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Design and Application of the In Situ Multimerization System for Enhanced Antibody-Receptor Internalization

  • 이상우 (Sangwoo Lee, 아주대학교 일반대학원)

초록/요약

Antibody-based therapeutics and antibody conjugates rely not only on antigen recognition but also on the post-binding fate of antibody–receptor complexes. After binding to cell-surface receptors, antibodies may remain at the plasma membrane, undergo internalization, recycle, or traffic to lysosomes for degradation. These processes strongly influence receptor downregulation, signaling modulation, and intracellular payload delivery. However, many antibody–receptor complexes are not efficiently internalized or degraded by antibody binding alone. Although multivalent or engineered antibody formats can enhance receptor clustering, genetically encoded formats often require construct-specific redesign, expression, and validation whenever the antibody, binding fragment, or target format is changed. This thesis addresses these challenges by developing and evaluating a site-specific antibody conjugation-based strategy for receptor-dependent in situ multimerization on cell surfaces. The strategy was first established using trastuzumab and HER2 as a model system. Trastuzumab was converted into complementary azide- and DBCO-functionalized conjugates through Fc-directed peptide-directed photocrosslinking （PEDIP）. The selected azide–DBCO click pair exhibited minimal background coupling under dilute antigen-free solution conditions, whereas HER2 engagement increased the local concentration of complementary conjugates and promoted cell-surface antibody multimerization. In HER2-expressing cells, in situ TRA–TRA multimerization enhanced antibody internalization, lysosomal trafficking, surface HER2 reduction, and total HER2 degradation. Inhibitor-based experiments suggested sensitivity to multiple endocytic perturbations, whereas downstream HER2 signaling analysis did not identify a clear additional signaling effect specifically attributable to click-induced multimerization under the tested conditions. The platform was then evaluated in a second antibody–antigen system using avelumab and PD-L1. AVE-based complementary conjugates were successfully prepared and tested in cell lines with different PD-L1 expression levels. Compared with the HER2 model, direct biochemical detection of cell-surface multimerization was limited, and the resulting effects on antibody internalization, lysosomal trafficking, and PD-L1 reduction were modest and cell-line dependent. The clearest responses were observed in HCC1954 cells, supporting the conclusion that the in situ multimerization principle can be transferred to an independent target system, while also indicating that receptor abundance and cellular context strongly constrain effect size. To strengthen activity in a less favorable PD-L1 context, heterotypic in situ multimerization was developed by pairing a PD-L1-targeting antibody with a HER2-targeting partner antibody. The click-reactive AVE–TRA pair promoted heterotypic antibody multimerization most clearly in HER2/PD-L1 double-positive HCC1954 cells. This condition increased AVE internalization, AVE–TRA co-distribution, endolysosomal trafficking, pHrodo-detectable delivery to acidic compartments, surface PD-L1 downregulation, and total PD-L1 reduction. These results support the concept that a poorly internalizing target can be redirected toward intracellular trafficking and protein loss by covalent association with a partner-receptor-bound antibody, although further validation is required to establish strict receptor-dependent selectivity. Finally, the chemical foundation of the platform was improved by engineering the PEDIP peptide itself. A CPX-based bacterial display screening workflow was established to enrich photoreactive Fc-binding peptides with enhanced UV-dependent IgG crosslinking activity. The selected B1 peptide improved solution-phase PEDIP efficiency while retaining Fc-directed site specificity at Met252 of the IgG heavy chain. B1-mediated PEDIP enabled preparation of fluorescent antibody conjugates and a trastuzumab–MMAE conjugate that retained HER2-dependent cytotoxicity. Collectively, this thesis connects site-specific antibody conjugation chemistry with receptor trafficking biology and establishes in situ multimerization as a modular strategy for controlling the post-binding fate of cell-surface receptors.

