KR20080011011A

KR20080011011A - A compound for differentiation from precusor natural killer cell to mature natural killer cell

Info

Publication number: KR20080011011A
Application number: KR1020060075450A
Authority: KR
Inventors: 김상현; 고창보; 김은미; 허영진; 심우영; 허성범; 윤지연; 이은희; 윤희정
Original assignee: 주식회사 굿셀라이프
Priority date: 2006-07-28
Filing date: 2006-08-10
Publication date: 2008-01-31

Abstract

A compound for inducing differentiation from a precursor natural killer cell to a mature natural killer cell is provided to increase the number of mature natural killer cells without using cancer cells or genes of other persons, so that therapeutic effects of the mature natural killer cell on cancer is enhanced. A method for producing a mature natural killer cell comprises the steps of: (i) treating a hematopoietic stem cell with interleukin-7, SCF(stem cell factor) and Flt3L(human soluble FMS(fibromyalgia syndrome)-like tyrosine kinase 3 ligand) to differentiate into a precursor natural killer cell; (ii) treating the precusor natural killer cell with a differentiation-regulating factor in the presence of human stroma cells; and (iii) treating the differentiated mature natural killer cell with interleukin-2 to activate it, wherein the hematopoietic stem cell is isolated from bone marrow, peripheral blood or umbilical cord blood of human.

Description

전구자연살해세포로부터 성숙자연살해세포로의 분화유도제{A Compound for Differentiation from Precusor Natural Killer Cell to Mature Natural Killer Cell} A Compound for Differentiation from Precusor Natural Killer Cell to Mature Natural Killer Cell}

본 발명은 전구자연살해세포(precusor natural killer cell, pNK)로부터 성숙자연살해세포(mature natural killer cell, mNK)로의 분화를 유도하는 화합물(이하, 분화유도제로 약칭할 수 있다)에 관한 것이다. 보다 자세하게는, 본 발명은 조혈모세포를 인터루킨-7, SCF(줄기세포인자) 및 Flt3L(Fms와 유사한 티로신 키나제 3 리간드)로 처리하여 전구자연살해세포 (precursor Natural killer cell, pNK)로 분화시키고, 전구자연살해세포를 상기 분화유도제로 처리하여 성숙자연살해세포로 분화시킴을 특징으로 하는, 성숙자연살해세포의 제조 방법에 관한 것이다.The present invention relates to compounds which induce differentiation from precursor natural killer cells (pNK) to mature natural killer cells (mNK) (hereinafter, abbreviated as differentiation inducing agents). More specifically, the present invention treats hematopoietic stem cells with interleukin-7, SCF (stem cell factor) and Flt3L (tymine tyrosine kinase 3 ligand similar to Fms) to differentiate into precursor natural killer cells (pNK), The present invention relates to a method for producing mature natural killer cells, characterized in that differentiation of progenitor natural killer cells with the differentiation-inducing agent to differentiate them into mature natural killer cells.

현재의 암 치료법들인 외과적 수술요법, 약물요법, 방사선치료법 등은 일시적으로 치료효과를 나타낼 수 있으나 내성, 재발 또는 여러 가지 부작용이 발생하는 문제점이 있어 많은 선진국에서 항암제나 진단시약을 개발하기 위해 면역반응을 조절할 수 있는 면역치료법을 이용하고자 하는 시도가 활발히 진행되고 있으나 암의 진단, 예방 및 치료법을 효율적으로 개발할 수 있는 면역세포치료법 개발은 아 직 초기단계로 알려져 있다. 자연살해세포(Natural Killer cell, NK)는 병원균, 암, 동종이계의(allogeneic) 세포 등을 제거하는 선천성면역 (innate immunity)에 관여하며, 인터페론-감마, 종양괴사인자-알파, 인터루킨-12 등의 사이토카인을 분비하여 적응성면역 (adaptive immunity)을 매개함으로써 암에 특이적인 기억 형성을 조절하는 기능을 가지고 있으며 여러 암들에서 자연살해세포의 기능 및 분화능력에 결함이 있는 것으로 보고되었다. 자연살해세포를 이용한 기존의 암 치료요법으로는 인터루킨-2로 자연살해세포를 활성화시켜 암세포에 대한 면역반응을 증진시키는 방법이 이용되어 왔는데 이러한 방법은 부작용과 함께 개인에 따른 치료효과의 지속성, 내성 및 치료효과의차이 등 많은 문제점을 가지고 있다고 알려져 있다. 자연살해세포의 암 살상 효과를 높이기 위해서는 무엇보다도 활성이 높은 자연살해세포를 대량으로 확보해야 한다. 자연살해세포는 암이나 난치성 바이러스 감염 등의 치료에 응용할 가능성이 있어 실제 임상에 사용하는 것은 100억 개 이상의 자연살해세포가 필요하다고 한다. 그러나 늘리는 것이 매우 어려운 세포로 지금까지 여러 가지 방법으로 시도되어 왔으나, 인터루킨-2나 인터루킨-15로는 많아야 수십 배가 한계였으나, 그 후 타인의 암세포를 섞으면 수백 배로 늘어나는 것이 밝혀졌다. 2005년에 발표된 가장 새로운 자연살해세포 증식 방법은 두 개의 유전자를 섞은 타인의 암세포를 섞어 배양하면 1000배가 된다고 하나 아직 연구단계에 있다. 적어도 수백 배 정도 이상을 늘리는 것은 타인의 암세포와 같이 섞어서 배양하는 것이 기본이다. 타인의 암세포와 섞거나 또한 유전자를 사용하는 것 등은 아직 해결되지 않은 안전상의 문제, 윤리적인 문제가 남겨져 있는 상황이다.Current cancer treatments, such as surgical surgery, drug therapy, and radiation therapy, may have a temporary therapeutic effect, but there are problems such as resistance, recurrence, or various side effects, so that many developed countries are immune to developing anticancer drugs or diagnostic reagents. Attempts have been made to use immunotherapies to modulate the response, but the development of immune cell therapies that can effectively develop the diagnosis, prevention and treatment of cancer is still known at an early stage. Natural Killer cells (NK) are involved in innate immunity that removes pathogens, cancers, allogeneic cells, interferon-gamma, tumor necrosis factor-alpha, interleukin-12, etc. It has been reported to have a function of regulating memory formation specific to cancer by secreting cytokines and mediating adaptive immunity, and defects in the function and differentiation of natural killer cells in several cancers. Conventional cancer therapy using natural killer cells has been used to enhance the immune response to cancer cells by activating natural killer cells with interleukin-2. These methods have side effects and persistence and resistance of individual therapeutic effects. And it is known that there are many problems such as the difference in the therapeutic effect. In order to enhance the cancer killing effect of natural killer cells, first of all, a large amount of highly active natural killer cells must be secured. Natural killer cells are likely to be applied to the treatment of cancer and intractable virus infections, so that the actual clinical use requires more than 10 billion natural killer cells. However, it has been tried to increase the number of cells very difficult, but interleukin-2 or interleukin-15 was limited to several tens of times, but after mixing cancer cells of others, it was found to increase several hundred times. The newest method of natural killer cell proliferation, released in 2005, is said to multiply and cultivate by culturing cancer cells of another person, which is a mixture of two genes. Increasing at least a hundredfold or more is basic to mix with other cancer cells. Mixing with other people's cancer cells or using genes has left unresolved safety and ethical issues.

본 발명의 목적은 전구자연살해세포로부터 성숙자연살해세포로의 분화를 유도하여 다량의 성숙자연살해세포를 제공하는데 있다. An object of the present invention is to provide a large amount of mature natural killer cells by inducing differentiation from pro-natural killer cells to mature natural killer cells.

본 발명의 다른 목적은 전구자연살해세포로부터 성숙자연살해세포로의 분화를 유도하는 물질을 제공하는데 있다. Another object of the present invention is to provide a substance for inducing differentiation from progenitor killer cells to mature killer cells.

본 발명의 또 다른 목적은 성숙자연살해세포를 저 용량의 인터루킨-2로 처리하여 활성화된 성숙자연살해세포를 제공하는데 있다. Another object of the present invention is to provide mature mature killer cells activated by treating mature natural killer cells with a low dose of interleukin-2.

본 발명의 또 다른 목적은 본 발명에 따라 분화되고 활성화된 성숙자연살해세포를 이용한 면역세포치료제를 제공하는데 있다. Another object of the present invention to provide an immune cell therapy using mature natural killer cells differentiated and activated according to the present invention.

상기의 기술적 과제를 달성하기 위하여, 본 발명자들은 기존의 대부분의 연구들에서 이용한 성숙자연살해세포와는 달리, 조혈모세포로부터 성숙자연살해세포로의 분화과정을 연구하였으며, 그 결과 분화를 촉진시키는 유전자를 발견하였다. 조혈모세포로부터 성숙자연살해세포로의 분화과정 중에 각각의 분화단계에 따라서 특이적으로 발현되는 유전자를 발굴하고자 각 분화단계별 세포들에 대하여 SAGE(유전자발현 수직 분석, Serial analysis of gene expression)를 수행하였다. 　SAGE는 양적인 게놈 상의 유전자 발현을 측정하기 위해 사용되는 기술로 다음의 세가지 조건을 충족해야한다. (1) 짧은 10개의 염기쌍 시퀀스 택(tag)은 전사를 확인할 수 있는 충분한 정보를 지니고 (2) 시퀀스 택은 서로 연결되어 long serial molecules (콘캐터머, concatemers)를 형성하고 (3) 관찰되는 택의 수를 셈으로써 그 대응되 는 전사의 발현 수준을 알 수 있어야 한다. 콘캐터머는 디지탈 택으로 구성되며, 대략 30-40개의 택을 포함하여 유전자은행(gene bank)의 데이타베이스와 비교하여 택의 빈도를 분석하여 세포내 대응하는 전사의 분량을 반영할 수 있다. 이러한 기법을 이용하여 성숙자연살해세포의 분화에 가장 큰 영향을 미칠 수 있는 전구자연살해세포에서 이들 분화단계별 발현 특이 유전자의 발현이 실제로 SAGE 결과와 일치하는지를 역전사중합효소연쇄반응으로 분석하였고 이들 특이유전자들을 성숙자연살해세포분화 유도 유전자 후보로 선정하였다. 이들 유전자는 자연살해세포의 발생 및 기능에 조절하는 역할이 있다는 것에 대하여 알려진 바가 없다. 본 발명자들은 전구자연살해세포로부터 성숙자연살해세포로 분화하는데 그들 유전자의 발현 산물이 필수적인 역할을 한다는 것을 발견하였고, 자연살해세포로의 분화조절제로서 작용하는 발현 산물의 발견을 기초로 하여, 사람 제대혈로부터 조혈모세포를 채취하여 이들 세포로부터 그러한 산물 자체 또는 이의 리간드를 이용하여 성숙자연살해세포로 분화시키는 시스템을 확립하여 세포독성 기능이 증강된 성숙자연살해세포를 대량으로 생성하는데 성공하였다.In order to achieve the above technical problem, the present inventors studied the differentiation process from hematopoietic stem cells to mature natural killer cells, unlike the mature natural killer cells used in most of the existing studies, and as a result genes that promote differentiation Found. During the differentiation process from hematopoietic stem cells to mature natural killer cells, SAGE (Serial Analysis of Gene Expression) was performed on the cells of each differentiation stage to identify genes that are specifically expressed according to differentiation stages. . SAGE is a technique used to measure gene expression on quantitative genomes and must meet three conditions: (1) short 10 base pair sequence tags have sufficient information to identify transcription; (2) sequence tags are linked together to form long serial molecules (concatemers) and (3) By counting, the expression level of the corresponding transcription should be known. Concatalmers are composed of digital tags and can include approximately 30-40 tags to analyze the frequency of the tag compared to the database of the gene bank to reflect the amount of corresponding transcription in the cell. Using this technique, we analyzed whether the expression of these expression-specific genes in different stages of proliferation, which may have the greatest effect on the differentiation of mature natural killer cells, by the transcriptase polymerase chain reaction. Were selected as candidate genes for inducing mature killer cell differentiation. It is not known that these genes play a role in regulating the development and function of natural killer cells. The inventors have found that the expression products of these genes play an essential role in differentiating from progenitor killer cells to mature killer cells, and based on the discovery of expression products that act as regulators of differentiation into natural killer cells, human cord blood By establishing a system for collecting hematopoietic stem cells from these cells and using these products themselves or their ligands to differentiate into mature natural killer cells, we successfully generated large numbers of mature natural killer cells with enhanced cytotoxic function.

이러한 성공에 따라, 한 가지 관점으로, 본 발명은 조혈모세포로부터 성숙자연살해세포로의 분화를 유도하는 화합물을 제공한다. 이러한 분화유도제는 유전자 발현 산물 자체가 세포 표면 수용체인 경우 그에 대한 항체 또는 리간드가 해당되며, 또한 유전자 발현 산물 자체가 리간드인 경우는 리간드 자체로 분화유도제로 사용할 수 있다. 본원에서 용어 "리간드"는 그의 수용체와 결합하여 작용성 반응을 나타내도록 하는 폴리펩타이드 또는 화합물을 의미한다. 따라서, 본 발명에 따른 리간드는 분화유도제로 사용될 수 있다. 용어 “분화”는 일반적으로 비교적 단순한 계(系)가 둘 이상의 질적으로 다른 부분계(部分系)로 분리되는 현상이다. 즉, 구조나 기능이 특수화하는 현상을 말한다. 성숙자연살해세포에서 용어 성숙은 자연살해세포가 세포의 고유 기능을 발휘할 수 있는 능력을 갖고 있음을 의미하며, 예를 들면 암세포를 인지하고 직접 암세포를 죽일 수 있는 능력을 가졌음을 가리킨다. 자연살해세포가 성숙되었는지의 여부는 발현 물질을 마커로 하여 확인할 수 있는데 마우스나 사람의 경우 잘 밝혀져 있다. 마우스의 마커로는 예를 들면 NK1.1, CD122, LY49 Family(Ly49A, Ly49C, Ly49D, Ly49E, Ly49F, Ly49G, Ly49H, Ly49I), NKG2A/C/E가 포함되며, 사람의 마커로는 예를 들면 NKG2A, NKG2D, NKp30, NKp44, NKp46, CD56, CD161이 포함된다. 이러한 마커들의 기능은 본 분야의 전문가에게는 잘 인지되어 있다. In accordance with this success, in one aspect, the present invention provides compounds that induce differentiation from hematopoietic stem cells to mature NK cells. Such a differentiation inducing agent is an antibody or a ligand thereof when the gene expression product itself is a cell surface receptor, and may be used as a differentiation inducing agent as the ligand itself when the gene expression product itself is a ligand. As used herein, the term “ligand” refers to a polypeptide or compound that binds to its receptor to produce a functional response. Thus, the ligands according to the invention can be used as differentiation inducers. The term “differentiation” is generally a phenomenon in which a relatively simple system is separated into two or more qualitatively different subsystems. In other words, it refers to a phenomenon in which a structure or a function is specialized. In mature natural killer cells, the term maturation means that natural killer cells have the ability to perform their own functions, for example, have the ability to recognize cancer cells and directly kill them. Whether natural killer cells are mature can be confirmed by using an expression material as a marker, which is well known in mice and humans. Mouse markers include, for example, NK1.1, CD122, LY49 Family (Ly49A, Ly49C, Ly49D, Ly49E, Ly49F, Ly49G, Ly49H, Ly49I), NKG2A / C / E. Examples include NKG2A, NKG2D, NKp30, NKp44 , NKp46, CD56, and CD161. The function of these markers is well recognized by those skilled in the art.

다른 관점으로서, 본 발명은 (i) 조혈모세포를 인터루킨-7, SCF 및 Flt3L로 처리하여 전구자연살해세포로 분화시키고, (ii) 전구자연살해세포를 분화조절제로 처리하여 성숙자연살해세포로 분화시켜 성숙자연살해세포를 수득함을 특징으로 하는, 성숙자연살해세포의 제조 방법을 제공한다. In another aspect, the present invention is characterized in that (i) hematopoietic stem cells are treated with interleukin-7, SCF and Flt3L to differentiate into progenitor killer cells, and (ii) progenitor killer cells are treated with differentiation regulators to differentiate into mature natural killer cells. It provides a method for producing mature natural killer cells, characterized in that to obtain mature natural killer cells.

한 가지 양태로, 본 발명은 조혈모세포를 사람의 골수, 말초혈액 또는 제대혈로부터 분리함을 특징으로 하여 사람 성숙자연살해세포를 제조하는 방법을 제공한다. In one embodiment, the present invention provides a method for producing human mature natural killer cells, characterized in that the hematopoietic stem cells are isolated from human bone marrow, peripheral blood or umbilical cord blood.

다른 양태로서, 본 발명은 전구자연살해세포를 사람 기질(stroma)세포의 존재하에 분화조절제로 처리함을 특징으로 하는 사람 성숙자연살해세포의 제조 방법 을 제공한다. In another aspect, the present invention provides a method for producing human mature natural killer cells, characterized in that the progenitor killer cells are treated with a differentiation regulator in the presence of human stroma cells.

또 다른 관점에서, 본 발명은 (i) 조혈모세포를 인터루킨-7, SCF 및 Flt3L로 처리하여 전구자연살해세포로 분화시키고, (ii) 전구자연살해세포를 분화조절제로 처리하여 성숙자연살해세포로 분화시켜 성숙자연살해세포를 수득하고, (iii) 분화된 성숙자연살해세포를 인터루킨-2로 처리하여 활성화시킴을 특징으로 하는, 활성화된 성숙자연살해세포의 제조 방법을 제공한다. 한 가지 양태로, 본 발명은 분화된 성숙자연살해세포를 약 8 내지 15ng/ml의 인터루킨-2로 처리함을 특징으로 하는 방법을 제공한다. In another aspect, the present invention (i) treatment of hematopoietic stem cells with interleukin-7, SCF and Flt3L to differentiate into pro-natural killer cells, (ii) treatment of pro-natural killer cells with differentiation regulators into mature natural killer cells Differentiation to obtain mature natural killer cells, and (iii) treating differentiated mature natural killer cells with interleukin-2 to activate them. In one embodiment, the invention provides a method characterized by treating differentiated mature natural killer cells with about 8-15 ng / ml of Interleukin-2.

또 다른 관점으로, 본 발명은 본 발명의 방법에 따라 제조된 활성화된 성숙자연살해세포를 포함함을 특징으로 하는 면역세포치료제를 제공한다. 본원에서 면역세포치료제는 각종 항암제 또는 면역증강제 등으로 사용될 수 있음을 의미한다. 활성화된 성숙자연살해세포에서 용어 “활성화”는 성숙자연살해세포가 IL-2에 의해 자극을 받음으로써 실질적인 세포독성 효능을 발휘한다는 것을 의미한다.In another aspect, the present invention provides an immune cell therapeutic agent comprising activated mature natural killer cells prepared according to the method of the present invention. Herein, the immune cell therapeutic agent may be used as various anticancer agents or immune enhancers. In activated mature killer cells, the term “activation” means that mature killer cells exert substantial cytotoxic efficacy by being stimulated by IL-2.

또 다른 관점으로서, 본 발명은 환자의 혈액 및/또는 골수로부터 조혈모세포를 추출하고, 조혈모세포를 인터루킨-7, SCF 및 Flt3L로 처리하여 전구자연살해세포로 분화시키고, 전구자연살해세포를 기질세포의 존재하에 분화조절제로 처리하여 성숙자연살해세포로 분화시키고, 분화된 성숙자연살해세포를 인터루킨-2로 처리하여 활성화시키고, 활성화된 성숙자연살해세포를 환자 자신에게 주입함을 특징으로 하는 자가면역세포치료요법을 제공한다. 본 발명에서 "자가면역세포치료요법"이란 환자의 몸에서 면역세포를 채취하여 혹은 환자 자신의 조혈모세포를 이용하여 시험 관 수준에서 면역세포로 분화시킨, 즉 체외에서의 배양을 통해 암세포만을 선택적으로 살해할 수 있는 면역세포의 수를 늘리거나 기능을 강화시킨 후 다시 체내로 주입하여 암을 치료하는 것을 말한다.In another aspect, the present invention is to extract the hematopoietic stem cells from the blood and / or bone marrow of the patient, to treat the hematopoietic stem cells with interleukin-7, SCF and Flt3L to differentiate into progenitor killer cells, the progenitor cells are stromal cells Autoimmune, characterized in that differentiation into mature natural killer cells by treatment with differentiation regulators in the presence of, and differentiated mature natural killer cells with interleukin-2 to activate them and inject the activated mature natural killer cells into the patient Provide cell therapy. In the present invention, "autoimmune cell therapy" refers to selectively collecting only cancer cells by taking an immune cell from the patient's body or using the patient's own hematopoietic stem cell to differentiate into immune cells at the in vitro level, that is, cultured in vitro. This means treating cancer by increasing the number of immune cells that can kill or enhancing their function and then injecting them back into the body.

또 다른 관점으로서, 본 발명은 사람 동종 제대혈, 공여 혈액 및/또는 골수로부터 조혈모세포를 추출하고, 조혈모세포를 인터루킨-7, SCF 및 Flt3L로 처리하여 전구자연살해세포로 분화시키고, 전구자연살해세포를 기질세포의 존재하에 분화조절제로 처리하여 성숙자연살해세포로 분화시키고, 분화된 성숙자연살해세포를 인터루킨-2로 처리하여 활성화시키고, 활성화된 성숙자연살해세포를 환자에게 주입함을 특징으로 하는 동종면역세포치료요법을 제공한다. 본 발명에서 "동종면역세포치료요법"이란 태아의 제대혈로부터 성체줄기세포를 이용하여 시험관 수준에서 면역세포로 분화시킨, 즉 체외에서의 배양을 통해 암세포만을 선택적으로 살해할 수 있는 면역세포의 수를 늘리거나 기능을 강화시킨 후 다시 체내로 주입하여 암을 치료하는 것을 말한다.In another aspect, the present invention extracts hematopoietic stem cells from human allogeneic umbilical cord blood, donor blood and / or bone marrow, and treats hematopoietic stem cells with interleukin-7, SCF and Flt3L to differentiate into progenitor killer cells, and progenitor killer cells. Is treated with a differentiation regulator in the presence of stromal cells to differentiate into mature natural killer cells, the differentiated mature natural killer cells are treated with interleukin-2 and activated, and the activated mature natural killer cells are injected into the patient. Homeoimmune cell therapy is provided. In the present invention, "homogenous immune cell therapy" refers to the number of immune cells capable of selectively killing only cancer cells through in vitro culture by differentiating them into immune cells at the in vitro level using adult stem cells from fetal cord blood. Increasing or enhancing function and then injected into the body to treat cancer.

자연살해세포는 특정 미생물 감염 및 신생물의 인식 및 파괴를 포함한 다양한 생물학적 기능을 갖는 선천적 면역계에 연루된 백혈구 림프구이다(Moretta, A., Bottino, C., Mingari, M.C., Biassoni, R. and Moretta, L., Nat. Immunol., 3, 6, 2002). 이들의 형태적 특징은 세포질 내에 조밀하게 분홍-보라색의 염색 과립의 존재를 근거로 거대한 과립 림프구(Large Granular Lymphocyte, LGL)-유사 형태를 보여준다. 이들은 정상적인 말초혈 림프구에서 약 10 내지 20%, 간 림프구에서 15 내지 25%, 비장 림프구에서 1 내지 5%를 포함한다. 휴면 자연살해세포는 혈중에 순환하나, 사이토카인에 의한 활성화 후 이들은 병원체-감염된 또는 악성 세포를 함유한 대부분의 조직 내로 유출 및 침윤할 수 있다 (Colucci, F., Di Santo, J.P. and Leibson, P.J., Nat. Immunol., 3, 807, 2002; Kelly J. M., Darcy P. K., Markby J. L., Godfrey D. I., Takeda K., Yagita H., Smyth M. J., Nat. Immunol., 3, 83 , 2002; Shi F. D., Wang H. B., Li H., Hong S., Taniguchi M., Link H., Van Kaer L., Ljunggren H. G., Nat. Immunol., 1, 245, 2000; Korsgren M., Persson C. G., Sundler F., Bjerke T., Hansson T., Chambers B. J., Hong S., Van Kaer L., Ljunggren H. G., Korsgren O., J. Exp. Med., 189, 553, 1999). 일반적으로, 자연살해세포의 표현형은 CD56 및 CD16(사람), NKR-P1C(마우스에서 NK1.1, 사람에서 CD161), DX5, Ly49(마우스; 이들은 특정 마우스 스트레인으로 한정된다) 표면 항원의 발현 및 CD3의 결여로 특징 지워진다. 사람 자연살해세포의 대부분(총 자연살해세포 중 90%)은 CD56의 저밀도 발현(CD56 dim 보다 더 세포독성을 나타냄)을 나타내고 고수준의 Fcγ 수용체 III(FcγRIII, CD16)를 발현하는 반면, 약 10%의 자연살해세포는 CD56^brightCD16^dim 또는 CD56^brightCD16^- 이다(Schattner, A. and Duggan, D. B., Arthritis Rheum., 27, 1072, 1984). 자연살해세포는 바이러스-감염된 세포, 종양 세포 및 일부 정상적인 동종 세포에서 사전 교육 없이 활성화 수용체와 이들의 리간드간의 상호작용을 통한 초기 숙주 방법에 핵심적인 역할을 한다. Natural killer cells are leukocyte lymphocytes involved in the innate immune system that have a variety of biological functions, including specific microbial infections and the recognition and destruction of neoplasms (Moretta, A., Bottino, C., Mingari, MC, Biassoni, R. and Moretta, L., Nat. Immunol., 3, 6, 2002). Their morphological features show large Granular Lymphocyte (LGL) -like morphology based on the presence of densely pink-purple stained granules in the cytoplasm. These include about 10-20% in normal peripheral blood lymphocytes, 15-25% in hepatic lymphocytes and 1-5% in splenic lymphocytes. Dormant killer cells circulate in the blood, but after activation by cytokines they can leak and infiltrate into most tissues containing pathogen-infected or malignant cells (Colucci, F., Di Santo, JP and Leibson, PJ). , Nat. Immunol., 3, 807, 2002; Kelly JM, Darcy PK, Markby JL, Godfrey DI, Takeda K., Yagita H., Smyth MJ, Nat. Immunol., 3, 83, 2002; Shi FD, Wang HB, Li H., Hong S., Taniguchi M., Link H., Van Kaer L., Ljunggren HG, Nat. Immunol., 1, 245, 2000; Korsgren M., Persson CG, Sundler F., Bjerke T , Hansson T., Chambers BJ, Hong S., Van Kaer L., Ljunggren HG, Korsgren O., J. Exp. Med., 189, 553, 1999). In general, phenotypes of natural killer cells are expressed by CD56 and CD16 (human), NKR-P1C (NK1.1 in mice, CD161 in humans), DX5, Ly49 (mouse; these are limited to specific mouse strains) surface antigen and Characterized by the lack of CD3. The majority of human killer cells (90% of total killer cells) exhibit low density expression of CD56 (which is more cytotoxic than CD56 dim) and express high levels of Fcγ receptor III (FcγRIII, CD16), while about 10% Natural killer cells are CD56 ^bright CD16 ^dim or CD56 ^bright CD16 ⁻ (Schattner, A. and Duggan, DB, Arthritis Rheum., 27, 1072, 1984). Natural killer cells play a key role in early host methods through interactions between activating receptors and their ligands, without prior training in virus-infected cells, tumor cells and some normal allogeneic cells.

자연살해세포는 주 조직적합성 복합체(Major Histocompatibility Complex, MHC) 부류 I 분자에 대해 특이적인 자연살해세포 억제 수용체를 통한 인식으로 인해 정상 세포와 주 조직적합성 복합체 부류 I 분자의 발현이 없는 세포를 구별하는 능력을 갖고 있다. 자연살해세포의 기능은 표적 세포에서 주 조직적합성 복합체 부류 I 또는 주 조직적합성 복합체 부류 I-연관된 분자와 같은 리간드와 상호작용하는 활성화 수용체와 억제 수용체간의 균형에 의해 조절된다(Rajaram, N., Tatake, R. J., Advani, S. H. and Gangal, S. G., Br. J. Cancer, 62, 205, 1990). 이들 수용체는 두 개의 구조적 과로 분류된다: 면역글로불린 상위과(백혈구 억제 수용체, 살해세포 Ig-유사 수용체 (KIR, CD158) 및 C-유형 렉틴-유사과(NKG2D, CD94/NKG2, 림프구 항원 49(LY49)). 자연살해세포는 세포-표면 수용체와 이들의 리간드사이의 상호작용 후 인터페론-감마 및 종양괴사인자-알파와 같은 몇 가지 사이토카인을 생성한다(Bryson, J. S. and Flanagan, D. L., J. Hematother. Stem Cell Res,. 9, 307, 2000). Natural killer cells differentiate normal cells from cells without expression of the major histocompatibility complex (MHC) class I molecules by recognition through natural killer cell inhibitory receptors. Have the power. The function of spontaneous killer cells is regulated by the balance between activating and inhibitory receptors that interact with ligands such as major histocompatibility complex class I or major histocompatibility complex class I-associated molecules (Rajaram, N., Tatake , RJ, Advani, SH and Gangal, SG, Br. J. Cancer, 62, 205, 1990). These receptors are classified into two structural families: the immunoglobulin superfamily (leukocyte inhibitory receptor, killer cell Ig-like receptor (KIR, CD158) and C-type lectin-like family (NKG2D, CD94 / NKG2, lymphocyte antigen 49 (LY49)) Natural killer cells produce several cytokines such as interferon-gamma and tumor necrosis factor-alpha after interaction between cell-surface receptors and their ligands (Bryson, JS and Flanagan, DL, J. Hematother. Stem). Cell Res ,. 9, 307, 2000).

자연살해세포의 작용기 기능은 인터루킨-2, 인터루킨-12, 인터루킨-15, 인터루킨-18, 인터루킨-21 및 I형 인터페론-αβ를 포함한 사이토카인과 세포 표면 수용체의 차별적인 개입과 조합하여 자극할 수 있는 반면, 이들은 인터페론-감마, 인터루킨-5, 인터루킨-10, 인터루킨-13, 인터페론-알파 및 과립구-대식세포 콜로니-자극 인자(GM-CSF)와 같은 면역조절 사이토카인뿐만 아니라 자연살해세포 수용체와 이들의 리간드간의 상호작용 후 많은 케모카인(Kemokine)을 생성한다(Shi F. D., Wang H. B., Li H., Hong S., Taniguchi M., Link H., Van Kaer L., Ljunggren H. G., Nat. Immunol., 1, 245, 2000). 사람의 경우, CD56^bright 자연살해세포 하위세트는 또한 인터페론-감마, 종양괴사인자-알파, 종양괴사인자-베타, 인터루킨-10 및 과립구-대식세포 콜로니-자극 인자(Lian R. H., Maeda M., Lohwasser S., Delcommenne M., Nakano T., Vance R. E., Raulet D. H., Takei F., J. Immunol ., 168, 4980, 2002)를 포함한 몇 가지 사이토카인을 생성하지만, CD56^dim 자연살해세포 하위세트는 이들 사이토카인을 생성하지 않는다(Colucci, F., Di Santo, J.P. and Leibson, P.J., Nat. Immunol., 3, 807, 2002; Shi F. D., Wang H. B., Li H., Hong S., Taniguchi M., Link H., Van Kaer L., Ljunggren H. G., Nat. Immunol., 1, 245, 2000). 비록 미성숙 자연살해세포가 인터루킨-5 및 인터루킨-13과 같은 Th2 사이토카인을 생성할 수 있을지라도, Th2 사이토카인을 생성하는 능력은 최종 분화시 상실된다; 대신 성숙자연살해세포는 인터페론-감마를 생성하는 능력을 획득한다(Van Beneden K., Stevenaert F., De Creus A., Debacker V., De Boever J., Plum J., Leclercq G., J . Immunol., 166, 4302, 2001). 자연살해세포는 인터루킨-15( Crosier, K. E. and Crosier, P. S., Pathology, 29, 131, 1997; Nakano, T., Kawamoto, K., Kishino, J., Nomura, K., Higashino, K. and Arita, H., J. Biochem., 323, 387, 1997; Fridell, Y. W., Villa, J., Jr, Attar, E. C. and Liu, E. T., J. Biol. Chem., 273, 7123, 1997) 또는 인터루킨-2(Goruppi, S., Ruaro, E. and Schneider, C., Oncogene, 12, 471, 1996)에 의해 부분적으로 조절되는 XCL1, CCL1, CCL3, CCL4, CCL5, CCL22, and CXCL8(Lundwall, A., Dackowski, W., Cohen, E., Shaffer, M., Mahr, A., Dahlback, B., Stenclrclr, J. and Wydro, R., Proc. Natl Acad. Sci., 83, 6716, 1986)를 포함한 많은 케모카인을 분비하고 반응한다. 이들 케모카인은 이차 림프구 조직에서 감염 및 신생 세포에 대한 자연살해세포 호밍(Homing)에 중요한 역할을 하며 그 조직에서 인터페론-감마의 생성이 T 세포 반응을 직접적으로 조절하는 역할을 할 수 있다(Lundwall, A., Dackowski, W., Cohen, E., Shaffer, M., Mahr, A., Dahlback, B., Stenclrclr, J. and Wydro, R., Proc. Natl Acad. Sci., 83, 6716, 1986). 휴면 CD56^dim/CD16⁺ 자연살해세포 하위세트는 CXCR1, CXCR2, CXCR3 및 CXCR4를 발현하는 한편, CD56^bright/CD16^-자연살해세포는 고수준의 CCR5 및 CCR7을 발현한다. 자연살해세포의 세포용해 활성은 CCL2, CCL3, CCL4, CCL5, CCL10 및 CXC3L1에 의해 자극된다. The functional functions of natural killer cells can be stimulated in combination with the differential intervention of cytokines and cell surface receptors, including interleukin-2, interleukin-12, interleukin-15, interleukin-18, interleukin-21 and type I interferon-αβ. In contrast, they are associated with natural killer cell receptors as well as immunomodulatory cytokines such as interferon-gamma, interleukin-5, interleukin-10, interleukin-13, interferon-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF). After interaction between these ligands, many chemokines are produced (Shi FD, Wang HB, Li H., Hong S., Taniguchi M., Link H., Van Kaer L., Ljunggren HG, Nat. Immunol. , 1, 245, 2000). In humans, the CD56 ^bright NK cell subset also contains interferon-gamma, tumor necrosis factor-alpha, tumor necrosis factor-beta, interleukin-10 and granulocyte-macrophage colony-stimulating factors (Lian RH, Maeda M., Lohwasser. S., Delcommenne M., Nakano T., Vance RE, Raulet DH, Takei F., J. Immunol., 168, 4980, 2002), but a subset of CD56 ^dim killer cells Does not produce these cytokines (Colucci, F., Di Santo, JP and Leibson, PJ, Nat. Immunol., 3, 807, 2002; Shi FD, Wang HB, Li H., Hong S., Taniguchi M. , Link H., Van Kaer L., Ljunggren HG, Nat. Immunol., 1, 245, 2000). Although immature killer cells can produce Th2 cytokines such as interleukin-5 and interleukin-13, the ability to produce Th2 cytokines is lost upon final differentiation; Instead, mature natural killer cells acquire the ability to produce interferon-gamma (Van Beneden K., Stevenaert F., De Creus A., Debacker V., De Boever J., Plum J., Leclercq G., J. Immunol., 166, 4302, 2001). Natural killer cells are interleukin-15 (Crosier, KE and Crosier, PS, Pathology, 29, 131, 1997; Nakano, T., Kawamoto, K., Kishino, J., Nomura, K., Higashino, K. and Arita , H., J. Biochem., 323, 387, 1997; Fridell, YW, Villa, J., Jr, Attar, EC and Liu, ET, J. Biol. Chem., 273, 7123, 1997) or interleukin- XCL1, CCL1, CCL3, CCL4, CCL5, CCL22, and CXCL8 (Lundwall, A., partially regulated by Goruppi, S., Ruaro, E. and Schneider, C., Oncogene, 12, 471, 1996). , Dackowski, W., Cohen, E., Shaffer, M., Mahr, A., Dahlback, B., Stenclrclr, J. and Wydro, R., Proc. Natl Acad. Sci., 83, 6716, 1986) Many chemokines, including, secrete and react. These chemokines play an important role in homing of killer cells against infectious and neoplastic cells in secondary lymphocyte tissues, and the production of interferon-gamma in the tissues can directly regulate T cell responses (Lundwall, A., Dackowski, W., Cohen, E., Shaffer, M., Mahr, A., Dahlback, B., Stenclrclr, J. and Wydro, R., Proc. Natl Acad. Sci., 83, 6716, 1986). The dormant CD56 ^dim / CD16 ⁺ natural killer cell subsets express CXCR1, CXCR2, CXCR3 and CXCR4, while the CD56 ^bright / CD16 ^- natural killer cells express high levels of CCR5 and CCR7. Cytolytic activity of natural killer cells is stimulated by CCL2, CCL3, CCL4, CCL5, CCL10 and CXC3L1.

자연살해세포가 암세포를 죽이는 기전에는 다음의 두 가지가 있다. 세포 수용체를 이용한 방법으로, 자연살해세포는 세포 표면에 세 가지 종류의 종양괴사인자 단백질들을 발현한다. 이는 파스리간드(FASL), 종양괴사인자 그리고 트레일(TRAIL)로 암세포에 있는 이들 수용체와 결합하여 암세포의 아포토시스를 유도하는 것으로 알려져 있다(Ashkenazi, A., Nature Rev. Cancer., 2, 420, 2002). 자연살해세포가 암세포를 죽이는 또 다른 방법은 퍼포린(Perforin)이나 그랜자임(Granzyme)과 같은 세포질성 과립물들을 통한 것이다. 이 물질들은 암세포의 세포막을 뚫어 암세포를 용해시키며 결국에는 암세포를 죽게 만드는 작용을 하게 된 다(Trapani, J. A., Davis, J., Sutton, V. R. and Smyth, M. J., Curr. Opin. Immunol., 12, 323, 2000). There are two mechanisms by which natural killer cells kill cancer cells. By using cell receptors, natural killer cells express three types of tumor necrosis factor proteins on the cell surface. It is known to induce apoptosis of cancer cells by binding to these receptors in cancer cells with parsligand (FASL), tumor necrosis factor and trail (TRAIL) (Ashkenazi, A., Nature Rev. Cancer., 2, 420, 2002 ). Another way natural killer cells kill cancer cells is through cellular granules such as Perforin and Granzyme. These substances penetrate the cell membranes of cancer cells to dissolve the cancer cells and eventually cause them to die (Trapani, JA, Davis, J., Sutton, VR and Smyth, MJ, Curr. Opin. Immunol., 12, 323, 2000).

자연살해세포가 만능 조혈모세포(혈액세포와 면역세포를 구성하는 근원이 되는 세포임)로부터 유래한다는 것은 잘 알려져 있지만 아직 그것의 발생과정에 대해서는 잘 알려져 있지 않다(Lian R. H., Maeda M., Lohwasser S., Delcommenne M., Nakano T., Vance R. E., Raulet D. H., Takei F., J. Immunol., 168, 4980, 2002). 조혈모세포는(사람에서는 Lin^-CD34⁺, 마우스에서는 Lin^-c-kit⁺Sca2⁺ ) 태아의 흉선, 태아의 간, 제대혈 그리고 성체 골수로부터 유도될 수 있고 이들은 T 세포나 자연살해세포로 분화할 수 있는 전구물질로(Lian R. H., Kumar V., Semin. Immunol., 14, 453, 2002; Douagi I., Colucci F., Di Santo JP., Cumano A., Blood, 99, 473, 2002) 시험관 수준에서 인터루킨-7, SCF, 그리고 flt3L과 반응하여 배양하면 전구자연살해세포로 분화할 수 있는 능력을 가진다고 알려져 있다(Williams N. S., Klem J., Puzanov I. J., Sivakumar P. V., Bennett M., Kumar V., J. Immunol,. 163, 2648, 1999). 여기서 분화는 일반적으로 비교적 단순한 계(系)가 둘 이상의 질적으로 다른 부분계(部分系)로 분리되는 현상이다. 즉, 구조나 기능이 특수화하는 현상을 말한다. 전구자연살해세포는 조혈모세포에서 성숙자연살해세포의 중간단계의 세포로 골수, 태아의 흉선, 혈액, 비장 그리고 간 등에 고루 퍼져 있다. 하지만 이들은 인터페론-감마를 생산하지 못하기 때문에 세포용해능력은 없다. 본 발명에서는 조혈모세포에 대한 마커를 c-kit⁺Lin^-(마우스), CD34⁺(사람)로 정의하였다.It is well known that natural killer cells are derived from pluripotent hematopoietic stem cells (the cells that constitute blood and immune cells), but are not yet well known about its development (Lian RH, Maeda M., Lohwasser S). , Delcommenne M., Nakano T., Vance RE, Raulet DH, Takei F., J. Immunol., 168, 4980, 2002). Hematopoietic stem cells (the human Lin ^- CD34 ^+, Mouse ^{^{^{Lin - c-kit + Sca2 +}}} ) can be derived from the fetal thymus, fetal liver, umbilical cord blood and adult bone marrow, and they can differentiate into T cells or natural killer cells In vitro (Lian RH, Kumar V., Semin. Immunol., 14, 453, 2002; Douagi I., Colucci F., Di Santo JP., Cumano A., Blood, 99, 473, 2002) Incubated with interleukin-7, SCF, and flt3L have been known to have the ability to differentiate into pro-NK cells (Williams NS, Klem J., Puzanov IJ, Sivakumar PV, Bennett M., Kumar V., J. Immunol ,. 163, 2648, 1999). Differentiation is generally a phenomenon in which a relatively simple system is separated into two or more qualitatively different subsystems. In other words, it refers to a phenomenon in which a structure or a function is specialized. Progenitor killer cells are intermediate cells of hematopoietic stem cells to mature killer cells that are spread throughout the bone marrow, fetal thymus, blood, spleen and liver. However, they do not produce interferon-gamma and thus have no cytolytic capacity. In the present invention the markers c-kit ⁺ Lin hematopoietic stem cells for the ^(mouse), CD34 ⁺ (person).

전구자연살해세포(사람에서는 CD56^-CD122⁺CD34⁺, 마우스에서는 CD122⁺ NK1.1^-DX5^-)는 인터루킨-15를 처리하여 배양시키면 미성숙자연살해세포(사람에서는 CD122⁺CD16l^-CD56^-KIR^-, 마우스에서는 CD122⁺CD2⁺NK1.1⁺DX5⁺Ly49^-)로 분화할 수 있다 . 전구자연살해세포를 인터루킨-15가 포함된 배지에서 지속적으로 배양을 하게 되면 조숙한(pseudomature) 세포용해성 자연살해(CD122⁺CD2⁺NK1.1⁺DX5⁺CD94/NKG2⁺Ly49^-) 세포로 분화할 수 있지만 이들의 세포용해능력은 거의 없는 것으로 알려져 있다. 기질세포는 여러 종류의 사이토카인과 발달 과정 중의 자연살해세포 표면 수용체와 직접적으로 반응할 수 있는 물질들을 분비하는 세포이다. 기질세포가 분비하는 이러한 물질들은 성숙자연살해세포의 성숙과정에 필수적인 요소로 성숙자연살해세포에 LyS49⁺가 발현되도록 해주는 기능을 가지는 것으로 보고되어 있다(Iizuka K., Chaplin D. D., Wang Y., Wu Q., Pegg L. E., Yokoyama W. M., Fu Y. X., Proc. Natl. Acad. Sci., 96, 6336, 1999; Briard D., Brouty-Boye D., Azzarone B., Jasmin C., J. Immunol., 168, 4326, 2002). 세포용해능을 가지는 Ly49⁺ 성숙자연살해세포(사람에서는 CD56⁺KIR⁺CD3^-, 마우스에서는 CD122⁺CD2⁺NK1.1⁺DX5⁺CD94/NKG2⁺Ly49⁺CD3^-)는 인터루킨-15 존재 하에서 전구자연살해세포와 기질세포의 공동배양을 통해 분화시킬 수 있다(Williams N. S., Klem J., Puzanov I. J., Sivakumar P. V., Bennett M., Kumar V., J. Immunol,. 163, 2648, 1999). 자연살해세포의 발달 단계 중 초기 발달 단계에는 CD94, NKG2A, NKG2C 와 Ly49B 가 발현되고 그 다음 단계에서는 Ly49G, Ly49C와 Ly49I가 발현되고 마지막 단계에서는 Ly49A, D, E와 F가 발현된다(Williams N. S., Kubota A., Bennett M., Kumar V., Takei F., Eur. J. Immunol., 30, 2074, 2000). 본 발명에서는 전구자연살해세포에 대한 마커를 CD122⁺ NK1.1^-(마우스)로 정의하였다.Progenitor natural killer cells (CD56 ^- CD122 ⁺ CD34 ^{+ in} humans, CD122 ⁺ NK1.1 ^- DX5 ^-in mice) were treated with interleukin-15 and cultured with immature killer cells (CD122 ⁺ CD16l ^- CD56 ^- KIR ^-in humans). In mice, CD122 ⁺ CD2 ⁺ NK1.1 ⁺ DX5 ⁺ Ly49 ⁻ ) can be differentiated. When the bulb continuously cultured NK cells in a medium containing IL-15 premature (pseudomature) soluble natural killer cells ^{^{^{(CD122 + CD2 + NK1.1 + DX5}}} + CD94 / NKG2 + Ly49 -) to differentiate into cells It is known that they have little cytolytic ability. Stromal cells are cells that secrete many types of cytokines and substances that can react directly with natural killer cell surface receptors during development. These substances secreted by stromal cells have been reported to have the function of expressing LyS49 ⁺ in mature natural killer cells as an essential factor in the maturation process of mature natural killer cells (Iizuka K., Chaplin DD, Wang Y., Wu). Q., Pegg LE, Yokoyama WM, Fu YX, Proc. Natl. Acad. Sci., 96, 6336, 1999; Briard D., Brouty-Boye D., Azzarone B., Jasmin C., J. Immunol., 168, 4326, 2002). Ly49 ⁺ mature natural killer cells (CD56 ⁺ KIR ⁺ CD3 ^{− in} humans, CD122 ⁺ CD2 ⁺ NK1.1 ⁺ DX5 ⁺ CD94 / NKG2 ⁺ Ly49 ⁺ CD3 ^{− in} cells) are pro-natural in the presence of interleukin-15 Differentiation is possible through coculture of killer and stromal cells (Williams NS, Klem J., Puzanov IJ, Sivakumar PV, Bennett M., Kumar V., J. Immunol ,. 163, 2648, 1999). CD94, NKG2A, NKG2C and Ly49B are expressed in the early stages of development of natural killer cells, Ly49G, Ly49C and Ly49I are expressed in the next stage, and Ly49A, D, E and F are expressed in the final stage (Williams NS, Kubota A., Bennett M., Kumar V., Takei F., Eur.J. Immunol., 30, 2074, 2000). In the present invention, a marker for precursor NK cells, CD122 ⁺ NK1.1 ^- was defined as (mouse).

