KR20100050198A - Pharmaceutical composition for treating immune disease and cancer - Google Patents

Pharmaceutical composition for treating immune disease and cancer Download PDF

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KR20100050198A
KR20100050198A KR1020080109371A KR20080109371A KR20100050198A KR 20100050198 A KR20100050198 A KR 20100050198A KR 1020080109371 A KR1020080109371 A KR 1020080109371A KR 20080109371 A KR20080109371 A KR 20080109371A KR 20100050198 A KR20100050198 A KR 20100050198A
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전병화
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육재민
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Abstract

PURPOSE: A pharmaceutical composition containing tat-ret-1 fusion protein is provided to effectively prevent and treat autoimmune diseases and cancer by controlling the development of HMGB1(high-mobility group box 1). CONSTITUTION: A pharmaceutical composition for treating autoimmune diseases and cancer contains tat-ref-1 fusion protein with the sequence number 1 as an active ingredient. The Tat-ref-1 fusion protein is produced by E.coli transformant BL 21(DE3)/pTat-ref-1 transformed as a development vector. The autoimmune disease includes sepsis, Churg-Strauss syndrome, Sjogren's syndrome, Systemic lupus erythematosus, or Rheumatoid arthritis. The cancer includes breast cancer, colon cancer, melanoma, prostate cancer, lung cancer or pancreatic cancer. The amount of each dose is 0.1~10 nM per day.

Description

면역질환 및 암 치료용 제약 조성물{Pharmaceutical composition for treating immune disease and cancer}Pharmaceutical composition for treating immune disease and cancer

본 발명은 융합단백질을 유효성분으로 함유하는 제약 조성물에 관한 것으로, 보다 상세하게는 HMGB1의 발현 및 활성을 억제하는 융합단백질을 유효성분으로 함유하여 면역질환과 암의 치료에 유용한 제약 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition containing the fusion protein as an active ingredient, and more particularly, to a pharmaceutical composition containing a fusion protein that inhibits the expression and activity of HMGB1 as an active ingredient and useful for the treatment of immune diseases and cancer. .

산화환원인자-1(redox factor-1, ref-1)은 다기능 단백질로서, DNA의 손상시 손상부위를 복구하는 어퓨리닉/어피리미디닉 엔도뉴클레아제 (apurinic/ apyrimidinic endonuclease)의 기능 및 AP-1, NF-kB와 같은 전사인자의 환원성 조절기능(redox function) 등을 갖는 것으로 알려져 있다. 전사인자에 대한 환원성 조절은 여러 전사인자(transcription factor)의 DNA 결합 영역(binding domain)에서 산화형 시스테인(cysteine) 잔기를 환원시키는 것에 의해 이루어지는 것으로 알려져 있다(Xanthoudakis S, Miao GG and Curran T, The redox and DNA-repair activities of Ref-1 are encoded by non-overlapping domains. Proc. Natl. Acad. Sci. U.S.A. 91 (1994), pp. 23~27).Redox factor-1 (ref-1) is a multifunctional protein that functions as apurinic / apyrimidinic endonuclease that repairs damaged areas of DNA. It is known to have a redox function of transcription factors such as AP-1 and NF-kB. Reducible regulation of transcription factors is known by reducing oxidative cysteine residues in the DNA binding domain of several transcription factors (Xanthoudakis S, Miao GG and Curran T, The redox and DNA-repair activities of Ref-1 are encoded by non-overlapping domains.Proc. Natl. Acad. Sci. USA 91 (1994), pp. 23-27).

최근에는 산화환원인자-1의 기능이 추가적으로 밝혀지고 있는데, 혈관내피세포에서 산화환원인자-1은 일산화질소의 생성을 증가시켜 혈압을 조절한다는 것 (Jeon et al, Apurinic/apyrimidinic endonuclease 1 regulates endothelial NO production and vascular tone. Circ Res. 2004 Oct 29;95(9):902-10)과 항염증기전(Kim et al, Apurinic/apyrimidinic endonuclease1/redox factor-1 inhibits monocyte adhesion in endothelial cells. Cardiovasc Res. 2006 Feb 1;69(2):520-6)이 보고된 바 있다. 즉, 재조합아데노바이러스를 이용하여 산화환원인자-1을 혈관내피세포에 과발현시키면 일산화질소의 생성이 증가되고 활성산소의 생성이 감소되는 것이 보고되었으며, 이러한 작용에 의해 혈압 조절에 중요한 역할을 한다고 알려져 있다. 또한 아데노바이러스를 이용하여 산화환원인자-1을 혈관내피세포에 과발현시킨 경우, tumor-necrosis factor-α에 의해 유도되는 VCAM-1 (vascular cell adhesion molecule-1)의 발현이 억제되고 혈관내피세포에 단핵구의 유착도 억제된다는 것이 밝혀진 바 있다.In recent years, the function of redox factor-1 has been further revealed that redox factor-1 regulates blood pressure by increasing production of nitric oxide (Jeon et al , Apurinic / apyrimidinic endonuclease 1 regulates endothelial NO). Circ Res. 2004 Oct 29; 95 (9): 902-10) and anti-inflammatory mechanisms (Kim et al , Apurinic / apyrimidinic endonuclease1 / redox factor-1 inhibits monocyte adhesion in endothelial cells.Cardiovasc Res. 2006 Feb 1; 69 (2): 520-6). In other words, overexpression of redox factor-1 in vascular endothelial cells using recombinant adenovirus has been reported to increase the production of nitric oxide and decrease the production of free radicals. have. In addition, when redox factor-1 is overexpressed in vascular endothelial cells using adenovirus, expression of vascular cell adhesion molecule-1 (VCAM-1) induced by tumor-necrosis factor-α is inhibited. It has been found that the adhesion of monocytes is also inhibited.

이에 본 발명자들은 특허출원 제10-2007-67150호를 통하여 서열번호 1의 세포투과성 산화환원인자 융합단백질 Tat-ref-1을 제조하고, Tat-ref-1이 VCAM-1의 발현 및 단핵구 유착을 효과적으로 억제하여 이를 함유하는 조성물이 항염증 질환의 치료에 유용함을 입증한 바 있다. 상기 특허출원에서 융합단백질 Tat-ref-1은 산화환원인자-1 단백질의 구조와 기능을 유지하면서 세포투과성을 획득하여 산화환원인자-1 단백질을 세포내로 직접 도입시키는 효과를 얻을 수 있어, 산화환원인자- 1 단백질의 기능에 의한 약학적 조성물로 사용되는 경우 보다 유용함을 확인할 수 있었다.Therefore, the inventors of the present invention through Patent Application No. 10-2007-67150 The cell permeable redox factor fusion protein Tat-ref-1 of SEQ ID NO: 1 was prepared, and the composition containing Tat-ref-1 effectively inhibits the expression and monocyte adhesion of VCAM-1, which is useful for the treatment of anti-inflammatory diseases. Proved. In the patent application, the fusion protein Tat-ref-1 obtains cell permeability while maintaining the structure and function of the redox factor-1 protein, thereby obtaining the effect of directly introducing the redox factor-1 protein into the cell. When used as a pharmaceutical composition by the function of the factor-1 protein was found to be more useful.

