, 단핵구세포와 IL-2로 자극한 림포카인 활성화 세포(LAC cell)에 비해 현저히 많은 양의 IFN-gamma를 생산하는 것으로 확인할 수 있었다.
As a result, as shown in Figure 5, it was confirmed that the production of a significant amount of IFN-gamma compared to monocytes and IL-2 stimulated lymphokine activated cells (LAC cells).
[실시예 6] 개 유래의 자연살해세포의 표적 종양세포에 대한 세포독성 평가Example 6 Cytotoxicity Evaluation on Target Tumor Cells from Natural Killer Cells Derived from Dogs
상기 실시예 2의 체외에서 증폭된 개 유래의 자연살해세포의 세포살상능을 조사하기 위해, 상기 실시예 2의 체외에서 증폭된 개 유래의 자연살해세포를 세척 후 효과세포(Effector cell) : 표적세포(Target cell) 비율(20:1, 10:1, 5:1, 2.5:1, 1.25:1)에 따라 표적세포인 51Cr-표지된 CTAC 세포(1×104/well)와 함께 96 웰 둥근 바닥 플레이트(well round bottom plate, Falcon, USA)에 넣고 37℃, 5% CO2 배양기에서 4시간 동안 배양하였다. 상기 배양 후, 상층액을 취하여 감마-카운터(γ-counter)로 51Cr 방출분석(release assay)을 수행하였다.In order to examine the cell killing ability of the natural killer cells derived from the dog amplified in vitro of Example 2, the effector cells after washing the natural killer cells derived from the amplified dog in vitro of Example 2 (Effector cell): target 96 with 51 Cr-labeled CTAC cells (1 × 10 4 / well) as target cells according to the target cell ratio (20: 1, 10: 1, 5: 1, 2.5: 1, 1.25: 1) The plate was placed in a well round bottom plate (Falcon, USA) and incubated for 4 hours in a 37 ° C., 5% CO 2 incubator. After the incubation, the supernatant was taken and subjected to a 51 Cr release assay with a gamma-counter.
상기 표적세포는 canine thyroid adenocarcinoma(CTAC) 세포, CF41.Mg(canine mammary gland tumor cell line) 세포, K9TCC-pu-AXC(canine transitional cell carcinoma cell line) 세포를 이용하였다.The target cells used canine thyroid adenocarcinoma (CTAC) cells, canine mammary gland tumor cell line (CF41.Mg) cells, and canine transitional cell carcinoma cell line (K9TCC-pu-AXC) cells.
배양 4시간 후, 개 NK-sensitive cell인 CTAC 세포에 대한 세포독성을 확인한 결과, 도 6에서도 확인할 수 있듯이 상기 실시예 2의 체외에서 증폭된 개 유래의 자연살해세포의 순도에 따라 60 내지 85%의 매우 높은 세포독성을 보였으며, 16시간 후, 효과세포 : 표적세포 비율이 20:1 비율일 때 세포독성은 80% 이상의 결과를 보여 체외에서 증폭된 개 유래의 자연살해세포가 매우 강력한 기능을 갖은 세포임을 확인할 수 있었다. After 4 hours of culture, as a result of confirming cytotoxicity against CTAC cells, which are dog NK-sensitive cells, as shown in FIG. 6, 60 to 85% according to the purity of natural killer cells derived from dogs amplified in vitro of Example 2 After 16 hours, cytotoxicity was more than 80% when the effect cell: target ratio was 20: 1, and natural killer cells derived from dogs amplified in vitro showed very strong functions. It was confirmed that the cells.
또한, 상기 체외에서 증폭된 개 유래의 자연살해세포로 CTAC를 공격시킨 후 세포를 염색하여 현미경으로 관찰한 결과, 도 7에서 확인할 수 있듯이 세포질 내 퍼포린(perforin)과 그랜자임(granzyme) B 과립을 함유한 개 유래의 자연살해세포가 표적 종양세포에 붙어 살상효과를 보이고 있음을 확인할 수 있었다. In addition, after the CTAC attack with natural killer cells derived from the in vitro amplified cells, the cells were stained and observed under a microscope. As shown in FIG. 7, perforin and granzyme B granules in the cytoplasm were observed. It was confirmed that natural killer cells derived from the dogs contained the killing effect attached to the target tumor cells.