전구자연살해세포는 PU.1, GATA3, Id2 그리고 Ets-1과 같은 전사 인자들을 발현한다(Boggs S. S., Trevisan M., Patrene K., Geogopoulos K., Nat. Immunol., 16, 137, 1998). 전사인자 ikaros, PU.1 (Colucci F., Samson S. I., DeKoter R. P., Lantz O., Singh H., Di Santo J. P., Blood, 97, 2625, 2001)과 Id2(Ikawa T., Fujimoto S., Kawamoto H., Katsura Y., Yokota Y., Proc. Natl. Acad. Sci,. 98, 5164, 2001)를 결핍시킨 마우스에서 전구자연살해세포 수의 감소가 관찰 된다. 조혈모세포는 myb 종양유전자(oncogene), c-myc 그리고 Oct 2b와 같은 전사 인자를 발현한다. 이들 인자들은 전구자연살해세포의 증식과 발달에 중요한 역할을 한다(Bar-Ner M., Messing L. T., Segal S., Immunobiology, 185, 150, 1992; Melotti P., Calabretta B., Blood, 87, 2221, 1996). 전구자연살해세포에서 Fc 수용체, 종양괴사인자 수용체, 인터루킨-7 수용체와 케모카인 수용체 그리고 CD36 와 같은 면역 조절제들은 전구자연살해세포 단계에서 중요한 역할을 하는 것 으로 알려져있다. Progenitor killer cells express transcription factors such as PU.1, GATA3, Id2 and Ets-1 (Boggs SS, Trevisan M., Patrene K., Geogopoulos K., Nat. Immunol., 16, 137, 1998) . Transcription factors ikaros, PU.1 (Colucci F., Samson SI, DeKoter RP, Lantz O., Singh H., Di Santo JP, Blood, 97, 2625, 2001) and Id2 (Ikawa T., Fujimoto S., Kawamoto H., Katsura Y., Yokota Y., Proc. Natl. Acad. Sci ,. 98, 5164, 2001) showed a decrease in the number of progenitor killer cells. Hematopoietic stem cells express transcription factors such as myb oncogene, c-myc and Oct 2b. These factors play an important role in the proliferation and development of progenitor killer cells (Bar-Ner M., Messing LT, Segal S., Immunobiology, 185, 150, 1992; Melotti P., Calabretta B., Blood, 87, 2221, 1996). In progenitor killer cells, Fc receptors, tumor necrosis factor receptors, interleukin-7 and chemokine receptors, and immunomodulators such as CD36 are known to play an important role in progenitor cell killing.

성숙자연살해세포에서 RGS(G 단백질 시그날 조절자, regulator of G protein signaling), 림프구 특이 단백질 티로신 키나아제와 Fyn 원종양유전자와 같은 시그날 전달 분자들은 자연살해세포의 성숙에 관여하는 물질들로 알려져 있다(Ikawa T., Fujimoto S., Kawamoto H., Katsura Y., Yokota Y., Proc. Natl. Acad. Sci,. 98, 5164, 2001; Ogasawara K., Hida S., Azimi N., Tagaya Y., Sato T., Yokochi-Fukuda T., Waldmann T. A., Taniguchi T., Taki S., Nature, 391, 700, 1998). 성숙자연살해세포는 암세포를 인지하고 직접 암세포를 죽일 수 있는 능력을 가진 세포를 말한다. 성숙자연살해세포에 인터루킨-2의 처리는 성숙자연살해세포의 증식과 활성을 유도하는 것으로 알려져 있다. 수용체 티로신 키나아제는 세포의 증식, 세포의 발달, 세포의 생존과 세포의 이동에 대한 세포 외부에서의 자극을 세포 안으로 시그날 전달하는 막을 통해서 생기는(transmembrane) 단백질로 구성된다(Ullrich, A., Schlessinger, J., Cell, 61, 203, 1990; Fantl, W. J., Johnson, D. E., Williams, L. T., Biochem., 62, 453, 1993; Heldin, C., Cell, 80, 213, 1995). 모든 수용체 티로신 키나아제는 공통적으로 세포질 키나아제 도메인을 가지고 있는 데 여기에 성장 인자가 결합을 하게 되면 수용체 티로신 키나아제는 활성화가 된다. 본 발명에서는 성숙자연살해세포에 대한 마커를 CD122⁺NK1.1⁺(마우스),CD34⁺(사람)로 정의하였고 성숙자연살해세포 중에서도 완전히 성숙한 세포들에 대한 마커로는 Family Ly49A⁺, Ly49C⁺, Ly49D⁺, Ly49E⁺, Ly49F⁺, Ly49G⁺, Ly49H⁺, Ly49I⁺, NKG2A/C/E⁺(마우스), CD56⁺, NKG2A⁺,CD161⁺, NKP46⁺,NKP30⁺,NKP44⁺, NKG20⁺(사람)로 정의하였다.In mature killer cells, signal transduction molecules such as GGS (regulator of G protein signaling), lymphocyte-specific protein tyrosine kinase and Fyn oncogenes are known to be involved in the maturation of NK cells. Ikawa T., Fujimoto S., Kawamoto H., Katsura Y., Yokota Y., Proc. Natl. Acad. Sci ,. 98, 5164, 2001; Ogasawara K., Hida S., Azimi N., Tagaya Y. , Sato T., Yokochi-Fukuda T., Waldmann TA, Taniguchi T., Taki S., Nature, 391, 700, 1998). Mature natural killer cells are cells that have the ability to recognize cancer cells and directly kill them. The treatment of interleukin-2 in mature natural killer cells is known to induce proliferation and activity of mature natural killer cells. Receptor tyrosine kinases are composed of transmembrane proteins that signal extracellular stimuli into cells in response to cell proliferation, cell development, cell survival, and cell migration (Ullrich, A., Schlessinger, J., Cell, 61, 203, 1990; Fantl, WJ, Johnson, DE, Williams, LT, Biochem., 62, 453, 1993; Heldin, C., Cell, 80, 213, 1995). All receptor tyrosine kinases have a common cytoplasmic kinase domain. When growth factors bind to them, receptor tyrosine kinases are activated. In the present invention, the marker for mature natural killer cells is CD122 ⁺ NK1.1 ⁺ (mouse), CD34 ⁺ as a marker for the (person) was defined as mature fully mature cells among NK cells ^{^{Family Ly49A +, Ly49C +, Ly49D}} +, Ly49E +, Ly49F +, Ly49G +, Ly49H +, Ly49I +, NKG2A / C / E ⁺ (mouse), CD56 ⁺ , NKG2A ⁺ , CD161 ⁺ , NKP46 ⁺ , NKP30 ⁺ , NKP44 ⁺ , NKG20 ⁺ (person).

본 발명에서 사용된 재조합 DNA 방법은 일반적으로 문헌[Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)] 및/또는 [Ausubel et al., eds., Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, N.Y. (1994)]에 기술된 것이다. 예를 들면, 아래 예시적으로 기술된 바와 같이 재조합 발현 기술을 통해 목적하는 단백질을 암호화한 핵산 서열을 적절한 벡터(Vector) 내로 삽입시킴으로써 목적하는 뉴클레오타이드 서열을 다량 생성할 수 있다. 그런 다음, 그 서열을 사용하여 검출 탐침 또는 증폭 프라이머를 생성할 수 있다. 또는, 단백질을 암호화한 폴리뉴클레오타이드를 발현벡터내로 삽입하고, 생성된 발현 벡터를 적절한 숙주내로 도입하여 배양함으로써 단백질을 다량으로 생성할 수 있다. 핵산 서열을 수득하는 다른 방법으로는 PCR(중합효소 연쇄 반응)이다. 역전사효소를 사용하여 폴리(A)+RNA 또는 전체 RNA로부터 cDNA를 준비한다. 전형적으로 폴리펩타이드의 아미노산 서열을 암호화한 cDNA의 두 개 개별 영역에 상보적인 두 프라이머를 Taq 중합효소와 같은 중합효소와 함께 cDNA에 첨가하고 중합효소가 두 프라이머 사이의 cDNA 영역을 증폭한다. 폴리펩타이드의 아미노산 서열을 암호화하는 핵산 분자는 원핵, 효모, 곤충(baculovirus systems) 및/또는 진핵 숙주 세포에서 증폭/발현시킬 수 있다(Meth. Enz. vol. 185 D. V. Goeddel ed., Academic Press, Sna Diego Calif., 1990). 전형적으로 발현벡터는 플라스미드 유지 및 외래 뉴클레오타이드 서열의 클로닝 및 발현을 위한 서열을 함유한다. 이러한 서열로는 프로모터, 인핸서, 복제원, 전사종결서열, 분비리더서열, 리보좀 결합 부위, 폴리아데닐화 서열, 발현될 폴리펩타이드의 암호화 핵산을 삽입하기 위한 폴리링커 및 선별 마커 등이 포함된다. 이러한 플랭킹 서열은 본 분야의 전문가에게는 잘 알려져 있으며 용이하게 선별적으로 사용할 수 있다.Recombinant DNA methods used in the present invention are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and / or Ausubel et al., Eds., Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, N.Y. (1994). For example, a large amount of the desired nucleotide sequence can be generated by inserting a nucleic acid sequence encoding the protein of interest into an appropriate vector through recombinant expression techniques as exemplarily described below. The sequence can then be used to generate detection probes or amplification primers. Alternatively, a large amount of protein can be produced by inserting a polynucleotide encoding a protein into an expression vector and introducing the resulting expression vector into an appropriate host and culturing. Another method of obtaining nucleic acid sequences is PCR (polymerase chain reaction). Reverse transcriptase is used to prepare cDNA from poly (A) + RNA or total RNA. Typically two primers complementary to two separate regions of the cDNA encoding the amino acid sequence of the polypeptide are added to the cDNA with a polymerase such as Taq polymerase and the polymerase amplifies the cDNA region between the two primers. Nucleic acid molecules encoding amino acid sequences of polypeptides can be amplified / expressed in prokaryotic, yeast, baculovirus systems and / or eukaryotic host cells (Meth. Enz. Vol. 185 DV Goeddel ed., Academic Press, Sna Diego Calif., 1990). Typically expression vectors contain sequences for plasmid maintenance and cloning and expression of foreign nucleotide sequences. Such sequences include promoters, enhancers, replicators, transcription termination sequences, secretory leader sequences, ribosomal binding sites, polyadenylation sequences, polylinkers for inserting the coding nucleic acid of the polypeptide to be expressed, and selection markers. Such flanking sequences are well known to those skilled in the art and can be readily used selectively.

포유동물 세포에서 단백질 코딩 DNA의 전사를 위해 적절한 프로모터의 예로는 SV40 프로모터(Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), MT-1(metallothionein gene) promoter(Palmiter et al., Science 222 (1983), 809-814), CMV 프로모터(Boshart et al., Cell 41:521-530, 1985) 또는 아데노바이러스 2 메이저 후기 프로모터(Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982)이다. 곤충 세포에서 적합하게 사용되는 프로모터의 예로는 폴리헤드린 프로모터 (미국특허 제4,745,051호; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), P10 프로모터 (J. M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), 백큘로바이러스 immediate early 유전자 1 프로모터(미국특허 제5,155,037호 및 제5,162,222호) 또는 백큘로바이러스 39K delayed-early 유전자 프로모터(미국특허 제5,155,037호 및 제5,162,222호)를 들 수 있다. 효모 숙주 세포에 적합한 프로모터로는 예를 들면 효모 글라이코라이시스 유전자 (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419-434) 또는 알코올 데하이드로게나제 유전자 (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982) 또는 TPI1(미국특허 제4,599,311호) 또는 ADH2-4c (Russell et al., Nature 304 (1983), 652-654) 프로모터가 포함된다. 진균 숙주 세포에 적합한 프로모터로는 ADH3 프로모터(McKnight et al., The EMBO J. 4 (1985), 2093-2099) 또는 tpiA 프로모터를 예로 들 수 있다. 다른 적절한 프로모터의 예로는 A. oryzae TAKA 아밀라제, Rhizomucor miehei 아스파틱 프로테이나제, A. niger 중성 알파-아밀라제, A. niger 산 안정한 알파-아밀라제, A. niger 또는 A. awamori 글루코아밀라제 (gluA), Rhizomucor miehei 리파제, A. oryzae 알카리성 프로테아제, A. oryzae 트리오즈 포스페이트 이소머라제 또는 A. nidulans 아세트아미다제 코딩 유전자로부터 유래 된 것이다.Examples of suitable promoters for transcription of protein encoding DNA in mammalian cells include the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), metallothionein gene (MT-1) promoter (Palmiter et). al., Science 222 (1983), 809-814), CMV promoter (Boshart et al., Cell 41: 521-530, 1985) or adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2 1304-1319, 1982). Examples of promoters suitably used in insect cells include polyhedrin promoters (US Pat. No. 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), P10 promoters (JM Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the baculovirus immediate early gene 1 promoter (US Pat. Nos. 5,155,037 and 5,162,222) or the baculovirus 39K delayed-early gene promoter (US Pat. No. 5,155,037 and 5,162,222). Suitable promoters for yeast host cells include, for example, the yeast glycolysis gene (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase gene (Young et al., In Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982) or TPI1 (US Patent 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654) promoter. Suitable promoters for fungal host cells include the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter. Examples of other suitable promoters include A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or A. awamori glucoamylase (gluA) , Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase coding gene.

DNA 서열은 필요한 경우 사람 성장 호르몬 터미네이터(Palmiter et al., Science 222, 1983, pp. 809-814) 또는 TPI1(Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) 또는 ADH3(McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099) 터미네이터에 작동적으로 연결될 수 있다. 발현 벡터는 또한 프로모터로부터 하류와 DNA 서열의 삽입 부위로부터 상류에 한 세트의 RNA 스플라이스 부위를 함유할 수 있다. 바람직한 RNA 스플라이스 부위는 아데노바이러스 및/또는 면역글로불린 유전자로부터 수득할 수 있다. 또한 발현 벡터에는 삽입 부위의 하류에 폴리아데닐화 시그날이 포함된다. 특히 바람직한 폴리아데닐화 시그날은 SV40으로부터 초기 또는 후기 폴리아데닐화 시그날, 아데노바이러스 5 Elb 영역으로부터 폴리아데닐화 시그날 또는 사람 성장 호르몬 유전자 터미네이터(DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981)이다. 발현 벡터는 또한 아데노바이러스 2 tripartite 리더와 같은 비암호 바이러스 리더 서열을 프로모터와 RNA 스플라이스 부위사이에 포함하고, SV40 인핸서와 같은 인핸서 서열을 함유할 수 있다.The DNA sequence can be used if necessary in human growth hormone terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or TPI1 (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434 ) Or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099) terminator. The expression vector may also contain a set of RNA splice sites downstream from the promoter and upstream from the insertion site of the DNA sequence. Preferred RNA splice sites can be obtained from adenovirus and / or immunoglobulin genes. The expression vector also includes a polyadenylation signal downstream of the insertion site. Particularly preferred polyadenylation signals are early or late polyadenylation signals from SV40, polyadenylation signals from adenovirus 5 Elb regions or human growth hormone gene terminators (DeNoto et al. Nucl. Acids Res. 9: 3719-3730, 1981 )to be. The expression vector may also contain a noncoding viral leader sequence, such as an adenovirus 2 tripartite leader, between the promoter and the RNA splice site and may contain an enhancer sequence, such as an SV40 enhancer.

본 발명에 따른 단백질을 숙주 세포의 분비 경로로 유도하기 위해, 분비 시그날 서열(리더 서열, prepro 서열 또는 pre 서열로도 알려져 있다)이 재조합 벡터에 제공될 수 있다. 분비 시그날 서열은 펩타이드 암호화 DNA 서열에 정확한 리딩 프레임으로 결합된다. 분비 시그날 서열은 흔히 펩타이드 암호화 서열의 5’에 위치한다. 분비 시그날 서열은 펩타이드와 정상적으로 결합 되거나 다른 분비 서열의 코딩 유전자로부터 유래 될 수 있다. 효모 세포로부터 분비되도록 하기 위해, 분비 시그날 서열은 세포의 분비 경로로 발현 폴리펩타이드의 충분한 유도를 보장하는 것은 어떠한 시그날 펩타이드도 코딩할 수 있다. 시그날 펩타이드는 천연 시그날 펩타이드 또는 이의 기능상 일부이거나 합성 펩타이드일 수 있다. 적합한 시그날 펩타이드는 알파-인자 시그날 펩타이드(미국특허 제4,870,008호), 마우스 타액 아밀라제(O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), 변형된 카르복시펩티다제 시그날 펩타이드(L. A. Valls et al., Cell 48, 1987, pp. 887-897), 효모 BAR1 신호 펩타이드(국제특허 WO 87/02670) 또는 효모 아스파트산 프로테아제 3(YAP3) 시그날 펩타이드(M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137)이다. 효모에서 충분한 분비를 위해, 리더 펩타이드 코딩 서열이 시그날 서열의 하류와 암호화 DNA 서열의 상류에 삽입될 수 있다. 리더 펩타이드의 기능은 배양 배지 로의 분비를 위해 발현된 펩타이드가 소포체(endoplasmic reticulum)로부터 골지체 및 분비낭으로 이동하도록 한다(즉, 세포벽을 따라 또는 세포막을 지나 세포질 주변으로 단백질의 분비). 리더 펩타이드는 효모 알파-인자 리더(미국특허 제4,546,082호, 미국특허 제4,870,008호, EP 123 294, EP 123 544 및 EP 163 529)일 수 있다. 대안으로, 리더 펩타이드는 합성 리더 펩타이드일 수 있으며, 예를 들면 국제특허 제WO89/02463호 또는 W92/11378호에 기술된 바와 같이 작제할 수 있다. 진균에서 사용하기 위한 시그날 펩타이드는 편리하게는 아스퍼질러스 종 아밀라제 또는 글루코아밀라제 코딩 유전자, Rhizomucor miehei 리파제 또는 프로테아제 또는 Humicola lanuginosa 리파제 코딩 유전자로부터 유래 될 수 있다. 곤충에서 사용하기 위한 시그날 펩타이드는 편리하게는 리피돕테란 Manduca sexta adipokinetic 호르몬 전구 시그날 펩타이드(미국특허 제5,023,328호)와 같은 곤충 유전자(국제 특허 제WO90/05783)로부터 유래 될 수 있다. In order to direct the proteins according to the invention into the secretory pathway of the host cell, secretory signal sequences (also known as leader sequences, prepro sequences or pre sequences) can be provided to the recombinant vector. The secretory signal sequence is bound to the peptide encoding DNA sequence in the correct reading frame. Secretory signal sequences are often located 5 'of the peptide coding sequence. Secretory signal sequences can be normally associated with the peptide or derived from coding genes of other secretory sequences. In order to be secreted from yeast cells, the secretory signal sequence can encode any signal peptide that ensures sufficient induction of the expression polypeptide into the cell's secretory pathway. The signal peptide may be a natural signal peptide or a functional part thereof or a synthetic peptide. Suitable signal peptides include alpha-factor signal peptide (US Pat. No. 4,870,008), mouse saliva amylase (O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), modified carboxypeptidase signal peptide (LA) Valls et al., Cell 48, 1987, pp. 887-897), yeast BAR1 signal peptide (WO 87/02670) or yeast aspartic acid protease 3 (YAP3) signal peptide (M. Egel-Mitani et al. , Yeast 6, 1990, pp. 127-137. For sufficient secretion in yeast, leader peptide coding sequences can be inserted downstream of the signal sequence and upstream of the coding DNA sequence. The function of the leader peptide allows the expressed peptide to migrate from the endoplasmic reticulum to the Golgi apparatus and secretory glands (ie, secretion of proteins along the cell wall or across the cell membrane around the cytoplasm) for secretion into the culture medium. The leader peptide may be a yeast alpha-factor leader (US Pat. No. 4,546,082, US Pat. No. 4,870,008, EP 123 294, EP 123 544 and EP 163 529). Alternatively, the leader peptide may be a synthetic leader peptide, and may be constructed as described, for example, in WO 89/02463 or W92 / 11378. Signal peptides for use in fungi may conveniently be derived from Aspergillus sp. Amylase or glucoamylase coding gene, Rhizomucor miehei lipase or protease or Humicola lanuginosa lipase coding gene. Signal peptides for use in insects may conveniently be derived from insect genes (International Patent No. WO90 / 05783), such as Lipidopteran Manduca sexta adipokinetic hormone precursor signal peptide (US Pat. No. 5,023,328).

포유동물에 DNA 서열을 형질감염 및 발현시키는 방법은 예를 들면 Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845에 기술되어 있다. 클로닝된 DNA 서열은 배양된 포유동물 세포내로 예를 들면 칼슘 포스페이트-매개된 형질감염(Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) 또는 전기천공(Neumann et al., EMBO J. 1:841-845, 1982)에 의해 도입된다. 외래 DNA를 발현하는 세포를 동정 및 선별하기 위해 선별성 표현형(선별 마커)를 제공하는 유전자는 일반적으로 해당 유전자 또는 cDNA와 함께 세포내로 도입된다. 바람직한 선별 마커는 네오마이신, 하이그로마이신 및 메토트렉세이트와 같은 약물에 대한 내성을 제공하는 유전자를 포함한다. 선별 마커는 증폭가능한 선별 마커일 수 있다. 바람직한 증폭성 선별 마커는 디하이드로폴레이트 리덕타제(DHFR) 서열이다. 선별 마커는 별도의 플라스미드로 해당 유전자와 동시에 세포내로 도입되거나 동일한 플라스미드로 도입될 수 있다. 동일한 플라스미드인 경우, 선별 마커와 해당 유전자는 다른 프로모터 또는 동일한 프로모터의 조절하에 있을 수 있는데, 후자 배열은 dicistronic 메시지를 생성한다. 이러한 유형의 작제물은 공지되어 있다 (미국특허 제4,713,339호). 또한, 전달체 DNA로 알려진 추가의 DNA를 세포내로 도입되는 혼합물에 추가하는 것이 유리할 수 있다.Methods for transfecting and expressing DNA sequences in mammals are described, for example, in Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845. The cloned DNA sequence is for example calcium phosphate-mediated transfection into cultured mammalian cells (Wigler et al., Cell 14: 725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603-616, 1981 Graham and Van der Eb, Virology 52d: 456-467, 1973) or electroporation (Neumann et al., EMBO J. 1: 841-845, 1982). Genes that provide a selective phenotype (selection marker) for identifying and selecting cells expressing foreign DNA are generally introduced into the cell along with the gene or cDNA. Preferred selectable markers include genes that provide resistance to drugs such as neomycin, hygromycin and methotrexate. The selectable marker may be an amplifiable selectable marker. Preferred amplifiable selectable markers are dihydrofolate reductase (DHFR) sequences. The selection marker may be introduced into the cell simultaneously with the gene of interest as a separate plasmid or into the same plasmid. In the same plasmid, the selection marker and the gene of interest may be under the control of another promoter or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known (US Pat. No. 4,713,339). It may also be advantageous to add additional DNA, known as carrier DNA, to the mixture that is introduced into the cell.

세포가 DNA를 흡수한 후 적절한 성장 배지에서 전형적으로 1-2일간 성장시켜 해당 유전자의 발현을 개시한다. 용어 적절한 성장 배지는 세포의 성장 및 해당 단백질의 발현에 필요한 영양소 및 기타 성분을 함유한 배지를 의미한다. 일반적으로 배지는 탄소원, 질소원, 필수아미노산, 필수 당, 비타민, 염, 인지질, 단백질 및 성장인자를 포함한다. 감마-카르복실화 단백질의 생성을 위해, 배지는 비타민 K, 바람직하게는 약 0.1 mug/ml 내지 약 5 mug/ml의 농도로 함유한다. 그런 다음, 약물 선별을 적용하여 선별 마커를 안정한 양상으로 발현하는 세포의 성장을 선별한 다. 증폭성 선별 마커로 형질감염된 세포의 경우, 약물 농도를 증가시켜 클로닝 된 서열의 증가 된 복제수를 선별할 수 있고, 그럼으로써 발현 수준을 증가시킨다. 이어서, 안정하게 형질감염된 세포의 클론을 해당 단백질의 발현에 대해 스크리닝한다.After the cells take up the DNA, they typically grow for 1-2 days in the appropriate growth medium to initiate expression of that gene. The term appropriate growth medium means a medium containing nutrients and other components necessary for the growth of cells and the expression of the protein of interest. In general, the medium includes a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, proteins and growth factors. For the production of gamma-carboxylated proteins, the medium contains vitamin K, preferably at a concentration of about 0.1 mug / ml to about 5 mug / ml. Drug screening is then applied to select the growth of cells expressing the selectable marker in a stable manner. For cells transfected with amplifiable selectable markers, the drug concentration can be increased to select for an increased copy number of the cloned sequence, thereby increasing the expression level. Clones of stably transfected cells are then screened for expression of the protein of interest.

해당 펩타이드의 코딩 DNA 서열이 도입되는 숙주 세포는 전사 후 변형 펩타이드를 생성할 수 세포면 어떠한 것이든 가능할 수 있으며, 효모, 진균 및 고등 진핵 세포가 포함된다. 본 발명에 사용되는 포유동물 세포주의 예로는 COS-1(ATCC CRL 1650), 베이비 햄스터 신장(BHK) 및 293(ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) 세포주를 들 수 있다. 바람직한 BHK 세포주는 tk-ts13 BHK 세포주(ATCC CRL 10314)이다(Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982). 또한 다른 세포주가 사용될 수 있는데 랫트 Hep I(랫트 간종양; ATCC CRL 1600), 랫트 Hep II(랫트 간종양; ATCC CRL 1548), TCMK(ATCC CCL 139), 사람 폐(ATCC HB 8065), NCTC 1469(ATCC CCL 9.1), CHO(ATCC CCL 61) and DUKX 세포(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980)가 포함된다. 적합한 효모의 예로는 사카로마이세스 종 또는 쉬조사카로마이세스 종의 세포, 특히 사카로마이세프 세레비지애 또는 사카로마이세스 클루이베리가 포함된다. 효모 세포를 이종 DNA로 형질전환시키고 그로부터 이종 폴리펩타이드를 생성하는 방법은 미국특허 제4,599,311, 제4,870,008, 제5,037,743 및 제4,845,075호에 기술되어 있다. 형질전환 세포는 선별 마커, 통상적으로 약물 내성 또는 특정 영양소, 예를 들면 루이신의 부재하에서 성장하는 능력에 의해 결정된 표현형에 의해 선별된다. 효모에 바람직한 벡터는 미국특허 제4,931,373호에 기술된 POT1 벡터이다. 다른 사상형 진균 세포의 예로는 아스퍼질러스 종, 뉴로스포라 종, Fusarium 종 또는 Trichoderma 종, 특히 A. oryzae, A. nidulans 또는 A. niger의 스트레인이다. 아스퍼질러스 종을 단백질 발현을 위해 사용한 것이 예를 들면 EP 272 277 및 EP 238 023, EP 184 438에 기술되어 있다. F. oxysporum의 형질전환은 예를 들면 문헌 Malardier et al., 1989, Gene 78: 147-156에 기술된 바와 같이 실시할 수 있다. Trichoderma 종의 형질전환은 EP 244 234에 기술된 바와 같이 실시할 수 있다. 사상형 진균이 숙주 세포로 사용될 때 편리하게는 DNA 작제물을 숙주 염색체에 통합시켜 재조합 숙주 세포를 수득할 수 있다. 이러한 통합은 일반적으로 DNA 서열이 세포에 안정하게 유지되기 때문에 장점으로 간주 된다. 숙주 염색체내로 DNA 작제물의 통합은 통상적인 방법, 예를 들면 동종 또는 이종 재The host cell into which the coding DNA sequence of the peptide is introduced may be any cell capable of producing post-transcriptional peptides, including yeast, fungal and higher eukaryotic cells. Examples of mammalian cell lines used in the present invention include COS-1 (ATCC CRL 1650), Baby Hamster Kidney (BHK) and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977) cell lines. Preferred BHK cell lines are tk-ts13 BHK cell lines (ATCC CRL 10314) (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982). Other cell lines may also be used: rat Hep I (rat liver tumor; ATCC CRL 1600), rat Hep II (rat liver tumor; ATCC CRL 1548), TCMK (ATCC CCL 139), human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220, 1980). Examples of suitable yeasts include cells of Saccharomyces sp. Or Schizocaromyces spp., In particular Saccharomyces cerevisiae or Saccharomyces cluiberg. Methods for transforming yeast cells with heterologous DNA and generating heterologous polypeptides therefrom are described in US Pat. Nos. 4,599,311, 4,870,008, 5,037,743 and 4,845,075. Transformed cells are selected by a phenotype determined by a selection marker, typically drug resistance or the ability to grow in the absence of certain nutrients such as leucine. Preferred vectors for yeast are the POT1 vectors described in US Pat. No. 4,931,373. Examples of other filamentous fungal cells are strains of Aspergillus spp., Neurospora spp., Fusarium spp. Or Trichoderma spp., In particular A. oryzae, A. nidulans or A. niger. The use of Aspergillus species for protein expression is described, for example, in EP 272 277 and EP 238 023, EP 184 438. Transformation of F. oxysporum can be carried out as described, for example, in Malardier et al., 1989, Gene 78: 147-156. Transformation of Trichoderma species can be carried out as described in EP 244 234. When filamentous fungi are used as host cells, the DNA construct can conveniently be integrated into the host chromosome to obtain a recombinant host cell. This integration is generally considered an advantage because the DNA sequence remains stable in the cell. Incorporation of the DNA construct into the host chromosome is a conventional method, for example homologous or heterologous recombination.

조합에 의해 실시될 수 있다. 곤충세포의 형질전환 및 이종 폴리펩타이드의 생성은 미국특허 제4,745,051호, 제4,879,236호, 제5,155,037호 및 제5,162,222호 및 EP 397,485호에 기술된 바와 같이 실시할 수 있다. 숙주로 사용된 곤충 세포주는 적합하게는 Spodoptera frugiperda 세포 또는 Trichoplusia ni 세포와 같은 Lepidoptera 세포주일 수 있다(미국특허 제5,077,214호). 배양 조건은 적합하게는 국제 특허 제WO89/01029 또는 WO89/01028에 기술된 바와 같을 수 있다.It can be carried out by a combination. Transformation of insect cells and generation of heterologous polypeptides can be carried out as described in US Pat. Nos. 4,745,051, 4,879,236, 5,155,037 and 5,162,222 and EP 397,485. Insect cell lines used as hosts may suitably be Lepidoptera cell lines such as Spodoptera frugiperda cells or Trichoplusia ni cells (US Pat. No. 5,077,214). Culture conditions may suitably be as described in WO89 / 01029 or WO89 / 01028.

상기된 형질전환 또는 형질감염 숙주 세포는 단백질의 발현을 허용하는 조건 하에서 적합한 영양 배지에서 배양한 후 생성된 단백질의 전체 또는 일부를 배양물로부터 회수할 수 있다. 세포를 배양하기 위해 사용된 배지는 숙주 세포를 성장시 키는데 적합한 통상적인 배지, 예를 들면 적절한 보충제를 함유한 최소 또는 복합 배지일 수 있다. 적합한 배지는 제조업체로부터 입수가능하거나 공지된 바에 따라 제조할 수 있다(예, ATCC의 카달로그). 이어서, 세포에 의해 생성된 단백질은 배양 배지로부터 통상적인 절차에 의해 회수할 수 있다. 통상의 절차는 숙주 세포를 원심분리 또는 여과에 의해 배지로부터 분리하고, 상청액 또는 여과액의 단백질수성 성분을 염(예, 황산암모늄)에 의해 침전시키며, 여러 크로마토그래피 절차(예, 이온 교환 크로마토그래피, 겔여과 크로마토그래피, 친화성 크로마토그래피 등)에 의해 정제하는 것 등을 포함한다.The above-described transformed or transfected host cells may be cultured in a suitable nutrient medium under conditions permitting the expression of the protein and then all or part of the resulting protein may be recovered from the culture. The medium used to culture the cells may be any conventional medium suitable for growing host cells, for example minimal or complex medium containing appropriate supplements. Suitable media can be obtained from the manufacturer or prepared as known (eg, catalog of ATCC). The protein produced by the cells can then be recovered from the culture medium by conventional procedures. Conventional procedures separate host cells from the medium by centrifugation or filtration, and precipitate the proteolytic components of the supernatant or filtrate with salts (e.g. ammonium sulfate) and perform several chromatography procedures (e.g. ion exchange chromatography). , Gel filtration chromatography, affinity chromatography, and the like).

본 발명에 따라 사용되는 Axl 항체는 폴리클로날(polyclonal) 또는 모노클로날(monoclonal) 어느 것이든 가능할 수 있다. Axl 항체는 Santa Cruz Biotech으로부터 구입할 수 있다. 또한 본 발명에서 사용되는 항체는 공지 방법에 따라 제조할 수 있다. Axl antibodies used in accordance with the present invention may be either polyclonal or monoclonal. Axl antibodies can be purchased from Santa Cruz Biotech. In addition, the antibody used in the present invention can be prepared according to a known method.

폴리클로날 항체는 항원과 보조제를 동물에 수회 피하 도는 복강내 주사하여 획득한다. 면역화할 종에서 면역원성인 단백질(예, 열쇠구멍삿갓조개 헤모시아닌, 혈청 유도화제 (예, 말레이미도벤조일 설포석신이미드 에스테르, N-하이드록시석신이미드, 글루타르알데하이드, 석신산 무수물, SOCl2 등)에 관련 항원을 결합시키는 유용할 수 있다. 동물은 항원, 면역원성 결합체 또는 유도체에 대하여 예를 들면 100mug 또는 5 mug의 단백질 또는 결합체(각각 토끼 또는 마우스의 경우)를 3회 용량의 프로인트 완전 부형제와 배합하고 용액을 여러 부위에 경피 주사함으로써 면역화 한다. 1개월 후 동물을 프로인트 완전 부형제중 펩타이드 또는 결합체의 최초 량의 1/5 내지 1/10로 여러 부위에 피하 주사 접종한다. 7 내지 14일 후 동물로부터 채혈하고 혈청을 항체 역가에 대해 검정한다. 역가 고저까지 동물을 접종한다. 바람직하게는, 동물을 동일한 항원의 결합체로 접종하지만, 다른 단백질 및/또는 다른 가교시약과 결합시키기도 한다. 또한 결합체는 단백질 융합물로서 재조합 세포 배양에서 만들 수 있다. 또한 알룸과 같은 응고제가 면역반응을 증강시키는데 적절히 사용된다.Polyclonal antibodies are obtained by several subcutaneous or intraperitoneal injections of antigen and adjuvant into an animal. Proteins that are immunogenic in the species to be immunized (eg keyhole hatch hemocyanin, serum inducing agents (eg maleimidobenzoyl sulfosuccinimide ester, N-hydroxysuccinimide, glutaraldehyde, succinic anhydride, SOCl2) Etc.) Animals may contain, for example, three doses of a protein or conjugate (for rabbits or mice, respectively) of 100 mug or 5 mug to an antigen, immunogenic binder or derivative. Immunized by combination with complete excipients and percutaneous injection of the solution at various sites One month later animals are inoculated subcutaneously at several sites with 1/5 to 1/10 of the initial amount of peptide or conjugate in Freund's complete excipients. Blood is drawn from the animals after 14 to 14 days and the serum is assayed for antibody titer Inoculate the animal to the titer height and preferably the animal is the same antigen Inoculates with a conjugate of, but also combines with other proteins and / or other crosslinking reagents, which can also be made in recombinant cell culture as protein fusions, and coagulants such as alum are suitably used to enhance immune responses.

모노클로날 항체는 실질적으로 균질한 항체의 군으로부터 수득 된다. 즉, 그 군을 포함한 개개 항체는 소량으로 존재할 수 있는 가능한 자연 발생 돌연변이를 제외하고 동일하다. 따라서, 변형 모노클로날은 개개 항체의 혼합물이 아닌 것으로서 항체의 특성을 가리킨다. 예를 들면, 모노클로날 항체는 문헌(Kohler et al., Nature, 256:495 (1975))에 기술된 하이브리도마 방법을 사용하여 제조하거나, 재조합 DNA 방법(미국특허 제4,816,567호)으로 제조할 수 있다. 하이브리도마 방법에서, 마우스 또는 다른 적절한 숙주 동물 (예, 햄스터)이 상기된 바와 같이 면역화 되어 면역화을 위해 사용된 단백질과 특이적으로 결합하는 항체를 생성하거나 생성할 수 있는 림프구를 유도한다. 다른 방법으로서, 림프구를 시험관에서 면역화할 수 있다. 그런 다음, 림프구를 폴리에틸렌 글리콜과 같은 적절한 융합제를 사용하여 골수종 세포와 융합시켜 하이브리도마 세포를 형성한다(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59 103 (Academic Press, 1986). 이에 따라 제조된 하이브리도마 세포를 바람직하게는 비융합된 모 골수종 세포의 성장 또는 생존을 억제하는 하나 이상의 물질을 함유하는 적합한 배양 배지에서 성장시 킨다. 예를 들면, 모 골수종 세포가 하이폭산틴 구아닌 포스포리보실 트랜스퍼라제(HGPRT 또는 HPRT)가 없다면, 하이브리도의 배양 배지는 전형적으로 하이폭산틴, 아미놉테린 및 티미딘(HAT 배지)를 포함할 것이다. Monoclonal antibodies are obtained from the group of substantially homogeneous antibodies. That is, the individual antibodies, including that group, are identical except for possible naturally occurring mutations that may be present in small amounts. Thus, modified monoclonal refers to the properties of the antibody as not being a mixture of individual antibodies. For example, monoclonal antibodies are prepared using the hybridoma method described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA method (US Pat. No. 4,816,567). can do. In hybridoma methods, mice or other suitable host animals (eg hamsters) are immunized as described above to induce lymphocytes that can produce or produce antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59 103 (Academic Press, 1986). The prepared hybridoma cells are grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Without poribosyl transferase (HGPRT or HPRT), the hybrido's culture medium will typically include hypoxanthine, aminopterin and thymidine (HAT medium).

이들 물질은 HGPRT-결핍 세포의 성장을 억제한다. 바람직한 골수종 세포는 효율적으로 융합하고, 선택된 항체-생성 세포에 의해 항체의 안정한 고수준 항체 생성을 지지하며, HAT와 같은 배지에 민감한 것이다. 이들 가운데, 바람직한 골수종 세포주는 마우스 골수종 세포주, 예를 들면 미국 캘리포니아 샌디에고, Salk Institute Cell Distribution Center에서 입수할 수 있는 MOPC-21 및 PMC-11 마우스 종양 및 미국 ATCC로부터 입수가능한 SP-2 또는 X63-Ag8-653 세포이다. 사람 골수종 및 마우스-사람 이종골수종 세포주가 또한 사람 모노클로날 항체의 생성을 위해 사용될 수 있다(Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 63 (Marcel Dekker, Inc., New York, 1987)). 하이브리도마 세포가 성장하는 배양 배지를 항원에 대한 모노클로날 항체의 생성에 대해 검정한다. 바람직하게는, 하이브리도마 세포에 의해 생성된 모노클로날 항체의 결합 특이성은 면역침강 도는 시험관 결합검정 예, 방사능면역검정 또는 효소-연결 면역흡착 검정)에 의해 결정한다. 모노클로날 항체의 결합 친화성은 예를 들면 문헌(Munson et al., Anal. Biochem., 107:220 (1980))의 Scatchard 분석으로 결정할 수 있다. 목적하는 특이성, 친화성 및/또는 활성을 갖는 항체를 생성하는 하이브리도마 세포를 동정한 후, 제한 희석 절차에 의해 아클로닝하여 클론을 얻고 표준 방법 (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59 103 (Academic Press, 1986))으로 성장시킨다. 이 목적에 적합한 배양 배지로는 예를 들면 D-MEM 또는 RPMI-1640 배지가 포함된다. 또한, 하이브리도마 세포는 동물에 복수 종양으로서 생체내에서 성장시킬 수 있다. 아클론에 의해 분비된 모노클로날 항체는 배양 배지, 복수액 또는 혈청으로부터 예를 들면 단백질 A-세파로즈, 하이드록실아파타이트 크로마토그래피, 겔 전기영동, 투석 또는 친화성 크로마토그래피와 같은 통상적인 항체 정제 절차에 의해 분리한다.These substances inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells are those that fuse efficiently, support stable high-level antibody production of antibodies by selected antibody-producing cells, and are sensitive to media such as HATs. Among these, preferred myeloma cell lines are mouse myeloma cell lines, eg, MOPC-21 and PMC-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8 available from ATCC, USA -653 cells. Human myeloma and mouse-human heteromyeloma cell lines can also be used for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51 63 (Marcel Dekker, Inc., New York, 1987)). Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay or enzyme-linked immunosorbent assay. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980). After identifying hybridoma cells that produce antibodies with the specificity, affinity, and / or activity of interest, they can be cloned by restriction dilution procedures to obtain clones and standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59 103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in an animal. Monoclonal antibodies secreted by aclones are conventional antibody tablets such as, for example, protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography from culture media, ascites fluid or serum. Separate by procedure.

조혈모세포는 자체 재생능이 있고 개인의 일생동안 수임된 선조세포의 기원이다. 원시뿐만 아니라 보다 수임된 조혈모세포의 일반적인 특징은 항-CD34 모노클로날 항체를 이용한 FACS(형광-활성화 세포 분류기) 분석에 의해 검출될 수 있는 CD34 항원의 발현이다. 조혈모세포는 골수, 말초혈액, 제대혈 등으로부터 유래된다. 제대혈은 가장 풍부한 조혈모세포의 근원으로 알려져 있다. 분만 후 직접 태반으로부터 수득 된 제대혈은 조혈모세포가 풍부하고 골수 및 말초혈로부터 얻은 세포보다 높은 증식능을 갖는다. 과립구-콜로니 자극 인자와 같은 조혈성장 인자의 주입은 CD34(+) 세포의 가동화를 상당히 촉진할 수 있다(Beyer J et al., Hematopoietic rescue after high-dose chemotherapy using autologous peripheral-blood progenitor cells or bone marrow: a randomized comparison. J Clin Oncol 1995;13:1328-1335; and Smith TJ et al., Economic analysis of a randomized clinical trial to compare filgrastim-mobilized peripheral-blood progenitor-cell trnasplantation and autologous bone marrow transplantation in patients with Hodgkin's and non-Hodgkin's lymphoma. J Clin Oncol 1997;15:5-10). 과립구-콜로니 자극 인자는 조혈모세포의 채취 기간 동안에 일일 300-960 ug의 용량으로 피하 투여된다. 조혈모세포를 분리하거나 정제하는데 사용되는 전략은 많이 있다. 이 가운데 형광(Preffer FI, et al., Lineage-negative side-population(SP) cells with restricted hematopoietic capacity circulate in normal human adult blood: immunophenotypic and functional characterization. STEM CELLS 2002; 20:417427) 또는 면역자기 기술(Przyborski SA. Isolation of human embryonal carcinoma stem cells by immunomagnetic sorting. Stem Cells 2001; 19:500504)을 이용하여 특정 세포 표면 마커에 따라 세포를 분류하거나, 배양 플라스틱에서 줄기세포의 차등 평판 효율을 활용하거나(Friedenstein AJ, et al., Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 1976; 4:267274), 컬럼-분리 기술(Huss R. Isolation of primary and immortalized CD34 hematopoietic and Mesenchymal stem cells from various sources. STEMCELLS 2000; 18:19)이 포함된다.Hematopoietic stem cells have their own regenerative capacity and are the origin of progenitor cells committed during the lifetime of an individual. A general feature of primitive as well as more committed hematopoietic stem cells is the expression of CD34 antigens that can be detected by FACS (fluorescence-activated cell sorter) analysis using anti-CD34 monoclonal antibodies. Hematopoietic stem cells are derived from bone marrow, peripheral blood, umbilical cord blood and the like. Umbilical cord blood is known to be the most abundant source of hematopoietic stem cells. Umbilical cord blood obtained directly from the placenta after delivery is rich in hematopoietic stem cells and has a higher proliferative capacity than cells obtained from bone marrow and peripheral blood. Infusion of hematopoietic growth factors, such as granulocyte-colony stimulating factor, can significantly promote the mobilization of CD34 (+) cells (Beyer J et al., Hematopoietic rescue after high-dose chemotherapy using autologous peripheral-blood progenitor cells or bone marrow : a randomized comparison.J Clin Oncol 1995; 13: 1328-1335; and Smith TJ et al., Economic analysis of a randomized clinical trial to compare filgrastim-mobilized peripheral-blood progenitor-cell trnasplantation and autologous bone marrow transplantation in patients with Hodgkin's and non-Hodgkin's lymphoma. J Clin Oncol 1997; 15: 5-10). Granulocyte-colony stimulating factor is administered subcutaneously at a dose of 300-960 ug per day during the harvest of hematopoietic stem cells. There are many strategies used to isolate or purify hematopoietic stem cells. Among them, fluorescence (Preffer FI, et al., Lineage-negative side-population (SP) cells with restricted hematopoietic capacity circulate in normal human adult blood: immunophenotypic and functional characterization. STEM CELLS 2002; 20: 417427) or immunomagnetic techniques ( Using Przyborski SA.Isolation of human embryonal carcinoma stem cells by immunomagnetic sorting.Stem Cells 2001; 19: 500504), cells can be sorted according to specific cell surface markers, or utilizing the differential plate efficiency of stem cells in cultured plastics (Friedenstein). AJ, et al., Fibroblast precursors in normal and irradiated mouse hematopoietic organs.Exp Hematol 1976; 4: 267274), Huss R. Isolation of primary and immortalized CD34 hematopoietic and Mesenchymal stem cells from various sources. STEMCELLS 2000 18:19).

