목차

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