KR101525856B1 - A inhibitor for transcription factor of fusion protein comprising DNA binding domain of transcription factor and protein transduction domain - Google Patents
A inhibitor for transcription factor of fusion protein comprising DNA binding domain of transcription factor and protein transduction domain Download PDFInfo
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- KR101525856B1 KR101525856B1 KR1020100080864A KR20100080864A KR101525856B1 KR 101525856 B1 KR101525856 B1 KR 101525856B1 KR 1020100080864 A KR1020100080864 A KR 1020100080864A KR 20100080864 A KR20100080864 A KR 20100080864A KR 101525856 B1 KR101525856 B1 KR 101525856B1
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Abstract
본 발명은 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 가지는 융합단백질을 가지는 전사인자 억제제 및 이의 제조방법에 관한 것이다. 또한,본 발명은 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 가지는 융합단백질, 이를 코딩하는 핵산, 이 핵산의 벡터 및 이 벡터로 형질전환된 숙주세포에 관한 것이다. 본 발명의 융합단백질을 통하여, 전사인자와 관련된 질병을 효과적으로 치료할 수 있다. The present invention relates to a transcription factor inhibitor having a fusion protein having a DNA binding domain and a protein transport domain of a transcription factor and a method for producing the same. The present invention also relates to a fusion protein having a DNA binding domain and a protein transport domain of a transcription factor, a nucleic acid encoding the same, a vector of the nucleic acid, and a host cell transformed with the vector. Through the fusion protein of the present invention, diseases associated with transcription factors can be effectively treated.
Description
본 발명은 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질을 가지는 전사인자 억제제 및 이의 제조방법에 관한 것이다.The present invention relates to a transcription factor inhibitor having a fusion protein comprising a DNA binding domain and a protein transduction domain of a transcription factor and a method for producing the same.
RORγt는 495개의 아미노산으로 이루어져 있고 크게 3개의 도메인으로 나누어져 있다. 아미노 말단에는 DNA와 결합할 수 있는 도메인, 카르복시 말단에는 다른 리간드와 결합할 수 있는 도메인, 그리고 그 사이를 연결하면서 그 구조를 형성해 주는 링커 도메인이 있다. 이 단백질은 CD4 T 세포의 한 종류인 T helper 17 (TH17) 세포로 분화할 수 있게 해주는 주요 전사인자이다. CD4 T 세포에는 현재까지 TH1, TH2, TH9, TH17, TfH 그리고 Treg 세포, 총 6종류의 세포로 분화가 가능한 것으로 알려져 있다. 그 중에서도 현재 자가면역질환에 가장 큰 영향력을 미친다고 알려져 있는 TH17에 관한 연구가 활발히 진행 중이고 이를 조절하는 물질을 찾는데 많은 연구가 이루어지고 있다. 선행 연구에서는 TH17으로의 분화를 유도하는 사이토카인들의 양을 조절하거나 제거함으로써 TH17으로의 분화를 막거나, TH17으로 분화가 이미 이루어진 후 TH17만이 분비하는 IL-17 사이토카인을 특정 항체를 이용하여 중화시키는 방법을 통하여 자가면역 질환 억제 연구가 이루어졌었다. 하지만 아직 까지 분화에 있어서 가장 중요하게 작용하는 전사인자인 RORγt를 타깃으로 RORγt 자체의 기능을 방해하거나 억제하는 연구는 진행되지 못하였다.
RORγt is composed of 495 amino acids and is divided into three domains. The amino terminus has a domain capable of binding to DNA, the carboxy terminus has a domain capable of binding with another ligand, and a linker domain that forms a linkage therebetween. This protein is a major transcription factor that allows differentiation into T helper 17 (TH17) cells, a class of CD4 T cells. CD4 T cells are known to be able to differentiate into six types of cells, TH1, TH2, TH9, TH17, TfH and Treg cells. Among them, TH17, which is known to have the greatest influence on autoimmune diseases, is currently being actively studied and many studies have been conducted to find a substance that regulates TH17. Previous studies have shown that by inhibiting the differentiation of TH17 by regulating or eliminating the amount of cytokines that induce TH17 differentiation, or by inhibiting the IL-17 cytokine secreted exclusively by TH17 after neutralization with TH17, The authors concluded that the inhibition of autoimmune diseases has been studied through the method of inhibition. However, studies on the inhibition or inhibition of the function of RORγt itself have not yet been conducted by targeting RORγt, the most important transcription factor in differentiation.
RORγt 전사인자와 관련된 다발성 경화증은 중추신경계에서 일어나는 가장 흔한 만성 염증성 질환의 일종이다. 보통 탈 수초화 현상에 의해 질환이 발생을 하는데 탈 수초화란 신경세포의 축삭 부분을 둘러싸고 있는 수초라는 절연물질이 어떤 요인에 의해 피괴가 되는데 파괴 정도가 심해지면 신경전달이 제대로 이루어지지 않고 더 심하면 둘러쌌던 신경세포가 죽게 된다. 지금까지 연구된 바로는 IL-17A에 의해 뇌실 주위의 백색질이나 척수 등에 염증세포를 모여들게 하여 중추신경계에 침투하게 되어 자가면역체계에 이상이 와서 자기 자신의 조직을 공격하게 되어 질환이 발병된다고 알려지고 있다. 따라서, IL-17A의 발현 억제가 가장 질환 치료에 핵심 열쇠가 되며, 이는 RORγt 전사인자 활성을 억제하여 해결할 수 있다.
Multiple sclerosis associated with RORγt transcription factors is one of the most common chronic inflammatory diseases that occur in the central nervous system. Degeneration is usually caused by dehydration phenomenon, de-herbalization is a herb that surrounds the axonal part of the nerve cell is destroyed by some factor, which is destroyed by the degree of destruction of the nerve is not properly transmitted, Nerve cells die. It is known that IL-17A causes IL-17A to infiltrate inflammation cells in the white matter and spinal cord around the ventricle and infiltrate into the central nervous system, resulting in an abnormality in the autoimmune system and attacking its own tissues. have. Thus, inhibition of IL-17A expression is the key to the treatment of most diseases, which can be overcome by inhibiting RORγt transcription factor activity.
단백질 운반 도메인(Protein Transduction Domain: PTD)은 1998년 HIV 바이러스에서 발견된 TAT 단백질의 일부분이 숙주세포 내로 생리활성분자를 침투시킬 수 있다는 것이 확인되면서 최초로 보고되었다. 이런 PTD는 120kDa 이상 되는 단백질들도 단시간 내에 세포 내로 전달할 수 있을 정도로 매우 효율적인 전달물질이며 7~50개의 아미노산으로 이루어질 만큼 크기가 굉장히 작다. 현재까지 약 50여 가지의 PTD가 알려지고 있고, 단백질뿐만 아니고 DNA나 RNA 또한 세포 내로 전달할 수 있다는 보고가 이루어지면서 활발한 연구가 진행되었다. 현재 본 발명자들도 2가지 PTD를 개발하였고 특허 등록하였다. (PCT/KR2003/000121 및 PCT/KR2003/000122)
The Protein Transduction Domain (PTD) was first reported in 1998, confirming that a portion of the TAT protein found in HIV viruses could penetrate physiologically active molecules into host cells. These PTDs are extremely efficient delivery materials that can deliver proteins over 120 kDa into the cells in a short time, and they are very small to be composed of 7 to 50 amino acids. Up to now, about 50 PTDs have been known, and active research has been carried out with reports that not only proteins but also DNA and RNA can be delivered into cells. At present, the present inventors also developed two PTDs and registered the patent. (PCT / KR2003 / 000121 and PCT / KR2003 / 000122)
따라서, 본 발명자들은 세포 내로 약물을 잘 전달할 수 있는 단백질 운반 도메인을 이용하여 리간드 결합 도메인이 없는 변형된 전사인자를 세포의 핵내로 전달함으로써, 전사인자의 활성을 억제하여 전사인자 관련 질병을 억제한다는 결과를 얻어 본 발명을 완성하였다. Therefore, the present inventors have succeeded in inhibiting the transcription factor related disease by inhibiting the transcription factor activity by transferring the modified transcription factor without the ligand binding domain into the nucleus of the cell using the protein transport domain capable of transferring the drug well into the cell The results were obtained and the present invention was completed.