성숙자연살해세포를 활성화하기 위해서 인터루킨-2를 사용하는 것은 잘 알려져 있다. 인터루킨-2의 처리 방법은 두 가지가 제시되고 있다. 첫 번째 방법으로 인터루킨-2를 직접 환자에 투여해서 투여된 인터루킨-2에 의해서 생체내에서 자연살해세포가 증식되고 활성화시키는 것이 있고, 두 번째 방법으로 환자로부터 채혈하여 혈액에서 성숙자연살해세포를 분리하여 인터루킨-2로 자극시켜 활성화시킨 후 활성화된 성숙자연살해세포를 다시 환자에게 투여하는 방법이 있다. 이들은 모두 인터루킨-2로 자연살해세포를 활성화시켜 활성화된 자연살해세포에 의해 암세포 파괴를 목적으로 하고 있다. 하지만 이러한 방법들은 고농도(약 150ng/ml)의 인터루킨-2가 사용되어 이에 따른 부작용이 문제점으로 대두 되고 있다. 이것에 대한 부작용으로는 강한 독성, 발열과 폐부종 그리고 쇼크의 유발이 있으며 이러한 효과는 인터루킨-2가 T림프구로 하여금 종양괴사인자나 인터페론-감마 와 같은 다른 사이토카인의 생산을 자극하여 이들 사이토카인이 혈관 내피와 다른 세포에 작용하기 때문이다. 이러한 문제점들을 해결하기 위해 적은 농도의 인터루킨-2 사용에 대한 연구들이 수행 중에 있지만 아직 만족할 만한 결과에 대해서는 보고되어 진 바가 없다(M.J. Smyth, Y. Hayakawa, K. Takeda, H. Yagita, Nat Rev Cancer. 2,850, 2002; M. A. Caligiuri,et al.,J. Exp. Med. 171, 1509,1990). It is well known to use interleukin-2 to activate mature natural killer cells. Two treatment methods for interleukin-2 have been proposed. In the first method, natural killer cells are proliferated and activated in vivo by interleukin-2 administered by directly administering interleukin-2 to the patient. In the second method, mature natural killer cells are isolated from blood by collecting blood from the patient. By stimulating with Interleukin-2, the activated mature natural killer cells are then administered back to the patient. All of them aim to destroy cancer cells by activating natural killer cells by activating natural killer cells with interleukin-2. However, these methods use a high concentration (about 150ng / ml) of Interleukin-2, which is a side effect of this problem has emerged as a problem. Side effects to this include strong toxicity, fever and pulmonary edema and shock. These effects can cause T-lymphocytes to stimulate the production of other cytokines, such as tumor necrosis factor or interferon-gamma, Because it acts on the vascular endothelium and other cells. Studies on the use of low levels of interleukin-2 have been conducted to address these problems, but no satisfactory results have been reported (MJ Smyth, Y. Hayakawa, K. Takeda, H. Yagita, Nat Rev Cancer). 2,850, 2002; MA Caligiuri, et al., J. Exp. Med. 171, 1509,1990).

본 발명에 따르면, 본 발명의 분화된 성숙자연살해세포를 약 8 내지 15ng/ml의 저용량 인터루킨-2로 처리하여도 성숙자연살해세포를 충분히 자극 활성화시킬 수 있는 것으로 밝혀졌다. 바람직한 인터루킨-2의 사용량은 약 10 ng/ml이다. 인터루킨-2의 그러한 저용량은 활성화된 성숙자연살해세포가 독성을 유발하지 않는 것으로 보인다. 세포 보존은 몇 가지 방법이 사용되고 있는데, 가장 잘 알려진 것은 초저온보존(cryopreservation)이다. 또한 HypoThermosolTM(BioLife Solutions Inc.) 계열 및 DMSO 용액(dimethyl sulfoxide)을 사용할 수 있다. 본 발명의 추가 관점으로서, 자연살해세포는 환자에게 투여 전후로 초저온보존될 수 있다. 초저온보존의 대표적인 방법이 미국특허 제60168991호에 기술되어 있다. 소규모 초저온보 온의 경우 세포는 사전냉장된 5% 사람 혈청 알부민(HAS)중에 200x10⁶/ml로 재현탁시킬 수 있다. 이어서, 상기 HAS 용액중에 20% 디메틸설폭사이드(DMSO)의 등량을 적가한다. 혼합물을 초저온바이알에 1 ml/바이알의 양으로 분취하고 초저온챔버(NalgeneTM)에서 -80℃로 밤새 동결시킨다. 대규모 초저온보존의 경우, 세포는 AIM V에 600x10⁶/ml로 재현탁할 수 있다. 그런 다음, 등량의 20% AIM V를 점증적으로 첨가한다. 혼합물은 속도제어 동결시스템(FormaTM)을 이용하여 20 ml/낭으로 동결용기(Cryocyte, Baxter)에 동결시킨다. 활성화된 성숙자연살해세포의 세포독성 유효량은 시험관 및 생체내 용도뿐만 아니라 이들 살해 세포의 궁극적인 표적이 되는 세포의 양 및 유형에 따라 다양할 수 있다. According to the present invention, it has been found that even when the differentiated mature killer cells of the present invention are treated with a low dose of Interleukin-2 at about 8-15 ng / ml, the mature natural killer cells can be sufficiently stimulated and activated. Preferred amount of interleukin-2 is about 10 ng / ml. Such low doses of interleukin-2 do not appear to cause toxic activated mature killer cells. Several methods are used for cell preservation, the best known of which is cryopreservation. HypoThermosol ™ (BioLife Solutions Inc.) series and DMSO solution (dimethyl sulfoxide) can also be used. As a further aspect of the invention, natural killer cells may be cryogenically stored before and after administration to a patient. Representative methods of cryogenic preservation are described in US Pat. No. 60,168,991. For small cryogenic insulation, cells can be resuspended at 200 × 10 ⁶ / ml in pre-chilled 5% human serum albumin (HAS). Subsequently, an equivalent amount of 20% dimethylsulfoxide (DMSO) is added dropwise to the HAS solution. The mixture is aliquoted in an cryogenic vial in an amount of 1 ml / vial and frozen overnight at −80 ° C. in a cryogenic chamber (Nalgene ™). For large scale cryogenic preservation, cells can be resuspended in AIM V at 600 × 10 ⁶ / ml. Then, an equivalent amount of 20% AIM V is added gradually. The mixture is frozen in freezer (Cryocyte, Baxter) at 20 ml / bag using a rate controlled freezing system (Forma ™). The cytotoxic effective amount of activated mature natural killer cells may vary depending on the in vitro and in vivo use as well as the amount and type of cells that are the ultimate target of these killer cells.

여기서 세포독성유효량은 성숙자연살해세포가 암세포를 살상할 수 있는(약리작용을 일으킬 수 있는) 약물의 분량으로 정의한다. 또한 유효량은 환자의 건강 상태 및 중증에 따라 변할 수 있기 때문에, 전문의가 모든 변수를 적절히 고려하여 결정해야 한다. 일반적으로, 성인 암환자에게 투여하는 양은 회당 10⁶ 내지 10¹²개 세포, 바람직하게는, 회당 10⁸ 내지 10¹¹개 세포, 더욱 바람직하게는 회당 10⁹ 내지 10¹⁰개 세포이다. 본 발명의 방법에 따라 증식된 성숙자연살해세포는 약제학적으로 허용되는 담체(예, 염수 용액)과 함께 치료를 위해 환자에게 피하, 근육내, 정맥내, 경막내로 투여될 수 있다. 다양한 생체 재료(세포 전달체)의 사용은 자연살해세포가 목(target)부위로 전달되는 효율과 자연살해세포에 의한 암살상 능력의 효 율을 높일 수 있다. 세포 전달체로는 다당류 계열의 메틸셀룰로오스(M.C. Tate, D.A. Shear, S.W. Hoffman, D.G. Stein, M.C. LaPlaca, Biomaterials 22, 1113, 2001), 키토산(Suh JKF, Matthew HWT. Biomaterials, 21, 2589, 2000; Lahiji A, Sohrabi A, Hungerford DS, et al., J Biomed Mater Res, 51, 586, 2000), N-이소프로필아크릴아마이드(isopropylacrylamide) 공중합체 계열의 P(NIPAM-co-AA)(Y.H. Bae, B. Vernon, C.K. Han, S.W. Kim, J. Control. Release 53, 249,1998 H. Gappa, M. Baudys, J.J. Koh, S.W. Kim, Y.H. Bae, Tissue Eng. 7, 35, 2001) 등이 있으며 이외에도 Poly(산화에틸렌)/poly(D,L-젖산-co-글리콜산) (B. Jeong, K.M. Lee, A. Gutowska, Y.H. An, Biomacromolecules 3, 865, 2002), P(PF-co-EG) (Suggs LJ, Mikos AG. Cell Trans, 8, 345, 1999), PEO/PEG (Mann BK, Gobin AS, Tsai AT, Schmedlen RH, West JL., Biomaterials, 22, 3045, 2001 Bryant SJ, Anseth KS. Biomaterials, 22, 619, 2001), PVA (Chih-Ta Lee, Po-Han Kung and Yu-Der Lee, Carbohydrate Polymers, 61, 348, 2005), 콜라겐 (Lee CR, Grodzinsky AJ, Spector M., Biomaterials 22, 3145, 2001), alginate (Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney DJ. Biotech Prog 17, 945, 2001 Smidsrd O, Skjak-Braek G., Trends Biotech, 8, 71, 1990) 등이 조직공학에 있어서 세포 치료에 대한 세포 전달체로써 사용되고 있다. An effective cytotoxicity is defined as the amount of drug that can kill the mature natural killer cells (which can cause pharmacological action). In addition, the effective amount may vary depending on the patient's state of health and severity, so the physician must determine all variables appropriately. Generally, the amount administered to an adult cancer patient is 10 ⁶ to 10 ¹² cells per time, preferably 10 ⁸ to 10 ¹¹ cells per time, more preferably 10 ⁹ to 10 ¹⁰ cells per time. Mature natural killer cells propagated according to the method of the present invention may be administered to the patient subcutaneously, intramuscularly, intravenously, or intradural for treatment with a pharmaceutically acceptable carrier (eg, saline solution). The use of various biomaterials (cell carriers) can increase the efficiency of natural killer cells being delivered to the target site and the ability of the natural killer cells to kill. Cell carriers include polysaccharide methyl cellulose (MC Tate, DA Shear, SW Hoffman, DG Stein, MC LaPlaca, Biomaterials 22, 1113, 2001), chitosan (Suh JKF, Matthew HWT. Biomaterials, 21, 2589, 2000; Lahiji A, Sohrabi A, Hungerford DS, et al., J Biomed Mater Res, 51, 586, 2000), P (NIPAM-co-AA) (YH Bae, B, based on N-isopropylacrylamide copolymer) Vernon, CK Han, SW Kim, J. Control.Release 53, 249,1998 H. Gappa, M. Baudys, JJ Koh, SW Kim, YH Bae, Tissue Eng. 7, 35, 2001). (Ethylene oxide) / poly (D, L-lactic acid-co-glycolic acid) (B. Jeong, KM Lee, A. Gutowska, YH An, Biomacromolecules 3, 865, 2002), P (PF-co-EG) ( Suggs LJ, Mikos AG.Cell Trans, 8, 345, 1999), PEO / PEG (Mann BK, Gobin AS, Tsai AT, Schmedlen RH, West JL., Biomaterials, 22, 3045, 2001 Bryant SJ, Anseth KS. Biomaterials , 22, 619, 2001), PVA (Chih-Ta Lee, Po-Han Kung and Yu-Der Lee, Carbohydrate Polymers, 61, 348, 2005), collagen (Lee CR, Grodzinsky AJ, Spector M., Biomaterials 22, 3145, 2001), alginate (Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney DJ.Biotech Prog 17, 945, 2001 Smidsrd O, Skjak-Braek G., Trends Biotech, 8, 71, 1990) are used as cell carriers for cell therapy in tissue engineering.

이하 본 발명은 상기의 도면을 참조하여 하기 실시예로 예시하고자 한다. 그러나, 이들 실시예가 본 발명을 한정하는 것으로 해석해서는 안 된다.Hereinafter, the present invention will be illustrated by the following examples with reference to the drawings. However, these examples should not be construed as limiting the present invention.

실시예Example 1. 마우스 자연살해세포 분화단계에 따라 발현되는 특정 유전자의 발굴 1. Identification of specific genes expressed according to differentiation stage of mouse natural killer cells

(A) 마우스의 골수(Bone marrow, BM)로부터 조혈모세포의 분리 방법과 자연살해세포의 분화 방법(A) Isolation of Hematopoietic Stem Cells from Bone Marrow (BM) and Differentiation of Natural Killer Cells

8-12 주령된 마우스(C57BL/6, Korea Reasearch Bioscience and Biotechnology에서 구입하여 무균상태에서 동물 관리 지침에 따라 관리하였음)의 전체 뼈를 분리하여 골수세포를 얻은 다음 세포용해 완충액(0.2% NaCl, 1.6% NaCl)으로 적혈구를 제거하여 세포를 2.4G2 상청액과 10분간 아이스에서 반응시키고 2mM EDTA 가 함유된 인산염 완충용액 (buffer A)에 세척하였다. 완충용액 A에 세포를 현탁시켜(1X108/500㎕) 바이오틴(biotin)이 붙여진 항체 칵테일(Mac-1, Gr-1, B220, NK 1.1, CD2, TER-119, Pharmingen)을 가하고 4℃에서 10분간 반응시킨 다음 세포를 완충용액 A로 세척하고, 1X10⁸세포에 900㎕의 완충용액 A와 100㎕의 스트렙트아비딘(streptavidin)-마이크로비즈(microbeads) (Miltenyi Biotec)를 가하여 4℃에서 15분간 반응시켰다. 세포를 세척한 후, MACS(마그네틱 비드-활성화 세포 분류기) 완충용액(2mM EDTA와 0.5% BSA가 함유된 인산염 완충용액)에 현탁시켜 나일론 메쉬(70㎛)로 여과하여 준비하였다. 마그테틱 컬럼(CS column, Miltenyi Biotech)을 슈퍼 MACS에 장치하고 마그네틱 비드가 표지된 세포를 통과시켜 MACS 완충용액으로 컬럼을 충분히 세척한 다음 컬럼을 통과한 액을 원심 분리하여 리니지 음성인(Lin^-) 세포를 얻었다. Lin^-세포(1X10⁷)에 FITC가 붙어 있는 항-c-kit (Pharmingen)을 가하고 4℃에서 10분간 반응시킨 후, 세척하고 항-FITC 마이크로 비즈 (Miltenyi Biotec)와 다시 4℃에서 15분간 반응시켜 500㎕의 MACS 완충용액에 현탁하고 마그네틱 필드내에서 MS 컬럼에 통과시켰다. 컬럼을 충분히 세척하고 1ml의 MACS 완충용액으로 밀어내어 c-kit+ 인 세포를 얻어 조혈모세포로 사용하였다. 이렇게 분리된 조혈모세포를 전구자연살해세포로 분화시키기 위해 SCF(30ng/ml, Biosource), 인터루킨-7 (0.5 ng/ml, PeproTech), Flt3l(50ng/ml,PeproTech), 인도메타신(2㎍/ml, Sigma), 젠타마이신(2㎍/ml, Sigma)이 함유된 RPMI(Gibco)배지로 3일마다 배지의 절반을 새롭게 갈아주면서 1X10⁶/ml의 농도로 24 웰 배양 용기(24 well plate)에서 배양하였다. 6일 동안 이들 사이토카인들을 첨가하여 배양 후 CD122-FITC 항체와 multisort 마이크로 비즈를 첨가시켜 반응시키고 MACS를 수행함으로써 CD122⁺한 전구자연살해세포를 얻었다. 전구자연살해세포를 성숙자연살해세포로 분화시키기 위해서 다시 6일 동안 인터루킨-15(20ng/ml), 인도메타신(2㎍/ml), 젠타마이신(2㎍/ml)이 첨가된 RPMI 배지를 이용하여 두 가지 방법으로 분화시켰는데, 기질세포(ATCC)와 공동배양(co-culture)하여 성숙자연살해세포(mature NK-2)로 분화시키는 방법과 공동배양하지 않고 덜 성숙자연살해세포(mature NK-1)로 분화시키는 방법을 수행하였으며 이들 세포를 수확하여 자연살해세포 표면분자들의 발현을 분석하였다. Whole bones of 8-12 week old mice (C57BL / 6, purchased from Korea Reasearch Bioscience and Biotechnology and administered according to animal care instructions under aseptic conditions) were isolated to obtain bone marrow cells, followed by cytolysis buffer (0.2% NaCl, 1.6 The cells were reacted with 2.4G2 supernatant for 10 min on ice and washed in phosphate buffer (buffer A) containing 2 mM EDTA. Suspend the cells in Buffer A (1 × 108/500 μl), add a biotin-attached antibody cocktail (Mac-1, Gr-1, B220, NK 1.1, CD2, TER-119, Pharmingen) and 10 at 4 ° C. After the reaction, the cells were washed with buffer A, and 900 µl of buffer A and 100 µl of streptavidin-microbeads (Miltenyi Biotec) were added to 1 × ^{10 8} cells for 15 minutes at 4 ° C. Reacted. The cells were washed and then suspended in MACS (Magnetic Bead-Activated Cell Sorter) buffer (phosphate buffer containing 2 mM EDTA and 0.5% BSA) and filtered through nylon mesh (70 μm). A magnetic column (CS column, Miltenyi Biotech) was placed in the super MACS, and the magnetic beads were passed through the labeled cells, the column was thoroughly washed with MACS buffer solution, and the solution passed through the column was centrifuged to give the Lineage negative (Lin ^−). ) Cells were obtained. Anti-c-kit (Pharmingen) with FITC attached to Lin ^- cells (1X10 ⁷ ) was reacted at 4 ° C for 10 minutes, washed, and reacted with anti-FITC microbeads (Miltenyi Biotec) again at 4 ° C for 15 minutes. And suspended in 500 μL MACS buffer and passed through an MS column in a magnetic field. The column was washed well and pushed out with 1 ml of MACS buffer to obtain c-kit + phosphorus cells to be used as hematopoietic stem cells. SCF (30 ng / ml, Biosource), Interleukin-7 (0.5 ng / ml, PeproTech), Flt3l (50 ng / ml, PeproTech), Indomethacin (2 ㎍) to differentiate the isolated hematopoietic stem cells into progenitor killer cells / ml, Sigma) and 24 well plate with RPMI (Gibco) medium containing gentamicin (2 μg / ml, Sigma) at a concentration of 1 × 10 ⁶ / ml Incubated). These cytokines were added for 6 days, followed by incubation with CD122-FITC antibody and multisort microbeads, followed by reaction and MACS. CD122 ⁺ progenitor cells were obtained. In order to differentiate progenitor killer cells into mature killer cells, RPMI medium containing interleukin-15 (20 ng / ml), indomethacin (2 µg / ml) and gentamicin (2 µg / ml) was added for 6 days. Differentiation was performed using two methods, co-culture with stromal cells (ATCC) to differentiate into mature NK-2 cells and less mature natural killer cells without co-culture. NK-1) was differentiated and these cells were harvested and analyzed for expression of surface cells of natural killer cells.

(B) SAGE를 통한 마우스 성숙자연살해세포의 분화 유도 물질 발굴(B) Discovery of differentiation inducer of mouse natural killer cells through SAGE

분리한 조혈모세포, 전구자연살해세포 그리고 두 가지의 성숙자연살해세포로부터 각각의 분화단계에 따라 특이적으로 발현되는 유전자를 발굴하고자 SAGE(Serial Analysis of gene expression)를 수행하였다. 각 분화 단계별 세포인 조혈모세포, 전구자연살해세포 그리고 두 가지의 성숙자연살해세포로부터 전체 RNA를 분리하고 각각의 RNA 5㎍을 oligo(dT)25 비드(Dynal A.S.)와 반응시켜 mRNA를 분리하였다. 각 분화 단계별 세포인 조혈모세포, 전구자연살해세포 그리고 성숙자연살해세포로부터 전체 RNA는 다음과 같은 방법으로 분리하였다. 각각의 세포를 인산염 완충용액으로 1회 세척한 후에 RNA 분리용액(RNAzol B, TEL-TEST) 500㎕를 각 시료에 넣고 부드럽게 피펫팅 하여 세포를 깨고 50㎕의 클로로포름을 넣고 잘 섞은 후 얼음에서 5분간 방치하였다. 이를 12,000rpm, 15분 동안 4℃에서 원심분리한 후 상청액을 취하고 동량의 이소프로필 알콜을 넣고 얼음에서 20분 동안 방치하였다. 다시 12,000rpm, 20분 동안 4℃에서 원심분리한 후 침전물을 80% 에탄올로 세척하고 건조시킨 후에 0.1% 디에틸 피로카보네이트(diethyl pyrocarbonate)가 포함된 물을 20㎕ 넣고 잘 녹였다. 이렇게 분리한 각각의 mRNA와 5'에 바이오틴이 붙어 있고 3‘위치에 올리고(dT)를 포함한 프라이머를 이용하여 cDNA 합성 키트(Life Technology)로 cDNA를 합성하였다. cDNA와 반응한 SAGE tag들은 T4 DNA 연결효소를 이용해 연결시켜 콘캐터머를 만들고 이 반응물은 제한효소 sphI(Roche)으로 처리한 pZero-1 벡터(Invitrogen)에 T4 DNA 연결효소(Roche)를 이용해 삽입하였다. M13 F 프라이머와 M13 R 프라이머를 이용해 중합효소 연쇄반응(PCR)을 수행하여 반응물을 얻었다. 중합효소 연쇄반응은 다음과 같은 방법으로 수행하였다. 위에서 얻은 제조물을 주형으로 하여 각각의 프라이머를 이용하여 중합효소 연쇄반응을 다음과 같이 수행하였다. 중합효소 연쇄반응 10 x 완충용액(Roche) 8㎕, 5' 프라이머 1㎕(20pmol), 3' 프라이머 1㎕(20pmol), 증류수 69㎕, Taq 중합효소(Takara) 1㎕(2.5U)의 반응물(총량 100㎕)을 95℃에서 5분간 방치하여 어떤 다른 효소활성을 저해시킨 후에 95℃에서 1분 30초, 55℃에서 1분, 72℃에서 2분 동안 반복하는 사이클을 30여 회 수행하고 마지막으로 95℃에서 1분 30초, 55℃에서 1분, 72℃에서 5분간 반응을 수행하였다. 이 반응물은 Big-Dye 시퀀싱 키트와 ABI1377 시퀀서(Perkin-elmer Applied Biosystems)로 염기서열 분석을 하였다. Tag 시퀀스는 SAGE 300 소프트웨어를 이용해 추출하였다. 레퍼런스 SAGE-tag 데이터베이스는 GenBank에 있는 기존에 알려진 마우스의 발현 시퀀스로 구성이 된 것이며 이들 시퀀스는 각 전사물의 오리엔테이션, polu(A) 시그날(AATAAA EH는 ATTAAA), poly(A) tail, 그리고 시퀀스 말단에 CATG 절단 사이트를 포함하였다. 컴퓨터 프로그램, GIST(Gene Identification and Sequence Topography)를 이용하여 SAGE tag 과 참조 SAGE 데이타 베이스를 이용하여 분석하였다. 그 결과, 전구자연살해세포에서만 발현되고 다른 분화단계에서는 발현되지 않는 유전자들 중 표1의 유전자들이 이들 세포에만 많은 수로 그리고 특이적으로 발현되고 있음을 알 수 있었다(표 1). 데이터의 통계 분석에 대한 유의 확률 (p-value)는 paired Student t test 소프트웨어 프로그램을 이용하여 분석하였고(실시예의 모든 데이타 적용) SAGE tag들의 발현에 대한 평가는 v2, Audic, 과 Claveric 방법(http://telethon.bio.unipd.it/bioinfo/ IDEG6_ form)을 이용하여 분석하였다. SAGE (Serial Analysis of Gene Expression) was performed to identify genes specifically expressed in differentiation stages from isolated hematopoietic stem cells, progenitor killer cells and two mature killer cells. Total RNA was isolated from hematopoietic stem cells, progenitor killer cells, and two mature spontaneous killer cells, each of the differentiation stages, and mRNA was isolated by reacting 5 ㎍ of each RNA with oligo (dT) 25 beads (Dynal A.S.). Total RNA was isolated from hematopoietic stem cells, progenitor killer cells and mature killer cells, which are cells of differentiation stages, as follows. Each cell was washed once with phosphate buffer solution, and 500 µl of RNA isolation solution (RNAzol B, TEL-TEST) was added to each sample and gently pipetted to break the cells. 50 µl of chloroform was added and mixed well. It was left for a minute. It was centrifuged at 12,000 rpm for 15 minutes at 4 ° C, the supernatant was taken, and the same amount of isopropyl alcohol was added and left on ice for 20 minutes. After centrifugation at 12,000 rpm for 20 minutes at 4 ° C., the precipitate was washed with 80% ethanol and dried, and 20 µl of water containing 0.1% diethyl pyrocarbonate was dissolved. Each of the mRNAs thus separated and the biotin attached to the 5 'and using a primer containing oligo (dT) at the 3' position cDNA was synthesized by cDNA synthesis kit (Life Technology). SAGE tags reacted with cDNA were concatenated using T4 DNA ligase, and the reaction was inserted into pZero-1 vector (Invitrogen) treated with restriction enzyme sphI (Roche) using T4 DNA ligase (Roche). . The polymerase chain reaction (PCR) was performed using M13 F primer and M13 R primer to obtain a reaction product. Polymerase chain reaction was performed in the following manner. Using the prepared product as a template, the polymerase chain reaction was performed using each primer as follows. Reaction of polymerase chain reaction 10 x buffer (Roche) 8 μl, 5 μl primer 1 μl (20 pmol), 3 ′ primer 1 μl (20 pmol), distilled water 69 μl, Taq polymerase (Takara) 1 μl (2.5U) (Total 100 μl) was allowed to stand at 95 ° C. for 5 minutes to inhibit any other enzymatic activity, followed by 30 cycles of 1 minute 30 seconds at 95 ° C., 1 minute at 55 ° C. and 2 minutes at 72 ° C. Finally, the reaction was performed at 95 ° C. for 1 minute 30 seconds, at 55 ° C. for 1 minute, and at 72 ° C. for 5 minutes. The reaction was sequenced with Big-Dye Sequencing Kit and ABI1377 Sequencer (Perkin-elmer Applied Biosystems). Tag sequences were extracted using SAGE 300 software. The reference SAGE-tag database consists of the expression sequences of known mice in GenBank, which consist of the orientation of each transcript, the polu (A) signal (AATAAA EH is ATTAAA), the poly (A) tail, and the end of the sequence. Included CATG cleavage site. A computer program, Gene Identification and Sequence Topography (GIST), was used to analyze SAGE tags and reference SAGE databases. As a result, it was found that the genes of Table 1 among the genes expressed only in progenitor killer cells but not in differentiation stages were expressed in a large number and specifically in these cells (Table 1). The significance probability (p-value) for statistical analysis of the data was analyzed using a paired Student t test software program (all data in the examples). The expression of SAGE tags was evaluated using the v2, Audic, and Claveric methods (http: //telethon.bio.unipd.it/bioinfo/IDEG6_ form).

전구자연살해세포에서 특이적으로 발현되는 유전자Genes Specific to Progenitor Natural Killer Cells No. No. 유전자 gene Unigene IDUnigene ID 1One 케모카인 수용체(chemokine receptor 1)Chemokine receptor 1 Mm.57051Mm.57051 22 콜로니 자극인자 요소 1 수용체(colony stimulating factor 1 receptor)Colony stimulating factor 1 receptor Mm.22574Mm.22574 33 형질전환 성장 인자 베타 수용체 II(Transforming growth factor, beta receptor II)Transforming growth factor (beta receptor II) Mm.172346Mm.172346 44 에스테로젠 수용체 베타 1(Estrogen related receptor beta like 1, Esrrbl1)Estrogen related receptor beta like 1, Esrrbl1 Mm.31355Mm.31355 55 c-src 타이로신 키나아제 c-src tyrosine kinase Mm.21974Mm.21974 66 Hmgb1(high mobility group box 1)High mobility group box 1 (Hmgb1) Mm.16421Mm.16421 77 페리틴(Ferritin) H 체인Ferritin H Chain Mm.1776Mm.1776 88 페리틴(Ferritin) L 체인Ferritin L chain Mm.7500Mm.7500 99 calgranulin Bcalgranulin B Mm.2128Mm.2128 1010 cell surface glycoprotein p91cell surface glycoprotein p91 Mm.3189Mm.3189 1111 Chloride channel 7Chloride channel 7 Mm.22727Mm.22727 1212 AdvillinAdvillin Mm.10739Mm.10739 1313 Cathepsin-LCathepsin-l Mm.930Mm.930 1414 Cathepsin-BCathepsin-b Mm.22753Mm.22753 1515 Cathepsin-HCathepsin-h Mm.2277Mm.2277 1616 EGF-like module-containing mucin-like hormone receptor-like 1EGF-like module-containing mucin-like hormone receptor-like 1 Mm.2254Mm.2254 1717 Triggering receptor expressed on myeloid cells 2Triggering receptor expressed on myeloid cells 2 Mm.261623Mm.261623 1818 SERPINA3GSERPINA3G Mm.15085Mm.15085 1919 LAPTm5LAPTm5 Mm.4554Mm.4554 2020 early growth response1early growth response1 Mm.181959Mm.181959 2121 calcium response factorcalcium response factor Mm.370325Mm.370325 2222 Apolipoprotein EApolipoprotein E Mm.168157Mm.168157 2323 Catenin betaCatenin beta Mm.291928Mm.291928 2424 LysozymeLysozyme Mm.45436Mm.45436 2525 BrevicanBrevican Mm.4598Mm.4598 2626 Matrix metalloproteinase 12Matrix metalloproteinase 12 Mm.2055Mm.2055 2727 Cellular repressor of EIA-stimulated geneCellular repressor of EIA-stimulated gene Mm.459Mm.459 2828 c-kit ligandc-kit ligand Mm.4235Mm.4235 2929 MPS1 proteinMPS1 protein Mm.3999Mm.3999 3030 Transglutaminase 2Transglutaminase 2 Mm.18843Mm.18843 3131 Serum and glucocorticoid-regulated protein kinaseSerum and glucocorticoid-regulated protein kinase Mm.28405Mm.28405 3232 Interferon-induced proteinInterferon-induced protein Mm.951Mm.951 3333 Milk fat globule membrane protein EGF factor 8Milk fat globule membrane protein EGF factor 8 Mm.1451Mm.1451 3434 Fc g receptorFc g receptor Mm.22119Mm.22119 3535 S100 calcium binding pretein A9 (calgranulin B)S100 calcium binding pretein A9 (calgranulin B) Mm.2128Mm.2128 3636 Arginase 1Arginase 1 Mm.154144Mm.154144 3737 Tumor necrosis factor receptor 1Tumor necrosis factor receptor 1 Mm.2666Mm.2666 3838 Retinoid-inducible serine carboxypeptidaseRetinoid-inducible serine carboxypeptidase Mm.34126Mm.34126 3939 Similar to hypothetical protein FLJ11000Similar to hypothetical protein FLJ11000 Mm.46356Mm.46356 4040 Interleukin-18 binding protein d precursorInterleukin-18 binding protein d precursor Mm.45579Mm.45579 4141 Chloride channel 7Chloride channel 7 Mm.22727Mm.22727 4242 CD36 antigenCD36 antigen Mm.18626Mm.18626 4343 Similar to putative zinc finger proteinSimilar to putative zinc finger protein Mm.205022Mm.205022 4444 Carbohydrate binding protein 35Carbohydrate binding protein 35 Mm.2970Mm.2970 4545 C-type calcium dependent, carbohydrateC-type calcium dependent, carbohydrate Mm.28263Mm.28263 4646 Lipoprotein lipaseLipoprotein lipase Mm.1514Mm.1514 4747 v-maf musculoaponeurotic fibrosarcoma oncogenev-maf musculoaponeurotic fibrosarcoma oncogene Mm.67919Mm.67919 4848 Interleukin-7 receptorInterleukin-7 receptor Mm.389Mm.389 4949 NeuropilinNeuropilin Mm.27448Mm.27448

5050 Peroxiredoxin 1Peroxiredoxin 1 Mm.30929Mm.30929 5151 la associated invariant chainla associated invariant chain Mm.248267Mm.248267 5252 Glycoprotein (transmembrane) nmbGlycoprotein (transmembrane) nmb Mm.23567Mm.23567 5353 Serum amyloid A (SAA) 3 proteinSerum amyloid A (SAA) 3 protein Mm.14277Mm.14277 5454 Sulfated glycoprotein (Sgp1)Sulfated glycoprotein (Sgp1) Mm.233010Mm.233010 5656 Response to metastatic cancers 1 (Rmcs1)Response to metastatic cancers 1 (Rmcs1) Mm.245067Mm.245067 5757 ATP-binding cassette, sub-family AATP-binding cassette, sub-family A Mm.369Mm.369 5858 Programmed cell death 1 ligand 1Programmed cell death 1 ligand 1 Mm.168681Mm.168681 5959 DnaJ (Hsp40)homolog (Dnajb11)DnaJ (Hsp40) homolog (Dnajb11) Mm.37516Mm.37516 6060 Fibronectin 1Fibronectin 1 Mm.193099Mm.193099 6161 Fatty acid binding protein 4Fatty acid binding protein 4 Mm.582Mm.582 6262 Complement component 1, q, gamma polypeptideComplement component 1, q, gamma polypeptide Mm.3453Mm.3453 6363 Fc g receptor, IgG, high affinity 1Fc g receptor, IgG, high affinity 1 Mm.150Mm.150 6464 IFN regulatory factor, IgG, high affinity 1IFN regulatory factor, IgG, high affinity 1 Mm.3233Mm.3233 6565 Complement component subunit Clq beta-chainComplement component subunit Clq beta-chain Mm.2570Mm.2570 6666 2'-5'oligoadenylate synthetase-like 22'-5'oligoadenylate synthetase-like 2 Mm.27162Mm.27162 6767 Chitinase 3-like 3 (Chi3l3)Chitinase 3-like 3 (Chi3l3) Mm.4571Mm.4571 6868 IFN-activated gene 203IFN-activated gene 203 Mm.245007Mm.245007 6969 Transforming growth factor, beta induced (Tgfbi)Transforming growth factor, beta induced (Tgfbi) Mm.14455Mm.14455 7070 Ig super family, member 7Ig super family, member 7 Mm.2699Mm.2699 7171 Chemokine (C-X-C motif) ligand 16Chemokine (C-X-C motif) ligand 16 Mm.46424Mm.46424 7272 Colony stimulating factor 2 receptor, beta 2, (Csf2rb2)Colony stimulating factor 2 receptor, beta 2, (Csf2rb2) Mm.1940Mm.1940 7373 H2-BfH2-Bf Mm.653Mm.653 7474 IFN-induced protein with tetratricopeptide repeats2IFN-induced protein with tetratricopeptide repeats2 Mm.2036Mm.2036 7575 RIKEN cDNA 2310005K03 geneRIKEN cDNA 2310005K03 gene Mm.20169Mm.20169 7676 RIKEN cDNA 3110037K17 geneRIKEN cDNA 3110037K17 gene Mm.25684Mm.25684 7777 RIKEN cDNA 5830458K16 geneRIKEN cDNA 5830458K16 gene Mm.180157Mm.180157 7878 Thymidylate kinase family LPS-inducible memberThymidylate kinase family LPS-inducible member Mm.1676Mm.1676 7979 Syntaxin binding protein 3Syntaxin binding protein 3 Mm.12155Mm.12155 8080 Fatty acid binding protein 5, epidermalFatty acid binding protein 5, epidermal Mm.741Mm.741 8181 IFN-stimulated gene 12 (Isg12)IFN-stimulated gene 12 (Isg12) Mm.46382Mm.46382 8282 Allograft inflammatory factor 1Allograft inflammatory factor 1 Mm.10747Mm.10747 8383 Biliverdin reductase B(flavin reductase(NADPH))Biliverdin reductase B (flavin reductase (NADPH)) Mm.24021Mm.24021 8484 Complement component 3a receptorComplement component 3a receptor Mm.2408Mm.2408 8585 Selenoprotein RSelenoprotein R Mm.28212Mm.28212 8686 IL-1 receptor antagonist (IL-1RA)IL-1 receptor antagonist (IL-1RA) Mm.882Mm.882 8787 Adenylosuccinate synthetaseAdenylosuccinate synthetase Mm.3440Mm.3440 8888 Hemopoietic cell kinaseHemopoietic cell kinase Mm.715Mm.715 8989 Death effector domain-containing DNA binding protein 2 (Dedd2)Death effector domain-containing DNA binding protein 2 (Dedd2) Mm.280594Mm.280594 9090 IFN induced transmembrane protein 2 likeIFN induced transmembrane protein 2 like Mm.175661Mm.175661 9191 GAVA-A receptor subunit 6GAVA-A receptor subunit 6 Mm.4915Mm.4915 9292 Selectin, platelet (p-selectin) ligand (Selpl)Selectin, platelet (p-selectin) ligand (Selpl) Mm.332590Mm.332590 9393 High mobility group box 1 (Hmgb1)High mobility group box 1 (Hmgb1) Mm.16421Mm.16421 9494 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptideTyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, epsilon polypeptide Mm.234700Mm.234700 9595 Serpinb6bSerpinb6b Mm.36526Mm.36526 9696 Regulator of G-protein signaling 16Regulator of G-protein signaling 16 Mm.181709Mm.181709 9797 RIKEN cDNA 6330406L22 gene (6330406L22Rik)RIKEN cDNA 6330406L22 gene (6330406L22Rik) Mm.243954Mm.243954 9898 Osteopontin precursorOsteopontin precursor Mm.321Mm.321 9999 Zinc finger protein 207Zinc finger protein 207 Mm.102253Mm.102253 100100 decay-accelerating factor GPI anchored mRNAdecay-accelerating factor GPI anchored mRNA Mm.101591Mm.101591 101101 Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting proteinAmyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein Mm.14255Mm.14255 102102 Glutaredoxin 1 (thioltransferase) (Glrx1)Glutaredoxin 1 (thioltransferase) (Glrx1) Mm.25844Mm.25844 103103 Coronin, actin binding protein 1A (Coro1a)Coronin, actin binding protein 1A (Coro1a) Mm.290482Mm.290482 104104 RIKEN cDNA 9630048M01 geneRIKEN cDNA 9630048M01 gene Mm.125242Mm.125242 105105 Dendritic cell protein GA17 (Ga17)Dendritic cell protein GA17 (Ga17) Mm.30078Mm.30078 106106 GTPase, IMAP family member 4 (Gimap4)GTPase, IMAP family member 4 (Gimap4) Mm.333048Mm.333048 107107 Upstream transcription factor 1Upstream transcription factor 1 Mm.8Mm.8 108108 RIKEN cDNA 1810015M01 gene (1810015M01Rik)RIKEN cDNA 1810015M01 gene (1810015M01Rik) Mm.27951Mm.27951 109109 Karyopherin (importin) alpha 6Karyopherin (importin) alpha 6 Mm.202358Mm.202358 110110 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 Mm.181933Mm.181933 111111 Kelch domain containing 2 (Klhdc2)Kelch domain containing 2 (Klhdc2) Mm.234368Mm.234368 112112 Structure specific recognition protein 1Structure specific recognition protein 1 Mm.219793Mm.219793 113113 Lymphocyte protein tyrosine kinaseLymphocyte protein tyrosine kinase Mm.293753Mm.293753 114114 F-box and leucine-rich repeat protein 14F-box and leucine-rich repeat protein 14 Mm.125477Mm.125477 115115 Osteoclastogenesis inhibitory factorOsteoclastogenesis inhibitory factor Mm.15383Mm.15383 116116 G protein-coupled receptor 171G protein-coupled receptor 171 Mm.123648Mm.123648 117117 RNA terminal phosphate cyclase domain 1RNA terminal phosphate cyclase domain 1 Mm.251228Mm.251228 118118 Cullin 2Cullin 2 Mm.291707Mm.291707 119119 CD160 antigenCD160 antigen Mm.34693Mm.34693 120120 Hypothetical RNA-binding domain, RBD structureHypothetical RNA-binding domain, RBD structure Mm.22379Mm.22379 121121 Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6DSema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D Mm.330536Mm.330536 122122 Activating signal cointegrator 1 complex subunit 3 like 1Activating signal cointegrator 1 complex subunit 3 like 1 Mm.215860Mm.215860 123123 Abl-interactor 1Abl-interactor 1 Mm.205647Mm.205647 124124 Hypothetical protein MGC36662Hypothetical protein MGC36662 Mm.61526Mm.61526 125125 RAP2C, member of RAS oncogene familyRAP2C, member of RAS oncogene family Mm.43152Mm.43152 126126 Valosin containing protein( Vcp)Valosin containing protein (Vcp) Mm.371711Mm.371711 127127 CD164 antigenCD164 antigen Mm.269815Mm.269815 128128 Similar to RIKEN cDNA 4933425K02 geneSimilar to RIKEN cDNA 4933425K02 gene Mm.85000Mm.85000 129129 AdvillinAdvillin Mm.10739Mm.10739 130130 Asparagine synthetaseAsparagine synthetase Mm.2942Mm.2942 131131 Zinc finger protein 259Zinc finger protein 259 Mm.17519Mm.17519 132132 Interleukin 12 receptor, beta 1Interleukin 12 receptor, beta 1 Mm.731Mm.731 133133 Growth arrest and DNA-damage-inducible 45 gammaGrowth arrest and DNA-damage-inducible 45 gamma Mm.9653Mm.9653 134134 Cytochrome b5 type BCytochrome b5 type B Mm.20242Mm.20242 135135 FMS-like tyrosine kinase 3 ligandFMS-like tyrosine kinase 3 ligand Mm.1658Mm.1658 136136 Programmed cell death 1 ligand 2Programmed cell death 1 ligand 2 Mm.116737Mm.116737 137137 S100 calcium binding protein A4S100 calcium binding protein A4 Mm.3925Mm.3925 138138 Metallothionein 2Metallothionein 2 Mm.142740Mm.142740 139139 Peptidylprolyl isomerase APeptidylprolyl isomerase A Mm.5246Mm.5246 140140 Osteopontin (OPN)Osteopontin (OPN) Mm.288474Mm.288474 141141 Chemokine (C-C motif) ligand 4Chemokine (C-C motif) ligand 4 Mm.1255Mm.1255 142142 T-cell receptor beta, variable 13 (Tcrb-V13)T-cell receptor beta, variable 13 (Tcrb-V13) Mm.333026Mm.333026 143143 Immune associated nucleotide 1Immune associated nucleotide 1 Mm.28395Mm.28395 144144 Interleukin 2 receptor, beta chain (Il2rb)Interleukin 2 receptor, beta chain (Il2rb) Mm.931Mm.931 145145 Cd28: CD28 antigenCD28: CD28 antigen Mm.255003Mm.255003 146146 Granzyme EGranzyme e Mm.14424Mm.14424 147147 serin protease inhibitorserin protease inhibitor Mm.45371Mm.45371 148148 Purine rich element binding protein A (Pura)Purine rich element binding protein A (Pura) Mm.259715Mm.259715 149149 Lymphocyte protein tyrosine kinase (Lck)Lymphocyte protein tyrosine kinase (Lck) Mm.142Mm.142 150150 Eukaryotic translation initiation factor 4 gamma, 3 (Eif4g3)Eukaryotic translation initiation factor 4 gamma, 3 (Eif4g3) Mm.268903Mm.268903 151151 Lamin A (Lmna)Lamin a (lmna) Mm.243014Mm.243014 152152 Raver1: Ribonucleoprotein, PTB-binding 1Raver1: Ribonucleoprotein, PTB-binding 1 Mm.196137Mm.196137 153153 Solute carrier family 6Solute carrier family 6 Mm.200518Mm.200518 154154 V-ral simian leukemia viral oncogene homolog A (ras related)V-ral simian leukemia viral oncogene homolog A (ras related) Mm.27348Mm.27348 155155 Fyn: Fyn proto-oncogeneFyn: Fyn proto-oncogene Mm.4848Mm.4848 156156 Expressed sequence AV024533Expressed sequence AV024533 Mm.46750Mm.46750 157157 Fhl2: Four and a half LIM domains 2Fhl2: Four and a half LIM domains 2 Mm.6799Mm.6799 158158 C1q and tumor necrosis factor related protein 6 (C1qtnf6)C1q and tumor necrosis factor related protein 6 (C1qtnf6) Mm.34776Mm.34776 159159 Zfp512: Zinc finger protein 512Zfp512: Zinc finger protein 512 Mm.259127Mm.259127 160160 P2ry10: Purinergic receptor P2Y, G-protein coupled 10P2ry10: Purinergic receptor P2Y, G-protein coupled 10 Mm.74639Mm.74639 161161 Protein phosphatase 1, regulatory (inhibitor) subunit 14B (Ppp1r14b)Protein phosphatase 1, regulatory (inhibitor) subunit 14B (Ppp1r14b) Mm.318969Mm.318969 162162 Strap: Serine/threonine kinase receptor associated proteinStrap: Serine / threonine kinase receptor associated protein Mm.22584Mm.22584 163163 Hdac3: Histone deacetylase 3Hdac3: Histone deacetylase 3 Mm.20521Mm.20521 164164 Ppp3cb: Protein phosphatase 3, catalytic subunit, beta isoformPpp3cb: Protein phosphatase 3, catalytic subunit, beta isoform Mm.274432Mm.274432 165165 Mmp9: Matrix metallopeptidase 9Mmp9: Matrix metallopeptidase 9 Mm.4406Mm.4406 166166 Ifnar2: Interferon (alpha and beta) receptor 2Ifnar2: Interferon (alpha and beta) receptor 2 Mm.6834Mm.6834 167167 Prefoldin 1Prefoldin 1 Mm.30184Mm.30184 168168 GTP-binding protein(mSara) homologue mRNAGTP-binding protein (mSara) homologue mRNA Mm.6698Mm.6698 169169 Copine 1(Rbm 12)Copine 1 (Rbm 12) Mm.27660Mm.27660 170170 Pretin phosphatase 3, catalytic subunit, gamma isoform (Ppp3cc)Pretin phosphatase 3, catalytic subunit, gamma isoform (Ppp3cc) Mm.1567Mm.1567 171171 Cyclin B2Cyclin B2 Mm.22592Mm.22592 172172 Pip5k1b: Phosphatidylinositol-4-phosphate 5-kinase, type 1 betaPip5k1b: Phosphatidylinositol-4-phosphate 5-kinase, type 1 beta Mm.296409Mm.296409 173173 Mlf2: Myeloid leukemia factor 2Mlf2: Myeloid leukemia factor 2 Mm.29737Mm.29737 174174 Retinoblastoma-like 2Retinoblastoma-like 2 Mm.235580Mm.235580 175175 Ppp6c: Protein phosphatase 6, catalytic subunitPpp6c: Protein phosphatase 6, catalytic subunit Mm.171389Mm.171389

실시예Example 2. 2. 케모카인Chemokines 수용체 1( Receptor 1 ( chemokinechemokine receptorreceptor 1)의 항체를 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using the antibody of 1) 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포로부터 자연살해세포의 분화 (A) Differentiation of natural killer cells from human cord blood-derived hematopoietic stem cells

제대혈로부터 다음의 방법을 이용하여 조혈모세포를 분리하였다. 50ml 튜브에 사람 제대혈을 25ml씩 나눠 담은 후(1팩=100ml) 1X PBS를 동량(25ml) 넣어주고 천천히 혼합하였다. 새 50ml 튜브에 Picoll을 20ml씩 나눠 담은 후 사람 제대혈과 1XPBS를 1:1로 섞은 것을 층이 훼손되지 않도록 부어 50ml까지 채웠다. 2000rpm, 25℃로 20분간 원심분리(브레이크를 푼 상태)하였다. 원심분리가 끝나고 나면 맨 위층인 노란색의 상층(혈청) 부분과 밑의 맑은 부분 사이에 있는 하얗게 보이는 층(세포층=약10ml)만 따로 얻어내서 1XPBS 20ml이 들어있는 50ml 튜브로 옮긴 후 2000rpm, 25℃에서 10분간 원심분리 하였다. 밑에 가라앉은 펠릿(Pellet)이 확인되면 상등액을 버리고 펠릿을 손으로 두드려 풀어준 다음, ACK 완충용액(0.15 M NH4Cl, 1mM KHCO3, 0.1 mM EDTA(disodium salt), pH 7.2)을 10ml 정도 넣어 섞은 후 37℃에 10분간 방치하였다. 2500rpm에서 4℃, 10분간 원심분리한 후에 상등액을 버리고 튜브를 손으로 쳐서 펠릿을 풀어 주었다. MACS 완충용액(2mM EDTA와 0.5% BSA를 1XPBS에 녹인 다음 여과함)을 약 2ml을 넣어서 피펫으로 부유시켜 새로운 50ml 튜브에 얹어진 여과기에 거른 후 세포가 담겨있던 튜브에 다시 MACS 완충용액을 10ml정도 넣어서 세척하고 여과기에 거르는 작업을 반복하여 50ml을 채웠다. Hematopoietic stem cells were isolated from umbilical cord blood using the following method. After 25ml of human cord blood was divided into 50ml tubes (1pack = 100ml), 1X PBS was added in the same amount (25ml) and mixed slowly. After dividing 20ml of Picoll into a new 50ml tube, a 1: 1 mixture of human cord blood and 1XPBS was poured to prevent damage to the layer and filled to 50ml. It was centrifuged at 2000 rpm and 25 degreeC for 20 minutes (brake | released). After the centrifugation, only a white layer (cell layer = about 10ml) between the upper yellow layer (serum) and the lower clear layer is separated and transferred to a 50ml tube containing 20ml of 1XPBS. Centrifuge for 10 minutes at. When the pellet is found to be underneath, discard the supernatant, tap the pellet to loosen it, and then mix with ACK buffer solution (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA (disodium salt), pH 7.2). It was left to stand at 37 degreeC for 10 minutes. After centrifugation at 2500 rpm for 4 minutes at 4 ° C, the supernatant was discarded and the pellet was struck by a tube. Add about 2ml of MACS buffer solution (2mM EDTA and 0.5% BSA dissolved in 1XPBS and filter), and pipet the mixture. Filter it with a new 50ml tube and filter it. Add 10 ml of MACS buffer to the tube containing cells. 50 ml was filled by repeating the filter and straining the filter.