High-mobility group box 1(HMGB1) 단백질은 모든 포유류의 핵에 고농도로 존재하며, DNA의 구조를 안정화 시키는 구조 단백질의 역할을 한다. HMGB1에 대한 초기 연구들은 대부분 패혈증에서의 역할에 치중되어 왔다. 패혈증은 세균 감염이나 외상적 충격 등으로 인체 면역시스템이 교란되어 지나칠 정도로 강한 면역 비상체제를 가동함으로써 생기는 일종의 '과다면역반응'이다. 정상인과 패혈증 환자의 혈청을 비교해 본 결과 정상인에 비해 패혈증 환자의 혈청에서 HMGB1의 농도가 크게 증가한 것을 알 수 있었다. 특히, 사망한 패혈증 환자의 경우 생존 패혈증 환자보다 HMGB1의 농도가 훨씬 더 높았다. 처그스트라우스증후군 (Churg Strauss syndrome) 및 류머티즘성 관절염 (rheumatoid arthritis) 환자의 혈청에서도 HMGB1의 농도가 건강인 보다 현저하게 높았으며, 처그스트라우스증후군 환자의 경우 HMGB1의 농도가 정상인의 32배 정도로 매우 높은 수치를 나타낸다고 보고되었다. 만성 자가면역성 질환인 쇼그렌증후군(Sogren's syndrome; SS) 환자의 침샘 조직검사에서 역시 건강인 보다 세포외로 분비된 HMGB1의 농도가 월등히 높게 나타났으며 전신성 면역계 질환인 전신성 홍반성 루프스 (systemic lupus erythematosus; SLE) 환자의 혈청에서도 건강인에 비해 HMGB1의 농도가 높게 나타났다. 이렇게 많은 면역질환과 자가면역 질환에서 HMGB1이 병인으로 중요한 역할을 하는 것이 밝혀지면서, 면역세포로 하여금 HMGB1의 분비를 차단하는 것에 의해 면역관용을 유도하는 것이 면역질환의 치료에 이용되고 있다.High-mobility group box 1 (HMGB1) protein is present in high concentrations in all mammalian nuclei and serves as a structural protein that stabilizes the structure of DNA. Early studies of HMGB1 have mostly focused on their role in sepsis. Sepsis is a type of 'hyperimmune reaction' caused by running an immune emergency system that is too strong to disturb the human immune system due to bacterial infection or traumatic shock. As a result of comparing sera of the normal and sepsis patients, the HMGB1 concentration was significantly increased in the serum of the sepsis patients compared to the normal. In particular, deceased sepsis patients had significantly higher concentrations of HMGB1 than patients with surviving sepsis. Serum levels of Chug Strauss syndrome and rheumatoid arthritis were significantly higher in HMGB1 concentrations than in healthy subjects, and HMGB1 levels were 32 times higher in healthy patients. Has been reported. Salivary gland biopsy of patients with Sogren's syndrome (SS), a chronic autoimmune disease, showed significantly higher levels of HMGB1 secreted extracellularly than healthy people. ) The serum levels of HMGB1 were higher than those of healthy subjects. As HMGB1 plays an important role as a etiology in many immune diseases and autoimmune diseases, inducing immune tolerance by blocking immune cell secretion of HMGB1 has been used in the treatment of immune diseases.

최근의 보고에 의하면 HMGB1은 암의 진행에서 역시 중요한 역할을 담당하며 다양한 유형의 암환자에서 정상인에 비해서 그 농도가 높게 나타난다. 대표적인 예로서, 일반인의 조직에 비해서 유방암 환자의 경우 HMGB1 단백질의 발현이 증가되는 것을 볼 수 있었고, 에스트로겐 수용체의 조절자 역할을 담당한다고 보고되었다. 대장암 모델에서는 HMGB1이 antiapoptotic oncoprotein으로써 작용함으로써, 암세포의 아포프토시스를 억제하고 대장암 세포의 이동성 및 침윤성을 증가시키는 것과 동시에 전이과정에도 중요한 역할을 담당한다. 이밖에도 흑색종, 전립선암, 폐암, 췌장암에도 관여한다고 보고되었다. 이제까지 알려진 바에 의하면 HMGB1은 E-selectin, TNF-α, 인슐린 수용체의 전사(transcription)과정에 관여하며, HMGB1과 그 수용체인 RAGE의 상호작용이 외부에서 이식된 C6 신경교종 암세포의 침윤, 이동, 성장 및 확산 등과 같은 다양한 작용을 매개하며, 상기 상호작용을 방해했을 때 이러한 작용들이 억제되었다. Recent reports suggest that HMGB1 also plays an important role in cancer progression and is higher in normalized levels in patients with various types of cancer. As a representative example, the expression of HMGB1 protein was increased in breast cancer patients compared to general tissues, and it was reported to play a role of a regulator of estrogen receptor. In the colorectal cancer model, HMGB1 acts as an antiapoptotic oncoprotein, inhibiting apoptosis of cancer cells, increasing the mobility and invasiveness of colorectal cancer cells, and playing an important role in metastasis. In addition, it has been reported to be involved in melanoma, prostate cancer, lung cancer and pancreatic cancer. To date, HMGB1 is involved in the transcription of E-selectin, TNF-α, and insulin receptors, and the interaction of HMGB1 with its receptor RAGE invades, migrates and grows externally transplanted C6 glioma cancer cells. And various actions such as diffusion and the like, and these actions were suppressed when they interfered with the interaction.

본 발명은 면역질환 및 암의 주요 병인으로 작용하는 HMGB1의 발현 및 활성을 억제하여 면역질환과 암의 치료에 유용한 제약 조성물을 제공하는 것을 그 목적으로 한다.It is an object of the present invention to provide a pharmaceutical composition useful for the treatment of immune diseases and cancers by inhibiting the expression and activity of HMGB1 which acts as a major etiology of immune diseases and cancers.

전술한 목적을 달성하기 위한 본 발명은 서열번호 1의 Tat-ref-1 융합단백질을 유효성분으로 함유하는 HMGB-1을 병인으로 하는 면역질환 및 암 치료용 제약 조성물인 것을 특징으로 한다.The present invention for achieving the above object is characterized in that the pharmaceutical composition for the treatment of immunological diseases and cancers with the etiology of HMGB-1 containing the Tat-ref-1 fusion protein of SEQ ID NO: 1 as an active ingredient.

ref-1 또는 Tat-ref-1 융합단백질 및 이들을 발현할 수 있는 발현벡터 또는 아데노바이러스를 사용하여, HMGB-1에 의해 야기되는 활성산소종(ROS) 및 각종 사이토카인의 발현 및 HMGB-1의 분비에 대한 산화환원인자-1 단백질의 영향을 확인하였다. 특허출원 10-2007-67150호에서 확인한 바와 마찬가지로 Tat-ref-1 융합단백질은 산화환원인자-1의 구조와 기능을 유지하면서 세포투과성을 갖는 단백질로서 제약학적 조성물로 보다 유용한 융합단백질이다. Expression of reactive oxygen species (ROS) and various cytokines caused by HMGB-1 and expression of HMGB-1 using ref-1 or Tat-ref-1 fusion proteins and expression vectors or adenoviruses capable of expressing them The effect of redox factor-1 protein on secretion was confirmed. As confirmed in Patent Application No. 10-2007-67150, Tat-ref-1 fusion protein is a protein having cell permeability while maintaining the structure and function of redox factor-1, and is a more useful fusion protein as a pharmaceutical composition.

본 발명의 실시예에서 확인할 수 있듯이 ref-1 및 Tat-ref-1은 면역질환이나 암의 존재 시 단핵구 내에 고농도로 존재하는 HMGB-1에 의해 야기되는 활성산소종(ROS)과 각종 사이토카인의 형성을 효과적으로 억제하여 부수적인 염증 반응을 억제하고 암세포의 형성을 방지한다. 또한, HMGB-1의 자극에 의해 유발되는 COX-2의 발현 역시 효과적으로 억제함을 알 수 있었다. COX-2는 유방암, 대장암, 췌장암 등 종양 성장에 중요한 역할을 담당하는 것으로 알려져 있다. As can be seen in the embodiments of the present invention, ref-1 and Tat-ref-1 are a combination of reactive oxygen species (ROS) and various cytokines caused by HMGB-1 present in high concentrations in monocytes in the presence of immune diseases or cancer. It effectively inhibits the formation of an inhibitory inflammatory response and prevents the formation of cancer cells. In addition, it was also found that the expression of COX-2 caused by the stimulation of HMGB-1 is also effectively suppressed. COX-2 is known to play an important role in tumor growth, such as breast cancer, colon cancer, pancreatic cancer.

