Dual-specific antibody co-targeting EGF receptor and Neuropilin-1 overcomes the resistance of tumors to EGFR-targeting antibody therapy
Dual-specific antibody co-targeting EGF receptor and Neuropilin-1 overcomes the resistance of tumors to EGFR-targeting antibody therapy
- 주제(키워드) Antibody
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000026765
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Therapeutic antibodies are one of the most successful therapeutics for patients with solid tumors. However, the abnormal physiological properties of tumor tissue, such as the incomplete blood vessels and extracellular matrix components, limit the penetration of mAbs from blood. In addition, the intrinsic properties of mAbs, containing the relatively large size and high-affinity antigen binding, also limit vascular extravasation and transport within tumor tissue. Although various approaches have attempt to enhance tumor penetration of mAb, promising promoter agent has been developed not yet. Besides limitation of tumor penetration, therapeutic mAbs exhibit low response rate in the treatment of cancer patients because of intrinsic or acquired resistance. Therefore, I conducted a study to increase the efficacy of mAbs by overcoming these limitations. In chapter 2, I report the development of immunoglobulin Fc-fused, neuropilin-1-specific peptide, which enhance tumor tissue penetration. An immunoglobulin Fc-fused peptide, Fc-TPP11, specifically binds to the VEGF-binding site of neuropilin-1 (NRP1) and increases the tumor penetration of co-injected anti-cancer drug or antibody. This is showing that Fc-TPP11 can be used as a tumor penetration-promoting agent. In chapter 3, I identified resistance mechanism to cetuximab (Ctx), anti-EGFR mAb, in pancreatic ductal adenocarcinoma cells (PDADs). Also, to overcome resistance, I generated EGFR and NRP1 dual targeting antibody, Ctx-TPP11. Ctx-TPP11 efficiently suppressed the growth of cetuximab-resistant PDACs, in vitro and in vivo. In chapter 4, I demonstrated that strategy for co-targeting of EGFR and NRP1 can overcome resistance to the five EGFR-targeted antibodies, including cetuximab, in non-small cell lung cancer (NSCLCs), too. Moreover, I found that resistance mechanism to cetuximab different from PDACs is integrin β3-driven bypass signaling in NSCLCs harboring Ras mutation. I validated that suppression of integrin β3-mediated signaling by targeting NRP1, while inhibiting EGFR signaling, restores cetuximab resistance in vitro. My conclusion is that the solid tumor-penetrating peptide (TPP) platform technology using peptide TPP11, targeting NRP1, can easily apply to anti-cancer drugs or antibodies and a strategy for co-targeting EGFR and NRP1 can be effective therapeutic strategy in PDACs and NSCLCs. Therefore, it will provided new insights into NRP1 biology and next generation therapeutic antibody.
more목차
CHAPTER 1. General introduction 1
1.1 Therapeutic antibodies in cancer 1
1.2 The role of Neuropilin-1 in vascular and tumor biology 4
1.3 Anti-EGFR targeted therapies in cancer 6
CHAPTER 2. Development of Immunoglobulin Fc-fused, Neuropilin-1-specfic peptide to enhance tumor tissue penetration 8
2.1 Abstract 8
2.2 Introduction 9
2.3 Materials and Methods 11
2.3.1 Fc-peptide library construction and screening 11
2.3.2 Construction, expression and purification of Fc-peptides and antibodies 11
2.3.3 Cell lines 11
