血管內皮生長因子之受體結合部位與人類免疫球蛋白G1之Fc片段的新型融合蛋白降低血管的新生

國 立 交 通 大 學

生物科技研究所

碩

士

論

文

血管內皮生長因子之受體結合部位與人類免疫球蛋白 G1 之 Fc 片

段的新型融合蛋白降低血管的新生

A novel fusion protein of the receptor binding domain of VEGF and

human IgG1 Fc portion reduces angiogenesis

研 究 生：鍾侑松

指導教授：廖光文博士

v

血管內皮生長因子之受體結合部位與人類免疫球蛋白 G1 之 Fc 片段的新型

融合蛋白降低血管的新生

A novel fusion protein of the receptor binding domain of VEGF and human

IgG1 Fc portion reduces angiogenesis

研 究 生：鍾侑松 Student：Yo-Shong Chung

指導教授：廖光文 Advisor：Kuang-Wen Liao

國 立 交 通 大 學

生 物 科 技 系

碩 士 論 文

A Thesis

Submitted to Institute of Biological Science and Technology

College of Biological Science and Technology

National Chiao Tung University

in partial Fulfillment of the Requirements

for the Degree of

Master

in

Biological Science and Technology

June 2007

vi

血管內皮生長因子之受體結合部位與人類免疫球蛋白 G1 之 Fc 片段的新型

融合蛋白降低血管的新生

研究生：鍾侑松 指導教授: 廖光文 博士

國立交通大學生物科技研究所

中文摘要

血管新生為在體內由原先已存在血管的基礎上衍生形成出新生血管的生理過程，有

別於從原位未分化的血管內皮前期細胞分化到內皮細胞。實質固態的腫瘤細胞生長可藉

由血管內皮生長因子激活血管的形成。第一型及第二型的血管內皮生長因子受體已被指

出，主要表現在正在增生的血管內皮細胞上。因此，阻礙這兩型受體的正常功能對於治

療癌症及一些血管新生依賴的疾病為一具有希望的策略。在此論文研究中，我們設計一

個結合血管內皮生長因子之受體結合部位 (胺基酸序列 8 到 109) 與具有良好免疫特性

的人類免疫球蛋白 G1 之 Fc 片段的新穎融合蛋白。RBDV-IgG1 Fc 嵌合式基因被選殖到

腺相關病毒表現系統上，作為表現融合蛋白的媒介。我們也呈現一種於人類腎臟上皮細

胞 (HEK-293) 的蛋白質表現系統，此法具有高產率及高純度的優勢。藉由酵素結合免

疫特性分析與流式細胞儀方法檢試 RBDV-IgG1 Fc 融合蛋白具有與受體結合的活性。並

且，由血管內皮細胞生長因子誘導的人類臍帶靜脈內皮細胞的增生也被此融合蛋白所抑

制，這說明了 RBDV-IgG1 Fc 在血管內皮細胞生長因子與其受體結合而引導的訊息傳導

途徑所扮演的頡抗作用。由這些結果顯示，RBDV-IgG1 Fc 融合蛋白在治療血管新生層

面的疾病具有發展的潛力。

vii

A novel fusion protein of the receptor binding domain of VEGF and human

IgG1 Fc portion reduces angiogenesis

Student: Yo-Shong Chung Advisor: Dr. Kuang-Wen Liao

Institute of Biological Science and Technology

National Chiao Tung University

ABSTRACT

Angiogenesis is a physiological process involving the growth of new blood vessels from

pre-existing vessels, rather than through in situ differentiation of undifferentiated precursor

cells to endothelial cells. Vascular endothelial growth factor (VEGF) is an angiogenic factor

that promotes the growth of solid tumor by inducing angiogenesis. The two VEGF receptors,

VEGFR-1 and VEGFR-2, have been shown to be expressed preferentially in the proliferating

endothelial cells. Thus, inhibiting these two VEGF receptors may be a promising strategy for

treatment of cancer and other angiogenesis-dependent diseases. In this study, we designed a

novel recombinant fusion protein composed of a targeting domain and an effector domain.

