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Title Page 1

ABSTRACT 4

Contents 6

PART Ⅰ. CDCP1-Targeted Antibody-Drug Conjugates with PNU-159682 for Potent Antitumor Activity in Pancreatic Cancer 15

Ⅰ. INTRODUCTION 16

Ⅱ. MATERIALS AND METHODS 20

1. Cell culture 20

2. Antibodies and reagents 23

3. Antibody generation 23

4. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 24

5. Immunoblotting 24

6. Immunohistochemistry (IHC) 25

7. Surface plasmon resonance (SPR) 25

8. Flow cytometry 26

9. Enzyme-linked immunosorbent assay (ELISA) 26

10. Target epitope identification and specific binding analysis 27

11. CDCP1 stability assay 28

12. Assessment of residual bound antibody internalization 28

13. Cellular imaging of antibody internalization 29

14. Generate for ADCs 29

15. Determination of drug-antibody ratio by LC/ESI-MS 30

16. Cytotoxicity assay 30

17. Apoptosis assay and cell cycle assay 31

18. In vivo efficacy study for ADC 32

19. In vivo efficacy study with chemotherapy combination 32

20. Analysis of RNA-sequencing data 33

21. Statistical analysis 34

Ⅲ. RESULTS 35

1. CDCP1 is expressed in various cancers 35

2. Generation of anti-CDCP1 antibodies 42

3. Characterization of specific high affinity 2G10 antibody 45

4. On-target and off-target binding of 2G10 antibodies 49

5. Characterization of 2G10 antibody mode of action 52

6. Rationale of the selective combination of the payload target gene for ADC 61

7. Production of 2G10 antibody conjugated with PNU-159682 71

8. Cytotoxic effect of 2G10-PNU159682 in vitro 77

9. In vivo antitumor efficacy of 2G10-PNU159682 in pancreatic cancer 82

Ⅳ. DISCUSSION 94

PART Ⅱ. Enhancing Anti-Tumor Immunity via TIGIT Blockade and CDCP1-Targeted Therapy in Pancreatic Cancer 99

Ⅰ. INTRODUCTION 100

Ⅱ. MATERIALS AND METHODS 103

1. Generation of antibody 103

2. Cell and culture 104

3. Enzyme-linked immunosorbent assay (ELISA) 104

4. Surface plasmon resonance (SPR) 105

5. TIGIT/PVR blockade assay 106

6. Flow cytometry 106

7. Immunoblotting and immunoprecipitation (IP) 107

8. Reverse transcription-quantitative polymerase chain reactions (RT-qPCR) 108

9. Cytotoxicity assay with NK-92 cells 109

10. Cytokine array 109

11. Isolation of human umbilical cord blood (UCB)-derived CD34⁺ HSCs 110

12. Generation of mouse models harboring human immune systems 110

13. In vivo efficacy study 111

14. Single-cell suspension from tumor tissue 111

15. Statistical analysis 114

Ⅲ. RESULTS 115

1. Screening for high-affinity and antagonistic anti-TIGIT antibodies 115

2. Chi4F11 exhibits superior binding affinity and antagonistic activity against TIGIT 120

3. Generation and characterization of chi2B5x4F11 bridging CDCP1⁺ and TIGIT⁺ cells 125

4. Chi2B5x4F11 promotes NK cell-mediated cytotoxicity by inhibiting TIGIT 131

5. TIGIT blockade enhances effector function and suppresses tumors in humanized PDAC models 136

Ⅳ. DISCUSSION 145

REFERENCES 156

ABBREVIATION 169

List of Tables 14

PART Ⅰ: CDCP1-Targeted Antibody-Drug Conjugates with PNU-159682 for Potent Antitumor Activity in Pancreatic Cancer 14

Table 1. Information on CDCP1 expression in various cancer cell lines 21

Table 2. The IHC analysis of CDCP1 expression using a tumor microarray of pancreatic... 41