얻어진 50ml 튜브 안에 담겨진 세포를 세포수 측정기(HEMOCYTOMETER, MARIENFELD)를 이용하여 세포수 측정을 하였다. 2000rpm으로 4℃에서 10분간 원심분리 한 다음 세포수를 측정해서 얻은 결과와 비교하여 1X10⁸을 한 번의 반응으로 계산해서 반응 수에 맞게 MACS 완충용액을 1ml씩 넣고 펠릿을 풀어준 뒤 반응 수에 맞게 준비된 1.5 ml 튜브에 나눠 담았다. 1700rpm, 3분씩 원심분리한 뒤 상등액을 버리고 완충용액 이용해 펠릿을 풀어주는 방법으로 펠릿을 씻어주는 것을 3번 반복하였다. 마지막엔 500㎕ MACS 완충용액를 넣어주고 펠릿을 풀어준 다음 각 1.5ml 튜브 별로 CD34⁺ micro bead-25㎕와 블로킹 시약-25㎕를 각각 넣고 섞어주었다. 4℃에서 30분간 반응한 후 튜브를 위아래로 5분마다 10회 뒤집어 주었다. 30분이 지나면 1700rpm으로 3분간 원심분리하고 상등액을 버리는 세척을 3번 반복하고 마지막에 MACS 완충용액 500㎕ 넣어준 후 펠릿을 풀어주었다. MACS 컬럼을 MACS에 설치한 후 MACS 완충용액 3ml로 세척해 주었다. 완충용액이 완전히 다 내려가기 전에 펠릿을 풀어준 것을 조심스럽게 부어주고 펠릿이 다 내려갔으면 다시 MACS 완충용액을 5ml 내려주고 다시 MACS 완충용액 2ml을 컬럼에 넣어 다시 걷어내었다. The cells contained in the obtained 50 ml tube were measured for cell number using a cell counting instrument (HEMOCYTOMETER, MARIENFELD). After centrifugation at 2000C for 10 minutes at 4 ℃, compare 1C10 ⁸ with one reaction and compare the results with the result of measuring the cell number. Divided into prepared 1.5 ml tubes. After centrifugation at 1700 rpm for 3 minutes, the supernatant was discarded and the pellet was washed three times by releasing the pellet using a buffer solution. Finally, 500 μl MACS buffer solution was added, the pellets were released, and each 1.5 ml tube was mixed with 25 μl CD34 ⁺ micro bead-25 μl and blocking reagent 25 μl. After reaction at 4 ° C. for 30 minutes, the tube was turned upside down 10 times every 5 minutes. After 30 minutes, centrifuged at 1700 rpm for 3 minutes, and the supernatant was discarded three times. Finally, 500 µl of MACS buffer solution was added, and the pellet was released. MACS column was installed in MACS and washed with 3 ml of MACS buffer. Carefully pour out the pellets before the buffer is completely down, and when the pellets are all down again, 5 ml of the MACS buffer was lowered again, and 2 ml of the MACS buffer was added to the column and again removed.

컬럼을 MACS에서 분리해 낸 다음, MACS 완충용액 5ml을 넣어주고 피스톤으로 15ml 튜브에 밀어내고 한 번 더 MACS 완충용액 5ml 넣어주고 밀어낸 다음 얻어낸 것을 세포수 측정을 해서 24 웰 배양용기에 조혈모세포 배양액(사람 SCF-30ng/ml(Pepro Tech), 사람 FLT3L50ng/ml(Pepro Tech), 사람 인터루킨-7 10ng/ml,(Pepro Tech)) 1ml에 웰당 1X10⁶ 세포/ml 씩 분주한 뒤 37℃, 5% CO₂ 배양기에서 배양하였다. 분리한 사람 조혈모세포로부터 전구자연살해세포로 분화시키기 위해 분리된 CD34⁺세포를 사람 SCF(30ng/ml), Flt-3L(50ng/ml), 인터루킨-7(10ng/ml)를 함유한 Myelocult H5100 (Gibco) 배지에 1x10⁶/ml의 농도로 현탁시켜 24 웰 배양용기에 배양하였다. 3일마다 절반의 배지를 신선한 배지로 바꾸어 주면서 계대배양 하였다. 이 세포들을 14일간 배양하여 사람 전구자연살해세포를 얻었다. 사람 전구자연살해세포로부터 성숙자연살해세포로 분화시키기 위해 배양 14일 후 생성된 전구자연살해세포를 사람 인터루킨-15(20ng/ml, Pepro Tech)를 함유하는 MyeloCult 배지(Gibco)에 제대혈로부터 분리한 기질세포(분리방법: 제대혈에서 조혈모세포 분리 후 CD34^-한 세포를 10%RPMI 배지에 평판하여 2 시간동안 5%(CO₂) 배양기에서 배양하였다. 배양액에 떠있는 세포만을 10%RPMI 배지를 이용하여 새로운 배양용기에 평판하였다. 4일, 7일 후 배양액을 교체한 후 8일째 되는 날 배지를 제거하고 1XPBS 3ml을 넣고배양기에서 5분간 방치하였다. 트립신-EDTA 1ml을 1분간 처리 한 후 10% RPMI 배지를 이용하여 세포를 세척하였다. 원심분리를 하여 세포를 모은 후 기질세포를 24 웰 당 4×10⁴씩 평판하였다)와 공동배양 하여 성숙자연살해세포를 획득하였다. 각 분화단계별 세포의 순도 (purity)를 다음과 같은 방법을 통해 확인하였다. 조혈모세포로부터 성숙자연살해세포 까지 분화단계별 세포들의 특성을 여러 가지 세포표면 분자들에 대한 항체를 이용하여 면역염색하기 위하여 1x10⁶ 세포로 조정하여 염색 완충용액(20mM HEPES, 3% 우태아혈청, 0.1% NaN3가 포함된 인산염 완충용액, pH 7.4)으로 1회 세척하였다. 이를 FITC(fluoresceinated isothiocyanate, 플루오레세인이 붙어 있는 이소티오시안산염) 또는 PE(phycoerythrin, 홍조소) 형광물질이 결합된 자연살해세포 분화단계별 마커 항체를 세포시료에 가하여 0℃에서 30분간 반응시키고 염색 완충용액으로 2회 세척한 후 FACS(BD/Aria)로 분석하였다. The column was separated from the MACS, and then 5 ml of MACS buffer was added and the piston was pushed out into a 15 ml tube, and then 5 ml of the MACS buffer was again pushed out, and the obtained cell number was measured. (Human SCF-30ng / ml (Pepro Tech), human FLT3L50ng / ml (Pepro Tech), human Interleukin-7 10ng / ml, (Pepro Tech)) in a 1ml aliquot of 1X10 ⁶ cells / ml per well, 37 ° C, 5 Cultured in a% CO ₂ incubator. CD34 ⁺ cells isolated from human hematopoietic stem cells to progenitor killer cells were Myelocult H5100 containing human SCF (30 ng / ml), Flt-3L (50 ng / ml) and interleukin-7 (10 ng / ml). (Gibco) medium was suspended in a concentration of 1x10 ⁶ / ml and cultured in a 24-well culture vessel. Every three days, half of the medium was subcultured with fresh medium. These cells were cultured for 14 days to obtain human progenitor killer cells. Progenitor killer cells generated after 14 days of culture to differentiate from human progenitor killer cells to mature killer cells were isolated from cord blood in MyeloCult medium (Gibco) containing human interleukin-15 (20 ng / ml, Pepro Tech). Stromal Cells (Isolation Method: CD34 ⁻ Cells After Separation of Hematopoietic Stem Cells from Umbilical Cord Blood and Plated in 10% RPMI Medium and Incubated in 5% (CO ₂ ) Incubator for 2 hours. After 4 days and 7 days, the medium was removed and the medium was removed on the 8th day, and 3 ml of 1XPBS was added and left in the incubator for 5 minutes. The cells were washed using RPMI medium, and cells were collected by centrifugation and co-cultured with stromal cells 4 × 10 ⁴ per 24 wells) to obtain mature natural killer cells. Purity of the cells for each differentiation stage was confirmed by the following method. Characterization of cells in differentiation stages from hematopoietic stem cells to mature natural killer cells was adjusted to 1x10 ⁶ cells for immunostaining using antibodies against various cell surface molecules, and staining buffer solution (20 mM HEPES, 3% fetal bovine serum, 0.1 Phosphate buffer with% NaN3, pH 7.4). It was reacted for 30 minutes at 0 ° C. by adding a marker antibody for each step of differentiating cell differentiation to which FITC (fluoresceinated isothiocyanate (fluorescein attached isothiocyanate) or PE (phycoerythrin) was added to the cell sample. After washing twice with solution it was analyzed by FACS (BD / Aria).

(B) 사람 제대혈 유래 조혈모세포에서 케모카인 수용체의 폴리클로날 항체를 이용한 성숙자연살해세포 분화법(B) Mature natural killer cell differentiation using polyclonal antibody of chemokine receptor in human cord blood-derived hematopoietic stem cells

(A)에서 얻은 전구자연살해세포를 (A)와 동일 조건에 인터루킨-15를 처리하는 대신 케모카인 수용체의 폴리클로날 항체 1㎍/ml(Santa Cruz)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 (A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. 사람에 대한 성숙자연살해세포 마커로는 NKG2A(Pharmingen), CD161(Pharmingen), NKP46(Pharmingen), NKP30(Pharmingen), NKP44 (Pharmingen), NKG20(Pharmingen) 그리고 CD56 (Pharmingen)을 사용하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 염소항체를 처리한 대조군에 비해 케모카인 수용체의 폴리클로날 항체를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Progenitor killer cells obtained in (A) were differentiated for 14 days by adding 1 µg / ml of polyclonal antibody of chemokine receptor (Santa Cruz) instead of interleukin-15 treatment under the same conditions as (A). Natural killer cells were analyzed by FACS in the same manner as in (A) to confirm their differentiation. The markers of mature natural killer cells for humans were NKG2A (Pharmingen), CD161 (Pharmingen), NKP46 (Pharmingen), NKP30 (Pharmingen), NKP44 (Pharmingen), NKG20 (Pharmingen) and CD56 (Pharmingen). CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 to 3 times higher differentiation rate when treated with polyclonal antibody of chemokine receptor compared to goat antibody treated control group.

추가로, 동일조건하에 전구자연살해세포를 케모카인 수용체의 폴리클로날 항체를 인터루킨-15(Pepro Tech)와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 케모카인 수용체의 폴리클로날 항체를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the proliferative killer cells were treated with a polyclonal antibody of chemokine receptor in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15 (Pepro Tech) to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the polyclonal antibody of chemokine receptor alone.

(C) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 케모카인 수용체의 폴리클로날 항체에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(C) Identification of anticancer activity by mature natural killer cells induced by differentiation by polyclonal antibody of chemokine receptor in human cord blood-derived hematopoietic stem cells in animal cancer model

케모카인 수용체의 폴리클로날 항체를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 5주령의 수컷 누드마우스(Nude Balb/c mouse, 면역기능이 결핍된 유전자변형 마우스, 오리엔트 바이오)를 이용하여 항암 활성을 측정해 보았다. 누드마우스는 무균시설이 갖추어진 동물실에서 동물 관리 지침에 따라 관리하였다. 사람 유래의 암세포(위암세포,KCLB no.00638; 자궁암세포,KCLB no.10002; 유방암세포, KCLB no.30022; 신장암 세포, KCLB no.30044; 흑색종 암세포, KCLB no.30068; 폐암 세포, KCLB no.30053; 난소암 세포, KCLB no.30077;를 포함한 여러 종류)를 10% 우태아 혈청이 포함된 RPMI 1640 세포 배양액(Gibco)을 이용하여 37℃, CO₂ 배양기에서 배양하였다. 배양한 암세포를 1XPBS로 세척한 후 트립신-EDTA(Gibco)를 처리하여 암세포를 회수하였다. 세포로부터 트립신-EDTA를 제거하고 1XPBS로 세포를 부유시켜 10⁸ 세포/0.1㎕이 되도록 세포수를 측정하여 준비하였다. 10⁸개의 암세포를 누드마우스의 피하에 인슐린 주사기(Pharmingen)를 이용해 주입하고 직경 약 0.8mm가 될 때까지 1주 정도 암세포를 키웠다. 형성된 암 조직에 케모카인 수용체의 폴리클로날 항체를 이용하여 분화시킨 사람 성숙자연살해세포(1x10⁶)를 사람 인터루킨-2(10ng/ml)로 활성화시킨 다음 암조직에 주입하였다. 시간 별로 암조직의 부피를 측정하여 관찰한 결과 염소항체를 처리하여 분화시킨 성숙자연살해세포를 주입한 대조군에 비해 케모카인 수용체의 폴리클로날 항체를 이용하여 분화시킨 사람 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. To examine whether human cancer cells are killed by human mature natural killer cells differentiated using polyclonal antibodies of chemokine receptors, 5 week old male nude mice (Nude Balb / c mouse, transgenic mice lacking immune function, Orient Bio) was used to measure the anticancer activity. Nude mice were administered in accordance with animal care guidelines in a sterile animal room. Human-derived cancer cells (gastric cancer cells, KCLB no.00638; uterine cancer cells, KCLB no.10002; breast cancer cells, KCLB no.30022; kidney cancer cells, KCLB no.30044; melanoma cancer cells, KCLB no.30068; lung cancer cells, KCLB no. 30053; various types including ovarian cancer cells, KCLB no. 30077;) were cultured in a CO ₂ incubator at 37 ° C. using RPMI 1640 cell culture medium (Gibco) containing 10% fetal bovine serum. The cultured cancer cells were washed with 1XPBS and then treated with trypsin-EDTA (Gibco) to recover the cancer cells. Trypsin-EDTA was removed from the cells, the cells were suspended in 1XPBS, and prepared by measuring the cell number to 10 ⁸ cells / 0.1 μl. 10 ⁸ cancer cells were injected subcutaneously with nude mice using an insulin syringe (Pharmingen), and cancer cells were grown for about 1 week until the diameter was about 0.8 mm. Human mature natural killer cells (1 × 10 ⁶ ) differentiated using polyclonal antibodies of chemokine receptors were activated into human interleukin-2 (10 ng / ml) and then injected into cancer tissues. As a result of measuring the volume of cancer tissues by time, the group injected with human natural killer cells differentiated using polyclonal antibody of chemokine receptor, compared to the control group injected with mature natural killer cells differentiated with goat antibody. Significant decreases in cancer tissue volume were observed in.

실시예Example 3. 3. 케모카인Chemokines 수용체의 Receptor 리간드인Ligand CCL3CCL3 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 케모카인 수용체의 리간드인 CCL3를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using CCL3, a ligand of chemokine receptor, in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 케모카인 수용체의 리간드인 CCL3를 1㎍/ml(R&D systems)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 케모카인 수용체의 리간드인 CCL3를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as in Example 2 (A), and then 14 days by adding 1 µg / ml (R & D systems) of CCL3, a ligand for the chemokine receptor, After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with CCL3, a ligand for the chemokine receptor, compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 케모카인 수용체의 리간드인 CCL3를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 케모카인 수용체의 리간드인 CCL3를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with CCL3, a ligand of the chemokine receptor, with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio to confirm the differentiation rate. As a result, it was shown that the differentiation rate was slightly increased compared to the case of treating CCL3, a ligand of the chemokine receptor alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 케모카인 수용체의 리간드인 CCL3에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation induced by CCL3, a ligand of chemokine receptor, in human cord blood-derived hematopoietic stem cells in animal cancer model

케모카인 수용체의 리간드인 CCL3를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 케모카인 수용체의 리간드인 CCL3를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using CCL3, a ligand of the chemokine receptor, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). In contrast to the injection of the NK cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using CCL3, a ligand of the chemokine receptor.

실시예Example 4. 4. 케모카인Chemokines 수용체의 Receptor 리간드인Ligand CCL5CCL5 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 케모카인 수용체의 리간드인 CCL5를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using CCL5, a ligand of chemokine receptor, in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 케모카인 수용체의 리간드인 CCL5를 1㎍/ml(R&D systems)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 케모카인 수용체의 리간드인 CCL5를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as in Example 2 (A), and then 14 days by adding 1 µg / ml (R & D systems) of CCL5, a ligand for the chemokine receptor, After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three fold increase in the differentiation rate when treated with CCL5, a ligand for the chemokine receptor, compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 케모카인 수용체의 리간드인 CCL5를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 케모카인 수용체의 리간드인 CCL5를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with CCL5, a ligand of the chemokine receptor, with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio to confirm the differentiation rate. As a result, it was shown that the differentiation rate was slightly increased compared to the case of treating CCL5, a ligand of chemokine receptor alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 케모카인 수용체의 리간드인 CCL5에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation induced by CCL5, a ligand of chemokine receptor, in human cord blood-derived hematopoietic stem cells in animal cancer model

케모카인 수용체의 리간드인 CCL5를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않고 분화시킨 성숙자연살해세포를 주입한 대조군에 비해 케모카인 수용체의 리간드인 CCL5를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using CCL5, a ligand of a chemokine receptor, the mature natural killer cells differentiated without treatment were treated in the same manner as in Example 2 (C). Compared to the control group injected with, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using CCL5, a ligand of the chemokine receptor.

실시예Example 5. 5. 콜로니Colony 자극인자Stimulus 요소 1 수용체( Factor 1 receptor ( colonycolony stimulatingstimulating factorfactor 1 One receptorreceptor )의 항체를 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using an antibody of 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 콜로니 자극인자 요소 1 수용체의 폴리클로날 항체를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using polyclonal antibody of colony stimulating factor element 1 receptor in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 콜로니 자극인자 요소 1 수용체의 폴리클로날 항체를 1㎍/ml(Santa Cruz)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 염소항체를 처리한 대조군에 비해 콜로니 자극인자 요소 1 수용체의 폴리클로날 항체를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 under the same conditions as Example 2 (A), the progenitor killer cells obtained in Example 2 (A) were treated with 1 μg / ml polyclonal antibody of colony stimulating factor element 1 receptor (Santa Cruz). After 14 days of differentiation with addition of human mature natural killer cells, the degree of differentiation was confirmed by FACS analysis in the same manner as in Example 2 (A). CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with colony-stimulating factor urea 1 polyclonal antibodies compared to goat-treated controls.

추가로, 동일조건하에 전구자연살해세포를 콜로니 자극인자 요소 1 수용체의 폴리클로날 항체를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 콜로니 자극인자 요소 1 수용체의 폴리클로날 항체를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with a polyclonal antibody of the colony stimulating factor element 1 receptor in an amount of 1 µg / ml at a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the polyclonal antibody of the colony stimulating factor element 1 receptor alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 콜로니 자극인자 요소 1 수용체의 폴리클로날 항체에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명 (B) Identification of anticancer activity by mature natural killer cells induced by differentiation by polyclonal antibody of colony stimulating factor element 1 receptor in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

콜로니 자극인자 요소 1 수용체의 폴리클로날 항체를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 염소항체를 처리하여 분화시킨 성숙자연살해세포를 주입한 대조군에 비해 콜로니 자극인자 요소 1 수용체의 폴리클로날 항체를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using polyclonal antibody of colony stimulating factor element 1 receptor, the goat antibody was treated in the same manner as in Example 2 (C). Compared to the control group injected with differentiated natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using polyclonal antibody of the colony stimulating factor element 1 receptor.

실시예Example 6. 6. 콜로니Colony 자극 인자 1 수용체의 Of stimulating factor 1 receptors 리간드인Ligand CSF1CSF1 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 콜로니 자극 인자 1 수용체의 리간드인 CSF1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using CSF1, a ligand of colony stimulating factor 1 receptor, in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 CSF1를 1㎍/ml(R&D systems)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 CSF1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (R & D systems) of CSF1 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with CSF1 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 CSF1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 CSF1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with CSF1 with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the CSF1 treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 콜로니 자극 인자 1 수용체의 리간드인 CSF1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation induced by CSF1, a ligand of colony stimulating factor 1 receptor, in human umbilical cord blood-derived hematopoietic stem cells

콜로니 자극 인자 1 수용체의 리간드인 CSF1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 CSF1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Maturation of human maturation cells differentiated using CSF1, a ligand of colony stimulating factor 1 receptor, in the same manner as in Example 2 (C) to observe whether human cancer cells are killed by natural killer cells. Compared with the injection of natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using CSF1.

실시예Example 7. 형질전환 성장 인자 베타 수용체 II( 7. Transforming Growth Factor Beta Receptor II ( TransformingTransforming growthgrowth factorfactor , , betabeta receptor receptor IIII )의 항체를 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using an antibody of 성숙자연살해세Mature Natural Killing 포의 분화 Eruption of guns

(A) 사람 제대혈 유래 조혈모세포에서 형질전환 성장 인자 베타 수용체 II의 폴리클로날 항체를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using polyclonal antibody of transforming growth factor beta receptor II in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 형질전환 성장 인자 베타 수용체 II의 폴리클로날 항체 1㎍/ml(Santa Cruz)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 염소항체를 처리한 대조군에 비해 형질전환 성장 인자 베타 수용체 II의 폴리클로날 항체를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. 1 μg / ml polyclonal antibody of transforming growth factor beta receptor II, instead of treating interleukin-15 under the same conditions as Example 2 (A), with the pro natural killer cells obtained in Example 2 (A) (Santa Cruz) After 14 days of differentiation with addition of human mature natural killer cells, the degree of differentiation was confirmed by FACS analysis in the same manner as in Example 2 (A). CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and One CD56 ⁺ NKG20 ⁺ cells showed a two to three fold increase in differentiation rate when treated with the polyclonal antibody of transforming growth factor beta receptor II compared to the goat antibody treated control group.

추가로, 동일조건하에 전구자연살해세포를 형질전환 성장 인자 베타 수용체 II의 폴리클로날 항체를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 형질전환 성장 인자 베타 수용체II의 폴리클로날 항체를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the proliferative killer cells were treated with a polyclonal antibody of transforming growth factor beta receptor II in an amount of 1 µg / ml at a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the polyclonal antibody of transforming growth factor beta receptor II alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 형질전환 성장 인자 베타 수용체II의 폴리클로날 항체에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명 (B) Identification of anticancer activity by mature natural killer cells induced by differentiation by polyclonal antibody of transforming growth factor beta receptor II in human cord blood-derived hematopoietic stem cells in animal cancer model

형질전환 성장 인자 베타 수용체II의 폴리클로날 항체를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 염소항체를 처리하여 분화시킨 성숙자연살해세포를 주입한 대조군에 비해 형질전환 성장 인자 베타 수용체II의 폴리클로날 항체를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using polyclonal antibody of transforming growth factor beta receptor II, goat antibodies were treated in the same manner as in Example 2 (C). Compared to the control group injected with the differentiated natural killer cells differentiated, a significant decrease in the volume of cancer tissue was observed in the group injected with the mature natural killer cells differentiated using the polyclonal antibody of transforming growth factor beta receptor II.

실시예Example 8. 8. 형질전환 성장 인자 베타Transformation Growth Factor Beta 수용체Receptor IIII 의of 리간드인Ligand TGFTGF -베타를 이용한 사람 제대혈 유래 조혈모세포로부터 From Beta Cord-derived Hematopoietic Stem Cells Using Beta 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 형질전환 성장인자 베타 수용체 II의 리간드인 TGF-베타를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using TGF-beta, a ligand of transforming growth factor beta receptor II, in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 TGF-베타를 1㎍/ml(R&D systems)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 CSF1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (R & D systems) of TGF-beta instead of interleukin-15 in the same conditions as in Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with CSF1 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 TGF-베타를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 TGF-베타를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with TGF-beta with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to TGF-beta alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 형질전환 성장인자 베타 수용체 II의 리간드인 TGF-베타에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation induced by TGF-beta, a ligand of transforming growth factor beta receptor II in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

형질전환 성장인자 베타 수용체 II의 리간드인 TGF-베타를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 TGF-베타를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using TGF-beta, a ligand of transforming growth factor beta receptor II, the experiment was performed in the same manner as in Example 2 (C). A significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using TGF-beta compared to the group injected with mature natural killer cells in the control group.

실시예Example 9. 9. 에스테로젠Esterogen 수용체 베타 1( Receptor beta 1 ( EstrogenEstrogen relatedrelated receptorreceptor betabeta likelike 1, Esrrbl1)의 항체를 이용한 사람 제대혈 유래 조혈모세포로부터 1, from human umbilical cord blood-derived hematopoietic stem cells using an antibody of Esrrbl1) 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Esrrbl1의 폴리클로날 항체를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using polyclonal antibody of Esrrbl1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Esrrbl1의 폴리클로날 항체 1㎍/ml(ABR-Affinity BioReagents)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 염소항체를 처리한 대조군에 비해 Esrrbl1의 폴리클로날 항체를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 μg / ml of Esrrbl1 polyclonal antibody (ABR-Affinity BioReagents) instead of treating Interleukin-15 under the same conditions as in Example 2 (A). After differentiation for one day, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with Esrrbl1 polyclonal antibody compared to goat-treated controls.

추가로, 동일조건하에 전구자연살해세포를 Esrrbl1의 폴리클로날 항체를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Esrrbl1의 폴리클로날 항체를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Esrrbl1 polyclonal antibody in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared with the polyclonal antibody of Esrrbl1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Esrrbl1의 폴리클로날 항체에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명 (B) Identification of anticancer activity by mature natural killer cells induced by differentiation by polyclonal antibody of Esrrbl1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Esrrbl1의 폴리클로날 항체를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 염소항체를 처리하여 분화시킨 성숙자연살해세포를 주입한 대조군에 비해 Esrrbl1의 폴리클로날 항체를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using polyclonal antibody of Esrrbl1, the mature natural killer killed by treatment with goat antibodies was tested in the same manner as in Example 2 (C). Compared to the control group injected with cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using Esrrbl1 polyclonal antibody.

실시예Example 10. 10. 에스테로젠Esterogen 수용체 베타 1( Receptor beta 1 ( EstrogenEstrogen relatedrelated receptorreceptor betabeta likelike 1, Esrrbl1) 1, Esrrbl1) 의of 리간드인Ligand 스테로이드성Steroid 인자-1Factor-1 (( steroidogenicsteroidogenic factorfactor -1)을 이용한 사람 제대혈 유래 조혈모세포로부터 -1) from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 스테로이드성 인자-1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using steroidal factor-1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 스테로이드성 인자-1을 1㎍/ml을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 스테로이드성 인자-1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml of steroidal factor-1 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with steroidal factor-1 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 스테로이드성 인자-1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 스테로이드성 인자-1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with steroidal factor-1 with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to steroidal factor-1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 스테로이드성 인자-1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of steroidal factor-1 in human cord blood-derived hematopoietic stem cells in animal cancer model

스테로이드성 인자-1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 스테로이드성 인자-1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using steroidal factor-1, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). Compared with the injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated with steroidal factor-1.

실시예Example 11. 11. c-c- srcsrc 타이로신Tyrosine 키나아제를Kinase 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 c-src 타이로신 키나아제를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using c-src tyrosine kinase in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 c-src 타이로신 키나아제 1㎍/ml(Calbiochem)를 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 염소항체를 처리한 대조군에 비해 c-src 타이로신 키나아제를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml of c-src tyrosine kinase (Calbiochem) instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when treated with c-src tyrosine kinase compared to control with goat antibody.

추가로, 동일조건하에 전구자연살해세포를 c-src 타이로신 키나아제를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 c-src 타이로신 키나아제를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating pro-natural killer cells with c-src tyrosine kinase in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate compared to the treatment with c-src tyrosine kinase alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 c-src 타이로신 키나아제에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명 (B) Identification of anticancer activity by mature natural killer cells induced by differentiation by c-src tyrosine kinase in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

c-src 타이로신 키나아제를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 염소항체를 처리하여 분화시킨 성숙자연살해세포를 주입한 대조군에 비해 c-src 타이로신 키나아제를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using c-src tyrosine kinase, the mature natural killer cells treated with goat antibodies were differentiated in the same manner as in Example 2 (C). Compared to the control group injected with, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with c-src tyrosine kinase.

실시예Example 12. 12. Hmgb1Hmgb1 (( highhigh mobilitymobility groupgroup boxbox 1)을 이용한 사람 제대혈 유래 조혈모세포로부터 From 1) human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Hmgb1을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Hmgb1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Hmgb1 1㎍/ml(Abnova Corporation)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Hmgb1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding Hmgb1 1µg / ml (Abnova Corporation) instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about two to three times increased differentiation when treated with Hmgb1 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Hmgb1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Hmgb1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, progenitor killer cells were treated with Hmgb1 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate under the same conditions. As a result, it showed a slightly increased differentiation rate compared to the case of Hmgb1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Hmgb1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Hmgb1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Hmgb1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Hmgb1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Hmgb1, the same procedure as in Example 2 (C) was conducted. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Hmgb1.

실시예Example 13. 페리틴( 13. Ferritin ( FerritinFerritin ) ) 헤비heavy 체인을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using Chains 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 페리틴 헤비 체인을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using ferritin heavy chain in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 페리틴 헤비 체인 1㎍/ml(Alpha Diagnostic International Inc )을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 페리틴 헤비 체인을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml of ferritin heavy chain (Alpha Diagnostic International Inc) instead of interleukin-15 under the same conditions as in Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and One CD56 ⁺ NKG20 ⁺ cells showed an approximately 2-3-fold increase in differentiation rate when ferritin heavy chains were treated compared to the no-treated controls.

추가로, 동일조건하에 전구자연살해세포를 페리틴 헤비 체인을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 페리틴 헤비 체인을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with ferritin heavy chains with interleukin-15 in an amount of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the ferritin heavy chain alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 페리틴 헤비 체인에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by ferritin heavy chain in human cord blood-derived hematopoietic stem cells in animal cancer model

페리틴 헤비 체인을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 페리틴 헤비 체인을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using ferritin heavy chain, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as Example 2 (C). In comparison with the fermentin heavy chains, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells.

실시예Example 14. 페리틴( 14. Ferritin ( FerritinFerritin ) 라이트 체인을 이용한 사람 제대혈 유래 조혈모세포로부터 ) From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using Light Chain 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 페리틴 라이트 체인을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using ferritin light chain in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 페리틴 라이트 체인 1㎍/ml(Alpha Diagnostic International Inc )을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 페리틴 라이트 체인을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Alpha Diagnostic International Inc) of ferritin light chain instead of treating Interleukin-15 under the same conditions as in Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about two to three times increased differentiation when ferritin light chains were treated compared to the no-treated controls.

추가로, 동일조건하에 전구자연살해세포를 페리틴 라이트 체인을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 페리틴 라이트 체인을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with ferritin light chains with interleukin-15 at a ratio of 1 μg / ml in order to confirm the differentiation rate. The results showed a slightly increased differentiation rate when treated with ferritin light chain alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 페리틴 라이트 체인에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by ferritin light chain in human cord blood-derived hematopoietic stem cells in animal cancer model

페리틴 라이트 체인을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 페리틴 라이트 체인을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using ferritin light chain, the mature natural killer cells of the control group treated with nothing were injected. Compared with the ferritin light chain, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells.

실시예Example 15. 15. 칼그라뉼린Cal granulin 비( ratio( calgranulincalgranulin B)를 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using B) 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 칼그라뉼린 비를 이용한 성숙자연살해세포 분화법 (A) Differentiation of Mature Natural Killer Cells Using Cal granulin Ratio in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 칼그라뉼린 비를 1㎍/ml(Abnova Corporation) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 칼그라뉼린 비를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Abnova Corporation) to the calcanulin ratio instead of treating Interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase in differentiation rate of 2 to 3 times when treated with the cal granulin ratio compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 칼그라뉼린 비를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 칼그라뉼린 비를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with an interleukin-15 ratio of 1 μg / ml in an amount of 1: 1 with interleukin-15 to confirm the differentiation rate. As a result, the rate of differentiation was slightly increased as compared with the treatment with cal granulin ratio alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 칼그라뉼린 비에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by cal granulin ratio in human cord blood-derived hematopoietic stem cells in animal cancer model

칼그라뉼린 비를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 칼그라뉼린 비를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using a cal granulin ratio, the mature natural killer cells of a control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with the control group, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using the cal granulin ratio.

실시예 16. 세포 표면 글라이코단백질 ( cell surface glycoprotein ) p91 의 폴리클로날 항체를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Example 16. Cell surface article lycopene protein (cell surface glycoprotein) differentiation of mature NK poly p91 from human cord blood-derived hematopoietic stem cells using a polyclonal antibody

(A) 사람 제대혈 유래 조혈모세포에서 세포 표면 글라이코단백질 p91의 폴리클로날 항체를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using polyclonal antibody of cell surface glycoprotein p91 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 자체 세포 표면 글라이코단백질 p91의 항체 1㎍/ml를 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 세포 표면 글라이코단백질 p91의 폴리클로날 항체를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml of the antibody of its own cell surface glycoprotein p91 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an approximately two- to three-fold increase in the differentiation rate when treated with polyclonal antibody of cell surface glycoprotein p91 compared to the control group treated with nothing.

추가로, 동일조건하에 전구자연살해세포를 세포 표면 글라이코단백질 p91의 폴리클로날 항체를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 세포 표면 글라이코단백질 p91의 폴리클로날 항체를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with a polyclonal antibody of cell surface glycoprotein p91 at an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. As a result, the cell surface glycoprotein p91 showed a slightly increased differentiation rate compared with the polyclonal antibody alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 세포 표면 글라이코단백질 p91의 폴리클로날 항체에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by polyclonal antibody of cell surface glycoprotein p91 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

세포 표면 글라이코단백질 p91의 폴리클로날 항체를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 세포 표면 글라이코단백질 p91의 폴리클로날 항체를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by mature human killer cells differentiated using polyclonal antibody of cell surface glycoprotein p91, the control group which did not process anything was tested in the same manner as in Example 2 (C). Compared with the injection of the mature natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group of the mature natural killer cells differentiated using the polyclonal antibody of cell surface glycoprotein p91.

실시예Example 17. 클로라이드 채널 7( 17. chloride channel 7 ( ChlorideChloride channelchannel 7)을 이용한 사람 제대혈 유래 조혈모세포로부터 7) from human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 클로라이드 채널 7을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using chloride channel 7 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 클로라이드 채널 7을 1㎍/ml(Biocompare) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 클로라이드 채널 7을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Biocompare) of chloride channel 7 and treated for 14 days instead of interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 to 3 fold increase in differentiation rate when treated with chloride channel 7 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 클로라이드 채널 7을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 클로라이드 채널 7을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with chloride channel 7 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate when treated with chloride channel 7 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 클로라이드 채널 7에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of chloride channel 7 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

클로라이드 채널 7을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 클로라이드 채널 7을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using chloride channel 7, the same method as in Example 2 (C) was used. Compared to the group injected with mature natural killer cells differentiated using chloride channel 7, significant decrease in cancer tissue volume was observed.

실시예Example 18. 18. 아드빌린(Advillin)을Advillin 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 아드빌린을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation method using avidin in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 아드빌린을 1㎍/ml 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 아드빌린을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml of adribine instead of interleukin-15 treatment under the same conditions as in Example 2 (A). Was analyzed by FACS in the same manner as in Example 2 (A) to determine the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with adribine compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 아드빌린을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 아드빌린을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with 1 ug / ml in a 1: 1 ratio of abilirin with interleukin-15 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the treatment with avidin alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 아드빌린에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of abiline in human cord blood-derived hematopoietic stem cells in animal cancer model

아드빌린을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 아드빌린을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated with avidin, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with the adiplin-derived mature natural killer cells, a significant decrease in the volume of cancer tissues was observed.

실시예Example 19. 19. 카텝신Cathepsin -- L(Cathepsin-L)을L (Cathepsin-L) 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 카텝신-L을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using cathepsin-L in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 카텝신-L을 1㎍/ml(R&D systems) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 카텝신-L을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml of cathepsin-L (R & D systems) instead of interleukin-15 under the same conditions as in Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an approximately 2-3-fold increase in differentiation rate when cathepsin-L was treated compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 카텝신-L을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 카텝신-L을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, differentiation rate was confirmed by treating protetracellular killer cells with cathepsin-L with interleukin-15 in an amount of 1 μg / ml in a ratio of 1: 1. As a result, it was shown that the differentiation rate slightly increased compared to the case of cathepsin-L alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 카텝신-L에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by cathepsin-L differentiation in human cord blood-derived hematopoietic stem cells in animal cancer model

카텝신-L을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 카텝신-L을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using cathepsin-L, the mature natural killer cells of the control group treated with none were injected as a result of the same method as in Example 2 (C). Compared to the other group, a significant reduction in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using cathepsin-L.

실시예Example 20. 20. 카텝신Cathepsin -B(-B ( CathepsinCathepsin -B)를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 -B) Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 카텝신-B를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using cathepsin-B in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 카텝신-B를 1㎍/ml(R&D systems) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 카텝신-B를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml of cathepsin-B (R & D systems) instead of interleukin-15 under the same conditions as in Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about two to three fold increase in differentiation rate when cathepsin-B was treated compared to the no-treated control group.

추가로, 동일조건하에 전구자연살해세포를 카텝신-B를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 카텝신-B를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, differentiation rate of pro natural killer cells was treated with cathepsin-B with interleukin-15 in an amount of 1 μg / ml at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared to the treatment with cathepsin-B alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 카텝신-B에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by cathepsin-B differentiation in human cord blood-derived hematopoietic stem cells in animal cancer model

카텝신-B를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 카텝신-B를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using cathepsin-B, the mature natural killer cells of the control group treated with nothing were injected as a result of the same experiment as in Example 2 (C). In contrast, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with cathepsin-B.

실시예Example 21. 21. 카텝신Cathepsin -H(-H ( CathepsinCathepsin -H)을 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 카텝신-H를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using cathepsin-H in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 카텝신-H를 1㎍/ml(R&D systems) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 카텝신-H를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The pro natural killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml of cathepsin-H (R & D systems) instead of interleukin-15 under the same conditions as in Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 to 3 times higher differentiation rate when cathepsin-H was treated compared to the control group which received no treatment.

추가로, 동일조건하에 전구자연살해세포를 카텝신-H를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 카텝신-H를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with cathepsin-H with interleukin-15 at a ratio of 1 μg / ml in a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the treatment with cathepsin-H alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 카텝신-H에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by cathepsin-H differentiation in human cord blood-derived hematopoietic stem cells in animal cancer model

카텝신-H을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 카텝신-H를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using cathepsin-H, the mature natural killer cells of the control group treated with nothing were injected in the same manner as in Example 2 (C). Compared to the other group, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with cathepsin-H.

실시예 22. Emr1( EGF - like module - containing mucin - like hormone receptor - like 1)의 항체를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Example 22 Emr1 ( EGF - like module - containing mucin - like hormone differentiation of mature NK cells from human cord blood-derived hematopoietic cells using antibodies like 1) - receptor

(A) 사람 제대혈 유래 조혈모세포에서 Emr1의 폴리클로날 항체를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Emr1 polyclonal antibody in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Emr1의 폴리클로날 항체 1㎍/ml(Santa Cruz)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 염소항체를 처리한 대조군에 비해 Emr1의 폴리클로날 항체를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as in Example 2 (A), and then 1 μg / ml of Emr1 polyclonal antibody (Santa Cruz) was added for 14 days. After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with polyclonal antibody of Emr1 compared to goat antibody treated control group.

추가로, 동일조건하에 전구자연살해세포를 Emr1의 폴리클로날 항체를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Emr1의 폴리클로날 항체를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with an Emr1 polyclonal antibody in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, it showed a slightly increased differentiation rate compared with the polyclonal antibody of Emr1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Emr1의 폴리클로날 항체에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명 (B) Characterization of anticancer activity by mature natural killer cells induced by differentiation by Emr1 polyclonal antibody in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Emr1의 폴리클로날 항체를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 염소항체를 처리하여 분화시킨 성숙자연살해세포를 주입한 대조군에 비해 Emr1의 폴리클로날 항체를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the polyclonal antibody of Emr1, the mature natural killer killed by treatment with goat antibodies was tested in the same manner as in Example 2 (C). Compared to the control group injected with the cells, a significant decrease in the volume of cancer tissues was observed in the group injected with the mature natural killer cells differentiated using the polyclonal antibody of Emr1.

실시예 23. Trem2 ( Triggering receptor expressed on myeloid cells 2)의 항체를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Example 23. Trem2 (Triggering receptor expressed on myeloid cells 2) differentiation of human cord blood-derived hematopoietic stem cells from mature NK cells using antibodies

(A) 사람 제대혈 유래 조혈모세포에서 trem2의 폴리클로날 항체를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using trem2 polyclonal antibody in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 trem2의 폴리클로날 항체 1㎍/ml(Santa Cruz)을 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 염소항체를 처리한 대조군에 비해 trem2의 폴리클로날 항체를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The pro natural killer cells obtained in Example 2 (A) were treated with interleukin-15 under the same conditions as in Example 2 (A), and then 1 μg / ml of trem2 polyclonal antibody (Santa Cruz) was added for 14 days. After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with trem2 polyclonal antibodies compared to goat-treated controls.

추가로, 동일조건하에 전구자연살해세포를 trem2의 폴리클로날 항체를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 trem2의 폴리클로날 항체를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, differentiation rate was confirmed by treating prom natural killer cells with trem2 polyclonal antibody in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, it showed a slightly increased differentiation rate compared with the trem2 polyclonal antibody alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 trem2의 폴리클로날 항체에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명 (B) Identification of anticancer activity by mature natural killer cells induced by differentiation by trem2 polyclonal antibody in human cord blood-derived hematopoietic stem cells in animal cancer model

trem2의 폴리클로날 항체를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 염소항체를 처리하여 분화시킨 성숙자연살해세포를 주입한 대조군에 비해 Trem2의 폴리클로날 항체를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using polyclonal antibody of trem2, the mature natural killer killed by treatment with goat antibodies was tested in the same manner as in Example 2 (C). Compared to the control group injected with the cells, a significant decrease in the volume of the cancer tissue was observed in the group injected with the mature natural killer cells differentiated using the polyclonal antibody of Trem2.

실시예Example 24. 24. SERPINA3GSERPINA3G 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 SERPINA3G를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using SERPINA3G in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 SERPINA3G를 1㎍/ml 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 SERPINA3G를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml of SERPINA3G instead of interleukin-15 treatment under the same conditions as in Example 2 (A). FACS analysis was performed in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about 2 to 3 times higher differentiation rate when treated with SERPINA3G than the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 SERPINA3G를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 SERPINA3G를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating SERPINA3G with interleukin-15 at a ratio of 1 µg / ml in a ratio of 1: 1. The results showed a slightly increased differentiation rate compared with SERPINA3G alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 SERPINA3G에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of SERPINA3G in human cord blood-derived hematopoietic stem cells in animal cancer model

SERPINA3G를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 SERPINA3G를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using SERPINA3G, experiments were performed in the same manner as in Example 2 (C). Significant decrease of cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with SERPINA3G.

실시예Example 25. 25. LAPTm5LAPTm5 유도물질 Inducer 레티논산Retinoic acid (( RetinoicRetinoic acidacid ) 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 LAPTm5의 유도물질 레티논산을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation method using retinonic acid inducing LAPTm5 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 레티논산을 1㎍/ml(Sigma) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 레티논산을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Sigma) of retinic acid instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with retinoic acid compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 레티논산를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 레티논산을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with retinic acid with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with retinoic acid alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 레티논산에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of retinoic acid in human cord blood-derived hematopoietic stem cells in animal cancer model

레티논산을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 레티논산을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using retinic acid, the same procedure as in Example 2 (C) was conducted. Compared with retinoic acid-derived mature natural killer cells, a significant decrease in cancer tissue volume was observed.

실시예Example 26. 26. Egr1Egr1 (( earlyearly growthgrowth response1response1 )을 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Egr1을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Egr1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Egr1을 1㎍/ml 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Egr1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml of Egr1 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). FACS analysis was performed in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with Egr1 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Egr1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Egr1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Egr1 with interleukin-15 at a ratio of 1 μg / ml in a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the treatment with Egr1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Egr1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by Egr1 differentiation in human cord blood-derived hematopoietic stem cells in animal cancer model

Egr1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Egr1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Egr1, the same procedure as in Example 2 (C) was performed. In the group injected with mature natural killer cells differentiated with Egr1, a significant decrease in cancer tissue volume was observed.

실시예Example 27. 27. CarfCarf (( calciumcalcium responseresponse factorfactor )를 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Carf를 이용한 성숙자연살해세포 분화법 (A) Differentiation of Mature Natural Killer Cells Using Carf in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Carf를 1㎍/ml(Novus Biological) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Carf를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding Carf to 1 μg / ml (Novus Biological) instead of treating Interleukin-15 under the same conditions as Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when Carf was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Carf를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Carf를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the proliferative killer cells were treated with Carf at an amount of 1 µg / ml at a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to Carf alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Carf에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Carf in human cord blood-derived hematopoietic stem cells in animal cancer model

Carf를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Carf를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Carf, the same procedure as in Example 2 (C) was performed. Significant decrease of cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Carf.

