이중특이 항체 기반기술을 위한 항체의 중쇄불변부위의 이종이중체 형성 변이체 개발
Development of heterodimeric Fc variants for bispecific antibody platform technology
- 주제(키워드) heterodimeric Fc , antibody engineering
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2015
- 학위수여년월 2015. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000020682
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Bispecific antibodies (BsAbs) bind to two different molecules and provide improved clinical benefits in the treatment of complicated diseases compared with conventional monoclonal antibodies (mAbs). BsAbs are developed most of the formats that deviate from the natural IgG architecture suffer from poor physicochemical properties, difficulties in large-scale manufacturing, and potential immunogenicity. To minimize these problems, heterodimeric Fc based bispecific heterodimeric IgG antibody formats that are as close as possible to human IgG have been developed. Heterodimeric Fc is designed by asymmetric mutations in the CH3 homodimeric interface of IgG heavy chains. In chapter 2, I report that newly designed heterodimeric Fc, called EW-RVT variant, by introducing asymmetric hydrohphobic interactions and asymmetric electrostatic interaction between two distinct CH3 domains. Designed heterodimeric Fc variants exhibit with more than 90% yield, retained the inherent FcRn and FcγRs interaction properties. Using the EW-RVT variant, heterodimeric Fc?based bsAbs (called bsVeMet) are constructed that simultaneously targets VEGFR-2 and Met (VEGFR-2×Met) in the format of scFv-Fc heterodimer. The bsVeMet simultaneously bound to both VEGFR-2 and Met in a noncompetitive manner, strongly inhibited the cell growth and greatly reduced the activations of the two targeted receptors. These results mean that EW-RVT can be applied to the production of bispecific antibody or Fc fusion protein. In chapter 3, to improved heterodimer formation and thermodynamic stability, I generated an inter-CH3 disulfide-bonded heterodimeric Fc variant, EW-RVTS?S. The EW-RVTS?S variant showed improved heterodimer yield by ~3% and higher thermodynamic stability of the CH3 domain by ~ 2.8℃ compared with the parent EW-RVT variant. To understand the molecular interactions at the CH3A-CH3B interface favoring heterodimer, determined the X-ray crystal structure of the EW-RVT and EW-RVTS?S. The overall three-dimensional structures of the EW-RVT and EW-RVTS?S Fc heterodimer are very closely superimposed with that of the wild type, including the regions responsible for the interactions with neonatal Fc and Fcγ receptors, indicating that the two Fc heterodimer-based bsAbs will have similar serum half-lives and effector functions to the conventional human IgG1. In chapter 4, I reported the heterodimeric Fc design by directed evolution using the yeast surface-displayed heterodimeric Fc. Heterodimeric Fc design using the directed evolution have the advantages, such as enable to high throughput screening and enable to isolation of unexpected type of pairs. Combinatorial heterodimeric Fc libraries are generated using the yeast mating. And heterodimeric Fc variants are obtained that exhibit high heterodimer formation yield and having unexpected interaction (e.g., cation-π interaction, hydrophobic interaction) through library screening. In conclusion, newly designed heterodimeric Fc variants exhibits high yield heterodimer formation, retained wild-type Fc biophysical properties and determine the X-ray crystal structures. These results will be useful for the design of heterodimeric Fcs and generation of IgG-like bsAbs. Additionally, developed heterodimeric Fc formation monitoring system are useful to screening of novel heterodimeric Fc variants. It will be provided new insights into how design the Fc heterodimer with high purity and biophysical properties.
more목차
CHAPTER 1. General introduction 1
1.1 Bispecific antibody (BsAb) 1
1.2 BsAb formats 4
1.3 Heterodimeric Fc 8
CHAPTER 2. Heterodimeric Fc-based bispecific antibody simultaneously targeting VEGFR-2
and Met exhibits potent antitumor activity 13
2.1 Abstract 13
2.2 Introductions 14
2.3 Materials and Methods 17
2.3.1 Cell lines and reagents 17
2.3.2 Design and evaluation of heterodimeric Fc 18
2.3.3. Construction, expression, and purification of antibodies 21
2.3.4. SEC analysis 22
2.3.5. Far-UV CD spectroscopy 22
2.3.6. Differential scanning calorimetry (DSC) 22
2.3.7. SPR analysis 23
2.3.8. Sandwich ELISA 23
2.3.9. Flow cytometry 24
2.3.10 Cell proliferation assay and western blotting 24
2.3.11 Tube formation assay 25
2.3.12 Animal experiments 26
2.3.13 Immunofluorescence of tumor tissues 26
2.4 Results 28
2.4.1 Design of the heterodimeric Fc variant 28
2.4.2 Evaluation of heterodimeric Fc formation 33
2.4.3 Biochemical characterization of heterodimeric Fc 37
2.4.4 Construction and biochemical characterization of a bsAb targeting VEGFR-2 and
Met 44
2.4.5 Biological activities of the bsAb targeting VEGFR-2 and Met in endothelial and
tumor cells 49
2.4.6 The in vivo anti-tumor efficacy of the VEGFR-2Met-targeting bsAb 57
2.5 Discussion 61
CHAPTER 3. Crystal structures of immunoglobulin Fc heterodimers reveal the molecular
basis for heterodimer formation 65
3.1 Abstract 65
3.2 Introductions 66
3.3 Material and Methods 68
3.3.1 Construction, expression, and purification of heterodimeric Fc variants 68
3.3.2 Differential scanning calorimetry 68
3.3.3 Guanidine hydrochloride unfolding assay 68
3.3.4 Crystallization and data collection 69
3.3.5 Structure determination and refinement 69
3.4 Results 72
3.4.1 Crystal structure of the EW-RVT heterodimeric Fc variant 72
3.4.2 Design and structural analysis of an inter-CH3 domain disulfide-bonded
heterodimeric Fc variant 78
3.4.3 Enhanced heterodimerization of the EW-RVTS-S Fc variant 81
3.4.4 Improved thermal stability of the EW-RVTS-S Fc variant 83
3.5 Discussion 88
CHAPTER 4. Engineering of heterodimeric Fc using yeast surface-displayed combinatorial
library sorting 90
4.1 Abstract 90
4.2 Introductions 91
4.3 Material and Methods 93
4.3.1 Yeast strains and media 93
4.3.2 Construction of heterodimeric Fc display systems 93
4.3.3 Heterodimeric Fc library construction and screening 96
4.3.4 Evaluation of heterodimeric Fc variants 96
4.3.5 Statistical Analysis 97
4.4 Results 98
4.4.1 Relationship between heterodimer yield and secreted Fc detection level 98
4.4.2 Generation of the heterodimeric Fc libraries by yeast mating 102
4.4.3 Isolation and evaluation of heterodimer formation favored CH3 variants from
heterodimeric Fc libraries 105
4.4.4 Secondary heterodimeric Fc libraries construction and isolation heterodimer
formation improved CH3 variants 114
4.4.5 Biochemical characterization of the CH3 variants from heterodimeric Fc libraries
120
4.5 Discussion 122
CONCLUSION 124
REFERENCES 126
ABSTRACT IN KOREAN 135
PUBLICATIONS 138