전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질을 가지는 전사인자 억제제 및 이의 제조방법을 제공하는데 목적이 있다.A transcription factor inhibitor having a fusion protein comprising a DNA binding domain and a protein transport domain of a transcription factor, and a method for producing the same.
또한, 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질, 이를 코딩하는 핵산, 이 핵산을 포함하는 벡터 및 이 벡터를 포함하는 숙주세포를 제공하는데 목적이 있다.It is also an object of the present invention to provide a fusion protein comprising a DNA binding domain and a protein transport domain of a transcription factor, a nucleic acid encoding the fusion protein, a vector comprising the nucleic acid, and a host cell comprising the vector.
또한, 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질 및 약제학적으로 허용되는 부형제를 포함하는 전사인자 관련 질병 치료용 약학 조성물 및 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질 및 약제학적으로 허용되는 부형제를 포함하는 약학 조성물을 제공하는데 목적이 있다.Also provided is a pharmaceutical composition for treating a transcription factor-related disease comprising a fusion protein comprising a DNA binding domain and a protein transduction domain of a transcription factor and a pharmaceutically acceptable excipient and a fusion composition comprising a DNA binding domain and a protein transduction domain of a transcription factor The present invention provides a pharmaceutical composition comprising a protein and a pharmaceutically acceptable excipient.
또한, 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질의 유효량을 전사인자 관련 질병을 가지는 환자에게 투여하는 것을 포함하는 전사인자 관련 질병을 치료하는 방법을 제공하는데 목적이 있다. It is also an object of the present invention to provide a method for treating a transcription factor-related disease comprising administering to a patient having a transcription factor-related disease an effective amount of a fusion protein comprising a DNA binding domain and a protein transduction domain of the transcription factor.
일 구체예에서, 본 발명의 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질을 가지는 전사인자 억제제를 제공한다. 다른 구체예에서, 상기 구체예의 전사인자는 RORγt (Retinoic acid-related orphan receptor gamma t), Tbet (T-box 21), GATA-3 (GATA binding protein 3), FOXP3 (Forkhead box P3), RORα4 (Retinoic acid-related orphan receptor alpha 4), HIF-1/2α (Hypoxia inducible factor 1/2α), STAT (Signal transducers and activators of transcription), PPARγ (peroxisome proliferator-activated receptor gamma), p53, AP-1, NFκB (nuclear factor kappa-B), NKx2.5 (NK2 transcription factor related, locus 5), FOXO (Forkhead box O), RELA (v-rel reticuloendotheliosis viral oncogene homolog A) 및 Runx (runt-related transcription factor)로 구성된 군으로부터 선택된 어느 하나임을 특징으로 하는 전사인자 억제제를 제공한다. 다른 구체예에서, 상기 구체예의 전사인자는 RORγt인 것을 특징으로 하는 전사인자 억제제를 제공한다. 다른 구체예에서, 상기 구체예의 단백질 운반 도메인은 Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia), Pep-1 (peptide), PTD-5, 11R (arginine), 7R 및 CTP(Cytoplamic transduction peptide)로 구성된 군으로부터 선택된 어느 하나임을 특징으로 하는 전사인자 억제제를 제공한다. 다른 구체예에서, 상기 구체예의 단백질 운반 도메인은 서열번호 1로 표시되는 서열을 가지는 것을 특징으로 하는 전사인자 억제제를 제공한다. 다른 구체예에서, 상기 구체예의 전사인자의 DNA 결합 도메인은 서열번호 2로 표시되는 서열을 가지는 것을 특징으로 하는 전사인자 억제제를 제공한다. 다른 구체예에서, 상기 구체예의 융합단백질은 서열번호 4로 표시되는 서열을 가지는 것을 특징으로 하는 전사인자 억제제를 제공한다.
In one embodiment, there is provided a transcription factor inhibitor having a fusion protein comprising a DNA binding domain and a protein delivery domain of a transcription factor of the invention. In another embodiment, the transcription factor of the above embodiment is selected from the group consisting of retinoic acid-related orphan receptor gamma (TOR), Tbet (T-box 21), GATA-3 (GATA binding protein 3), FOXP3 (Forkhead box P3) (HIF-1 / 2α), STAT (signal transducers and activators of transcription), PPARγ (peroxisome proliferator-activated receptor gamma), p53, AP-1, (Nuclear factor kappa-B), NKx2.5 (NK2 transcription factor related, locus 5), FOXO (Forkhead box O), RELA (v rel relucentendotheliosis viral oncogene homolog A) and Runx Or a pharmaceutically acceptable salt thereof. In another embodiment, a transcription factor inhibitor is provided, wherein the transcription factor of the embodiment is ROR gamma t. In another embodiment, the protein delivery domain of the above embodiment is selected from the group consisting of Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia), Pep- And CTP (Cytoplamic Transduction Peptide). The present invention also provides a transcription factor inhibitor. In another embodiment, there is provided a transcription factor inhibitor characterized in that the protein transport domain of the embodiment has the sequence shown in SEQ ID NO: 1. In another embodiment, there is provided a transcription factor inhibitor characterized in that the DNA binding domain of the transcription factor of this embodiment has the sequence of SEQ ID NO: 2. In another embodiment, the fusion protein of the above embodiment has a sequence represented by SEQ ID NO: 4.
일 구체예에서, 본 발명의 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질 및 이의 제조방법을 제공한다. 다른 구체예에서, 상기 구체예의 전사인자는 RORγt (Retinoic acid-related orphan receptor gamma t), Tbet (T-box 21), GATA-3 (GATA binding protein 3), FOXP3 (Forkhead box P3), RORα4 (Retinoic acid-related orphan receptor alpha 4), HIF-1/2α (Hypoxia inducible factor 1/2α), STAT (Signal transducers and activators of transcription), PPARγ (peroxisome proliferator-activated receptor gamma), p53, AP-1, NFκB (nuclear factor kappa-B), NKx2.5 (NK2 transcription factor related, locus 5), FOXO (Forkhead box O), RELA (v-rel reticuloendotheliosis viral oncogene homolog A) 및 Runx (runt-related transcription factor)로 구성된 군으로부터 선택된 어느 하나임을 특징으로 하는 융합단백질 및 이의 제조방법을 제공한다. 다른 구체예에서, 상기 구체예의 전사인자는 RORγt인 것을 특징으로 하는 융합단백질 및 이의 제조방법을 제공한다. 다른 구체예에서, 상기 구체예의 단백질 운반 도메인은 Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia), Pep-1 (peptide), PTD-5, 11R (arginine), 7R 및 CTP(Cytoplamic transduction peptide)로 구성된 군으로부터 선택된 어느 하나임을 특징으로 하는 융합단백질 및 이의 제조방법을 제공한다. 다른 구체예에서, 상기 구체예의 단백질 운반 도메인은 서열번호 1로 표시되는 서열을 가지는 것을 특징으로 하는 융합단백질 및 이의 제조방법을 제공한다. 다른 구체예에서, 상기 구체예의 전사인자의 DNA 결합 도메인은 서열번호 2로 표시되는 서열을 가지는 것을 특징으로 하는 융합단백질 및 이의 제조방법을 제공한다. 다른 구체예에서, 상기 구체예의 융합단백질은 서열번호 4로 표시되는 서열을 가지는 것을 특징으로 하는 융합단백질 및 이의 제조방법을 제공한다.
