DGIST Scholar: Engineering precision immunomodulators: Primed MSC-EVs for enhanced immune regulatory function & Tandem bispecific IL-7R agonist antibody for selective T cell expansion

Title

Engineering precision immunomodulators: Primed MSC-EVs for enhanced immune regulatory function & Tandem bispecific IL-7R agonist antibody for selective T cell expansion

Alternative Title

정밀 면역 조절제 엔지니어링: 향상된 면역 조절 기능을 위한 프라이밍된 MSC-EV 및 선택적 T 세포 확장을 위한 탠덤 이중 특이성 IL-7R 작용제 항체

DGIST Authors

Jun-Kook Park ; Kyungmoo Yea ; Il-Kyu Choi

Advisor

예경무

Co-Advisor(s)

Il-Kyu Choi

Issued Date

2026

Awarded Date

2026-02-01

Type

Thesis

Description

Precision immunomodulation, Engineered biologics, Mesenchymal stem cell-derived extracellular vesicles, IL-7 receptor agonist antibody

Abstract

면역 체계의 항상성 붕괴는 과도한 면역 반응을 보이는 자가면역질환, 급성 폐 손상(ALI)과 같은 면역 과활성 질환과, 암이나 만성 바이러스 감염처럼 면역 반응이 불충분한 면역 결핍 질환을 야기합니다. 기존의 면역 치료법은 전신에 영향을 미치는 부작용이 크고, 근본적인 면역 조절 기능의 회복보다는 증상 완화에 초점을 맞추는 한계가 있었습니다. 본 학위 논문은 이러한 한계를 극복하기 위해 생명공학 기술을 이용하여 면역 체계의 항상성을 정밀하게 복원하는 두 가지 혁신적인 면역조절 치료제 개발 연구를 제시합니다.
첫 번째 연구는 사이토카인으로 기능이 강화된 중간엽 줄기세포 유래 세포외소포(MSC-EVs)를 이용한 급성 폐 손상 치료에 관한 것입니다. 염증성 사이토카인(IFN-γ, TNF-α)으로 자극한 중간엽 줄기세포에서 분리한 세포외소포(P-MEVs)는 기존 세포외소포보다 항염증 및 조직 재생 능력이 월등히 뛰어났습니다. 동물 실험 결과, 이 강화된 세포외소포는 급성 폐 손상 모델에서 폐의 염증과 손상을 효과적으로 완화했으며, 이는 세포외소포 내 면역 조절 기능이 강화된 마이크로RNA(miRNA) 덕분임을 확인했습니다. 이 연구는 세포 공학 기술을 통해 세포외소포의 치료 효능을 극대화하여 면역 과활성 질환에 대한 새로운 치료 전략을 제시합니다.
두 번째 연구는 T 세포 결핍을 극복하기 위한 새로운 항체 기반의 IL-7 수용체 작용제 개발에 중점을 둡니다. 기존의 IL-7 치료제는 짧은 반감기와 좁은 치료 범위의 한계가 있었습니다. 이를 해결하기 위해, IL-7 수용체의 두 소단위체(IL-7Rα, γc)에 동시에 결합하는 완전 인간 이중 특이 항체(TB4)를 설계 및 제작했습니다. 이 항체는 기존 IL-7보다 약효가 오래 지속될 뿐만 아니라, 특정 T 세포 아형인 항바이러스 유전자 특성을 가진 CD4⁺ 기억 T 세포를 선택적으로 증식시키는 독특한 기능을 보였습니다. 이 결과는 분자 공학 기술을 통해 면역 결핍 상태에서 특정 면역 세포의 기능을 정밀하게 조절할 수 있는 차세대 면역 치료제 개발의 가능성을 입증합니다.
결론적으로, 본 논문은 세포 및 분자 공학 기술을 활용하여 각각 면역 과활성 및 면역 결핍이라는 상반된 면역 불균형 상태를 조절하는 두 가지 정밀 면역치료제를 성공적으로 개발했습니다. 이러한 연구 결과들은 면역 질환의 근본적인 원인을 해결하고, 인체의 자연적인 면역 항상성을 회복시키는 차세대 바이오 의약품 개발에 중요한 과학적 토대를 제공합니다.
