Enhanced anticancer effects of multimerized [B3(Fab)-PE38] antibody immunotoxin utilizing repeats of the Fab binding domain of protein G
- 주제(키워드) antibody-toxin
- 발행기관 고려대학교 대학원
- 지도교수 최무현
- 발행년도 2014
- 학위수여년월 2014. 8
- 학위구분 석사
- 학과 일반대학원 생명과학과
- 세부전공 분자생명과학전공
- 원문페이지 66 p
- 실제URI http://www.dcollection.net/handler/korea/000000053001
- 본문언어 영어
- 제출원본 000045808806
초록/요약
The recombinant Fab-toxin is a hybrid cytotoxic protein which consists of the cancer-specific Fab domain and the cytotoxic domain of a toxin, Pseudomonas exotoxin A. The Fd-ext-PE38 (long chain) and light chain (short chain) are covalently linked by a disulfide bond. The Fab-ext-PE38 made in this study targets the carbohydrate antigen, LewisY, on the surface of cancer cells and catalyzes ADP-ribosylation on elongation factor 2. As a result, it inhibits the protein synthesis of cancer cells and finally induces apoptosis. In this study, I have focused on making multivalent antibody-toxin using GR protein (Protein G Domain Ⅲ) that has affinity to CH1 domain of Fab fragment to initiate disulfide-dimer antibody-toxin. Domain Ⅲ of Streptococcal protein G was constructed up to 20 repeats to associate with Fab-ext-PE38. The cytotoxicity of associated Fab-ext-PE38 with GR proteins were higher than Fab-ext-PE38 control on carcinoma cell lines A431 (Humanepidermoid), MCF7 (Breast) and CRL-1739 (Stomach). It is considered that non-covalent bounded Fab-ext-PE38 on GR protein is consisted of several hydrogen bonds and is less stable than the disulfide-dimer antibody-toxin which is connected by covalent disulfide-bond. Previously studied divalent antibody-toxins are dimerized form of Fab-ext-PE38 molecule by disulfide-bond located in the modified hinge regions. Disulfide-bond is occurred solely depends on the random collision during refolding procedure between two Fab-ext-PE38s. To facilitate disulfide-bond dimer antibody-toxin, Domain Ⅲ of streptococcal protein G will help to form disulfide-dimer antibody-toxin. Associated Fab-ext-PE38 with GR proteins was reduced and oxidized with 2--mercaptoethanol and glutathione oxidized form to facilitate disulfide-dimer. The yield of disulfide-dimer was significantly increased approximate 17-fold by redox suffling experiment in GR10 association with Fab-ext-PE38. It is because high local concentration of Fab-ext-PE38 on GR protein (Protein G Domain Ⅲ) facilitates to form disulfide-bond dimer antibody-toxin. These results suggest that the activity of long half-life in blood circulation, such as a dimer having the characteristics of antibody-toxin to be able to produce a high yield expected.
