KR20220040092A - Exosomes Derived From Canine For Inhibiting Proliferation of Canine Solid Cancer - Google Patents
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Abstract
Description
본 발명은 개과동물로부터 유래된 엑소좀을 포함하는 개과 동물의 암 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating cancer in canines comprising exosomes derived from canines.
현재 성체줄기세포 치료법은 불치/난치 질환을 치료하기 위한 새로운 방법으로 다양하게 임상 적용되고 있으며, 성공적인 치료 사례들이 다수 보고되고 있다. 그러나, 공여자로부터 추출, 선별된 줄기세포를 증식시키기 위해 체외 배양하는 과정 중 발생할 수 있는 이종 혈청 (xenogenic serum; 예를 들어, fetal bovine/calf serum)의 internalization에 의한 인수전염(zoonosis)의 문제, 줄기세포가 체내 이식되었을 때 왕성한 증식력과 상대적으로 큰 세포 사이즈 등의 줄기세포 특성으로 인해 발생할 수 있는 종양 형성(tumor formation) 문제, 혈관 폐쇄 유발 경색 (vascular occlusion causing infarcts) 등의 위험 요소가 존재한다.Currently, adult stem cell therapy is being applied clinically as a new method to treat incurable/incurable diseases, and many successful treatment cases have been reported. However, the problem of zoonosis due to internalization of xenogenic serum (eg, fetal bovine/calf serum) that may occur during in vitro culturing to proliferate the stem cells extracted and selected from the donor, When stem cells are transplanted into the body, there are risk factors such as tumor formation problems and vascular occlusion causing infarcts that can occur due to stem cell characteristics such as strong proliferation and relatively large cell size. .
또한, 자가유래 줄기세포를 이용한 치료법 적용 시, 줄기세포의 체외 증식 능력, 병변으로의 이동 능력, 치료인자 분비 능력 등의 줄기세포의 재생 치료 효능이 환자마다 차이가 있어, 줄기세포 치료법의 범용성에 한계가 있다.In addition, when autologous stem cell therapy is applied, stem cell regeneration treatment efficacy such as in vitro proliferation ability of stem cells, ability to migrate to lesions, and ability to secrete therapeutic factors differs from patient to patient. There are limits.
따라서, 앞서 언급한 살아있는 줄기세포를 직접 이용한 치료에서 발생할 수 있는 다양한 위험 요소 혹은 문제들을 회피하는 방안이 활발히 연구되고 있으며, 그 일환으로 특정세포로부터 유래된 세포외 소포체가(Extracellular Vesicle, EV) 줄기세포 치료 기능을 대신할 수 있다는 연구 결과들이 나오고 있다. Therefore, methods for avoiding various risk factors or problems that may occur in the aforementioned treatment using live stem cells are actively being studied. Research results are coming out that it can replace the function of cell therapy.
EV는 세포에서 분비되는 이중 지질막 구조의 물질로 크기에 따라 엑소좀(exosomes)(40-200 nm), 마이크로베시클(microvesicles)(200-1,000 nm), 라지 온코좀(large oncosome)(1-10 μm)으로 나뉘며, 이 중 엑소좀(exosomes)은 세포에서 분비되는 100 나노미터 전후 크기의 작은 소포체로서, 세포의 성질과 상태를 대변하는 역할을 한다.EV is a double lipid membrane-structured material secreted from cells, depending on the size of exosomes (40-200 nm), microvesicles (200-1,000 nm), and large oncosomes (1- 10 μm), and among them, exosomes are small vesicles with a size of about 100 nanometers secreted from cells, and play a role in representing the properties and status of cells.
즉, 엑소좀(exosome)은 세포 내에서 자연적으로 만들어지는 나노 크기의 소포체로서, 단백질 및 유전적 정보를 포함하고 있어 세포 밖으로 유전정보를 포함한 다양한 신호를 다른 세포에 전달하여 발달과정, 증식, 분화, 면역조절, 혈관생성, 다양한 질병의 진행 등에 관여하고 있다. 생체 나노소포체(nanovesicle)인 엑소좀의 경우 면역반응을 피할 수 있고, 인체 적합성이 뛰어나며, 약물탑재 기능, 특정세포로의 표적전달 효과, 혈액 내 안정성 등의 장점을 통해 최근 약물 전달체로도 큰 관심을 받고 있다. That is, exosomes are nano-sized endoplasmic reticulum that are naturally created within cells, and contain proteins and genetic information. , immune regulation, angiogenesis, and the progression of various diseases. In the case of exosomes, which are biological nanovesicles, it is of great interest as a drug delivery agent in recent years due to its advantages such as avoidance of immune response, excellent human compatibility, drug loading function, target delivery effect to specific cells, and blood stability. are receiving
또한, 엑소좀은 원(origin) 세포의 특성에 따라 엑소좀이 포장하는 내용물(protein, mRNA, miRNA)이 달라지며, 어떤 내용물을 가지고 있느냐에 따라 타겟 세포로 이동 후, 타겟 세포의 운명이 달라진다. 실제로 여러 연구 그룹에서 엑소좀의 표면 단백질이나 내부의 단백질, RNAs가 타겟 세포로 이동하여 재프로그래밍화한다는 보고들이 있다. In addition, the contents (protein, mRNA, miRNA) packaged by the exosome vary according to the characteristics of the original cell, and the fate of the target cell after moving to the target cell depends on what contents it has. . In fact, there are reports from several research groups that exosome surface proteins or internal proteins and RNAs move to target cells and reprogram them.
이러한 엑소좀이 어떤 분자적 기작에 의해 만들어지는지 확실히 밝혀진 바가 없으나, 적혈구 세포뿐만 아니라, B-림프구, T-림프구, 수지상 세포, 혈소판, 대식 세포 등을 포함한 다양한 종류의 면역 세포들과 종양 세포, 줄기세포 등도 살아 있는 상태에서 엑소좀을 생산하여 분비한다고 알려져 있다. 특히, 줄기세포에서 유래된 엑소좀은 수용체 및 단백질뿐 아니라 핵 성분을 함유하고 있어 세포 간 커뮤니케이션 역할을 하는 것으로 알려져 있다.Although it is not clear by what molecular mechanism these exosomes are made, various types of immune cells including red blood cells, B-lymphocytes, T-lymphocytes, dendritic cells, platelets, macrophages, and tumor cells, It is known that stem cells also produce and secrete exosomes in a living state. In particular, stem cell-derived exosomes contain not only receptors and proteins, but also nuclear components, and are known to play a role in intercellular communication.
또한, 줄기세포에서 유래된 엑소좀은 줄기세포에 비해 동물 혈청을 상대적으로 적게 함유하고 있어 동물 혈청 감염에 의한 증상(zoonosis)의 위험성 역시 배재할 수 있다.In addition, since the stem cell-derived exosomes contain relatively less animal serum compared to stem cells, the risk of zoonosis caused by animal serum infection can also be excluded.
이에, 살아있는 줄기세포의 직접 주입/이식 치료법을 대체하는, 특정 세포, 예컨대, 줄기세포로부터 유래된 세포외 소포체를 이용한 치료법이 대두되어 많은 전임상 시험에서 효능을 보이고 있으며, 몇몇 질병치료에 있어 임상 단계에 진입한 사례도 보고되고 있다.Accordingly, a treatment using a specific cell, for example, an extracellular vesicle derived from a stem cell, has emerged as an alternative to the direct injection/transplantation therapy of living stem cells, and has shown efficacy in many preclinical trials, and is a clinical step in the treatment of several diseases. Cases of entry into
그러나, 이러한 엑소좀을 이용한 연구들은, 동물 종양 연구에는 적용된 바가 없다.However, studies using these exosomes have not been applied to animal tumor studies.
동물모델은 손상된 또는 질환을 가진 조직의 치유 및 기능회복을 위한 재생의학에 있어 그 중요성이 대두되고 있다. 이중에서도 개과동물은 현재 370 여종 이상의 유전질환이 보고되어 있고 대다수가 인간에서 발생하는 질환 및 기능장애와 닮아있다. 따라서, 인간의 유전질환의 기전 및 병인 연구 특히 인간에게서 직접 연구가 어려운 X-연관 중증 복합 면역결핍 및 듀시엔형 근이영양증과 같은 복합 유전성 희귀 열성 질환 연구를 위한 동물모델로서 개과동물의 중요성이 증가하고 있다. 이와 같이 개과 동물은 고형장기 이식뿐 아니라 암, 심혈관질환, 골재생, 당뇨병, 백혈병, 척수손상 등의 인간 병리학 기전 연구와 새로운 치료법을 시험하기 위한 유용한 동물모델이다. 더불어 줄기세포 이식 및 유전자 치료와 같은 다양한 치료방법을 연구하기 위한 이상적인 대형동물모델이다.Animal models are gaining importance in regenerative medicine for the healing and functional recovery of damaged or diseased tissues. Among these, more than 370 genetic diseases have been reported in canine animals, and most of them resemble diseases and dysfunctions that occur in humans. Therefore, the importance of canine animals as an animal model for the study of mechanisms and pathogenesis of genetic diseases in humans is increasing, especially for complex genetic rare recessive diseases such as X-linked severe combined immunodeficiency and Duchenne muscular dystrophy, which are difficult to study directly in humans. there is. As described above, canine animals are useful animal models to study human pathological mechanisms such as cancer, cardiovascular disease, bone regeneration, diabetes, leukemia, and spinal cord injury as well as solid organ transplantation and to test new treatments. In addition, it is an ideal large animal model to study various therapeutic methods such as stem cell transplantation and gene therapy.
