Development of cell-penetrating antibody targeting intracellular activated Ras proteins to block the interaction with effector proteins
Development of cell-penetrating antibody targeting intracellular activated Ras proteins to block the interaction with effector proteins
- 주제(키워드) antibody , cell-penetrating antibody , anti-cancer
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2017
- 학위수여년월 2017. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000025734
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Antibody is very powerful therapeutic reagents targeting protein-protein interactions (PPIs) associated with disease and cancer. However, their target proteins are limited to cell surface receptors and some secreted proteins, because of their hydrophilicity and large size. Impairment of these interactions is more efficient with antibodies than with small molecules because of the large and hydrophilic interaction surfaces of antibodies. Although some approaches have succeeded in delivering antibodies into the cytosol of live cells, many problems, including difficulty in systemic administration, nonspecific cytotoxicity, and loss of stability, remain unresolved. In chapter 2, I report the development of cytotransmabs, full-length human IgG format cell-penetrating antibodies, which penetrate the cytosol of live cells. When VL of IgG format antibodies was replaced with a humanized cell-penetrating VL domain, all cytotransmabs were efficiently internalized and localized in the cytosol. Cytotransmabs that escaped from early endosomes were degraded by the proteasome pathway but not by the lysosome pathway. Furthermore, a human lysyl-tRNA synthetase (KRS)-targeting cytotransmab, called KT4, bound to cytosolic KRS proteins when it was treated to live cells, showing that cytotransmabs can target proteins in the cytosol. In chapter 3, I successfully developed RT11, full-length human IgG format cell-penetrating antibody, which selectively bound to activated Ras proteins. RT11 penetrated into the cytosolic region of live cells and interfered with interactions of effector proteins, thereby suppressing downstream signaling and in vitro growth of oncogenic Ras-mutated cancer cells. Moreover, systemic administration of integrin ανβ3- and ανβ5-binding peptide fused RT11, exhibited anti-tumor activity in oncogenic Ras-mutated tumor xenograft mouse models but not in Ras wild type tumors. In chapter 4, I demonstrated that TRIM21 can degrade a full-length human IgG format cell-penetrating antibody without pathogen infection and affect the anti-tumor efficacy of a Ras·GTP-targeting iMab. I generated an Fc variant of the Ras·GTP-targeting iMab by substituting N434 with aspartic acid to reduce the interaction with TRIM21 and the rate of degradation in the cytosol. Compared with the Ras·GTP-targeting iMab with wild-type Fc, Fc variants exhibited enhanced anti-tumor efficacy in oncogenic Ras-mutated human colorectal and lung cancer cells, without noticeable cytotoxicity in Ras wild-type cancer cells. My conclusion is that the iMab platform technology has the potential of generating human IgG format antibodies for the inhibition of intracellular PPIs associated with disease, using systemic administration such as conventional therapeutic antibody regimens. Therefore, the iMab can extend the antibody target space to proteins in cytosolic region from the current limit of only extracellular and secreted proteins.
more목차
CHAPTER 1. General introduction 1
1.1 Molecular undruggable targets in disease and cancer 1
1.2 Oncogenic Ras mutation in cancer 5
1.3 Intracellular Fc receptor TRIM21 9
CHAPTER 2. A novel platform of cell-penetrating antibodies of human full-length IgG format directly targeting intracellular proteins 11
2.1 Abstract 11
2.2 Introduction 12
2.3 Materials and Methods 14
2.3.1 Cell lines 14
2.3.2 Generation, expression and purification of light chain variable domain antibodies 14
2.3.3 Antibody Construction of full-length human IgG cytotransmabs 14
2.3.4 Generation of KT4 cytotransmab targeting cytosolic KRS 15
2.3.5 Expression and purification of human IgG antibodies 15
2.3.6 Confocal immunofluorescence microscopy 15
2.3.7 Live cell imaging 16
2.3.8 Flow cytometry 17
2.3.9 Cell proliferation assay 17
2.3.10 Immunoprecipitation and western blotting 17
2.3.11 Enzyme-linked immunosorbent assay (ELISA) 18
2.4 Results
2.4.1 Engineering of cell-penetrating light chain variable domain antibodies 19
2.4.2 Generation of cell-penetrating full-length IgG antibodies 21
2.4.3 Internalization and endocytosis mechanism of cytotransmab 25
2.4.4 Pulse-chase trafficking and cytosolic stability of cytotransmab 28
2.4.5 Anti-KRS cytotransmab can directly target the cytosolic KRS 33
2.5 Discussion 36
CHAPTER 3. Cell-penetrating antibody targeting interactions between the activated Ras and effector proteins to inhibit the growth of Ras-mutant tumors 38
3.1 Abstract 38
3.2 Introduction 39
3.3 Materials and Methods 41
3.3.1 Antibodies and reagents 41
3.3.2 Cell lines 41
3.3.3 Expression and purification of antibodies 42
3.3.4 ELISA 42
3.3.5 Confocal immunofluorescence microscopy 43
3.3.6 Western blotting 43
3.3.7 Immunoprecipitation (IP) 43
3.3.8 Cell proliferation assay 44
3.3.9 Flow cytometry 45
3.3.10 Intracellular KRasG12V-cRafRBD interaction inhibition assay 45
3.3.11 Xenograft tumor models 45
3.3.12 Immunohistochemical analysis of tumor tissues 46
3.3.13 Pharmacokinetic studies in mice 47
3.3.14 Biodistribution imaging in vivo 47
3.4 Results
3.4.1 Generation of cell-penetrating activated Ras targeting iMab 49
3.4.2 RT11 penetrates the cytosolic region and interaction with activated Ras 51
3.4.3 RT11 inhibits the in vitro growth of Ras mutant tumor cells 54
3.4.4 RT11 blocks active Ras-effector protein interactions 56
3.4.5 Generation RGD10 peptide-fused Ras·GTP-targeting iMab, RT11-i 59
3.4.6 Pharmacokinetics and biodistribution of RT11-i 64
3.4.7 RT11-i inhibits the in vivo growth of Ras mutant tumors 68
3.4.8 RT11-i overcomes KRas mutation-driven cetuximab resistance 73
3.5 Discussion 76
CHAPTER 4. Engineering the Fc region of Ras·GTP-targeting iMab to improve its stability in cytosol of live cells 78
4.1 Abstract 78
4.2 Introduction 79
4.3 Materials and Methods 81
4.3.1 Cell lines 81
4.3.2 Construction of SA-GFP1-10 expressing stable cell line 81
4.3.3 Construction of GFP11-SBP2-fused antibody 81
4.3.4 Expression and purification of antibody 82
4.3.5 Confocal immunofluorescence microscopy 82
4.3.6 TRIM21 knockdown 82
4.3.7 Pulse-chase quantification of cytosolic RT22-ep59-GFP11-SBP2 antibody 83
4.3.8 ELISA 83
4.3.9 Flow cytometry 83
4.3.10 Cell proliferation assay 84
4.3.11 Soft agar colony formation assay 84
4.4 Results
4.4.1 TRIM21 degrades the cytosolic localized Ras·GTP iMab 85
4.4.2 Generation of Fc variant to escape from TRIM21-mediated degradation 89
4.4.3 Fc variant of EpCAM targeting Ras·GTP iMab exhibits the improved anti-proliferative efficacy in colorectal cancer 93
4.4.4 Fc variant of integrin targeting Ras·GTP iMab exhibits the improved anti-proliferative efficacy in lung cancer 96
4.5 Discussion 99
CONCLUSION 101
REFERENCES 103
ABSTRACT IN KOREAN 110