|The disruption of immune homeostasis leads to a spectrum of disorders, from hyper-inflammatory conditions like autoimmune diseases and acute lung injury (ALI) to immunodeficient states such as cancer and chronic viral infections. Conventional immunotherapies are often limited by systemic side effects and tend to alleviate symptoms rather than restore the underlying immune regulatory functions. This dissertation presents two studies on the development of innovative immunomodulators using bioengineering principles to precisely restore immune homeostasis. The first study focuses on the treatment of acute lung injury using mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) enhanced with cytokine priming. MSC-EVs isolated from MSCs stimulated with inflammatory cytokines (IFN-γ and TNF-α) demonstrated significantly superior anti-inflammatory and tissue-regenerative capabilities compared to EVs from unprimed cells. In a preclinical model of ALI, these primed MSC-EVs effectively mitigated lung inflammation and damage. This enhanced function was attributed to the enrichment of immunoregulatory microRNAs within the EVs. This research presents a novel therapeutic strategy for hyper-inflammatory diseases by maximizing the therapeutic efficacy of EVs through cellular engineering. The second study centers on the development of a novel antibody-based IL-7 receptor agonist to overcome T cell deficiency. Conventional IL-7 therapy is hampered by a short half-life and a narrow therapeutic window. To address this, a fully human tandem bispecific antibody was engineered to co-engage the two subunits of the IL-7 receptor (IL-7Rα and γc). This antibody not only exhibited a longer duration of action than recombinant IL-7 but also uniquely induced the selective expansion of CD4⁺ memory T cells with a potent antiviral gene signature. These findings demonstrate the potential of molecular engineering to develop next-generation immunotherapeutics capable of precisely modulating specific immune cell functions in states of immune deficiency. In conclusion, this dissertation successfully demonstrates the development of two distinct precision immunomodulators that address opposite ends of the immune dysregulation spectrum, such as immune hyperactivation and insufficiency, through cellular and molecular engineering strategies. These findings provide a critical scientific foundation for the development of next-generation biologics designed to resolve the root causes of immune diseases and restore natural immune homeostasis.