아울러 개에서 암과 같은 난치성 질환의 발생이 현저히 증가하고 있어 이들 난치성 질환에 대한 새로운 치료제 개발 역시 매우 필요하다.In addition, since the incidence of intractable diseases such as cancer in dogs is significantly increasing, the development of new therapeutic agents for these intractable diseases is also very necessary.
이와 같은 생물학적 유사성 때문에 개에서 암에 관한 연구는 사람 또는 실험동물만을 대상으로 한 암 연구와 차별화되는 귀중한 정보를 제공하며, 이와 같은 가치에 대한 인식은 암-관련 유전자 확인 연구를 비롯하여 환경성 위험요소, 암의 생물학적 특성 이해, 그리고 특히 새로운 암치료법 개발 및 효능 평가 등의 연구 분야에서 현저히 높아지고 있다. Because of these biological similarities, research on cancer in dogs provides valuable information that is different from cancer research on humans or experimental animals only. The research field, such as understanding the biological properties of cancer, and in particular, the development of new cancer treatment methods and efficacy evaluation, is increasing significantly.
그러나, 아직까지 개에서 세계적으로 이를 이용한 암치료에 관련된 보고가 없다.However, there is no report related to cancer treatment using it worldwide in dogs.
따라서, 개의 여러 조직에서 특정 세포를 분리하고 특성화하는 것이 세포 연구분야의 주요과제이다. 인간과 쥐 유래의 줄기세포는 여러 방면으로 종래 기술에서 시도되어 왔으나, 개과동물의 경우, 위와 같이 인간 질환 연구를 위한 유용한 대형동물모델임에도 불구하고 아직은 그 연구가 미진한 상황이다. 지방조직, 골수, 제대혈 등으로부터 줄기세포의 분리가 가능하나 그 수득량에 제한이 있고, 지방조직 또는 골수를 개체로부터 획득하는 방법이 침습적이며, 고통을 유발한다는 점에서 한계가 있다.Therefore, the isolation and characterization of specific cells from various tissues of dogs is a major task in the field of cell research. Human and mouse-derived stem cells have been tried in the prior art in various ways, but in the case of canines, despite being a useful large animal model for human disease research, the research is still incomplete. Although it is possible to isolate stem cells from adipose tissue, bone marrow, umbilical cord blood, etc., the yield is limited, and a method for obtaining adipose tissue or bone marrow from an individual is invasive and has limitations in that it causes pain.
한편, 성체 줄기세포 중 하나인 조혈줄기세포(hematopoietic stem cell)는 혈액을 구성하는 모든 세포(적혈구, 백혈구, 혈소판 및 림프구)로 분화할 수 있는 세포로서, 주로 골수에 있는 조혈줄기세포로부터 생체의 면역체계를 구성하는 세포가 지속적으로 자가 재생된다. 이들 면역체계를 구성하는 세포들 중 특히, 자연살해세포(natural killer cell, 이하 'NK 세포')는 비특이적으로 암세포 및 바이러스 감염세포를 살상할 수 있는 능력이 밝혀지면서 많은 연구가 진행되었으며, NK 세포의 분화와 활성의 결함은 유방암(Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), 흑색종암(Ryuke Y, et al., Melanoma Res., 13: 349-356, 2003), 폐암(Villegas FR, et al., Lung Cancer, 35: 23-28, 2002) 등 다양한 질병들과 관련되어 있음이 밝혀졌다. 이러한 연구들을 바탕으로 암과 같은 난치성질환 치료에 NK 세포를 이용하는 NK 세포 치료법이 대두되고 있다. On the other hand, hematopoietic stem cells, one of the adult stem cells, are cells capable of differentiating into all cells (red blood cells, white blood cells, platelets and lymphocytes) constituting the blood, and are mainly derived from hematopoietic stem cells in the bone marrow. Cells that make up the immune system continuously self-renew. Among the cells constituting these immune systems, natural killer cells (hereinafter 'NK cells') have been found to have the ability to non-specifically kill cancer cells and virus-infected cells, and many studies have been conducted. Defects in differentiation and activity of breast cancer (Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), melanoma cancer (Ryuke Y, et al., Melanoma Res., 13: 349- 356, 2003), lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002) has been found to be associated with various diseases. Based on these studies, NK cell therapy using NK cells to treat intractable diseases such as cancer is emerging.
자연살해세포(natural killer cell, NK cell)는 선천면역세포 중의 하나로 암세포에 대해 선택적인 세포독성을 보이는 세포이다. 자연살해세포는 다른 면역세포와 달리 암세포를 즉각적으로 감지하여 바로 제거할 수 있는데 이것은 자연살해세포 표면에 존재하는 다양한 면역수용체(immune receptor)를 통해 암세포와 정상세포를 구분할 수 있기 때문이다. 많은 암환자의 경우 자연살해세포의 수나 항암활성에 결함이 발견되고 있으며 자연살해세포의 기능 이상은 이러한 암의 발생과 밀접한 관련을 가지고 있다고 알려져 있다.Natural killer cells (NK cells) are cells that show selective cytotoxicity against cancer cells as one of the innate immune cells. Unlike other immune cells, natural killer cells can instantly detect and remove cancer cells because they can differentiate cancer cells from normal cells through various immune receptors on the surface of natural killer cells. In many cancer patients, defects are found in the number or anticancer activity of natural killer cells, and it is known that abnormalities in the function of natural killer cells are closely related to the occurrence of these cancers.
자연살해세포가 세포를 살해하는 과정은 퍼포린(perforin) 및 그랜자임(granzyme)을 포함하는 세포질 과립의 배출과 FasL 및 TRAIL을 포함하는 과정과 관련되어 있다. 자연살해세포는 IFN-γ, INF-α, GM-CSF 및 IL-10과 같은 다양한 사이토카인을 분비하고, 세포 표면에 여러 수용체를 발현하는데 이들 수용체는 세포 흡착, 세포 살해 능력의 활성화, 또는 세포 살해능력의 억제에 관여한다.The process in which natural killer cells kill cells is related to the release of cytoplasmic granules containing perforin and granzyme and processes containing FasL and TRAIL. Natural killer cells secrete various cytokines, such as IFN-γ, INF-α, GM-CSF, and IL-10, and express several receptors on the cell surface. It is involved in suppressing the killing ability.
따라서, 동물에 적용된 바 없는 "엑소좀 이용 종양 억제"는 동물 질병 분야에서 향후 차세대 세포치료제로의 발전 가능성과 다양한 질병 관련 매개 연구에 중요한 기초 정보를 제공할 수 있으며, 이러한 엑소좀의 특성을 고려할 때 성체줄기세포로부터 분리한 엑소좀을 이용한 세포치료법은 안전하면서도 효과적으로 암을 치료할 수 있을 뿐만 아니라, 기존의 줄기세포 치료법의 한계를 극복할 수 있는 새로운 패러다임이 될 것으로 기대된다.Therefore, "tumor suppression using exosomes", which has not been applied to animals, can provide important basic information for the potential for future development of next-generation cell therapies in the field of animal diseases and various disease-related mediation studies. Cell therapy using exosomes isolated from adult stem cells is expected to become a new paradigm that can not only safely and effectively treat cancer, but also overcome the limitations of existing stem cell therapies.
