Overcoming the limitations of anti-VEGF therapy using a VEGFA and NRP1 dual-targeting bispecific antibody
- 주제(키워드) VEGFA , NRP1 , angiogenesis , cancer
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030466
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Numerous pathways participate in angiogenesis, which is the fundamental process involved in physiological and pathological conditions. Vascular endothelial growth factor A (VEGFA), the leading player in this process, binds VEGF receptors and activates downstream signaling pathways related to cell proliferation, migration, and permeability. Since VEGF was identified, isolated, and cloned by Ferrara over three decades ago, several studies have investigated the VEGF family and VEGF receptors' roles in pathological diseases such as cancers and ocular neovascular diseases. Due to these efforts, anti-VEGF agents have become a standard treatment for solid tumors and wet age-related macular degeneration. Currently, many studies have demonstrated that VEGFA is a multifunctional factor that plays a role in cancer cell and cancer stem cell proliferation and immunity via the activation of VEGFR1 and VEGFR2 in addition to angiogenesis. Thus, anti-VEGF agents have been developed in various forms, such as monoclonal antibodies, small molecules, and recombinant proteins in cancer therapy. However, their efficacy is transient and unsatisfactory, although these anti-VEGF agents successfully block tumor angiogenesis in-clinic. There are several mechanisms of resistance and non-responsiveness to anti-VEGF agents. Targeting neuropilin-1 (NRP1) may be one potential strategy to overcome these limitations of anti-VEGF agents. NRP1 is a multifunctional receptor involved in development, various processes related to tumors, and immune responses. NRP1 enhances signaling pathways related to angiogenesis by interacting with multiple ligands and their cognate receptors, such as fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), tumor growth factor-β1 (TGF-β1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and the VEGF family. It was recently reported that NRP1 coordinates different cellular functions other than angiogenesis via the above interactions. Thus, NRP1 inhibitors may block alternative pro-angiogenic growth factors, improve penetration within tumor tissue, and transform from immunosuppressive to immunosusceptible conditions of the tumor microenvironment, leading to enhanced anticancer efficacy with conventional drugs such as chemotherapy and immunotherapy. Based on these aspects, this study was performed to overcome the resistance to anti-VEGF therapy by dual-targeting VEGF and NRP1. I generated and tested an anti-VEGFA and anti-NRP1 dual-targeting bispecific antibody (named IDB0076), an anti-human VEGFA monoclonal antibody linked to an NRP-targeting peptide to the C-terminus of the heavy chain. This dual-targeting antibody simultaneously binds to VEGFA and NRP1 in vitro. Additionally, this molecule inhibited VEGFA-induced tube formation and suppressed VEGFB- and PlGF-induced migration in HUVECs via NRP1 internalization. IDB0076 showed superior anticancer efficacy than that of bevacizumab in a pancreatic xenograft model. Moreover, this dual-targeting antibody did not produce significant systemic adverse effects in a preliminary toxicity study of cynomolgus monkeys. In particular, IDB0076 showed no noticeable nephrotoxicity, which has previously been reported for the combination therapy of bevacizumab and an anti-NRP1 antibody. My conclusion is that the dual-targeting of VEGFA and NRP1 by genetic fusion of an NRP1 specific peptide to the C-terminus of an anti-human VEGFA monoclonal antibody could be a promising anticancer agent with manageable toxicity to overcome the limitations of existing anti-VEGF therapeutic drugs.
more목차
CHAPTER 1. General introduction 1
1.1 Angiogenesis and VEGF 1
1.1.1 Anti-VEGF treatments for wetAMD 3
1.1.2 Anti-VEGF treatments for cancers 5
1.2 Limitations of anti-VEGF treatments for cancers 8
1.3 Neuropilin 1 (NRP1) 11
CHAPTER 2. Overcoming the limitations of anti-VEGF therapy using a VEGFA and NRP1 dual-targeting bispecific antibody 14
2.1 Abstract 14
2.2 Introduction 15
2.3 Materials & Methods 17
2.3.1 Cell lines and Reagents 17
2.3.2 Protein expression and purification 17
2.3.3 Affinity measurement 17
2.3.4 HUVEC migration assay 18
2.3.5 HUVEC permeability assay 18
2.3.6 NRP1 internalization assay 18
2.3.7 HUVEC tube formation assay 18
2.3.8 Pharmacokinetic assay 19
2.3.9 BxPC-3 xenograft model 19
2.3.10 Preliminary repeated dose toxicity study 20
2.3.11 Statistical analysis 21
2.4 Results 22
2.4.1 IDB0076 is successfully produced in CHO-DG44 cells. 22
2.4.2 IDB0076 is a VEGFA and NRP1 dual-targeting antibody 24
2.4.3 Dual-targeting activity of IDB0076 sustains in vivo. 28
2.4.4 IDB0076 suppresses the signal pathways of alternative proangigenic growth factors. 31
2.4.5 IDB0076 is superior to bevacizumab in a BxPC-3 xenograft model 34
2.4.6 IDB0076 is well tolerated in cynomolgus monkeys 36
2.5 Discussion 49
CONCLUSIONS 51
REFERENCES 52
ABSTRACT IN KOREAN 60
