KR102632530B1 - Stomach cancer specific targeting exosome composition as a drug anticancer delivery and use thereof - Google Patents
Stomach cancer specific targeting exosome composition as a drug anticancer delivery and use thereof Download PDFInfo
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- KR102632530B1 KR102632530B1 KR1020210065918A KR20210065918A KR102632530B1 KR 102632530 B1 KR102632530 B1 KR 102632530B1 KR 1020210065918 A KR1020210065918 A KR 1020210065918A KR 20210065918 A KR20210065918 A KR 20210065918A KR 102632530 B1 KR102632530 B1 KR 102632530B1
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- gastric cancer
- exosomes
- adipose
- cells
- stem cells
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Abstract
본 발명은 줄기세포의 세포외소포체로 발현되는 유전자 내에 위암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자가 삽입된 재조합 발현벡터, 상기 발현벡터로 형질전환된 세포외소포체를 포함하는 약학적 조성물 및 위암 예방 또는 치료용 발현벡터 제조방법에 대한 것으로, 본 발명은 세포외소포체로서 엑소좀을 약물전달물질로서 활용하여 표적 세포인 위암 세포에 선별적으로 결합하여 위암을 표적으로 특이적 작용이 가능한 이점이 있다.The present invention provides a recombinant expression vector in which a gene expressing a ligand that binds to a receptor of gastric cancer cells is inserted into a gene expressed in the extracellular vesicles of stem cells, a pharmaceutical composition comprising extracellular vesicles transformed with the expression vector, and The present invention relates to a method of manufacturing an expression vector for preventing or treating gastric cancer. The present invention utilizes exosomes as extracellular vesicles as drug delivery substances to selectively bind to gastric cancer cells, which are target cells, and has the advantage of having a specific action targeting gastric cancer. There is.
Description
본 발명은 재조합 발현벡터로 형질전환된 지방유래줄기세포로부터 수득된 엑소좀을 유효성분으로 포함하는 위암 특이적 표적 엑소좀 조성물 및 이의 용도에 관한 것이다.The present invention relates to a gastric cancer-specific targeting exosome composition containing as an active ingredient exosomes obtained from adipose-derived stem cells transformed with a recombinant expression vector and its use.
위암은 가장 빈번하게 발생하는 암 중 하나이며 한국과 아시아에서 암 사망의 주요 원인에 해당한다. 현재 보고된 바에 따르면 위암의 원인은 유전적 요인, 식이 습관, 흡연, 알코올 소비 및 헬리코박터 파일로리 감염을 들 수 있으며, 지난 수십 년 동안 조기 진단 및 개선된 치료 기술로 인해 위암의 5년 생존율이 유의하게 증가하였지만, 후기 발견의 어려움과 높은 재발률로 인해 후기 생존율은 여전히 10% 미만으로 알려져 있다. 최근 암 조기검진사업이 시행됨에 따라 위암이 조기에 진단되는 경우가 늘고 있고, 조기위암 중에서 림프절 전이가 없는 경우에는 내시경적 절제술로 완치가 가능하지만, 림프절 전이를 완벽하게 예측할 수 없기 때문에 내시경적 절제술의 대상 환자를 선별하는데 한계가 있다.Gastric cancer is one of the most frequently occurring cancers and is the leading cause of cancer death in Korea and Asia. Currently, it has been reported that the causes of stomach cancer include genetic factors, dietary habits, smoking, alcohol consumption, and Helicobacter pylori infection. Due to early diagnosis and improved treatment techniques over the past few decades, the 5-year survival rate of stomach cancer has significantly decreased. Although it has increased, the late survival rate is still known to be less than 10% due to the difficulty of late detection and high recurrence rate. With the recent implementation of early cancer screening programs, the number of cases of stomach cancer being diagnosed at an early stage is increasing. Among early stomach cancers, cases without lymph node metastasis can be cured through endoscopic resection, but because lymph node metastasis cannot be perfectly predicted, endoscopic resection is recommended. There are limitations in selecting target patients.
현재 사용중인 항암제로서 5-FU, 시스플라틴, 독소루비신, 마이토마이신 등이 있으나, 상기 물질은 장기간 과량을 사용하면 신장 및 간독성을 나타내며 여러 부작용이 있어 암세포에 특이적으로 전달되지 아니하여 생체에 적용하기에는 한계가 있다.Anticancer drugs currently in use include 5-FU, cisplatin, doxorubicin, mitomycin, etc. However, these substances exhibit kidney and liver toxicity when used in excessive amounts for a long period of time and have various side effects, so they are not specifically delivered to cancer cells, making them unsuitable for application to living bodies. There are limits.
한편, 엑소좀은 그 크기가 30-100nm에 불과하지만, 원세포에 들어있는 단백질, 핵산, 지질 등 여러 물질을 포함하고 있는 물질로서, 면역세포나 줄기세포 등 우리 지방유래줄기세포가 분비하는 나노입자이다. 엑소좀의 세포간 정보 전달체 역할을 하는 기능이 알려지면서 차세대 항암물질로 주목받고 있다.On the other hand, exosomes are only 30-100 nm in size, but they contain various substances such as proteins, nucleic acids, and lipids contained in the original cell, and are nano particles secreted by our adipose-derived stem cells such as immune cells and stem cells. It is a particle. As the function of exosomes as intercellular information carriers became known, they are attracting attention as next-generation anticancer substances.
이에, 본 발명자들은 약물전달물질로서의 위암 특이적 표적 세포외소포체를 개발하기 위하여 예의 노력한 결과, 지방유래줄기세포로부터 추출된 엑소좀이 위암 표적 항암제의 기능이 있음을 확인함으로써, 본 발명을 완성하였다.Accordingly, the present inventors made diligent efforts to develop a stomach cancer-specific targeting extracellular vesicle as a drug delivery material, and as a result confirmed that exosomes extracted from adipose-derived stem cells have the function of a stomach cancer-targeting anticancer agent, thereby completing the present invention. .