이러한 상황하에서, 본 발명자들은 개과 동물(특히, 개)의 암을 치료하기 위한 효과적이고 안전한 치료법을 예의 연구 노력하였다. 그 결과, 본 발명자들은 개의 성체줄기세포인 지방조직-유래 중간엽줄기세포(canine adipose tissue-derived mesenchymal stem cells)로부터 세포외소포체(extracellular vesicles)인 엑소좀(exosomes)을 분리하고; 개의 말초혈액단핵세포(peripheral blood mononuclear cells)-유래 자연살해세포(natural killer cells, NK cells)로부터 세포외소포체(extracellular vesicles)인 엑소좀(exosomes)을 각각 분리하여 그 특성을 분석하였고, 상기 개 MSC-유래 엑소좀 및 NK 세포-유래 엑소좀을 유선종양 모델에 적용했을 때, 몸무게 감소없이 유선종양에 대한 항종양 효과가 나타나는 것을 규명함으로써, 안전하고 효과적인 암 치료제로서의 가능성을 제공하여 본 발명을 완성하였다.Under these circumstances, the present inventors intensively researched and endeavored to find an effective and safe treatment method for treating cancer in canines (especially in dogs). As a result, the present inventors separated the exosomes, which are extracellular vesicles, from canine adipose tissue-derived mesenchymal stem cells, which are canine adult stem cells; Exosomes, which are extracellular vesicles, from canine peripheral blood mononuclear cells-derived natural killer cells (NK cells), respectively, were isolated and their characteristics were analyzed, and the characteristics were analyzed. When MSC-derived exosomes and NK cell-derived exosomes were applied to a mammary gland tumor model, the present invention was developed by providing the potential as a safe and effective cancer treatment agent by identifying the antitumor effect on mammary gland tumors without weight loss. completed.
따라서, 본 발명의 일 목적은 개과 동물(canine)로부터 유래된 엑소좀(exosome)을 유효성분으로 포함하는 개과 동물의 암 예방 또는 치료용 약학적 조성물을 제공하는 데 있다. Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating canine cancer, which includes an exosome derived from canine as an active ingredient.
또한, 본 발명의 다른 목적은 개과 동물(canine)로부터 유래된 엑소좀(exosome)을 유효성분으로 포함하는 개과 동물의 암 예방 또는 개선용 사료 조성물을 제공하는 것이다.In addition, another object of the present invention is to provide a feed composition for preventing or improving cancer in canines, which includes exosomes derived from canine animals as an active ingredient.
또한, 본 발명의 또 다른 목적은 개과 동물(canine)로부터 유래된 엑소좀(exosome)을 유효성분으로 포함하는 조성물을 개과 동물에게 투여하는 단계를 포함하는, 개과 동물의 암 예방 또는 치료 방법을 제공하는 데 있다.In addition, another object of the present invention is to provide a method for preventing or treating canine cancer, comprising administering to the canine a composition comprising an exosome derived from canine as an active ingredient to the canine animal is to do
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 명세서에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terminology used herein is used for the purpose of description only, and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, component, part, or a combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not
이하, 본 발명에 대하여 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태에 따르면, 본 발명은 개과 동물(canine)로부터 유래된 엑소좀(exosome)을 유효성분으로 포함하는 개과 동물의 암 예방 또는 치료용 약학적 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating canine cancer, comprising an exosome derived from canine as an active ingredient.
본 발명의 주요한 특징은 개과 동물(canine)로부터 유래된 엑소좀(exosome)에 의해 암 크기가 억제되는 항 종양 효과가 발휘되어 암 질환에 대하여 치료 효과를 달성할 수 있게 된다는 것이다.A major feature of the present invention is that an anti-tumor effect of suppressing cancer size is exhibited by exosomes derived from canine animals, thereby achieving a therapeutic effect on cancer diseases.
본 발명의 용어, "엑소좀(exosome)"은 다양한 세포들로부터 분비되는 막 구조의 작은 소낭을 의미하며, 다낭체와 원형질막의 융합이 일어나 세포 밖 환경으로 방출되는 소낭을 의미한다. 엑소좀의 지름은 40 내지 200 nm의 지름을 가진 것일 수 있으나, 본 발명의 엑소좀이 목적 효과를 발휘하는 한, 이에 제한되는 것은 아니다.As used herein, the term “exosome” refers to a small vesicle with a membrane structure secreted from various cells, and refers to a vesicle that is released into the extracellular environment after fusion of the polycystic body and the plasma membrane. The diameter of the exosome may be one having a diameter of 40 to 200 nm, but as long as the exosome of the present invention exerts the desired effect, it is not limited thereto.
본 발명에서 엑소좀을 공여하는 세포는 진핵세포라면 제한되지 않는다.Cells donating exosomes in the present invention are not limited as long as they are eukaryotic cells.
본 발명에 있어서, 엑소좀을 공여하는 세포는 진핵세포라면 제한되지 않으며, 상기 엑소좀은 특정 세포를 배양함으로써 상기 세포로부터 자연적으로 분비되어 분리, 추출 및/또는 농축된 것일 수 있으며, 상기 특정 세포는 성체줄기세포(Adult Stem Cell) 또는 자연살해세포(natural killer cells, NK 세포)를 포함할 수 있다.In the present invention, the cell donating the exosome is not limited as long as it is a eukaryotic cell, and the exosome may be naturally secreted from the cell by culturing a specific cell and separated, extracted and/or concentrated, and the specific cell may include adult stem cells (Adult Stem Cell) or natural killer cells (NK cells).
본 발명의 용어, "줄기세포"는 미분화된 세포로서 자기 복제 능력을 가지면서 두 개 이상의 서로 다른 종류의 세포로 분화하는 능력을 갖는 세포를 말한다. 본 발명의 줄기세포는 자가 또는 동종 유래 줄기세포일 수 있으며, 인간 및 비인간 포유류를 포함한 임의 유형의 동물 유래일 수 있고, 상기 줄기세포가 성체로부터 유래된 것이든 배아로부터 유래된 것이든 이에 한정되지 않는다. As used herein, the term "stem cell" refers to a cell having the ability to differentiate into two or more different types of cells while having the ability to self-replicate as an undifferentiated cell. The stem cells of the present invention may be autologous or allogeneic stem cells, and may be derived from any type of animal including humans and non-human mammals, and whether the stem cells are adult or embryonic, but not limited thereto. does not
본 발명의 줄기세포는 배아 줄기세포 또는 성체 줄기세포를 포함할 수 있으며, 바람직하게는 성체 줄기세포이다.The stem cells of the present invention may include embryonic stem cells or adult stem cells, preferably adult stem cells.
상기 성체 줄기세포는, 개과 동물 조직 유래 중간엽 줄기세포(Mesenchymal Stem Cells, MSC), 개과 동물 조직 유래 조혈줄기세포(Hematopoietic Stem Cells, HSC), 개과 동물 조직 유래 중간엽 기질세포(mesenchymal stromal cell), 다분화능 줄기세포 또는 양막상피세포일 수 있으며, 바람직하게는 개과 동물 조직 유래 중간엽 줄기세포 또는 개과 동물 조직 유래 조혈줄기세포이나, 이에 한정되지 않는다. The adult stem cells, canine tissue-derived mesenchymal stem cells (Mesenchymal Stem Cells, MSC), canine tissue-derived hematopoietic stem cells (Hematopoietic Stem Cells, HSC), canine tissue-derived mesenchymal stromal cells (mesenchymal stromal cell) , may be pluripotent stem cells or amniotic epithelial cells, preferably canine tissue-derived mesenchymal stem cells or canine tissue-derived hematopoietic stem cells, but is not limited thereto.
상기 중간엽 줄기세포는 개과 동물의 지방조직, 혈액, 뇌, 제대, 제대혈, 골수, 근육, 신경, 피부, 양막 및 태반으로 이루어진 군에서 선택된 1 종 이상으로부터 유래된 중간엽 줄기세포일 수 있으나, 이에 한정되지 않는다.The mesenchymal stem cells may be mesenchymal stem cells derived from one or more selected from the group consisting of canine adipose tissue, blood, brain, umbilical cord, umbilical cord blood, bone marrow, muscle, nerve, skin, amniotic membrane and placenta, However, the present invention is not limited thereto.
본 발명에서, 바람직하게는 상기 성체줄기세포는 개과 동물의 지방조직에서 분리된 중간엽줄기세포(Mesenchymal Stem Cells, MSC)인 지방조직-유래 중간엽줄기세포(canine adipose tissue-derived mesenchymal stem cells)이다.In the present invention, preferably, the adult stem cells are mesenchymal stem cells (MSC) isolated from canine adipose tissue-derived mesenchymal stem cells (canine adipose tissue-derived mesenchymal stem cells) am.
본 발명에서, 상기 NK 세포는 개과 동물의 말초혈액, 골수, 림프절, 비장, 폐 및 간장으로 이루어진 군에서 선택된 1 종 이상으로부터 분리될 수 있고, 바람직하게는, 말초혈액으로부터 분리될 수 있다.In the present invention, the NK cells may be isolated from one or more selected from the group consisting of canine peripheral blood, bone marrow, lymph node, spleen, lung and liver, and preferably, may be isolated from peripheral blood.
본 발명의 일 실시예에 따르면, 본 발명의 상기 NK 세포는 개과 동물의 말초혈액에서 분리된 말초혈액 단핵세포(peripheral blood mononuclear cells, PBMC)로부터 유래된다.According to one embodiment of the present invention, the NK cells of the present invention are derived from peripheral blood mononuclear cells (PBMC) isolated from canine peripheral blood.
