목차

1. Introduction 1
1.1 Delivery of antibodies into the cytosol 1
1.2 Cytosol-penetrating antibody (Cytotransmab) 5
1.3 Amino acids for endosomal escape 8
1.4 Structural characteristics of antibody constant regions 9
2. Materials and methods 12
2.1 Cell lines 12
2.2 Construction of antibody expression plasmids 12
2.3 Construction of SA-GFP1-10-expressing stable cell lines 13
2.4 Structural analysis of antibody constant regions 14
2.5 Expression and purification of IgG antibodies 14
2.6 Size Exclusion Chromatography (SEC) 15
2.7 Enzyme-linked immunosorbent assay (ELISA) 15
2.8 Flow cytometry 16
2.9 Bio-layer interferometry (BLI) 16
2.10 Western blot 17
2.11 Split-GFP complementation assay 18
2.12 Split-luciferase complementation assay 18
2.13 Trypan blue uptake assay 19
2.14 Confocal immunofluorescence microscopy 20
2.15 Statistical analysis 21
3. Results 22
3.1 Generation of cytotransmabs with CH3-mediated endosomal escape ability 22
3.1.1 Design of endosomal escape motifs in the CH3 domain of IgG antibodies 22
3.1.2 Characterization of biophysical properties of CH3 variants 31
3.1.3 Validation of endosomal escape ability of CH3 variants 38
3.2 Generation of cytotransmabs with CL-mediated endosomal escape ability 43
3.2.1 Grafting of endosomal escape motifs from the CH3 into the CL domain 43
3.2.2 in2C21 has CL-mediated endosomal escape ability 45
3.3 Generation of cytotransmabs with CH3 and CL-mediated endosomal escape ability 48
3.3.1 Characterization of biophysical properties of CH3-CL variants 48
3.3.2 in2C41 possesses improved cytosol-penetrating ability 52
3.4 Generation of intracellular protein-targeting cytotransmabs 58
4. Discussion 64
REFERENCES 68
ABSTRACT IN KOREAN 72