Development of cytosol-penetrating antibody platform technology for inhibition of intracellular protein-protein interaction in living cells
Development of cytosol-penetrating antibody platform technology for inhibition of intracellular protein-protein interaction in living cells
- 주제(키워드) antibody , cell penetrating , Ras , affinity maturation
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2016
- 학위수여년월 2016. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000022737
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Protein–protein interactions (PPIs) are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Targeting the interfaces between intracellular proteins has huge therapeutic potential, but discovering antagonistic drugs that disrupt PPIs is an enormous challenge. Antibodies are appropriate for antagonizing PPIs with high specificity and affinity for targeted proteins and have large binding interfaces, but they cannot penetrate into the cytosol of living cells because of their large size and hydrophilicity. Many attempts have been made to directly deliver antibodies into the cytosol. Although this research has had some success in delivering antibodies into living cells, many issues, including nucleus localization, loss of antibody stability, and difficulties with systemic administration, remain unresolved. Here I report an antibody technology platform using full-length IgG for penetrating the cytosol and targeting intracellular PPIs. In chapter 2, I report on a cytosol-penetrating antibody called cytotransmab, in the form of a full-length IgG that is created by incorporating a cytosol-penetrating light chain variable domain (VL) into light chains (LCs) and then co-expressing the LCs with the heavy chains (HCs). Cytotransmab was internalized into living cells through a physiological endocytic pathway and then reached the cytosol regions. In chapter 3, I report the application of cytotransmab to directly develop a Ras·guanosine triphosphate (GTP)-specific cytotransmab, called Ras·GTP iMab (internalizing and interfering monoclonal antibody). Ras·GTP iMab was generated by incorporating a Ras·GTP-specific heavy chain variable domain (VH) into the HC of cytotransmab, which has a VL that is able to penetrate into the cytosol of living cells. Ras·GTP iMab efficiently blocked the interaction between Ras·GTP and effector molecules, thus inhibiting downstream cell growth and survival signaling pathways such as Raf-MEK-Erk1/2 and PI3K/Akt. In addition, tumor-expressed integrin-targeted Ras·GTP iMab significantly impaired in vivo tumor growth in human xenograft tumor-bearing mice. In chapter 4, to improve the therapeutic potency of Ras·GTP iMab, I performed affinity maturation using complementarity determining region (CDR)-focused randomization as predicted by computational homology modeling and molecular docking. Isolated variants had a ~5-fold increased affinity against active Ras. My conclusion was that the cytosol-penetrating antibody technology platform has the potential to deliver antibodies into the cytosol of living cells and to directly target the “undruggable” intracellular PPIs. Therefore, this technology platform should be widely applicable to the study of diverse biologic questions, making it attractive for clinical applications.
more목차
1. General introduction 13
1.1 Intracellular protein-protein interactions as therapeutic targets in cancer 13
1.2 Cancer target molecules: oncogenic KRas mutation 17
2. Development of intact full-length IgG antibody that penetrate into the cytosol of living cells 21
2.1 Abstract 21
2.2 Introductions 22
2.3 Materials and Methods 24
2.3.1 Cell lines and reagents 24
2.3.2 Modeling of humanized VL single domain antibodies 24
2.3.3 Construction, expression and purification of humanized VL single domain antibodies 24