본 발명의 목적은 줄기세포의 세포외소포체로 발현되는 유전자 내에 위암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자가 삽입된 재조합 발현벡터로 형질전환된 지방유래줄기세포로부터 수득된 세포외소포체를 유효성분으로 포함하는 위암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.The purpose of the present invention is to validate extracellular vesicles obtained from adipose-derived stem cells transformed with a recombinant expression vector in which a gene expressing a ligand that binds to a receptor on gastric cancer cells is inserted into a gene expressed in the extracellular vesicles of stem cells. The aim is to provide a pharmaceutical composition for preventing or treating gastric cancer, including the composition as an ingredient.
본 발명의 또 다른 목적은 (a) 지방유래줄기세포의 세포외소포체로 발현되는 유전자 내에 위암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자를 도입하는 단계; (b) 상기 유전자가 도입된 세포를 배지에서 배양하여 세포외소포체를 수득하는 단계; 및 (c) 상기 수득된 세포외소포체에 HSP90 억제제를 도입시키는 단계;를 포함하는 위암의 예방 또는 치료용 약학적 조성물 제조방법을 제공하는 것이다.Another object of the present invention is (a) introducing a gene that expresses a ligand that binds to a receptor in gastric cancer cells into a gene expressed in the extracellular vesicles of adipose-derived stem cells; (b) culturing the cells into which the gene has been introduced in medium to obtain extracellular endoplasmic reticulum; and (c) introducing an HSP90 inhibitor into the obtained extracellular vesicles.
본 출원에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다.Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.
본 발명의 일 측면은 줄기세포의 세포외소포체로 발현되는 유전자 내에 위암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자가 삽입된 재조합 발현벡터로 형질전환된 지방유래줄기세포로부터 수득된 세포외소포체를 유효성분으로 포함하는 위암의 예방 또는 치료용 약학적 조성물을 제공한다.One aspect of the present invention is an extracellular vesicle obtained from adipose-derived stem cells transformed with a recombinant expression vector in which a gene expressing a ligand that binds to a receptor of gastric cancer cells is inserted into a gene expressed in the extracellular vesicle of the stem cell. Provided is a pharmaceutical composition for the prevention or treatment of gastric cancer containing it as an active ingredient.
본 발명에서의 "발현벡터"는 선형 DNA 벡터, 플라스미드 DNA 벡터 및 재조합 바이러스 벡터로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니며 당업계에서 형질전환을 위해 사용되는 통상적인 벡터들은 모두 본 발명의 방법에 사용될 수 있다. 본 발명의 일 구체예에 있어서, 상기 발현벡터는 pDisplay 벡터일 수 있다.The "expression vector" in the present invention may be selected from the group consisting of linear DNA vectors, plasmid DNA vectors, and recombinant viral vectors, but is not limited thereto, and all common vectors used for transformation in the art are those of the present invention. It can be used in the method of. In one embodiment of the present invention, the expression vector may be a pDisplay vector.
본 발명에서의 "리간드"는 줄기세포의 세포외소포체로 발현되는 유전자 내에 내에 삽입되어 세포외소포체로 발현 시 위암 세포의 수용체와 결합하는 물질을 의미한다. 바람직하게, 상기 리간드는 서열번호 1의 염기 서열로 암호화될 수 있다.In the present invention, “ligand” refers to a substance that is inserted into a gene expressed in the extracellular vesicles of stem cells and binds to the receptor of gastric cancer cells when expressed in the extracellular vesicles. Preferably, the ligand may be encoded with the nucleotide sequence of SEQ ID NO: 1.
본 발명에서의 "세포외소포체"는 세포에서 유래되는 막으로 둘러싸인 작은 구체를 의미하는 것으로, 상기 세포외소포체는 자연계, 예컨대 식물, 동물, 미생물로부터 유래된 세포외 소포체 또는 인공적으로 제조된 세포외소포체일 수 있다. 또한, 상기 세포는 자연계 생물 개체로부터 분리된 세포인 것일 수 있다.In the present invention, “extracellular vesicle” refers to a small sphere surrounded by a membrane derived from a cell. The extracellular vesicle is an extracellular vesicle derived from the natural world, such as plants, animals, and microorganisms, or an artificially manufactured extracellular vesicle. It may be the endoplasmic reticulum. Additionally, the cells may be cells isolated from natural organisms.
상기 "세포외소포체"는 엑소좀(exosome), 엑토솜(ectosome) 또는 미세소포(microvesicle)일 수 있으나, 본 발명의 일 구체예에 있어서, 상기 "세포외소포체"는 엑소좀일 수 있다.The “extracellular vesicle” may be an exosome, ectosome, or microvesicle, but in one embodiment of the present invention, the “extracellular vesicle” may be an exosome.
본 발명에서의 "엑소좀"은 다양한 세포들로부터 분비되는 막 구조의 작은 소낭으로서, 다낭체와 원형질막의 융합이 일어나 세포 밖 환경으로 방출되는 소낭을 의미한다. 본 발명의 일 구체예에 있어서, 상기 엑소좀은 지방유래줄기세포로부터 유래 또는 분리한 것일 수 있다. In the present invention, “exosome” refers to a small vesicle with a membrane structure secreted from various cells, and is released into the extracellular environment after fusion of a multivesicular body and the plasma membrane. In one embodiment of the present invention, the exosomes may be derived from or isolated from adipose-derived stem cells.
