아주대학교

목차

1. Introduction 1
1.1 Cytosol-penetrating antibodies as a next-generation therapeutic modality 1
1.2 The endosomal escape bottleneck 2
1.3 Overcoming the endosomal escape bottleneck via pH-dependent antibodies 4
2. Materials and methods 6
2.1 Construction of expression plasmids 6
2.2 In silico structural modeling 6
2.3 Protein expression and purification 6
2.4 Size exclusion chromatography (SEC) 7
2.5 Biolayer Interferometry (BLI) 7
2.6 Enzyme-linked immunosorbent assay (ELISA) 8
2.7 Cell lines 8
2.8 Flow cytometry 8
2.9 Split-GFP complementation assay 9
2.10 Split luciferase endosomal escape quantification (SLEEQ) assay 9
2.11 Statistical analysis 9
3. Results 11
3.1 Engineering pH-dependent anti-EGFR antibody 11
3.2 Screening of the optimized Anti-EGFR pCT clone via histidine scanning 14
3.3 In silico structural analysis of engineered pH-dependent antibody 16
3.4 Development of a universal pH-dependent cytotransmab platform 18
3.4.1 Modular extension of the pH-switch module to multiple TAAs 18
3.4.2 Biophysical characterization and developability 20
3.5 Quantitative evaluation of pH-dependent binding kinetics 22
3.6 Verification of enhanced endosomal escape efficiency 25
3.6.1 Characterization of antigen expression on reporter cell lines 25
3.6.2 Visualizing cytosolic delivery via split-GFP complementation assay 27
3.6.3 Quantifying endosomal escape efficiency via SLEEQ assay 29
4. Discussion 32
CONCLUSION 34
REFERENCES 35
ABSTRACT IN KOREAN 36