본 발명에 있어서 '말초혈액 단핵세포(PBMC, Peripheral Blood Mononuclear Cell)'란, 말초혈액내에 존재하는 단핵의 세포로서, B cell, T cell, Macrophage, Dendritic cell, NK cell(natural killer cell) 등의 면역세포, 호염기구(basophil), 호산구(eosinophil), 호중구(neutrophil)와 같은 과립구세포(granulocytes) 등을 포함하고 있다. 상기 PBMC는 통상의 제조방법, 예를 들어 Ficoll-Paque을 이용한 비중 원침법으로 분리할 수 있다(Blood, 1998, 92: 2989-93 등).In the present invention, 'Peripheral Blood Mononuclear Cell (PBMC)' is a mononuclear cell present in peripheral blood, such as B cell, T cell, Macrophage, Dendritic cell, NK cell (natural killer cell), etc. It contains immune cells, basophils, eosinophils, and granulocytes such as neutrophils. The PBMC can be separated by a conventional manufacturing method, for example, specific gravity centrifugation using Ficoll-Paque (Blood, 1998, 92: 2989-93, etc.).
본 명세서에서 사용되는 "개과 동물(canine)"은 개, 늑대, 여우, 코요테, 자칼, 승냥이 등의 잡식성 동물로서, 발가락으로 걷는 지행동물을 의미한다. As used herein, the term "canine" is an omnivorous animal such as a dog, a wolf, a fox, a coyote, a jackal, and a feral cat, and refers to a walking animal that walks on its toes.
상기 개과동물은 크게 개족과 여우족으로 나뉜다. 개족은 개, 갈기늑대, 가로무늬자칼, 검은 등자칼, 딩고, 붉은늑대, 에티오피아늑대, 인도늑대, 코요태, 황금자칼, 게잡이여우속, 들개속, 승냥이, 아프리카들개, 작은귀개, 포클랜드늑대, 안데스여우, 다윈여우, 아르헨티나회색여우, 팜파스여우, 페루사막여우, 흰여우 등을 포함한다.The canines are largely divided into dogs and foxes. Dogs, maned wolf, striped jackal, black stirrup jackal, dingo, red wolf, Ethiopian wolf, Indian wolf, coyote, golden jackal, crab-eating vulpes, wild canis, feral cat, African wild dog, small-eared dog, Falkland wolf, Andean fox, Darwin fox, Argentinian gray fox, pampas fox, Peruvian desert fox, white fox, etc.
여우족은 북극여우, 붉은여우, 스위프트여우, 키트여우, 코사크여우, 케이프여우, 검은꼬리모래여우, 벵골여우, 티베트모래여우, 블랜포드여우, 흰꼬리모래여우, 페넥여우, 회색여우, 섬여우, 코즈멜여우, 큰귀여우 및 너구리 등을 포함한다.Foxes include arctic fox, red fox, swift fox, kit fox, cossack fox, cape fox, black-tailed sand fox, Bengal fox, Tibetan sand fox, Blanford fox, white-tailed sand fox, fennec fox, gray fox, island fox , cozumel fox, big-eared fox, and raccoon dog.
본 발명의 엑소좀이 효과를 발휘하는 한, 상기 개과 동물의 대상은 이에 제한되지 않으나, 본 발명에서 개과 동물은, 바람직하게는, 개를 포함하며, 예를 들어, 순종 및/또는 잡종 반려견(companion dog), 쇼견(show dog), 사역견(working dog), 목양견(herding dog), 수렵견(hunting dog), 호위견(guard dog), 경찰견(police dog), 경주견(racing dog) 및/또는 실험견(laboratory dog)과 같은 개들, 특히 사육견도 포함한다.As long as the exosome of the present invention exerts an effect, the subject of the canine animal is not limited thereto, but in the present invention, the canine animal, preferably, includes a dog, for example, purebred and/or hybrid dog ( companion dog, show dog, working dog, herding dog, hunting dog, guard dog, police dog, racing dog and/or Also includes dogs such as laboratory dogs, especially domestic dogs.
본 발명의 바람직한 구현예에 따르면, 본 발명의 엑소좀은, (i) 개 지방조직에서 유래된 중간엽줄기세포(Mesenchymal Stem Cells, MSC)로부터 분리된 엑소좀(exosome)(MSC-EXO); 또는 According to a preferred embodiment of the present invention, the exosomes of the present invention include (i) exosomes isolated from mesenchymal stem cells (MSC) derived from canine adipose tissue (MSC-EXO); or
(ii) 개 말초혈액 단핵세포(peripheral blood mononuclear cells, PBMCs) 에서 유래된 자연살해세포(natural killer cells, NK 세포)로부터 분리된 엑소좀(exosome)(NK-EXO)이다.(ii) exosomes (NK-EXO) isolated from natural killer cells (NK cells) derived from canine peripheral blood mononuclear cells (PBMCs).
본 발명에 따르면, 상기 암은 유방암, 편평상피세포암, 자궁암, 자궁경부암, 전립선암, 두경부암, 췌장암, 뇌종양, 간암, 피부암, 식도암, 고환암, 신장암, 대장암, 직장암, 위암, 방광암, 난소암, 담관암 및 담낭암으로 이루어지는 군으로부터 선택되는 1종 이상이고, 바람직하게는 유방암이다.According to the present invention, the cancer is breast cancer, squamous cell cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, It is at least one selected from the group consisting of ovarian cancer, bile duct cancer, and gallbladder cancer, preferably breast cancer.
본 발명의 일 실시예에 있어서, 본 발명의 엑소좀은 암의 크기는 감소시키면서, 대상 객체의 정상 체중은 유지되도록 하므로, 이는 본 발명의 엑소좀이 암세포만을 타겟으로 하여 다른 정상세포에는 영향을 미치지 않는, 안전하고 탁월한 항암 효과를 발휘한다는 것을 제시한다.In one embodiment of the present invention, the exosome of the present invention reduces the size of cancer while maintaining the normal body weight of the target object, so that the exosome of the present invention targets only cancer cells and affects other normal cells It is suggested that it is safe and exhibits excellent anticancer effect.
또한, 본 발명에 따른 약학적 조성물은 약학적으로 허용 가능한 담체를 추가적으로 포함할 수 있다. 상기 약학적으로 허용 가능한 담체는 제제시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 사이클로덱스트린, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 등을 포함하지만 이에 한정되지 않으며, 필요에 따라 항산화제, 완충액 등 다른 통상의 첨가제를 더 포함할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립, 또는 정제로 제제화할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제화에 관해서는 각 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학적 조성물은 제형에 특별한 제한은 없으나 주사제, 흡입제, 피부 외용제, 또는 경구 섭취제 등으로 제제화할 수 있다.In addition, the pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives, such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets. With respect to a suitable pharmaceutically acceptable carrier and formulation, it may be preferably formulated according to each component. The pharmaceutical composition of the present invention is not particularly limited in the formulation, but may be formulated as an injection, inhalant, external preparation for skin, or oral ingestion.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 피부, 비강, 기도에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, dermally, nasally, or applied to the respiratory tract) according to a desired method, and the dosage may vary depending on the patient's condition and body weight, disease Although it varies depending on the degree, drug form, administration route, and time of administration, it may be appropriately selected by those skilled in the art.
본 발명에 따른 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art.
구체적으로, 본 발명에 따른 조성물의 유효량은 대상 개과 동물의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.001 내지 150 mg, 바람직하게는 0.01 내지 100 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the subject canine animal, and generally 0.001 to 150 mg per kg body weight, preferably 0.01 to 100 mg per kg body weight, is administered daily or every other day Or it can be administered in divided
본 발명의 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체를 더 포함할 수 있다. 예를 들어, 주사제의 경우에는 보존제, 무통화제, 가용화제 또는 안정화제 등을, 국소 투여용 제제의 경우에는 기제(base), 부형제, 윤활제 또는 보존제 등을 추가로 포함할 수 있다.The composition of the present invention may further include an appropriate carrier commonly used in the preparation of pharmaceutical compositions. For example, in the case of an injection, a preservative, an analgesic agent, a solubilizer or a stabilizer, etc., and in the case of a formulation for topical administration, a base, an excipient, a lubricant, or a preservative may be further included.
본 발명의 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있으며, 바람직하게는 치료가 필요한 개체의 직접 생착 또는 이식하는 것이 가능하나 이에 한정되지는 않는다.The composition of the present invention may be administered to an individual by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine or intracerebrovascular injection, preferably by direct engraftment or intracerebrovascularization of the subject in need of treatment. Transplantation is possible, but not limited thereto.
본 발명의 조성물은 암 치료를 위하여 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for cancer treatment.
또한, 본 발명의 다른 양태에 따르면, 본 발명은 개과 동물(canine)로부터 유래된 엑소좀(exosome)을 유효성분으로 포함하는 개과 동물의 암 예방 또는 개선용 사료 조성물을 제공한다.In addition, according to another aspect of the present invention, the present invention provides a feed composition for preventing or improving canine cancer, which includes an exosome derived from canine as an active ingredient.