본 발명의 일 구체예에 있어서, 상기 엑소좀은 50 내지 150 nm의 직경을 갖는 것일 수 있다. 또한, 상기 엑소좀은 1 내지 50 μL/ml의 농도로 포함될 수 있다.In one embodiment of the present invention, the exosome may have a diameter of 50 to 150 nm. Additionally, the exosomes may be included at a concentration of 1 to 50 μL/ml.
본 발명에서의 "줄기세포(stem cells)"는 미분화 상태에서 적절한 조건을 맞춰주면 다양한 조직 세포로 분화될 수 있는 만능세포로서 다양한 유래의 세포를 의미한다.In the present invention, “stem cells” refer to cells of various origins, which are pluripotent cells that can be differentiated into various tissue cells when appropriate conditions are met in an undifferentiated state.
상기 줄기세포는 골수유래줄기세포, 제대혈유래줄기세포 또는 지방유래줄기세포일 수 있다. 본 발명의 일 구체예에 있어서, 상기 줄기세포는 지방유래줄기세포일 수 있다. 또한, 상기 골수유래줄기세포, 제대혈유래줄기세포 또는 지방유래줄기세포는 인체 또는 동물유래 줄기세포인 것일 수 있다.The stem cells may be bone marrow-derived stem cells, umbilical cord blood-derived stem cells, or adipose-derived stem cells. In one embodiment of the present invention, the stem cells may be adipose-derived stem cells. Additionally, the bone marrow-derived stem cells, umbilical cord blood-derived stem cells, or adipose-derived stem cells may be human- or animal-derived stem cells.
본 발명에서의 "지방유래줄기세포"는 지방 조직으로부터 유래한 줄기세포로서, 다분화능 및 자기증식능을 가진 세포를 의미하며, 본 발명의 지방유래줄기세포는 지방흡입술 및 다양한 외과적 수술을 통해 수득할 수 있으나, 이에 한정되지 않는다.In the present invention, “adipose-derived stem cells” are stem cells derived from adipose tissue and refer to cells with multipotency and self-proliferation ability, and the adipose-derived stem cells of the present invention are obtained through liposuction and various surgical procedures. It can be done, but it is not limited to this.
본 발명의 일 구체예에 있어서, 상기 엑소좀은 줄기세포 등 호스트세포의 엑소좀으로 발현하는 유전자 내에 위암 세포의 수용체와 결합하는 리간드를 발현시키도록 형질전환된 줄기세포로부터 유래된 것으로서 위암 세포에 표적으로 작용할 수 있는 것을 의미한다.In one embodiment of the present invention, the exosome is derived from a stem cell transformed to express a ligand that binds to a receptor of gastric cancer cells within a gene expressed as an exosome of a host cell, such as a stem cell, and is transmitted to gastric cancer cells. It means something that can act as a target.
또한, 본 발명의 일 구체예에 있어서, 상기 엑소좀은 HSP90 억제제를 추가로 장착한 것일 수 있다.Additionally, in one embodiment of the present invention, the exosome may be additionally loaded with an HSP90 inhibitor.
본 발명에서의 열충격 단백질군(heat-shock protein families; HSPs)은 분자샤페론으로 ATP 의존적인 구조 변화를 통해 클라이언트 단백질(client protein)의 접힘을 조절하여 초기 단백질(nascent proteins)의 활성화, 손상된 단백질의 재접힘이나 분해를 도와주는 단백질을 의미한다. 또한, 클라이언트 단백질은 분자샤페론과의 결합을 통해 집합 현상(aggregation)을 회피하며 이들의 결합은 클라이언트 단백질의 막 이동(membrane translocation)을 통해 세포내 퇴적(intracellular deposition)을 도와주는 단백질을 의미한다.Heat-shock protein families (HSPs) in the present invention are molecular chaperones that regulate the folding of client proteins through ATP-dependent structural changes to activate nascent proteins and repair damaged proteins. It refers to a protein that helps with refolding or decomposition. In addition, client proteins avoid aggregation through binding to molecular chaperones, and their binding refers to proteins that assist intracellular deposition through membrane translocation of client proteins.
한편, 상기 열충격 단백질군의 하나인 Hsp90의 분자 샤페론 기능은 세포의 신호 전달 경로에 관계 있는 다양한 클라이언트 단백질의 안정성과 활성화에 필요하다고 알려져 있다. 클라이언트 단백질의 암유발 돌연변이는 더 높은 수준의 Hsp90 기능을 요구하고 Hsp90의 과발현으로 이어지게 되며, 정상 조직과 대비하여 과발현된 Hsp90은 암세포에서 나타나는 공통된 특징이다.Meanwhile, it is known that the molecular chaperone function of Hsp90, one of the heat shock protein family, is required for the stability and activation of various client proteins related to cellular signal transduction pathways. Cancer-causing mutations in client proteins require a higher level of Hsp90 function and lead to overexpression of Hsp90. Compared to normal tissues, overexpressed Hsp90 is a common feature in cancer cells.
상기 HSP90 클라이언트 단백질과 연관된 암의 속성으로서, HER2, RAF, KIT 및 MET는 자가 성장 촉진, CDK4, CDK6 및 CyclinD는 성장 억제 신호에 대한 반응성 저하, IGF-1R 및 AKT는 세포 사멸과정으로부터의 회피, hTERT는 무한 증식능력의 획득, HIF, MET, Src 및 VEGF는 지속적인 혈관 증식, MET, MMP2 및 Urokinase은 암세포의 전이에 해당하는 신호 전달 경로에 영향을 주어 상관관계가 있다고 알려져 있다As properties of cancer associated with the HSP90 client protein, HER2, RAF, KIT, and MET promote self-growth, CDK4, CDK6, and CyclinD reduce responsiveness to growth inhibitory signals, and IGF-1R and AKT avoid cell death processes. It is known that hTERT acquires unlimited proliferation ability, HIF, MET, Src, and VEGF have continuous vascular proliferation, and MET, MMP2, and Urokinase affect the signal transduction pathway corresponding to cancer cell metastasis and are thus correlated.