이와 같이 Tat-ref-1은 면역질환이나 암 환자에 정상인에 비해 고농도로 존재하는 HMGB-1에 의해 생산되는 염증 및 암의 진행과 관련된 인자들의 발현 수준을 크게 낮출 뿐 아니라, 전염증성 사이토카인에 의한 HMGB-1 자체의 세포외 분비 수 준을 억제하여 면역질환 및 암의 치료제로서의 효용성을 확인하였다.As such, Tat-ref-1 significantly lowers the expression level of factors related to inflammation and cancer progression produced by HMGB-1, which is present in high concentrations in immune disease or cancer patients, as well as in proinflammatory cytokines. Inhibiting the extracellular secretion level of HMGB-1 itself to confirm its efficacy as a therapeutic agent for immune diseases and cancer.

Tat-ref-1 융합단백질을 유효성분으로 함유하는 약제학적 조성물은 약제학적 분야에 통상적으로 허용되는 담체, 부형제, 희석제 등과 함께 배합하여 피부도말제제, 경구, 정맥주사 및 근육주사제 형태로 제형화할 수 있다. Pharmaceutical compositions containing Tat-ref-1 fusion protein as an active ingredient may be formulated in the form of skin smears, oral, intravenous and intramuscular injections, in combination with carriers, excipients, diluents, and the like, commonly accepted in the pharmaceutical arts. Can be.

본 발명에 따른 유효성분의 투여량은 체내에서 활성성분의 흡수도, 활성화율 및 배설속도, 환자의 연령, 성별 및 상태, 치료할 질병의 중증정도 등에 따라 적절히 선택되나, 일반적으로 0.1~10 nM이 바람직하며 1회 내지 수회로 나누어 투여할 수 있다.The dosage of the active ingredient according to the present invention is appropriately selected depending on the absorption rate, activation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, but generally 0.1 to 10 nM Preferred and can be administered once to several times.

이상과 같이 본 발명의 Tat-ret-1 융합단백질을 유효성분으로 함유하는 제약 조성물은 HMGB1의 발현을 효과적으로 억제하여 HMGB1의 발현과 관련한 면역질환, 자가면역질환 및 암의 예방 및 치료에 유용하게 이용될 수 있다. As described above, the pharmaceutical composition containing the Tat-ret-1 fusion protein of the present invention as an active ingredient effectively inhibits the expression of HMGB1 and is useful for the prevention and treatment of immune diseases, autoimmune diseases and cancers related to the expression of HMGB1. Can be.

이하 실시예를 통하여 본 발명을 상세하게 설명한다. 그러나, 이들 실시예는 예시적인 목적일 뿐 본 발명이 이에 한정되는 것은 아니다. The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

실시예Example

실시예 1 : Tat-ref-1 발현벡터의 제조 및 형질전환Example 1 Preparation and Transformation of Tat-ref-1 Expression Vector

서열번호 1의 Tat-ref-1 발현벡터는 특허출원 10-2007-67150호에 기재된 방법에 의해 제조하였으며, 대장균 형질전환체 BL21(DE3)/pTat-ref-1로부터 융합단백질 Tat-ref-1을 발현시켜 얻었다. The Tat-ref-1 expression vector of SEQ ID NO: 1 was prepared by the method described in Patent Application No. 10-2007-67150, and the fusion protein Tat-ref-1 from E. coli transformant BL21 (DE3) / pTat-ref-1 It was obtained by expression.

ref-1의 산화/환원 활성에는 65번 및 93번의 cysteine 잔기가 관여하는 것으로 알려져 있다. 이에, ref-1 대신 ref-1의 65번과 93번 cyteine 잔기가 alanine으로 치환된 [C65A/C93A] 돌연변이 ref-1(muref-1) (피츠버그의과대학 Kaikobad Irani교수가 기증함)을 이용하여 특허출원 10-2007-67150호에 기재된 것과 동일한 방법에 의해 대조군으로 Tat-muref-1 발현벡터를 제조하고 대장균 BL21(DE3)에 형질전환한 대장균 형질전환체 및 융합단백질 Tat-muref-1을 얻었다.It is known that cysteine residues 65 and 93 are involved in the oxidation / reduction activity of ref-1. Thus, using the [C65A / C93A] mutant ref-1 (muref-1) (contributed by Professor Kaikobad Irani, Pittsburgh Medical School) in which cyteine residues 65 and 93 of ref-1 were replaced with alanine instead of ref-1. A Tat-muref-1 expression vector was prepared as a control by the same method as described in Patent Application No. 10-2007-67150, and E. coli transformant and fusion protein Tat-muref-1 transformed to E. coli BL21 (DE3) were obtained. .

실시예 2 : 재조합 아데노바이러스의 생산Example 2 Production of Recombinant Adenovirus

Ad-null은 control 아데노바이러스로서 Clontech사 (Mountain, CA, USA)에서 구입하였으며, ref-1 유전자를 함유하는 재조합 아데노바이러스 Ad-ref-1은 피츠버그의과대학 Kaikobad Irani교수로부터 기증받았다. ref-1의 65번과 93번 cyteine 잔기가 alanine으로 치환된 [C65A/C93A] 돌연변이 ref-1(muref-1)유전자(피츠버그의과대학 Kaikobad Irani교수가 기증)를 함유하는 재조합 아데노바이러스는 Labfrontier Co (Korea)에서 주문 제작하였다. Ad-null was purchased from Clontech (Mountain, CA, USA) as a control adenovirus, and the recombinant adenovirus Ad-ref-1 containing the ref-1 gene was donated by Professor Kaikobad Irani of the University of Pittsburgh School of Medicine. Recombinant adenoviruses containing the [C65A / C93A] mutant ref-1 (muref-1) gene (donated by Professor Kaikobad Irani, University of Pittsburgh Medical School) in which cyteine residues 65 and 93 of ref-1 were substituted with alanine were labfrontier co. Made to order from (Korea).

Ad-null, Adref-1 혹은 Ad-muref-1 아데노바이러스는 HEK 293 세포주에서 증폭을 하였으며, 증폭된 아데노바이러스는 Clontech사 (Mountain View, CA)에서 구입한 Adeno-X virus purification kit을 이용하여 제공된 방법에 따라 정제하였다. 정제된 재조합 아데노바이러스는 THP-1 세포주에 18시간 동안 감염시켰다. Ad-null, Adref-1 or Ad-muref-1 adenovirus was amplified in the HEK 293 cell line, and the amplified adenovirus was provided using an Adeno-X virus purification kit purchased from Clontech (Mountain View, CA). Purification was according to the method. Purified recombinant adenovirus was infected with THP-1 cell line for 18 hours.

실시예 3 : 인간 단핵구에서 HMGB1에 의해 유도되는 세포내 활성산소족(ROS)에 대한 ref-1의 영향 측정 Example 3 Determination of the Effect of ref-1 on Intracellular Reactive Oxygen (ROS) Induced by HMGB1 in Human Monocytes

HMGB1에 의한 ROS 생성이 유도되는지 확인하기 위하여, 다음과 같이 사람 primary monocytes에 HMGB1을 처리한 후에 superoxide 생성을 측정하였다.In order to confirm that ROS production by HMGB1 was induced, superoxide production was measured after HMGB1 treatment in human primary monocytes as follows.