Table 3. Isotyping for anti-CDCP1 antibodies using monoclonal hybridoma culture... 44

Table 4. Effect on cytotoxicity with the linker-payload complex 66

Table 5. Effect on cytotoxicity with various types of 2G10-ADCs 70

Table 6. List of ADCs under preclinical or clinical trial for pancreatic cancer 72

Table 7. In vitro cytotoxicity assay 81

PART Ⅱ. Enhancing Anti-Tumor Immunity via TIGIT Blockade and CDCP1-Targeted Therapy in Pancreatic Cancer 14

Table 1. Antibodies are used for flow cytometry 113

List of Figures 11

PART Ⅰ. CDCP1-Targeted Antibody-Drug Conjugates with PNU-159682 for Potent Antitumor Activity in Pancreatic Cancer 11

Figure 1. The SOCs for pancreatic cancer patients 19

Figure 2. CDCP1 expression across various cancer types 37

Figure 3. Correlation between in silico CDCP1 expression and KRAS mutation status in... 39

Figure 4. CDCP1 expression in PDX tissues from PDAC patients by IHC staining 40

Figure 5. Screening of the selection for candidate antibody targeting CDCP1 43

Figure 6. The 2G10 antibody binds to CDCP1 with high affinity 46

Figure 7. The 2G10 specifically binds to human CDCP1 47

Figure 8. Identification of the CDCP1 binding domain of 2G10 antibody 48

Figure 9. Identification of on-target expressions in normal cells 50

Figure 10. Identification of off-target binding protein for 2G10 antibody through HuProt™... 51

Figure 11. Degradation of cellular CDCP1 protein by the 2G10 antibody 53

Figure 12. 2G10 antibody-induced internalization of CDCP1 in pancreatic cancer cells 59

Figure 13. Schematic overview of ADC design and development 60

Figure 14. Expression of genes associated with ADC sensitivity or resistance in PDAC 64

Figure 15. Screening of potent payload for ADC application in pancreatic cancer 65

Figure 16. Generation of four different 2G10-ADCs conjugated with various linker-payload... 68

Figure 17. Comparison of cytotoxicity using four different 2G10-ADCs 69

Figure 18. The 2G10-PNU159682 exhibited a DAR of 4.95 75

Figure 19. Binding comparability between the naked 2G10 antibody and the conjugated... 76

Figure 20. Cell cycle and apoptosis analysis with 2G10-PNU159682 78

Figure 21. In vitro cytotoxicity of 2G10-PNU159682 and chemotherapy agents 80

Figure 22. Therapeutic efficacy of 2G10-PNU159682 in PANC-1 xenograft 84

Figure 23. Therapeutic efficacy of 2G10-PNU159682 in MIA PaCa-2 xenograft 86

Figure 24. Therapeutic efficacy of 2G10-PNU159682 in HL-60 xenograft 88

Figure 25. Combination therapy 2G10-PNU159682 with standard chemotherapy in PANC-... 90

Figure 26. Combination therapy 2G10-PNU159682 with standard chemotherapy in AsPC-... 92

Figure 27. PTEN expression level in prostate cancer cell line 93

PART Ⅱ. Enhancing Anti-Tumor Immunity via TIGIT Blockade and CDCP1-Targeted Therapy in Pancreatic Cancer 12

Figure 1. Selection of anti-TIGIT antibodies with high and competitive binding to human... 118

Figure 2. Binding affinity and specificity of anti-TIGIT antibodies to TIGIT and PVR 119

Figure 3. Generation and characterization of chimeric anti-TIGIT antibody, chi4F11 122

Figure 4. The chi4F11 antibody time-dependently inhibited TIGIT-mediated signal... 123

Figure 5. Binding affinity and competitive binding of chi4F11 124

Figure 6. Binding specificity of parental chi2B5 antibody targeting human CDCP1 127

