Title

Development of targeted therapeutics for improving drug delivery and supportive care in chemotherapy : sEV-based modular platform and G-CSFR agonist antibody

Alternative Title

화학요법에서의 약물 전달 개선 및 지원 치료를 위한 표적 치료제 개발: sEV 기반 모듈형 플랫폼 및 G-CSFR 작용제 항체

DGIST Authors

Jiwon Shin ; Kyungmoo Yea ; Daeha Seo

Advisor

예경무

Co-Advisor(s)

Daeha Seo

Issued Date

2025

Awarded Date

2025-08-01

Type

Thesis

Description

Chemotherapy, Drug delivery system, Small extracellular vesicle, Surface engineering, Granulocyte-colony stimulating factor, Agonist antibody, Hematopoietic stem/progenitor cell, Mobilization

Table Of Contents

Abstract i
Table of Contents ii
List of Figures v

I. Introduction 1
1.1 The evolution of cancer therapy 1
1.2 Chemotherapy 3
1.3 Improving chemotherapy efficacy: Stable and targeted delivery 3
1.4 Mitigating chemotherapy-induced toxicity: Hematopoietic stem cell transplantation 4
1.5 Research object 5

II. Modular and Nondisturbing Chimeric Adaptor Protein Facilitating Surface Engineering of small Extracellular Vesicles 7
2.1 Abstract 7
2.2 Introduction 8
2.2.1. Extracellular vesicles 8
2.2.2 Methods for surface modification of EVs and limitations 8
2.2.3 Aims and objectives 8
2.3 Methods 10
2.3.1 Cells 10
2.3.2 Plasmid construction 10
2.3.3 Expression and purification of antibody 10
2.3.4 Production of stable cell lines 11
2.3.5 Optical microscopy 11
2.3.6 Sample preparation for microscopy 11
2.3.7 Quantitative analysis of EGFP expression at both single-cellular and single-sEV levels 12
2.3.8 Evaluation of expression level and binding efficiency to BG on the cellular membrane 12
2.3.9 Single-molecule tracking 12
2.3.10 Isolation of sEVs 12
2.3.11 Size and concentration analysis of sEVs 13
2.3.12 Western blot analysis 13
2.3.13 Fixation of sEVs for transmission electron microscopy (TEM) analysis 14
2.3.14 Validation of BG-Cet conjugation using SDS-PAGE 14
2.3.15 Surface charge measurement 14
2.3.16 Surface engineering of sEV-CAP 14
2.3.17 Quantification of CAP and conjugated functionalized molecules (X) on a single sEV-X 15
2.3.18 Linear regression analysis of CAP with X on a single sEV-X 16
2.3.19 Analysis of Pearson’s correlation between CAP and conjugated X on a single sEV-X 16
2.3.20 Loading Dox into sEV-CAP 16
2.3.21 In vitro assay 17
2.3.22 Animal experiments 18
2.3.23 Immunofluorescence and histology analysis 18
2.3.24 Imaging data and statistical analysis 19
2.4 Results 21
2.4.1 The sEV-CAP: Surface modification and characterization of sEVs 21
2.4.2 The sEV-X: Stepwise modular engineering of sEVs 31
2.4.3 The sEV-X: Functional enhancement via molecular conjugation 41
2.4.4 sEV(Dox)-Y: Augmented therapeutic potential of sEVs in vivo 45
2.5 Discussion 50

III. Granulocyte-Colony Stimulating Factor Receptor Agonist Antibody, a Potent Mobilizer of
Hematopoietic Stem and Progenitor Cells 52
3.1 Abstract 52
3.2 Introduction 53
3.2.1 Challenges in peripheral blood stem cell mobilization 53
3.2.2 Limitations of current mobilization agents 53
3.2.3 Aims and objectives 53
3.3 Methods 55
3.3.1 Cells 55
3.3.2 Construction of random mutant phage library 55
3.3.3 Antibody selection 55
3.3.4 Antibody expression and purification 56
3.3.5 ELISA 56
3.3.6 Surface plasmon resonance analysis 56
3.3.7 Cell proliferation assay 57
3.3.8 CCLE data mining for CSF3R expression 57
3.3.9 Western blot analysis 57
3.3.10 PB-CD34⁺ cell isolation 58
3.3.11 Granulocyte differentiation analysis 58
3.3.12 In vivo experiments 59
3.3.13 Immunostaining and analysis of hematopoietic and blood cells 59
3.3.14 Splenocytes transplantation assay 59
3.3.15 Single-cell RNA library preparation and sequencing 60
3.3.16 scRNA-seq data processing 60
3.3.17 Cell type annotation 61
3.3.18 Differential gene expression and functional enrichment 61
3.3.19 Gene set scoring and cell cycle analysis 61
3.3.20 Statistical analysis 61
3.4 Results 63
3.4.1 Enhanced agonistic potency of anti-G-CSFR antibody by affinity maturation 63
3.4.2 Potent HSPC mobilization by optimized anti-G-CSFR antibody 71
3.4.3 Transcriptional changes in hematopoietic cells in BM induced by C9 76
3.4.4 Transcriptional reprogramming and niche remodeling in G-CSFR-expressing populations by C9 79
3.4.5 Transcriptomic changes in T/B cells by C9 85
3.4.6 Reconstitution capacity and lymphocyte recovery by C9-mobilized cells 87
3.5 Discussion 90

IV. Conclusion and future perspective 93

References 94
Summary in Korean 108

URI

https://scholar.dgist.ac.kr/handle/20.500.11750/59762
http://dgist.dcollection.net/common/orgView/200000892471

DOI

10.22677/THESIS.200000892471

Degree

Doctor

Department

Department of New Biology

Publisher

DGIST

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