또한, 상기 사료 조성물은 사료에 추가적인 첨가제로 이용할 수 있다.In addition, the feed composition may be used as an additional additive to the feed.
본 발명에서 사용되는 용어, "개선"이란, 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated, for example, the severity of symptoms.
본 발명의 사료 조성물은 유효성분을 사료에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 조성물은 원료에 대하여 15 중량%이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있다.The feed composition of the present invention may be used as it is by adding the active ingredient to the feed or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the intended use thereof. In general, in the production of food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be less than or equal to the above range.
본 발명의 사료 조성물은 지시된 비율로 필수 성분으로서 상기 유효성분을 함유하는 것 외에 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 수크로오스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.The feed composition of the present invention is not particularly limited in other ingredients other than containing the active ingredient as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the selection of a person skilled in the art.
상기 외에 본 발명의 사료 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다.In addition to the above, the feed composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.
또한, 본 발명의 또 다른 양태에 따르면, 개과 동물(canine)로부터 유래된 엑소좀(exosome)을 유효성분으로 포함하는 조성물을 개과 동물에게 투여하는 단계를 포함하는, 개과 동물의 암 예방 또는 치료 방법을 제공한다.In addition, according to another aspect of the present invention, a method for preventing or treating canine cancer, comprising administering to the canine a composition comprising an exosome derived from canine as an active ingredient to the canine animal provides
상술한 개과 동물(canine)로부터 유래된 엑소좀(exosome)을 유효성분으로 포함하는 조성물은, 개과 동물로부터 유래된 엑소좀(exosome)을 분리하여 제조한 개과 동물(canine)의 세포 치료제를 의미한다.The composition comprising the above-described canine-derived exosome as an active ingredient refers to a canine-derived cell therapeutic agent prepared by separating the canine-derived exosome. .
본 발명의 용어 "세포치료제"란, 대상으로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 의미한다.As used herein, the term "cell therapy agent" refers to cells and tissues manufactured through separation, culture, and special manipulation from a subject, and is a drug used for treatment, diagnosis, and prevention purposes (US FDA regulations), and the function of cells or tissues is It refers to a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological properties of cells in other ways to restore them.
바람직하게, 상기 세포치료제는 개과동물의 암 치료용인 세포 치료제일 수 있다.Preferably, the cell therapy agent may be a cell therapy agent for canine cancer treatment.
따라서, 본 발명의 상기 세포 치료제를 이를 필요로 하는 개체에게 투여하는 단계를 포함하는, 개과동물을 치료하는 방법을 제공한다.Accordingly, there is provided a method of treating a canine animal, comprising administering the cell therapeutic agent of the present invention to an individual in need thereof.
상기 본 발명의 용어 "치료"란 본 발명의 조성물의 투여로 개과동물의 질환 예컨대, 암 세포에 의해 유도되는 질환의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.The term "treatment" of the present invention refers to any action in which the symptoms of a disease induced by cancer cells are improved or beneficially changed in a canine animal by administration of the composition of the present invention.
본 발명의 방법은 본 발명의 엑소좀을 유효성분으로 포함하는 조성물을 이용하므로, 이와 중복된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the method of the present invention uses a composition comprising the exosome of the present invention as an active ingredient, the overlapping content will be omitted in order to avoid excessive complexity of the present specification.
본 발명에 따른 개과 동물(canine)로부터 유래된 엑소좀은 암세포를 표적화하여 암의 크기를 효과적으로 감소시킬 수 있으므로 개과 동물의 암 예방 또는 치료 용도로 유용하게 사용될 수 있다.The exosome derived from canine according to the present invention can effectively reduce the size of cancer by targeting cancer cells, and thus can be usefully used for the prevention or treatment of canine cancer.
도 1a는 본 발명의 개-조직 유래 엑소좀의 분리 과정을 개략적으로 나타낸다. 도 1b는 본 발명의 개 조직-유래 엑소좀의 전자현미경 및 나노사이트 분석 결과를 보여준다. 도 1c는 본 발명의 개 조직-유래 엑소좀의 나노입자추적시스템 분석 결과를 보여준다. 도 1d는 본 발명의 개 조직-유래 엑소좀에서 웨스턴 블롯을 통해 Flotillin-1, CD63, CD81, Alix, HSP70 및 TSG101와 같은 엑소좀 마커들의 발현을 확인한 결과를 보여준다.
도 2a는 유선종양세포주 REM134에 본 발명의 개 자연살해세포 유래 엑소좀(NK-EXO)를 처리했을 때 항 종양 효과를 이미지로 보여주며, 도 2b는 이를 그래프화한 것이다. 도 2c는 대조군인 유선종양세포주 REM134만을 처리한 결과를 그래프화한 것이다. 도 2d는 주간별로 마우스의 몸무게를 측정한 결과를 보여준다.
도 3a는 유선종양세포주 REM134에 본 발명의 개 성체줄기세포 유래 엑소좀(MSC-EXO)을 처리했을 때 항종양 효과를 이미지로 보여주며, 도 3b는 이를 그래프화한 것이다. 도 3c는 대조군인 유선종양세포주 REM134만을 처리한 결과를 그래프화한 것이다. 도 3d는 주간별로 마우스의 몸무게를 측정한 결과를 보여준다.Figure 1a schematically shows the isolation process of the dog-tissue-derived exosomes of the present invention. Figure 1b shows the results of electron microscopy and nanosite analysis of dog tissue-derived exosomes of the present invention. Figure 1c shows the analysis results of the nanoparticle tracking system of the dog tissue-derived exosomes of the present invention. Figure 1d shows the results of confirming the expression of exosome markers such as Flotillin-1, CD63, CD81, Alix, HSP70 and TSG101 through Western blot in the dog tissue-derived exosomes of the present invention.
Figure 2a shows the anti-tumor effect as an image when the mammary tumor cell line REM134 is treated with the canine natural killer cell-derived exosomes (NK-EXO) of the present invention, and Figure 2b is a graph showing this. Figure 2c is a graph showing the results of processing only the mammary tumor cell line REM134 as a control. Figure 2d shows the results of measuring the weight of the mouse for each week.
Figure 3a shows the antitumor effect as an image when the mammary tumor cell line REM134 is treated with the individual adult stem cell-derived exosomes (MSC-EXO) of the present invention, and Figure 3b is a graph showing this. 3c is a graph showing the results of treatment with only the mammary tumor cell line REM134 as a control. Figure 3d shows the results of measuring the weight of the mouse for each week.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.
실시예 1. 개 조직-유래 엑소좀 분리 및 특성 분석Example 1. Canine tissue-derived exosome isolation and characterization
1-1. 개 말초혈액 단핵세포(peripheral blood mononuclear cells, PBMCs) 에서 유래된 자연살해세포(natural killer cells, NK 세포)로부터 엑소좀(exosome)(NK-EXO)의 분리1-1. Isolation of exosomes (NK-EXO) from natural killer cells (NK cells) derived from canine peripheral blood mononuclear cells (PBMCs)
NK 세포는 다양한 면역 수용체를 표면에 발현한다. 면역 수용체는 활성화 수용체(activating receptor)와 억제 수용체(inhibitory receptor)로 나눠지는 데, 이들 수용체의 조절에 의해 NK 세포는 정상 세포와 암 세포를 구분한다. NK cells express various immune receptors on their surface. Immune receptors are divided into activating receptors and inhibitory receptors, and the regulation of these receptors allows NK cells to differentiate between normal cells and cancer cells.
이러한 NK 세포는 인간의 경우 말초혈액 림프구의 약 10~15%의 비율을 차지하나, 개는 훨씬 적은 비율인 약 5%를 차지한다. These NK cells make up about 10-15% of peripheral blood lymphocytes in humans, but make up a much smaller percentage, about 5%, in dogs.
이에, 본 발명자들은, 개의 암 치료에 적용하기 위하여, 먼저, 개 말초혈액 단핵세포(peripheral blood mononuclear cells, PBMCs)에서 자연살해세포(natural killer cells, NK 세포)를 분리 및 증식시킨 후, 상기 증폭된 개 말초혈액 단핵세포(peripheral blood mononuclear cells, PBMCs)에서 유래된 자연살해세포(natural killer cells, NK 세포)로부터 엑소좀(exosome)(NK-EXO)을 분리 및 농축하였다(도 1a).Accordingly, the present inventors, in order to apply to cancer treatment in dogs, first, natural killer cells (NK cells) from canine peripheral blood mononuclear cells (PBMCs) isolate and proliferate, and then the amplification Exosomes (NK-EXO) were isolated and concentrated from natural killer cells (NK cells) derived from canine peripheral blood mononuclear cells (PBMCs) ( FIG. 1A ).