Figure 7. Generation of bispecific antibody, chi2B5x4F11 128

Figure 8. Dual targeting for CDCP1 and TIGIT using chi2B5x4F11 129

Figure 9. Bridging with chi2B5x4F11 between human CDCP1 and TIGIT assessed by SPR 130

Figure 10. Expression of checkpoint molecules in effector cells and CDCP1 in target cells 132

Figure 11. Enhanced antitumor cytotoxicity by dual targeting of TIGIT and CDCP1 with... 133

Figure 12. Assessment of the cytotoxic activity of chi2B5x4F11 in the NK-92 and PANC-1... 134

Figure 13. Time-dependent transcriptional changes induced by chi2B5x4F11 treatment 135

Figure 14. Generation of the humanized mouse model with UCB-derived CD34+ HSCs 140

Figure 15. TIGIT blockade remodels immune responses and restrains tumor progression... 141

Figure 16. Immune cell profiling and checkpoint molecule expression following... 143

초록보기

 Pancreatic ductal adenocarcinoma (PDAC) remains one of the most treatment-refractory solid tumors, with a five-year survival rate below 10%. It is characterized by late diagnosis, rapid progression, and resistance to existing chemotherapies and immunotherapies. A defining molecular feature of PDAC is the high prevalence of oncogenic RAS mutations, which drive tumor growth and contribute to an aggressive phenotype and poor prognosis. However, direct targeting of RAS mutations has proven challenging, underscoring the need for alternative therapeutic strategies. CUB domain-containing protein 1 (CDCP1), a transmembrane protein upregulated downstream of RAS activation, has emerged as a promising therapeutic target due to its high expression in PDAC.

In the first part of this study, a CDCP1-targeted antibody-drug conjugate (ADC), 2G10-PNU159682, was developed, which incorporates a topoisomerase Ⅱ inhibitor payload. CDCP1 overexpression significantly correlated with Ras mutations in pancreatic cancer. 2G10-PNU159682 demonstrated potent anti-tumor activity both in vitro and in vivo, outperforming selective RAS inhibitors MRTX1133 (targeting KRASG12D) and sotorasib (targeting KRASG12C). In a mouse xenograft model, 2G10-PNU159682 achieved durable tumor regression and complete remission lasting up to 100 days, even after relapse following gemcitabine or FOLFIRINOX, highlighting its therapeutic potential.

Given the critical role of the immunosuppressive tumor microenvironment in PDAC resistance, immune modulation was investigated as an independent therapeutic strategy. The second part of the study developed a high-affinity anti-TIGIT monoclonal antibody (chi4F11) and a bispecific antibody (chi2B5×4F11) that simultaneously targets TIGIT and CDCP1. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T and NK cells that promotes immune dysfunction in PDAC by engaging with its ligand, PVR, which is abundantly expressed in the tumor milieu. Functional analyses using in vitro NK-92 cell assays and an in vivo humanized mouse model engrafted with CDCP1+ PANC-1 cells demonstrated robust anti­tumor immune responses. The parental chi4F11 antibody effectively blocked the TIGIT-PVR axis, restored AKT-FOXO1 signaling, and preserved CD226 expression. The bispecific antibody chi2B5×4F11 further enhanced NK-92 cell-mediated cytotoxicity and cytokine release. In vivo, treatment with chi2B5×4F11 reduced the proportion of TIGIT expression in CD4+ T cells, CD8+ T cells, and NK cells in circulation, while increasing the frequency of TIGIT- cells within the CD226+ population, thereby enhancing anti-tumor responses.

These findings suggest a novel dual therapeutic strategy in PDAC, employing CDCP1-targeted cytotoxic delivery and CDCP1-TIGIT dual-targeting immune modulation. The approach employs two distinct strategies involving ADCs carrying topoisomerase Ⅱ inhibitors for direct tumor killing and bispecific antibodies targeting both CDCP1 and TIGIT to overcome immune suppression. These complementary strategies collectively provide a promising framework to address oncogenic drivers and immune resistance in pancreatic cancer.

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