1) 단핵구 준비 및 세포 배양1) Monocyte Preparation and Cell Culture

건강인 공혈자로부터 정맥혈을 제공받아 Histopaque-1077(Sigma-Aldrich)을 이용한 밀도구배 원심분리로 말초혈액세포 (peripheral blood mononuclear cells)를 수집하였다. 수집한 말초혈액단핵세포는 배양액에 2×106/ml의 농도로 부유시켜 37℃, 5% CO2 조건에서 24-well culture plate에 부착시켰다. 1시간 후에 부착되지 않은 상층액은 제거하고 새 배지로 채워 plate에 부착된 단핵구만 실험에 사용하였다. 분리된 단핵구의 순도는 항 CD14 항체를 이용한 유세포 측정으로 95% 이상임을 확인하였다. Peripheral blood mononuclear cells were collected by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich). The collected peripheral blood mononuclear cells were suspended in culture at a concentration of 2 × 10 6 / ml and attached to 24-well culture plates at 37 ° C. and 5% CO 2 . After 1 hour, the unattached supernatant was removed and filled with fresh medium, and the monocytes attached to the plate were used for the experiment. Purity of the isolated monocytes was confirmed to be 95% or more by flow cytometry using an anti-CD14 antibody.

하기 단핵구를 이용한 모든 실시예의 실험은 별도의 언급이 없는 한 본 방법에 의해 얻어진 단핵구를 이용하였다.Experiments of all the examples using the following monocytes used monocytes obtained by the present method unless otherwise stated.

2) 아데노바이러스의 도입 및 HMGB1 자극2) Introduction of Adenovirus and HMGB1 Stimulation

1)에서 준비한 단핵구 내에 실시예 2에서 제조한 대조군 아데노 바이러스(Ad-null), ref-1이 암호화된 재조합 아데노 바이러스(Ad-ref-1) 또는 돌연변이 muref-1을 암호화하는 재조합 아데노 바이러스(Ad-muref-1)을 200pfu씩 LipofectAMINE 2000 (Invitrogen, Carlsbad, CA)을 이용하여 세포내로 도입시켰다. 정상 배양액에서 24시간 배양한 후, 산화효소(oxidase) 억제제인 diphenyleneiodium(DPI, Calbiotech) 20 μM로 처리 또는 처리되지 않은 도입된 세포들을 HMGB1(Sigma) 100 ng/ml로 15분 동안 자극시켰다. In the monocytes prepared in 1), the control adenovirus (Ad-null) prepared in Example 2, the recombinant adenovirus (Ad-ref-1) encoded by ref-1 or the recombinant adenovirus (Ad-encoded mutated muref-1 (Ad-null) -muref-1) were introduced into cells using LipofectAMINE 2000 (Invitrogen, Carlsbad, Calif.) at 200 pfu. After incubation for 24 hours in normal culture, introduced cells treated with or without treatment with 20 μM of diphenyleneiodium (DPI, Calbiotech), an oxidase inhibitor, were stimulated with HMGB1 (Sigma) 100 ng / ml for 15 minutes.

3) ROS 형성 측정3) ROS formation measurement

생성된 ROS는 산화적 형광 염색시약인 dihydroethidium(DHE, Calbiothech)을 사용하여 유세포 분석기 또는 형광현미경으로 측정하고(J. Exp. Med. 204 583-594 (2007), Cell Microbiology 10 741-754 (2007)) 그 결과를 도 1의 A와 B에 각각 도시하였다. The generated ROS was measured by flow cytometry or fluorescence microscopy using an oxidative fluorescent dye dihydroethidium (DHE, Calbiothech) (J. Exp. Med. 204 583-594 (2007), Cell Microbiology 10 741-754 (2007) The results are shown in FIGS. 1A and 1B, respectively.

유세포 분석은 HMGB1 (100 ng/ml)으로 처리한 단핵구를 DHE (20 μM)로 30분 동안 반응시켜 염색한 후 세척하고 FACS Calibur(BD Biosciences, San Jose, CA)에서 분석하였으며, 데이터들은 CellQuest software(BD Biosciences)를 이용하여 그래프로 나타내었다. Flow cytometry was performed by staining the monocytes treated with HMGB1 (100 ng / ml) with DHE (20 μM) for 30 minutes, staining, washing and analyzing in FACS Calibur (BD Biosciences, San Jose, Calif.). (BD Biosciences) is shown graphically.

현광현미경 측정은 ROS 형성은 HMGB1 (100 ng/ml)으로 처리한 단핵구를 30분 동안 DHE (20 μM)으로 반응시키고 공초점현미경으로 분석하였다. 살아있는 세포들은 혈청이 들어있지 않은 배지로 세척하여 레이저 주사 현미경으로 분석하였다. 도 1의 B에서 바의 길이는 50㎛이다.Light microscopy was measured ROS formation was reacted with DHE (20 μM) for 30 minutes in monocytes treated with HMGB1 (100 ng / ml) and analyzed by confocal microscopy. Living cells were washed with medium without serum and analyzed by laser scanning microscope. The length of the bar in B of FIG. 1 is 50 μm.

도 1의 A와 B에서 확인할 수 있듯이 HMGB1 자극 15분에 세포내 활성산소족 분비가 증가되었고, 이 효과는 NADPH 산화효소 (oxidase) 억제제인 DPI에 의해 억제되었다. Ad-null 혹은 Ad-muref-1을 도입한 primary monocytes에서는 HMGB1에 반응하여 ROS 생성이 증가되었으나, Ad-ref-1을 도입한 primary monocytes에서는 ROS 생성이 DPI를 전처리한 세포에서의 수준처럼 거의 완전히 저해되었다.As can be seen from A and B of FIG. 1, 15 minutes of HMGB1 stimulation increased intracellular reactive oxygen secretion, and this effect was inhibited by DPI, a NADPH oxidase inhibitor. In primary monocytes in which Ad-null or Ad-muref-1 was introduced, ROS production was increased in response to HMGB1, but in primary monocytes in which Ad-ref-1 was introduced, ROS production was almost completely as level as in DPI pretreated cells. Inhibited.

4) NADPH 활성에 대한 ref-1의 영향 측정 4) Determining the Effect of ref-1 on NADPH Activity

HMGB1에 의해 유도되는 ROS 생성은 DPI에 의해 억제되기 때문에, ref-1이 HMGB1에 의해 조절되는 NADPH(Nicotinamide adenine dinucleotide phosphate) 활성에 영향을 주는지를 조사하였다.Since ROS production induced by HMGB1 is inhibited by DPI, we investigated whether ref-1 affects nicotinamide adenine dinucleotide phosphate (NADPH) activity regulated by HMGB1.

NADPH 활성은 lucigenin 염색시약을 이용한 발광 (luminescence)법으로 측정하고(Cell Microbiol. 9 382-396 (2007), J. Leukoc. Biol. 81 59-66 (2007)) 그 결과를 도 2에 나타내었다. 도 2에서 D는 대조군으로서 0.1% DMSO를 처리한 세포, U는 Ad-null이 도입된 세포, wt는 Ad-ref-1이 도입된 세포, mu는 Ad-muref-1이 도입된 세포를 각각 나타낸다. NADPH activity was measured by luminescence using lucigenin staining reagent (Cell Microbiol. 9 382-396 (2007), J. Leukoc. Biol. 81 59-66 (2007)) and the results are shown in FIG. . In FIG. 2, D is a control cell treated with 0.1% DMSO, U is Ad-null-induced cell, wt is Ad-ref-1-introduced cell, and mu is Ad-muref-1-introduced cell, respectively. Indicates.