In one embodiment, there is provided a fusion protein comprising a DNA binding domain and a protein transport domain of a transcription factor of the invention and a method of producing the same. In another embodiment, the transcription factor of the above embodiment is selected from the group consisting of retinoic acid-related orphan receptor gamma (TOR), Tbet (T-box 21), GATA-3 (GATA binding protein 3), FOXP3 (Forkhead box P3) (HIF-1 / 2α), STAT (signal transducers and activators of transcription), PPARγ (peroxisome proliferator-activated receptor gamma), p53, AP-1, (Nuclear factor kappa-B), NKx2.5 (NK2 transcription factor related, locus 5), FOXO (Forkhead box O), RELA (v rel relucentendotheliosis viral oncogene homolog A) and Runx And a method for producing the fusion protein. In another embodiment, the fusion protein of the above embodiment is a ROR gamma t and a method for producing the fusion protein. In another embodiment, the protein delivery domain of the above embodiment is selected from the group consisting of Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia), Pep- And CTP (Cytoplamic Transduction Peptide), and a method for producing the fusion protein. In another embodiment, the protein transport domain of the above embodiment has a sequence represented by SEQ ID NO: 1, and a method for producing the fusion protein. In another embodiment, the DNA binding domain of the transcription factor of the above embodiment has a sequence represented by SEQ ID NO: 2, and a method for producing the fusion protein. In another embodiment, the fusion protein of the above embodiment has a sequence represented by SEQ ID NO: 4, and a method for producing the fusion protein.
일 구체예에서, 본 발명은 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질을 코딩하는 핵산을 제공한다. 다른 구체예에서, 상기 구체예의 단백질 운반 도메인은 서열번호 1로 표시되는 서열을 가지는 것을 특징으로 하는 핵산을 제공한다. 다른 구체예에서, 상기 구체예의 전사인자의 DNA 결합 도메인은 서열번호 2로 표시되는 서열을 가지는 것을 특징으로 하는 핵산을 제공한다. 다른 구체예에서, 상기 구체예의 융합단백질은 서열번호 4로 표시되는 서열을 가지는 것을 특징으로 하는 핵산을 제공한다.
In one embodiment, the invention provides a nucleic acid encoding a fusion protein comprising a DNA binding domain and a protein transport domain of a transcription factor. In another embodiment, the protein delivery domain of the embodiment has a sequence as set forth in SEQ ID NO: 1. In another embodiment, the DNA binding domain of the transcription factor of the embodiment has a sequence as set forth in SEQ ID NO: 2. In another embodiment, the fusion protein of the above embodiment provides a nucleic acid having the sequence represented by SEQ ID NO: 4.
일 구체예에서, 본 발명은 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질을 코딩하는 핵산의 벡터를 제공한다. 다른 구체예에서, 상기 구체예의 단백질 운반 도메인은 서열번호 1로 표시되는 서열을 가지는 것을 특징으로 하는 핵산의 벡터를 제공한다. 다른 구체예에서, 상기 구체예의 전사인자의 DNA 결합 도메인은 서열번호 2로 표시되는 서열을 가지는 것을 특징으로 하는 핵산의 벡터를 제공한다. 다른 구체예에서, 상기 구체예의 융합단백질은 서열번호 4로 표시되는 서열을 가지는 것을 특징으로 하는 핵산의 벡터를 제공한다. 다른 구체예에서, 상기 구체예의 벡터를 포함하는 숙주세포를 제공한다.
In one embodiment, the invention provides a vector of nucleic acids encoding a fusion protein comprising a DNA binding domain and a protein transport domain of a transcription factor. In another embodiment, the protein delivery domain of the embodiment has a sequence of SEQ ID NO: 1. In another embodiment, the DNA binding domain of the transcription factor of this embodiment has a sequence of SEQ ID NO: 2. In another embodiment, the fusion protein of the above embodiment provides a vector of nucleic acids characterized by having the sequence shown in SEQ ID NO: 4. In another embodiment, there is provided a host cell comprising a vector of the above embodiments.
일 구체예에서, 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질 및 약제학적으로 허용되는 부형제를 포함하는 약학 조성물을 제공한다.
In one embodiment, there is provided a pharmaceutical composition comprising a fusion protein comprising a DNA binding domain and a protein transport domain of a transcription factor and a pharmaceutically acceptable excipient.
일 구체예에서, 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질의 유효량을 전사인자 관련 질병을 가지는 환자에게 투여하는 것을 포함하는 전사인자 관련 질병을 치료하는 방법을 제공한다.
In one embodiment, there is provided a method of treating a transcription factor related disease comprising administering to a patient having a transcription factor related disease an effective amount of a fusion protein comprising a DNA binding domain and a protein transduction domain of a transcription factor.
일 구체예에서, 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질 및 약제학적으로 허용되는 부형제를 포함하는 전사인자 관련 질병 치료용 약학 조성물을 제공한다. 상기 전사인자가 NKx2.5 (NK2 transcription factor related, locus 5) 또는 HIF-1α (Hypoxia inducible factor 1α)인 경우 전사인자 관련 질병은 심장질환이다. 상기 전사인자가 HIF-1α (Hypoxia inducible factor 1α)인 경우 전사인자 관련 질병은 뇌질환이다. 상기 전사인자가 PPARγ (peroxisome proliferator-activated receptor gamma)인 경우 전사인자 관련 질병은 당뇨병이다. 상기 전사인자가 RORγt (Retinoic acid-related orphan receptor gamma t), RORα4 (Retinoic acid-related orphan receptor alpha 4), Tbet (T-box 21), FOXP3 (Forkhead box P3) 또는 FOXO (Forkhead box O)인 경우 전사인자 관련 질병은 자가면역질환이다. 상기 전사인자가 HIF-2α (Hypoxia inducible factor 2α) 또는 STAT (Signal transducers and activators of transcription)인 경우 전사인자 관련 질병은 염증질환이다. 상기 전사인자가 AP-1 또는 NFκB (nuclear factor kappa-B)인 경우 전사인자 관련 질병은 혈관질환이다. 상기 전사인자가 p53, SP1 또는 RELA (v-rel reticuloendotheliosis viral oncogene homolog A)인 경우 전사인자 관련 질병은 암이다. 상기 전사인자가 GATA-3 (GATA binding protein 3)인 경우 전사인자 관련 질병은 피부질환이다. 또한, 상기 전사인자가 RORγt인 경우 전사인자 관련 질병은 다발성 경화증이다.
In one embodiment, there is provided a pharmaceutical composition for treating a transcription factor-related disease comprising a fusion protein comprising a DNA binding domain and a protein transport domain of a transcription factor and a pharmaceutically acceptable excipient. If the transcription factor is NKx2.5 (NK2 transcription factor related, locus 5) or HIF-1α (Hypoxia
일 구체예에서, 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인을 포함하는 융합단백질을 포함하는 전사인자 관련 질병 치료용 제제를 제공한다.
In one embodiment, there is provided a pharmaceutical composition for treating a transcription factor-related disease comprising a fusion protein comprising a DNA binding domain and a protein transport domain of a transcription factor.
일 구체예에서, 서열번호 4로 표시되는 융합단백질 및 약제학적으로 허용되는 부형제를 포함하는 다발성 경화증 치료용 약학 조성물을 제공한다.
In one embodiment, there is provided a pharmaceutical composition for the treatment of multiple sclerosis comprising the fusion protein of SEQ ID NO: 4 and a pharmaceutically acceptable excipient.
단백질 운반 도메인(Protein Transduction Domain: PTD)은 이에 제한되는 것은 아니지만, 7~50개의 아미노산으로 이루어져 있고, 120kDa 이상 되는 단백질뿐만 아니라, DNA나 RNA를 세포 내로 전달할 수 있는 도메인으로 예를 들면, Hph-1, Mph-1 및 Sim-2과 같은 것을 말한다.
