아주대학교

목차

PART I: CDCP1-Targeted Antibody-Drug Conjugates with PNU-159682 for Potent Antitumor Activity in Pancreatic Cancer 1
I. INTRODUCTION 2
II. MATERIALS AND METHODS 6
1. Cell culture 6
2. Antibodies and reagents 9
3. Antibody generation 9
4. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 10
5. Immunoblotting 10
6. Immunohistochemistry (IHC) 11
7. Surface plasmon resonance (SPR) 11
8. Flow cytometry 12
9. Enzyme-linked immunosorbent assay (ELISA) 12
10. Target epitope identification and specific binding analysis 13
11. CDCP1 stability assay 14
12. Assessment of residual bound antibody internalization 14
13. Cellular imaging of antibody internalization 15
14. Generate for ADCs 15
15. Determination of drug–antibody ratio by LC/ESI-MS 16
16. Cytotoxicity assay 16
17. Apoptosis assay and cell cycle assay 17
18. In vivo efficacy study for ADC 18
19. In vivo efficacy study with chemotherapy combination 18
20. Analysis of RNA-sequencing data 19
21. Statistical analysis 20
III. RESULTS 21
1. CDCP1 is expressed in various cancers 21
2. Generation of anti-CDCP1 antibodies 28
3. Characterization of specific high affinity 2G10 antibody 31
4. On-target and off-target binding of 2G10 antibodies 35
5. Characterization of 2G10 antibody mode of action 38
6. Rationale of the selective combination of the payload target gene for ADC 47
7. Production of 2G10 antibody conjugated with PNU-159682 57
8. Cytotoxic effect of 2G10-PNU159682 in vitro 63
9. In vivo antitumor efficacy of 2G10-PNU159682 in pancreatic cancer 68
IV. DISCUSSION 80
PART II: Enhancing Anti-Tumor Immunity via TIGIT Blockade and CDCP1-Targeted Therapy in Pancreatic Cancer 85
I. INTRODUCTION 86
II. MATERIALS AND METHODS 89
1. Generation of antibody 89
2. Cell and culture 90
3. Enzyme-linked immunosorbent assay (ELISA) 90
4. Surface plasmon resonance (SPR) 91
5. TIGIT/PVR blockade assay 92
6. Flow cytometry 92
7. Immunoblotting and immunoprecipitation (IP) 93
8. Reverse transcription-quantitative polymerase chain reactions (RT-qPCR) 94
9. Cytotoxicity assay with NK-92 cells 95
10. Cytokine array 95
11. Isolation of human umbilical cord blood (UCB)-derived CD34+ HSCs 96
12. Generation of mouse models harboring human immune systems 96
13. In vivo efficacy study 97
14. Single-cell suspension from tumor tissue 97
15. Statistical analysis 100
III. RESULTS 101
1. Screening for high-affinity and antagonistic anti-TIGIT antibodies 101
2. Chi4F11 exhibits superior binding affinity and antagonistic activity against TIGIT 106
3. Generation and characterization of chi2B5×4F11 bridging CDCP1⁺ and TIGIT⁺ cells 111
4. Chi2B5×4F11 promotes NK cell-mediated cytotoxicity by inhibiting TIGIT 117
5. TIGIT blockade enhances effector function and suppresses tumors in humanized PDAC models 122
IV. DISCUSSION 131

REFERENCES 142
ABBREVIATION 155