목차

1. Introduction 1
1.1 Intracellular protein delivery 1
1.2 Endocytic pathway 3
1.3 Endosome environment sensitive affinity change 4
2. Materials and methods 5
2.1 Expression plasmids Construction 5
2.2 Expression and purification of antigens and Antiboies in mammalian cell 5
2.3 Modeling of Alphafold2 6
2.4 Size Exclusion Chromatography (SEC) 6
2.5 Flow cytometry 7
2.6 Bio-layer Interferometry (BLI) 7
2.7 Cell lines 8
2.8 Stable cell line construction 8
2.9 Western blot 9
2.10 Split-GFP Complementation assay 9
2.11 Monobody library construction and screening by Yeast surface display 10
2.12 Expression and purification of Immunotoxin 11
2.13 Enzyme-linked immunosorbent assay (ELISA) 11
2.14 in vitro cytotoxicity assay 12
2.15 Statistical analysis 12
3. Results 13
3.1 Construction and validation of tumor associated antigen 13
3.2 Design and engineering of anti-tumor associated antigen monobody 16
3.3 Generation of anti-tumor associated antigen monobody-fused CT99 20
3.3.1 Validating biophysical property of monobody-fused CT99 20
3.3.2 Evaluating antigen binding on reducing condition of monobody-fused CT99 23
3.3.3 Evaluating endosomal escape efficiency of monobody-fused CT99 using Split-GFP assay 26
3.4 Affinity maturation of monobody using yeast surface display 30
3.5 Generation and Evaluation monobody-fused PE24 immunotoxin 35
3.5.1 Evaluating antigen binding on reducing condition of monobody-fused PE24 35
3.5.2 Validating cytosolic localization of monobody-fused PE24 in vitro cytotoxicity assay 40
3.6 Affinity matured monobody-PE24 exhibits expanded therapeutic window in vitro 44
3.7 Validating endosomal escape efficiency of affinity matured monobody-fused CT99 48
4. Discussion 50
CONCLUSION 51
REFERENCES 53
ABSTRACT IN KOREAN 57