2.3.4 Xenograft tumor models 12
2.3.5 Immunofluorescence microscopy of tumor tissues 12
2.3.6 Ex vivo tumor penetration assay 13
2.3.7 In vitro transendothelial permeability assay 13
2.3.8 Quantification of DOX in tumors 13
2.3.9 Biodistribution 14
2.3.10 Pharmacokinetics 14
2.3.11 Matrigel plug assay 15
2.3.12 Tumor metastasis model 15
2.4 Results
2.4.1 Generation of NRP1-specific, Fc-fused peptides 16
2.4.2 Fc-TPP11 homes onto tumor vessels and extravasates into tumor tissues by downregulating NRP1-mediated VE-cadherin 21
2.4.3 Fc-TPP11 penetrates ex vivo tumor tissues through NRP1-mediated E-cadherin downregulation 25
2.4.4 Fc-TPP11 enhances anti-tumor effect of co-injected drug by promoting the tumor tissue penetration 27
2.4.5 Cetuximab-TPP11 penetrates into tumor tissues more significantly than cetuximab 27
2.4.6 Pharmacokinetics of Fc-TPP11 30
2.4.7 Fc-TPP11 suppresses VEGF-mediated angiogenesis 32
2.4.8 Fc-TPP11 shows in vivo anti-tumor efficacy 35
2.4.9 Fc-TPP11 does not promote tumor metastasis 37
2.5 Discussion 40
CHAPTER 3. Dual-targeting of EGF receptor and Neuropilin-1 overcomes cetuximab resistance in pancreatic ductal adenocarcinoma 43
3.1 Abstract 43
3.2 Introduction 44
3.3 Materials and Methods 46
3.3.1 Antibodies and reagents 46
3.3.2 Cell lines 46
3.3.3 Cell 47
3.3.4 RNA interference 47
3.3.5 Flow cytometry 47
3.3.6 Western blot analysis 48
3.3.7 Cell adhesion assay 48
3.3.8 Xenograft tumor models 49
3.3.9 Confocal microscopy of cells and tissues 49
3.3.10 Statistical analysis 50
3.4 Results
3.4.1 Cetuximab resistance in PDADs is associated with integrin β1 overexpression and its downstream Src-Akt signaling axis 51
3.4.2 Ctx-TPP11, an EGFR-NRP1-co-targeting antibody, overcomes integrin β1-mediated cetuximab resistance in CtxR PDACs 54
3.4.3 Dual targeting of EGFR and NRP1 suppresses integrin β1-mediated CtxR signaling in CtxR PDACs 58
3.4.4 NRP1 targeting of Fc-TPP11 and Ctx-TPP11 reduces the amount of cell surface-expressed active integrin β1 through NRP1-coupled internalization 60
3.4.5 Dual targeting of EGFR and NRP1 shows in vivo anti-tumor activity and suppression of Src and Akt activation in CtxR PDAC xenografts 63
3.5 Discussion 66
CHAPTER 4. Co-targeting of EGFR and Neuropilin-1 overcomes resistance to the EGFR-directed therapeutic antibody in Non-Small Cell Lung Cancer with KRas mutation 69
4.1 Abstract 69
4.2 Introduction 70
4.3 Materials and Methods 72
4.3.1 Reagents and antibodies 72
4.3.2 Cell lines 72
4.3.3 Construction of antibody plasmids 73
4.3.4 Expression and purification of antibodies 73
4.3.5 Cell proliferation assay 73
4.3.6 RNA interference 73
4.3.7 Western blotting 74
4.3.8 Immunoprecipitation 74
4.3.9 Confocal microscopy of cells and tissues 75
4.4 Results
4.4.1 An EGFR-NRP1-co-targeting antibody overcomes resistance to EGFR- targeted mAbs in NSCLCs 76
4.4.2 Expression of NRP1 and integrin β3 mediates cetuximab resistance of NSCLCs harboring KRas mutation 80
4.4.3 NRP1 interacts with integrin β3 and form integrin β3-mediated KRas complex 83
4.4.4 Integrin β3-mediated pathway, KRas-TBK1 and Src-Akt, is critical for cetuximab resistance in NSCLCs harboring KRas mutation 85
4.4.5 Ctx-TPP11 downregulate the cell surface levels of integrin β3 by NRP1-coupled internalization and inhibits integrin β3-mediated CtxR signaling 87
4.5 Discussion 90
CONCLUSION 92
REFERENCES 94
ABSTRACT IN KOREAN 105
PUBLICATIONS 107