실시예Example 28. 28. ApoApo -E(-E ( ApolipoproteinApolipoprotein E)를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using E)

(A) 사람 제대혈 유래 조혈모세포에서 Apo-E를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Apo-E in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Apo-E를 1㎍/ml(Alpha Diagnostic International Inc) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Carf를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Alpha Diagnostic International Inc) of Apo-E instead of interleukin-15 under the same conditions as in Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when Carf was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Apo-E를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Apo-E를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Apo-E with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of Apo-E alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Apo-E에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by Apo-E differentiation in human cord blood-derived hematopoietic stem cells in animal cancer model

Apo-E를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Apo-E를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Apo-E, the mature natural killer cells of the control group treated with nothing were injected. In comparison with the Apo-E-induced mature natural killer cells, a significant decrease in the volume of cancer tissues was observed.

실시예Example 29. 베타 29. Beta 카테닌(Catenin beta)를Catenin beta 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 베타 카테닌을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using beta-catenin in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 베타 카테닌을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 베타 카테닌을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of beta-catenin instead of interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about 2-3 times increased differentiation rate when treated with beta-catenin compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 베타 카테닌을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 베타 카테닌을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with beta-catenin with interleukin-15 at a ratio of 1 µg / ml in a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to beta catenin alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 베타 카테닌에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of beta-catenin in human cord blood-derived hematopoietic stem cells in animal cancer model

베타 카테닌을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 베타 카테닌을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using beta-catenin, the result of experiments in the same manner as in Example 2 (C) was obtained. Compared with the beta-catenin-induced mature natural killer cells, a significant decrease in cancer tissue volume was observed.

실시예Example 30. 30. 라이소자임Lysozyme (( LysozymeLysozyme )을 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 라이소자임을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using lysozyme in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 라이소자임을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 라이소자임을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of lysozyme instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about 2 to 3 times higher differentiation rate when treated with lysozyme compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 라이소자임을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과라이소자임을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with lysozyme with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to lysozyme alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 라이소자임에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by lysozyme in human cord blood-derived hematopoietic stem cells in animal cancer model

라이소자임을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 라이소자임을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using lysozyme, the same procedure as in Example 2 (C) was performed. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using lysozyme.

실시예Example 31. 31. 브레비칸(Brevican)을Brevican 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 브레비칸을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Brevican in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 브레비칸을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 브레비칸을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Brevican instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2-3 times differentiation rate when treated with Brevican compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 브레비칸을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 브레비칸을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Brevican with Interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of brevican alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 브레비칸에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Brevican in human cord blood-derived hematopoietic stem cells in animal cancer model

브레비칸을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 브레비칸을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Brevican, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as Example 2 (C). Compared with the Brevican-induced mature natural killer cells, a significant decrease in the volume of cancer tissues was observed.

실시예Example 32. 32. MatrixMatrix metalloproteinasemetalloproteinase 12을12 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Matrix metalloproteinase 12을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using matrix metalloproteinase 12 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Matrix metalloproteinase 12을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Matrix metalloproteinase 12을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Matrix metalloproteinase 12 to the same conditions as Example 2 (A) instead of treating Interleukin-15. Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 times higher differentiation rate when Matrix metalloproteinase 12 was treated than the control group.

추가로, 동일조건하에 전구자연살해세포를 Matrix metalloproteinase 12을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Matrix metalloproteinase 12을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, differentiation rate of progenitor killer cells was treated with Matrix metalloproteinase 12 with interleukin-15 in a 1: 1 µg / ml amount. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with Matrix metalloproteinase 12 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Matrix metalloproteinase 12에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Matrix metalloproteinase 12 in human cord blood-derived hematopoietic stem cells in animal cancer model

Matrix metalloproteinase 12을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Matrix metalloproteinase 12을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Matrix metalloproteinase 12, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). In contrast, the tumor cell volume was significantly decreased in the group injected with mature natural killer cells differentiated using Matrix metalloproteinase 12.

실시예Example 33. 33. CellularCellular repressorrepressor ofof EIAEIA -- stimulatedstimulated genegene 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Cellular repressor of EIA-stimulated gene을 이용한 성숙자연살해세포 분화법 (A) Mature Natural Killer Differentiation Using Cellular Repressor of EIA-stimulated Gene in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Cellular repressor of EIA-stimulated gene을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Cellular repressor of EIA-stimulated gene을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 in the same conditions as Example 2 (A), the progenitor killer cells obtained in Example 2 (A) were added with 1 µg / ml (Upstat) of Cellular repressor of EIA-stimulated gene for 14 days. After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with the cellular repressor of EIA-stimulated gene compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Cellular repressor of EIA-stimulated gene을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Cellular repressor of EIA-stimulated gene을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating the progenitor killer cells with the cellular repressor of EIA-stimulated gene in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate compared to the case of treatment with the cellular repressor of EIA-stimulated gene alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Cellular repressor of EIA-stimulated gene에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by cellular repressor of EIA-stimulated gene in human cord blood-derived hematopoietic stem cells in animal cancer model

Cellular repressor of EIA-stimulated gene을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Cellular repressor of EIA-stimulated gene을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the cellular repressor of EIA-stimulated gene, the mature natural killer of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared with the cell injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using the cellular repressor of EIA-stimulated gene.

실시예Example 34. c- 34. c- kitkit ligandligand 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 c-kit ligand를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using c-kit ligand in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 c-kit ligand를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 c-kit ligand를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of c-kit ligand instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with c-kit ligand compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 c-kit ligand를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 c-kit ligand를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the proliferation of NK cells was treated with c-kit ligand with interleukin-15 in a ratio of 1 ㎍ / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the c-kit ligand alone treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 c-kit ligand에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by c-kit ligand in human cord blood-derived hematopoietic stem cells in animal cancer model

c-kit ligand를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 c-kit ligand를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using a c-kit ligand, the mature natural killer cells of a control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to that, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using c-kit ligand.

실시예Example 35. 35. MPS1MPS1 단백질을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using Protein 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 MPS1 단백질을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using MPS1 protein in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 MPS1 단백질을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 MPS1 단백질을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of MPS1 protein instead of interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when MPS1 protein was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 MPS1 단백질을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 MPS1 단백질을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with MPS1 protein with interleukin-15 at a ratio of 1 µg / ml in a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the MPS1 protein alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 MPS1 단백질에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by MPS1 protein in human cord blood-derived hematopoietic stem cells in animal cancer model

MPS1 단백질을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 MPS1 단백질을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using MPS1 protein, the result of experiments in the same manner as in Example 2 (C) was obtained. Compared to the group injected with mature natural killer cells differentiated using MPS1 protein, a significant decrease in cancer tissue volume was observed.

실시예Example 36. 36. TransglutaminaseTransglutaminase 2를 이용한 사람 제대혈 유래 조혈모세포로부터 From human umbilical cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Transglutaminase 2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Transglutaminase 2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Transglutaminase 2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Transglutaminase 2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Transglutaminase 2 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 times higher differentiation rate when Transglutaminase 2 was treated compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Transglutaminase 2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Transglutaminase 2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Transglutaminase 2 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1 μg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the case of Transglutaminase 2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Transglutaminase 2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Transglutaminase 2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Transglutaminase 2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Transglutaminase 2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using transglutaminase 2, the mature natural killer cells of the control group treated with nothing were injected. Compared with the transglutaminase 2-derived mature natural killer cells, the cancer tissue volume was significantly decreased.

실시예Example 37. 37. SerumSerum andand glucocorticoidglucocorticoid -- regulatedregulated proteinprotein kinasekinase 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Serum and glucocorticoid-regulated protein kinase를 이용한 성숙자연살해세포 분화법 (A) Differentiation of Mature Natural Killer Cells Using Serum and Glucocorticoid-regulated Protein Kinase in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Serum and glucocorticoid-regulated protein kinase를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Serum and glucocorticoid-regulated protein kinase를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 in the same conditions as in Example 2 (A), progenitor killer cells obtained in Example 2 (A) were added with 1 μg / ml (Upstat) of Serum and glucocorticoid-regulated protein kinase for 14 days. After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with Serum and Glucocorticoid-regulated protein kinase compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Serum and glucocorticoid-regulated protein kinase를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Serum and glucocorticoid-regulated protein kinase를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, progenitor killer cells were treated with Serum and glucocorticoid-regulated protein kinase with interleukin-15 at a ratio of 1 ㎍ / ml in a 1: 1 ratio to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the treatment with Serum and Glucocorticoid-regulated protein kinase alone.

실시예Example 38. 38. InterferonInterferon -- inducedinduced 단백질을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using Protein 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Interferon-induced 단백질을 이용한 성숙자연살해세포 분화법 (A) Differentiation of Mature Natural Killer Cells Using Interferon-induced Protein in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Interferon-induced 단백질을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Interferon-induced 단백질을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Interferon-induced protein instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with Interferon-induced protein compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Interferon-induced 단백질을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Interferon-induced 단백질을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating interferon-induced protein with Interleukin-15 at a ratio of 1 μg / ml in a ratio of 1: 1. The results showed a slightly increased differentiation rate compared to the treatment with Interferon-induced protein alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Interferon-induced 단백질에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Interferon-induced protein in human cord blood-derived hematopoietic stem cells in animal cancer model

Interferon-induced 단백질을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Interferon-induced 단백질을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using interferon-induced protein, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to that, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Interferon-induced protein.

실시예Example 39. 39. MilkMilk fatfat globuleglobule membranemembrane proteinprotein EGFEGF factorfactor 8을 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Milk fat globule membrane protein EGF factor 8을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using milk fat globule membrane protein EGF factor 8 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Milk fat globule membrane protein EGF factor 8을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Milk fat globule membrane protein EGF factor 8을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as in Example 2 (A), and then added with 1 μg / ml (Upstat) of milk fat globule membrane protein EGF factor 8 for 14 days. After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and The CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when treated with the milk fat globule membrane protein EGF factor 8 compared to the no-treated control group.

추가로, 동일조건하에 전구자연살해세포를 Milk fat globule membrane protein EGF factor 8을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Milk fat globule membrane protein EGF factor 8을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating the progenitor killer cells with milk fat globule membrane protein EGF factor 8 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, milk fat globule membrane protein EGF factor 8 showed a slightly increased differentiation rate.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Milk fat globule membrane protein EGF factor 8에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by milk fat globule membrane protein EGF factor 8 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Milk fat globule membrane protein EGF factor 8을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Milk fat globule membrane protein EGF factor 8을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Human mature cells differentiated with milk fat globule membrane protein EGF factor 8 To observe whether human cancer cells are killed by human natural killer cells, the same method as in Example 2 (C) was conducted. Compared with the injection of killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using milk fat globule membrane protein EGF factor 8.

실시예Example 40. 40. FcFc g 수용체를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using g-receptor 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Fc g 수용체를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Fc g receptor in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Fc g 수용체를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Fc g 수용체를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Fc g receptor instead of treating Interleukin-15 under the same conditions as Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with Fc g receptor compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Fc g 수용체를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Fc g 수용체를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Fc g receptors with interleukin-15 at a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate when treated with Fc g receptor alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Fc g 수용체에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Fc g receptor in human cord blood-derived hematopoietic stem cells in animal cancer model

Fc g 수용체를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Fc g 수용체를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Fc g receptor, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). In comparison with the Fc g receptor-induced mature natural killer cells, a significant decrease in cancer tissue volume was observed.

실시예Example 41. 41. S100S100 calciumcalcium bindingbinding preteinpretein A9를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using A9 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 S100 calcium binding pretein A9를 이용한 성숙자연살해세포 분화법 (A) Differentiation of mature natural killer cells using S100 calcium binding pretein A9 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 S100 calcium binding pretein A9를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 S100 calcium binding pretein A9를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of S100 calcium binding pretein A9 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with S100 calcium binding pretein A9 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 S100 calcium binding pretein A9를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 S100 calcium binding pretein A9를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate of progenitor killer cells was treated with S100 calcium binding pretein A9 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate compared to the S100 calcium binding pretein A9 treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 S100 calcium binding pretein A9에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by S100 calcium binding pretein A9 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

S100 calcium binding pretein A9를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 S100 calcium binding pretein A9를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using S100 calcium binding pretein A9, the mature natural killer cells of the control group which did not process anything were tested in the same manner as in Example 2 (C). Compared with the injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated with S100 calcium binding pretein A9.

실시예Example 42. 42. ArginaseArginase 1를1 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Arginase 1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Arginase 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Arginase 1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Arginase 1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Arginase 1 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when Arginase 1 was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Arginase 1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Arginase 1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Arginase 1 with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with Arginase 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Arginase 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Arginase 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Arginase 1를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Arginase 1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Arginase 1, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with Arginase 1-derived mature natural killer cells, significant decreases in cancer tissue volume were observed.

실시예Example 43. 43. TumorTumor necrosisnecrosis factorfactor receptorreceptor 1를1 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Tumor necrosis factor receptor 1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Tumor necrosis factor receptor 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Tumor necrosis factor receptor 1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Tumor necrosis factor receptor 1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Tumor necrosis factor receptor 1 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with Tumor necrosis factor receptor 1 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Tumor necrosis factor receptor 1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Tumor necrosis factor receptor 1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Tumor necrosis factor receptor 1 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1 μg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the treatment with Tumor necrosis factor receptor 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Tumor necrosis factor receptor 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Tumor necrosis factor receptor 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Tumor necrosis factor receptor 1를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Tumor necrosis factor receptor 1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Tumor necrosis factor receptor 1, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). Compared with the injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using Tumor necrosis factor receptor 1.

실시예Example 44. 44. RetinoidRetinoid -- inducibleinducible serineserine carboxypeptidasecarboxypeptidase 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Retinoid-inducible serine carboxypeptidase를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Retinoid-inducible serine carboxypeptidase in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Retinoid-inducible serine carboxypeptidase를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Retinoid-inducible serine carboxypeptidase를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Retinoid-inducible serine carboxypeptidase instead of interleukin-15 under the same conditions as in Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with Retinoid-inducible serine carboxypeptidase compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Retinoid-inducible serine carboxypeptidase를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Retinoid-inducible serine carboxypeptidase를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating Retinoid-inducible serine carboxypeptidase with Interleukin-15 at a ratio of 1 µg / ml in a 1: 1 ratio. The results showed a slightly increased differentiation rate compared to the treatment with Retinoid-inducible serine carboxypeptidase alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Retinoid-inducible serine carboxypeptidase에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Retinoid-inducible serine carboxypeptidase in human cord blood-derived hematopoietic stem cells in animal cancer model

Retinoid-inducible serine carboxypeptidase를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Retinoid-inducible serine carboxypeptidase를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated by using retinoid-inducible serine carboxypeptidase, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). Compared with the injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated with Retinoid-inducible serine carboxypeptidase.

실시예Example 45. 45. SimilarSimilar toto hypotheticalhypothetical proteinprotein FLJ11000FLJ11000 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Similar to hypothetical protein FLJ11000를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using similar to hypothetical protein FLJ11000 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Similar to hypothetical protein FLJ11000를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Similar to hypothetical protein FLJ11000를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of similar to hypothetical protein FLJ11000, instead of treating Interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with similar to hypothetical protein FLJ11000 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Similar to hypothetical protein FLJ11000를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Similar to hypothetical protein FLJ11000를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with Similar to hypothetical protein FLJ11000 with interleukin-15 in a 1: 1 μg / ml amount. The results showed a slightly increased differentiation rate compared to the treatment of Similar to hypothetical protein FLJ11000 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Similar to hypothetical protein FLJ11000에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by hypothetical protein FLJ11000 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Similar to hypothetical protein FLJ11000를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Similar to hypothetical protein FLJ11000를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Similar to hypothetical protein FLJ11000, the experiment was performed in the same manner as in Example 2 (C) to observe whether human cancer cells were killed by human mature natural killer cells differentiated using FLJ11000. A significant decrease in the volume of cancerous tissue was observed in the group injected with mature natural killer cells differentiated using similar to hypothetical protein FLJ11000.

실시예Example 46. 46. InterleukinInterleukin -18 -18 bindingbinding proteinprotein d d precursorprecursor 를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 Interleukin-18 binding protein d precursor를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Interleukin-18 binding protein d precursor in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Interleukin-18 binding protein d precursor를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Interleukin-18 binding protein d precursor를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 at 1 µg / ml (Upstat) for 14 days instead of treating Interleukin-15 under the same conditions as in Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells exhibited a two- to three-fold increase in differentiation rate when treated with Interleukin-18 binding protein d precursor compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Interleukin-18 binding protein d precursor를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Interleukin-18 binding protein d precursor를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating interleukin-18 binding protein d precursor with Interleukin-15 in an amount of 1 µg / ml at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared to the treatment with Interleukin-18 binding protein d precursor alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Interleukin-18 binding protein d precursor에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Interleukin-18 binding protein d precursor in human cord blood-derived hematopoietic stem cells in animal cancer model

Interleukin-18 binding protein d precursor를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Interleukin-18 binding protein d precursor를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Interleukin-18 binding protein d precursor, the mature natural killer of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared to the cells injected, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using Interleukin-18 binding protein d precursor.

실시예Example 47. 47. ChlorideChloride channelchannel 7을 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Chloride channel 7을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Chloride channel 7 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Chloride channel 7을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Chloride channel 7을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding Chloride channel 7 to 1 μg / ml (Upstat) instead of interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 times higher differentiation rate when treated with Chloride channel 7 than the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Chloride channel 7을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Chloride channel 7을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, differentiation rate was confirmed by treating Chloride channel 7 with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio. As a result, it showed a slightly increased differentiation rate when treated with Chloride channel 7 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Chloride channel 7에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Chloride channel 7 in human cord blood-derived hematopoietic stem cells in animal cancer model

Chloride channel 7을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Chloride channel 7을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Chloride channel 7, the mature natural killer cells of the control group treated with nothing were injected. Compared with the group injected with mature natural killer cells differentiated using Chloride channel 7, significant decreases in cancer tissue volume were observed.

실시예Example 48. 48. CD36CD36 antigenantigen 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 CD36 antigen을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using CD36 antigen in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 CD36 antigen을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 CD36 antigen을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of CD36 antigen instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with CD36 antigen compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 CD36 antigen을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 CD36 antigen을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with CD36 antigen with interleukin-15 at a ratio of 1 ㎍ / ml in a 1: 1 ratio to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the CD36 antigen alone treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 CD36 antigen에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of CD36 antigen in human cord blood-derived hematopoietic stem cells in animal cancer model

CD36 antigen을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 CD36 antigen을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using CD36 antigen, the result of the experiment in the same manner as in Example 2 (C) was obtained. Compared with CD36 antigen-induced mature natural killer cells, significant decreases in cancer tissue volume were observed.

실시예Example 41. 41. SimilarSimilar toto putativeputative zinczinc fingerfinger proteinprotein 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서Similar to putative zinc finger protein을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using similar to putative zinc finger protein in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Similar to putative zinc finger protein을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Similar to putative zinc finger protein을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of similar to putative zinc finger protein instead of interleukin-15 in the same conditions as in Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with similar to putative zinc finger protein.

추가로, 동일조건하에 전구자연살해세포를 Similar to putative zinc finger protein을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Similar to putative zinc finger protein을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with a similar to putative zinc finger protein at a ratio of 1 μg / ml at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate compared to the treatment with Similar to putative zinc finger protein alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Similar to putative zinc finger protein에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by similar to putative zinc finger protein in human cord blood-derived hematopoietic stem cells in animal cancer model

Similar to putative zinc finger protein을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Similar to putative zinc finger protein을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using similar to putative zinc finger protein, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). There was a significant decrease in the volume of cancer tissues in the group injected with mature natural killer cells differentiated using similar to putative zinc finger protein.

실시예Example 50. 50. CarbohydrateCarbohydrate bindingbinding proteinprotein 35를 이용한 사람 제대혈 유래 Derived from human cord blood using 35 조혈모세Hematopoietic Moses 포로부터 From Pau 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Carbohydrate binding protein 35를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using carbohydrate binding protein 35 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Carbohydrate binding protein 35를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Carbohydrate binding protein 35를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. After progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as in Example 2 (A), and then differentiated for 14 days by adding 1 μg / ml (Upstat) of Carbohydrate binding protein 35 Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a 2 ~ 3 fold increase in differentiation rate when treated with Carbohydrate binding protein 35 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Carbohydrate binding protein 35를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Carbohydrate binding protein 35를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, carbohydrate binding protein 35 was treated with carbohydrate binding protein 35 in an amount of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed that the carbohydrate binding protein 35 had a slightly increased differentiation rate compared to the single treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Carbohydrate binding protein 35에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by carbohydrate binding protein 35 in human cord blood-derived hematopoietic stem cells in animal cancer model

Carbohydrate binding protein 35를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Carbohydrate binding protein 35를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using carbohydrate binding protein 35, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). In contrast, significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Carbohydrate binding protein 35.

실시예Example 51. C- 51.C- typetype calciumcalcium dependentdependent , , carbohydratecarbohydrate 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 C-type calcium dependent, carbohydrate를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using C-type calcium dependent, carbohydrate in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 C-type calcium dependent, carbohydrate를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 C-type calcium dependent, carbohydrate를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with C-type calcium dependent, carbohydrate (1 μg / ml (Upstat)) for 14 days instead of treating Interleukin-15 under the same conditions as Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a 2 ~ 3 fold increase in differentiation rate when treated with C-type calcium dependent and carbohydrate compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 C-type calcium dependent, carbohydrate를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 C-type calcium dependent, carbohydrate를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, C-type calcium dependent and carbohydrates were treated with 1 ug / ml in a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the C-type calcium dependent and carbohydrate treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 C-type calcium dependent, carbohydrate에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by C-type calcium dependent, carbohydrate in human cord blood-derived hematopoietic stem cells in animal cancer model

C-type calcium dependent, carbohydrate를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 C-type calcium dependent, carbohydrate를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using C-type calcium dependent, carbohydrate, the mature natural killer of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared to the cells injected, significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using C-type calcium dependent and carbohydrate.

실시예Example 52. 52. LipoproteinLipoprotein lipaselipase 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Lipoprotein lipase를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Lipoprotein lipase in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Lipoprotein lipase를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Lipoprotein lipase를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding Lipoprotein lipase 1 µg / ml (Upstat) instead of interleukin-15 in the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold more differentiation when treated with Lipoprotein lipase than the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Lipoprotein lipase를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Lipoprotein lipase를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Lipoprotein lipase with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the treatment with Lipoprotein lipase alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Lipoprotein lipase에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Lipoprotein lipase in human cord blood-derived hematopoietic stem cells in animal cancer model

Lipoprotein lipase를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Lipoprotein lipase를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using lipoprotein lipase, the mature natural killer cells of the control group treated with nothing were injected. Compared with the injection of mature natural killer cells differentiated with Lipoprotein lipase, the cancer tissue volume was significantly decreased.

실시예Example 53. v- 53.v- mafmaf musculoaponeuroticmusculoaponeurotic fibrosarcomafibrosarcoma oncogeneoncogene 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 v-maf musculoaponeurotic fibrosarcoma oncogene을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using v-maf musculoaponeurotic fibrosarcoma oncogene in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 v-maf musculoaponeurotic fibrosarcoma oncogene을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 v-maf musculoaponeurotic fibrosarcoma oncogene을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of v-maf musculoaponeurotic fibrosarcoma oncogene instead of interleukin-15 treatment under the same conditions as in Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with v-maf musculoaponeurotic fibrosarcoma oncogene compared to the control group that received no treatment.

추가로, 동일조건하에 전구자연살해세포를 v-maf musculoaponeurotic fibrosarcoma oncogene을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 v-maf musculoaponeurotic fibrosarcoma oncogene을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with v-maf musculoaponeurotic fibrosarcoma oncogene with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the v-maf musculoaponeurotic fibrosarcoma oncogene alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 v-maf musculoaponeurotic fibrosarcoma oncogene에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by v-maf musculoaponeurotic fibrosarcoma oncogene in human cord blood-derived hematopoietic stem cells in animal cancer model

v-maf musculoaponeurotic fibrosarcoma oncogene을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 v-maf musculoaponeurotic fibrosarcoma oncogene을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. To examine whether human cancer cells are killed by human mature natural killer cells differentiated using v-maf musculoaponeurotic fibrosarcoma oncogene Compared with the injection of MGF, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using v-maf musculoaponeurotic fibrosarcoma oncogene.

실시예Example 54. 54. InterleukinInterleukin -7 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Interleukin-7 을 이용한 성숙자연살해세포 분화법 (A) Interleukin-7 in Human Cord Blood-derived Hematopoietic Stem Cells Mature natural killer cell differentiation using

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Interleukin-7 을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Interleukin-7 을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Proleukin killer cells obtained in Example 2 (A) were treated with Interleukin-7 instead of interleukin-15 under the same conditions as in Example 2 (A). Differentiation was performed for 14 days by adding 1 μg / ml (Upstat) and human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells treated with Interleukin-7 compared to the control without any treatment When treated, the rate of differentiation was increased by 2 to 3 times.

추가로, 동일조건하에 전구자연살해세포를 Interleukin-7 을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Interleukin-7 을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, proleukin killing cells were treated with Interleukin-7 under the same conditions. Differentiation rate was confirmed by treatment with interleukin-15 at 1 μg / ml in a ratio of 1: 1. As a result, Interleukin-7 It was shown that the rate of differentiation was slightly increased as compared with the treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Interleukin-7에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Interleukin-7 in human cord blood-derived hematopoietic stem cells in animal cancer model

Interleukin-7 을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Interleukin-7 을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다.In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Interleukin-7, the mature natural killer cells of the control group treated with nothing were injected. Compared to Interleukin-7 Significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using the cells.

실시예Example 55. 55. NeuropilinNeuropilin 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Neuropilin을 이용한 성숙자연살해세포 분화법 (A) Mature Natural Killer Differentiation Using Neuropilin in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Neuropilin을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Neuropilin을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Neuropilin instead of interleukin-15 treatment under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with Neuropilin compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Neuropilin을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Neuropilin을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Neuropilin with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with neuropilin alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Neuropilin에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Neuropilin in human cord blood-derived hematopoietic stem cells in animal cancer model

Neuropilin을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Neuropilin을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using neuropilin, the experiments were performed in the same manner as in Example 2 (C). Significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using neuropilin.

실시예Example 56. 56. PeroxiredoxinPeroxiredoxin 1을 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Peroxiredoxin 1을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Peroxiredoxin 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Peroxiredoxin 1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Peroxiredoxin 1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Peroxiredoxin 1 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold more differentiation when treated with Peroxiredoxin 1 than the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Peroxiredoxin 1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Peroxiredoxin 1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Peroxiredoxin 1 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1 μg / ml to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the treatment with Peroxiredoxin 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Peroxiredoxin 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Peroxiredoxin 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Peroxiredoxin 1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Peroxiredoxin 1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Peroxiredoxin 1, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with the injection of mature natural killer cells differentiated with Peroxiredoxin 1, a significant decrease in cancer tissue volume was observed.

실시예Example 57. 57. lala associatedassociated invariantinvariant chainchain 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 la associated invariant chain을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using la associated invariant chain in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 la associated invariant chain을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 la associated invariant chain을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of la associated invariant chain instead of interleukin-15 under the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed differentiation rate by 2-3 times when treated with la associated invariant chain compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 la associated invariant chain을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 la associated invariant chain을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating pro-natural killer cells with la associated invariant chain with interleukin-15 in a 1: 1 ratio. As a result, the rate of differentiation was slightly increased as compared with the treatment with la associated invariant chain alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 la associated invariant chain에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by la associated invariant chain in human cord blood-derived hematopoietic stem cells in animal cancer model

la associated invariant chain을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 la associated invariant chain을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the la associated invariant chain, the mature natural killer cells of the control group treated with none were injected as a result of the same method as in Example 2 (C). Compared with one group, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using la associated invariant chain.

실시예Example 58. 58. GlycoproteinGlycoprotein ( ( transmembranetransmembrane ) ) nmbnmb 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Glycoprotein (transmembrane) nmb를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Glycoprotein (transmembrane) nmb in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Glycoprotein (transmembrane) nmb를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Glycoprotein (transmembrane) nmb를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Glycoprotein (transmembrane) nmb instead of interleukin-15 in the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with Glycoprotein (transmembrane) nmb compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Glycoprotein (transmembrane) nmb를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Glycoprotein (transmembrane) nmb를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Glycoprotein (transmembrane) nmb with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. As a result, it was shown that the differentiation rate was slightly increased compared with the treatment with glycoprotein (transmembrane) nmb alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Glycoprotein (transmembrane) nmb에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Glycoprotein (transmembrane) nmb in human cord blood-derived hematopoietic stem cells in animal cancer model

Glycoprotein (transmembrane) nmb를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Glycoprotein (transmembrane) nmb를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using glycoprotein (transmembrane) nmb, the mature natural killer cells of the control group which did not process anything were tested in the same manner as in Example 2 (C). Compared with the injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using Glycoprotein (transmembrane) nmb.

실시예Example 59. 59. SerumSerum amyloidamyloid A ( A ( SAASAA ) 3 ) 3 proteinprotein 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Serum amyloid A (SAA) 3 protein을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Serum amyloid A (SAA) 3 protein in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Serum amyloid A (SAA) 3 protein을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Serum amyloid A (SAA) 3 protein을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of Serum amyloid A (SAA) 3 protein instead of the treatment of interleukin-15 under the same conditions as in Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and The CD56 ⁺ NKG20 ⁺ cells showed a two to three fold increase in differentiation rate when treated with Serum amyloid A (SAA) 3 protein compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Serum amyloid A (SAA) 3 protein을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Serum amyloid A (SAA) 3 protein을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the differentiation rate of progenitor killer cells was treated with Serum amyloid A (SAA) 3 protein in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, it showed a slightly increased differentiation rate compared to the treatment with Serum amyloid A (SAA) 3 protein alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Serum amyloid A (SAA) 3 protein에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Serum amyloid A (SAA) 3 protein in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Serum amyloid A (SAA) 3 protein을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Serum amyloid A (SAA) 3 protein을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Human mature natural cells differentiated with Serum amyloid A (SAA) 3 protein to observe whether human cancer cells are killed by natural killer cells. Compared with the injection of killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using Serum amyloid A (SAA) 3 protein.

실시예Example 60. 60. Sgp1Sgp1 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Sgp1을 이용한 성숙자연살해세포 분화법 (A) Spontaneous natural killer cell differentiation using Sgp1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Sgp1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Sgp1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Sgp1 to the same conditions as Example 2 (A) instead of interleukin-15 treatment. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase in differentiation rate of 2 to 3 times when Sgp1 was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Sgp1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Sgp1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, progenitor killer cells were treated with Sgp1 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate under the same conditions. The results showed a slightly increased differentiation rate compared to the Sgp1 treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Sgp1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Sgp1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Sgp1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Sgp1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Experiments were carried out in the same manner as in Example 2 (C) to observe whether human cancer cells were killed by human mature natural killer cells differentiated using Sgp1. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Sgp1.

실시예Example 61. 61. Rmcs1Rmcs1 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Rmcs1을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Rmcs1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Rmcs1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Rmcs1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Rmcs1 to the same conditions as Example 2 (A) instead of interleukin-15 treatment. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when Rmcs1 was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Rmcs1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Rmcs1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Rmcs1 with interleukin-15 in a ratio of 1 µg / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with Rmcs1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Rmcs1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Rmcs1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Rmcs1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Rmcs1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Rmcs1, the same method as in Example 2 (C) was performed. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Rmcs1.

실시예Example 62. 62. ATPATP -- bindingbinding cassettecassette , , subsub -- familyfamily A를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using A 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 ATP-binding cassette, sub-family A를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using ATP-binding cassette, sub-family A in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 ATP-binding cassette, sub-family A를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 ATP-binding cassette, sub-family A를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 μg / ml (Upstat) of ATP-binding cassette and sub-family A, instead of treating Interleukin-15 under the same conditions as Example 2 (A). After differentiation for 1 day, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when ATP-binding cassette and sub-family A were treated compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 ATP-binding cassette, sub-family A를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 ATP-binding cassette, sub-family A를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, ATP-binding cassette and sub-family A were treated with 1 µg / ml in a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the treatment with ATP-binding cassette and sub-family A alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 ATP-binding cassette, sub-family A에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by ATP-binding cassette, sub-family A in human cord blood-derived hematopoietic stem cells in animal cancer model

ATP-binding cassette, sub-family A를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 ATP-binding cassette, sub-family A를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Human maturation differentiated using ATP-binding cassette, sub-family A. In order to observe whether human cancer cells are killed by natural killer cells, the same method as in Example 2 (C) was used. Compared with the injection of natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using ATP-binding cassette and sub-family A.

실시예Example 63. 63. ProgrammedProgrammed cellcell deathdeath 1 One ligandligand 1을 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Programmed cell death 1 ligand 1을 이용한 성숙자연살해세포 분화법 (A) Programmed cell death 1 ligand 1 in human cord blood-derived hematopoietic stem cells Mature natural killer cell differentiation using

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Programmed cell death 1 ligand 1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Programmed cell death 1 ligand 1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with Programmed cell death 1 ligand 1 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Differentiation was performed for 14 days by adding 1 μg / ml (Upstat) and human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ 1 cells contained Programmed cell death 1 ligand 1 compared to control group When treated, the rate of differentiation was increased by 2 to 3 times.

추가로, 동일조건하에 전구자연살해세포를 Programmed cell death 1 ligand 1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Programmed cell death 1 ligand 1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Programmed cell death 1 ligand 1 Differentiation rate was confirmed by treatment with interleukin-15 at 1 μg / ml in a ratio of 1: 1. As a result, Programmed cell death 1 ligand 1 It was shown that the rate of differentiation was slightly increased as compared with the treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Programmed cell death 1 ligand 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Programmed cell death 1 ligand 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Programmed cell death 1 ligand 1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Programmed cell death 1 ligand 1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다.Programmed cell death 1 Maturated natural killer cells of the control group treated with nothing after experimenting in the same manner as in Example 2 (C) to observe whether human cancer cells are killed by human mature natural killer cells differentiated using ligand 1 Compared with the injection of Programmed cell death 1 ligand 1 Significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using the cells.

실시예Example 64. 64. Dnajb11Dnajb11 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Dnajb11을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Dnajb11 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Dnajb11을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Dnajb11을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Dnajb11 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about 2 to 3 times more differentiation rate when treated with Dnajb11 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Dnajb11을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Dnajb11을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Dnajb11 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it was shown that the differentiation rate was slightly increased compared to the case of treatment with Dnajb11 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Dnajb11에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Dnajb11 in human cord blood-derived hematopoietic stem cells in animal cancer model

Dnajb11을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Dnajb11을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Dnajb11, the same method as in Example 2 (C) was used. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using DNAB11.

실시예Example 65. 65. Fibronectin1Fibronectin1 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Fibronectin1을 이용한 성숙자연살해세포 분화법 (A) Fibronectin1 in human cord blood-derived hematopoietic stem cells Mature natural killer cell differentiation using

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Fibronectin1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Fibronectin1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Fibronectin1 was treated with the progenitor killer cells obtained in Example 2 (A) instead of interleukin-15 under the same conditions as in Example 2 (A). Differentiation was performed for 14 days by adding 1 μg / ml (Upstat) and human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells received Fibronectin1 compared to the control group When treated, the rate of differentiation was increased by 2 to 3 times.

추가로, 동일조건하에 전구자연살해세포를 Fibronectin1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Fibronectin1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, Fibronectin1 was induced to progenitor killer cells under the same conditions. Differentiation rate was confirmed by treatment with interleukin-15 at 1 μg / ml in a ratio of 1: 1. As a result Fibronectin1 It was shown that the rate of differentiation was slightly increased as compared with the treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Fibronectin1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Fibronectin1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Fibronectin1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Fibronectin1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다.In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using fibronectin1, the same method as in Example 2 (C) was performed. Fibronectin1 Significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using the cells.

실시예Example 66. 66. FattyFatty acidacid bindingbinding proteinprotein 4를 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 4 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Fatty acid binding protein 4를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Fatty acid binding protein 4 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Fatty acid binding protein 4를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Fatty acid binding protein 4를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Fatty acid binding protein 4 instead of interleukin-15 under the same conditions as in Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed differentiation rate of 2 ~ 3 fold when Fatty acid binding protein 4 was treated compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 Fatty acid binding protein 4를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Fatty acid binding protein 4를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Fatty acid binding protein 4 with interleukin-15 at a ratio of 1 ㎍ / ml in a 1: 1 ratio to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with Fatty acid binding protein 4 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Fatty acid binding protein 4에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Fatty acid binding protein 4 in human cord blood-derived hematopoietic stem cells in animal cancer model

Fatty acid binding protein 4를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Fatty acid binding protein 4를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using fatty acid binding protein 4, the mature natural killer cells of the control group treated with nothing were tested as in Example 2 (C). Compared with the injection, a significant decrease in the volume of the cancer tissue was observed in the group injected with mature natural killer cells differentiated using Fatty acid binding protein 4.

실시예Example 67. 67. ComplementComplement componentcomponent 1, q, 1, q, gammagamma polypeptidepolypeptide 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Complement component 1, q, gamma polypeptide를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Complement component 1, q, gamma polypeptides in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Complement component 1, q, gamma polypeptide를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Complement component 1, q, gamma polypeptide를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as in Example 2 (A), and then 14 days of addition of 1 μg / ml (Upstat) of Complement component 1, q, and gamma polypeptides. After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with Complement component 1, q, and gamma polypeptides compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Complement component 1, q, gamma polypeptide를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Complement component 1, q, gamma polypeptide를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with Complement component 1, q, and gamma polypeptides at a ratio of 1: 1 with interleukin-15 at a ratio of 1: 1. As a result, it showed a slightly increased differentiation rate compared with the treatment with Complement component 1, q, gamma polypeptide alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Complement component 1, q, gamma polypeptide에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Complement component 1, q, gamma polypeptides in human cord blood-derived hematopoietic stem cells in animal cancer model

Complement component 1, q, gamma polypeptide를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Complement component 1, q, gamma polypeptide를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the Complement component 1, q and gamma polypeptides, the mature nature of the control group which was not treated with the same method as in Example 2 (C) Compared with the injection of killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using the Complement component 1, q and gamma polypeptides.

실시예Example 68. 68. FcFc g g receptorreceptor , , IgGIgG , , highhigh affinityaffinity 1을 이용한 사람 제대혈 유래 Derived from human cord blood using 1 조혈모Hematopoietic stem 세포로부터 From the cell 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Fc g receptor, IgG, high affinity 1을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Fc g receptor, IgG, high affinity 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Fc g receptor, IgG, high affinity 1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Fc g receptor, IgG, high affinity 1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of Fc g receptor, IgG and high affinity 1 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). After differentiation for 1 day, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when treated with Fc g receptor, IgG, and high affinity 1 compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 Fc g receptor, IgG, high affinity 1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Fc g receptor, IgG, high affinity 1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Fc g receptor, IgG and high affinity 1 with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with Fc g receptor, IgG, and high affinity 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Fc g receptor, IgG, high affinity 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Fc g receptor, IgG, high affinity 1 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Fc g receptor, IgG, high affinity 1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Fc g receptor, IgG, high affinity 1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Human maturation differentiated using Fc g receptor, IgG, high affinity 1 In order to observe whether human cancer cells are killed by natural killer cells, the same method as in Example 2 (C) was used. Compared with the injection of natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using Fc g receptor, IgG and high affinity 1.

실시예Example 69. 69. IFNIFN regulatoryregulatory factorfactor 를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 IFN regulatory factor를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using IFN regulatory factor in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 IFN regulatory factor를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 IFN regulatory factor를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of IFN regulatory factor instead of interleukin-15 in the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase in differentiation rate of 2 to 3 times when IFN-regulated factor was treated compared to the non-treated control group.

추가로, 동일조건하에 전구자연살해세포를 IFN regulatory factor를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 IFN regulatory factor를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with IFN regulatory factor in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate compared to the IFN alone treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 IFN regulatory factor에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of IFN regulatory factors in human cord blood-derived hematopoietic stem cells in animal cancer model

IFN regulatory factor를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 IFN regulatory factor를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using IFN regulatory factor, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as Example 2 (C). In contrast, significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using IFN regulatory factors.

실시예Example 70. 70. ComplementComplement componentcomponent subunitsubunit ClqClq betabeta -- chainchain 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Complement component subunit Clq beta-chain을 이용한 성숙자연살해세포 분화법 (A) Mature Natural Killer Cell Differentiation Using Complement Component Subunit Clq Beta Chain in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Complement component subunit Clq beta-chain을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Complement component subunit Clq beta-chain을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as in Example 2 (A), and then added with 1 µg / ml (Upstat) of Complement component subunit Clq beta-chain for 14 days. After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with the Complement component subunit Clq beta-chain compared to the control without any treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Complement component subunit Clq beta-chain에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Complement component subunit Clq beta-chain in human cord blood-derived hematopoietic stem cells in animal cancer model

Complement component subunit Clq beta-chain을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Complement component subunit Clq beta-chain을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated by using the Complement component subunit Clq beta-chain, the mature natural killer of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared to the cell injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using the Complement component subunit Clq beta-chain.

실시예Example 71. 2'-5' 71. 2'-5 ' oligoadenylateoligoadenylate synthetasesynthetase -- likelike 2를 이용한 사람 제대혈 유래 조혈모세포로부터 From human umbilical cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 2'-5' oligoadenylate synthetase-like 2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using 2'-5 'oligoadenylate synthetase-like 2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 2'-5' oligoadenylate synthetase-like 2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 2'-5' oligoadenylate synthetase-like 2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of 2'-5 'oligoadenylate synthetase-like 2 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). After differentiation for 14 days, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a 2 ~ 3 fold increase in differentiation rate when treated with 2'-5 'oligoadenylate synthetase-like 2 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 2'-5' oligoadenylate synthetase-like 2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 2'-5' oligoadenylate synthetase-like 2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating 2'-5 'oligoadenylate synthetase-like 2 with interleukin-15 at a ratio of 1 µg / ml in a 1: 1 ratio. The results showed a slightly increased differentiation rate when treated with 2'-5 'oligoadenylate synthetase-like 2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 2'-5' oligoadenylate synthetase-like 2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by 2'-5 'oligoadenylate synthetase-like 2 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

2'-5' oligoadenylate synthetase-like 2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 2'-5' oligoadenylate synthetase-like 2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells were killed by human mature natural killer cells differentiated using 2'-5 'oligoadenylate synthetase-like 2, the same procedure as in Example 2 (C) was performed. Compared with the injection of mature natural killer cells, a significant decrease in the volume of cancer tissues was observed in the injection of mature natural killer cells differentiated with 2'-5 'oligoadenylate synthetase-like 2.

실시예Example 72. 72. Chi3l3Chi3l3 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Chi3l3를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Chi3l3 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Chi3l3를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Chi3l3를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Chi3l3 to the same conditions as Example 2 (A) instead of interleukin-15 treatment. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation when treated with Chi3l3 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Chi3l3를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Chi3l3를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, progenitor killer cells were treated with Chi3l3 with interleukin-15 at a ratio of 1 μg / ml in a ratio of 1 μg / ml to confirm the differentiation rate. As a result, it was shown that the differentiation rate slightly increased compared to the case of treatment with Chi3l3 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Chi3l3에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Chi3l3 in human cord blood-derived hematopoietic stem cells in animal cancer model

Chi3l3를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Chi3l3를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Chi3l3, the experiments were performed in the same manner as in Example 2 (C). Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Chi3l3.

실시예Example 73. 73. IFNIFN -- activatedactivated genegene 203을 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using 203

(A) 사람 제대혈 유래 조혈모세포에서 IFN-activated gene 203을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using IFN-activated gene 203 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 IFN-activated gene 203을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 IFN-activated gene 203을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of IFN-activated gene 203 instead of interleukin-15 under the same conditions as in Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold more differentiation when treated with IFN-activated gene 203 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 IFN-activated gene 203을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 IFN-activated gene 203을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating IFN-activated gene 203 with interleukin-15 at a ratio of 1 μg / ml in a ratio of 1: 1. The results showed a slightly increased differentiation rate compared with IFN-activated gene 203 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 IFN-activated gene 203에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by IFN-activated gene 203 in human cord blood-derived hematopoietic stem cells in animal cancer model

IFN-activated gene 203을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 IFN-activated gene 203을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using IFN-activated gene 203, the mature natural killer cells of the control group treated with nothing were tested as in Example 2 (C). Compared with the injection, a significant decrease in the volume of the cancer tissue was observed in the group injected with mature natural killer cells differentiated using IFN-activated gene 203.

실시예Example 74. 74. TgfbiTgfbi 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Tgfbi를 이용한 성숙자연살해세포 분화법 (A) Differentiation of Mature Natural Killer Cells Using Tgfbi in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Tgfbi를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Tgfbi를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Tgfbi instead of interleukin-15 treatment under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Tgfbi compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Tgfbi를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Tgfbi를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Tgfbi with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to Tgfbi alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Tgfbi에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Tgfbi in human cord blood-derived hematopoietic stem cells in animal cancer model

Tgfbi를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Tgfbi를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Tgfbi, the same method as in Example 2 (C) was used. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Tgfbi.

실시예Example 75. 75. IgIg supersuper familyfamily , , membermember 7를7 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Ig super family, member 7를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Ig super family, member 7 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Ig super family, member 7를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Ig super family, member 7를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Ig super family, member 7, instead of interleukin-15 treatment under the same conditions as Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold more differentiation when treated with Ig super family, member 7, compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Ig super family, member 7를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Ig super family, member 7를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with Ig super family, member 7 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate when treated with Ig super family, member 7 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Ig super family, member 7에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Ig super family, member 7 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Ig super family, member 7를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Ig super family, member 7를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Ig super family, member 7, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). In comparison with the injection of Ig super family, member 7 injected with mature natural killer cells, a significant decrease in the volume of cancer tissues was observed.

실시예Example 76. 76. ChemokineChemokine (C-X-C (C-X-C motifmotif ) ) ligandligand 16를16 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Chemokine (C-X-C motif) ligand 16를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Chemokine (C-X-C motif) ligand 16 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Chemokine (C-X-C motif) ligand 16를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Chemokine (C-X-C motif) ligand 16를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 14 μg / ml (Upstat) of Chemokine (CXC motif) ligand 16 for 14 days instead of treating Interleukin-15 under the same conditions as Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with Chemokine (CXC motif) ligand 16 compared to the untreated control group.

추가로, 동일조건하에 전구자연살해세포를 Chemokine (C-X-C motif) ligand 16를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Chemokine (C-X-C motif) ligand 16를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Chemokine (C-X-C motif) ligand 16 with interleukin-15 at a ratio of 1 ㎍ / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the Chemokine (C-X-C motif) ligand 16 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Chemokine (C-X-C motif) ligand 16에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Chemokine (C-X-C motif) ligand 16 in human cord blood-derived hematopoietic stem cells in animal cancer model

Chemokine (C-X-C motif) ligand 16를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Chemokine (C-X-C motif) ligand 16를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Chemokine (CXC motif) ligand 16, the mature natural killer of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared to the cell injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using Chemokine (CXC motif) ligand 16.