Protein Transduction Domain (PTD) includes, but is not limited to, a protein consisting of 7 to 50 amino acids and capable of transferring DNA or RNA into cells as well as a protein having a size of 120 kDa or more. For example, Hph- 1, Mph-1 and Sim-2.
전사인자(Transcription factor)는 DNA의 프로모터 영역에 결합하여 전사를 조절하는 인자로서, 이에 한정하지 않으나, RORγt (Retinoic acid-related orphan receptor gamma t), Tbet (T-box 21), GATA-3 (GATA binding protein 3), FOXP3 (Forkhead box P3), RORα4 (Retinoic acid-related orphan receptor alpha 4), HIF-1/2α (Hypoxia inducible factor 1/2α), STAT (Signal transducers and activators of transcription), PPARγ (peroxisome proliferator-activated receptor gamma), p53, AP-1, NFκB (nuclear factor kappa-B), NKx2.5 (NK2 transcription factor related, locus 5), FOXO (Forkhead box O), RELA (v-rel reticuloendotheliosis viral oncogene homolog A) 및 Runx (runt-related transcription factor) 등을 말한다.
The transcription factor is a factor that binds to the promoter region of the DNA to regulate transcription, and is not limited thereto. However, RORγt, Tbet (T-box 21), GATA-3 GATA binding protein 3), FOXP3 (Forkhead box P3), RORα4 (Retinoic acid-related orphan receptor alpha 4), HIF-1 / 2α (Hypoxia
전사인자 유도체는 DNA의 프로모터 영역에 결합하여 전사를 조절하는 전사인자를 변형한 형태로, 이에 한정하지 않으나, DNA 결합 도메인 또는 그 일부의 서열이 변형하거나, 추가하거나, 제거된 것을 말한다.A transcription factor derivative is a modified form of a transcription factor that binds to a promoter region of a DNA to regulate transcription. It refers to a DNA binding domain or a part of a sequence thereof modified, added, or deleted.
본 발명의 전사인자의 DNA 결합 도메인 및 단백질 운반 도메인(PTD)을 가지는 융합단백질을 통하여 전사인자와 관련된 질병을 효과적으로 치료할 수 있다. A fusion protein having a DNA binding domain and a protein transport domain (PTD) of the transcription factor of the present invention can effectively treat a transcription factor-related disease.
도 1은 본 발명의 일 구체예로 사용된 RγD와 Hph-1-PTD의 융합단백질(이하, HH-RγD라고 함)이다.
도 2는 발명의 일 구체예로 사용된 HH-RγD 융합단백질에 대한 코마시 블루 염색 사진이다.
도 3은 발명의 일 구체예로 사용된 HH-RγD 융합단백질이 절켓 T 세포에 농도별, 시간별로 전달된 것을 확인한 웨스턴 블롯 사진이다.
도 4는 발명의 일 구체예로 사용된 HH-RγD 융합 단백질이 HeLa 세포에 전달된 것을 형광현미경을 통해서 본 사진이다.
도 5는 발명의 일 구체예로 사용된 HH-RγD 융합 단백질이 비장세포에서 CD3와 CD28에 의해 활성화되어 발현하는 IL-17A, IFN-γ, IL-4, IL-2의 양을 조절했다는 것을 나타내는 그래프이다.
도 6은 발명의 일 구체예로 사용된 HH-RγD 융합 단백질이 CD4+CD25-CD62L+ 미성숙 T 세포가 각 TH 세포로 분화되는 것을 억제할 수 있는지 IL-17A, IL-17F, IFN-γ, IL-13의 발현양을 통하여 확인한 그래프이다.
도 7은 발명의 일 구체예로 사용된 HH-RγD 융합 단백질이 다발성 경화증 동물모델인 EAE에서 얼마나 치료효과를 나타내는지 행동학적 점수를 통해 나타낸 그래프이다.
도 8은 발명의 일 구체예로 사용된 HH-RγD 융합 단백질이 다발성 경화증 동물모델인 EAE에서 얼마나 치료효과를 나타내는지 세포학적 측면을 ELISA를 통해 확인한 그래프이다.
도 9는 발명의 일 구체예로 사용된 HH-RγD 융합 단백질이 다발성 경화증 동물모델인 EAE에서 얼마나 치료효과를 나타내는지 세포학적 측면에서 FACS를 통해 확인한 그래프이다.
도 10은 발명의 일 구체예로 사용된 HH-RγD 융합 단백질이 다발성 경화증 동물모델인 EAE에서 얼마나 치료효과를 나타내는지 조직학적 측면에서 룩솔 패스트 블루(Luxol Fast Blue) 염색법으로 확인한 사진이다.
도 11은 발명의 일 구체예로 사용된 HH-RγD 융합 단백질이 다발성 경화증 동물모델인 EAE에서 얼마나 치료효과를 나타내는지 조직학적 측면에서 해마톡실 & 에오신(Hematoxylin & Eosin) 염색법으로 확인한 사진이다.
도 12는 발명의 일 구체예로 사용된 HH-RγD 융합 단백질이 다발성 경화증 동물모델인 EAE에서 얼마나 치료효과를 나타내는지 조직학적 측면에서 페리오딕 산 쉬프(Periodic acid Schiff) 염색법으로 확인한 사진이다.Fig. 1 is a fusion protein of RγD and Hph-1-PTD (hereinafter referred to as HH-RγD) used as one embodiment of the present invention.
2 is a chromosome blue photograph of the HH-R? D fusion protein used as one embodiment of the invention.
FIG. 3 is a Western blot photograph showing that the HH-RγD fusion protein used as one embodiment of the invention is delivered to the cuticle T cells by concentration and time.
FIG. 4 is a photograph showing that the HH-RγD fusion protein used as an embodiment of the invention is transferred to HeLa cells through a fluorescence microscope.
FIG. 5 shows that the HH-RγD fusion protein used as an embodiment of the invention regulates the amount of IL-17A, IFN-γ, IL-4 and IL-2 expressed by activation of CD3 and CD28 in spleen cells FIG.
FIG. 6 is a graph showing that the HH-RγD fusion protein used as an embodiment of the present invention inhibits the differentiation of CD4 + CD25 - CD62L + immature T cells into TH cells, or IL-17A, IL-17F, IFN- IL-13 < / RTI >
FIG. 7 is a graph showing a behavioral score showing how the HH-R? D fusion protein used as an embodiment of the invention shows therapeutic effect in EAE, which is an animal model of multiple sclerosis.
FIG. 8 is a graph showing the cytological aspect of the HH-R? D fusion protein used as an embodiment of the invention by EIA, which is an animal model of multiple sclerosis, through ELISA.
FIG. 9 is a graph showing the effect of HH-R? D fusion protein used as an embodiment of the invention on EAE, an animal model of multiple sclerosis, through FACS in terms of cytology.
FIG. 10 is a photograph of the HH-R? D fusion protein used as an embodiment of the invention, which shows the therapeutic effect of EAE, which is an animal model of multiple sclerosis, in terms of histology by Luxol Fast Blue staining method.
FIG. 11 is a photograph showing hematoxylin & eosin staining of HH-R? D fusion protein used as an embodiment of the present invention in terms of histology showing the therapeutic effect of EAE, which is an animal model of multiple sclerosis.
FIG. 12 is a photograph of the HH-R? D fusion protein used as an embodiment of the invention, which shows the therapeutic effect in EAE, which is an animal model of multiple sclerosis, in terms of histological staining by periodic acid Schiff staining.
이하, 본 발명을 하기의 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.