도 2에서 확인할 수 있듯이, HMGB1으로 Ad-null이 도입된 세포들을 자극한 것과 비교했을 때, Ad-ref-1이 도입된 사람의 단핵구에서는 NADPH oxidase 활성은 감소되었지만 Ad-muref-1이 도입된 세포들에서는 거의 변화가 없었다. 이 결과들로 단핵구/대식세포에서 ref-1은 HMGB1에 의해 유도되는 NADPH oxidase의 활성화와 ROS 생성을 억제시킴을 알 수 있다.As can be seen in Figure 2, compared with the stimulation of Ad-null-induced cells with HMGB1, N-ADPH oxidase activity was reduced in the monocytes of human Ad-ref-1 introduced but Ad-muref-1 was introduced There was little change in the cells. These results indicate that ref-1 inhibits the activation of NADPH oxidase and ROS production by HMGB1 in monocytes / macrophages.

실시예 4 : HMGB1 유도 전염증성 사이토카인 형성에 대한 Tat-ref-1 또는 ref-1의 영향Example 4 Effect of Tat-ref-1 or ref-1 on HMGB1 Induced Proinflammatory Cytokine Formation

1) 단핵구에서 사이토카인의 생성 억제1) inhibition of cytokine production in monocytes

이전 연구들로 단핵구에서 HMGB1의 처리는 TNF-α, IL-6, IL-1α, 그리고 IL-1β를 포함한 전염증성 사이토카인을 분비한다는 것을 알 수 있다. 이에, HMGB1에 의해 유도되는 전염증성 사이토카인 형성이 Tat-ref-1에 의해 조절되는지를 조사하였다. Previous studies have shown that treatment of HMGB1 in monocytes secretes pro-inflammatory cytokines including TNF-α, IL-6, IL-1α, and IL-1β. Therefore, we investigated whether proinflammatory cytokine formation induced by HMGB1 is regulated by Tat-ref-1.

실시예 3의 1)의 방법으로 얻은 단핵구에 HMGB1 (100 ng/ml) 또는 LPS(Escherichia coli 026:B6, Sigma) 100 ng/ml를 첨가하여 RPMI 1640 배지[10% fetal bovine serum, sodium pyruvate, non-essential amino acids, penicillin G (100 IU/ml) and streptomycin (100 μg/ml)이 들어있는]에서 배양하였다. 세포 배양 상층액을 도 3의 그래프에 기재된 시간별로 채취하여 TNF-α 와 IL-6의 양을 PharMingen사의 Duoset antibody pairs를 이용하여 ELISA 방법에 의해 측정하고 그 결과를 도 3에 나타내었다. HMGB1 (100 ng / ml) or LPS (Escherichia coli 026: B6, Sigma) 100 ng / ml was added to the monocytes obtained by the method of Example 3), and then RPMI 1640 medium [10% fetal bovine serum, sodium pyruvate, incubated with non-essential amino acids, penicillin G (100 IU / ml) and streptomycin (100 μg / ml). Cell culture supernatants were collected for each time described in the graph of FIG. 3 and the amounts of TNF-α and IL-6 were measured by ELISA using PharMingen's Duoset antibody pairs, and the results are shown in FIG. 3.

상기 사이토카인 생성에 미치는 Tat-ref-1 융합단백질의 영향을 확인하기 위하여 HMGB1 자극전에 Tat-ref-1 또는 돌연변이 Tat-muref-1을 0.1, 1, 10 nM로 미리 1시간 동안 처리한 것을 제외하고는 상기와 동일한 방법에 의해 HMGB1 100 ng/ml을 넣고 18시간동안 배양하였다. 배양 후 세포배양 상층액으로부터 TNF-α 와 IL-6의 양을 동일한 방법에 의해 측정하고 그 결과를 도 4에 나타내었다. 도 4에서 U는 전처리를 하지 않은 대조군을, D는 0.1% DMSO를, REF1 및 muREF1은 각각 Tat-ref-1 및 Tat-muref-1을 처리한 군을 나타내며 왼쪽으로부터 처리 농도가 0.1, 1, 10nM에 해당한다. Tat-ref-1 or mutant Tat-muref-1 was treated with 0.1, 1, 10 nM for 1 hour before HMGB1 stimulation to confirm the effect of Tat-ref-1 fusion protein on the cytokine production Then, in the same manner as above, HMGB1 100 ng / ml was added and incubated for 18 hours. After culture, the amount of TNF-α and IL-6 was measured from the cell culture supernatant by the same method, and the results are shown in FIG. 4. In FIG. 4, U represents a control group without pretreatment, D represents 0.1% DMSO, and REF1 and muREF1 treat Tat-ref-1 and Tat-muref-1, respectively. It corresponds to 10 nM.

도 3에서 확인할 수 있듯이 단핵구에서 HMGB1에 의한 TNF-α 와 IL-6 발현은 자극 후 18시간에서 최고점이었다. 그러나 Tat-ref-1 단백질을 전처리한 경우, HMGB1에 의해 자극된 human primary monocytes에서 TNF-α 생성이 HMGB1의 농도 의존적으로 확실하게 억제되는 것을 확인할 수 있었다(도 4). TNF-α 억제 효과와 비교하여, Tat-ref-1을 도입한 경우 HMGB1에 의한 IL-6 생성은 약간 억제되었다. 이에 반해, TAT-Tat-muref-1을 도입한 경우에는 TNF-α 나 IL-6의 발현을 전혀 억제하지 못하였다.As can be seen in FIG. 3, TNF-α and IL-6 expression by HMGB1 in monocytes peaked at 18 hours after stimulation. However, when the Tat-ref-1 protein was pretreated, it was confirmed that TNF-α production was surely suppressed in a concentration dependent manner of HMGB1 in human primary monocytes stimulated by HMGB1 (FIG. 4). Compared with the TNF-α inhibitory effect, the introduction of Tat-ref-1 slightly inhibited IL-6 production by HMGB1. In contrast, the introduction of TAT-Tat-muref-1 did not inhibit the expression of TNF-α or IL-6.

2) THP-1 세포에서 사이토카인의 생성 억제2) Inhibition of Cytokine Production in THP-1 Cells

THP-1 cells에서 ref-1 효과를 조사하기 위해, THP-1 세포를 4 nM PMA (phorbol-12-myristate-13-acetate)를 24시간 처리하여 대식세포로 분화시킨 후 PBS로 세 번 세척하여 사용하였다. THP-1 세포에 empty vector control (mock), ref-1을 암호화하는 재조합 벡터(ref-1) 및 기능을 갖지 못하는 ref-1를 암호화하는돌연변이 벡터(muref-1)를 세포에 도입하였다. 24시간 후 도입된 세포들을 HMGB1 (100 ng/ml)으로 18시간 동안 자극시켰으며 상층액을 모아 ELISA 방법으로 TNF-α 와 IL-6 수준을 분석하고 그 결과를 도 5에 도시하였다.To investigate the ref-1 effect in THP-1 cells, THP-1 cells were treated with 4 nM PMA (phorbol-12-myristate-13-acetate) for 24 hours to differentiate into macrophages and washed three times with PBS. Used. Into THP-1 cells, empty vector control (mock), recombinant vector (ref-1) encoding ref-1, and mutant vector (muref-1) encoding ref-1 having no function were introduced into the cells. The cells introduced after 24 hours were stimulated with HMGB1 (100 ng / ml) for 18 hours. The supernatants were collected and analyzed for TNF-α and IL-6 levels by ELISA method and the results are shown in FIG. 5.