실시예Example 77. 77. Csf2rb2Csf2rb2 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Csf2rb2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Csf2rb2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Csf2rb2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Csf2rb2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Csf2rb2 to the same conditions as Example 2 (A) instead of treating Interleukin-15. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about two to three times increased differentiation when treated with Csf2rb2 compared to the no-treated control group.

추가로, 동일조건하에 전구자연살해세포를 Csf2rb2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Csf2rb2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Csf2rb2 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it was shown that the differentiation rate slightly increased compared to the case of treatment with Csf2rb2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Csf2rb2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Csf2rb2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Csf2rb2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Csf2rb2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Csf2rb2, the experiments were performed in the same manner as in Example 2 (C). Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Csf2rb2.

실시예Example 78. 78. H2H2 -- BfBf 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 H2-Bf를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using H2-Bf in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 H2-Bf를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 H2-Bf를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of H2-Bf instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about 2 to 3 times higher differentiation rate when treated with H2-Bf compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 H2-Bf를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 H2-Bf를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with H2-Bf in a ratio of 1 µg / ml in a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to H2-Bf alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 H2-Bf에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by H2-Bf in human cord blood-derived hematopoietic stem cells in animal cancer model

H2-Bf를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 H2-Bf를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using H2-Bf, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to the group injected with mature natural killer cells differentiated with H2-Bf, a significant decrease in the volume of cancer tissue was observed.

실시예Example 79. 79. IFNIFN -- inducedinduced proteinprotein withwith tetratricopeptidetetratricopeptide repeats2repeats2 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 IFN-induced protein with tetratricopeptide repeats2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using IFN-induced protein with tetratricopeptide repeats2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 IFN-induced protein with tetratricopeptide repeats2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 IFN-induced protein with tetratricopeptide repeats2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 under the same conditions as in Example 2 (A), the progenitor killer cells obtained in Example 2 (A) were added with 1 μg / ml (Upstat) of IFN-induced protein with tetratricopeptide repeats2 for 14 days. After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with IFN-induced protein with tetratricopeptide repeats2 compared to the untreated control group.

추가로, 동일조건하에 전구자연살해세포를 IFN-induced protein with tetratricopeptide repeats2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 IFN-induced protein with tetratricopeptide repeats2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating IFN-induced protein with tetratricopeptide repeats2 with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio. The results showed a slightly increased differentiation rate compared with IFN-induced protein with tetratricopeptide repeats2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 IFN-induced protein with tetratricopeptide repeats2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by IFN-induced protein with tetratricopeptide repeats2 in human cord blood-derived hematopoietic stem cells in animal cancer model

IFN-induced protein with tetratricopeptide repeats2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 IFN-induced protein with tetratricopeptide repeats2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using IFN-induced protein with tetratricopeptide repeats2, the mature natural killer of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared to the cells injected, significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using IFN-induced protein with tetratricopeptide repeats2.

실시예Example 80. 80. RIKENRIKEN cDNAcDNA 2310005 2310005 K03K03 genegene 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 RIKEN cDNA 2310005K03 gene를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using RIKEN cDNA 2310005K03 gene in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 RIKEN cDNA 2310005K03 gene를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 RIKEN cDNA 2310005K03 gene를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of RIKEN cDNA 2310005K03 gene instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with the RIKEN cDNA 2310005K03 gene compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 RIKEN cDNA 2310005K03 gene를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 RIKEN cDNA 2310005K03 gene를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with RIKEN cDNA 2310005K03 gene at a ratio of 1: 1 with interleukin-15 at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared with the RIKEN cDNA 2310005K03 gene alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 RIKEN cDNA 2310005K03 gene에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by RIKEN cDNA 2310005K03 gene in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

RIKEN cDNA 2310005K03 gene를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 RIKEN cDNA 2310005K03 gene를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using RIKEN cDNA 2310005K03 gene, the mature natural killer cells of the control group treated with nothing were injected as a result of the same experiment as in Example 2 (C). Compared with the RIKEN cDNA 2310005K03 gene, a significant reduction in cancer tissue volume was observed in the group injected with mature natural killer cells.

실시예Example 81. 81. RIKENRIKEN cDNAcDNA 3110037 3110037 K17K17 genegene 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 RIKEN cDNA 3110037K17 gene를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using RIKEN cDNA 3110037K17 gene in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 RIKEN cDNA 3110037K17 gene를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 RIKEN cDNA 3110037K17 gene를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of RIKEN cDNA 3110037K17 gene instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with the RIKEN cDNA 3110037K17 gene compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 RIKEN cDNA 3110037K17 gene를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 RIKEN cDNA 3110037K17 gene를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with RIKEN cDNA 3110037K17 gene in a ratio of 1: 1 with interleukin-15 at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared with the RIKEN cDNA 3110037K17 gene alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 RIKEN cDNA 3110037K17 gene에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by RIKEN cDNA 3110037K17 gene in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

RIKEN cDNA 3110037K17 gene를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 RIKEN cDNA 3110037K17 gene를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using RIKEN cDNA 3110037K17 gene, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with the RIKEN cDNA 3110037K17 gene, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells.

실시예Example 82. 82. RIKENRIKEN cDNAcDNA 5830458 5830458 K16K16 genegene 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 RIKEN cDNA 5830458K16 gene를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using RIKEN cDNA 5830458K16 gene in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 RIKEN cDNA 5830458K16 gene를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 RIKEN cDNA 5830458K16 gene를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of RIKEN cDNA 5830458K16 gene instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with the RIKEN cDNA 5830458K16 gene compared to the no-treated control group.

추가로, 동일조건하에 전구자연살해세포를 RIKEN cDNA 5830458K16 gene를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 RIKEN cDNA 5830458K16 gene를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with RIKEN cDNA 5830458K16 gene in an amount of 1: 1 with interleukin-15 in a ratio of 1: 1. The results showed a slightly increased differentiation rate compared with the RIKEN cDNA 5830458K16 gene alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 RIKEN cDNA 5830458K16 gene에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by RIKEN cDNA 5830458K16 gene in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

RIKEN cDNA 5830458K16 gene를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 RIKEN cDNA 5830458K16 gene를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using RIKEN cDNA 5830458K16 gene, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with the RIKEN cDNA 5830458K16 gene, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells.

실시예Example 83. 83. ThymidylateThymidylate kinasekinase familyfamily LPSLPS -- inducibleinducible membermember 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Thymidylate kinase family LPS-inducible member를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Thymidylate kinase family LPS-inducible member in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Thymidylate kinase family LPS-inducible member를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Thymidylate kinase family LPS-inducible member를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of Thymidylate kinase family LPS-inducible member for 14 days instead of treating Interleukin-15 under the same conditions as Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with Thymidylate kinase family LPS-inducible member compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Thymidylate kinase family LPS-inducible member를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 RIKEN cDNA 5830458K16 gene를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, the proliferative killer cells were treated with Thymidylate kinase family LPS-inducible member with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio to confirm the differentiation rate under the same conditions. The results showed a slightly increased differentiation rate compared with the RIKEN cDNA 5830458K16 gene alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Thymidylate kinase family LPS-inducible member에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Thymidylate kinase family LPS-inducible member in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Thymidylate kinase family LPS-inducible member를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Thymidylate kinase family LPS-inducible member를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Thymidylate kinase family LPS-inducible member, the mature natural killer of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared with the cell injection, a significant decrease in the volume of cancer tissue was observed in the group injected with the mature natural killer cells differentiated using the Thymidylate kinase family LPS-inducible member.

실시예Example 84. 84. SyntaxinSyntaxin bindingbinding proteinprotein 3를3 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Syntaxin binding protein 3를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Syntaxin binding protein 3 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Syntaxin binding protein 3를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 RIKEN cDNA 5830458K16 gene를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. After progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as in Example 2 (A), 1 µg / ml (Upstat) of Syntaxin binding protein 3 was added and differentiated for 14 days. Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with the RIKEN cDNA 5830458K16 gene compared to the no-treated control group.

추가로, 동일조건하에 전구자연살해세포를 Syntaxin binding protein 3를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Syntaxin binding protein 3를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating Syntaxin binding protein 3 with Interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with Syntaxin binding protein 3 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Syntaxin binding protein 3에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Syntaxin binding protein 3 in human cord blood-derived hematopoietic stem cells in animal cancer model

Syntaxin binding protein 3를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Syntaxin binding protein 3를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Syntaxin binding protein 3, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to that, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Syntaxin binding protein 3.

실시예Example 85. 85. FattyFatty acidacid bindingbinding proteinprotein 5를 이용한 사람 제대혈 유래 조혈모세포로부터 From human umbilical cord blood-derived hematopoietic stem cells using 5 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Fatty acid binding protein 5를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Fatty acid binding protein 5 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Fatty acid binding protein 5를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Fatty acid binding protein 5를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Fatty acid binding protein 5 instead of interleukin-15 under the same conditions as in Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 times higher differentiation rate when Fatty acid binding protein 5 was treated compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 Fatty acid binding protein 5를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Fatty acid binding protein 5를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with Fatty acid binding protein 5 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, it was shown that the differentiation rate was slightly increased compared to the case of fatty acid binding protein 5 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Fatty acid binding protein 5에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Fatty acid binding protein 5 in human cord blood-derived hematopoietic stem cells in animal cancer model

Fatty acid binding protein 5를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Fatty acid binding protein 5를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using fatty acid binding protein 5, the mature natural killer cells of the control group treated with nothing were tested as in Example 2 (C). Compared with the injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated with Fatty acid binding protein 5.

실시예Example 82. 82. Isg12Isg12 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Isg12를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Isg12 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Isg12를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Isg12를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Isg12 instead of interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with Isg12 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Isg12를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Isg12를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Isg12 with interleukin-15 in a ratio of 1 μg / ml in a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with Isg12 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Isg12에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Isg12 in human cord blood-derived hematopoietic stem cells in animal cancer model

Isg12를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Isg12를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Isg12, the result of experiments in the same manner as in Example 2 (C) was compared with that injected with mature natural killer cells of the control group which had not been treated. In the group injected with mature natural killer cells differentiated using Isg12, a significant decrease in cancer tissue volume was observed.

실시예 87. Allograft inflammatory factor 1을 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Example 87. Allograft inflammatory differentiation of mature NK cells from human cord blood-derived stem cells with factor 1

(A) 사람 제대혈 유래 조혈모세포에서 Allograft inflammatory factor 1을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using allograft inflammatory factor 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Allograft inflammatory factor 1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다.CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Allograft inflammatory factor 1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Allograft inflammatory factor 1 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold higher differentiation rate when Allograft inflammatory factor 1 was treated compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Allograft inflammatory factor 1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Allograft inflammatory factor 1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Allograft inflammatory factor 1 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. As a result, allograft inflammatory factor 1 showed a slightly increased differentiation rate compared to the treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Allograft inflammatory factor 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by allograft inflammatory factor 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Allograft inflammatory factor 1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Allograft inflammatory factor 1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using allograft inflammatory factor 1, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to that, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Allograft inflammatory factor 1.

실시예Example 88. 88. BiliverdinBiliverdin reductasereductase B를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using B

(A) 사람 제대혈 유래 조혈모세포에서 Biliverdin reductase B를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Biliverdin reductase B in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Biliverdin reductase B를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Biliverdin reductase B를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Biliverdin reductase B instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold more differentiation when treated with Biliverdin reductase B compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Biliverdin reductase B를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Biliverdin reductase B를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating Biliverdin reductase B with Interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio. As a result, it showed a slightly increased differentiation rate compared with biliverdin reductase B alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Biliverdin reductase B에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Biliverdin reductase B in human cord blood-derived hematopoietic stem cells in animal cancer model

Biliverdin reductase B를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Biliverdin reductase B를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Biliverdin reductase B, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to the group injected with mature killer cells differentiated using Biliverdin reductase B, a significant decrease in the volume of cancer tissue was observed.

실시예Example 89. 89. ComplementComplement componentcomponent 3a 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using 3a 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Complement component 3a를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Complement component 3a in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Complement component 3a를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Complement component 3a를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Complement component 3a, instead of treating Interleukin-15 under the same conditions as Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Complement component 3a compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Complement component 3a를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Complement component 3a를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating the progenitor killer cells with Complement component 3a in a ratio of 1 μg / ml in a ratio of 1: 1 with interleukin-15. As a result, it showed a slightly increased differentiation rate compared to the case of treating Complement component 3a alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Complement component 3a에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Complement component 3a in human cord blood-derived hematopoietic stem cells in animal cancer model

Complement component 3a를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Complement component 3a를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the Complement component 3a, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to the group injected with mature natural killer cells differentiated using Complement component 3a, a significant decrease in the volume of cancer tissue was observed.

실시예Example 90. 90. SelenoproteinSelenoprotein R를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using R 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Selenoprotein R를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Selenoprotein R in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Selenoprotein R를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 RIKEN cDNA 5830458K16 gene를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Selenoprotein R instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with the RIKEN cDNA 5830458K16 gene compared to the no-treated control group.

추가로, 동일조건하에 전구자연살해세포를 Selenoprotein R를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Selenoprotein R를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate of progenitor killer cells was treated with Selenoprotein R with interleukin-15 in a 1: 1 µg / ml amount. The results showed a slightly increased differentiation rate compared with Selenoprotein R alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Selenoprotein R에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Selenoprotein R in human cord blood-derived hematopoietic stem cells in animal cancer model

Selenoprotein R를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Selenoprotein R를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated by using Selenoprotein R, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with Selenoprotein R, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells.

실시예Example 91. 91. ILIL -1-One RARA 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 IL-1RA를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using IL-1RA in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 IL-1RA를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 IL-1RA를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of IL-1RA instead of interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with IL-1RA compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 IL-1RA를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 IL-1RA를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, progenitor killer cells were treated with IL-1RA with interleukin-15 in a ratio of 1 μg / ml in a ratio of 1 μg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with IL-1RA alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 IL-1RA에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by IL-1RA in human cord blood-derived hematopoietic stem cells in animal cancer model

IL-1RA를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 IL-1RA를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using IL-1RA, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to the group injected with mature natural killer cells differentiated using IL-1RA, a significant decrease in the volume of cancer tissue was observed.

실시예Example 92. 92. AdenylosuccinateAdenylosuccinate synthetasesynthetase 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Adenylosuccinate synthetase를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Adenylosuccinate synthetase in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Adenylosuccinate synthetase를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Adenylosuccinate synthetase를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Adenylosuccinate synthetase instead of interleukin-15 in the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 to 3 times higher differentiation rate when Adenylosuccinate synthetase was treated compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Adenylosuccinate synthetase를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 RIKEN cDNA 5830458K16 gene를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, Adenylosuccinate synthetase was treated with Aleukin-15 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the RIKEN cDNA 5830458K16 gene alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Adenylosuccinate synthetase에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Adenylosuccinate synthetase in human cord blood-derived hematopoietic stem cells in animal cancer model

Adenylosuccinate synthetase를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Adenylosuccinate synthetase를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Adenylosuccinate synthetase, the same method as in Example 2 (C) was used. Compared with Adenylosuccinate synthetase, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells.

실시예Example 82. 82. HemopoieticHemopoietic cellcell kinasekinase 를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 Hemopoietic cell kinase를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using hemopoietic cell kinase in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Hemopoietic cell kinase를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Hemopoietic cell kinase를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. After progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as in Example 2 (A), 1 μg / ml (Upstat) of Hemopoietic cell kinase was added and differentiated for 14 days. Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with hemopoietic cell kinase compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Hemopoietic cell kinase를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Hemopoietic cell kinase를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with Hemopoietic cell kinase in an amount of 1 μg / ml in a ratio of 1: 1 with interleukin-15. As a result, the hemopoietic cell kinase alone showed slightly increased differentiation rate.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Hemopoietic cell kinase에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by hemopoietic cell kinase in human cord blood-derived hematopoietic stem cells in animal cancer model

Hemopoietic cell kinase를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Hemopoietic cell kinase를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using hemopoietic cell kinase, the mature natural killer cells of the control group treated with nothing were injected. Compared with the hemopoietic cell kinase, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells.

실시예Example 94. 94. Dedd2Dedd2 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Dedd2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Dedd2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Dedd2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Dedd2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Dedd2 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with Dedd2 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Dedd2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Dedd2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Dedd2 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the case of treatment with Dedd2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Dedd2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Dedd2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Dedd2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Dedd2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Dedd2, the same method as in Example 2 (C) was performed. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Dedd2.

실시예Example 95. 95. IFNIFN inducedinduced transmembranetransmembrane proteinprotein 2 를2 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 IFN induced transmembrane protein 2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using IFN induced transmembrane protein 2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 IFN induced transmembrane protein 2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 IFN induced transmembrane protein 2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of IFN induced transmembrane protein 2 instead of interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with IFN induced transmembrane protein 2 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 IFN induced transmembrane protein 2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 IFN induced transmembrane protein 2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating IFN induced transmembrane protein 2 with Interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio. The results showed a slightly increased differentiation rate compared with IFN induced transmembrane protein 2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 IFN induced transmembrane protein 2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of IFN induced transmembrane protein 2 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

IFN induced transmembrane protein 2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 IFN induced transmembrane protein 2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using IFN-induced transmembrane protein 2, the mature natural killer cells of the control group treated with nothing were tested as in Example 2 (C). Compared with the injection, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using IFN induced transmembrane protein 2.

실시예Example 96. 96. GAVAGAVA -A -A receptorreceptor subunitsubunit 6를6 이용한 사람 제대혈 유래 Derived from human cord blood 조혈모세포로부From hematopoietic stem cells 터 foundation 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 GAVA-A receptor subunit 6를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using GAVA-A receptor subunit 6 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 GAVA-A receptor subunit 6를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 GAVA-A receptor subunit 6를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of GAVA-A receptor subunit 6 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 to 3 fold more differentiation when treated with GAVA-A receptor subunit 6 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 GAVA-A receptor subunit 6를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 GAVA-A receptor subunit 6를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with GAVA-A receptor subunit 6 with interleukin-15 in a ratio of 1 μg / ml at a ratio of 1 μg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the GAVA-A receptor subunit 6 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 GAVA-A receptor subunit 6에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by GAVA-A receptor subunit 6 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

GAVA-A receptor subunit 6를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 GAVA-A receptor subunit 6를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using GAVA-A receptor subunit 6, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). In comparison with the injection of the NK cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using GAVA-A receptor subunit 6.

실시예Example 97. 97. SelplSelpl 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Selpl를 이용한 성숙자연살해세포 분화법 (A) Differentiation of mature natural killer cells using Selpl in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Selpl를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Selpl를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Selpl instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Selpl compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Selpl를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Selpl를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate of progenitor killer cells was treated with 1 μg / ml of Selpl at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate compared to Selpl alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Selpl에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Selpl in human cord blood-derived hematopoietic stem cells in animal cancer model

Selpl를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Selpl를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Selpl, experiments were carried out in the same manner as in Example 2 (C). Significant decrease of cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Selpl.

실시예Example 98. 98. Hmgb1Hmgb1 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Hmgb1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Hmgb1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Hmgb1 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about two to three times increased differentiation when treated with Hmgb1 compared to the control group without any treatment.

실시예Example 99. 99. TyrosineTyrosine 3- 3- monooxygenasemonooxygenase /Of tryptophantryptophan 5- 5- monooxygenasemonooxygenase activationactivation protein, protein, epsilonepsilon polypeptidepolypeptide 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein and epsilon polypeptide in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 under the same conditions as in Example 2 (A), the progenitor killer cells obtained in Example 2 (A) were treated with Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein and epsilon polypeptide in 1 μg / ml ( Upstat) was added and differentiated for 14 days, and then human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein and epsilon polypeptide compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, progenitor killer cells were treated with Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein and epsilon polypeptide with interleukin-15 in a 1: 1 ratio of 1 µg / ml under the same conditions to confirm the differentiation rate. . The results showed a slightly increased differentiation rate compared to the treatment with tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein and epsilon polypeptide alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, epsilon polypeptide in human cord blood-derived hematopoietic stem cells

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, epsilon Polypeptide differentiated with human mature natural killer cells to observe whether human cancer cells are killed by the same method as in Example 2 (C) to treat nothing A significant decrease in the volume of the cancer tissue was observed in the group injected with mature natural killer cells differentiated with Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein and epsilon polypeptide, compared with the control group with mature natural killer cells. .

실시예Example 100. 100. Serpinb6bSerpinb6b 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Serpinb6b를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Serpinb6b in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Serpinb6b를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Serpinb6b를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Serpinb6b instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Serpinb6b compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Serpinb6b를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Serpinb6b를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating Serpinb6b with Interleukin-15 in an amount of 1 µg / ml in a ratio of 1: 1. The results showed a slightly increased differentiation rate compared to the treatment with Serpinb6b alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Serpinb6b에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Serpinb6b in human cord blood-derived hematopoietic stem cells in animal cancer model

Serpinb6b를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Serpinb6b를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using serpinb6b, the same procedure as in Example 2 (C) was performed. In the group injected with mature natural killer cells differentiated using serpinb6b, a significant decrease in the volume of cancer tissue was observed.

실시예Example 101. 101. RegulatorRegulator ofof G- G- proteinprotein signalingsignaling 16를16 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Regulator of G-protein signaling 16를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Regulator of G-protein signaling 16 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Regulator of G-protein signaling 16를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Regulator of G-protein signaling 16를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 14 μg / ml (Upstat) of Regulator of G-protein signaling 16 for 14 days instead of interleukin-15 treatment under the same conditions as in Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when treated with the Regulator of G-protein signaling 16, compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Regulator of G-protein signaling 16를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Regulator of G-protein signaling 16를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Regulator of G-protein signaling 16 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1 μg / ml to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the treatment with the regulator of G-protein signaling 16 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Regulator of G-protein signaling 16에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Regulator of G-protein signaling 16 in human cord blood-derived hematopoietic stem cells in animal cancer model

Regulator of G-protein signaling 16를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Regulator of G-protein signaling 16를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using a regulator of G-protein signaling 16, the mature natural killer of a control group treated with nothing was tested in the same manner as in Example 2 (C). Compared to the cell injection, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using the Regulator of G-protein signaling 16.

실시예Example 102. 6330406 102. 6330406 L22RikL22Rik 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 6330406L22Rik를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using 6330406L22Rik in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 6330406L22Rik를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 6330406L22Rik를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1µg / ml (Upstat) of 6330406L22Rik instead of treating Interleukin-15 under the same conditions as Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with 6330406L22Rik compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 6330406L22Rik를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 6330406L22Rik를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with 6330406L22Rik at a rate of 1: 1 with interleukin-15 at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared to the 6330406L22Rik treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 6330406L22Rik에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by 6330406L22Rik in human cord blood-derived hematopoietic stem cells in animal cancer model

6330406L22Rik를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 6330406L22Rik를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using 6330406L22Rik, experiments were performed in the same manner as in Example 2 (C). Significant decrease of cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using 6330406L22Rik.

실시예Example 103. 103. OsteopontinOsteopontin precursorprecursor 를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 Osteopontin precursor를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Osteopontin precursor in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Osteopontin precursor를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Osteopontin precursor를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Osteopontin precursor instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with Osteopontin precursor compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Osteopontin precursor를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Osteopontin precursor를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Osteopontin precursor with interleukin-15 in a ratio of 1 ㎍ / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it was shown that the differentiation rate was slightly increased as compared with the treatment with Osteopontin precursor alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Osteopontin precursor에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Osteopontin precursors in human cord blood-derived hematopoietic stem cells in animal cancer model

Osteopontin precursor를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Osteopontin precursor를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated by using osteopontin precursor, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as Example 2 (C). Compared to the group injected with mature natural killer cells differentiated with Osteopontin precursor, a significant decrease in cancer tissue volume was observed.

실시예Example 104. 104. ZincZinc fingerfinger proteinprotein 207를207 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Zinc finger protein 207를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using zinc finger protein 207 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Zinc finger protein 207를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Zinc finger protein 207를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Zinc finger protein 207 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when zinc finger protein 207 was treated compared to the control group which had no treatment.

추가로, 동일조건하에 전구자연살해세포를 Zinc finger protein 207를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Zinc finger protein 207를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Zinc finger protein 207 with interleukin-15 in a ratio of 1 ㎍ / ml in a 1: 1 ratio to confirm the differentiation rate. As a result, zinc finger protein 207 alone showed slightly increased differentiation rate.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Zinc finger protein 207에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Zinc finger protein 207 in human cord blood-derived hematopoietic stem cells in animal cancer model

Zinc finger protein 207를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Zinc finger protein 207를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using zinc finger protein 207, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to the other group, significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Zinc finger protein 207.

실시예Example 105. 105. decaydecay -- acceleratingaccelerating factorfactor GPIGPI 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 decay-accelerating factor GPI를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using decay-accelerating factor GPI in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 decay-accelerating factor GPI를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 decay-accelerating factor GPI를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of decay-accelerating factor GPI instead of interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with decay-accelerating factor GPI compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 decay-accelerating factor GPI를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 decay-accelerating factor GPI를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating decay-accelerating factor GPI with interleukin-15 in a ratio of 1 ㎍ / ml at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared with the decay-accelerating factor GPI alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 decay-accelerating factor GPI에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by decay-accelerating factor GPI in human cord blood-derived hematopoietic stem cells in animal cancer model

decay-accelerating factor GPI를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 decay-accelerating factor GPI를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the decay-accelerating factor GPI, the mature natural killer cells of the control group treated with nothing were tested as in Example 2 (C). Compared with the injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using decay-accelerating factor GPI.

실시예Example 106. 106. AmyloidAmyloid betabeta (A4) (A4) precursorprecursor proteinprotein -- bindingbinding , , familyfamily B, B, membermember 1 interacting 1 interacting proteinprotein 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 in the same conditions as Example 2 (A), the progenitor killer cells obtained in Example 2 (A) were 1 μg of Amyloid beta (A4) precursor protein-binding, family B, and member 1 interacting protein. After 14 days of differentiation by addition of / ml (Upstat), human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with Amyloid beta (A4) precursor protein-binding, family B, and member 1 interacting protein.

추가로, 동일조건하에 전구자연살해세포를 Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein with interleukin-15 at a ratio of 1 μg / ml in a ratio of 1 μg / ml. It was confirmed. As a result, it was shown that the differentiation rate was slightly increased compared with the treatment with Amyloid beta (A4) precursor protein-binding, family B and member 1 interacting protein alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein in human cord blood-derived hematopoietic stem cells

Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Experiments were carried out in the same manner as in Example 2 (C) to observe whether human cancer cells are killed by mature human killer cells differentiated using amyloid beta (A4) precursor protein-binding, family B, and member 1 interacting protein. Results Compared with the injection of mature natural killer cells from the control group treated with nothing, cancer tissue volume in the group injected with mature natural killer cells differentiated using Amyloid beta (A4) precursor protein-binding, family B, and member 1 interacting protein. A significant decrease of was observed.

실시예Example 107. 107. Glrx1Glrx1 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Glrx1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Glrx1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Glrx1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Glrx1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Glrx1 to the same conditions as Example 2 (A) instead of interleukin-15 treatment. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 to 3 fold increase in differentiation rate when treated with Glrx1 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Glrx1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Glrx1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Glrx1 with interleukin-15 at a ratio of 1 μg / ml in a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the treatment with Glrx1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Glrx1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Glrx1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Glrx1를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Glrx1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Glrx1, experiments were performed in the same manner as in Example 2 (C). Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Glrx1.

실시예Example 108. 108. Coro1aCoro1a 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Coro1a를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Coro1a in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Coro1a를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Coro1a를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Coro1a instead of interleukin-15 in the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increased rate of differentiation of 2 to 3 times when Coro1a was treated compared to the non-treated controls.

추가로, 동일조건하에 전구자연살해세포를 Coro1a를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Coro1a를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Coro1a with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1: 1 to confirm the differentiation rate. As a result, it was shown that the differentiation rate slightly increased compared to the case of treatment with Coro1a alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Coro1a에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Coro1a in human cord blood-derived hematopoietic stem cells in animal cancer model

Coro1a를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Coro1a를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Coro1a, experiments were carried out in the same manner as in Example 2 (C). Significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using Coro1a.

실시예Example 109. 109. RIKENRIKEN cDNAcDNA 9630048 9630048 M01M01 genegene 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 RIKEN cDNA 9630048M01 gene를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using RIKEN cDNA 9630048M01 gene in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 RIKEN cDNA 9630048M01 gene를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 RIKEN cDNA 9630048M01 gene를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of RIKEN cDNA 9630048M01 gene instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with the RIKEN cDNA 9630048M01 gene compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 RIKEN cDNA 9630048M01 gene를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 RIKEN cDNA 9630048M01 gene를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with RIKEN cDNA 9630048M01 gene at a ratio of 1: 1 with interleukin-15 at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared with the RIKEN cDNA 9630048M01 gene alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 RIKEN cDNA 9630048M01 gene에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by RIKEN cDNA 9630048M01 gene in human cord blood-derived hematopoietic stem cells in animal cancer model

RIKEN cDNA 9630048M01 gene를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 RIKEN cDNA 9630048M01 gene를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the RIKEN cDNA 9630048M01 gene, the mature natural killer cells of the control group treated with none were injected as a result of the same method as in Example 2 (C). Compared with the RIKEN cDNA 9630048M01 gene, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells.

실시예Example 110. 110. DendriticDendritic cellcell proteinprotein GA17GA17 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Dendritic cell protein GA17를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Dendritic cell protein GA17 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Dendritic cell protein GA17를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Dendritic cell protein GA17를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Dendritic cell protein GA17 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed differentiation rate by 2 to 3 times when treated with Dendritic cell protein GA17 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Dendritic cell protein GA17를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Dendritic cell protein GA17를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Dendritic cell protein GA17 with interleukin-15 in a ratio of 1 µg / ml at a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate when treated with Dendritic cell protein GA17 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Dendritic cell protein GA17에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Dendritic cell protein GA17 in human cord blood-derived hematopoietic stem cells in animal cancer model

Dendritic cell protein GA17를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Dendritic cell protein GA17를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using dendritic cell protein GA17, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). In contrast, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Dendritic cell protein GA17.

실시예Example 111. G 111.G proteinprotein -- coupledcoupled receptorreceptor 171를171 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 G protein-coupled receptor 171를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using G protein-coupled receptor 171 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 G protein-coupled receptor 171를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 G protein-coupled receptor 171를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of G protein-coupled receptor 171 instead of treating Interleukin-15 under the same conditions as Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with G protein-coupled receptor 171 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 G protein-coupled receptor 171를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 G protein-coupled receptor 171를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with G protein-coupled receptor 171 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate when treated with G protein-coupled receptor 171 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 G protein-coupled receptor 171에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by G protein-coupled receptor 171 in human cord blood-derived hematopoietic stem cells in animal cancer model

G protein-coupled receptor 171를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 G protein-coupled receptor 171를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using G protein-coupled receptor 171, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). In contrast to the injection of the NK cells, the tumor volume was significantly decreased in the group injected with mature natural killer cells differentiated using G protein-coupled receptor 171.

실시예Example 112. 112. UpstreamUpstream transcriptiontranscription factorfactor 1를1 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Upstream transcription factor 1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using upstream transcription factor 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Upstream transcription factor 1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Upstream transcription factor 1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Upstream transcription factor 1 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and One CD56 ⁺ NKG20 ⁺ cells showed a two to three fold increase in the differentiation rate when treated with Upstream transcription factor 1 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Upstream transcription factor 1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Upstream transcription factor 1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rates of progenitor killer cells were treated with Upstream transcription factor 1 in an amount of 1 µg / ml at a ratio of 1: 1 with interleukin-15. As a result, it showed a slightly increased differentiation rate when treated with Upstream transcription factor 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Upstream transcription factor 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by upstream transcription factor 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Upstream transcription factor 1를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Upstream transcription factor 1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using upstream transcription factor 1, the mature natural killer cells of the control group treated with none were injected as a result of the same method as in Example 2 (C). In contrast, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Upstream transcription factor 1.

실시예Example 113. 1810015 113.1810015 M01RikM01Rik 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 1810015M01Rik를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using 1810015M01Rik in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 1810015M01Rik를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 1810015M01Rik를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of 1810015M01Rik instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about two to three times increased differentiation when treated with 1810015M01Rik compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 1810015M01Rik를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 1810015M01Rik를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with 1810015M01Rik at a ratio of 1: 1 with interleukin-15 at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared to the 1810015M01Rik treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 1810015M01Rik에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by 1810015M01Rik in human cord blood-derived hematopoietic stem cells in animal cancer model

1810015M01Rik를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 1810015M01Rik를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using 1810015M01Rik, experiments were carried out in the same manner as in Example 2 (C). Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using 1810015M01Rik.

실시예Example 114. 114. KaryopherinKaryopherin ( ( importinimportin ) ) alphaalpha 6를6 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Karyopherin (importin) alpha 6를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Karyopherin (importin) alpha 6 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Karyopherin (importin) alpha 6를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Karyopherin (importin) alpha 6를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Karyopherin (importin) alpha 6 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three fold increase in differentiation rate when treated with Karyopherin (importin) alpha 6 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Karyopherin (importin) alpha 6를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Karyopherin (importin) alpha 6를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Karyopherin (importin) alpha 6 with interleukin-15 in a ratio of 1 ㎍ / ml at a ratio of 1 ㎍ / ml to confirm the differentiation rate. As a result, the rate of differentiation was slightly increased as compared with the treatment with Karyopherin (importin) alpha 6 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Karyopherin (importin) alpha 6에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Karyopherin (importin) alpha 6 in human cord blood-derived hematopoietic stem cells in animal cancer model

Karyopherin (importin) alpha 6를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Karyopherin (importin) alpha 6를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Karyopherin (importin) alpha 6, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). In comparison with the injection of NK, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using Karyopherin (importin) alpha 6.

실시예 115. Ubiquinol - cytochrome c reductase , Rieske iron - sulfur polypeptide 1를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Example 115. Ubiquinol - cytochrome c reductase , Rieske iron - sulfur polypeptide Differentiation of mature NK cells from human cord blood-derived hematopoietic stem cells using one

(A) 사람 제대혈 유래 조혈모세포에서 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Ubiquinol-cytochrome c reductase and Rieske iron-sulfur polypeptide 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 under the same conditions as Example 2 (A), the progenitor killer cells obtained in Example 2 (A) were treated with 1 μg / ml of Ubiquinol-cytochrome c reductase and Rieske iron-sulfur polypeptide 1 (Upstat). After addition and differentiation for 14 days, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with Ubiquinol-cytochrome c reductase and Rieske iron-sulfur polypeptide 1 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, probiotic killer cells were treated with Ubiquinol-cytochrome c reductase and Rieske iron-sulfur polypeptide 1 together with interleukin-15 in a ratio of 1 ㎍ / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate when treated with Ubiquinol-cytochrome c reductase and Rieske iron-sulfur polypeptide 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using ubiquinol-cytochrome c reductase and Rieske iron-sulfur polypeptide 1, no treatment was performed in the same manner as in Example 2 (C). Compared with the control group of mature natural killer cells, there was a significant decrease in the volume of cancer tissue in the group of mature natural killer cells differentiated using Ubiquinol-cytochrome c reductase and Rieske iron-sulfur polypeptide 1.

실시예Example 116. 116. KelchKelch domaindomain containingcontaining 2 를2 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Kelch domain containing 2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Kelch domain containing 2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Kelch domain containing 2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Kelch domain containing 2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Kelch domain containing 2 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold more differentiation when treated with Kelch domain containing 2 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Kelch domain containing 2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Kelch domain containing 2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Kelch domain containing 2 with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1: 1 to confirm the differentiation rate. As a result, it was shown that the differentiation rate slightly increased compared to the case of Kelch domain containing 2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Kelch domain containing 2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Kelch domain containing 2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Kelch domain containing 2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Kelch domain containing 2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Kelch domain containing 2, the mature natural killer cells of the control group treated with nothing were injected in the same manner as in Example 2 (C). Compared to the one injected with mature natural killer cells differentiated with Kelch domain containing 2, a significant decrease in the volume of cancer tissue was observed.

실시예Example 117. 117. StructureStructure specificspecific recognitionrecognition proteinprotein 1를1 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Structure specific recognition protein 1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using structure specific recognition protein 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Structure specific recognition protein 1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Structure specific recognition protein 1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Structure specific recognition protein 1 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when treated with Structure specific recognition protein 1 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Structure specific recognition protein 1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Structure specific recognition protein 1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Structure specific recognition protein 1 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared with the structure specific recognition protein 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Structure specific recognition protein 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Structure specific recognition protein 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Structure specific recognition protein 1를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Structure specific recognition protein 1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using structure specific recognition protein 1, the mature natural killer cells of the control group treated with nothing were tested as in Example 2 (C). Compared with the injection, a significant decrease in the volume of the cancer tissue was observed in the group injected with mature natural killer cells differentiated using Structure specific recognition protein 1.

실시예Example 118. 118. LymphocyteLymphocyte proteinprotein tyrosinetyrosine kinasekinase 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Lymphocyte protein tyrosine kinase를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Lymphocyte protein tyrosine kinase in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Lymphocyte protein tyrosine kinase를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Lymphocyte protein tyrosine kinase를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Lymphocyte protein tyrosine kinase, instead of treating Interleukin-15 under the same conditions as Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate with Lymphocyte protein tyrosine kinase compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Lymphocyte protein tyrosine kinase를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Lymphocyte protein tyrosine kinase를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating Lymphocyte protein tyrosine kinase with 1 μg / ml in a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate compared with Lymphocyte protein tyrosine kinase alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Lymphocyte protein tyrosine kinase에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Lymphocyte protein tyrosine kinase in human cord blood-derived hematopoietic stem cells in animal cancer model

Lymphocyte protein tyrosine kinase를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Lymphocyte protein tyrosine kinase를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using lymphocyte protein tyrosine kinase, the mature natural killer cells of the control group treated with nothing were injected in the same manner as in Example 2 (C). Compared to that, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Lymphocyte protein tyrosine kinase.

실시예Example 119. F- 119.F- boxbox andand leucineleucine -- richrich repeatrepeat proteinprotein 14를 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 14 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 F-box and leucine-rich repeat protein 14를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using F-box and leucine-rich repeat protein 14 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 F-box and leucine-rich repeat protein 14를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 F-box and leucine-rich repeat protein 14를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 in the same conditions as Example 2 (A), the progenitor killer cells obtained in Example 2 (A) were added with 1 µg / ml (Upstat) of F-box and leucine-rich repeat protein 14 After differentiation for 14 days, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when F-box and leucine-rich repeat protein 14 were treated compared to the non-treated control group.

추가로, 동일조건하에 전구자연살해세포를 F-box and leucine-rich repeat protein 14를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 F-box and leucine-rich repeat protein 14를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with F-box and leucine-rich repeat protein 14 with interleukin-15 at a ratio of 1 ㎍ / ml in a 1: 1 ratio to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the F-box and leucine-rich repeat protein 14 treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 F-box and leucine-rich repeat protein 14에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by F-box and leucine-rich repeat protein 14 in human cord blood-derived hematopoietic stem cells in animal cancer model

F-box and leucine-rich repeat protein 14를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 F-box and leucine-rich repeat protein 14를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated with F-box and leucine-rich repeat protein 14, the same procedure as in Example 2 (C) was performed. Compared with the injection of mature natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using F-box and leucine-rich repeat protein 14.

실시예Example 120. 120. OsteoclastogenesisOsteoclastogenesis inhibitoryinhibitory factorfactor 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Osteoclastogenesis inhibitory factor를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Osteoclastogenesis inhibitory factor in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Osteoclastogenesis inhibitory factor를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Osteoclastogenesis inhibitory factor를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Osteoclastogenesis inhibitory factor instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with the Osteoclastogenesis inhibitory factor compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Osteoclastogenesis inhibitory factor를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Osteoclastogenesis inhibitory factor를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, progenitor killer cells were treated with Osteoclastogenesis inhibitory factor with interleukin-15 in a ratio of 1 ㎍ / ml at a ratio of 1: 1 to confirm the differentiation rate under the same conditions. The results showed a slightly increased differentiation rate compared to the treatment with the Osteoclastogenesis inhibitory factor alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Osteoclastogenesis inhibitory factor에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Osteoclastogenesis inhibitory factor in human cord blood-derived hematopoietic stem cells in animal cancer model

Osteoclastogenesis inhibitory factor를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Osteoclastogenesis inhibitory factor를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using an osteostelastogenesis inhibitory factor, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). In contrast, significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using Osteoclastogenesis inhibitory factor.

실시예Example 121. G 121.G proteinprotein -- coupledcoupled receptorreceptor 171을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Cord Blood-derived Hematopoietic Stem Cells Using 171 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 G protein-coupled receptor 171을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using G protein-coupled receptor 171 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 G protein-coupled receptor 171을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 G protein-coupled receptor 171을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of G protein-coupled receptor 171 instead of treating Interleukin-15 under the same conditions as Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with G protein-coupled receptor 171 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 G protein-coupled receptor 171을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 G protein-coupled receptor 171을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with G protein-coupled receptor 171 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1 μg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate when treated with G protein-coupled receptor 171 alone.

G protein-coupled receptor 171을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 G protein-coupled receptor 171을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using G protein-coupled receptor 171, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). In comparison with the injection of the NK cells, a significant decrease in the volume of the cancer tissue was observed in the group injected with mature natural killer cells differentiated using G protein-coupled receptor 171.

실시예Example 122. 122. RNARNA terminalterminal phosphatephosphate cyclasecyclase domaindomain 1를1 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 RNA terminal phosphate cyclase domain 1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using RNA terminal phosphate cyclase domain 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 RNA terminal phosphate cyclase domain 1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 RNA terminal phosphate cyclase domain 1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of RNA terminal phosphate cyclase domain 1 instead of treating Interleukin-15 under the same conditions as Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold higher differentiation rate when RNA terminal phosphate cyclase domain 1 was treated compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 RNA terminal phosphate cyclase domain 1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 RNA terminal phosphate cyclase domain 1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with RNA terminal phosphate cyclase domain 1 with interleukin-15 in a ratio of 1 ㎍ / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared with the RNA terminal phosphate cyclase domain 1 treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 RNA terminal phosphate cyclase domain 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by RNA terminal phosphate cyclase domain 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

RNA terminal phosphate cyclase domain 1를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 RNA terminal phosphate cyclase domain 1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using RNA terminal phosphate cyclase domain 1, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). In comparison with the injection of the NK cells, a significant decrease in the volume of the cancer tissue was observed in the group injected with mature natural killer cells differentiated using RNA terminal phosphate cyclase domain 1.

실시예Example 123. 123. CullinCullin 2를 이용한 사람 제대혈 유래 조혈모세포로부터 From human umbilical cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Cullin 2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Cullin 2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Cullin 2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Cullin 2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Cullin 2 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 to 3 times higher differentiation rate when Cullin 2 was treated than the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Cullin 2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Cullin 2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Cullin 2 with interleukin-15 in a ratio of 1 ug / ml in a ratio of 1 µg / ml to confirm the differentiation rate. As a result, it was shown that the differentiation rate was slightly increased as compared with the treatment with Cullin 2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Cullin 2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Cullin 2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Cullin 2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 culin 2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Cullin 2, the mature natural killer cells of the control group treated with nothing were injected. Compared with the injection of mature natural killer cells differentiated with culin 2, a significant decrease in the volume of cancer tissue was observed.

실시예Example 124. 124. CD160CD160 antigenantigen 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 CD160 antigen를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using CD160 antigen in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 CD160 antigen를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 CD160 antigen를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of CD160 antigen instead of interleukin-15 treatment under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 times higher differentiation rate when treated with CD160 antigen than the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 CD160 antigen를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 CD160 antigen를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with CD160 antigen with interleukin-15 at a ratio of 1 ㎍ / ml at a ratio of 1 ㎍ / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the CD160 antigen alone treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 CD160 antigen에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of CD160 antigen in human cord blood-derived hematopoietic stem cells in animal cancer model

CD160 antigen를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 CD160 antigen를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using CD160 antigen, the control group was treated with mature natural killer cells without any treatment. Compared with the injection of mature natural killer cells differentiated with CD160 antigen, significant decreases in cancer tissue volume were observed.

실시예Example 125. 125. HypotheticalHypothetical RNARNA -- bindingbinding domaindomain 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Hypothetical RNA-binding domain을 이용한 성숙자연살해세포 분화법 (A) Mature Natural Killer Cell Differentiation Using Hypothetical RNA-binding Domain in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Hypothetical RNA-binding domain을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Hypothetical RNA-binding domain을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of the Hypothetical RNA-binding domain instead of treating Interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold more differentiation when treated with the hypothetical RNA-binding domain than the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Hypothetical RNA-binding domain을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Hypothetical RNA-binding domain을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the differentiation rate of progenitor killer cells was treated with the interleukin-15 at 1 μg / ml in a ratio of 1: 1 with interleukin-15. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with the hypothetical RNA-binding domain alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Hypothetical RNA-binding domain에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by hypothetical RNA-binding domain in human cord blood-derived hematopoietic stem cells in animal cancer model

Hypothetical RNA-binding domain을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Hypothetical RNA-binding domain을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the hypothetical RNA-binding domain, the mature natural killer cells of the control group which did not process anything were tested in the same manner as in Example 2 (C). Compared with the injection, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using the hypothetical RNA-binding domain.

실시예Example 126. 126. semaphorinsemaphorin 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 semaphorin를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using semaphorin in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 semaphorin를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 semaphorin를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of semaphorin instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 times higher differentiation rate when semaphorin was treated than the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 semaphorin를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 semaphorin를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate of progenitor killer cells was treated with 1 μg / ml of semaphorin at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate when semaphorin was treated alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 semaphorin에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of semaphorin in human cord blood-derived hematopoietic stem cells in animal cancer model

semaphorin를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 semaphorin를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using semaphorin, the experiment was performed in the same manner as in Example 2 (C). Significant decrease of cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with semaphorin.

실시예Example 127. 127. ActivatingActivating signalsignal cointegratorcointegrator 1 One complexcomplex subunitsubunit 3 3 likelike 1를1 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Activating signal cointegrator 1 complex subunit 3 like 1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using activating signal cointegrator 1 complex subunit 3 like 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Activating signal cointegrator 1 complex subunit 3 like 1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Activating signal cointegrator 1 complex subunit 3 like 1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of activating signal cointegrator 1 complex subunit 3 like 1 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). After differentiation for one day, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Activating signal cointegrator 1 complex subunit 3 like 1 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Activating signal cointegrator 1 complex subunit 3 like 1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Activating signal cointegrator 1 complex subunit 3 like 1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating NK cell activating signal cointegrator 1 complex subunit 3 like 1 with interleukin-15 in a 1: 1 ratio. The results showed a slightly increased differentiation rate compared to the activating signal cointegrator 1 complex subunit 3 like 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Activating signal cointegrator 1 complex subunit 3 like 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by activating signal cointegrator 1 complex subunit 3 like 1 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Activating signal cointegrator 1 complex subunit 3 like 1를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Activating signal cointegrator 1 complex subunit 3 like 1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Human maturation differentiated using Activating signal cointegrator 1 complex subunit 3 like 1 As a result of experiments in the same manner as in Example 2 (C) to observe whether human cancer cells are killed by natural killer cells Compared with the injection of natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using Activating signal cointegrator 1 complex subunit 3 like 1.