실시예Example 1: 전사인자 유도체 및 단백질 운반 도메인의 1: Transcription factor derivatives and protein transport domain 융합단백질의Of the fusion protein 제조 Produce
단백질 운반 도메인인 Hph-1(서열번호: 1)을 전사인자 유도체인 RγD(서열번호: 2, 전사인자의 DNA 결합 도메인) 및 RγL(서열번호: 3, 전사인자의 리간드 결합 도메인)과 클로닝하여 Hph-1-RγD 및 Hph-1-RγL과 같은 재조합 DNA를 제작하였다. 이 2개의 재조합 DNA와 RγD를 pRSETb 벡터에 클로닝하고, 3개의 재조합 벡터로 BL21 star (DE3) pLys S 균주를 형질전환시켰다. 형질전환 균주를 배양한 후 1mM IPTG (isopropyl-β-D-thiogalactopyranoside)를 넣어서 8시간 동안 단백질을 발현하도록 유도하였다. 그 후 세포만 걷어서 분해용 완충액 (10mM 이미다졸, 300mM NaCl, 50mM NaH2PO4, pH 8.0)으로 용해시킨 후 분쇄기로 세포를 분해시켰다. 단백질 앞 부분에 인위적으로 결합시켜 놓은 6개의 히스티딘(Histidine)을 이용하여 Ni-NTA 비드를 이용하여 결합시켰다. 컬럼에 단백질을 넣고 세척용 완충액 (30mM 이미다졸, 300mM NaCl, 50mM NaH2PO4, pH 8.0)으로 충분히 세척하고, 용출용 완충액 (3M 이미다졸, 300mM NaCl, 50mM NaH2PO4, pH 8.0)로 단백질을 분리하였다. PD-10을 이용하여 완충액을 10% 글리세롤 PBS로 바꾸어주면서 NaCl과 이미다졸을 제거하였다. 수득된 단백질에는 LPS와 같은 엔도톡신이 존재하므로 이를 제거하기 위해 SP비드를 통하여 한번 더 정제를 하였다. 이를 다시 결합용 완충액 (300mM NaCl, 50mM NaH2PO4, pH 7.2)으로 결합시킨 후 이를 컬럼에 넣고 용출용 완충액(2M NaCl, 50mM NaH2PO4, pH 8.0)으로 단백질을 분리하였다. 마지막으로 PD-10을 통하여 NaCl을 제거하고 완충액을 PBS로 바꾸어준 후 실험 전까지 -80℃에서 보관하였다.
The protein transport domain Hph-1 (SEQ ID NO: 1) was cloned with the transcription factor derivative RγD (SEQ ID NO: 2, the DNA binding domain of the transcription factor) and RγL (SEQ ID NO: 3, ligand binding domain of the transcription factor) Recombinant DNA such as Hph-1-R? D and Hph-1-R? L was prepared. These two recombinant DNAs and RγD were cloned into a pRSETb vector, and the BL21 star (DE3) pLysS strain was transformed with three recombinant vectors. The transformant strain was cultured and 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to induce protein expression for 8 hours. Then, the cells were solubilized with a digestion buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 8.0), and then the cells were disrupted using a pulverizer. Six histidine (Histidine) artificially bound to the front of the protein was used to bind using Ni-NTA beads. Into the protein in the column wash buffer for (
실시예Example 2: 융합 단백질의 세포 내 전달 확인-단백질 운반 도메인의 기능확인 2: Identification of intracellular delivery of fusion protein - Identification of protein transport domain function
<2-1. <2-1. 웨스턴Western 블롯을Blot 이용하여 using 세포내Intracellular 전달 확인> Delivery confirmation>
절켓 T 세포와 C57BL/6 쥐의 비장세포를 이용하여 농도별 (0, 100nM, 200nM, 500nM, 1μM, 2μM, 5μM)로 또는 시간별 (0, 1, 3, 6, 12, 24, 48h)로 융합단백질 1시간씩 함께 배양하고 웨스턴 블롯으로 단백질 전달 여부를 확인하였다(도 3 참조). 농도에 따라서 잘 전달되는 것을 확인하였고, 1시간 만에 대부분 세포 내로 전달이 되었으며, 점점 세포 내에서 분해가 일어나면서 48시간이 되면 약 90%정도가 분해되는 것을 확인하였다. 단백질이 오랫동안 분해가 되지 않아 생겨나는 부작용은 발생하지 않을 것임을 확인하였다.
(0, 1, 3, 6, 12, 24, 48h) by concentration (0, 100 nM, 200 nM, 500 nM, 1 μM, 2 μM, 5 μM) The fusion protein was incubated for 1 hour and protein transfer was confirmed by Western blotting (see FIG. 3). It was confirmed that the cells were well transduced according to the concentration and most of the cells were transferred to the cells within 1 hour and gradually decomposed in the cells and about 90% were decomposed at 48 hours. It was confirmed that the side effects that would occur due to the protein not decomposing for a long time would not occur.
<2-2. 항체를 이용하여 세포의 핵까지 전달되는지 여부 확인><2-2. Determine if it is delivered to the nucleus of the cell using antibodies>
HH-RγD 융합단백질 500nM을 1시간 동안 HeLa 세포와 함께 배양을 하고 PBS로 씻어 준 후 Triton X-100을 이용하여 세포에 틈을 만들어준 후 그 사이로 형광 표지 항체를 상기 융합단백질에 결합시켰다. 그 다음에 DAPI 염색제를 이용하여 세포의 핵을 염색한 후, 형광현미경을 통해서 형광의 위치를 확인하여 융합단백질이 전달된 위치를 확인하였다. 그 결과 단백질은 PTD의 성질에 의해 세포의 핵까지 전달되었다는 것을 확인하였다(도 4 참조).
500 nM of HH-R? D fusion protein was incubated with HeLa cells for 1 hour, washed with PBS, and then made into a cell with Triton X-100 to bind the fluorescent label antibody to the fusion protein therebetween. Then, the nuclei of the cells were stained using DAPI stain, and the position of the fluorescence was confirmed through a fluorescence microscope to confirm the location of the fusion protein. As a result, it was confirmed that the protein was transferred to the nucleus of the cell by the property of PTD (see FIG. 4).
실시예Example 3: 3: 융합단백질의Of the fusion protein 전사인자의 특이적 억제 효과 확인: 전사인자 결합 도메인의 기능확인 Identification of specific inhibitory effects of transcription factors: Identification of transcription factor binding domain function
<3-1. 사이토카인 <3-1. Cytokine ILIL -17A 의 생성 억제>-17A < / RTI >
비장세포에 항 CD3 및 CD28 항체를 이용하여 T 세포만을 활성화시켜 생성되는 사이토카인 분비를 확인하였다. 7주령 된 암컷 C57BL/6 쥐의 비장에서 비장세포를 분리하고 이 세포를 항 CD3 항체 1μg/ml로 코팅한 12웰(well)에 항 CD28 항체 0.5μg/ml과 함께 72시간 동안 배양하였다. 그 후 배양액에 존재하는 사이토카인을 ELISA를 이용하여 그 양을 측정하였다. TH1을 대표하는 IFN-γ와 TH2를 대표하는 IL-4, TH17을 대표하는 IL-17A 및 T 세포의 활성화가 되었음을 알 수 있는 IL-2의 양을 확인하였다. 모두 항 CD3 및 CD28 항체를 통해서 활성화시킴으로써 분비가 증가한다는 것을 알았다. The secretion of cytokines produced by activating only T cells using anti-CD3 and CD28 antibodies was confirmed in spleen cells. Splenocytes were isolated from the spleen of 7-week-old female C57BL / 6 mice, and the cells were cultured for 72 hours with 0.5 μg / ml of anti-CD28 antibody in 12 wells coated with 1 μg / ml of anti-CD3 antibody. After that, the amount of cytokine present in the culture solution was measured by ELISA. IL-17 representing representative TH1, IL-4 representative of TH2, IL-17 representing representative TH17, and IL-2 level indicating activation of T cells were confirmed. All were found to be secreted by activation through anti-CD3 and CD28 antibodies.
