Engineering of Bispecific Antibodies Simultaneously Targeting Human Interleukin 4 Receptor Alpha (IL-4Rα) and Interleukin 5 Receptor Alpha (IL-5Rα) for Treatment of Severe Eosinophilic Asthma
- 주제(키워드) Antibody engineering , Bispecific antibody , asthma
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030567
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Asthma is a heterogeneous disease driven by many inflammatory mediators and the severity is threatened a patient’s quality of life. Particularly, most patients with severe asthma showed persistent eosinophilia in blood and airway tissue and diagnosed with severe eosinophilic asthma (SEA). A variety of type 2 cytokines such as interleukin (IL)-4, IL-5 and IL-13 involve in pathways leading to eosinophilia. With the Increased understanding of the biological mechanism of asthma, monoclonal antibodies (mAbs) targeting a specific type 2 inflammatory pathway have been developed. Up to now, five mAbs (omalizumab, mepolizumab, reslizumab, dupilumab and benralizumab) have been licensed for the treatment of patients with severe eosinophilic asthma. However, due to the chronic and multifactorial nature of asthma, the demand for alternative biologics with more potent than conventional mAbs remains high. In chapter 2, I developed mAbs against IL-4 receptor α (IL-4Rα) because it mediates the type 2 inflammatory response of both IL-4 and IL-13 cytokines on a broad range of cells such as immune cells, smooth muscle cells, and endothelial cells. In chapter 3, I generated humanized anti-IL-5Rα mAbs to directly target eosinophils which is a hallmark of severe asthma. To enhance the affinity and antagonistic activity, I engineered complementary-determining regions (CDRs) using yeast surface display technology. Combining proper library construction strategies and screening methods, I successfully generated mAbs with improved affinity and potent antagonistic activity. In these studies, I demonstrated that affinity and binding epitope of mAbs are critical determinants of biological activities. Additionally, I provided potential candidates, 4R.N6 (anti-IL-4Rα mAb) and 5R65.7 (anti-IL-5Rα mAb), as alternative therapeutics for patients with SEA. In chapter 4, I suggested a therapeutic concept using bispecific antibodies (BiAbs) having potential with more potent therapeutic efficacy than mAbs for patients with SEA. Since the heterogeneous and functional redundancy of type 2 cytokines, targeting a specific pathway by mAbs still showed unsatisfactory outcomes to some patients with SEA. In this context, simultaneous targeting both IL-4Rα and IL-5Rα using BiAbs would be more efficacious by concurrently inhibiting multiple type 2 inflammatory pathways. Further, BiAbs also might elicit enhanced antibody-dependent cellular cytotoxicity activity by binding to both IL-4Rα and IL-5Rα expressed on the same cells (e.g. eosinophils). I designed three different formats of bispecific antibodies (BiAbs) using the antigen-binding domain of 4R.N6 and 5R65.7 to explore an optimal format with favorable properties in developability and biological efficacy. The three different formats of BiAbs including bispecific IgG (BsIgG), IgG single-chain Fv (IgG-scFv), and dual-variable domain IgG (DVD-IgG), have distinguishing features such as binding valency for each antigen, the geometry of antigen-binding sites. Since the unnatural format of BiAbs often is an obstacle to further study, I initially analyzed of biochemical properties of BiAbs. The three different formats of BiAbs showed good purification yield and stability. They exhibited different binding kinetics for each antigen, which would lead to different functionality in further studies performing on in vitro and ex vivo.