자연살해세포는 CD8+ cytotoxic T cell(CTL)과 달리 종양의 특이 항원에 대한 사전 인식 없이 종양세포를 공격하여 사멸시킬 수 있기 때문에 상기 실시예 1의 체외에서 증폭된 개 유래의 자연살해세포가 완전한 자연살해세포임을 확인하기 위하여 CTAC 세포와 완전히 다른 개 유선종양세포(CF41-Mg)와 방광이행상피종 (K9TCC-pu-AXC)에 대한 세포독성을 동시에 확인하였다.Unlike natural CD8 + cytotoxic T cells (CTLs), natural killer cells can attack and kill tumor cells without prior recognition of the specific antigens of the tumors. In order to confirm that they are killer cells, cytotoxicity against CTAC cells and completely different canine mammary tumor cells (CF41-Mg) and bladder metastatic epithelial cells (K9TCC-pu-AXC) were simultaneously confirmed.
그 결과 도 8에서 확인할 수 있듯이, CF41.Mg 세포 및 K9TCC-pu-AXC 세포에 대한 세포독성을 확인한 결과, 도 8에서 확인할 수 있듯이 4시간 후, 효과세포 : 표적세포 비율이 20:1 비율일 때 세포독성은 CF41.Mg 세포의 경우 73.6±2.3%, K9TCC-pu-AXC 세포의 경우 66.6±2.0%의 세포독성을 나타내었고 반면, 배양 시작일(0일)의 단핵구세포의 경우 0.5 내지 30%의 독성을 보이는 것을 확인할 수 있었다.As a result, as shown in FIG. 8, the cytotoxicity of CF41.Mg cells and K9TCC-pu-AXC cells was confirmed. As shown in FIG. 8, after 4 hours, the effect cell: target cell ratio was 20: 1. Cytotoxicity was 73.6 ± 2.3% for CF41.Mg cells and 66.6 ± 2.0% for K9TCC-pu-AXC cells, whereas 0.5-30% for monocytes at the start of culture (day 0). It was confirmed that the toxicity of.
이는 상기 종양에 대한 항원 인식 없이 매우 높은 살상효과를 보임을 통해 실시예 1의 체외에서 증폭된 개 유래의 자연살해세포가 강력한 자연살해세포로서의 특성을 갖가지고 있음을 확인한 결과이다.
This is a result of confirming that the natural killer cells derived from the dog amplified in vitro in Example 1 has the characteristics as a strong natural killer cells by showing a very high killing effect without antigen recognition for the tumor.
상기의 결과로부터 본 발명의 개로부터 유래된 자연살해세포(natural killer cell, NK cell)의 분화 및 증폭방법은 그 형태, 표현형 및 기능적인 측면을 볼 때 체외(In vitro)에서 자연살해세포로 성공적으로 배양할 수 있고, 나이가 임상단계에서 자연살해세포를 안전하고, 효율적으로 배양할 수 있는 closed system에도 적용이 가능할 것으로 기대된다.From the above results, the method for differentiation and amplification of natural killer cells (NK cells) derived from dogs of the present invention was successful as natural killer cells in vitro in view of its morphology, phenotype and functional aspects. It is expected to be applicable to a closed system that can safely and efficiently cultivate natural killer cells at an early clinical stage.
Claims (9)
상기 유효성분은 IL-2 10 내지 500 U/㎖ 및 IL-15 0.1 내지 100 U/㎖를 함유하는 것을 특징으로 하는 조성물.The method of claim 1,
The active ingredient is a composition characterized in that it contains IL-2 10 to 500 U / ㎖ and IL-15 0.1 to 100 U / ㎖.
2) 상기 분리된 단핵구세포를 피콜(Ficoll)로 정제하는 단계;
3) 피더 세포(Feeder cell) 존재 하에, 제 1항 또는 제 2항에 따른 배지 조성물을 상기 2)에서 정제된 단핵구세포(mononuclear cells)와 공조 배양하는 단계:
를 포함하는 것을 특징으로 하는, 개로부터 유래된 자연살해세포(natural killer cell, NK cell)의 분화 및 증폭방법.1) superimposing monocytes derived from dogs on Ficoll-Hypaque and centrifuging;
2) purifying the isolated monocytes with Ficoll;
3) co-culturing the medium composition according to claim 1 or 2 with mononuclear cells purified in 2) in the presence of a feeder cell:
Method for differentiation and amplification of natural killer cells derived from dogs (natural killer cells, NK cells) comprising a.
상기 1) 단계의 피콜-하이파크(Ficoll-Hypaque) 비중은 1.077 내지 1.119 g/㎖인 것을 특징으로 하는 분화 및 증폭방법.The method of claim 3,
Ficoll-Hypaque specific gravity of step 1) is characterized in that the differentiation and amplification method is 1.077 to 1.119 g / ㎖.