more초록/요약
재조합 항체-독소 융합 단백질은 암세포에 특이적으로 반응하는 항체에 Pseudomonas exotoxin A 라는 세포살상 인자를 유전적으로 연결하여 만든 인공물질이다. Fd-ext-PE38 (긴사슬)과 L사슬(짧은사슬)은 이황화 결합에 의해 공유결합으로 연결되어 있다. 본 연구에서 Fd-ext-PE38은 고형암 세포표면에 발현하는 LewisY 계열의 당 중합체 항원을 인식하도록 만들었으며 EF-2에 ADP-리보실화를 촉진하여 단백질의 합성을 저해하여 세포살상을 유도한다. 본 연구에서는 Fab의 CH1 도메인에 친화력있는GR 단백질(Protein G의 도메인Ⅲ)을 이용하여 단량체 항체-독소의 이황화 결합을 촉진하여 항체-독소 다량체를 만드는 것에 중점을 두었다. Streptococcal protein G의 도메인 Ⅲ 는 항체-독소 단량체와 결합하기 위해 20반복사슬까지 제작되었다. A431 (Humanepidermoid), MCF7 (Breast) 및 CRL01739 (Stomach) 암세포주에 대한 GR과 결합된 항체-독소의 세포독성은 항체-독소 단량체 보다 더 높은 것을 확인하였다. 그러나 GR단백질과 항체-독소 단량체는 몇 개의 수소결합으로 이루어진 비공유 결합으로 결합되어있어 공유결합인 이황화결합 이량체보다 덜 안정할 것으로 사려된다. 변형된 경첩영역에서 단량체가 이황화 결합에 의해 이량체 항체-독소가 형성 되는 것을 이전에 연구했다. 두 개의 항체-독소 단량체 사이에 이황화 결합은 재접힘 과정 동안 오로지 임의적 충돌에 의해서만 일어난다. GR단백질과 항체-독소 단량체를 결합시킨후 2-mercaptoethaol로 환원 시키고 이어서 GSSG로 산화 시키면 복합체 내에서 이황화 결합 가교 이량체가 생성됨을 관찰하였다. GR10과 항체-독소 단량체의 리독스 셔플링 실험결과 이황화 결합 항체-독소 이량체 수득률이 약17배로 상당히 증가하는 것을 확인 할 수 있었다. 이는 GR반복사슬 골격이 항체-독소 단량체와 접착함으로서 복합체내의 단량체의 국소농도 (Local concentration)가 증가하는 것이 이유가 되며 그 결과로 단량체 내부의 사슬내 짝이 없는 시스테인 잔기와 이웃한 다른 단량체의 사슬내 짝이 없는 시스테인 사이의 접근빈도가 증가하여 이황화 결합 가교의 형성이 촉진되는 것으로 보여진다. 이러한 결과는 혈액순환계에서의 긴 활성도 반감기 등의 특성을 지닌 이량체 항체-독소를 높은 수득률로 생산할 수 있을 것으로 기대된다.
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Contents
Abstract•••••••••••••••••••••••••••••••••••••••••••••••••••••••••i
Contents•••••••••••••••••••••••••••••••••••••••••••••••••••••••iv
List of tables••••••••••••••••••••••••••••••••••••••••••••••••••vi
List of Figures •••••••••••••••••••••••••••••••••••••••••••••• vii
List of Abbreviations••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• iiv
1. Introduction ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••1
2. Materials and Methods ••••••••••••••••••••••••••••••••••••••••••••••6
2.1 Bacterial strain and plasmids ••••••••••••••••••••••••••••••••••••••••••••••6
2.2 Isolation of the Inclusion Bodies of [B3(Fd)-ext-PE38] and [B3(L)) •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••6
2.3 Refolding, and Purification of [B3(Fab)-ext-PE38] •••••••••••••••••••8
2.4 Anion exchange column••••••••••••••••••••••••••••••••••••••••••••••••••••9
2.5 Affinity column••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••10
2.6 Size exclusion column •••••••••••••••••••••••••••••••••••••••••••••••••••••11
2.7 Cell culture•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••12
2.8 Treatment of atCW1 with ProteinG •••••••••••••••••••••••••••••••••••••12
2.9 Lactate dehydrogenase (LDH) release••••••••••••••••••••••••••••••••••13
2.10 Redox suffling of associated Fab-monomer with GR proteins•••••14
3. Results and Discussion•••••••••••••••••••••••••••••••••••••••••••••••15
3.1 pCW1 and pMCH75 expression and purification of inclusion body •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••15
3.2 Refolding and Purification of [B3(Fab)-ext-PE38] ••••••••••••••••••16
3.3 Cytotoxicity of associated Fab-toxin with region 8 repeat peptide toward B3-antigen expressing cancer cell••••••••••••••••••••••••••••••17
3.4 Disulfide-dimer, [B3(Fab)-ext-PE38]2, formation between [B3(Fab)-ext-PE38], monomers that associated to constructs of GR protein•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••19
4.References••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••42
Abstract in Korean •••••••••••••••••••••••••••••••••••••••••••••••••••49