more목차
CHAPTER 1. General introduction 1
1.1 Biology of eosinophilic asthma 1
1.2 Monoclonal antibodies for the treatment of eosinophilic asthma 4
1.3 Bispecific antibodies (BiAbs) 7
CHAPTER 2. Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity 11
2.1 Abstract 11
2.2 Introduction 12
2.3 Materials and Methods 14
2.3.1 Protein expression and purification 14
2.3.2 Screening of yeast human synthetic Fab library against IL-4Rα 15
2.3.3 Affinity maturation of Abs 15
2.3.4 ELISA 16
2.3.5 Size-exclusion chromatography 17
2.3.6 Determination of Ab affinity by bio-layer interferometry 18
2.3.7 HEK-BlueTM IL-4/IL-13 reporter assay 18
2.3.8 Binding specificity to cell surface-expressed IL-4Rα 19
2.3.9 Human PBMCs 19
2.3.10 Inhibition of IL-4-dependent T cell proliferation 20
2.3.11 Inhibition of IL-4-dependent TH2 cell differentiation 20
2.3.12 Statistical analysis 21
2.4 Results 22
2.4.1 Screening and isolation of anti-IL4Rα Abs from yeast surface-displayed human Fab library 22
2.4.2 Affinity maturation of 4R34 Ab by engineering complementarity-determining regions (CDRs) 28
2.4.3 Engineering of 4R34.1 to generate more potent IL-4Rα-blocking Ab 33
2.4.4 Specificity assessment of 4R34.1.19 37
2.4.5 Epitope mapping of anti-IL-4Rα Abs 40
2.4.6 4R34.1.19 exerts potent inhibitory activity on IL-4-dependent T cell proliferation and TH2 differentiation 44
2.5 Discussion 47
CHAPTER 3. Engineering of humanized antibodies against human interleukin 5 receptor alpha subunit that cause potent antibody-dependent cell-mediated cytotoxicity 50
3.1 Abstract 50
3.2 Introduction 52
3.3 Materials and Methods 54
3.3.1 Isolation of murine Abs against human IL-5Rα 54
3.3.2 Expression and purification of Abs and proteins 55
3.3.3 Cell Cultures 56
3.3.4 Establishment of TF-1 cells stably expressing IL-5Rα 56
3.3.5 A TF-1/IL-5Rα cell proliferation assay 57
3.3.6 Affinity maturation of Abs 57
3.3.7 An ELISA 58
3.3.8 Determination of Ab affinity by bio-layer interferometry 59
3.3.9 Size-exclusion chromatography (SEC) analysis of Abs 59
3.3.10 Epitope mapping of Abs 60
3.3.11 Flow cytometry 60
3.3.12 Isolation of peripheral eosinophils and NK Cells 61
3.3.13 An eosinophil proliferation assay 62
3.3.14 An ADCC assay 62
3.3.15 Statistical analysis 63
3.4 Results 64
3.4.1 Generation of murine Abs against IL-5Rα by immunization 64
3.4.2 Humanization of Anti–IL-5Rα m2B7 68
3.4.3 Affinity Maturation of hu2B7 to Generate 5R65 72
3.4.4 Engineering of 5R65 to Generate a More Potent Ab, 5R65.7 77
3.4.5 Stability assessment and domain mapping of the engineered anti–IL-5Rα Abs 81
3.4.6 5R65.7 impedes robust antagonistic activity on human eosinophils 85
3.4.7 5R65.7 has potent ADCC activity on human eosinophils the benralizumab analogue 89
3.5 Discussion 92
CHAPTER 4. Engineering of bispecific antibodies for effective dual targeting IL-4Rα and IL-5Rα 96
4.1 Abstract 96
4.2 Introduction 98
4.3 Materials and Methods 100
4.3.1 Engineering of anti-IL-4Rα Abs 100
4.3.2 IL-4Rα-blocking assay using HEK-BlueTM IL-4/IL-13 reporter cells 101
4.3.3 Construction, expression, and purification of BiAbs 101
4.3.4 SDS-PAGE and Size-exclusion chromatography analysis 102
4.3.5 Enzyme-linked immunosorbent assay (ELISA) 103
4.3.6 Bio-layer interferometry 103
4.3.7 Statistical analysis 104
4.4 Results 105
4.4.1 Engineering of 4R34.1.19 to generate anti-IL-4Rα mAbs with enhanced antagonistic activity 105
4.4.2 Design of BiAbs 110
4.4.3 Expression and stability analysis of BiAbs 113
4.4.4 Binding analysis of BiAbs 116
4.5 Discussion 118
CONCLUSION 121
REFERENCES 123
ABSTRACT IN KOREAN 136
