아주대학교

목차

1. Introduction 1
1.1 Antibody–drug conjugates as intracellular delivery-based cancer therapeutics 1
1.2 CEACAM6 as a delivery-limited ADC target in pancreatic cancer 3
1.3 Delivery-aware antibody selection and normalized internalization degree 5
2. Materials and methods 6
2.1 Cell lines 6
2.2 scRNA-seq dataset and preprocessing 6
2.3 CEACAM6-based stratification and differential gene expression 6
2.4 Gene set enrichment analysis and activity scoring 6
2.5 Survival analysis 7
2.6 Preparation of recombinant protein 7
2.7 Generation of CEACAM6-overexpressing SNU-2466 and SNU-2469 cells 7
2.8 Mouse immunization 8
2.9 Optofluidic selection of antigen-specific plasma B cells and BCR sequence analysis 9
2.10 Construction of antibody expression plasmids 9
2.11 Expression and purification of antibodies 10
2.12 ELISA 10
2.13 Flow cytometry 11
2.14 Bio-layer interferometry 11
2.15 Epitope mapping 12
2.16 Internalization 12
2.17 Antibody–drug conjugation and analysis of drug-to-antibody ratio (DAR) 12
2.18 Cytotoxicity assay 13
2.19 Quantification of internalized antibodies using a pH-sensitive dye 13
2.20 In vivo antitumor efficacy study 13
2.21 Statistical analysis 14
3. Results 15
3.1 Transcriptomic and clinical profiling supports CEACAM6 as a therapeutically relevant target in pancreatic cancer 15
3.2 Generation and prioritization of anti-CEACAM6 mouse antibodies 18
3.2.1 Discovery and specificity profiling of anti-CEACAM6 mouse antibodies 18
3.2.2 Epitope mapping and internalization profiling of anti-CEACAM6 mouse antibodies 21
3.2.3 Binding kinetic characterization of selected anti-CEACAM6 mouse antibodies 24
3.3 Humanization and characterization of selected anti-CEACAM6 antibodies 26
3.3.1 Humanization of selected anti-CEACAM6 antibodies 26
3.3.2 Characterization of humanized anti-CEACAM6 antibodies preserving parental binding properties 28
3.4 Generation and cytotoxic evaluation of CEACAM6-Targeting ADCs 33
3.4.1 Generation of CEACAM6-targeting MMAE conjugates 33
3.4.2 In vitro cytotoxicity and therapeutic window of CEACAM6-targeting ADCs 35
3.5 Evaluation of normalized internalization degree as a delivery-aware ADC potency metric 42
3.5.1 Surface engagement and internalization alone are insufficient to explain ADC potency 42
3.5.2 Definition of NID as an integrated delivery metric 46
3.5.3 Evaluation of NID as a predictor of ADC potency 48
3.6 pHrodo-based tracking of intracellular ADC accumulation 51
3.7 In vivo antitumor efficacy of h9738-vc-MMAE in a Capan-1 xenograft model 53
4. Discussion 56
CONCLUSION 60
REFERENCES 61
ABSTRACT IN KOREAN 64