본 발명에서의 "HSP90 억제제"는 종양인자의 활성을 유도하는 HSP90의 발현 및 활성을 억제하는 물질로서, 위암에서 활성도가 증가되어 있는 HSP90를 억제하는 물질을 의미한다.In the present invention, “HSP90 inhibitor” refers to a substance that inhibits the expression and activity of HSP90, which induces the activity of tumor factors, and inhibits HSP90, whose activity is increased in gastric cancer.
본 발명에서의 "HSP90 억제제"는 라디시콜(Radicicol) 유도체, 겔다나마이신(Geldanamycin) 유도체, 노보비오신(Novobiocin) 유도체, 또는 퓨린(Purine)을 기본으로 한 인공적으로 합성된 물질 등일 수 있으며, 구체적으로 STA-9090(Ganetespib), NVP-AUY922(Luminespib), 17-AAG(Tanespimycin), 17-DMAG(Alvespimycin), STA-4783(Elesclomol) 및 AT13387(Onlaespib)으로 이루어진 군에서 선택될 수 있으나, 본 발명의 일 구체예에 있어서, 상기 "HSP90 억제제"는 17-DMAG일 수 있다. The “HSP90 inhibitor” in the present invention may be a Radicicol derivative, a Geldanamycin derivative, a Novobiocin derivative, or an artificially synthesized substance based on Purine. , specifically, may be selected from the group consisting of STA-9090 (Ganetespib), NVP-AUY922 (Luminespib), 17-AAG (Tanespimycin), 17-DMAG (Alvespimycin), STA-4783 (Elesclomol), and AT13387 (Onlaespib). , In one embodiment of the present invention, the “HSP90 inhibitor” may be 17-DMAG.
또한, 본 발명의 또 다른 측면은 (a) 지방유래줄기세포의 세포외소포체로 발현되는 유전자 내에 위암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자를 도입하는 단계; (b) 상기 유전자가 도입된 세포를 배지에서 배양하여 세포외소포체를 수득하는 단계; 및 (c) 상기 수득된 세포외소포체에 HSP90 억제제를 도입시키는 단계;를 포함하는 위암 예방 또는 치료용 약학적 조성물 제조방법을 제공한다.In addition, another aspect of the present invention includes the steps of (a) introducing a gene that expresses a ligand that binds to a receptor of gastric cancer cells into a gene expressed in the extracellular vesicles of adipose-derived stem cells; (b) culturing the cells into which the gene has been introduced in medium to obtain extracellular endoplasmic reticulum; and (c) introducing an HSP90 inhibitor into the obtained extracellular vesicles.
본 발명에서의 "세포외소포체"는 엑소좀(exosome), 엑토솜(ectosome) 또는 미세소포(microvesicle)일 수 있으나, 본 발명의 일 구체예에 있어서, 상기 "세포외소포체"는 엑소좀일 수 있다.In the present invention, “extracellular vesicles” may be exosomes, ectosomes, or microvesicles, but in one embodiment of the present invention, the “extracellular vesicles” may be exosomes. there is.
본 발명에서의 "배지"는 지방유래줄기세포 배양배지인 것일 수 있다. 본 발명의 일 구체예에 있어서, 상기 지방유래줄기세포 배양배지는 지방유래줄기세포를 FBS(inactivated fetal bovine serum) 및 P/S(penicillin-streptomycin)을 첨가한 DMEM high low glucose 배지 또는 FBS-Free DMEM low glucose로 배지일 수 있다.“Medium” in the present invention may be an adipose-derived stem cell culture medium. In one embodiment of the present invention, the adipose-derived stem cell culture medium is DMEM high low glucose medium supplemented with FBS (inactivated fetal bovine serum) and P/S (penicillin-streptomycin) or FBS-Free. The medium may be DMEM low glucose.
본 발명에서의 “예방”은 본 발명에 따른 약학적 조성물의 투여에 의해 위암을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.“Prevention” in the present invention refers to all actions that suppress or delay the onset of stomach cancer by administering the pharmaceutical composition according to the present invention.
본 발명에서의 “치료”는 본 발명에 따른 약학적 조성물의 투여에 의해 위암에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.“Treatment” in the present invention refers to any action in which symptoms of stomach cancer are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.
본 발명에서의 "위암(gastric cancer)"은 위에 생기는 악성 종양으로, 위 점막상피에서 생기는 위선암과 점막하층에서 생기는 악성림프종, 근육육종, 간질성 종양 등을 의미하나, 이에 한정되지 않는다.In the present invention, “gastric cancer” refers to a malignant tumor that occurs in the stomach, and includes, but is not limited to, gastric adenocarcinoma that occurs in the gastric mucosal epithelium, malignant lymphoma, myosarcoma, and interstitial tumor that occur in the submucosal layer.
본 발명의 줄기세포의 세포외소포체로 발현되는 유전자 내에 위암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자가 삽입된 재조합 발현벡터, 상기 발현벡터로 형질전환된 줄기세포로부터 수득된 세포외소포체를 포함하는 약학적 조성물은 세포외소포체로서 엑소좀을 약물전달물질로서 활용하여 표적 세포인 위암 세포에 선별적으로 결합하여 위암에 특이적으로 작용할 수 있다. 따라서, 상기 표적 엑소좀은 위암 치료 분야, 특히 임상적 적용 기술로 응용될 수 있다.A recombinant expression vector in which a gene expressing a ligand that binds to a receptor of gastric cancer cells is inserted into a gene expressed in the extracellular vesicles of the stem cells of the present invention, including extracellular vesicles obtained from stem cells transformed with the expression vector The pharmaceutical composition is an extracellular vesicle that utilizes exosomes as a drug delivery material to selectively bind to gastric cancer cells, which are target cells, and can act specifically on gastric cancer. Therefore, the targeting exosome can be applied in the field of gastric cancer treatment, especially as a clinical application technology.
