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이중특이 항체 기반기술을 위한 항체의 중쇄불변부위의 이종이중체 형성 변이체 개발
- Alternative Title
- Development of heterodimeric Fc variants for bispecific antibody platform technology
- Author(s)
- 최혜지
- Alternative Author(s)
- Hye-Ji Choi
- Advisor
- 김용성
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2015-08
- Language
- eng
- Keyword
- heterodimeric Fc; antibody engineering
- Alternative Abstract
- 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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