간략하게는 다음과 같다:Briefly:
(1) 먼저, 개의 혈액 유래 말초혈액 단핵세포(peripheral blood mononuclear cells, PBMCs)로부터 NK 세포를 선택적으로 증식하고자, CD5 (MCA1037PE, Bio-Rad, USA) 항체를 30분간 염색한 다음, anti-PE microbeads (Miltenyi Biotec, USA)로 2차 염색하여 MACS system (Miltenyi Biotec)을 통해 CD5 음성세포만을 분리하였다. 상기 CD5 음성세포(PBMCs-유래 NK 세포) 4Х106개를 500 U/mL human IL-2, 10 ng/mL human IL-15 및 5 ng/mL canine IL-21이 첨가된 배양액에서 7일 간격으로 계대하여 총 21일간 37℃, 5% CO2 조건에서 배양함으로써 PBMCs-유래 NK 세포를 증식시켰다.(1) First, in order to selectively proliferate NK cells from canine blood-derived peripheral blood mononuclear cells (PBMCs), CD5 (MCA1037PE, Bio-Rad, USA) antibody was stained for 30 minutes, and then, anti-PE Only CD5-negative cells were isolated through the MACS system (Miltenyi Biotec) by secondary staining with microbeads (Miltenyi Biotec, USA). The above CD5-negative cells (PBMCs-derived NK cells) 4Х10 6 cells were cultured with 500 U/mL human IL-2, 10 ng/mL human IL-15, and 5 ng/mL canine IL-21 at intervals of 7 days. PBMCs-derived NK cells were proliferated by passage and culture at 37° C., 5% CO 2 conditions for a total of 21 days.
(2) 상기 증식된 PBMCs-유래 NK 세포를, 다음 날 1% P/S가 첨가된 DPBS로 세척한 다음, exosome-depleted FBS를 포함한 세포 배양액(20 mL)으로 교체하여 3일간 배양함으로써 PBMCs-유래 NK 세포로부터 엑소좀(exosome)(NK-EXO)을 분리하였다.(2) The proliferated PBMCs-derived NK cells were washed with DPBS supplemented with 1% P/S the next day, replaced with a cell culture medium (20 mL) containing exosome-depleted FBS, and cultured for 3 days. Exosomes (NK-EXO) were isolated from the derived NK cells.
(3) 50 mL용 tube에 세포 배양액을 수집하여 연속적으로 원심분리하였다. 연속 원심분리는 1,500 g에서 3분, 2,000 g에서 15분, 3,000 g에서 20분을 수행하였다. 이 때 각 단계마다 상층액을 새로운 tube에 옮겨 담았다.(3) The cell culture solution was collected in a 50 mL tube and centrifuged continuously. Continuous centrifugation was performed at 1,500 g for 3 minutes, at 2,000 g for 15 minutes, and at 3,000 g for 20 minutes. At this time, the supernatant was transferred to a new tube for each step.
(4) 마지막 원심분리 후 0.22 μm filter로 상층액을 걸러주었다.(4) After the last centrifugation, the supernatant was filtered with a 0.22 μm filter.
(5) 10 kDa 농축필터 tube를 이용하여 (4)의 상층액을 농축하였다. (5) The supernatant of (4) was concentrated using a 10 kDa concentration filter tube.
4,000 g에서 15분간 원심분리하여 tube 아래 액체는 버리고 농축필터에 남은 액체는 새로운 tube에 옮겨 담았다.After centrifugation at 4,000 g for 15 minutes, the liquid under the tube was discarded and the liquid remaining in the concentration filter was transferred to a new tube.
(6) 상기 (5)의 과정을 반복하여 세포 배양액을 농축하였다.(6) The process of (5) above was repeated to concentrate the cell culture solution.
(7) 농축된 세포 배양액에 0.22 μm filter로 여과한 PBS를 첨가하여 전체 11 mL을 맞추었다. (7) PBS filtered with a 0.22 μm filter was added to the concentrated cell culture solution to make a total of 11 mL.
(8) 초고속 원심분리전용 tube에 (7)을 옮겨 담고, 모든 tube의 무게를 소수점 2번째 자리까지 맞춘 다음, 100,000 g에서 90분간 초고속 원심분리하여 상층액을 제거하였다.(8) Transfer (7) to a tube for ultra-high speed centrifugation, adjust the weight of all tubes to the second decimal place, and ultra-high speed centrifugation at 100,000 g for 90 minutes to remove the supernatant.
(9) 0.22 μm filter로 여과한 PBS (200~500 μL)를 넣어 tube 바닥에 있는 침전물을 잘 녹였다. (9) Put PBS (200~500 μL) filtered through a 0.22 μm filter to dissolve the precipitate at the bottom of the tube.
(10) 최종적으로 농축된 NK-EXO를, 새로운 micro-tube에 옮겨 담고, 냉동 보관하였다.(10) Finally, the concentrated NK-EXO was transferred to a new micro-tube and stored frozen.
1-2. 개 지방조직 유래-중간엽줄기세포(Mesenchymal Stem Cells, MSC)로부터 엑소좀(exosome)(MSC-EXO)의 분리1-2. Isolation of exosomes (MSC-EXO) from canine adipose tissue-derived mesenchymal stem cells (MSC)
본 발명자들은, 개의 암 치료에 적용하기 위하여, 성체줄기세포(Canine Adult Stem Cells)인 개 지방조직 유래-중간엽줄기세포(Mesenchymal Stem Cells, MSC)로부터 엑소좀(MSC-EXO)을 분리 및 농축하였다(도 1a).The present inventors, in order to apply to the treatment of cancer in dogs, canine adult stem cells (Canine Adult Stem Cells) derived from dog adipose tissue-mesenchymal stem cells (MSC) from exosomes (MSC-EXO) isolate and concentrate (Fig. 1a).
간략하게는, 다음과 같다:Briefly, it is as follows:
(1) 먼저, 개 지방조직으로부터 MSC를 획득하였다.(1) First, MSCs were obtained from dog adipose tissue.
정상 개의 복부 지방조직을 수집하여 페니실린과 스트렙토마이신이 포함된 PBS로 세척한 다음 수술용 가위를 이용하여 잘게 잘랐다. 0.1% 콜라게나제(collagenase) 유형 I 용액을 넣고 37℃ 교반조(shaking water bath)에서 1시간 동안 반응시켰다. 100 ㎛ cell strainer를 이용하여 조직 부유물을 거르고 1500 rpm에서 5분간 원심분리하여 상층액을 제거한 다음 10% FBS가 포함된 DMEM 배지를 넣어 T175 플라스크에 배양하였다. 분리한 최초의 MSC는 7일 이내에 colony를 형성하여 섬유아세포(fibroblast-like) 모양으로 부착하여 잘 자라는 것을 확인하였다. 이후 해당 MSC의 성체줄기세포 성능을 확인하고자 CD29, CD34, CD44 등의 세포 표면 단백질 발현 유무와 중배엽세포로의 분화능을 확인하였다.Abdominal adipose tissue from normal dogs was collected, washed with PBS containing penicillin and streptomycin, and then chopped using surgical scissors. A 0.1% collagenase type I solution was added and reacted for 1 hour in a shaking water bath at 37°C. The tissue suspension was filtered using a 100 μm cell strainer, centrifuged at 1500 rpm for 5 minutes to remove the supernatant, and then DMEM medium containing 10% FBS was added and cultured in a T175 flask. It was confirmed that the first isolated MSCs formed colonies within 7 days, attached to them in a fibroblast-like shape, and grew well. Afterwards, to confirm the adult stem cell performance of the MSC, the presence or absence of expression of cell surface proteins such as CD29, CD34, and CD44 and the ability to differentiate into mesodermal cells were checked.
175 T flask에 4×106 개의 MSC를 부착하여 1일간 배양하였다. 4×10 6 MSCs were attached to a 175 T flask and cultured for 1 day.
(2) 상기 MSC를, 다음 날 1% P/S가 첨가된 DPBS로 세척한 다음, exosome-depleted FBS를 포함한 세포 배양액(20 mL)으로 교체하여 3일간 배양함으로써 개 지방조직-유래 MSC로부터 엑소좀(exosome)(MSC-EXO)을 분리하였다.(2) The MSCs were washed with DPBS supplemented with 1% P/S the next day, and then replaced with a cell culture medium (20 mL) containing exosome-depleted FBS and cultured for 3 days. Exosomes (MSC-EXO) were isolated.
(3) 50 mL용 tube에 세포 배양액을 수집하여 연속적으로 원심분리하였다. 연속 원심분리는 1,500 g에서 3분, 2,000 g에서 15분, 3,000 g에서 20분을 수행하였다. 이 때 각 단계마다 상층액을 새로운 tube에 옮겨 담았다.(3) The cell culture solution was collected in a 50 mL tube and centrifuged continuously. Continuous centrifugation was performed at 1,500 g for 3 minutes, at 2,000 g for 15 minutes, and at 3,000 g for 20 minutes. At this time, the supernatant was transferred to a new tube for each step.