2.3.4 Construction of intact IgG cytotransmabs 25
2.3.5 Expression and purification of IgG antibodies 25
2.3.6 Size exclusion chromatography (SEC) 25
2.3.7 Confocal immunofluorescence microscopy 26
2.3.8 Cell viability assay 27
2.3.9 Enzyme-linked immunosorbent assay (ELISA) 27
2.3.10 DNA hydrolyzing assay 27
2.3.11 Pulse-chase experiment to monitor intracellular trafficking of cytotransmab 28
2.3.12 Generation of anti-KRS KT4 cytotransmab 28
2.3.13 Surface plasmon resonance (SPR) 28
2.4 Results
2.4.1 Generation of a cytosol-penetrating antibody containing humanized VL single domain 30
2.4.2 Design of cytotransmab by incorporation of humanized, cytosol-penetrating VL 35
2.4.3 Generation of cytosol-penetrating cytotransmab 39
2.4.4 Endocytic mechanism underlying cytotransmab internalization 44
2.4.5 Intracellular trafficking and stability of internalized cytotransmab 46
2.4.6 Cytotransmab penetrates and directly binds to a targeted cytosolic protein 48
2.5 Discussion 51
3. Development of Cytosol-penetrating antibody inhibiting Ras-effector interactions suppresses the growth of Ras-addicted tumors 54
3.1 Abstract 54
3.2 Introductions 55
3.3 Materials and Methods 57
3.3.1 Cell lines and reagents 57
3.3.2 Construction of recombinant protein expression plasmids 58
3.3.3 Protein expression and purification 58
3.3.4 Preparation of GppNHp or GDP-loaded Ras proteins 59
3.3.5 Screening of yeast HC library against KRasG12DGppNHp 60
3.3.6 Library construction and screening for affinity maturation of RT4 61
3.3.7 Construction of antibody expression plasmid 62
3.3.8 Expression and purification of iMab 62
3.3.9 Enzyme-linked immunosorbent assay (ELISA) 63
3.3.10 Competition ELISA with cRafRBD and RalGDSRBD 63
3.3.11 Surface plasmon resonance (SPR) 63
3.3.12 Enhanced split GFP complementation assay 64
3.3.13 Confocal immunofluorescence microscopy 65
3.3.14 Immunoprecipitation and western blot analysis 65
3.3.15 Cell proliferation assay 66
3.3.16 cRafRBD localization assay 66
3.3.17 cRafRBD binding assay 66
3.3.18 Cell surface binding assay (FACS) 67
3.3.19 Xenograft tumor models 67
3.3.20 Immunofluorescence microscopy of tumor tissues 67
3.3.21 Statistical analysis 68
3.4 Results
3.4.1 Generation of KRasGTP specific iMab, RT11 69
3.4.2 RT11 internalizes into the cytosol of living cells and specifically binds to the active KRasGTP form 79
3.4.3 RT11 inhibits tumor cell growth by interfering the PPI between KRas and effector proteins 88
3.4.4 Generation of tumor-associated integrin targeting, KRasGTP specific iMab, RT11-i 91
3.4.5 RT11-i suppresses in vivo growth of KRas-mutant tumor xenografts in mice 94
3.5 Discussion 97
4. Affinity maturation of RasGTP specific iMab, RT11 99
4.1 Abstract 99
4.2 Introductions 100
4.3 Materials and Methods 101
4.3.1 Cell lines and reagents 101
4.3.2 Homology modeling and docking 101
4.3.3 Library construction of RT11 VH 101
4.3.4 Construction, expression and purification of avi-KRasG12DGppNHp 102
4.3.5 Gel shift assay 103
4.3.6 Isolation of affinity improved single clones form scFab library 103
4.3.7 Construction of RT11 variants expression plasmids 104
4.3.8 Expression and purification of RT11 iMab variants 104
4.3.9 Enzyme-linked immunosorbent assay (ELISA) 104
4.3.10 Surface plasmon resonance (SPR) 105
4.3.11 Cell proliferation assays 105
4.4 Results
4.4.1 Strategy and construction of RT11 scFab library 106
4.4.2 Screening and isolation of affinity matured RT11 variants 108
4.4.3 Binding analysis of affinity improved RT22-i3 112
4.4.4 RT11-i3 variants interacts with intracellular active KRas and inhibits the growth of oncogenic Ras-addicted tumor cells 115
4.5 Discussion 118
CONCLUSION 120
REFERENCE 122
ABSTRACT IN KOREAN 130
PUBLICATIONS AND PATENTS 132
