Title

Development of bio-therapeutics for intractable disease: GFRAL antagonist antibody for alleviating chemotherapy-induced cachexia & Primed MSC-sEV for amelioration of COVID-19-induced ARDS

Alternative Title

난치 질환의 치료를 위한 바이오 의약품 개발: 화학항암제 유발 악액질 완화 항체 및 COVID-19 유발 급성 호흡곤란 증후군 개선 작은 세포외 소포체

DGIST Authors

Jongwon Jeong ; Kyungmoo Yea ; Jaemin Lee

Advisor

예경무

Co-Advisor(s)

Jaemin Lee

Issued Date

2024

Awarded Date

2024-08-01

Citation

Jongwon Jeong. (2024). Development of bio-therapeutics for intractable disease: GFRAL antagonist antibody for alleviating chemotherapy-induced cachexia & Primed MSC-sEV for amelioration of COVID-19-induced ARDS. doi: 10.22677/THESIS.200000798073

Type

Thesis

Description

Intractable disease, Bio-therapeutics, Antibody, Small extracellular vesicle

Table Of Contents

I. Introduction 1
1.1 Intractable disease 1
1.2 Small-molecule drugs 1
1.3 Bio-therapeutics 2
1.4 Antibody therapeutics 4
1.5 Small extracellular vesicles (sEVs) therapeutics 5
1.6 Research objectives and chapter outlines 6
II. GFRAL antagonist antibody for alleviating chemotherapy-induced cachexia 8
2.1 Introduction 8
2.1.1 Chemotherapy-induced cachexia 8
2.1.2 Growth differentiation factor 15 (GDF15) 8
2.1.3 GDF15/GFRAL/RET axis 9
2.1.4 Aims and objectives 9
2.2 Materials & Methods 10
2.2.1 Biopanning 10
2.2.2 Enzyme-linked immunosorbent assay (ELISA) 10
2.2.3 Antibody purification 11
2.2.4 Surface plasmon resonance (SPR) spectrometry 11
2.2.5 Cells and cell culture 12
2.2.6 Luciferase reporter assay 12
2.2.7 Flow cytometry analysis 12
2.2.8 Immunofluorescence analysis 13
2.2.9 Western blot analysis 13
2.2.10 Animals and in vivo experiments 14
2.2.11 Quantitative real-time polymerase chain reaction (qPCR) 15
2.2.12 Statistical analysis 15
2.3 Results 16
2.3.1 GFRAL antagonist antibody was selected by biopanning and cell-based reporter assay 16
2.3.2 The antibody A11 binds to GFRAL and potently suppresses the GDF15/GFRAL/RET axis 20
2.3.3 The antibody A11 attenuates cisplatin-induced cachexia through inhibition of the GDF15/GFRAL/RET axis in vivo 26
2.3.4 The antibody A11 alleviates cisplatin-induced cachexia in melanoma-bearing mice 32
2.4 Discussion 39
III. Primed MSC-sEV for amelioration of COVID-19-induced ARDS 42
3.1 Introduction 42
3.1.1 COVID-19-induced ARDS 42
3.1.2 MSCs for COVID-19 therapy 42
3.1.3 MSC-derived sEVs 42
3.1.4 MSC priming 43
3.1.5 Aims and objectives 44
3.2 Materials & Methods 45
3.2.1 Cell culture 45
3.2.2 Inflammatory priming of hADMSC with IFN-γ and TNF-α 45
3.2.3 Isolation of hADMSC-derived sEVs 45
3.2.4 Nanoparticle tracking analysis (NTA) 46
3.2.5 Western blot analysis 46
3.2.6 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 47
3.2.7 In vitro studies with THP-1 macrophage-like cells and A549 lung epithelial cells 47
3.2.8 Cell viability assay 48
3.2.9 In vitro epithelial cell permeability assay 48
3.2.10 Animals and mouse model of LPS-induced ARDS 48
3.2.11 Lung wet-to-dry (W/D) ratio 49
3.2.12 Evans blue dye extravasation 49
3.2.13 Hematoxylin and eosin (H&E) staining 50
3.2.14 SARS-CoV-2 infection with Vero E6 cells 50
3.2.15 Small RNA isolation from sEVs 50
3.2.16 miRNA library preparation and sequencing 50
3.2.17 miRNA data analysis 51
3.2.18 Statistical analysis 51
3.3 Results 53
3.3.1 Effect of inflammatory priming of hADMSC with cytokines IFN-γ and TNF-α 53
3.3.2 Characterization of hADMSC-derived sEVs after stimulation by pro-inflammatory cytokines 55
3.3.3 Effects of MSC-sEVs and LPS on cell viability 57
3.3.4 PE ameliorate the LPS-induced damages in macrophages and lung epithelial cells 59
3.3.5 PE reverse the body weight loss in a mouse model of acute lung injury 62
3.3.6 PE alleviate the LPS-induced inflammation in the lungs 64
3.3.7 PE mitigate LPS-induced lung injury, pulmonary edema, and vascular leakage 67
3.3.8 PE ameliorate the SARS-CoV-2-induced damages in SARS-CoV-2 infected cells 69
3.3.9 PE contain enriched miRNA profiles involved in immune response and tissue regeneration 71
3.4 Discussion 73
References 76
Summary in Korean 90

URI

http://hdl.handle.net/20.500.11750/57569
http://dgist.dcollection.net/common/orgView/200000798073

DOI

10.22677/THESIS.200000798073

Degree

Doctor

Department

Department of New Biology

Publisher

DGIST

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