IL-17A를 발현하게 하는 RORγt의 작용을 HH-RγD 융합단백질이 억제할 수 있는지 확인하기 위하여 CD3 및 CD28 항체로 활성화시키기 이전에 비장세포와 200nM HH-RγD 융합단백질을 1시간 동안 먼저 배양시켜 단백질을 세포 내로 전달시킨 후, 전달되지 못한 단백질들은 세척하고 상기와 동일한 방법으로 항체를 이용하여 활성화시켜서 싸이토카인의 양을 확인하였다. 그 결과 IFN-γ, IL-4, IL-2 모두 HH-RγD를 전달에 의하여 변화가 없었지만, IL-17A의 양은 50%가량 줄어들었음을 확인하였다. 이를 통해 HH-RγD 융합단백질이 TH17세포에만 특이적으로 작용한다는 것을 확인하였다(도 5 참조).
To confirm that the HH-RγD fusion protein can inhibit the action of RORγt which causes IL-17A expression, spleen cells and 200 nM HH-RγD fusion protein were first cultured for 1 hour before activation with CD3 and CD28 antibodies, Was transferred into the cells, and the proteins that had not been transferred were washed and activated with the antibody in the same manner as above to confirm the amount of cytokine. As a result, it was confirmed that IFN-γ, IL-4 and IL-2 were not changed by HH-RγD but IL-17A was reduced by 50%. As a result, it was confirmed that the HH-RγD fusion protein specifically functions in TH17 cells (see FIG. 5).
<3-2. <3-2. TH17TH17 으로의 분화 억제 효과>≪ RTI ID = 0.0 >
실시예 3-1의 쥐에서 비장세포를 분리한 후 MACS(Magnetic Cell Sorting)를 이용하여 어떠한 항원에도 노출되지 않은 상태인 CD4+ CD25- CD62L+ 미성숙 T 세포를 분리하였다. 미성숙 CD4 T 세포에 HH-RγD 융합단백질을 100nM 및 100fM을 1시간 동안 배양시킨 후, 항 CD3 항체 1μg/ml가 코팅되어있는 12웰에 항 CD28 항체 0.5μg/ml와 각 TH 세포로 분화할 수 있는 싸이토카인을 함께 넣고 72시간 동안 배양하였다. TH1로 분화시키기 위하여 IL-12 10ng/ml 및 항 IL-4 항체를 첨가하였고, TH2로 분화시키기 위하여 IL-4 5ng/ml 및 항-IFN-γ항체를 첨가하였으며, TH17로 분화시키기 위하여 TGFβ1 3ng/ml, IL-6 30ng/ml 및 IL-21 100ng/ml와 항 IL-4 항체 및 항-IFN-γ항체를 첨가하였다. 72시간 뒤에 배양액을 ELISA로 각 싸이토카인의 양을 확인해본 결과 TH1, TH2의 대표적인 싸이토카인은 아무런 변화가 없는 반면, TH17의 대표적인 싸이토카인인 IL-17A는 확연히 줄어들었음을 확인하였다. 또한 IL-17F도 RORγt에 의하여 발현이 되는데 이 또한 줄어들었음을 확인하였다. 이 결과로부터 TH17으로의 분화과정에서도 HH-RγD 융합단백질이 억제기능을 할 수 있다는 것을 알 수 있었다(도 6 참조).
After splenocytes were isolated from the rat of Example 3-1, CD4 + CD25 - CD62L + immature T cells isolated from any antigen were isolated using MACS (Magnetic Cell Sorting). The immature CD4 T cells were cultured with HH-RγD fusion protein at 100 nM and 100 fM for 1 hour. Then, 0.5 μg / ml of anti-CD28 antibody and 12 μl of anti-CD3 antibody were coated on each 1 μg / The cytokines were incubated for 72 hours. In order to differentiate into TH1, 10 ng / ml of IL-12 and anti-IL-4 antibody were added, and 5 ng / ml of IL-4 and anti-IFN-y antibody were added to differentiate into TH2. / ml, 30 ng / ml IL-6, 100 ng / ml IL-21 and anti-IL-4 and anti-IFN-y antibodies. After 72 hours, the amount of each cytokine was determined by ELISA. As a result, it was confirmed that the representative cytokine of TH1 and TH2 did not change, while that of IL-17A, a representative cytokine of TH17, was significantly reduced. In addition, IL-17F was also expressed by RORγt, which was also reduced. From these results, it was found that the HH-R? D fusion protein can also inhibit the function of differentiation into TH17 (see FIG. 6).
<3-3. <3-3. HHHH -RγL -R? L 융합단백질의Of the fusion protein TH17TH17 으로의 분화 억제 효과>≪ RTI ID = 0.0 >
RORγt의 카르복실 말단에 위치하는 리간드 결합 도메인의 기능을 확인하기 위하여 HH-RγL 융합단백질 100nM로 실시예 3-2와 동일한 실험을 수행하였다. 그 결과 TH1, TH2, TH17세포 모두에서 어떠한 변화도 일어나지 않았다. 이 결과를 통해 HH-RγD만이 특이적으로 TH17 분화에 억제효과를 나타낼 수 있다는 것을 확인하였다. 하지만 Hph-1이 없는 RγD또한 세포 내에 전달이 되지 않기 때문에 같은 100nM를 전달하여도 아무런 효과가 없다는 것을 함께 확인하였다.
In order to confirm the function of the ligand binding domain located at the carboxyl terminus of RORγt, the same experiment as in Example 3-2 was performed with 100 nM of HH-RγL fusion protein. As a result, no change was observed in all the TH1, TH2 and TH17 cells. These results confirm that only HH-RγD can specifically inhibit TH17 differentiation. However, RγD without Hph-1 is not also transferred into the cell, so that it is confirmed that the same 100 nM is not effective.
실시예Example 4: 다발성 경화증 동물모델에서의 4: Multiple sclerosis in animal models HHHH -RγD -R? D 융합단백질의Of the fusion protein 효과 effect
<4-1. 다발성 경화증의 동물모델에서 효과><4-1. Effect on animal models of multiple sclerosis>
다발성 경화증의 동물모델로 실험용 자가면역 뇌척수염 (EAE)라 하는 모델을 사용하였다. 6주령 된 암컷 C57BL/6 쥐를 2주 동안 안정기를 준 후 0 일과 2일째에 페르투시스 톡신(Pertussis Toxin) 500ng씩을 복강에 주입하고, 0일과 7일째에 MOG 펩타이드 150ng+CFA 200μg을 각각 피하지방에 각각 주입하였다. 그러면 일반적으로 9일째부터 쥐의 행동학적 측면에서 반응이 오기 시작하였다. 가장 먼저 꼬리에 마비가 오고 그 다음 뒷다리, 그 후 심하면 앞다리까지 마비가 오게 되는데 그 정도를 점수화하여 질환 정도를 평가하였다. (1점: 꼬리에 약간의 마비, 2점: 꼬리에 완전한 마비와 뒷다리에 약간의 마비, 3점: 한쪽 뒷다리 완전 마비, 4점: 양쪽 뒷다리 완전 마비, 5점: 뒷다리 완전 마비와 앞다리 약간/완전 마비, 6점: 죽음) 이러한 질환 동물 모델에 2일째부터 일주일에 3차례씩 50μg의 HH-RγD 융합단백질을 복강에 투여하였다. 그 결과 HH-RγD 융합단백질을 투여한 쥐의 경우 발병율이 50%로 줄었고 그 심한 정도 또한 미비할 정도로 질환이 완화되었다. 아무것도 투여하지 않은 쥐의 경우 발병율은 100%이고, 심한 정도 또한 3.05±0.23임에 반해 HH-RγD투여 쥐는 0.55±0.27이였다(도 7 참조).