도 1은 발현벡터를 통하여 엑소좀에 타겟팅 리간드를 발현시켜 표적 세포인 암세포에 엑소좀이 결합하는 모습을 나타낸 모식도이다. 위암 표적 엑소좀을 처리 시 17-DMAG가 도입됨에 따라 정상세포 대비 위암세포를 특이적으로 공격할 수 있다.
도 2는 본 발명의 재조합 단백질의 발현을 위한 pDisplay에서의 벡터 구성을 나타낸 것이다.
도 3은 엑소좀에 대한 구조 분석을 위하여 투과전자현미경(TEM) 및 유세포분석기 (FACS)을 이용하여 분석한 결과이다.
도 4는 MTT assay 방법을 통하여 엑소좀의 세포 독성 평가한 결과이다.
도 5는 위암세포주인 AGS에서의 산화 스트레스에 반응하는 단백질인 Ho-1, 세포사멸 인자(pro-apoptotic factor)인 cleaved PARP 및 caspase3의 발현 수준 및 항-세포사멸 인자(anti-apoptotic factor)인 Bcl-xl의 발현 수준을 확인한 결과이다.
도 6은 위암세포주인 AGS에서의 산화 스트레스에 반응하는 단백질인 Ho-1, 세포사멸 인자(pro-apoptotic factor)인 cleaved PARP 및 caspase3의 발현 수준 및 항-세포사멸 인자(anti-apoptotic factor)인 Bcl-xl의 발현 수준을 확인한 결과이다.
도 7은 위암세포주인 AGS에서의 효소인 Catalase, 활성산소 방어인자인 SOD(superoxide dismutase)와 GPx(glutathione peroxidase)의 발현 수준을 확인한 결과이다.
도 8은 BALB/C Nude mouse에 위암세포주인 AGS을 이식하여 구축된 위암 동물 모델에서의 외형 관찰 결과를 나타낸 것이다.Figure 1 is a schematic diagram showing the binding of exosomes to cancer cells, which are target cells, by expressing a targeting ligand in exosomes through an expression vector. As 17-DMAG is introduced when processing stomach cancer targeting exosomes, it can specifically attack stomach cancer cells compared to normal cells.
Figure 2 shows the vector configuration in pDisplay for expression of the recombinant protein of the present invention.
Figure 3 shows the results of analysis using transmission electron microscopy (TEM) and flow cytometry (FACS) for structural analysis of exosomes.
Figure 4 shows the results of evaluating the cytotoxicity of exosomes through the MTT assay method.
Figure 5 shows the expression levels of Ho-1, a protein that responds to oxidative stress, cleaved PARP and caspase3, which are pro-apoptotic factors, and the expression levels of caspase3, which is an anti-apoptotic factor, in AGS, a gastric cancer cell line. This is the result of confirming the expression level of Bcl-xl.
Figure 6 shows the expression levels of Ho-1, a protein that responds to oxidative stress, cleaved PARP and caspase3, which are pro-apoptotic factors, and the expression levels of caspase3, which is an anti-apoptotic factor, in AGS, a gastric cancer cell line. This is the result of confirming the expression level of Bcl-xl.
Figure 7 shows the results of confirming the expression levels of the enzyme Catalase and the free radical defense factors SOD (superoxide dismutase) and GPx (glutathione peroxidase) in the gastric cancer cell line AGS.
Figure 8 shows the results of external observation in a gastric cancer animal model constructed by transplanting AGS, a gastric cancer cell line, into BALB/C Nude mouse.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
실시예 1. 지방유래줄기세포 유래의 표적 엑소좀 제작 및 확인Example 1. Production and confirmation of target exosomes derived from adipose-derived stem cells
본 발명의 위암에 특이적으로 작용하는 표적 엑소좀을 제작하기 위하여 지방유래줄기세포를 사용하여 엑소좀을 추출하였다. 참고적으로, 본 발명에서는 표적 엑소좀을 생산하기 위한 donor cells로서 지방유래줄기세포를 사용하였지만, NK세포와 같은 면역세포도 가능하므로, 이에 한정하지 않는다.In order to produce target exosomes that specifically act on gastric cancer of the present invention, exosomes were extracted using adipose-derived stem cells. For reference, in the present invention, adipose-derived stem cells were used as donor cells to produce target exosomes, but immune cells such as NK cells can also be used, so the method is not limited thereto.
지방유래줄기세포를 제조사의 지침에 따라 형질감염시약(lipofectamine, invitrogen)을 사용하여 pDisplay 벡터(invitrogen)에 위암세포 표적 리간드를 인코딩하는 염기 서열(GTT GAA ACT TCA CAG TAT TTC AGA GGG ACA CTG TCA)을 삽입시켰다. 상기 벡터를 클로닝한 후 해당 벡터가 클로닝된 결과를 확인하였으며(도 2), 지방유래줄기세포에 삽입하여 표적 엑소좀을 제작하였다.Adipose-derived stem cells were transfected into the pDisplay vector (invitrogen) using a transfection reagent (lipofectamine, invitrogen) according to the manufacturer's instructions. The base sequence encoding the gastric cancer cell targeting ligand (GTT GAA ACT TCA CAG TAT TTC AGA GGG ACA CTG TCA) was inserted. After cloning the vector, the results of cloning the vector were confirmed (Figure 2), and target exosomes were produced by inserting them into adipose-derived stem cells.