실시예Example 128. 128. AblAbl -- interactorinteractor 1을 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Abl-interactor 1을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Abl-interactor 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Abl-interactor 1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Abl-interactor 1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Abl-interactor 1 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when Abl-interactor 1 was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Abl-interactor 1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Abl-interactor 1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, Abl-interactor 1 was treated with Aleukin-15 in an amount of 1 μg / ml in a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. As a result, it was shown that the differentiation rate was slightly increased compared to the case of treatment with Abl-interactor 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Abl-interactor 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Abl-interactor 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Abl-interactor 1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Abl-interactor 1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Abl-interactor 1, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to the one injected with the Abl-interactor 1, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells.

실시예Example 129. 129. HypotheticalHypothetical proteinprotein MGC36662MGC36662 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Hypothetical protein MGC36662를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Hypothetical protein MGC36662 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Hypothetical protein MGC36662를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Hypothetical protein MGC36662를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Hypothetical protein MGC36662 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with Hypothetical protein MGC36662 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Hypothetical protein MGC36662를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Hypothetical protein MGC36662를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under different conditions, differentiation rate of progenitor killer cells was treated with Hypothetical protein MGC36662 with interleukin-15 in an amount of 1 µg / ml at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared to the treatment with Hypothetical protein MGC36662 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Hypothetical protein MGC36662에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Hypothetical protein MGC36662 in human cord blood-derived hematopoietic stem cells in animal cancer model

Hypothetical protein MGC36662를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Hypothetical protein MGC36662를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the hypothetical protein MGC36662, the mature natural killer cells of the control group treated with nothing were injected. Compared to the group injected with the mature natural killer cells differentiated with the hypothetical protein MGC36662, a significant decrease in the volume of the cancer tissue was observed.

실시예Example 130. 130. RAP2CRAP2C , , membermember ofof RASRAS oncogeneoncogene familyfamily 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 RAP2C, member of RAS oncogene family를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using RAP2C, member of RAS oncogene family in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 RAP2C, member of RAS oncogene family를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 RAP2C, member of RAS oncogene family를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of RAP2C, member of RAS oncogene family for 14 days instead of treatment with interleukin-15 under the same conditions as in Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when treated with the RAP2C, member of RAS oncogene family, compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 RAP2C, member of RAS oncogene family를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 RAP2C, member of RAS oncogene family를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the proliferation of NK cells was treated with RAP2C, a member of RAS oncogene family with interleukin-15 at a ratio of 1 ㎍ / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the RAP2C, member of RAS oncogene family.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 RAP2C, member of RAS oncogene family에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by RAP2C, member of RAS oncogene family in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

RAP2C, member of RAS oncogene family를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 RAP2C, member of RAS oncogene family를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using RAP2C, a member of RAS oncogene family, mature natural killing of a control group treated with nothing was performed in the same manner as in Example 2 (C). Compared with the cell injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using RAP2C, a member of RAS oncogene family.

실시예Example 131. 131. ValosinValosin containingcontaining proteinprotein 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Valosin containing protein을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Valosin containing protein in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Valosin containing protein을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Valosin containing protein을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of Valosin containing protein and treated for 14 days instead of interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed differentiation rate of 2 ~ 3 fold when treated with Valosin containing protein compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Valosin containing protein을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Valosin containing protein을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating the progenitor killer cells with Valosin containing protein at a ratio of 1: 1 with interleukin-15 at a ratio of 1: 1. The results showed a slightly increased differentiation rate compared to the valosin containing protein alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Valosin containing protein에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Valosin containing protein in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Valosin containing protein을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Valosin containing protein을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Valosin containing protein, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to the group injected with mature natural killer cells differentiated with Valosin containing protein, a significant decrease in cancer tissue volume was observed.

실시예Example 132. 132. CD164CD164 antigenantigen 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 CD164 antigen을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using CD164 antigen in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 CD164 antigen을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 CD164 antigen을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of CD164 antigen instead of treating Interleukin-15 under the same conditions as Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three fold increase in differentiation rate when treated with CD164 antigen compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 CD164 antigen을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 CD164 antigen을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with CD164 antigen with interleukin-15 at a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with the CD164 antigen alone treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 CD164 antigen에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of CD164 antigen in human cord blood-derived hematopoietic stem cells in animal cancer model

CD164 antigen을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 CD164 antigen을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using CD164 antigen, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as Example 2 (C). Compared with the CD164 antigen-induced mature natural killer cells, significant decreases in cancer tissue volume were observed.

실시예Example 133. 133. SimilarSimilar toto RIKENRIKEN cDNAcDNA 4933425 4933425 K02K02 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Similar to RIKEN cDNA 4933425K02를 이용한 성숙자연살해세포 분화법 (A) Similar to RIKEN cDNA 4933425K02 in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Similar to RIKEN cDNA 4933425K02를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Similar to RIKEN cDNA 4933425K02를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of similar to RIKEN cDNA 4933425K02 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with Similar to RIKEN cDNA 4933425K02 compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 Similar to RIKEN cDNA 4933425K02를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Similar to RIKEN cDNA 4933425K02를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating pro-natural killer cells with Similar to RIKEN cDNA 4933425K02 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, it showed a slightly increased differentiation rate compared to the case treated with Similar to RIKEN cDNA 4933425K02 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Similar to RIKEN cDNA 4933425K02에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by RIKEN cDNA 4933425K02 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Similar to RIKEN cDNA 4933425K02를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Similar to RIKEN cDNA 4933425K02를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Similar to RIKEN cDNA 4933425K02 In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using RIKEN cDNA 4933425K02 experiments were performed in the same manner as in Example 2 (C) as a result Significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using Similar to RIKEN cDNA 4933425K02.

실시예Example 134. 134. AdvillinAdvillin 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Advillin를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Advillin in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Advillin를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Advillin를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Advillin instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Advillin compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Advillin를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Advillin를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Advillin with interleukin-15 at a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the treatment with Advillin alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Advillin에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Advillin in human cord blood-derived hematopoietic stem cells in animal cancer model

Advillin를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Advillin를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Advillin, experiments were performed in the same manner as in Example 2 (C). Significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated with Advillin.

실시예Example 135. 135. AsparagineAsparagine synthetasesynthetase 를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 Asparagine synthetase를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Asparagine synthetase in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Asparagine synthetase를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Asparagine synthetase를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Asparagine synthetase in the same condition as Example 2 (A) instead of interleukin-15 treatment. Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 times higher differentiation rate when treated with Asparagine synthetase than the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Asparagine synthetase를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Asparagine synthetase를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Asparagine synthetase with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the treatment with Asparagine synthetase alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Asparagine synthetase에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Asparagine synthetase in human cord blood-derived hematopoietic stem cells in animal cancer model

Asparagine synthetase를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Asparagine synthetase를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Asparagine synthetase, the mature natural killer cells of the control group treated with nothing were injected. Compared with the injection of mature natural killer cells differentiated with Asparagine synthetase, the cancer tissue volume was significantly decreased.

실시예Example 136. 136. ZincZinc fingerfinger proteinprotein 259를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using 259 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Zinc finger protein 259를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using zinc finger protein 259 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Zinc finger protein 259를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Zinc finger protein 259를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Zinc finger protein 259 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when zinc finger protein 259 was treated compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Zinc finger protein 259를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Zinc finger protein 259를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Zinc finger protein 259 with interleukin-15 in a ratio of 1 ㎍ / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, the zinc finger protein 259 alone showed slightly increased differentiation rate.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Zinc finger protein 259에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Zinc finger protein 259 in human cord blood-derived hematopoietic stem cells in animal cancer model

Zinc finger protein 259를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Zinc finger protein 259를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using zinc finger protein 259, the mature natural killer cells of the control group treated with nothing were injected in the same manner as in Example 2 (C). Compared to the ones injected with zinc finger protein 259, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells.

실시예Example 137. 137. InterleukinInterleukin 12를 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 12 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Interleukin 12를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Interleukin 12 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Interleukin 12를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Interleukin 12를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Interleukin 12 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 times more differentiation rate when Interleukin 12 was treated than the control group.

추가로, 동일조건하에 전구자연살해세포를 Interleukin 12를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Interleukin 12를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating proleukin killer cells with Interleukin 12 with Interleukin-15 in a 1: 1 μg amount. The results showed a slightly increased differentiation rate compared to the treatment with Interleukin 12 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Interleukin 12에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Interleukin 12 in human cord blood-derived hematopoietic stem cells in animal cancer model

Interleukin 12를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Interleukin 12를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Interleukin 12, the mature natural killer cells of the control group treated with nothing were injected. Compared with the injection of mature natural killer cells differentiated using Interleukin 12, a significant decrease in cancer tissue volume was observed.

실시예Example 138. 138. GrowthGrowth arrestarrest andand DNADNA -- damagedamage -- inducibleinducible 45 45 gammagamma 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Growth arrest and DNA-damage-inducible 45 gamma를 이용한 성숙자연살해세포 분화법 (A) Differentiation of mature natural killer cells using growth arrest and DNA-damage-inducible 45 gamma in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Growth arrest and DNA-damage-inducible 45 gamma를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Growth arrest and DNA-damage-inducible 45 gamma를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 in the same conditions as Example 2 (A), the progenitor killer cells obtained in Example 2 (A) were added with 1 µg / ml (Upstat) of growth arrest and DNA-damage-inducible 45 gamma. After differentiation for 14 days, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with Growth arrest and DNA-damage-inducible 45 gamma compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Growth arrest and DNA-damage-inducible 45 gamma를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Growth arrest and DNA-damage-inducible 45 gamma를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with growth arrest and DNA-damage-inducible 45 gamma at a ratio of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, the growth arrest and DNA-damage-inducible 45 gamma treatment alone showed slightly increased differentiation rate.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Growth arrest and DNA-damage-inducible 45 gamma에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by growth arrest and DNA-damage-inducible 45 gamma in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Growth arrest and DNA-damage-inducible 45 gamma 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Growth arrest and DNA-damage-inducible 45 gamma를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Growth arrest and DNA-damage-inducible 45 Gamma Human maturation Differentiated using natural killer cells to observe whether human cancer cells are killed by the same method as in Example 2 (C) Compared with the injection of natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using growth arrest and DNA-damage-inducible 45 gamma.

실시예Example 139. 139. CytochromeCytochrome b5b5 typetype B를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using B

(A) 사람 제대혈 유래 조혈모세포에서 Cytochrome b5 type B를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Cytochrome b5 type B in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Cytochrome b5 type B를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Cytochrome b5 type B를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding Cytochrome b5 type B to 1 µg / ml (Upstat) instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase in differentiation rate of 2 to 3 times when Cytochrome b5 type B was treated compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 Cytochrome b5 type B를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Cytochrome b5 type B를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Cytochrome b5 type B with interleukin-15 in a ratio of 1 µg / ml at a ratio of 1 µg / ml to confirm the differentiation rate. As a result, it was shown that the differentiation rate slightly increased compared to the case of treatment with Cytochrome b5 type B alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Cytochrome b5 type B에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Cytochrome b5 type B in human cord blood-derived hematopoietic stem cells in animal cancer model

Cytochrome b5 type B를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Cytochrome b5 type B를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using cytochrome b5 type B, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with one group, significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated with Cytochrome b5 type B.

실시예Example 140. 140. FMSFMS -- likelike tyrosinetyrosine kinasekinase 3 3 ligandligand 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 FMS-like tyrosine kinase 3 ligand를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using FMS-like tyrosine kinase 3 ligand in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 FMS-like tyrosine kinase 3 ligand를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 FMS-like tyrosine kinase 3 ligand를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of FMS-like tyrosine kinase 3 ligand for 14 days instead of treating Interleukin-15 under the same conditions as Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in differentiation rate when treated with FMS-like tyrosine kinase 3 ligand compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 FMS-like tyrosine kinase 3 ligand를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 FMS-like tyrosine kinase 3 ligand를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with FMS-like tyrosine kinase 3 ligand with interleukin-15 in a ratio of 1 ㎍ / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate when treated with FMS-like tyrosine kinase 3 ligand alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 FMS-like tyrosine kinase 3 ligand에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by FMS-like tyrosine kinase 3 ligand in human cord blood-derived hematopoietic stem cells in animal cancer model

FMS-like tyrosine kinase 3 ligand를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 FMS-like tyrosine kinase 3 ligand를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using FMS-like tyrosine kinase 3 ligand, the mature natural killer of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared to the cells injected, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using FMS-like tyrosine kinase 3 ligand.

실시예Example 141. 141. ProgrammedProgrammed cellcell deathdeath 1 One ligandligand 2를 이용한 사람 제대혈 유래 조혈모세포로부터 From human umbilical cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Programmed cell death 1 ligand 2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Programmed cell death 1 ligand 2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Programmed cell death 1 ligand 2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Programmed cell death 1 ligand 2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Programmed cell death 1 ligand 2 instead of treating Interleukin-15 under the same conditions as Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with Programmed cell death 1 ligand 2 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Programmed cell death ligand 2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Programmed cell death ligand 2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Interleukin-15 in a ratio of 1 μg / ml at a ratio of 1: 1 with Programmed cell death ligand 2 to confirm the differentiation rate. The results showed a slightly increased differentiation rate when treated with Programmed cell death ligand 2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Programmed cell death ligand 2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Programmed cell death ligand 2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Programmed cell death ligand 2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Programmed cell death ligand 2를이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Programmed cell death ligand 2, the mature natural killer cells of the control group treated with nothing were tested as in Example 2 (C). Compared with the injection, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using Programmed cell death ligand 2.

실시예Example 142. 142. S100S100 calciumcalcium bindingbinding proteinprotein A4를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using A4 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 S100 calcium binding protein A4를 이용한 성숙자연살해세포 분화법 (A) Differentiation of mature natural killer cells using S100 calcium binding protein A4 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 S100 calcium binding protein A4를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 S100 calcium binding protein A4를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of S100 calcium binding protein A4 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Later, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with S100 calcium binding protein A4 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 S100 calcium binding protein A4를인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 S100 calcium binding protein A4를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with S100 calcium binding protein A4 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate compared to the S100 calcium binding protein A4 treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 S100 calcium binding protein A4에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by S100 calcium binding protein A4 in human cord blood-derived hematopoietic stem cells in animal cancer model

S100 calcium binding protein A4를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 S100 calcium binding protein A4를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using S100 calcium binding protein A4, the mature natural killer cells of the control group treated with nothing were tested as in Example 2 (C). Compared with the injection, a significant decrease in the volume of the cancer tissue was observed in the group injected with mature natural killer cells differentiated with S100 calcium binding protein A4.

실시예Example 143. 143. MetallothioneinMetallothionein 2를 이용한 사람 제대혈 유래 조혈모세포로부터 From human umbilical cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Metallothionein 2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using metallothionein 2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Metallothionein 2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Metallothionein 2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Metallothionein 2 instead of interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a 2 ~ 3 fold increase in differentiation rate when treated with Metallothionein 2 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Metallothionein 2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Metallothionein 2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Metallothionein 2 with interleukin-15 in a ratio of 1 ㎍ / ml in a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the treatment with Metallothionein 2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Metallothionein 2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by metallothionein 2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Metallothionein 2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Metallothionein 2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using metallothionein 2, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with the metallothionein 2-derived mature natural killer cells, a significant decrease in the volume of cancer tissues was observed.

실시예Example 144. 144. PeptidylprolylPeptidylprolyl isomeraseisomerase A를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using A 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Peptidylprolyl isomerase A를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using peptidylprolyl isomerase A in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Peptidylprolyl isomerase A를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Peptidylprolyl isomerase A를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Peptidylprolyl isomerase A instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in the differentiation rate when Peptidylprolyl isomerase A was treated compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Peptidylprolyl isomerase A를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Peptidylprolyl isomerase A를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating Peptidylprolyl isomerase A with Interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio. As a result, it showed a slightly increased differentiation rate compared with the treatment with Peptidylprolyl isomerase A alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Peptidylprolyl isomerase A에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Peptidylprolyl isomerase A in human cord blood-derived hematopoietic stem cells in animal cancer model

Peptidylprolyl isomerase A를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Peptidylprolyl isomerase A를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using peptidylprolyl isomerase A, the mature natural killer cells of the control group treated with nothing were injected. Compared to the group injected with mature killer cells differentiated with Peptidylprolyl isomerase A, a significant decrease in the volume of cancer tissue was observed.

실시예Example 145. 145. OsteopontinOsteopontin 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Osteopontin를 이용한 성숙자연살해세포 분화법 (A) in human umbilical cord blood-derived hematopoietic stem cells Differentiation of Mature Natural Killer Cells Using Osteopontin

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Osteopontin를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Osteopontin를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Osteopontin instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Osteopontin compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Osteopontin를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Osteopontin를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Osteopontin with interleukin-15 in a ratio of 1 ㎍ / ml in a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the treatment with Osteopontin alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Osteopontin에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Osteopontin in human cord blood-derived hematopoietic stem cells in animal cancer model

Osteopontin를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Osteopontin를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using osteoblastin, the same method as in Example 2 (C) was performed. Significant decrease of cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using osteopontin.

실시예Example 146. 146. ChemokineChemokine (C-C (C-C motifmotif ) ) ligandligand 4를 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 4 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Chemokine (C-C motif) ligand 4를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Chemokine (C-C motif) ligand 4 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Chemokine (C-C motif) ligand 4를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Chemokine (C-C motif) ligand 4를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 14 μg / ml (Upstat) of Chemokine (CC motif) ligand 4 for 14 days instead of treating Interleukin-15 under the same conditions as Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when treated with Chemokine (CC motif) ligand 4 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Chemokine (C-C motif) ligand 4를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Chemokine (C-C motif) ligand 4를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Chemokine (C-C motif) ligand 4 with interleukin-15 at a ratio of 1 ㎍ / ml in a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with Chemokine (C-C motif) ligand 4 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Chemokine (C-C motif) ligand 4에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Chemokine (C-C motif) ligand 4 in human cord blood-derived hematopoietic stem cells in animal cancer model

Chemokine (C-C motif) ligand 4를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Chemokine (C-C motif) ligand 4를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Chemokine (CC motif) ligand 4, the mature natural killer of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared with the cell injection, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated with Chemokine (CC motif) ligand 4.

실시예Example 147. 147. TcrbTcrb -- V13V13 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Tcrb-V13를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Tcrb-V13 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Tcrb-V13를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Tcrb-V13를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Tcrb-V13 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation when treated with Tcrb-V13 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Tcrb-V13를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Tcrb-V13를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Tcrb-V13 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of Tcrb-V13 alone.

Tcrb-V13를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Tcrb-V13를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Tcrb-V13, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). In comparison with the TCRb-V13-derived mature natural killer cells, a significant decrease in the volume of cancer tissues was observed.

실시예Example 148. 148. ImmuneImmune associatedassociated nucleotidenucleotide 1를1 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Immune associated nucleotide 1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Immune associated nucleotide 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Immune associated nucleotide 1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Immune associated nucleotide 1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Immune associated nucleotide 1 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 to 3 times higher differentiation rate when treated with Immune associated nucleotide 1 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Immune associated nucleotide 1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Immune associated nucleotide 1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progenitor killer cells with Immune associated nucleotide 1 at a ratio of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with Immune associated nucleotide 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Immune associated nucleotide 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Immune associated nucleotide 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Immune associated nucleotide 1 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Immune associated nucleotide 1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Immune associated nucleotide 1, the mature natural killer cells of the control group treated with nothing were injected. Compared to the group injected with mature natural killer cells differentiated with Immune associated nucleotide 1, a significant decrease in cancer tissue volume was observed.

실시예Example 149. 149. Il2rbIl2rb 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Il2rb를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Il2rb in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Il2rb를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Il2rb를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Il2rb instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with Il2rb compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Il2rb를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Il2rb를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, progenitor killer cells were treated with Il2rb with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of treating Il2rb alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Il2rb에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Il2rb in human cord blood-derived hematopoietic stem cells in animal cancer model

Il2rb를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Il2rb를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Experiments were carried out in the same manner as in Example 2 (C) to observe whether human cancer cells were killed by human mature natural killer cells differentiated using Il2rb. Significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using Il2rb.

실시예Example 150. 150. Cd28Cd28 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Cd28를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Cd28 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Cd28를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Cd28를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Cd28 to the same conditions as Example 2 (A) instead of treating Interleukin-15. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about two to three times increased differentiation when treated with Cd28 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Cd28를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Cd28를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Cd28 with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the Cd28 alone treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Cd28 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Cd28 in human cord blood-derived hematopoietic stem cells in animal cancer model

Cd28를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Cd28를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Experiments were performed in the same manner as in Example 2 (C) to observe whether human cancer cells were killed by human mature natural killer cells differentiated using Cd28. Significant decreases in cancer tissue volume were observed in the group injected with mature natural killer cells differentiated using Cd28.

실시예Example 151. 151. GranzymeGranzyme E를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using E 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Granzyme E를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Granzyme E in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Granzyme E를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Granzyme E를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Granzyme E instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Granzyme E compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Granzyme E를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Granzyme E를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Granzyme E with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared with the single treatment with Granzyme E.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Granzyme E에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Granzyme E in human cord blood-derived hematopoietic stem cells in animal cancer model

Granzyme E를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Granzyme E를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Granzyme E, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to granzyme E-induced mature natural killer cells, significant decreases in cancer tissue volume were observed.

실시예Example 152. 152. Serpinb9bSerpinb9b , , SerineSerine (( oror cysteinecysteine ) ) peptidasepeptidase inhibitorinhibitor , , cladeclade B, member 9b를 이용한 사람 제대혈 유래 조혈모세포로부터 B, from human cord blood-derived hematopoietic stem cells using member 9b 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Serpinb9b, Serine(or cysteine) peptidase inhibitor, clade B, member 9b를 이용한 성숙자연살해세포 분화법 (A) Differentiation of mature natural killer cells using Serpinb9b, Serine (or cysteine) peptidase inhibitor, clade B, and member 9b in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Serpinb9b, Serine(or cysteine) peptidase inhibitor, clade B, member 9b를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Serpinb9b, Serine(or cysteine) peptidase inhibitor, clade B, member 9b를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. Instead of treating interleukin-15 under the same conditions as Example 2 (A), the progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml of Serpinb9b, Serine (or cysteine) peptidase inhibitor, clade B, and member 9b. After adding (Upstat) to differentiate for 14 days, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold more differentiation with Serpinb9b, Serine (or cysteine) peptidase inhibitor, clade B, and member 9b compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 Serpinb9b, Serine(or cysteine) peptidase inhibitor, clade B, member 9b를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Serpinb9b, Serine(or cysteine) peptidase inhibitor, clade B, member 9b를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating Serpinb9b, Serine (or cysteine) peptidase inhibitor, clade B, and member 9b together with interleukin-15 at a ratio of 1 ㎍ / ml in a 1: 1 ratio. It was. The results showed a slightly increased differentiation rate compared to the treatment with Serpinb9b, Serine (or cysteine) peptidase inhibitor, clade B, and member 9b alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Serpinb9b, Serine(or cysteine) peptidase inhibitor, clade B, member 9b에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Serpinb9b, Serine (or cysteine) peptidase inhibitor, clade B, member 9b in human cord blood-derived hematopoietic stem cells

Serpinb9b, Serine(or cysteine) peptidase inhibitor, clade B, member 9b를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Serpinb9b, Serine(or cysteine) peptidase inhibitor, clade B, member 9b를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using serpinb9b, Serine (or cysteine) peptidase inhibitor, clade B, and member 9b, the result of the experiment was the same as in Example 2 (C). Compared with the injection of mature natural killer cells of the untreated control group, cancer tissue volume was significantly decreased in the group injected with mature natural killer cells differentiated using Serpinb9b, Serine (or cysteine) peptidase inhibitor, clade B, and member 9b. Was observed.

실시예Example 153. 153. PurinePurine richrich elementelement bindingbinding proteinprotein A를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using A 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Purine rich element binding protein A를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Purine rich element binding protein A in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Purine rich element binding protein A를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Purine rich element binding protein A를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Purine rich element binding protein A instead of treating Interleukin-15 under the same conditions as Example 2 (A). After the human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A), the degree of differentiation was confirmed. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 times higher differentiation rate when treated with Purine rich element binding protein A than the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Similar to RIKEN cDNA 4933425K02를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Purine rich element binding protein A를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating pro-natural killer cells with Similar to RIKEN cDNA 4933425K02 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, it was shown that the differentiation rate was slightly increased compared to the case of treating with Purine rich element binding protein A alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Purine rich element binding protein A에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Purine rich element binding protein A in human cord blood-derived hematopoietic stem cells in animal cancer model

Purine rich element binding protein A를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Purine rich element binding protein A를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Purine rich element binding protein A, the mature natural killer cells of the control group treated with nothing were tested in the same manner as in Example 2 (C). Compared to the injection of A, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using Purine rich element binding protein A.

실시예Example 154. 154. LymphocyteLymphocyte proteinprotein tyrosinetyrosine kinasekinase 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

실시예Example 155. 155. Eif4g3Eif4g3 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Eif4g3를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Eif4g3 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Eif4g3를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Eif4g3를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Eif4g3 to Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when Eif4g3 was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Eif4g3를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Eif4g3를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, Eif4g3 was treated with 1 μg / ml amount of Eif4g3 with interleukin-15 at a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with Eif4g3 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Eif4g3에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Eif4g3 in human cord blood-derived hematopoietic stem cells in animal cancer model

Eif4g3를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Eif4g3를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Eif4g3, the same procedure as in Example 2 (C) was performed. In the group injected with mature natural killer cells differentiated with Eif4g3, a significant decrease in the volume of cancer tissue was observed.

실시예Example 156. 156. LaminLamin A를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using A 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Lamin A를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Lamin A in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Lamin A를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Lamin A를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Lamin A instead of interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about 2-3 times increased differentiation rate when treated with Lamin A compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Lamin A를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Lamin A를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, progenitor killer cells were treated with Lamin A with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the treatment with Lamin A alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Lamin A에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Lamin A in human cord blood-derived hematopoietic stem cells in animal cancer model

Lamin A를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Lamin A를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Lamin A, the mature natural killer cells of the control group treated with nothing were injected. Compared with Lamin A-derived mature natural killer cells, a significant decrease in cancer tissue volume was observed.

실시예Example 157. 157. Raver1Raver1 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Raver1를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Raver1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Raver1를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Raver1를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Raver1 to the same conditions as Example 2 (A) instead of interleukin-15 treatment. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when treated with Raver1 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Raver1를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Raver1를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, progenitor killer cells were treated with Raver1 with interleukin-15 at a ratio of 1 µg / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of Raver1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Raver1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Raver1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Raver1를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Raver1를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Raver1, the same procedure as in Example 2 (C) was performed. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Raver1.

실시예Example 158. 158. SoluteSolute carriercarrier familyfamily 6를6 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Solute carrier family 6를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Solute carrier family 6 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Solute carrier family 6를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Solute carrier family 6를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. After progenitor killer cells obtained in Example 2 (A) were treated with Interleukin-15 under the same conditions as Example 2 (A), and then differentiated for 14 days by adding 1 µg / ml (Upstat) of Solute carrier family 6 Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Solute carrier family 6 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Solute carrier family 6를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Solute carrier family 6를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating the progenitor killer cells with Solute carrier family 6 in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. As a result, it was shown that the differentiation rate was slightly increased compared to the case of treating Solute carrier family 6 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Solute carrier family 6에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Solute carrier family 6 in human cord blood-derived hematopoietic stem cells in animal cancer model

Solute carrier family 6를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Solute carrier family 6를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using the solute carrier family 6, the mature natural killer cells of the control group treated with nothing were injected in the same manner as in Example 2 (C). Compared to the one injected with mature natural killer cells differentiated using Solute carrier family 6, a significant decrease in cancer tissue volume was observed.

실시예Example 159. V- 159.V- ralral simiansimian leukemialeukemia viralviral oncogeneoncogene homologhomolog A를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using A 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 V-ral simian leukemia viral oncogene homolog A를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using V-ral simian leukemia viral oncogene homolog A in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 V-ral simian leukemia viral oncogene homolog A를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 V-ral simian leukemia viral oncogene homolog A를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The pro-natural killer cells obtained in Example 2 (A) were treated with 1 μg / ml (Upstat) of V-ral simian leukemia viral oncogene homolog A instead of the treatment of interleukin-15 under the same conditions as in Example 2 (A). After differentiation for one day, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three-fold increase in the differentiation rate when treated with V-ral simian leukemia viral oncogene homolog A compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 V-ral simian leukemia viral oncogene homolog A를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 V-ral simian leukemia viral oncogene homolog A를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating V-ral simian leukemia viral oncogene homolog A with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio. The results showed a slightly increased differentiation rate compared to the V-ral simian leukemia viral oncogene homolog A alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 V-ral simian leukemia viral oncogene homolog A에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by V-ral simian leukemia viral oncogene homolog A in human umbilical cord blood-derived hematopoietic stem cells

V-ral simian leukemia viral oncogene homolog A를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 V-ral simian leukemia viral oncogene homolog A를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Human maturation differentiated using V-ral simian leukemia viral oncogene homolog A. Experiments were performed in the same manner as in Example 2 (C) to observe whether human cancer cells were killed by natural killer cells. Compared with the injection of natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using V-ral simian leukemia viral oncogene homolog A.

실시예Example 160. 160. FynFyn protoproto -- oncogeneoncogene 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Fyn proto-oncogene를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Fyn proto-oncogene in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Fyn proto-oncogene를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Fyn proto-oncogene를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Fyn proto-oncogene instead of interleukin-15 in the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold higher differentiation rate when Fyn proto-oncogene was treated than the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Fyn proto-oncogene를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Fyn proto-oncogene를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating progeny killer cells with Fyn proto-oncogene in an amount of 1 μg / ml at a ratio of 1: 1 with interleukin-15. The results showed a slightly increased differentiation rate when treated with Fyn proto-oncogene alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Fyn proto-oncogene에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Fyn proto-oncogene in human cord blood-derived hematopoietic stem cells in animal cancer model

Fyn proto-oncogene를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Fyn proto-oncogene를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated with Fyn proto-oncogene, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to that, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Fyn proto-oncogene.

실시예Example 161. 161. ExpressedExpressed sequencesequence AV024533AV024533 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Expressed sequence AV024533을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Expressed sequence AV024533 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Expressed sequence AV024533을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Expressed sequence AV024533을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Expressed sequence AV024533 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). Mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an approximately 2-3-fold increase in the differentiation rate when treated with Expressed sequence AV024533 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Expressed sequence AV024533을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Expressed sequence AV024533을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating the Expressed Sequence AV024533 with Interleukin-15 in an amount of 1 µg / ml at a ratio of 1: 1. As a result, the rate of differentiation was slightly increased compared to the case of the Expressed sequence AV024533 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Expressed sequence AV024533에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Expressed sequence AV024533 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

Expressed sequence AV024533을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Expressed sequence AV024533을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Expressed sequence AV024533, the mature natural killer cells of the control group treated with nothing were injected. In contrast, in the group injected with mature natural killer cells differentiated using Expressed sequence AV024533, a significant decrease in the volume of cancer tissue was observed.

실시예Example 162. 162. Fhl2Fhl2 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Fhl2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Fhl2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Fhl2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Fhl2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 μg / ml (Upstat) of Fhl2 instead of interleukin-15 treatment under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed about 2-3 times increased differentiation rate when treated with Fhl2 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Fhl2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Fhl2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, progenitor killer cells were treated with Fhl2 with interleukin-15 at a ratio of 1 μg / ml in a ratio of 1 μg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the Fhl2 treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Fhl2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Fhl2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Fhl2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Fhl2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Experiments were performed in the same manner as in Example 2 (C) to observe whether human cancer cells were killed by human mature natural killer cells differentiated using Fhl2. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Fhl2.

실시예Example 163. 163. C1qC1q andand tumortumor necrosisnecrosis factorfactor relatedrelated proteinprotein 6 를6 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 C1q and tumor necrosis factor related protein 6 를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using C1q and tumor necrosis factor related protein 6 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 C1q and tumor necrosis factor related protein 6 를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 C1q and tumor necrosis factor related protein 6 를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of C1q and tumor necrosis factor related protein 6 instead of treatment with interleukin-15 under the same conditions as in Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two- to three-fold increase in differentiation rate when treated with C1q and tumor necrosis factor related protein 6 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 C1q and tumor necrosis factor related protein 6 를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 C1q and tumor necrosis factor related protein 6 를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, the proliferative killer cells were treated with C1q and tumor necrosis factor related protein 6 with interleukin-15 in a 1: 1 ratio of 1 µg / ml under the same conditions to confirm the differentiation rate. The results showed that the C1q and tumor necrosis factor related protein 6 had a slightly increased differentiation rate compared to the single treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 C1q and tumor necrosis factor related protein 6 에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by C1q and tumor necrosis factor related protein 6 in human umbilical cord blood-derived hematopoietic stem cells in animal cancer model

C1q and tumor necrosis factor related protein 6 를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 C1q and tumor necrosis factor related protein 6 를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. The mature nature of the control group treated with nothing was tested in the same manner as in Example 2 (C) to observe whether human cancer cells are killed by human natural killer cells differentiated using C1q and tumor necrosis factor related protein 6 Compared with the injection of killer cells, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using C1q and tumor necrosis factor related protein 6.

실시예Example 164. 164. ZincZinc fingerfinger proteinprotein 512를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using 512 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Zinc finger protein 512를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using zinc finger protein 512 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Zinc finger protein 512를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Zinc finger protein 512를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Zinc finger protein 512 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed differentiation rate of 2 ~ 3 fold when Zinc finger protein 512 was treated compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 Zinc finger protein 512를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Zinc finger protein 512를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Zinc finger protein 512 with interleukin-15 in a ratio of 1 ㎍ / ml in a ratio of 1: 1 to confirm the differentiation rate. As a result, zinc finger protein 512 was slightly increased compared to the treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Zinc finger protein 512에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Zinc finger protein 512 in human cord blood-derived hematopoietic stem cells in animal cancer model

Zinc finger protein 512를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Zinc finger protein 512를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using zinc finger protein 512, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to the other group, significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Zinc finger protein 512.

실시예Example 165. 165. P2ry10P2ry10 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 P2ry10를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using P2ry10 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 P2ry10를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 P2ry10를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of P2ry10 in the same condition as Example 2 (A) instead of treating Interleukin-15. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when P2ry10 was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 P2ry10를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 P2ry10를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, P2ry10 was treated with P2ry10 with interleukin-15 in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the case of P2ry10 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 P2ry10에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of P2ry10 in human cord blood-derived hematopoietic stem cells in animal cancer model

P2ry10를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 P2ry10를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using P2ry10, the experiments were performed in the same manner as in Example 2 (C). Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using P2ry10.

실시예Example 166. 166. Ppp1r14bPpp1r14b 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Ppp1r14b를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Ppp1r14b in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Ppp1r14b를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Ppp1r14b를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Ppp1r14b instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation when Ppp1r14b was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Ppp1r14b를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Ppp1r14b를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. Further, under the same conditions, PPP1r14b was treated with 1 µg / ml in a ratio of 1: 1 with interleukin-15 to confirm the differentiation rate. As a result, it was shown that the differentiation rate was slightly increased compared to the case of treatment with Ppp1r14b alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Ppp1r14b에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Ppp1r14b in human cord blood-derived hematopoietic stem cells in animal cancer model

Ppp1r14b를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Ppp1r14b를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Ppp1r14b, experiments were performed in the same manner as in Example 2 (C). Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Ppp1r14b.

실시예Example 167. 167. StrapStrap 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Strap를 이용한 성숙자연살해세포 분화법 (A) Mature Natural Killer Cell Differentiation Using Strap in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Strap를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Strap를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Strap instead of interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increased rate of differentiation of 2 to 3 times when treated with Strap compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Strap를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Strap를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case treated with Strap alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Strap에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by strap differentiation in human cord blood-derived hematopoietic stem cells in animal cancer model

Strap를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Strap를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated by using straps, the same experiment as in Example 2 (C) was conducted. Significant decrease of cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using strap.

실시예Example 168. 168. Hdac3Hdd3 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Hdac3를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Hdac3 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Hdac3를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Hdac3를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Hdac3 in the same condition as Example 2 (A) instead of interleukin-15 treatment. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an approximately two- to three-fold increase in the differentiation rate when Hdac3 was treated compared to the no-treated control group.

추가로, 동일조건하에 전구자연살해세포를 Hdac3를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Hdac3를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Hdac3 with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared with Hdac3 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Hdac3에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Hdac3 in human cord blood-derived hematopoietic stem cells in animal cancer model

Hdac3를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Hdac3를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Experiments were performed in the same manner as in Example 2 (C) to observe whether human cancer cells were killed by human mature natural killer cells differentiated using Hdac3. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Hdac3.

실시예Example 169. 169. Ppp3cbPpp3cb 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Ppp3cb를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Ppp3cb in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Ppp3cb를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Ppp3cb를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Ppp3cb to the same conditions as Example 2 (A) instead of treating Interleukin-15. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ one cells showed a 2-3-fold increase in differentiation rate when treated with Ppp3cb compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Ppp3cb를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Ppp3cb를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the proliferative killer cells were treated with Ppp3cb with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm the differentiation rate. The results showed a slightly increased differentiation rate compared to the Ppp3cb treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Ppp3cb에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Ppp3cb in human cord blood-derived hematopoietic stem cells in animal cancer model

Ppp3cb를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Ppp3cb를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Ppp3cb, the same method as in Example 2 (C) was performed. Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Ppp3cb.

실시예Example 170. 170. Mmp9Mmp9 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Mmp9를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Mmp9 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Mmp9를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Mmp9를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Mmp9 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation when treated with Mmp9 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Mmp9를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Mmp9를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, progenitor killer cells were treated with Mmp9 with interleukin-15 at a ratio of 1 μg / ml at a ratio of 1: 1 to confirm differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with Mmp9 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Mmp9에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Mmp9 in human cord blood-derived hematopoietic stem cells in animal cancer model

Mmp9를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Mmp9를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Mmp9, the same method as in Example 2 (C) was performed. In the group injected with mature natural killer cells differentiated with Mmp9, a significant decrease in the volume of cancer tissues was observed.

실시예Example 171. 171. PrefoldinPrefoldin 1을 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Prefoldin 1을 이용한 성숙자연살해세포 분화법 (A) Differentiation of Mature Natural Killer Cells Using Prefoldin 1 in Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Prefoldin 1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Prefoldin 1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Prefoldin 1 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Prefoldin 1 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Prefoldin 1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Prefoldin 1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Prefoldin 1 with interleukin-15 at a ratio of 1 μg / ml in a ratio of 1 μg / ml to confirm the differentiation rate. As a result, it was shown that the differentiation rate was slightly increased compared to the case of treatment with Prefoldin 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Prefoldin 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Prefoldin 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Prefoldin 1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Prefoldin 1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using prefoldin 1, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared with the prepopin 1-derived mature natural killer cells, a significant decrease in the volume of cancer tissues was observed.

실시예Example 173. 173. GTPGTP -- bindingbinding proteinprotein (( mSaramSara ) ) homologuehomologue 을 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 GTP-binding protein(mSara) homologue을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using GTP-binding protein (mSara) homologue in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 GTP-binding protein(mSara) homologue을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 GTP-binding protein(mSara) homologue을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with GTP-binding protein (mSara) homologue by adding 1 μg / ml (Upstat) to 14 days instead of interleukin-15 under the same conditions as Example 2 (A). After differentiation, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm the degree of differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a two to three fold increase in differentiation rate when treated with GTP-binding protein (mSara) homologue compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 GTP-binding protein(mSara) homologue을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 GTP-binding protein(mSara) homologue을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating GTP-binding protein (mSara) homologue with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio. The results showed a slightly increased differentiation rate compared to the GTP-binding protein (mSara) homologue alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 GTP-binding protein(mSara) homologue에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by GTP-binding protein (mSara) homologue in human cord blood-derived hematopoietic stem cells in animal cancer model

GTP-binding protein(mSara) homologue을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 GTP-binding protein(mSara) homologue을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using GTP-binding protein (mSara) homologue, the mature nature of the control group treated with nothing was tested in the same manner as in Example 2 (C). Compared with the injection of killer cells, a significant decrease in the volume of cancer tissue was observed in the group injected with mature natural killer cells differentiated using GTP-binding protein (mSara) homologue.

실시예Example 174. 174. CopineCopine 1을 이용한 사람 제대혈 유래 조혈모세포로부터 From human cord blood-derived hematopoietic stem cells using 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Copine 1을 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Copine 1 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Copine 1을 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Copine 1을 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Copine 1 instead of treating Interleukin-15 under the same conditions as Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increased rate of differentiation of 2 to 3 times when Copine 1 was treated compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Copine 1을 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Copine 1을 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating propine killer cells with Copine 1 in an amount of 1 µg / ml at a ratio of 1: 1 with interleukin-15. As a result, it showed a slightly increased differentiation rate compared to the case of treatment with Copine 1 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Copine 1에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Copine 1 in human cord blood-derived hematopoietic stem cells in animal cancer model

Copine 1을 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Copine 1을 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Copine 1, the mature natural killer cells of the control group treated with nothing were injected. Compared with Copine 1-derived mature natural killer cells, significant decreases in cancer tissue volume were observed.

실시예Example 175. 175. PretinPretin phosphatasephosphatase 3, 3, catalyticcatalytic subunitsubunit , , gammagamma isoformisoform 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Pretin phosphatase 3, catalytic subunit, gamma isoform를 이용한 성숙자연살해세포 분화법 (A) Differentiation of mature killer cells using pretin phosphatase 3, catalytic subunit and gamma isoform in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Pretin phosphatase 3, catalytic subunit, gamma isoform를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Pretin phosphatase 3, catalytic subunit, gamma isoform를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were treated with 1 µg / ml (Upstat) of Pretin phosphatase 3, catalytic subunit, and gamma isoform instead of treating Interleukin-15 under the same conditions as in Example 2 (A). After differentiation for one day, human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 to 3 times higher differentiation rate when treated with Pretin phosphatase 3, catalytic subunit, and gamma isoform compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Pretin phosphatase 3, catalytic subunit, gamma isoform를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Pretin phosphatase 3, catalytic subunit, gamma isoform를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, differentiation rate was confirmed by treating pretin phosphate 3, catalytic subunit, and gamma isoform with interleukin-15 in an amount of 1 μg / ml in a 1: 1 ratio. The results showed a slightly increased differentiation rate compared to the treatment with pretin phosphatase 3, catalytic subunit and gamma isoform alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Pretin phosphatase 3, catalytic subunit, gamma isoform에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by pretin phosphatase 3, catalytic subunit, gamma isoform in human cord blood-derived hematopoietic stem cells in animal cancer model

Pretin phosphatase 3, catalytic subunit, gamma isoform를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Pretin phosphatase 3, catalytic subunit, gamma isoform를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. Human maturation differentiated using pretin phosphatase 3, catalytic subunit, and gamma isoform. In order to observe whether human cancer cells are killed by natural killer cells, the maturation of the control group without any treatment was performed in the same manner as in Example 2 (C). Compared with the injection of natural killer cells, a significant decrease in the volume of cancer tissues was observed in the group injected with mature natural killer cells differentiated using pretin phosphatase 3, catalytic subunit and gamma isoform.

실시예Example 176. 176. CyclinCyclin B2를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells Using B2 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Cyclin B2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Cyclin B2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Cyclin B2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Cyclin B2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Cyclin B2 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of 2 to 3 times differentiation rate when treated with Cyclin B2 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Cyclin B2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Cyclin B2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, the progenitor killer cells were treated with Cyclin B2 with interleukin-15 at a ratio of 1 μg / ml in a 1: 1 ratio to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared with the Cyclin B2 treatment alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Cyclin B2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Cyclin B2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Cyclin B2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Cyclin B2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Cyclin B2, the mature natural killer cells of the control group treated with nothing were tested as in Example 2 (C). Compared with the Cyclin B2-derived mature natural killer cells, a significant decrease in cancer tissue volume was observed.

실시예Example 177. 177. Pip5k1bPip5k1b 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Pip5k1b를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Pip5k1b in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Pip5k1b를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Pip5k1b를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Pip5k1b instead of interleukin-15 treatment under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation rate when Pip5k1b was treated compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Pip5k1b를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Pip5k1b를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Pip5k1b with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared to the case of Pip5k1b alone treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Pip5k1b에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Pip5k1b in human cord blood-derived hematopoietic stem cells in animal cancer model

Pip5k1b를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Pip5k1b를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Pip5k1b, experiments were performed in the same manner as in Example 2 (C). Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Pip5k1b.

실시예Example 178. 178. Mlf2Mlf2 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Mlf2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Mlf2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Mlf2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Mlf2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Mlf2 to the same conditions as Example 2 (A) instead of treating Interleukin-15. The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed an increase of about 2 to 3 times differentiation when treated with Mlf2 compared to the control without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Mlf2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Mlf2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Mlf2 with interleukin-15 in a ratio of 1 μg / ml in a ratio of 1 μg / ml to confirm the differentiation rate. As a result, it showed a slightly increased differentiation rate compared with the case of Mlf2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Mlf2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation of Mlf2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Mlf2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Mlf2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Mlf2, the same procedure as in Example 2 (C) was performed. In the group injected with mature natural killer cells differentiated with Mlf2, a significant decrease in the volume of cancer tissue was observed.

실시예Example 179. 179. RetinoblastomaRetinoblastoma -- likelike 2를 이용한 사람 제대혈 유래 조혈모세포로부터 성숙자연살해세포의 분화 Differentiation of Mature Natural Killer Cells from Human Umbilical Cord Blood-derived Hematopoietic Stem Cells

(A) 사람 제대혈 유래 조혈모세포에서 Retinoblastoma-like 2를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Retinoblastoma-like 2 in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Retinoblastoma-like 2를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Retinoblastoma-like 2를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Retinoblastoma-like 2 instead of treating Interleukin-15 under the same conditions as in Example 2 (A). Human mature natural killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed about 2 ~ 3 fold more differentiation when treated with Retinoblastoma-like 2 compared to the control group without any treatment.