상기에서 사용한 발현벡터의 제조를 위하여 특허출원 10-2007-67150호에 기재된 방법에 의해 ref-1 유전자의 전체 염기서열을 암호화하는 야생형 (wild-type) ref-1 유전자를 분리하였다. EcoRI 및 HindIII 제한효소를 사용하여 pCMV-Tag2B (Stratagene, USA) 벡터를 절단한 다음 상기 ref-1 cDNA 또는 muref-1 cDNA를 삽입하여 ref-1 발현벡터와 muref-1 발현벡터를 각각 완성하여 사용하였다. In order to prepare the expression vector used above, a wild-type ref-1 gene encoding the entire nucleotide sequence of the ref-1 gene was isolated by the method described in Patent Application 10-2007-67150. The pCMV-Tag2B (Stratagene, USA) vector was digested using EcoRI and HindIII restriction enzymes, and then the ref-1 cDNA or muref-1 cDNA were inserted to complete the ref-1 expression vector and the muref-1 expression vector. It was.

도 5에서 확인할 수 있듯이, mock control 혹은 muref-1이 도입된 세포에서는 HMGB1의 자극으로 사이토카인 분비가 활발히 유도되었다. 그러나, ref-1이 도입된 THP-1 cells 에서는 HMGB1에 의한 TNF-a 와 IL-6의 발현이 완전히 억제되었다. As can be seen in FIG. 5, cytokine secretion was actively induced by stimulation of HMGB1 in mock control or muref-1 cells. However, in THP-1 cells with ref-1, expression of TNF-a and IL-6 by HMGB1 was completely inhibited.

이와 같은 결과로부터 단핵구/대식세포에서 Tat-ref-1은 HMGB1의 자극에 대한 전염증성 반응의 강력한 억제자임을 알 수 있었다.These results indicate that Tat-ref-1 is a potent inhibitor of proinflammatory responses to stimulation of HMGB1 in monocytes / macrophages.

실시예 5 : HMGB1로 자극된 단핵구에서 COX-2 발현에 대한 Tat-ref-1의 영향Example 5 Effect of Tat-ref-1 on COX-2 Expression in Monocytes Stimulated with HMGB1

HMGB1으로 자극된 단핵구에서 COX-2 발현에 대한 Tat-ref-1의 영향을 확인하기 위하여 먼저 HMGB1에 의해 조절되는 COX-2 발현을 조사하고 그 결과를 도 6의 A에 나타내었다. 즉, 사람 단핵구에 HMGB1 100 ng/ml를 도 6의 A에 기재된 시간별로 처리한 후 하였다. 세포들을 모은 후 항-COX-2 항체를 이용하여 Western blot을 수행하였다. 동일한 막에 붙어 있는 항체들은 떼어내고 β-actin 항체를 붙여 단백질 양을 비교 분석하였다. In order to confirm the effect of Tat-ref-1 on COX-2 expression in monocytes stimulated with HMGB1, COX-2 expression regulated by HMGB1 was first investigated and the results are shown in FIG. 6A. That is, human monocytes were treated with 100 ng / ml of HMGB1 for each time described in FIG. 6A. After the cells were collected, Western blot was performed using anti-COX-2 antibody. Antibodies attached to the same membrane were detached and β-actin antibody was added to compare protein levels.

도 6의 A에서 확인할 수 있듯이, 단핵구에서 HMGB1 자극은 시간 의존적으로 COX-2 발현을 활발히 유도하였으며, HMGB1으로 자극 후 18시간 내에 COX-2 발현이 최고였고, 이후부터 발현이 감소되었다. As can be seen in Figure 6A, HMGB1 stimulation in monocytes actively induced COX-2 expression in a time-dependent manner, COX-2 expression was the highest within 18 hours after stimulation with HMGB1, after which expression decreased.

다음으로 HMGB1에 의한 COX-2 발현이 Tat-ref-1에 의해 조절되는지를 조사하기 위하여 단핵구들은 1 nM TAT-Tat-ref-1 (wt) 또는 돌연변이 TAT-Tat-ref-1 (Tat-muref-1) 단백질로 1시간 동안 전처리한 후 18시간 동안 HMGB1 (100 ng/ml)으로 자극하였다. 자극된 세포들을 모아, 항-COX-2 항체로 Western blot에 의해 활성화된 COX-2 수준을 측정하고 그 결과를 도 6의 B에 도시하였다. Next, to examine whether COX-2 expression by HMGB1 is regulated by Tat-ref-1, monocytes were either 1 nM TAT-Tat-ref-1 (wt) or mutant TAT-Tat-ref-1 (Tat-muref). -1) pretreated with protein for 1 hour and then stimulated with HMGB1 (100 ng / ml) for 18 hours. Stimulated cells were collected and measured for COX-2 levels activated by Western blot with an anti-COX-2 antibody and the results are shown in FIG. 6B.

이와 별도로, 단핵구에 empty adenoviral vector control (Ad-null), ref-1이 암호화된 재조합 아데노바이러스(Ad-ref-1) 또는 돌연변이 ref-1을 암호화하는 재조합 아데노바이러스 (Ad-muref-1) 각각을 200 moi씩 세포내로 도입시켰다. 각 아데노바이러스가 도입된 세포들을 정상 배지에서 24시간 동안 배양한 후 18시간 동안 HMGB1(100 ng/ml)으로 자극시켰다. 18시간 후 세포들을 15분 동안 4% p-formaldehyde로 고정하였고 15분 동안 0.1% Triton X-100으로 삼투화시킨 다음 3% BSA가 포함된 PBS로 1시간 동안 반응시켜 반응을 중지시켰다. COX-2 염색을 위해, 세포들을 항-COX-2 항체 (Cell Signaling) (1:100)로 3시간 동안 염색하였고, 세척한 다음 rhodamine (TRITC)가 결합된 anti-rabbit IgG로 1시간 동안 반응시켰다. laser-scanning confocal microscope (LSM 510, version 2.3; Carl Zeiss, Inc., Thornwood, NY)을 사용하여 COX-2 면역형광 사진을 관찰하고 그 결과를 도 7에 나타내었다.Separately, empty adenoviral vector control (Ad-null), a recombinant recombinant adenovirus (Ad-ref-1) that encodes ref-1, or a recombinant adenovirus (Ad-muref-1) that encodes mutant ref-1, respectively, in monocytes. Were introduced into the cells at 200 moi. Each adenovirus introduced cells were incubated in normal medium for 24 hours and then stimulated with HMGB1 (100 ng / ml) for 18 hours. After 18 hours, the cells were fixed with 4% p- formaldehyde for 15 minutes, osmosis with 0.1% Triton X-100 for 15 minutes, and then reacted with PBS containing 3% BSA for 1 hour to stop the reaction. For COX-2 staining, cells were stained with anti-COX-2 antibody (Cell Signaling) (1: 100) for 3 hours, washed and then reacted with rhodamine (TRITC) bound anti-rabbit IgG for 1 hour. I was. COX-2 immunofluorescence photographs were observed using a laser-scanning confocal microscope (LSM 510, version 2.3; Carl Zeiss, Inc., Thornwood, NY) and the results are shown in FIG. 7.

도 6의 B와 도 7로부터 HMGB1에 의한 COX-2 발현이 Tat-ref-1 단백질 또는 Ad-ref-1 아데노바이러스를 처리한 단핵구에서 점차적으로 감소되는 것을 확인할 수 있었다. 그러나, Tat-muref-1 혹은 mock control protein 과 Ad-muref-1 혹은 Ad-null 아데노바이러스 처리된 단핵구에서는 COX-2의 발현이 조절되지 않았다.6 and 7, COM-2 expression by HMGB1 was gradually decreased in monocytes treated with Tat-ref-1 protein or Ad-ref-1 adenovirus. However, the expression of COX-2 was not regulated in Tat-muref-1 or mock control proteins and Ad-muref-1 or Ad-null adenovirus treated monocytes.