제작된 표적 엑소좀을 추출하기 위하여 지방유래줄기세포를 10% FBS(inactivated fetal bovine serum) 및 1% P/S(penicillin-streptomycin)을 첨가한 DMEM low glucose 배지에서 37℃, 5% CO2 조건으로 배양하였다.To extract the produced target exosomes, adipose-derived stem cells were grown in DMEM low glucose medium supplemented with 10% FBS (inactivated fetal bovine serum) and 1% P/S (penicillin-streptomycin) at 37°C and 5% CO 2 conditions. It was cultured.
24시간 경과 후 상기 배지를 FBS-Free DMEM low glucose로 배지를 교환하였으며, 배지 교환 후 24시간 경과 후 지방유래줄기세포의 배양 배지를 걷어 분별원심분리(differential centrifugation) 방법으로 1000rpm 조건으로 원심분리하여 세포를 제거하였다.After 24 hours, the medium was exchanged with FBS-Free DMEM low glucose, and 24 hours after the medium exchange, the culture medium of adipose-derived stem cells was removed and centrifuged at 1000 rpm using differential centrifugation. Cells were removed.
상기 세포가 제거된 배양 배지를 thermo exosome 추출 kit(Thermo Fisher scientific)을 사용하여 제조사의 지침에 따라 위암 표적 엑소좀을 확보하였다. The culture medium from which the cells were removed was used to obtain gastric cancer targeting exosomes using a thermo exosome extraction kit (Thermo Fisher scientific) according to the manufacturer's instructions.
여기에, 상기 과정을 통하여 확보한 표적 엑소좀에 Neon(invitrogen)을 사용하여 HSP90 억제제로서 17-DMAG을 전기천공법(electroporation)을 사용하여 표적 엑소좀 내에 삽입하여 HSP90 억제제를 도입한 엑소좀을 추가적으로 제작하였다.Here, 17-DMAG as an HSP90 inhibitor was inserted into the target exosome obtained through the above process using Neon (invitrogen) using electroporation to create an exosome into which the HSP90 inhibitor was introduced. It was produced additionally.
실시예 2. 지방유래줄기세포 유래의 표적 엑소좀의 특성 분석Example 2. Characteristic analysis of target exosomes derived from adipose-derived stem cells
본 발명의 위암에 특이적으로 작용하는 표적 엑소좀에 대한 구조 분석을 위하여 투과전자현미경(TEM) 및 유세포분석기 (FACS)을 이용하여 분석하였다. 이에 대한, 엑소좀의 FACs, TEM 분석 결과를 도 3에 나타내었다.To analyze the structure of the target exosome that specifically acts on gastric cancer of the present invention, transmission electron microscopy (TEM) and flow cytometry (FACS) were used to analyze the structure. Regarding this, the results of FACs and TEM analysis of exosomes are shown in Figure 3.
지방유래줄기세포 배양액에서 분리된 엑소좀은 나노미터 단위의 미세한 구형 구조를 갖는 것을 확인할 수 있었다. 또한, 유세포분석기 (FACS)를 이용하여 엑소좀 표면 마커인 CD81, CD63, 및 CD9의 발현이 확인됨에 따라 분리된 엑소좀은 전형적인 엑소좀의 특성을 갖는 것을 알 수 있다(도 3).It was confirmed that exosomes isolated from adipose-derived stem cell culture medium had a fine spherical structure in the nanometer scale. In addition, the expression of exosome surface markers CD81, CD63, and CD9 was confirmed using flow cytometry (FACS), showing that the isolated exosomes had typical exosome characteristics (Figure 3).
실시예 3. 지방유래줄기세포 유래의 표적 엑소좀의 세포 독성 평가Example 3. Evaluation of cytotoxicity of target exosomes derived from adipose-derived stem cells
위암 세포주인 AGS에 표적 엑소좀(t-Exo) 및 HSP90 억제제로서 17-DMAG을 도입한 표적 엑소좀(t-Exo+17-DMAG)에 MTT assay 방법을 통하여 24시간, 48시간 동안 처리 시의 세포독성을 측정하였다. 표적 엑소좀(t-Exo)은 0 및 12.5ul의 농도로, HSP90 억제제로서 17-DMAG을 도입한 표적 엑소좀(t-Exo+17-DMAG)은 0, 10, 25, 50, 100 및 200nM의 농도로 처리하였다.The target exosome (t-Exo) and the target exosome (t-Exo+17-DMAG) introduced with 17-DMAG as an HSP90 inhibitor into AGS, a gastric cancer cell line, were treated for 24 and 48 hours using the MTT assay method. Cytotoxicity was measured. Targeting exosomes (t-Exo) are at concentrations of 0 and 12.5ul, and target exosomes (t-Exo+17-DMAG) incorporating 17-DMAG as an HSP90 inhibitor are at concentrations of 0, 10, 25, 50, 100, and 200nM. It was treated at a concentration of .
이 후, cell viability를 측정한 결과, 17-DMAG의 농도 의존적으로 cell viability가 감소하는 경향을 확인할 수 있었다(도 4).Afterwards, as a result of measuring cell viability, it was confirmed that cell viability tended to decrease depending on the concentration of 17-DMAG (Figure 4).
실시예 4. 위암세포주인 AGS에서의 지방유래줄기세포 유래 표적 엑소좀의 효능 평가(1)Example 4. Evaluation of the efficacy of adipose-derived stem cell-derived targeting exosomes in AGS, a gastric cancer cell line (1)
지방유래줄기세포 엑소좀과 지방유래줄기세포 위암 표적 엑소좀에 17-DMAG를 도입한 엑소좀을 AGS 위암 세포주에 처리 후, apoptosis 관련인자의 발현 양상을 확인하였다.After treating the AGS gastric cancer cell line with exosomes containing 17-DMAG in adipose-derived stem cell exosomes and adipose-derived stem cell gastric cancer targeting exosomes, the expression patterns of apoptosis-related factors were confirmed.