(4) 마지막 원심분리 후 0.22 μm filter로 상층액을 걸러주었다.(4) After the last centrifugation, the supernatant was filtered with a 0.22 μm filter.
(5) 10 kDa 농축필터 tube를 이용하여 (4)의 상층액을 농축하였다. (5) The supernatant of (4) was concentrated using a 10 kDa concentration filter tube.
4,000 g에서 15분간 원심분리하여 tube 아래 액체는 버리고 농축필터에 남은 액체는 새로운 tube에 옮겨 담았다.After centrifugation at 4,000 g for 15 minutes, the liquid under the tube was discarded and the liquid remaining in the concentration filter was transferred to a new tube.
(6) 상기 (5)의 과정을 반복하여 세포 배양액을 농축하였다.(6) The process of (5) above was repeated to concentrate the cell culture solution.
(7) 농축된 세포 배양액에 0.22 μm filter로 여과한 PBS를 첨가하여 전체 11 mL을 맞추었다. (7) PBS filtered with a 0.22 μm filter was added to the concentrated cell culture solution to make a total of 11 mL.
(8) 초고속 원심분리전용 tube에 (7)을 옮겨 담고, 모든 tube의 무게를 소수점 2번째 자리까지 맞춘 다음, 100,000 g에서 90분간 초고속 원심분리하여 상층액을 제거하였다.(8) Transfer (7) to a tube for ultra-high speed centrifugation, adjust the weight of all tubes to the second decimal place, and ultra-high speed centrifugation at 100,000 g for 90 minutes to remove the supernatant.
(9) 0.22 μm filter로 여과한 PBS (200~500 μL)를 넣어 tube 바닥에 있는 침전물을 잘 녹였다. (9) Put PBS (200~500 μL) filtered through a 0.22 μm filter to dissolve the precipitate at the bottom of the tube.
(10) 최종적으로 농축된 MSC-EXO를, 새로운 micro-tube에 옮겨 담고, 냉동 보관하였다.(10) Finally, the concentrated MSC-EXO was transferred to a new micro-tube and stored frozen.
실시예 2. 개 조직-유래 엑소좀의 특성 분석Example 2. Characterization of canine tissue-derived exosomes
본 발명자들은, 상기 실시예 1에서 획득한 본 발명의 개 조직-유래 엑소좀(MSC-EXO)의 고유한 특성을 분석하기 위하여, 전자현미경, 나노사이트, 나노입자추적시스템 및 웨스턴 블롯을 실시하였다. The present inventors performed electron microscopy, nanosite, nanoparticle tracking system and Western blot to analyze the unique properties of the dog tissue-derived exosome (MSC-EXO) of the present invention obtained in Example 1 above. .
그 결과, 도 1b에 나타낸 바와 같이, 전자현미경 및 나노사이트 분석을 통해서, 본 발명의 개 조직-유래 엑소좀의 크기는 평균 145 nm의 사이즈인 것을 확인하였다.As a result, as shown in Figure 1b, through electron microscopy and nanosite analysis, it was confirmed that the dog tissue-derived exosomes of the present invention had an average size of 145 nm.
또한, 도 1c에 나타낸 바와 같이, 현탁액에서 개개의 입자의 브라운 운동(Brownian motion)을 기록하여 입자의 농도, 크기 및 크기 분포를 확인할 수 있는 나노입자추적시스템(NTA; Nanoparticle tracking analysis)을 통하여, 본 발명의 엑소좀은 50 내지 200nm의 직경을 가지는 것을 확인하였다.In addition, as shown in Fig. 1c, by recording the Brownian motion of individual particles in suspension, the concentration, size, and size distribution of the particles can be confirmed through a nanoparticle tracking system (NTA; Nanoparticle tracking analysis), It was confirmed that the exosomes of the present invention have a diameter of 50 to 200 nm.
또한, 도 1d에 나타낸 바와 같이, 웨스턴 블롯을 통해서 본 발명의 엑소좀 에서 Flotillin-1, CD63, CD81, Alix, HSP70 및 TSG101와 같은 엑소좀 마커들이 발현되는 것을 확인하였다. In addition, as shown in FIG. 1d, it was confirmed that exosome markers such as Flotillin-1, CD63, CD81, Alix, HSP70 and TSG101 were expressed in the exosomes of the present invention through Western blot.
실시예 3. 개 조직-유래 엑소좀에 의한 항종양 효과Example 3. Anti-tumor effect by canine tissue-derived exosomes
본 발명자들은 실시예 1에서 획득한 본 발명의 개 조직 유래의 엑소좀들(NK-EXO 및 MSC-EXO)이 실제 암세포에 대해 억제 효과를 나타내는 지 확인하기 위해, 마우스 유선종양 모델에 처리한 후 관찰하였다.In order to confirm whether the exosomes (NK-EXO and MSC-EXO) derived from the dog tissue of the present invention obtained in Example 1 show an inhibitory effect on actual cancer cells, after treatment in a mouse mammary tumor model observed.
먼저, 생후 5~6 주령 마우스(BALB/c-nude, female)를 특정 병원체 부재 무균 사육실(온도_22 ± 3℃ 습도_50 ± 5%, 명암주기_12시간 낮, 12시간 밤)의 표준사육 환경에서 1 주 동안 적응시켰다. 또한, 스페어가 형성된 개 유선종양세포주 REM134를 배양한 후, 1Х103/60㎕/DPBS로 준비하고 매트리겔(60㎕)과 1:1로 혼합하였다. 1 주 후, 마우스를 마취시킨 다음 21G 주사기를 이용하여 오른쪽 유선 지방 지지체(right mammary fat pad of mice) 부위의 피하로 상기 준비된 스페어 형성 유선종양세포를 서서히 주입하여 유선종양 모델을 제작하였다.First, a 5- to 6-week-old mouse (BALB/c-nude, female) was treated in a specific pathogen-free sterile breeding room (temperature_22 ± 3℃ humidity_50 ± 5%, light-dark cycle_12 hours daytime, 12 hours night) standard. They were acclimatized for 1 week in a captive environment. In addition, after culturing the canine mammary tumor cell line REM134 in which the spare was formed, it was prepared with 1Х10 3 /60 μl/DPBS and mixed 1:1 with Matrigel (60 μl). After 1 week, the mice were anesthetized, and then, using a 21G syringe, the prepared spare-forming mammary tumor cells were slowly injected subcutaneously into the right mammary fat pad of mice to prepare a mammary tumor model.
이어서, 유선종양 모델 마우스에서 종양이 형성되도록 2 주간 두고, 육안으로 형성된 종양을 확인한 다음, 1주에 2회 종양 부위 또는 혈관으로 실시예 1에서 획득한 본 발명의 NK 세포-유래 엑소좀 NK-EXO(100 ㎍)를 투여하고 종양 크기를 1주일 간격으로 측정하였다.Then, the NK cell-derived exosome NK- of the present invention obtained in Example 1 as a tumor site or blood vessel twice a week after leaving the mammary tumor model mouse for 2 weeks to form a tumor, visually confirming the formed tumor, and EXO (100 μg) was administered and the tumor size was measured at weekly intervals.
이때, 대조군으로 유선종양 모델 마우스에 엑소좀을 처리하지 않은 그룹(REM134_1Х103)을 이용하였다(도 2c).At this time, a group (REM134_1Х10 3 ) not treated with exosomes in mammary tumor model mice was used as a control group ( FIG. 2c ).
그 결과, 도 2a 및 2b에 나타낸 바와 같이, 8 주 후 대조군인 유선종양세포주 REM134만을 처리한 경우(도 2c)에 비해, 엑소좀 NK-EXO를 처리했을 때 종양 크기가 80% 이상 현저하게 감소한 것을 확인하였다.As a result, as shown in FIGS. 2a and 2b, after 8 weeks, when treated with exosome NK-EXO, compared to the case where only the control mammary gland tumor cell line REM134 was treated ( FIG. 2c ), the tumor size was significantly reduced by 80% or more. confirmed that.
또한, 주간별로 마우스의 몸무게를 측정했을 때, 도 2d에 나타낸 바와 같이, NK 세포-유래 엑소좀 투여군의 마우스 몸무게는 아무것도 처리하지 않은 정상 대조군(control)과 유사하게 유지하는 것을 확인하였다.In addition, when the body weight of the mice was measured weekly, as shown in FIG. 2d , it was confirmed that the weight of the mouse in the NK cell-derived exosome-administered group was maintained similar to that of the normal control group that was not treated with anything.
