Development of tumor-specific, cytosol-penetrating antibody with improved endosome-escape efficacy
- 주제(키워드) 항체 , 세포질침투항체
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2020
- 학위수여년월 2020. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029542
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Therapeutic antibodies are the most widely researched because of high specificity, high affinity and long serum half-life. However, since antibodies can’t directly penetrate cell membranes as a result of their large molecular size and hydrophilicity, they can only target cell surface expressed receptors and secreted proteins. My group previously reported full-length human IgG format antibodies, called cytotransmabs, which penetrate into the cytoplasm of live cells. To further improve the practical applicability of cytoplasm-penetrating antibodies, I attempted to improve the cytoplasmic delivery of cytotransmab. In chapter 2, to enhance the endosome-escape efficacy of cytotransmab, I first investigated the endosome-escape mechanism of cytotransmab and defined the endosome-escape motif and engineered w/ increasing the interaction of lipid and endosome-escape motif. The local conformational changes of the endosome-escape motif of cytotransmab in response to the acidified pH of endosome were crucial for the membrane pore formation, which allows the cytotransmab to escape into the cytoplasm. Ultimately, I was able to developed the cytotransmab w/ 3-fold improved endosome-escape efficacy. In chapter 3, I successfully generated the VH domain which has ability to localize in cytoplasm by grafting the endosome-escape motif into VH-CDR3 and the VL domain which specifically binds to EpCAM receptor instead of HSPG receptor. Combined the VH and VL domain, I developed a tumor-specific cytoplasm-penetrating cytotransmab w/ improved endosome-escape efficacy. In chapter 4, I demonstrate that immunotoxin, consist of targeting moiety and toxin molecule, can exhibit high cytotoxicity and low side-effect because of tumor-specific delivery of toxins. Because toxin molecules derived from bacteria/plant cause immunogenicity, I generated fully humanized immunotoxin using cytotransmab and hpRNase, which exhibited cytotoxicity in EpCAM-positive human colorectal cancer cells, w/o noticeable cytotoxicity in EpCAM-negative cancer cells. Consequently, cytotransmab technology holds the applicability to develop human IgG format antibodies which can deliver of exogenous cytotoxic molecules, using tumor-specific cytotransmab w/ improved endosome-escape efficacy. Therefore, engineering an antibodies to have efficient endosome-escape ability can overcome limitation of antibodies and provide an opportunity to design an antibodies for antibodies-based agents which carry a wide applicability of bioactive cargos into the cytoplasm.
more목차
1. Overall introduction 1
1.1 Delivery of antibodies into the cytoplasm 1
1.2 Endosome-escape activity and the mechanism 4
1.3 Immunotoxin 6
2. Acidic endosome pH-induced conformational changes mediate endosome-escape of a cytoplasm-penetrating antibody 8
2.1 Abstract 8
2.2 Introduction 9
2.3 Materials and Methods 11
2.3.1 Cells 11
2.3.2 Construction of SA-GFP 1-10-expressing stable cell line by lentiviral transduction 11
2.3.3 Construction of TMab4 mutants expression plasmids 11
2.3.4 Antibodies production 12
2.3.5 Immunofluorescence analysis 12
2.3.6 Trypan blue uptake assay 13
2.3.7 Flow cytometric analysis 13
2.3.8 Modeling of TMab4 variable fragment 14
2.3.9 Cytoplasmic calcein release assay 14
2.3.10 Quantification of cytoplasmic TMab4-GFP 11- SBP2 antibodies 14
2.3.11 Quantitative Western blotting 15
2.3.12 Co-localization of α-tubulin with anti-α-tubulin cytotransmabs 15
2.3.13 Statistical analysis 16
2.4 Results 17
2.4.1 TMab4 induces membrane pore formation at an endosome acidic pH 17
2.4.2 TMab4 destabilizes phospholipid membranes at an endosome acidic pH 21
2.4.3 Acidic pH-induced conformational changes of TMab4 trigger endosome-escape 23
2.4.4 The endosome-escape structural motif resides in VL-CDR3 of TMab4 25
2.4.5 Rational design of TMab4 mutants with enhanced endosome-escape efficacy 28
2.4.6 Quantitative assessment of cellular uptake and cytoplasmic access of TMab4 by an enhanced split GFP complementation assay 32
2.4.7 Generation of an intracellular antigen-targeting cytotransmab w/ enhanced endosome-escape efficacy 37
2.5 Discussion 39
3. Engineering of a tumor cell-specific, cytoplasm-penetrating antibodies with high endosome-escape efficacy 42
3.1 Abstract 42
3.2 Introduction 43
3.3 Materials and Methods 45
3.3.1 Cells 45
3.3.2 Construction of cytotransmab expression plasmids 45
3.3.3 Antibodies production 45
3.3.4 Immunofluorescence analysis 46
3.3.5 High performance liquid chromatography (HPLC) 46
3.3.6 Flow cytometric analysis 47
3.3.7 Quantification of cytoplasmic cytotransmab 47
3.3.8 Modeling of endosome-escape motif-grafted VH and VL domain 48
3.3.9 Trypan blue uptake assay 48
3.3.10 Cytoplasmic calcein release assay 48
3.3.11 Quantitative Western blotting 49
3.4 Results 50
3.4.1 Engineering TMab4-WYW to abolish the HSPG binding activity 50
3.4.2 Generation of a cytotransmab internalized through EpCAM-mediated endocytosis 52
3.4.3 Generation of a cytotransmab with a VH-dependent endosome-escape activity 56
3.4.4 Generation of a cytotransmab with endosome-escape motifs embedded in both the VH and VL domain 59
3.5 Discussion 63
4. Development of new-generation fully-humanized immunotoxin using cytotransmab 66
4.1 Abstract 66
4.2 Introduction 67
4.3 Materials and Methods 68
4.3.1 Cells 68
4.3.2 Construction of hpRNase-fused antibodies 68
4.3.3 Antibodies production 68
4.3.4 SMAC 69
4.3.5 Pharmacokinetics experiments on mice 69
4.3.6 Flow cytometric analysis 69
4.3.7 Ribonucleolytic activity assay 70
4.3.8 Immunofluorescence analysis 70
4.3.9 Cell proliferation assay 71
4.3.10 Cell line-derived xenograft mouse models 71
4.3.11 Immunohistochemistry analysis of tumor tissues 72
4.4 Results 73
4.4.1 Generation of EpCAM-specific immunoRNase w/ cytotransmab, epCT95-R 73
4.4.2 Pharmacokinetics of epCT95-R 75
4.4.3 The cellular internalization of epCT95-R via EpCAM-specific endocytosis 77
4.4.4 epCT95-R inhibits the in vitro growth of EpCAM expressed tumor cells by hpRNase enzyme activity 79
4.4.5 epCT95-R suppresses in vivo growth of EpCAM-overexpressed tumor xenografts in mice 83
4.5 Discussion 86
5. CONCLUSION 88
6. REFERENCES 89
7. ABSTRACT IN KOREAN 92
