Engineering of "E3 ubiquitin ligase recruiting ligand fused to cytosol-penetrating antibody" to degrade cytosolic β-catenin
- 주제(키워드) β-catenin , cytosol penetrating antibody , E3 ubiquitin ligase
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2020
- 학위수여년월 2020. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030362
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Oncogenic protein degradation approach has emerged as a new concept to overcome the limitation of classical monoclonal antibody therapy. Some researches on cytosolic protein degradation using small molecule or polypeptides has been conducted, but not complete IgG-format. Here, for the first time, I report eliminating cytosolic β-catenin using cytosol-penetrating antibody in human IgG1 format. I engineered β-catenin specific antibody (inBC) with improved anti-tumor activity by fusion with E3 ubiquitin ligase, termed inBC-E3. I found that inBC-VHL recruits Cul2 E3 complex in the proximity of the neo-substrate β-catenin, thus entering to ubiquitin proteasome pathway for degradation. inBC-VHL exhibits tumor growth inhibition in vitro and in vivo according to β-catenin depletion by conferring β-catenin degradation activity to inBC, which had no tumor suppressive effect. My results identified that engagement of ubiquitination cascade to the cancer-related protein for degradation offers potent therapeutic effects over traditional drug development strategies.
more목차
1. Introduction 1
1.1 Oncogenic cytosolic protein, β-catenin 1
1.2 Cytosol-penetrating antibody 4
1.3 Ubiquitin proteasome system (UPS) 5
1.4 Target protein degradation 6
2. Materials and methods 7
2.1 Cell lines 7
2.2 Construction of expression plasmids 7
2.3 Expression and purification of IgG antibodies 7
2.4 Size Exclusion Chromatography (SEC) 8
2.5 Enzyme-linked immunosorbent assay (ELISA) 8
2.6 Confocal immunofluorescence microscopy 8
2.7 Modeling of VHL fusion linker 9
2.8 Flow cytometry 9
2.9 Western blot 9
2.10 Immunoprecipitation (IP) 9
2.11 in vitro cytotoxicity assay 10
2.12 Xenograft tumor models 10
2.13 Statistical analysis 10
3. Results 12
3.1 Development of β-catenin degradation antibody 12
3.1.1 Design of β-catenin degradation antibody, inBC-E3 12
3.1.2 Proposed model of inBC-E3 ligase system 13
3.2 Engineering of E3 ligase fused antibody 15
3.2.1 Design of various inBC-E3 classes 15
3.2.2 Validation of various inBC-E3 classes 17
3.3 Engineering of VHL fusion linker 19
3.3.1 Modeling of VHL fusion linker 19
3.3.2. Design of VHL fusion linker variants 22
3.3.3 Validation of VHL fusion linker variants 23
3.4 inBC-VHL interacts with both β-catenin and Cul2 E3 ligase complex 26
3.5 inBC-VHL degrades cytosolic β-catenin 28
3.6 Mechanism of action of inBC-VHL 30
3.6.1 Kinetics of inBC-VHL 30
3.6.2 inBC-VHL induces Cul2 E3 complex recruitment and proteasomal degradation 31
3.7 inBC-VHL targets cytosolic β-catenin through ubiquitin-conjugated proteolysis 33
3.8 inBC-VHL inhibits CRC cell growth in vitro 35
3.9 inBC-VHL inhibits CRC cell growth in vivo 37
3.10 Fusion of other E3 ubiquitin ligase recruiting ligands 39
4. Discussion 41
5. CONCLUSION 42
6. REFERENCES 43