Table Of Contents

List of Contents
Abstract i
List of Contents ii
List of Figures v
List of Tables vi

I. Introduction 1
1.1 Immune homeostasis and dysregulation 1
1.2 Limitations of conventional immunotherapies 1
1.3 The emergence of precision immunomodulation 2
1.4 Engineering strategies for immunomodulation 3
1.5 Conceptual framework of this dissertation 3

II. Primed MSC-EVs for enhanced immune regulatory function 5
2.1 Introduction 5
2.1.1 Pathophysiology and clinical challenge of acute lung injury 5
2.1.2 Therapeutic potential and limitations of MSC-derived extracellular vesicles 5
2.1.3 Cytokine priming as an engineering strategy to enhance MSC-EVs Immunomodulation 6
2.1.4 Study overview: Evaluating cytokine-primed MSC-EVs for ALI and viral lung injury 6
2.2 Materials & Methods 7
2.2.1 Cell culture 7
2.2.2 Inflammatory priming of hADMSC with IFN-γ and TNF-α 7
2.2.3 Isolation of hADMSC-derived sEVs 7
2.2.4 Nanoparticle tracking analysis (NTA) 8
2.2.5 Transmission electron microscopy (TEM) 8
2.2.6 Western blot analysis 9
2.2.7 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 10
2.2.8 In vitro studies with THP-1 macrophage-like cells and A549 lung epithelial cells 10
2.2.9 Enzyme-linked immunosorbent assay (ELISA) 10
2.2.10 Cell viability assay 11
2.2.11 In vitro epithelial cell permeability assay 11
2.2.12 Animals and LPS-induced ALI mouse model 12
2.2.13 Flow cytometry 12
2.2.14 Lung W/D ratio 13
2.2.15 Evans blue dye extravasation 13
2.2.16 Hematoxylin and eosin (H&E) staining 13
2.2.17 Virus amplification 14
2.2.18 SARS-CoV-2 infection in Vero E6 cells 14
2.2.19 Small RNA isolation from EVs 15
2.2.20 miRNA library preparation and sequencing 15
2.2.21 miRNA data analysis 15
2.2.22 Statistical analysis 16
2.3 Results 19
2.3.1 Characterization of hADMSC-derived EVs 19
2.3.2 P-MEVs ameliorate LPS-induced inflammation and damage in macrophages and lung epithelial cells 24
2.3.3 P-MEVs alleviate inflammation in an LPS-induced ALI mouse model 29
2.3.4 P-MEVs attenuate LPS-induced lung injury, pulmonary edema, and vascular leakage 32
2.3.5 P-MEVs ameliorate the SARS-CoV-2-induced damage in SARS-CoV-2-infected cells 32
2.3.6 miRNAs enriched in P-MEVs suppress LPS-induced inflammation in macrophages and enhance
therapeutic potential against ALI 34
2.4 Discussion 43

III. Tandem bispecific IL-7R agonist antibody for selective T cell expansion 48
3.1 Introduction 48
3.1.1 Essential role of IL-7 in T cell development and homeostasis 48
3.1.2 Therapeutic potential of IL-7 in selective T cell restoration 48
3.1.3 Limitations of recombinant IL-7 and the need for improved therapeutics 49
3.1.4 Study overview: Development of a tandem bispecific IL-7R agonist antibody (TB4) 49
3.2 Materials & Methods 50
3.2.1 Cells and cell culture 50
3.2.2 hPBMC 50
3.2.3 Biopanning 50
3.2.4 ELISA 51
3.2.5 Functional screening for bispecific IL-7R agonists 52
3.2.6 Antibody purification 52
3.2.7 SDS-PAGE 53
3.2.8 Human IL-7 reporter assay 53
3.2.9 Construction and expression of tandem scFv-Fc antibodies 53
3.2.10 BLI analysis 54
3.2.11 Cell-based binding assay 54
3.2.12 In silico structural prediction 55
3.2.13 STAT5 phosphorylation assay 55
3.2.14 Western blotting 56
3.2.15 Antibody internalization 57
3.2.16 T cell survival 57
3.2.17 T cell proliferation 58
3.2.18 T cell subset population 59
3.2.19 RNA-Seq and Data Processing 59
3.2.20 Statistical analysis 60
3.3 Results 62
3.3.1 An engineered tandem bispecific antibody, TB4, functions as a potent IL-7R agonist 62
3.3.2 TB4 demonstrates potent bispecific binding to IL-7Rα and γc, enhanced by the tandem format 68
3.3.3 TB4 is predicted to engage IL-7Rα and γc through distinct molecular interfaces 72
3.3.4 TB4 induces sustained STAT5 signaling and undergoes slow internalization kinetics 77
3.3.5 TB4 promotes the survival and proliferation of T cells, but differentially modulates CD4⁺ and CD8⁺ T
cell subsets 80
3.3.6 Transcriptomic analysis reveals a distinct antiviral signature induced by TB4 83
3.4 Discussion 88

References 92
Summary in Korean 101

URI

https://scholar.dgist.ac.kr/handle/20.500.11750/59603
http://dgist.dcollection.net/common/orgView/200000942451

DOI

10.22677/THESIS.200000942451

Degree

Doctor

Department

Department of New Biology

Publisher

DGIST

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Engineering precision immunomodulators: Primed MSC-EVs for enhanced immune regulatory function & Tandem bispecific IL-7R agonist antibody for selective T cell expansion

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