실시예 6: 전염증성 사이토카인들에 의한 HMGB1의 세포외 분비에 대한 Tat-ref-1의 영향Example 6: Effect of Tat-ref-1 on Extracellular Secretion of HMGB1 by Proinflammatory Cytokines

단핵구와 대식세포는 선천·적응면역 반응을 강하게 유도하는 TLR ligand와 상호작용으로 HMGB1를 분비한다고 알려져 있다. TLR4 (LPS, 100 ng/ml) 혹은 TLR3 (poly I:C, 20 μg/ml) 리간드에 의한 HMGB1의 세포외 분비가 Tat-ref-1에 의해 조절되는지를 검증하였다. Monocytes and macrophages are known to secrete HMGB1 by interacting with TLR ligands that strongly induce an innate and adaptive immune response. It was verified whether the extracellular secretion of HMGB1 by TLR4 (LPS, 100 ng / ml) or TLR3 (poly I: C, 20 μg / ml) ligand was regulated by Tat-ref-1.

사람의 단핵구들은 도 8의 A에 기재된 시간별로 100 ng/ml LPS (InvivoGen) (왼쪽)와 20 μg/ml ploy I:C (InvivoGen) (오른쪽)로 자극하였다. 세포배양 상층액을 모아 동일한 양의 단백질들이 포함된 상층액에 세척된 50 μl 단백질-G Sepharose beads를 넣고 4℃에서 하루 밤동안 반응시켜 면역침강을 수행하였다. 샘플들은 용출용액 (lysis buffer)으로 세 번 세척한 후 항-HMGB1 항체로 Western blot 분석을 수행한 후 그 결과를 도 8의 A에 나타내었다. 단핵구에서 HMGB1의 세포외 분비는 LPS 혹은 poly I:C를 처리한 18시간 후에 가장 높게 나타났다. Human monocytes were stimulated with 100 ng / ml LPS (InvivoGen) (left) and 20 μg / ml ploy I: C (InvivoGen) (right) at the time described in FIG. 8A. The cell culture supernatant was collected, and 50 μl protein-G Sepharose beads washed in the supernatant containing the same amount of protein were reacted at 4 ° C. overnight to perform immunoprecipitation. Samples were washed three times with lysis buffer and then subjected to Western blot analysis with anti-HMGB1 antibody. The results are shown in FIG. Extracellular secretion of HMGB1 in monocytes was highest after 18 hours of LPS or poly I: C treatment.

다음으로, Tat-ref-1가 TLR4/LPS에 의해 유도되는 HMGB1의 세포외 분비를 조절하는지를 조사하였다. THP-1 세포에 실시예 4의 2)에서 사용한 FLAG가 붙여져 있는 ref-1 (ref-1), 돌연변이 ref-1 (muref-1) 또는 빈 control (mock) 벡터 각각을 도입시켰다. 24시간 후 도입된 세포들은 혈청이 없는 배지에 배양하여 굶긴 후, 24시간 동안 LPS (100 ng/ml)로 자극시켰다. 세포배양 상층액을 모아, 상기와 마찬가지 방법에 의해 면역침강법과 Western blot 분석을 수행하여 도 8의 B에 도시하였으며, 면역형광을 측정하여 도 9에 나타내었다. 세포들은 면역형광 염색을 위해 항-HMGB1 항체로 배양하였다. 핵들은 20 μg/ml propidium iodide로 15분 동안 반응시킨 후 공초점현미경으로 관찰하였다. 도 9에서 Bar의 길이는 20 μm이다.Next, it was examined whether Tat-ref-1 regulates the extracellular secretion of HMGB1 induced by TLR4 / LPS. THP-1 cells were introduced with ref-1 (ref-1), mutant ref-1 (muref-1), or empty control (mock) vectors to which FLAG was attached in Example 4). Cells introduced after 24 hours were starved by incubation in medium without serum and then stimulated with LPS (100 ng / ml) for 24 hours. The cell culture supernatants were collected, and immunoprecipitation and Western blot analysis were performed by the same method as above, and shown in FIG. 8B. The immunofluorescence was measured and shown in FIG. 9. Cells were incubated with anti-HMGB1 antibody for immunofluorescence staining. Nuclei were reacted with 20 μg / ml propidium iodide for 15 minutes and then observed by confocal microscopy. In Fig. 9 the length of the bar is 20 μm.

도 8의 B로부터 LPS에 의한 HMGB1의 세포외 분비는 야생형 ref-1을 과발현시킨 세포에서 유의하게 억제되었으나, 돌연변이형인 muref-1 혹은 mock empty vector가 도입된 세포에서는 억제되지 않았음을 확인할 수 있었다.From B of FIG. 8, extracellular secretion of HMGB1 by LPS was significantly inhibited in cells overexpressing wild type ref-1, but not in cells in which the mutant muref-1 or mock empty vector was introduced. .

도 9로부터 mock empty 혹은 muref-1 vector를 도입한 THP-1 세포에서는 HMGB1이 핵으로부터 세포질로 이동한 반면에, ref-1을 도입한 세포에서는 HMGB1 단백질이 세포질로의 이동이 충분히 저해되어 단핵구/대식세포에서 ref-1은 HMGB1이 핵에 보존되게 하는 것을 확인할 수 있었다.In FIG. 9, in THP-1 cells into which a mock empty or muref-1 vector was introduced, HMGB1 migrated from the nucleus to the cytoplasm, whereas in cells introduced with ref-1, the HMGB1 protein was sufficiently inhibited to move into the cytoplasm. In macrophages, ref-1 caused HMGB1 to be conserved in the nucleus.

도 1은 HMGB1 유도 전염증성 사이토카인 형성에 대한 ref-1의 영향을 보여주는 그래프 및 사진.1 is a graph and photograph showing the effect of ref-1 on HMGB1 induced proinflammatory cytokine formation.

도 2는 NADPH oxidase 활성에 대한 ref-1의 영향을 보여주는 그래프.2 is a graph showing the effect of ref-1 on NADPH oxidase activity.

도 3은 단핵구의 HMGB1 및 LPS 자극에 의해 사이토카인 생성이 증가하는 것을 보여주는 그래프.3 is a graph showing increased cytokine production by HMGB1 and LPS stimulation of monocytes.

도 4는 단핵구에서 HMGB1 유도 전염증성 사이토카인 형성에 대한 Tat-ref-1의 영향을 보여주는 그래프.4 is a graph showing the effect of Tat-ref-1 on HMGB1 induced proinflammatory cytokine formation in monocytes.

도 5는 THP-1 세포에서 HMGB1 유도 전염증성 사이토카인 형성에 대한 Tat-ref-1의 영향을 보여주는 그래프.5 is a graph showing the effect of Tat-ref-1 on HMGB1 induced proinflammatory cytokine formation in THP-1 cells.

도 6은 HMGB1로 자극된 단핵구에서 COX-2 발현에 대한 Tat-ref-1의 영향을 보여주는 웨스턴 블럿 결과. 6 Western blot results showing the effect of Tat-ref-1 on COX-2 expression in HMGB1 stimulated monocytes.

도 7은 HMGB1로 자극된 단핵구에서 COX-2 발현에 대한 ref-1의 영향을 보여주는 사진 및 그래프. 7 is a photograph and graph showing the effect of ref-1 on COX-2 expression in monocytes stimulated with HMGB1.