지방유래줄기세포 유래 엑소좀 대비 본 발명에 따른 재조합 벡터가 클로닝된 지방유래줄기세포 유래 위암 표적 엑소좀의 항암 효능을 평가하기 위하여 위암세포주인 AGS에 대조군으로서의 엑소좀(ct), neon electroporation 진행하지 않은 표적 엑소좀(0), neon electroporation 진행한 표적 엑소좀(Ele) 및 neon electroporation을 진행하여 HSP90 억제제로서 17-DMAG을 도입한 표적 엑소좀(Ele+DMAG)을 24시간, 48시간 동안 처리하여 결과를 관찰하였다.In order to evaluate the anticancer efficacy of adipose-derived stem cell-derived gastric cancer-targeting exosomes cloned with the recombinant vector according to the present invention compared to adipose-derived stem cell-derived exosomes, neon electroporation of exosomes (ct) as a control was performed on AGS, a gastric cancer cell line. Target exosomes (0), target exosomes that underwent neon electroporation (Ele), and target exosomes that underwent neon electroporation and introduced 17-DMAG as an HSP90 inhibitor (Ele+DMAG) were treated for 24 and 48 hours. The results were observed.
TNF-alpha가 유도하는 apoptosis를 억제할 수 있는 산화 스트레스에 반응하는 단백질인 Ho-1(Heme oxygenase-1), 세포사멸 기전에 관여하는 세포사멸 인자(pro-apoptotic factor)인 cleaved PARP 및 caspase3의 발현 수준 및 항-세포사멸 인자(anti-apoptotic factor)인 Bcl-xl의 발현 수준을 비교함으로써, 변화를 관찰하였다(도 5 및 도 6).Ho-1 (Heme oxygenase-1), a protein that responds to oxidative stress that can suppress apoptosis induced by TNF-alpha, cleaved PARP and caspase3, which are pro-apoptotic factors involved in the cell death mechanism. Changes were observed by comparing the expression level and the expression level of Bcl-xl, an anti-apoptotic factor (Figures 5 and 6).
그 결과, 도 5 및 도 6에 나타낸 바와 같이, HSP90 억제제인 17-DMAG를 도입한 엑소좀을 AGS 위암 세포주에 처리한 군에서 위암 세포주의 사멸을 억제하는데 관여하는 HO-1의 발현이 감소되는 경향을 확인할 수 있었으며, pro-apoptosis에 관여하는 인자인 caspase3, cleaved PARP의 발현은 증가하고, anti-apoptosis에 관여하는 인자인 Bcl-xl의 발현은 감소하는 경향을 확인할 수 있었다.As a result, as shown in Figures 5 and 6, the expression of HO-1, which is involved in suppressing the death of gastric cancer cell lines, was reduced in the group treated with exosomes containing 17-DMAG, an HSP90 inhibitor, in AGS gastric cancer cell lines. A trend was confirmed, and the expression of caspase3 and cleaved PARP, factors involved in pro-apoptosis, tended to increase, and the expression of Bcl-xl, a factor involved in anti-apoptosis, tended to decrease.
실시예 5. 위암세포주인 AGS에서의 지방유래줄기세포 유래 표적 엑소좀의 효능 평가(2)Example 5. Evaluation of the efficacy of adipose-derived stem cell-derived targeting exosomes in AGS, a gastric cancer cell line (2)
지방유래줄기세포 엑소좀과 지방유래줄기세포 위암 표적 엑소좀에 17-DMAG를 도입한 엑소좀을 AGS 위암 세포주에 처리 후, 항산화 관련 인자의 발현을 확인하였다.After treating the AGS gastric cancer cell line with exosomes containing 17-DMAG in adipose-derived stem cell exosomes and adipose-derived stem cell gastric cancer targeting exosomes, the expression of antioxidant-related factors was confirmed.
지방유래줄기세포 유래 엑소좀 대비 본 발명에 따른 재조합 벡터가 클로닝된 지방유래줄기세포 유래 위암 표적 엑소좀의 항암 효능을 평가하기 위하여 위암세포주인 AGS에 대조군으로서의 엑소좀(con), 표적 엑소좀(t-EXO), neon electroporation 진행한 표적 엑소좀(t-EXO-ele) 및 neon electroporation 진행하여 HSP90 억제제로서 17-DMAG을 도입한 표적 엑소좀(t-EXO-ele+DMAG)을 24시간, 48시간 동안 처리하여 결과를 관찰하였다.In order to evaluate the anticancer efficacy of adipose-derived stem cell-derived gastric cancer targeting exosomes cloned with the recombinant vector according to the present invention compared to adipose-derived stem cell-derived exosomes, exosomes (con) as a control and target exosomes ( t-EXO), target exosomes that underwent neon electroporation (t-EXO-ele), and target exosomes that introduced 17-DMAG as an HSP90 inhibitor by neon electroporation (t-EXO-ele+DMAG) for 24 hours, 48 hours. The results were observed after processing for some time.
인체의 조직과 세포를 공격하고 산화시켜 세포노화를 촉진시키는 활성산소를 분해하는데 관여하고 과산화 수소를 물과 산소로 바꾸는 효소인 Catalase, 활성산소 방어인자인 SOD(superoxide dismutase)와 GPx(glutathione peroxidase)의 발현 수준을 비교함으로써, 변화를 관찰하였다(도 7).Catalase, an enzyme involved in decomposing free radicals that attack and oxidize human tissues and cells and promote cellular aging, and converts hydrogen peroxide into water and oxygen, and SOD (superoxide dismutase) and GPx (glutathione peroxidase), which are free radical defense factors. By comparing the expression levels, changes were observed (Figure 7).
