Engineering of Interleukin12 (IL12)-Based Immunocytokines with Improved Biophysical Properties
- 주제(키워드) Immunocytokine , Engineering
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2021
- 학위수여년월 2021. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031099
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Interleukin 12 (IL12), a heterodimeric cytokine, is composed of p35 and p40 and elicits potent anti-tumor immunity. However, its short serum half-life and high-dose-related toxicities hamper successful clinical studies of recombinant IL12. In this study, I constructed a heterodimeric Fc-based tumor-targeting antibody-fused IL12 (Ab-mono-IL12) for long-acting and tumor-homing interleukin-12 (IL12) while maintaining the naturally occurring monovalent. Using anti-Her2 herceptin (HCT) as a model of tumor targeting IgG antibody, I constructed various formats varying heterodimeric Fc scaffold pairs with immunoglobulin γ1 or γ4 isotype and length of linker for the fusion of p35 and p40 to each C-terminus of heterodimeric Fc pair and tested the expression propensity in the correctly assembled monomeric form. I further rationally designed IL12 variants with mutations introduced into the interfaces of p35 and p40 subunit with aim of improving the natural IL12 heterodimeric assembly while preventing undesirable oligomer formation. In serial engineering studies of IL12, I found that HCT(L60×L70)-mono-IL12(DDYp40×EKKp35) with mutations of C177D, S183D, L184Y on p40 and S44E, H49E, C74K on p35 was the best format to prevent undesirable oligomer formation. HCT(L60×L70)-mono-IL12(DDYp40×EKKp35) retained IL12 receptor binding activity and biological activities comparable to the wild-type. The IL12 with the mutations of DDYp40×EKKp35 also manifested the least formation of oligomeric species in the context of other IgG-mono-hIL12 formats, anti-EDA F8-mono-IL12, and showed comparable IL12 receptor binding activity and biological activity to wild type. These results demonstrate the potential of IgG(L60×L70)-mono-IL12(DDYp40×EKKp35) as a novel platform to replace conventional IL12-based immunotherapy.
more목차
1. Introduction 1
1.1 Structure and function of interleukin12(IL12) 1
1.2 Previous studies of IL12 based Immunocytokines and their limitation 3
1.3 Heterodimeric Fc 5
2. Material and methods 7
3. Results 11
3.1 Evaluation of heterodimeric Fc variants in IgG format. 11
3.2 Optimization of antibody format and purification condition. 14
3.3 Linker optimization of HCT-mono-IL12 18
3.4 Rationale of IL12 interface engineering for improved biophysical properties 22
3.5 Generation and characterization of HCT-mono-IL12(Dp40×Kp35) variant 26
3.6 Combination of best linker and Dp40×Kp35 mutation for HCT-mono-IL12 30
3.7 Generation and characterization of HCT-mono-IL12(DDp40×Kp35) 32
3.8 Generation and characterization of HCT-mono-IL12(DDYp40×EKKp35) 36
3.9 In vitro biochemical and biological activities of HCT-mono-IL12 (DDYp40×EKKp35) 40
3.10 Evaluation of DDYp40×EKKp35 mutation in other IgG-mono-IL12 42
4. Discussion 44
5. References 47
6. Abstract in Korean 51