추가로, 동일조건하에 전구자연살해세포를 Retinoblastoma-like 2를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Retinoblastoma-like 2를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, progenitor killer cells were treated with Retinoblastoma-like 2 with interleukin-15 in a ratio of 1 µg / ml in a ratio of 1 µg / ml to confirm the differentiation rate. The results showed a slightly increased differentiation rate when treated with Retinoblastoma-like 2 alone.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Retinoblastoma-like 2에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Retinoblastoma-like 2 in human cord blood-derived hematopoietic stem cells in animal cancer model

Retinoblastoma-like 2를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Retinoblastoma-like 2를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Retinoblastoma-like 2, the mature natural killer cells of the control group treated with nothing were injected as a result of the same method as in Example 2 (C). Compared to the other group, a significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated using Retinoblastoma-like 2.

실시예Example 180. 180. Ppp6cPpp6c 를 이용한 사람 제대혈 유래 조혈모세포로부터 From Human Umbilical Cord Blood-derived Hematopoietic Stem Cells 성숙자연살해세포의Mature natural killer cells 분화 differentiation

(A) 사람 제대혈 유래 조혈모세포에서 Ppp6c를 이용한 성숙자연살해세포 분화법 (A) Mature natural killer cell differentiation using Ppp6c in human cord blood-derived hematopoietic stem cells

실시예 2(A)에서 얻은 전구자연살해세포를 실시예 2(A)와 동일 조건에 인터루킨-15를 처리하는 대신 Ppp6c를 1㎍/ml(Upstat) 첨가하여 14일 동안 분화시킨 후 사람 성숙자연살해세포를 실시예 2(A)와 같은 방법으로 FACS 분석하여 그 분화 정도를 확인하였다. CD56⁺NKG2A⁺ _, CD56⁺CD161⁺ _,CD56⁺NKP46⁺ _,CD56⁺NKP30⁺ _,CD56⁺NKP44⁺그리고CD56⁺NKG20⁺한 세포들은 아무것도 처리하지 않은 대조군에 비해 Ppp6c를 처리하였을 경우 약 2~3배 증가된 분화율을 보였다. The progenitor killer cells obtained in Example 2 (A) were differentiated for 14 days by adding 1 µg / ml (Upstat) of Ppp6c instead of treating Interleukin-15 under the same conditions as in Example 2 (A). The killer cells were analyzed by FACS in the same manner as in Example 2 (A) to confirm their differentiation. CD56 ⁺ NKG2A ⁺ _, ^{^{_{^{CD56 + CD161 +, CD56 + NKP46}}}} +, CD56 + NKP30 +, CD56 + NKP44 + and CD56 ⁺ NKG20 ⁺ cells showed a differentiation rate of 2 to 3 times when Ppp6c was treated compared to the control group.

추가로, 동일조건하에 전구자연살해세포를 Ppp6c를 인터루킨-15와 함께 1:1의 비율로 1㎍/ml 량으로 처리하여 분화율을 확인하였다. 그 결과 Ppp6c를 단독으로 처리한 경우에 비하여 약간 증가된 분화율을 보이는 것으로 나타났다. In addition, under the same conditions, PPP6c was treated with 1 µg / ml in a ratio of 1: 1 with interleukin-15, and the differentiation rate was confirmed. The results showed a slightly increased differentiation rate compared to the Ppp6c alone treatment.

(B) 동물 암 모델에서 사람 제대혈 유래 조혈모세포에서 Ppp6c에 의해 분화 유도된 성숙자연살해세포에 의한 항암 활성 규명(B) Identification of anticancer activity by mature natural killer cells induced by differentiation by Ppp6c in human cord blood-derived hematopoietic stem cells in animal cancer model

Ppp6c를 이용하여 분화시킨 사람 성숙자연살해세포에 의해 사람 암세포가 사멸되는지 관찰해 보기 위해 실시예 2(C)와 동일한 방법으로 실험한 결과 아무것도 처리하지 않은 대조군의 성숙자연살해세포를 주입한 것에 비해 Ppp6c를 이용하여 분화시킨 성숙자연살해세포를 주입한 군에서 암조직 부피의 현저한 감소가 관찰되었다. In order to observe whether human cancer cells are killed by human mature natural killer cells differentiated using Ppp6c, experiments were performed in the same manner as in Example 2 (C). Significant decrease in cancer tissue volume was observed in the group injected with mature natural killer cells differentiated with Ppp6c.

Claims

전구자연살해세포로부터 성숙자연살해세포로의 분화를 유도하는데 사용되는, 케모카인 수용체 1(chemokine receptor 1)의 항체, 케모카인 수용체의 리간드인 CCL3, 케모카인 수용체의 리간드인 CCL5, 콜로니 자극인자 요소 1 수용체(colony stimulating factor 1 receptor)의 항체, 콜로니 자극 인자 1 수용체의 리간드인 CSF1, 형질전환 성장 인자 베타 수용체 II(Transforming growth factor, beta receptor II)의 항체, 형질전환 성장 인자 베타 수용체II의 리간드인 TGF-베타, 에스테로젠 수용체 베타 1(Estrogen related receptor beta like 1, Esrrbl1)의 항체, 에스테로젠 수용체 베타 1(Estrogen related receptor beta like 1, Esrrbl1)의 리간드인 스테로이드성 인자-1(steroidogenic factor-1), c-src 타이로신 키나 아제, Hmgb1(high mobility group box 1), 페리틴(Ferritin) 헤비 체인, 페리틴(Ferritin) 라이트 체인, 칼그라뉼린 비(calgranulin B), 세포 표면 글라이코단백질(cell surface glycoprotein) p91의 폴리클로날 항체, 클로라이드 채널 7(Chloride channel 7), 아드빌린(Advillin), 카텝신-L(Cathepsin-L), 카텝신-B(Cathepsin-B), 카텝신-H(Cathepsin-H), Emr1(EGF-like module-containing mucin-like hormone receptor-like 1)의 항체, Trem2(Triggering receptor expressed on myeloid cells 2)의 항체, SERPINA3G, LAPTm5 유도물질 레티논산(Retinoic acid), Egr1(early growth response1), Carf(calcium response factor), Apo-E(Apolipoprotein E), 베타 카테닌(Catenin beta), lysozyme, Brevican, Matrix metalloproteinase 12, cellular repressor of EIA-stimulated gene, c-kit ligand, MPS1 protein, transglutaminase 2, serum and glucocorticoid regulated protein kinase, interferon induced protein, milk fat globule membrane protein EGF factor 8, Fc g receptor, S100 calcium binding protein A9, Arginase1, TNF1, retinoid inducible serine carboxypeptidase, Interleukin-18 binding protein d precursor, Chloride channel 7, CD36 antigen, Similar to putative zinc finger protein, Carbohydrate binding protein 35, C-type calcium dependent, carbohydrate, Lipoprotein lipase, v-maf musculoaponeurotic fibrosarcoma oncogene, Interleukin-7, Neuropilin, Peroxiredoxin 1la associated invariant chainGlycoprotein (transmembrane) nmbSerum amyloid A (SAA) 3 proteinSulfated glycoprotein (Sgp1)Response to metastatic cancers 1 (Rmcs1)ATP-binding cassette, sub-family AProgrammed cell death 1 ligand 1DnaJ (Hsp40)homolog (Dnajb11)Fibronectin 1Fatty acid binding protein 4Complement component 1, q, gamma polypeptideFc g receptor, IgG, high affinity 1IFN regulatory factor, IgG, high affinity 1Complement component subunit Clq beta-chain2'-5'oligoadenylate synthetase-like 2Chitinase 3-like 3 (Chi3l3)IFN-activated gene 203Transforming growth factor, beta induced (Tgfbi)Ig super family, member 7Chemokine (C-X-C motif) ligand 16Colony stimulating factor 2 receptor, beta 2, (Csf2rb2)H2-BfIFN-induced protein with tetratricopeptide repeats2RIKEN cDNA 2310005K03 geneRIKEN cDNA 3110037K17 geneRIKEN cDNA 5830458K16 geneThymidylate kinase family LPS-inducible memberSyntaxin binding protein 3Fatty acid binding protein 5, epidermalIFN-stimulated gene 12 (Isg12)Allograft inflammatory factor 1Biliverdin reductase B(flavin reductase(NADPH))Complement component 3a receptorSelenoprotein RIL-1 receptor antagonist (IL-1RA)Adenylosuccinate synthetaseHemopoietic cell kinaseDeath effector domain-containing DNA binding protein 2 (Dedd2)IFN induced transmembrane protein 2 likeGAVA-A receptor subunit 6Selectin, platelet (p-selectin) ligand (Selpl)High mobility group box 1 (Hmgb1)Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptideSerpinb6bRegulator of G-protein signaling 16RIKEN cDNA 6330406L22 gene (6330406L22Rik)Osteopontin precursorZinc finger protein 207decay- accelerating factor GPI anchored mRNAAmyloid beta (A4) precursor protein-binding, family B, member 1 interacting proteinGlutaredoxin 1 (thioltransferase) (Glrx1)Coronin, actin binding protein 1A (Coro1a)RIKEN cDNA 9630048M01 geneDendritic cell protein GA17 (Ga17)GTPase, IMAP family member 4 (Gimap4)Upstream transcription factor 1RIKEN cDNA 1810015M01 gene (1810015M01Rik)Karyopherin (importin) alpha 6Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1Kelch domain containing 2 (Klhdc2)Structure specific recognition protein 1Lymphocyte protein tyrosine kinaseF-box and leucine-rich repeat protein 14Osteoclastogenesis inhibitory factorG protein-coupled receptor 171RNA terminal phosphate cyclase domain 1Cullin 2CD160 antigenHypothetical RNA-binding domain, RBD structureSema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6DActivating signal cointegrator 1 complex subunit 3 like 1Abl-interactor 1Hypothetical protein MGC36662RAP2C, member of RAS oncogene familyValosin containing protein(Vcp)CD164 antigenSimilar to RIKEN cDNA 4933425K02 geneAdvillinAsparagine synthetaseZinc finger protein 259Interleukin 12 receptor, beta 1Growth arrest and DNA-damage-inducible 45 gammaCytochrome b5 type BFMS-like tyrosine kinase 3 ligandProgrammed cell death 1 ligand 2S100 calcium binding protein A4Metallothionein 2Peptidylprolyl isomerase AOsteopontin (OPN)Chemokine (C-C motif) ligand 4T-cell receptor beta, variable 13 (Tcrb-V13)Immune associated nucleotide 1Interleukin 2 receptor, beta chain (Il2rb)Cd28: CD28 antigenGranzyme Eserin protease inhibitorPurine rich element binding protein A (Pura)Lymphocyte protein tyrosine kinase (Lck)Eukaryotic translation initiation factor 4 gamma, 3 (Eif4g3)Lamin A (Lmna)Raver1: Ribonucleoprotein, PTB-binding 1Solute carrier family 6V-ral simian leukemia viral oncogene homolog A (ras related)Fyn: Fyn proto-oncogeneExpressed sequence AV024533Fhl2: Four and a half LIM domains 2C1q and tumor necrosis factor related protein 6 (C1qtnf6)Zfp512: Zinc finger protein 512P2ry10: Purinergic receptor P2Y, G-protein coupled 10Protein phosphatase 1, regulatory (inhibitor) subunit 14B (Ppp1r14b)Strap: Serine/threonine kinase receptor associated proteinHdac3: Histone deacetylase 3Ppp3cb: Protein phosphatase 3, catalytic subunit, beta isoformMmp9: Matrix metallopeptidase 9Ifnar2: Interferon (alpha and beta) receptor 2Prefoldin 1GTP-binding protein(mSara) homologue mRNACopine 1(Rbm 12)Pretin phosphatase 3, catalytic subunit, gamma isoform (Ppp3cc)Cyclin B2Pip5k1b: Phosphatidylinositol-4-phosphate 5-kinase, type 1 betaMlf2: Myeloid leukemia factor 2Retinoblastoma-like 2Ppp6c: Protein phosphatase 6, catalytic subunit으로 이루어진 그룹중에서 선택된 화합물.Antibodies of chemokine receptor 1, CCL3, a ligand for chemokine receptor, CCL5, a ligand for chemokine receptor, and colony stimulating factor element 1 receptor, which are used to induce differentiation from progenitor killer cells to mature killer cells. antibody of colony stimulating factor 1 receptor, CSF1, ligand of colony stimulating factor 1 receptor, antibody of transforming growth factor beta receptor II, TGF-, ligand of transforming growth factor beta receptor II Beta, an antibody of Estrogen related receptor beta like 1, Esrrbl1, steroidogenic factor-1, a ligand of Estrogen related receptor beta like 1, Esrrbl1, c-src tyrosine kinase, high mobility group box 1 (Hmgb1), Ferritin heavy chain, Ferritin light chain, calgranulin B ), Polyclonal antibody of cell surface glycoprotein p91, chloride channel 7, Advillin, Catepsin-L, Catepsin-B (Cathepsin) -B), Catepsin-H (Cathepsin-H), antibody of EGF-like module-containing mucin-like hormone receptor-like 1 (EmR1), antibody of Triggering receptor expressed on myeloid cells 2 (SERPINA3G, LAPTm5) Inducer Retinoic acid, Early growth response1, Egr1 (calcium response factor), Apo-E (Apolipoprotein E), beta catenin (Catenin beta), lysozyme, Brevican, Matrix metalloproteinase 12, cellular repressor of EIA -stimulated gene, c-kit ligand, MPS1 protein, transglutaminase 2, serum and glucocorticoid regulated protein kinase, interferon induced protein, milk fat globule membrane protein EGF factor 8, Fc g receptor, S100 calcium binding protein A9, Arginase1, TNF1, retinoid inducible serine carboxypeptidase, Interleukin-18 binding protein d precursor, Chloride ch annel 7, CD36 antigen, Similar to putative zinc finger protein, Carbohydrate binding protein 35, C-type calcium dependent, carbohydrate, Lipoprotein lipase, v-maf musculoaponeurotic fibrosarcoma oncogene, Interleukin-7, Neuropilin, Peroxiredoxin 1la associated invariant chain Glycoprotein (transmembrane) nmbSerum amyloid A (SAA) 3 proteinSulfated glycoprotein (Sgp1) Response to metastatic cancers 1 (Rmcs1) ATP-binding cassette, sub-family AProgrammed cell death 1 ligand 1DnaJ (Hsp40) homolog (Dnajb11) Fibronectin 1Fatty acid binding protein 4Complement component 1, q, gamma polypeptideFg g receptor, IgG, high affinity 1 IFN regulatory factor, IgG, high affinity 1 Complement component subunit Clq beta-chain2'-5'oligoadenylate synthetase-like 2Chitinase 3-like 3 (Chi3l3) IFN-activated gene 203Transforming growth factor, beta induced (Tgfbi) Ig super family, member 7 Chemokine (CXC motif) ligand 16Colony stimulating factor 2 receptor, beta 2, (Csf2rb2) H2-BfIFN-induced protein with tetratricopept ide repeats2RIKEN cDNA 2310005K03 geneRIKEN cDNA 3110037K17 geneRIKEN cDNA 5830458K16 geneThymidylate kinase family LPS-inducible memberSyntaxin binding protein 3Fatty acid binding protein 5, epidermalIFN-stimulated gene 12 (Isg12) Allograft inflammatory factor reductase Base RIL-1 receptor antagonist (IL-1RA) Adenylosuccinate synthetase Hemopoietic cell kinaseDeath effector domain-containing DNA binding protein 2 (Dedd2) IFN induced transmembrane protein 2 likeGAVA-A receptor subunit 6Selectin, platelet (p-selectin) ligand (Selpl) box 1 (Hmgb1) Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, epsilon polypeptide , family B, member 1 interacting protein doxin 1 (thioltransferase) (Glrx1) Coronin, actin binding protein 1A (Coro1a) RIKEN cDNA 9630048M01 geneDendritic cell protein GA17 (Ga17) GTPase, IMAP family member 4 (Gimap4) Upstream transcription factor 1RIKEN cDNA 1810015M01 gene (1810015M01pherin) alpha 6 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1Kelch domain containing 2 (Klhdc2) Structure specific recognition protein 1Lymphocyte protein tyrosine kinase RNA-binding domain, RBD structureSema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6DActivating signal cointegrator 1 complex subunit 3 like 1Abl-interactor 1Hypothetical protein MGC36662RAP2C, member of RAS oncogene familyValosin containing protein (Vcp) CD164 antigenSimilar to RIKEN cDNA 4933425K02 geneAdvillinAsparagine synthetaseZinc finger protein 259 Interleukin 12 receptor, beta 1 Growth arrest and DNA-damage-inducible 45 gammaCytochrome b5 type BFMS-like tyrosine kinase 3 ligandProgrammed cell death 1 ligand 2S100 calcium binding protein A4Metallothionein 2Peptidylprolyl isomerase AOsteopontin (OPN) Chemokine 4 (CC-cell receptor) beta, variable 13 (Tcrb-V13) Immune associated nucleotide 1 Interleukin 2 receptor, beta chain (Il2rb) Cd28: CD28 antigenGranzyme Eserin protease inhibitorPurine rich element binding protein A (Pura) Lymphocyte protein tyrosine kinase (Lck) Eukaryotic translation initiation factor 4 gamma, 3 (Eif4g3) Lamin A (Lmna) Raver1: Ribonucleoprotein, PTB-binding 1Solute carrier family 6V-ral simian leukemia viral oncogene homolog A (ras related) Fyn: Fyn proto-oncogeneExpressed sequence AV024533Fhl2: Four and a half LIM domains 2C1q and tumor necrosis factor related protein 6 (C1qtnf6) Zfp512: Zinc finger protein 512P2ry10: Purinergic receptor P2Y, G-protein coupled 10 Protein phosphatase 1, regul atory (inhibitor) subunit 14B (Ppp1r14b) Strap: Serine / threonine kinase receptor associated protein Hdac3: Histone deacetylase 3 Ppp3cb: Protein phosphatase 3, catalytic subunit, beta isoformMmp9: Matrix metallopeptidase 9 mSara) homologue mRNACopine 1 (Rbm 12) Pretin phosphatase 3, catalytic subunit, gamma isoform (Ppp3cc) Cyclin B2Pip5k1b: Phosphatidylinositol-4-phosphate 5-kinase, type 1 betaMlf2: Myeloid leukemia factor 2 A compound selected from the group consisting of subunits.

(i) 조혈모세포를 인터루킨-7, SCF 및 Flt3L로 처리하여 전구자연살해세포로 분화시키고, (ii) 전구자연살해세포를 케모카인 수용체 1(chemokine receptor 1)의 항체, 케모카인 수용체의 리간드인 CCL3, 케모카인 수용체의 리간드인 CCL5, 콜로니 자극인자 요소 1 수용체(colony stimulating factor 1 receptor)의 항체, 콜로니 자극 인자 1 수용체의 리간드인 CSF1, 형질전환 성장 인자 베타 수용체 II(Transforming growth factor, beta receptor II)의 항체, 형질전환 성장 인자 베타 수용체II의 리간드인 TGF-베타, 에스테로젠 수용체 베타 1(Estrogen related receptor beta like 1, Esrrbl1)의 항체, 에스테로젠 수용체 베타 1(Estrogen related receptor beta like 1, Esrrbl1)의 리간드인 스테로이드성 인자-1(steroidogenic factor-1), c-src 타이로신 키나아제, Hmgb1(high mobility group box 1), 페리틴(Ferritin) 헤비 체인, 페리틴(Ferritin) 라이트 체인, 칼그라뉼린 비(calgranulin B), 세포 표면 글라이코단백질(cell surface glycoprotein) p91의 폴리클로날 항체, 클로라이드 채널 7(Chloride channel 7), 아드빌린(Advillin), 카텝신-L(Cathepsin-L), 카텝신-B(Cathepsin-B), 카텝신-H(Cathepsin-H), Emr1(EGF-like module-containing mucin-like hormone receptor-like 1)의 항체, Trem2(Triggering receptor expressed on myeloid cells 2)의 항체, SERPINA3G, LAPTm5 유도물질 레티논산(Retinoic acid), Egr1(early growth response1), Carf(calcium response factor), Apo-E(Apolipoprotein E), 베타 카테닌(Catenin beta)lysozyme, Brevican, Matrix metalloproteinase 12, cellular repressor of EIA-stimulated gene, c-kit ligand, MPS1 protein, transglutaminase 2, serum and glucocorticoid regulated protein kinase, interferon induced protein, milk fat globule membrane protein EGF factor 8, Fc g receptor, S100 calcium binding protein A9, Arginase1, TNF1, retinoid inducible serine carboxypeptidase, Interleukin-18 binding protein d precursor, Chloride channel 7, CD36 antigen, Similar to putative zinc finger protein, Carbohydrate binding protein 35, C-type calcium dependent, carbohydrate, Lipoprotein lipase, v-maf musculoaponeurotic fibrosarcoma oncogene, Interleukin-7, Neuropilin, Peroxiredoxin 1la associated invariant chainGlycoprotein (transmembrane) nmbSerum amyloid A (SAA) 3 proteinSulfated glycoprotein (Sgp1)Response to metastatic cancers 1 (Rmcs1)ATP-binding cassette, sub-family AProgrammed cell death 1 ligand 1DnaJ (Hsp40)homolog (Dnajb11)Fibronectin 1Fatty acid binding protein 4Complement component 1, q, gamma polypeptideFc g receptor, IgG, high affinity 1IFN regulatory factor, IgG, high affinity 1Complement component subunit Clq beta-chain2'-5'oligoadenylate synthetase-like 2Chitinase 3-like 3 (Chi3l3)IFN-activated gene 203Transforming growth factor, beta induced (Tgfbi)Ig super family, member 7Chemokine (C-X-C motif) ligand 16Colony stimulating factor 2 receptor, beta 2, (Csf2rb2)H2-BfIFN-induced protein with tetratricopeptide repeats2RIKEN cDNA 2310005K03 geneRIKEN cDNA 3110037K17 geneRIKEN cDNA 5830458K16 geneThymidylate kinase family LPS-inducible memberSyntaxin binding protein 3Fatty acid binding protein 5, epidermalIFN-stimulated gene 12 (Isg12)Allograft inflammatory factor 1Biliverdin reductase B(flavin reductase(NADPH))Complement component 3a receptorSelenoprotein RIL-1 receptor antagonist (IL-1RA)Adenylosuccinate synthetaseHemopoietic cell kinaseDeath effector domain-containing DNA binding protein 2 (Dedd2)IFN induced transmembrane protein 2 likeGAVA-A receptor subunit 6Selectin, platelet (p-selectin) ligand (Selpl)High mobility group box 1 (Hmgb1)Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptideSerpinb6bRegulator of G-protein signaling 16RIKEN cDNA 6330406L22 gene (6330406L22Rik)Osteopontin precursorZinc finger protein 207decay-accelerating factor GPI anchored mRNAAmyloid beta (A4) precursor protein-binding, family B, member 1 interacting proteinGlutaredoxin 1 (thioltransferase) (Glrx1)Coronin, actin binding protein 1A (Coro1a)RIKEN cDNA 9630048M01 geneDendritic cell protein GA17 (Ga17)GTPase, IMAP family member 4 (Gimap4)Upstream transcription factor 1RIKEN cDNA 1810015M01 gene (1810015M01Rik)Karyopherin (importin) alpha 6Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1Kelch domain containing 2 (Klhdc2)Structure specific recognition protein 1Lymphocyte protein tyrosine kinaseF-box and leucine-rich repeat protein 14Osteoclastogenesis inhibitory factorG protein-coupled receptor 171RNA terminal phosphate cyclase domain 1Cullin 2CD160 antigenHypothetical RNA-binding domain, RBD structureSema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6DActivating signal cointegrator 1 complex subunit 3 like 1Abl-interactor 1Hypothetical protein MGC36662RAP2C, member of RAS oncogene familyValosin containing protein(Vcp)CD164 antigenSimilar to RIKEN cDNA 4933425K02 geneAdvillinAsparagine synthetaseZinc finger protein 259Interleukin 12 receptor, beta 1Growth arrest and DNA-damage-inducible 45 gammaCytochrome b5 type BFMS-like tyrosine kinase 3 ligandProgrammed cell death 1 ligand 2S100 calcium binding protein A4Metallothionein 2Peptidylprolyl isomerase AOsteopontin (OPN)Chemokine (C-C motif) ligand 4T-cell receptor beta, variable 13 (Tcrb-V13)Immune associated nucleotide 1Interleukin 2 receptor, beta chain (Il2rb)Cd28: CD28 antigenGranzyme Eserin protease inhibitorPurine rich element binding protein A (Pura)Lymphocyte protein tyrosine kinase (Lck)Eukaryotic translation initiation factor 4 gamma, 3 (Eif4g3)Lamin A (Lmna)Raver1: Ribonucleoprotein, PTB-binding 1Solute carrier family 6V-ral simian leukemia viral oncogene homolog A (ras related)Fyn: Fyn proto-oncogeneExpressed sequence AV024533Fhl2: Four and a half LIM domains 2C1q and tumor necrosis factor related protein 6 (C1qtnf6)Zfp512: Zinc finger protein 512P2ry10: Purinergic receptor P2Y, G-protein coupled 10Protein phosphatase 1, regulatory (inhibitor) subunit 14B (Ppp1r14b)Strap: Serine/threonine kinase receptor associated proteinHdac3: Histone deacetylase 3Ppp3cb: Protein phosphatase 3, catalytic subunit, beta isoformMmp9: Matrix metallopeptidase 9Ifnar2: Interferon (alpha and beta) receptor 2Prefoldin 1GTP-binding protein(mSara) homologue mRNACopine 1(Rbm 12)Pretin phosphatase 3, catalytic subunit, gamma isoform (Ppp3cc)Cyclin B2Pip5k1b: Phosphatidylinositol-4-phosphate 5-kinase, type 1 betaMlf2: Myeloid leukemia factor 2Retinoblastoma-like 2Ppp6c: Protein phosphatase 6, catalytic subunit으로 이루어진 그룹중에서 선택된 화합물중 하나 또는 둘 이상으로 처리하여 성숙자연살해세포로 분화시켜 성숙자연살해세포를 수득함을 특징으로 하는, 성숙자연살해세포의 제조 방법.(i) hematopoietic stem cells were treated with interleukin-7, SCF and Flt3L to differentiate into progenitor killer cells, (ii) progenitor killer cells were antibodies of chemokine receptor 1, chemokine receptor ligand CCL3, CCL5, a ligand of the chemokine receptor, an antibody of the colony stimulating factor 1 receptor, CSF1, a ligand of the colony stimulating factor 1 receptor, and a transforming growth factor, beta receptor II Antibody, TGF-beta, a ligand of transforming growth factor beta receptor II, an antibody of Estrogen related receptor beta like 1 (Esrrbl1), and an estrogen related receptor beta like 1 (Esrrbl1) Ligands steroidogenic factor-1, c-src tyrosine kinase, high mobility group box 1 (Hmgb1), ferritin heavy chains, ferritin lite Chain, calgranulin B, polyclonal antibody of cell surface glycoprotein p91, chloride channel 7, advillin, cathepsin-L (Cathepsin-L) L), Catepsin-B (Cathepsin-B), Catepsin-H (Cathepsin-H), Emr1 (EGF-like module-containing mucin-like hormone receptor-like 1) antibodies, Trem2 (Triggering receptor expressed on myeloid Antibodies of cells 2), SERPINA3G, LAPTm5 inducer Retinoic acid, Egr1 (early growth response1), Carf (calcium response factor), Apo-E (Apolipoprotein E), beta catenin (Catenin beta) lysozyme, Brevican, Matrix metalloproteinase 12, cellular repressor of EIA-stimulated gene, c-kit ligand, MPS1 protein, transglutaminase 2, serum and glucocorticoid regulated protein kinase, interferon induced protein, milk fat globule membrane protein EGF factor 8, Fc g receptor, S100 calcium binding protein A9, Arginase1, TNF1, retinoid inducible serine carboxypeptidase, Interleukin-18 binding protein d precursor, Chloride channel 7, CD36 antigen, Similar to putative zinc finger protein, Carbohydrate binding protein 35, C-type calcium dependent, carbohydrate, Lipoprotein lipase, v-maf musculoaponeurotic fibrosarcoma oncogene, Interleukin-7, Neuropilin, Peroxiredoxin 1la associated invariant chainGlycoprotein (transmembrane) nmbSerum amyloid A (SAA) 3 proteinSulfated glycoprotein (Sgp1) Response to metastatic cancers 1 (Rmcs1) ATP-binding cassette, sub-family AProgrammed cell death 1 ligand 1DnaJ (Hsp40) homolog (Dnajb11) Fitty acid binding protein 4Complement component 1, q, gamma polypeptideFg g receptor, IgG, high affinity 1IFN regulatory factor, IgG, high affinity 1Complement component subunit Clq beta-chain2'-5'oligoadenylate synthetase-like 2Chitinase 3-like 3 (Chi3l3) IFN- activated gene 203 Transforming growth factor, beta induced (Tgfbi) Ig super family, member 7 Chemokine (CXC motif) ligand 16Colony stimulating factor 2 receptor, beta 2, (Csf2rb2) H2-B fIFN-induced protein with tetratricopeptide repeats2RIKEN cDNA 2310005K03 geneRIKEN cDNA 3110037K17 geneRIKEN cDNA 5830458K16 geneThymidylate kinase family LPS-inducible memberSyntaxin binding protein 3 Complement component 3a receptorSelenoprotein RIL-1 receptor antagonist (IL-1RA) Adenylosuccinate synthetase Hemopoietic cell kinaseDeath effector domain-containing DNA binding protein 2 (Dedd2) IFN induced transmembrane protein 2 likeGAVA-A receptor subunit 6Selectin, platelet (p-selectin) ligand () Selpl) High mobility group box 1 (Hmgb1) Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, epsilon polypeptide ) precursor protein-binding, famil y B, member 1 interacting protein Glutaredoxin 1 (thioltransferase) (Glrx1) Coronin, actin binding protein 1A (Coro1a) RIKEN cDNA 9630048M01 geneDendritic cell protein GA17 (Ga17) GTPase, IMAP family member 4 (Gimap4) Karyopherin (importin) alpha 6Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1Kelch domain containing 2 (Klhdc2) Structure specific recognition protein 1Lymphocyte protein tyrosine kinaseF-box and leucine-rich repeat protein 14Osteoclastogenesis inhibitory factor171 protein171-coupled receptor phosphate cyclase domain 1Cullin 2CD160 antigenHypothetical RNA-binding domain, RBD structureSema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) (Vcp) CD164 antigenSimilar to RIKEN cDNA 4933425K02 gen eAdvillinAsparagine synthetaseZinc finger protein 259Interleukin 12 receptor, beta 1Growth arrest and DNA-damage-inducible 45 gammaCytochrome b5 type BFMS-like tyrosine kinase 3 ligand Cell receptor beta, variable 13 (Tcrb-V13) Immune associated nucleotide 1 Interleukin 2 receptor, beta chain (Il2rb) Cd28: CD28 antigenGranzyme Eserin protease inhibitor Purine rich element binding protein A (Pura) Lymphocyte protein tyrosine kinase (Lck) Eukaryotic translation initiation factor 4 gamma, 3 (Eif4g3) Lamin A (Lmna) Raver1: Ribonucleoprotein, PTB-binding 1Solute carrier family 6V-ral simian leukemia viral oncogene homolog A (ras related) Fyn: Fyn proto-oncogeneExpressed sequence AV024533Fhl2: Four and a half LIM domains 2C1q and tumor necrosis factor related protein 6 (C1qtnf6) Zfp512: Zinc finger protein 512P2ry10: Purinergic receptor P2Y, G-protei n coupled 10 Protein phosphatase 1, regulatory (inhibitor) subunit 14B (Ppp1r14b) Strap: Serine / threonine kinase receptor associated protein Hdac3: Histone deacetylase 3Ppp3cb: Protein phosphatase 3, catalytic subunit, beta isoformMmp9: Matrix metallopeptidase 9Ifnar2: Interferonalpha and 2 Prefoldin 1GTP-binding protein (mSara) homologue mRNA Copine 1 (Rbm 12) Pretin phosphatase 3, catalytic subunit, gamma isoform (Ppp3cc) Cyclin B2P5P1kb: : Protein phosphatase 6, a method for producing mature natural killer cells, characterized in that differentiation into mature natural killer cells by treatment with one or two or more of the compounds selected from the group consisting of catalytic subunits.

제2항에 있어서, 조혈모세포가 사람의 골수, 말초혈액 및 제대혈로 이루어진 그룹중에서 선택된 어느 하나로부터 유래된 것인 사람 성숙자연살해세포의 제조 방법.The method of claim 2, wherein the hematopoietic stem cells are derived from any one selected from the group consisting of human bone marrow, peripheral blood and umbilical cord blood.

제3항에 있어서, 전구자연살해세포를 사람 기질세포의 존재하에 화합물로 처리하는 사람 성숙자연살해세포의 방법.4. The method of claim 3, wherein the progenitor killer cells are treated with a compound in the presence of human stromal cells.

(i) 조혈모세포를 인터루킨-7, SCF 및 Flt3L로 처리하여 전구자연살해세포로 분화시키고, (ii) 전구자연살해세포를 케모카인 수용체 1(chemokine receptor 1)의 항체, 케모카인 수용체의 리간드인 CCL3, 케모카인 수용체의 리간드인 CCL5, 콜로니 자극인자 요소 1 수용체(colony stimulating factor 1 receptor)의 항체, 콜로 니 자극 인자 1 수용체의 리간드인 CSF1, 형질전환 성장 인자 베타 수용체 II(Transforming growth factor, beta receptor II)의 항체, 형질전환 성장 인자 베타 수용체II의 리간드인 TGF-베타, 에스테로젠 수용체 베타 1(Estrogen related receptor beta like 1, Esrrbl1)의 항체, 에스테로젠 수용체 베타 1(Estrogen related receptor beta like 1, Esrrbl1)의 리간드인 스테로이드성 인자-1(steroidogenic factor-1), c-src 타이로신 키나아제, Hmgb1(high mobility group box 1), 페리틴(Ferritin) 헤비 체인, 페리틴(Ferritin) 라이트 체인, 칼그라뉼린 비(calgranulin B), 세포 표면 글라이코단백질(cell surface glycoprotein) p91의 폴리클로날 항체, 클로라이드 채널 7(Chloride channel 7), 아드빌린(Advillin), 카텝신-L(Cathepsin-L), 카텝신-B(Cathepsin-B), 카텝신-H(Cathepsin-H), Emr1(EGF-like module-containing mucin-like hormone receptor-like 1)의 항체, Trem2(Triggering receptor expressed on myeloid cells 2)의 항체, SERPINA3G, LAPTm5 유도물질 레티논산(Retinoic acid), Egr1(early growth response1), Carf(calcium response factor), Apo-E(Apolipoprotein E), 베타 카테닌(Catenin beta), lysozyme, Brevican, Matrix metalloproteinase 12, cellular repressor of EIA-stimulated gene, c-kit ligand, MPS1 protein, transglutaminase 2, serum and glucocorticoid regulated protein kinase, interferon induced protein, milk fat globule membrane protein EGF factor 8, Fc g receptor, S100 calcium binding protein A9, Arginase1, TNF1, retinoid inducible serine carboxypeptidase, Interleukin-18 binding protein d precursor, Chloride channel 7, CD36 antigen, Similar to putative zinc finger protein, Carbohydrate binding protein 35, C-type calcium dependent, carbohydrate, Lipoprotein lipase, v-maf musculoaponeurotic fibrosarcoma oncogene, Interleukin-7, Neuropilin, Peroxiredoxin 1la associated invariant chainGlycoprotein (transmembrane) nmbSerum amyloid A (SAA) 3 proteinSulfated glycoprotein (Sgp1)Response to metastatic cancers 1 (Rmcs1)ATP-binding cassette, sub-family AProgrammed cell death 1 ligand 1DnaJ (Hsp40)homolog (Dnajb11)Fibronectin 1Fatty acid binding protein 4Complement component 1, q, gamma polypeptideFc g receptor, IgG, high affinity 1IFN regulatory factor, IgG, high affinity 1Complement component subunit Clq beta-chain2'-5'oligoadenylate synthetase-like 2Chitinase 3-like 3 (Chi3l3)IFN-activated gene 203Transforming growth factor, beta induced (Tgfbi)Ig super family, member 7Chemokine (C-X-C motif) ligand 16Colony stimulating factor 2 receptor, beta 2, (Csf2rb2)H2-BfIFN-induced protein with tetratricopeptide repeats2RIKEN cDNA 2310005K03 geneRIKEN cDNA 3110037K17 geneRIKEN cDNA 5830458K16 geneThymidylate kinase family LPS-inducible memberSyntaxin binding protein 3Fatty acid binding protein 5, epidermalIFN-stimulated gene 12 (Isg12)Allograft inflammatory factor 1Biliverdin reductase B(flavin reductase(NADPH))Complement component 3a receptorSelenoprotein RIL-1 receptor antagonist (IL-1RA)Adenylosuccinate synthetaseHemopoietic cell kinaseDeath effector domain-containing DNA binding protein 2 (Dedd2)IFN induced transmembrane protein 2 likeGAVA-A receptor subunit 6Selectin, platelet (p-selectin) ligand (Selpl)High mobility group box 1 (Hmgb1)Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptideSerpinb6bRegulator of G-protein signaling 16RIKEN cDNA 6330406L22 gene (6330406L22Rik)Osteopontin precursorZinc finger protein 207decay-accelerating factor GPI anchored mRNAAmyloid beta (A4) precursor protein-binding, family B, member 1 interacting proteinGlutaredoxin 1 (thioltransferase) (Glrx1)Coronin, actin binding protein 1A (Coro1a)RIKEN cDNA 9630048M01 geneDendritic cell protein GA17 (Ga17)GTPase, IMAP family member 4 (Gimap4)Upstream transcription factor 1RIKEN cDNA 1810015M01 gene (1810015M01Rik)Karyopherin (importin) alpha 6Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1Kelch domain containing 2 (Klhdc2)Structure specific recognition protein 1Lymphocyte protein tyrosine kinaseF-box and leucine-rich repeat protein 14Osteoclastogenesis inhibitory factorG protein-coupled receptor 171RNA terminal phosphate cyclase domain 1Cullin 2CD160 antigenHypothetical RNA-binding domain, RBD structureSema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6DActivating signal cointegrator 1 complex subunit 3 like 1Abl-interactor 1Hypothetical protein MGC36662RAP2C, member of RAS oncogene familyValosin containing protein(Vcp)CD164 antigenSimilar to RIKEN cDNA 4933425K02 geneAdvillinAsparagine synthetaseZinc finger protein 259Interleukin 12 receptor, beta 1Growth arrest and DNA-damage-inducible 45 gammaCytochrome b5 type BFMS-like tyrosine kinase 3 ligandProgrammed cell death 1 ligand 2S100 calcium binding protein A4Metallothionein 2Peptidylprolyl isomerase AOsteopontin (OPN)Chemokine (C-C motif) ligand 4T-cell receptor beta, variable 13 (Tcrb-V13)Immune associated nucleotide 1Interleukin 2 receptor, beta chain (Il2rb)Cd28: CD28 antigenGranzyme Eserin protease inhibitorPurine rich element binding protein A (Pura)Lymphocyte protein tyrosine kinase (Lck)Eukaryotic translation initiation factor 4 gamma, 3 (Eif4g3)Lamin A (Lmna)Raver1: Ribonucleoprotein, PTB-binding 1Solute carrier family 6V-ral simian leukemia viral oncogene homolog A (ras related)Fyn: Fyn proto-oncogeneExpressed sequence AV024533Fhl2: Four and a half LIM domains 2C1q and tumor necrosis factor related protein 6 (C1qtnf6)Zfp512: Zinc finger protein 512P2ry10: Purinergic receptor P2Y, G-protein coupled 10Protein phosphatase 1, regulatory (inhibitor) subunit 14B (Ppp1r14b)Strap: Serine/threonine kinase receptor associated proteinHdac3: Histone deacetylase 3Ppp3cb: Protein phosphatase 3, catalytic subunit, beta isoformMmp9: Matrix metallopeptidase 9Ifnar2: Interferon (alpha and beta) receptor 2Prefoldin 1GTP-binding protein(mSara) homologue mRNACopine 1(Rbm 12)Pretin phosphatase 3, catalytic subunit, gamma isoform (Ppp3cc)Cyclin B2Pip5k1b: Phosphatidylinositol-4-phosphate 5-kinase, type 1 betaMlf2: Myeloid leukemia factor 2Retinoblastoma-like 2Ppp6c: Protein phosphatase 6, catalytic subunit으로 이루어진 그룹중에서 선택된 화합물중 어느 하나 또는 둘 이상으로 처리하여 성숙자연살해세포로 분화시켜 성숙자연살해세포를 수득하고, (iii) 분화된 성숙자연살해세포를 인터루킨-2로 처리하여 활성화시킴을 특징으로 하는, 활성화된 성숙자연살해세포의 제조 방법.(i) hematopoietic stem cells were treated with interleukin-7, SCF and Flt3L to differentiate into progenitor killer cells, (ii) progenitor killer cells were antibodies of chemokine receptor 1, chemokine receptor ligand CCL3, CCL5, a ligand of chemokine receptor, antibody of colony stimulating factor 1 receptor, CSF1, ligand of colony stimulating factor 1 receptor, transforming growth factor, beta receptor II TGF-beta, a ligand for transforming growth factor beta receptor II, an antibody of Estrogen related receptor beta like 1 (Esrrbl1), and an estrogen related receptor beta like 1 (Esrrbl1) Steroidogenic factor-1, c-src tyrosine kinase, high mobility group box 1 (Hmgb1), ferritin heavy chain, ferritin light Chain, calgranulin B, polyclonal antibody of cell surface glycoprotein p91, chloride channel 7, advillin, cathepsin-L (Cathepsin-L) L), Catepsin-B (Cathepsin-B), Catepsin-H (Cathepsin-H), Emr1 (EGF-like module-containing mucin-like hormone receptor-like 1) antibodies, Trem2 (Triggering receptor expressed on myeloid Antibodies of cells 2), SERPINA3G, LAPTm5 inducer Retinoic acid, Egr1 (early growth response1), Carf (calcium response factor), Apo-E (Apolipoprotein E), beta catenin (Catenin beta), lysozyme, Brevican , Matrix metalloproteinase 12, cellular repressor of EIA-stimulated gene, c-kit ligand, MPS1 protein, transglutaminase 2, serum and glucocorticoid regulated protein kinase, interferon induced protein, milk fat globule membrane protein EGF factor 8, Fc g receptor, S100 calcium binding protein A9, Arginase1, TNF1, retinoid inducible serine carboxypeptidase, Interleukin-1 8 binding protein d precursor, Chloride channel 7, CD36 antigen, Similar to putative zinc finger protein, Carbohydrate binding protein 35, C-type calcium dependent, carbohydrate, Lipoprotein lipase, v-maf musculoaponeurotic fibrosarcoma oncogene, Interleukin-7, Neuropilin, Peroxiredoxin 1la associated invariant chain Glycoprotein (transmembrane) nmbSerum amyloid A (SAA) 3 proteinSulfated glycoprotein (Sgp1) Response to metastatic cancers 1 (Rmcs1) ATP-binding cassette, sub-family AProgrammed cell death 1 ligand 1DnaJ (Hsp40) homolog (Dnajb11) Fittyectin acid binding protein 4 Complement component 1, q, gamma polypeptideFg g receptor, IgG, high affinity 1IFN regulatory factor, IgG, high affinity 1Complement component subunit Clq beta-chain2'-5'oligoadenylate synthetase-like 2Chitinase 3-like 3 (Chi3l3) IFN -activated gene 203Transforming growth factor, beta induced (Tgfbi) Ig super family, member 7 Chemokine (CXC motif) ligand 16 Colony stimulating factor 2 receptor, beta 2, (Csf2rb2) H2 -BfIFN-induced protein with tetratricopeptide repeats2RIKEN cDNA 2310005K03 geneRIKEN cDNA 3110037K17 geneRIKEN cDNA 5830458K16 gene Complement component 3a receptorSelenoprotein RIL-1 receptor antagonist (IL-1RA) Adenylosuccinate synthetase Hemopoietic cell kinaseDeath effector domain-containing DNA binding protein 2 (Dedd2) IFN induced transmembrane protein 2 likeGAVA-A receptor subunit 6Selectin, platelet (p-selectin) ligand (Selpl) High mobility group box 1 (Hmgb1) Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, epsilon polypeptide A4) precursor protein-binding, fam ily B, member 1 interacting protein Glutaredoxin 1 (thioltransferase) (Glrx1) Coronin, actin binding protein 1A (Coro1a) RIKEN cDNA 9630048M01 gene Karyopherin (importin) alpha 6Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1Kelch domain containing 2 (Klhdc2) Structure specific recognition protein 1Lymphocyte protein tyrosine kinaseF-box and leucine-rich repeat protein 14Osteoclastogenesis inhibitory factor171 protein171-coupled receptor phosphate cyclase domain 1Cullin 2CD160 antigenHypothetical RNA-binding domain, RBD structureSema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6DActivating signal cointegrator 1 complex subunit 3 like 1Abl-interactor 1Hypothetical protein MGC36662RAP2C, member of alosin protein (Vcp) CD164 antigenSimilar to RIKEN cDNA 4933425K02 g Zinc finger protein 259 Interleukin 12 receptor, beta 1 Growth arrest and DNA-damage-inducible 45 gamma Cytochrome b5 type BFMS-like tyrosine kinase 3 ligand Programmed cell death 1 ligand 2 Cell receptor beta, variable 13 (Tcrb-V13) Immune associated nucleotide 1 Interleukin 2 receptor, beta chain (Il2rb) Cd28: CD28 antigenGranzyme Eserin protease inhibitorPurine rich element binding protein A (Pura) Lymphocyte protein tyrosine kinase (Lck) Eukaryotic translation initiation factor 4 gamma, 3 (Eif4g3) Lamin A (Lmna) Raver1: Ribonucleoprotein, PTB-binding 1Solute carrier family 6V-ral simian leukemia viral oncogene homolog A (ras related) Fyn: Fyn proto-oncogeneExpressed sequence AV024533Fhl2: Four and a half LIM domains 2C1q and tumor necrosis factor related protein 6 (C1qtnf6) Zfp512: Zinc finger protein 512P2ry10: Purinergic receptor P2Y, G-prot ein coupled 10 Protein phosphatase 1, regulatory (inhibitor) subunit 14B (Ppp1r14b) Strap: Serine / threonine kinase receptor associated proteinHdac3: Histone deacetylase 3Ppp3cb: Protein phosphatase 3, catalytic subunit, beta isoformMmp9: Matrix metallopeptidase 9Ifnar2 and betata (alpha) 2 Prefoldin 1GTP-binding protein (mSara) homologue mRNA Copine 1 (Rbm 12) Pretin phosphatase 3, catalytic subunit, gamma isoform (Ppp3cc) Cyclin B2P5P1kb: : Treated with one or two or more compounds selected from the group consisting of Protein phosphatase 6 and catalytic subunit to differentiate into mature natural killer cells to obtain mature natural killer cells, and (iii) to differentiate the mature mature killer cells into interleukin-2 Method for producing an activated mature natural killer cells, characterized in that the activation by treatment with.

제5항에 따라 제조된 활성화된 성숙자연살해세포를 포함함을 특징으로 하는 면역세포치료제.Immune cell therapy comprising the activated mature natural killer cells prepared according to claim 5.

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