Engineering of a Cell-Penetrating Degrading Antibody to Degrade Cytosolic β-catenin by Recruiting E3 Ubiquitin Ligase
- 주제(키워드) β-catenin , Colorectal cancer , Targeted Protein Degradation , Cullin RING E3 ligase
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2022
- 학위수여년월 2022. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031619
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Abnormal activation of Wnt signaling is oncogenic and caused by mutations in the destruction complex components. β-catenin is mutated in ~80% of human Colorectal Cancers (CRC) and is one of the undruggable proteins due to its flat surfaces that lack deep binding pockets. Despite the discovery of small molecules that target the Wnt pathway, β-catenin is rarely inhibited. As same as the Targeted Protein Degradation (TPD) approach, I designed a cytosol-penetrating/degrading IgG antibody, Degrobody, which is directly bound to cytosolic β-catenin and selectively removed it, thereby suppressing its CRC cell/tumor growth. Degrobody bound to the C-terminal domain of β-catenin and recruited the CUL5 E3 ligase to induce degradation. Degrobody showed potent anti-cancer effects on a CRC xenograft model. This study offers a promising β-catenin degrading antibody that could be an effective mAb-based strategy in overcoming the limitations of existing TPD approaches.
more목차
1. Introduction 1
1.1 Cytosolic onco-protein, β-catenin 1
1.2 Cytosol-penetrating/interfering antibody 3
1.3 Targeted Protein Degradation (TPD) 4
1.4 Ubiquitin-Proteasome System (UPS) 5
2. Materials and methods 6
2.1 Cell lines 6
2.2 Construction of DNA plasmids 6
2.3 Construction of HEK293T stable cell lines 6
2.4 Generation of IgG antibodies 7
2.5 Size Exclusion Chromatography (SEC) 7
2.6 Western blot 7
2.7 Structural analysis of VHL/SOCS2 mutant fusion variants 8
2.8 Enzyme-linked immunosorbent assay (ELISA) 8
2.9 Confocal immunofluorescence microscopy 8
2.10 Flow cytometry 8
2.11 NanoLuc assay 9
2.12 Immunoprecipitation (IP) 9
2.13 Cytotoxicity assay 9
2.14 Mouse xenograft models 10
2.15 Statistical analysis 10
3. Results 11
3.1 Development of β-catenin degrading antibody 11
3.1.1 Design of β-catenin degrading antibody, Degrobody 11
3.1.2 Mechanism of action of Degrobody 12
3.2 Engineering of E3 recruiter fused antibody 14
3.2.1 Design and optimization of various inBC2-E3 recuiters 14
3.3 Engineering of VHL/SOCS2 mutant fusion variants 18
3.3.1 Design of VHL/SOCS2 mutant fusion variants 18
3.3.2 Validation of VHL/SOCS2 mutant fusion variants 21
3.4 Identification of inBC2-SOCS2-7 as a β-catenin degrader 23
3.5 inBC2-SOCS2-7 degrades cytosolic β-catenin 25
3.5.1 Characterization of inBC2-SOCS2-7 using NanoLuc assay 25
3.5.2 Cytosolic β-catenin degradation in colorectal cancer cell lines 27
3.6 Mechanism of action of inBC2-SOCS2-7 29
3.7 inBC2-SOCS2-7 degrades β-catenin using Ubiquitin-Proteasome System 31
3.8 inBC2-SOCS2-7 inhibits CRC cell growth in vitro and in vivo. 33
4. Discussion 36
CONCLUSION 37
REFERENCES 38
ABSTRACT IN KOREAN 41