A model called experimental autoimmune encephalomyelitis (EAE) was used as an animal model of multiple sclerosis. Six-week-old female C57BL / 6 rats were infused into the abdominal cavity with 500 ng of pertussis toxin (Pertussis Toxin) on
<4-2. 치료된 다발성 경화증의 동물모델에서 효과><4-2. Effect on animal models of treated multiple sclerosis>
치료된 쥐의 비장세포를 분리하고 항원으로 작용했던 MOG 펩타이드를 이용하여 CD4 T 세포들을 재활성화시켰다. 이 경우 쥐의 체내에서 이미 항원에 노출되었기 때문에 체외에서 다시 자극을 주면 더 강한 활성이 일어나 분화를 마친 CD4 T세포들이 각자의 대표 싸이토카인을 분비하게 된다. HH-RγD 융합단백질을 주입해준 쥐의 경우 질환 유도 후 10일째 되는 날에 세포를 얻어내어 40μg/ml의 MOG 펩타이드로 48시간 동안 재활성화시켰다. 배양액에 존재하는 싸이토카인의 양을 ELISA를 통하여 확인해본 결과 IL-17A가 확연하게 줄어들었음을 확인하였다. ELISA 결과뿐만이 아니라 싸이토카인을 세포 밖으로 분비하지 못하게 Golgi PLUG라는 억제제를 처리하고 세포내의 싸이토카인에 직접 형광표지된 항체로 그 양을 FACS로 측정하여도 같은 결과를 얻었다(도 8 및 9 참조).
Splenocytes from the treated mice were isolated and reactivated with CD4 T cells using MOG peptide that acted as antigen. In this case, since the mice are already exposed to the antigen in the rat, stimulation in vitro stimulates stronger activity, and the differentiated CD4 T cells secrete their respective representative cytokines. In the case of mice injected with the HH-RγD fusion protein, cells were harvested on
<4-3. 다발성 경화증의 동물모델에서 조직학적 효과><4-3. Histological Effects in Animal Models of Multiple Sclerosis>
질환 유도 후 가장 쥐의 상태가 좋지 않은 21일째에 쥐의 척수를 얻어 수초의 파괴 정도와 염증 세포들의 침투 정도를 확인하였다. 수초의 파괴정도를 확인하기 위하여 록솔 패스트 블루(Luxol Fast Blue) 염색제를 사용하였다. 이는 수초부위를 염색해 주는 염색제로 질환이 많이 진행된 경우 염색이 제대로 이루어 지지 않는다. 하지만 HH-RγD 융합단백질을 투여했던 쥐의 척수는 정상적임을 확인하였다. 또한 본 발명자들은 면역조직화학법을 이용하여 염증세포들을 염색하여 확인해본 결과 질환이 많이 진행된 경우 수초 쪽으로 많은 세포가 침투했다는 것을 확인하였고, 마찬가지로 HH-RγD를 투여한 쥐의 경우는 정상과 비슷할 정도의 세포들만 존재한다는 것을 확인하였다. 이런 모든 결과로 볼 때 HH-RγD는 IL-17A의 발현을 줄여줌으로써 다발성 경화증이 유발되지 않도록 막아주는 역할을 한다는 것을 입증하였다(도 10 참조).
On the 21st day after the induction of the disease, the spinal cord of the rat was obtained and the degree of destruction of the myelin and the penetration of the inflammatory cells were confirmed. A luxol fast blue stain was used to determine the degree of destruction of aquatic plants. This is a dye that stains a plant part. If the disease progresses a lot, it is not stained properly. However, the spinal cord of rats receiving the HH-RγD fusion protein was found to be normal. In addition, the present inventors confirmed by immunohistochemistry that inflammatory cells were stained and confirmed that many cells infiltrated into a few seconds when the disease progressed. Similarly, in the case of mice administered with HH-RγD, Of the cells were present. All these results demonstrate that HH-RγD plays a role in preventing the induction of multiple sclerosis by decreasing the expression of IL-17A (see FIG. 10).
실시예Example 5: 5: HHHH -RγD -R? D 융합단백질의Of the fusion protein 급성독성실험Acute Toxicity Experiment
대한실험공급센터에서 공급받은 6주령의 특정병원체부재(specific pathogen-free, SPF) SD계 랫트를 사용하여 급성독성실험을 하기와 같이 실시하였다.The acute toxicity test was carried out as described below using specific pathogen-free (SPF) SD rats of 6 weeks old supplied from the experimental supply center.
각 그룹당 2마리씩의 동물에 본 발명의 HH-RγD 융합단백질을 1 g/㎏의 용량으로 1회 경구 투여 후, 동물의 폐사여부, 임상증상 및 체중변화를 관찰하고 혈액학적 검사와 혈액생화학적 검사를 실시하였으며, 부검하여 육안으로 강장기와 흉강 장기의 이상 여부를 관찰하였다.After two doses of the HH-RγD fusion protein of the present invention were orally administered to two animals per group at a dose of 1 g / kg once, the mortality, clinical symptoms, and weight changes of the animals were observed, and hematological tests and blood biochemical tests , And autopsy was performed to observe whether or not the organs and thoracic organs were abnormal.
실험결과, 실험 물질을 투여한 모든 동물에서 특이할 만한 임상증상이나 폐사된 동물은 없었으며, 체중변화, 혈액검사, 혈액생화학 검사 및 부검 소견 등에서도 독성변화는 관찰되지 않았다. 이상의 결과, 본 발명의 HH-RγD 융합단백질은 랫트에서 각각 1 g/㎏까지도 독성변화를 나타내지 않았으며, 경구투여 최소치사량(LD50)은 1 g/㎏이상인 안전한 물질로 판단됨을 확인할 수 있었다.
As a result of the experiment, there were no clinical symptoms or dead animals in all animals treated with the test substance, and no toxic change was observed in weight change, blood test, blood biochemical test, and autopsy findings. As a result, it was confirmed that the HH-R? D fusion protein of the present invention did not show any toxicity at 1 g / kg or more in rats, and that the oral LDL was at least 1 g / kg.
이상에서 실시예를 들어 본 발명을 더욱 상세하게 설명하였으나, 본 발명은 반드시 이러한 실시예로 국한되는 것은 아니고, 본 발명의 기술사상을 벗어나지 않는 범위 내에서 다양하게 변형 실시될 수 있다. 따라서 본 발명에 개시된 실시예들은 본 발명의 기술적 사상을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시예에 의하여 본 발명의 기술적 사상의 범위가 한정되는 것은 아니다. 본 발명의 보호범위는 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술적 사상은 본 발명의 권리범위에 포함되는 것으로 해석되어야 할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not to be limited to the details thereof, and various changes and modifications may be made without departing from the spirit and scope of the invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but are intended to illustrate and not limit the scope of the technical spirit of the present invention. The scope of protection of the present invention should be construed according to the claims, and all technical ideas which are within the scope of the same should be interpreted as being included in the scope of the present invention.