도 8은 전염증성 사이토카인들에 의한 HMGB1의 세포외 분비에 대한 ref-1의 영향을 보여주는 웨스턴 블럿 결과. FIG. 8 is a Western blot result showing the effect of ref-1 on extracellular secretion of HMGB1 by proinflammatory cytokines.

도 9는 전염증성 사이토카인들에 의한 HMGB1의 세포외 분비에 대한 ref-1의 영향을 보여주는 면역형광 사진.9 is an immunofluorescence photograph showing the effect of ref-1 on extracellular secretion of HMGB1 by proinflammatory cytokines.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Pharmaceutical composition for treating immune disease and cancer <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 355 <212> PRT <213> Artificial Sequence <220> <223> Tat-ref-1 <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser 20 25 30 Asp Pro Asn Ser Met Pro Lys Arg Gly Lys Lys Gly Ala Val Ala Glu 35 40 45 Asp Gly Asp Glu Leu Arg Thr Glu Pro Glu Ala Lys Lys Ser Lys Thr 50 55 60 Ala Ala Lys Lys Asn Asp Lys Glu Ala Ala Gly Glu Gly Pro Ala Leu 65 70 75 80 Tyr Glu Asp Pro Pro Asp Gln Lys Thr Ser Pro Ser Gly Lys Pro Ala 85 90 95 Thr Leu Lys Ile Cys Ser Trp Asn Val Asp Gly Leu Arg Ala Trp Ile 100 105 110 Lys Lys Lys Gly Leu Asp Trp Val Lys Glu Glu Ala Pro Asp Ile Leu 115 120 125 Cys Leu Gln Glu Thr Lys Cys Ser Glu Asn Lys Leu Pro Ala Glu Leu 130 135 140 Gln Glu Leu Pro Gly Leu Ser His Gln Tyr Trp Ser Ala Pro Ser Asp 145 150 155 160 Lys Glu Gly Tyr Ser Gly Val Gly Leu Leu Ser Arg Gln Cys Pro Leu 165 170 175 Lys Val Ser Tyr Gly Ile Gly Glu Glu Glu His Asp Gln Glu Gly Arg 180 185 190 Val Ile Val Ala Glu Phe Asp Ser Phe Val Leu Val Thr Ala Tyr Val 195 200 205 Pro Asn Ala Gly Arg Gly Leu Val Arg Leu Glu Tyr Arg Gln Arg Trp 210 215 220 Asp Glu Ala Phe Arg Lys Phe Leu Lys Gly Leu Ala Ser Arg Lys Pro 225 230 235 240 Leu Val Leu Cys Gly Asp Leu Asn Val Ala His Glu Glu Ile Asp Leu 245 250 255 Arg Asn Pro Lys Gly Asn Lys Lys Asn Ala Gly Phe Thr Pro Gln Glu 260 265 270 Arg Gln Gly Phe Gly Glu Leu Leu Gln Ala Val Pro Leu Ala Asp Ser 275 280 285 Phe Arg His Leu Tyr Pro Asn Thr Pro Tyr Ala Tyr Thr Phe Trp Thr 290 295 300 Tyr Met Met Asn Ala Arg Ser Lys Asn Val Gly Trp Arg Leu Asp Tyr 305 310 315 320 Phe Leu Leu Ser His Ser Leu Leu Pro Ala Leu Cys Asp Ser Lys Ile 325 330 335 Arg Ser Lys Ala Leu Gly Ser Asp His Cys Pro Ile Thr Leu Tyr Leu 340 345 350 Ala Leu *** 355 <110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Pharmaceutical composition for treating immune disease and cancer <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 355 <212> PRT <213> Artificial Sequence <220> <223> Tat-ref-1 <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser              20 25 30 Asp Pro Asn Ser Met Pro Lys Arg Gly Lys Lys Gly Ala Val Ala Glu          35 40 45 Asp Gly Asp Glu Leu Arg Thr Glu Pro Glu Ala Lys Lys Ser Lys Thr      50 55 60 Ala Ala Lys Lys Asn Asp Lys Glu Ala Ala Gly Glu Gly Pro Ala Leu  65 70 75 80 Tyr Glu Asp Pro Pro Asp Gln Lys Thr Ser Pro Ser Gly Lys Pro Ala                  85 90 95 Thr Leu Lys Ile Cys Ser Trp Asn Val Asp Gly Leu Arg Ala Trp Ile             100 105 110 Lys Lys Lys Gly Leu Asp Trp Val Lys Glu Glu Ala Pro Asp Ile Leu         115 120 125 Cys Leu Gln Glu Thr Lys Cys Ser Glu Asn Lys Leu Pro Ala Glu Leu     130 135 140 Gln Glu Leu Pro Gly Leu Ser His Gln Tyr Trp Ser Ala Pro Ser Asp 145 150 155 160 Lys Glu Gly Tyr Ser Gly Val Gly Leu Leu Ser Arg Gln Cys Pro Leu                 165 170 175 Lys Val Ser Tyr Gly Ile Gly Glu Glu Glu His Asp Gln Glu Gly Arg             180 185 190 Val Ile Val Ala Glu Phe Asp Ser Phe Val Leu Val Thr Ala Tyr Val         195 200 205 Pro Asn Ala Gly Arg Gly Leu Val Arg Leu Glu Tyr Arg Gln Arg Trp     210 215 220 Asp Glu Ala Phe Arg Lys Phe Leu Lys Gly Leu Ala Ser Arg Lys Pro 225 230 235 240 Leu Val Leu Cys Gly Asp Leu Asn Val Ala His Glu Glu Ile Asp Leu                 245 250 255 Arg Asn Pro Lys Gly Asn Lys Lys Asn Ala Gly Phe Thr Pro Gln Glu             260 265 270 Arg Gln Gly Phe Gly Glu Leu Leu Gln Ala Val Pro Leu Ala Asp Ser         275 280 285 Phe Arg His Leu Tyr Pro Asn Thr Pro Tyr Ala Tyr Thr Phe Trp Thr     290 295 300 Tyr Met Met Asn Ala Arg Ser Lys Asn Val Gly Trp Arg Leu Asp Tyr 305 310 315 320 Phe Leu Leu Ser His Ser Leu Leu Pro Ala Leu Cys Asp Ser Lys Ile                 325 330 335 Arg Ser Lys Ala Leu Gly Ser Asp His Cys Pro Ile Thr Leu Tyr Leu             340 345 350 Ala Leu ***         355  

Claims (3)

서열번호 1의 Tat-ref-1 융합단백질을 유효성분으로 함유하는 HMGB-1을 병인으로하는 면역질환 및 암 치료용 제약 조성물.A pharmaceutical composition for the treatment of immune diseases and cancers, comprising HMGB-1 containing the Tat-ref-1 fusion protein of SEQ ID NO. 1 as an active ingredient. 제 1 항에 있어서,The method of claim 1, 상기 면역질환은 패혈증, 처그스트라우스 증후군, 쇼그랜 증후군, 전신 홍반성 루프스 또는 류머티즘성 관절염인 것을 특징으로 하는 면역질환 및 암 치료용 조성물.The immune disease is sepsis, Chug Strauss syndrome, Shogran syndrome, systemic lupus erythematosus or rheumatoid arthritis, characterized in that the composition for the treatment of immune diseases and cancer. 제 1 항에 있어서,The method of claim 1, 상기 암은 유방암, 대장암, 흑색종, 전립선암, 폐암 또는 췌장암인 것을 특징으로 하는 면역질환 및 암 치료용 조성물. The cancer is breast cancer, colorectal cancer, melanoma, prostate cancer, lung cancer or pancreatic cancer, characterized in that the composition for the treatment of immune diseases and cancer.
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