The targeting domain is the receptor binding domain of human VEGF (residues 8-109) and

the effector domain is the Fc region of a human IgG1 immunoglobulin that can induce a

cytolytic immune response against the targeted cells. The chimeric gene, RBDV-IgG1 Fc, was

subcloned into AAV expression vector to produce the fusion protein. We also present an

approach for the purification of the fusion protein with high yield and high purity from

HEK-293 expression system. The binding of RBDV-IgG1 Fc fusion protein to the VEGF

receptors was examined by ELISA and flow cytometry. Furthermore, the proliferation of

VEGF-induced human umbilical vein endothelial cells (HUVECs) was inhibited by the

RBDV-IgG1 Fc, suggesting an antagonistic role in VEGF/VEGF receptors signal pathway.

These results showed that RBDV-IgG1 Fc fusion protein is potential for the suppression of

angiogenesis in vivo.

viii

Acknowledgements

這一段旅程能夠走完，承蒙許多人的恩惠，在此無法一一詳述，但吾將由衷地感恩著。首 先要感謝廖光文老師，在這兩年研究生活中，能提供良好的實驗資源，不論在儀器設備上或著 材料的購買，也都願意給予我們大膽去嘗試與操作，難能可貴；而對於實驗的瓶頸也能秉著熱 心一起商量對策，很多時候都讓我當頭棒喝，豁然開朗之感。再來要感謝的是詩涵學姐，和我 討論與指導了許多實驗該如何進行，也分享了許多實驗的甘苦。這本論文能夠完成還必須感謝 劉柯俊博士、怡玫與家瑋學姐們、財木學長在內皮細胞相關實驗上的熱心援助與技術上的指導， 本論文才能得以完整實現。感謝上知、弘育與俊元學長們，在基礎實驗上給予很多建議與耐心 教導，不但在自己本身實驗忙的灰頭土臉時，還替我這學弟輩份慷慨準備了無數次的便當。同 時，感謝我的同學韻如和懷堯，能互相督促實驗的進度，共同度過與感受同屬於這一屆的許多 苦澀和喜樂。另外還要感謝俊毅學長、琦豔學姐、榮樺、瑞萍同學，對於我每一次突如其來的 期刊文獻來源與統計問題，都不厭其煩地願意空出時間替我尋找與指導，使本論文增色不少。 在此，要謝謝宛誼在質體的先行建構，也由於她的認真，使得我在接下來的工作能夠順利許多。 當然，我要特別向實驗室成員表達感謝之意，包括國欽學長、亦涵、靜宜、于玲、鈺珊學姐們、 其翰、昱丞、源庭、彥谷、依穎、正晟、沛芸學弟妹們在這段時間內在諸多小地方上的建議與 指導，實驗才致臻完善，並且也花了不少心思去打點口試的餐點，在在都讓我感到無比窩心。 感謝口試委員吳彰哲老師與袁俊傑老師在最後階段能撥允這麼多寶貴的時間給予建議改進事項 並對論文作嚴謹的批改。 最後，我心中要特別感恩我每一位親愛的家人，因為有家人的關心、照顧與支持，才使我 有勇氣與信心去面對眼前無數的挫折挑戰，也能以更寬廣成熟心胸去接受接踵而至的困境！生 活中的每一次互動與歡笑，都讓我備感溫馨，而今日因為有了家人滿滿暖暖的祝福，我才能如 期順利地完成學業。如今我更知道，家人是我所擁有的最珍貴的恩賜。同樣地，我也忘不了我 的好友們，在我無助時願意傾聽，在我不如意時替我求神拜佛，每一次的鼓勵與打氣，我都感 恩在心頭，在我內心深處，有著一份不盡地情誼。有這麼真誠的家人與良友，實乃三生有幸！

ix

TABLE OF CONTENTS

中文摘要 vi Abstract vii Acknowledgements viii List of Figures xii List of Tables xiii List of Abbreviations xiv

CHAPTER ONE: INTRODUCTION

1.1 Angiogenesis 2 1.2 Vascular endothelial growth factor 3 1.2.1 Biological activities of VEGF 4 1.2.2 Properties of VEGF gene and isoforms 6 1.2.3 Regulation of VEGF gene expression 7 1.3 VEGF receptors 8