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목차

CHAPTER 1. General Introduction 1
1.1 Antibody-based therapeutics and antibody conjugates 2
1.2 Cell-surface receptor fate after antibody binding 4
1.3 Receptor internalization and lysosomal trafficking in antibody-based therapy 6
1.4 Receptor organization and clustering as regulators of receptor fate 9
1.4.1 Comparison with existing receptor-redirection strategies 11
1.5 Challenges in constructing well-defined antibody conjugates 13
1.6 Affinity-guided photocrosslinking as an enabling technology 15
1.7 Aims of this thesis 17
1.8 General materials and methods 19
1.8.1 Cell lines and cell culture 19
1.8.2 Antibody expression and purification 20
1.8.3 Fc-directed PEDIP photocrosslinking 20
1.8.4 Preparation of azide- and DBCO-functionalized antibody conjugates 21
1.8.5 Size-exclusion chromatography and analytical characterization 21
1.8.6 In situ click reaction assays 22
1.8.7 Western blot analysis 23
1.8.8 Flow cytometry 23
1.8.9 Confocal microscopy and lysosomal colocalization analysis 24
1.8.10 pHrodo-based analysis of acidified intracellular compartments 24
1.8.11 Quantification and statistical analysis 25
CHAPTER 2. In situ Multimerization: System Design and Functional Characterization 26
2.1 Introduction 27
2.2 Chapter-specific experimental procedures 30
2.2.1 Antigen-free solution-phase click reactivity assay 30
2.2.2 Analysis of downstream HER2 signaling 31
2.2.3 Preliminary inhibitor-based analysis of endocytic pathway involvement 31
2.3 Results and discussion 32
2.3.1 Design of the in situ multimerization system 32
2.3.2 Construction and characterization of TRA-based click-reactive conjugates 36
2.3.3 Evaluation of solution-phase background reactivity and cell-surface multimerization 39
2.3.4 Effect of in situ TRA–TRA multimerization on antibody internalization and lysosomal trafficking 43
2.3.5 Effect of in situ multimerization on surface HER2 reduction and receptor degradation 49
2.3.6 Preliminary mechanistic observations on endocytic pathway involvement and downstream HER2 signaling 52
2.4 Conclusion 56
CHAPTER 3. Generalization of in situ Multimerization Using Avelumab and PD-L1 57
3.1 Introduction 58
3.2 Chapter-specific experimental procedures 60
3.2.1 AVE-based click-reactive conjugates 60
3.2.2 Evaluation of PD-L1-associated AVE responses and receptor-fate analysis 60
3.3 Results and discussion 62
3.3.1 Construction of AVE-based click-reactive conjugates for platform generalization 62
3.3.2 PD-L1 expression profiling and limited biochemical detection under low surface-binding conditions 64
3.3.3 Modest and cell-line-dependent effects of click-reactive AVE conditions on internalization and lysosomal trafficking 66
3.3.4 Modest and cell-line-dependent effects of click-reactive AVE conditions on surface PD-L1 reduction and receptor signal 71
3.4 Conclusion 74
CHAPTER 4. Heterotypic in situ multimerization redirects PD-L1 toward internalization and degradation 76
4.1 Introduction 77
4.2 Chapter-specific experimental procedures 79
4.2.1 Heterotypic AVE–TRA in situ reaction assay 79
4.2.2 Analysis of heterotypic internalization, lysosomal trafficking, and PD-L1 reduction 80
4.3 Results and discussion 81
4.3.1 Design and validation of heterotypic click-reactive antibody pairs 81
4.3.2 Internalization and endolysosomal trafficking of AVE–TRA heterotypic complexes 85
4.3.3 Surface downregulation and total PD-L1 protein reduction 91
4.4 Conclusion 93
CHAPTER 5. Optimization of Fc-directed Photocrosslinking for Site-specific Antibody Conjugation 95
5.1 Introduction 96
5.2 Materials and Methods 99
5.2.1 Bacterial display of PEDIP peptide constructs 99
5.2.2 Photocrosslinking assay of bacterially displayed peptides 104
5.2.3 Construction and screening of PEDIP peptide libraries 104
5.2.4 Solution-phase characterization of B1-mediated PEDIP 105
5.2.5 Preparation of fluorescent antibody conjugates and ADCs 106
5.2.6 LC–MS/MS site mapping and ADC characterization 107
5.2.7 In vitro cytotoxicity assay 108
5.3 Results and Discussion 109
5.3.1 Establishment of a bacterial display system and design of photoreactive Fc-binding peptide libraries 109
5.3.2 Screening and identification of B1 as an enhanced photocrosslinking peptide 113
5.3.3 Characterization of B1-mediated photocrosslinking activity 119
5.3.4 Preparation and evaluation of site-specific ADCs using B1-mediated PEDIP 122
5.4 Conclusion 126
CHAPTER 6. Summary 128
References 131

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