또한, 상기 유선종양 모델 마우스에서 종양이 형성되도록 2 주간 두고, 육안으로 형성된 종양을 확인한 다음, 1주에 2회 종양 부위 또는 혈관으로 실시예 1에서 획득한 본 발명의 MSC 유래 엑소좀 MSC-EXO(100 ㎍)를 투여하고 종양 크기 및 마우스 몸무게를 1주일 간격으로 측정하였다.In addition, the MSC-derived exosome MSC-EXO of the present invention obtained in Example 1 as a tumor site or blood vessel twice a week after leaving the mammary tumor model mouse for 2 weeks to form a tumor, confirming the formed tumor with the naked eye, (100 μg) was administered, and tumor size and mouse weight were measured at weekly intervals.
이때, 대조군으로 유선종양 모델 마우스에 엑소좀을 처리하지 않은 그룹(REM134_1Х103)을 이용하였다(도 3c).In this case, a group (REM134_1Х10 3 ) that was not treated with exosomes in mammary tumor model mice was used as a control group ( FIG. 3c ).
그 결과, 도 3a 및 3b에 나타낸 바와 같이, 8 주 후 대조군인 유선종양세포주 REM134만을 처리한 경우(도 3c)에 비해, 엑소좀 MSC-EXO를 처리했을 때 종양 크기가 50% 이상 현저하게 감소한 것을 확인할 수 있었다.As a result, as shown in FIGS. 3A and 3B , after 8 weeks, the tumor size was significantly reduced by more than 50% when treated with exosome MSC-EXO compared to the case where only the control mammary gland tumor cell line REM134 was treated ( FIG. 3C ). could confirm that
또한, 주간별로 마우스의 몸무게를 측정했을 때, 도 3d에 나타낸 바와 같이, MSC-유래 엑소좀 투여군의 마우스 몸무게는 아무것도 처리하지 않은 정상 대조군(control)과 유사하게 유지하는 것을 확인하였다.In addition, when the body weight of the mice was measured weekly, as shown in FIG. 3d , it was confirmed that the mouse weight of the MSC-derived exosome-administered group was maintained similar to that of the normal control group that was not treated with anything.
따라서, 상기 결과들은 본 발명의 개 지방조직 유래-MSC로부터 분리된 엑소좀(MSC-EXO) 및 개 PBMCs에서 유래된 NK 세포로부터 분리된 엑소좀(exosome)(NK-EXO)이, 암세포만을 타겟으로 하여 다른 정상세포에는 영향을 미치지 않고 안전하게, 고형암을 억제하는 탁월한 항암 효과를 발휘한다는 것을 의미한다. Therefore, the above results show that the present invention's canine adipose tissue-derived exosome isolated from MSC (MSC-EXO) and canine PBMCs-derived NK cell-derived exosome (NK-EXO) target cancer cells only. This means that it exerts an excellent anticancer effect that safely suppresses solid cancer without affecting other normal cells.
Claims (13)
A pharmaceutical composition for preventing or treating cancer in canines, comprising an exosome derived from canine animals as an active ingredient.
상기 엑소좀은 성체줄기세포(Adult Stem Cell) 또는 자연살해세포(natural killer cells, NK 세포)로부터 분리된 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
According to claim 1,
The exosome is characterized in that it is separated from adult stem cells (Adult Stem Cell) or natural killer cells (NK cells),
A pharmaceutical composition for preventing or treating cancer in canine animals.
상기 성체줄기세포는 개과 동물의 지방조직, 혈액, 뇌, 제대, 제대혈, 골수, 근육, 신경, 피부, 양막 및 태반으로 이루어진 군에서 선택된 1 종 이상으로부터 분리되는 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
3. The method of claim 2,
The adult stem cells are characterized in that it is isolated from one or more selected from the group consisting of adipose tissue, blood, brain, umbilical cord, umbilical cord blood, bone marrow, muscle, nerve, skin, amniotic membrane and placenta of canine animals,
A pharmaceutical composition for preventing or treating cancer in canine animals.
상기 성체줄기세포는 상기 개과 동물의 지방조직에서 분리된 중간엽줄기세포(Mesenchymal Stem Cells, MSC)인 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
4. The method of claim 3,
The adult stem cells are characterized in that the mesenchymal stem cells (Mesenchymal Stem Cells, MSC) isolated from the adipose tissue of the canine animal,
A pharmaceutical composition for preventing or treating cancer in canine animals.
상기 NK 세포는 개과 동물의 말초혈액, 골수, 림프절, 비장, 폐 및 간장으로 이루어진 군에서 선택된 1 종 이상으로부터 분리되는 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
3. The method of claim 2,
The NK cells are characterized in that it is isolated from one or more selected from the group consisting of canine peripheral blood, bone marrow, lymph node, spleen, lung and liver,
A pharmaceutical composition for preventing or treating cancer in canine animals.
상기 NK 세포는 상기 개과 동물의 말초혈액에서 분리된 말초혈액 단핵세포(peripheral blood mononuclear cells, PBMC)로부터 유래되는 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
6. The method of claim 5,
The NK cells are characterized in that derived from peripheral blood mononuclear cells (PBMC) isolated from the peripheral blood of the canine animal,
A pharmaceutical composition for preventing or treating cancer in canine animals.
상기 개과 동물은 개, 갈기늑대, 가로무늬자칼, 검은 등자칼, 딩고, 붉은늑대, 에티오피아늑대, 인도늑대, 코요태, 황금자칼, 게잡이여우속, 들개속, 승냥이, 아프리카들개, 작은귀개, 포클랜드늑대, 안데스여우, 다윈여우, 아르헨티나회색여우, 팜파스여우, 페루사막여우, 흰여우, 여우족은 북극여우, 붉은여우, 스위프트여우, 키트여우, 코사크여우, 케이프여우, 검은꼬리모래여우, 벵골여우, 티베트모래여우, 블랜포드여우, 흰꼬리모래여우, 페넥여우, 회색여우, 섬여우, 코즈멜여우, 큰귀여우 및 너구리로 이루어진 군으로부터 선택되는 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
According to claim 1,
The canines include dogs, maned wolves, striped jackals, black stirrup jackals, dingoes, red wolves, Ethiopian wolves, Indian wolves, coyotes, golden jackals, crab-eating vulpes, wild dog genus, seungnyangyi, African wild dog, small-eared dog, Falklands. Wolf, Andean fox, Darwin fox, Argentine gray fox, pampas fox, Peruvian desert fox, white fox, arctic fox, red fox, swift fox, kit fox, cossack fox, cape fox, black-tailed sand fox, bengal fox , Tibetan sand fox, Blanford fox, white-tailed sand fox, fennec fox, gray fox, island fox, cozumel fox, large eared fox and raccoon dog, characterized in that it is selected from the group consisting of,
A pharmaceutical composition for preventing or treating cancer in canines.
상기 엑소좀은, (i) 개 지방조직에서 유래된 중간엽줄기세포(Mesenchymal Stem Cells, MSC)로부터 분리된 엑소좀(exosome); 또는
(ii) 개 말초혈액 단핵세포(peripheral blood mononuclear cells, PBMCs) 에서 유래된 자연살해세포(natural killer cells, NK 세포)로부터 분리된 엑소좀(exosome)인 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
According to claim 1,
The exosome is, (i) an exosome isolated from mesenchymal stem cells (MSC) derived from dog adipose tissue; or
(ii) characterized in that it is an exosome isolated from natural killer cells (NK cells) derived from dog peripheral blood mononuclear cells (PBMCs),
A pharmaceutical composition for preventing or treating cancer in canine animals.
상기 엑소좀은 40 내지 200 nm의 지름을 가진 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
According to claim 1,
The exosome is characterized in that it has a diameter of 40 to 200 nm,
A pharmaceutical composition for preventing or treating cancer in canine animals.
상기 암은 유방암, 편평상피세포암, 자궁암, 자궁경부암, 전립선암, 두경부암, 췌장암, 뇌종양, 간암, 피부암, 식도암, 고환암, 신장암, 대장암, 직장암, 위암, 방광암, 난소암, 담관암 및 담낭암으로 이루어지는 군으로부터 선택되는 1종 이상인 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
According to claim 1,
The cancer is breast cancer, squamous cell cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colorectal cancer, rectal cancer, stomach cancer, bladder cancer, ovarian cancer, bile duct cancer and characterized in that at least one selected from the group consisting of gallbladder cancer,
A pharmaceutical composition for preventing or treating cancer in canine animals.
상기 엑소좀은 암의 크기를 감소시키는 것을 특징으로 하는,
개과 동물의 암 예방 또는 치료용 약학적 조성물.
According to claim 1,
The exosome is characterized in that it reduces the size of cancer,
A pharmaceutical composition for preventing or treating cancer in canine animals.
A feed composition for preventing or improving cancer in canines, comprising exosomes derived from canine animals as an active ingredient.
A method for preventing or treating canine cancer, comprising administering to the canine a composition comprising an exosome derived from a canine animal as an active ingredient.
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