그 결과, 도 7에 나타낸 바와 같이, HSP90 억제제인 17-DMAG를 도입한 엑소좀을 AGS 위암 세포주에 처리한 군에서 catalase의 발현이 감소되는 경향을 확인할 수 있었으며, SOD 및 GPx과 같은 활성산소 방어인자들의 발현도 감소하는 경향을 확인할 수 있었다.As a result, as shown in Figure 7, it was confirmed that the expression of catalase tended to decrease in the group treated with exosomes introduced with 17-DMAG, an HSP90 inhibitor, into the AGS gastric cancer cell line, and that the expression of catalase tended to decrease, and the expression of catalase tended to decrease, and the expression of catalase decreased A tendency to decrease the expression of factors was confirmed.
실시예 6. 위암세포주 AGS 이식에 따른 위암 동물 모델을 이용한 지방유래줄기세포 유래 표적 엑소좀의 효능 평가Example 6. Evaluation of the efficacy of adipose-derived stem cell-derived targeted exosomes using a gastric cancer animal model following transplantation of the gastric cancer cell line AGS
BALB/C Nude mouse(in vivo)의 둔부에 위암세포주인 AGS을 5x105으로 이식하여 위암을 유도한 뒤, 지방유래줄기세포 유래 엑소좀을 이용한 위암의 치료 효능을 확인하고자 하였다.After inducing gastric cancer by transplanting 5x10 5 AGS, a gastric cancer cell line, into the buttocks of a BALB/C Nude mouse (in vivo), we attempted to confirm the efficacy of treating gastric cancer using exosomes derived from adipose-derived stem cells.
대조군으로서의 엑소좀(ct), 표적 엑소좀(tEXO), neon electroporation 진행한 표적 엑소좀(tEXO-ec) 및 neon electroporation 진행하여 HSP90 억제제로서 17-DMAG을 도입한 표적 엑소좀(tEXO-eDMAG) 각각을 상기 구축된 종양모델에 Tail vein injection 방식으로 2주간 주입한 후, 마우스를 희생하여 위암의 항 종양효과에 엑소좀이 미치는 영향을 확인하였다.Exosomes as control (ct), target exosomes (tEXO), target exosomes subjected to neon electroporation (tEXO-ec), and target exosomes introduced by neon electroporation with 17-DMAG as an HSP90 inhibitor (tEXO-eDMAG), respectively. After being injected into the tumor model constructed above by tail vein injection for 2 weeks, mice were sacrificed to confirm the effect of exosomes on the anti-tumor effect of gastric cancer.
상기 BALB/C Nude mouse(in vivo)에 위암세포주인 AGS을 5x105으로 이식하여 구축된 위암 동물 모델에서의 외형 관찰 결과에 의하여 17-DMAG 도입 표적 엑소좀 처리한 군에서 종양의 크기가 억제되는 경향을 확인할 수 있었다(도 8).Based on the external observation results in the gastric cancer animal model constructed by transplanting 5x10 5 AGS, a gastric cancer cell line, into the BALB/C Nude mouse (in vivo), the size of the tumor was suppressed in the group treated with 17-DMAG targeted exosomes. A trend could be confirmed (Figure 8).
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.
<110> The Catholic University of Korea Industry-Academic Cooperation Foundation <120> Stomach cancer specific targeting exosome composition as a drug anticancer delivery and use thereof <130> DPC212404 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> incoding sequence <400> 1 gttgaaactt cacagtattt cagagggaca ctgtca 36 <110> The Catholic University of Korea Industry-Academic Cooperation Foundation <120> Stomach cancer specific targeting exosome composition as a drug anticancer delivery and use thereof <130>DPC212404 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> encoding sequence <400> 1 gttgaaactt cacagtattt cagagggaca ctgtca 36
Claims (10)
상기 유전자는 서열번호 1을 포함하는 것을 특징으로 하고,
상기 엑소좀은 HSP90 억제제를 추가적으로 더 포함하는 것을 특징으로 하고,
상기 HSP90 억제제는 17-DMAG(Alvespimycin)인 것을 특징으로 하는, 위암의 예방 또는 치료용 약학적 조성물.Contains as an active ingredient exosomes obtained from adipose-derived stem cells transformed with a recombinant vector inserted with a gene that expresses a ligand that binds to a receptor on gastric cancer cells,
The gene is characterized in that it contains SEQ ID NO: 1,
The exosome is characterized in that it additionally contains an HSP90 inhibitor,
A pharmaceutical composition for preventing or treating gastric cancer, wherein the HSP90 inhibitor is 17-DMAG (Alvespimycin).
(b) 상기 유전자가 도입된 지방유래줄기세포를 배지에서 배양하여 엑소좀을 수득하는 단계; 및
(c) 상기 수득된 엑소좀에 HSP90 억제제를 도입시키는 단계로서,
상기 HSP90 억제제는 17-DMAG(Alvespimycin)인 것을 특징으로 하는, 단계;
를 포함하는 위암 예방 또는 치료용 약학적 조성물 제조방법.
(a) transforming adipose-derived stem cells with a recombinant vector into which a gene containing SEQ ID NO: 1 is inserted, which expresses a ligand that binds to a receptor on gastric cancer cells;
(b) culturing the adipose-derived stem cells into which the gene has been introduced in a medium to obtain exosomes; and
(c) introducing an HSP90 inhibitor into the obtained exosomes,
wherein the HSP90 inhibitor is 17-DMAG (Alvespimycin);
A method for producing a pharmaceutical composition for preventing or treating stomach cancer, comprising:
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