상기 2) 단계의 정제는 75%(비중 1.057) 내지 80%(비중 1.063)농도의 Lymphoprep, 100%(비중 1.077) 농도의 Lymphoprep 및 100% Histopaque(비중 1.119)의 불연속 밀도구배(discontinuous density gradients) 원심분리를 이용하는 것을 특징으로 하는 분화 및 증폭방법.The method of claim 3,
The purification of step 2) is performed by Lymphoprep at 75% (specific gravity 1.057) to 80% (specific gravity 1.063) concentration, Lymphoprep at 100% concentration (1.077 specific gravity) and discontinuous density gradients of 100% Histopaque (specific gravity 1.119). Differentiation and amplification method using centrifugation.
상기 2) 단계의 정제는 76%(비중 1.058) 내지 77%(비중 1.059) 농도의 Lymphoprep, 100%(비중1.077) 농도의 Lymphoprep 및 100% Histopaque(비중 1.119)의 불연속 밀도구배(discontinuous density gradients) 원심분리를 이용하는 것을 특징으로 하는 분화 및 증폭방법.6. The method of claim 5,
Purification of step 2) is performed by Lymphoprep at a concentration of 76% (1.058 specific gravity) to 1.059 (specific gravity 1.059), Lymphoprep at a concentration of 100% (1.077 specific gravity) and discontinuous density gradients of 100% Histopaque (specific gravity 1.119). Differentiation and amplification method using centrifugation.
상기 3) 단계는 K562 피더 세포(Feeder cell) 존재 하에, 상기 2)의 정제된 단핵구세포를 IL-2 10 내지 500 U/㎖ 및 IL-15 0.1 내지 100 U/㎖를 포함하는 배지에서 10 내지 25일 동안 공조 배양하는 것을 특징으로 하는 분화 및 증폭방법.The method of claim 3,
Step 3) is in the presence of K562 feeder cells (Feeder cell), the purified mononuclear cells of 2) 10-2 in a medium containing IL-2 10 to 500 U / ㎖ and IL-15 0.1 to 100 U / ㎖ Differentiation and amplification method characterized in that the co-culture for 25 days.
상기 방법은 CD16, NKG2D, NKp30, NKp44, 및 Ly49로 이루어진 활성 수용체의 발현을 증가시키는 것을 특징으로 하는 분화 및 증폭방법.The method according to any one of claims 4 to 7, wherein
The method of differentiation and amplification, characterized in that for increasing the expression of the active receptor consisting of CD16, NKG2D, NKp30, NKp44, and Ly49.
상기 방법은 자연살해세포는 순도가 95% 이상인 것을 특징으로 하는 분화 및 증폭방법.The method of claim 8,
The method is characterized in that the natural killer cells have a purity of 95% or more.
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| WO2019221463A1 (en) * | 2018-05-16 | 2019-11-21 | 고려대학교 산학협력단 | Expansion culture method for human-derived natural killer cells by using hdac inhibitor |
| WO2020213972A1 (en) * | 2019-04-17 | 2020-10-22 | 주식회사 차바이오텍 | Natural killer cells exhibiting increased anticancer activity and immunotherapeutic use thereof |
| WO2023038476A1 (en) * | 2021-09-10 | 2023-03-16 | 한국생명공학연구원 | Method for producing natural killer cell and use thereof for preventing or treating infectious diseases |
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| CN115094037B (en) * | 2022-07-19 | 2023-11-10 | 百欧派(天津)生物技术有限公司 | Composition with canine natural killer cell in-vitro activation function matched with autologous plasma for use and application thereof |
| CN115125204B (en) * | 2022-07-19 | 2023-08-22 | 百欧派(天津)生物技术有限公司 | Composition with canine natural killer cell in-vitro activation function and in-vitro culture method |
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| WO2019221463A1 (en) * | 2018-05-16 | 2019-11-21 | 고려대학교 산학협력단 | Expansion culture method for human-derived natural killer cells by using hdac inhibitor |
| WO2020213972A1 (en) * | 2019-04-17 | 2020-10-22 | 주식회사 차바이오텍 | Natural killer cells exhibiting increased anticancer activity and immunotherapeutic use thereof |
| WO2023038476A1 (en) * | 2021-09-10 | 2023-03-16 | 한국생명공학연구원 | Method for producing natural killer cell and use thereof for preventing or treating infectious diseases |
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