<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A inhibitor for transcription factor of fusion protein comprising DNA binding domain of transcription factor and protein transduction domain <130> IPDC-39367 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Hph-1 <400> 1 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg 1 5 10 <210> 2 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> RgammaD <400> 2 Met Arg Thr Gln Ile Glu Val Ile Pro Cys Lys Ile Cys Gly Asp Lys 1 5 10 15 Ser Ser Gly Ile His Tyr Gly Val Ile Thr Cys Glu Gly Cys Lys Gly 20 25 30 Phe Phe Arg Arg Ser Gln Gln Cys Asn Val Ala Tyr Ser Cys Thr Arg 35 40 45 Gln Gln Asn Cys Pro Ile Asp Arg Thr Ser Arg Asn Arg Cys Gln His 50 55 60 Cys Arg Leu Gln Lys Cys Leu Ala Leu Gly Met Ser Arg Asp Ala Val 65 70 75 80 Lys Phe Gly Arg Met Ser Lys Lys Gln Arg Asp Ser Leu His Ala Glu 85 90 95 Val Gln Lys <210> 3 <211> 203 <212> PRT <213> Artificial Sequence <220> <223> RgammaL <400> 3 Met Trp Glu Arg Cys Ala His His Leu Thr Glu Ala Ile Gln Tyr Val 1 5 10 15 Val Glu Phe Ala Lys Arg Leu Ser Gly Phe Met Glu Leu Cys Gln Asn 20 25 30 Asp Gln Ile Ile Leu Leu Lys Ala Gly Ala Met Glu Val Val Leu Val 35 40 45 Arg Met Cys Arg Ala Tyr Asn Ala Asn Asn His Thr Val Phe Phe Glu 50 55 60 Gly Lys Tyr Gly Gly Val Glu Leu Phe Arg Ala Leu Gly Cys Ser Glu 65 70 75 80 Leu Ile Ser Ser Ile Phe Asp Phe Ser His Phe Leu Ser Ala Leu Cys 85 90 95 Phe Ser Glu Asp Glu Ile Ala Leu Tyr Thr Ala Leu Val Leu Ile Asn 100 105 110 Ala Asn Arg Pro Gly Leu Gln Glu Lys Arg Arg Val Glu His Leu Gln 115 120 125 Tyr Asn Leu Glu Leu Ala Phe His His His Leu Cys Lys Thr His Arg 130 135 140 Gln Gly Leu Leu Ala Lys Leu Pro Pro Lys Gly Lys Leu Arg Ser Leu 145 150 155 160 Cys Ser Gln His Val Glu Lys Leu Gln Ile Phe Gln His Leu His Pro 165 170 175 Ile Val Val Gln Ala Ala Phe Pro Pro Leu Tyr Lys Glu Leu Phe Ser 180 185 190 Thr Asp Val Glu Ser Pro Glu Gly Leu Ser Lys 195 200 <210> 4 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> HH-RgammaD <400> 4 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Ser Tyr Ala 1 5 10 15 Arg Val Arg Arg Arg Gly Pro Arg Arg Glu Phe Arg Thr Gln Ile Glu 20 25 30 Val Ile Pro Cys Lys Ile Cys Gly Asp Lys Ser Ser Gly Ile His Tyr 35 40 45 Gly Val Ile Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Gln 50 55 60 Gln Cys Asn Val Ala Tyr Ser Cys Thr Arg Gln Gln Asn Cys Pro Ile 65 70 75 80 Asp Arg Thr Ser Arg Asn Arg Cys Gln His Cys Arg Leu Gln Lys Cys 85 90 95 Leu Ala Leu Gly Met Ser Arg Asp Ala Val Lys Phe Gly Arg Met Ser 100 105 110 Lys Lys Gln Arg Asp Ser Leu His Ala Glu Val Gln Lys 115 120 125 <110> Industry-Academic Cooperation Foundation, Yonsei University <120> A inhibitor for transcription factor of fusion protein DNA binding domain of transcription factor and protein 전도 도 <130> IPDC-39367 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Hph-1 <400> 1 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg 1 5 10 <210> 2 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> RgammaD <400> 2 Met Arg Thr Gln Ile Glu Val Ile Pro Cys Lys Ile Cys Gly Asp Lys 1 5 10 15 Ser Ser Gly Ile His Tyr Gly Val Ile Thr Cys Glu Gly Cys Lys Gly 20 25 30 Phe Phe Arg Arg Ser Ser Gln Gln Cys Asn Val Ala Tyr Ser Cys Thr Arg 35 40 45 Gln Gln Asn Cys Pro Ile Asp Arg Thr Ser Arg Asn Arg Cys Gln His 50 55 60 Cys Arg Leu Gln Lys Cys Leu Ala Leu Gly Met Ser Ser Asp Ala Val 65 70 75 80 Lys Phe Gly Arg Met Ser Lys Lys Gln Arg Asp Ser Leu His Ala Glu 85 90 95 Val Gln Lys <210> 3 <211> 203 <212> PRT <213> Artificial Sequence <220> <223> RgammaL <400> 3 Met Trp Glu Arg Cys Ala His His Leu Thr Glu Ala Ile Gln Tyr Val 1 5 10 15 Val Glu Phe Ala Lys Arg Leu Ser Gly Phe Met Glu Leu Cys Gln Asn 20 25 30 Asp Gln Ile Ile Leu Leu Lys Ala Gly Ala Met Glu Val Val Leu Val 35 40 45 Arg Met Cys Arg Ala Tyr Asn Ala Asn Asn His Thr Val Phe Phe Glu 50 55 60 Gly Lys Tyr Gly Gly Val Glu Leu Phe Arg Ala Leu Gly Cys Ser Glu 65 70 75 80 Leu Ile Ser Ser Ile Phe Asp Ser Ser Phe Ser Ser Ala Leu Cys 85 90 95 Phe Ser Glu Asp Glu Ile Ala Leu Tyr Thr Ala Leu Val Leu Ile Asn 100 105 110 Ala Asn Arg Pro Gly Leu Gln Glu Lys Arg Arg Val Glu His Leu Gln 115 120 125 Tyr Asn Leu Glu Leu Ala Phe His His His Leu Cys Lys Thr His Arg 130 135 140 Gln Gly Leu Leu Ala Lys Leu Pro Pro Lys Gly Lys Leu Arg Ser Leu 145 150 155 160 Cys Ser Gln His Val Glu Lys Leu Gln Ile Phe Gln His Leu His Pro 165 170 175 Ile Val Val Gln Ala Ala Phe Pro Pro Leu Tyr Lys Glu Leu Phe Ser 180 185 190 Thr Asp Val Glu Ser Pro Glu Gly Leu Ser Lys 195 200 <210> 4 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> HH-RgammaD <400> 4 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Ser Tyr Ala 1 5 10 15 Arg Val Arg Arg Arg Gly Pro Arg Arg Glu Phe Arg Thr Gln Ile Glu 20 25 30 Val Ile Pro Cys Lys Ile Cys Gly Asp Lys Ser Ser Gly Ile His Tyr 35 40 45 Gly Val Ile Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Gln 50 55 60 Gln Cys Asn Val Ala Tyr Ser Cys Thr Arg Gln Gln Asn Cys Pro Ile 65 70 75 80 Asp Arg Thr Ser Arg Asn Arg Cys Gln His Cys Arg Leu Gln Lys Cys 85 90 95 Leu Ala Leu Gly Met Ser Ser Asp Ala Val Lys Phe Gly Arg Met Ser 100 105 110 Lys Lys Gln Arg Asp Ser Leu His Ala Glu Val Gln Lys 115 120 125
Claims (50)
단백질 운반 도메인인 Hph-1은 서열번호 1로 구성되는 것을 특징으로 하는 다발성 경화증의 치료 또는 예방용 약학 조성물. 45. The method of claim 44,
1. A pharmaceutical composition for treating or preventing multiple sclerosis, wherein the protein transport domain, Hph-1, comprises SEQ ID NO: 1.
전사인자 RORγt의 DNA 결합 도메인은 서열번호 2로 구성되는 것을 특징으로 하는 다발성 경화증의 치료 또는 예방용 약학 조성물. 45. The method of claim 44,
Wherein the DNA binding domain of the transcription factor RORγt is comprised of SEQ ID NO: 2.
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| EP18188839.7A EP3424946B1 (en) | 2010-08-20 | 2011-08-19 | A fusion protein having transcription modulating domain and a protein transduction domain, and an inhibitor for transcription factor function comprising the same |
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