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Distinct Therapeutic Modalities in Pancreatic Cancer via CDCP1-Targeted Antibody-Drug Conjugates and CDCP1×TIGIT Bispecific Antibodies

  • 엄윤정 (아주대학교 일반대학원)

초록/요약

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), 2G10PNU159682, was developed, which incorporates a topoisomerase II inhibitor payload. CDCP1 overexpression significantly correlated with Ras mutations in pancreatic cancer. 2G10PNU159682 demonstrated potent anti-tumor activity both in vitro and in vivo, outperforming selective RAS inhibitors MRTX1133 (targeting KRAS G12D) and sotorasib (targeting KRAS G12C). 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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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

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