1.3.1 VEGFR-1 9

1.3.2 VEGFR-2 11 1.3.3 VEGFR-3 and neuropilin 12 1.4 Angiogenesis inhibitors 13 1.5 Strategies to inhibit VEGF signaling 14 1.5.1 Anti-VEGF antibodies 14 1..5.2 Anti-VEGFR-2 antibodies 15 1.5.3 Soluble VEGF receptors 16 1.5.4 Small molecular substances 17

x

1.6 Antibody based therapy 18 1.6.1 Immune responses induced by Fc fragement 19 1.7 Research rationale and objectives 21

CHAPTER TWO: MATERIALS & METHODS

2.1 Materials 2.1.1 Chemicals 27 2.1.2 Kits 30 2.1.3 Primers 30 2.1.4 Antibodies 31 2.1.5 Cells 31

2.1.6 Buffers and media 31 2.2 Methods

2.2.1 Contruction of the pAAV-MCS/IgG1 and pAAV-MCS/RBDV-IgG1 (summary) 34 2.2.2 IgG1 Fc and RBDV-IgG1 Fc fragments preparation 35 2.2.3 Ligation and transformation 36 2.2.4 Polymerase chain reaction (PCR) and bacterial E coli colony PCR 37 2.2.5 Mini preparation 38 2.2.6 Midi preparation 39 2.2.7 Cell culture 40 2.2.8 Transfecting HEK-293 cells 40 2.2.9 Expression and purification of chimeric proteins 41 2.2.10 SDS-PAGE and Western blot 42 2.2.11 In vitro receptor binding assay 43 2.2.12 Cell surface binding assay 43 2.2.13 HUVECs proliferation assay 44 2.2.14 Tube formation assay 45 2.2.15 NK killing assay 45 2.2.16 Statistic analysis 46

xi

CHAPTER THREE: RESULTS

3.1 Construction of PpAAV-MCS/IgG1 Fc expression plasmid 48 3.2 Construction of PpAAV-MCS/RBDV-IgG1 Fc expression plasmid 49 3.3 Expression and characterization of chimeric gene 50 3.4 Chimeric proteins purification 51 3.5 The activity of RBDV-IgG1 Fc binding to human VEGF receptors 51 3.6 The activity of RBDV-IgG1 Fc binding to HUVECs cell surface 52 3.7 In vitro potency and efficacy of RBDV-IgG1 Fc in inhibiting HUVECs proliferation 52 3.8 Effect of blockade of VEGF receptors on in vitro tube formation 54 3.9 IL-2 activated human NK cytotoxicity 55

CHAPTER FOUR: DISCUSSION

75

References

81

xii

List of figures

Figure 1.

Tube representation of the dimeric structure of the receptor binding domain of VEGF 8-109

24

Figure 2.

Diagram of different receptor binding sites of VEGF for KDR (VEGFR-2) and FLT-1 (VEGFR-1)

25

Figure 3.

Scheme of the chimeric construction 56

Figure 4.

Restriction enzyme digestion of the IL2 LS IgGr1/pcDNA3.1 plasmid 57

Figure 5.

Restriction enzyme digestion of the RBDV-IgGr1/pcDNA3.1 plasmid 58

Figure 6.

Bacterial colony PCR assay for pAAV-MCS/IgG1 Fc construct 59

Figure 7.

Bacterial colony PCR assay for pAAV-MCS/RBDV-IgG1 Fc construct 60

Figure 8.

PCR screeming of pAAV-MCS/IgG1 Fc and pAAV-MCS/RBDV-IgG1 Fc 61

Figure 9.

Restriction enzyme digestion of the pAAV-MCS/IgG1 Fc and pAAV-MCS/RBDV-IgG1 Fc

62

Figure 10.

The fluorescence expression in HEK-293 cell 63

Figure 11.

Dection of the chimeric proteins 64

Figure 12.

Diagram of procedure for the purification of chimeric proteins in this study 65

Figure 13.

SDS-PAGE analysis of IgG1 Fc proteins showing the purification results 66

Figure 14.

SDS-PAGE analysis of the purified chimeric proteins 67

Figure 15.

Characterization of HisTrap column purified chimric proteins 68

Figure 16.

Receptor binding activities of purified RBDV-IgG1 Fc 69

Figure 17.

Cell surface binding activity 70

Figure 18.

RBDV-IgG1 Fc inhibits the VEGF-induced proliferation of HUVECs in a dose-dependent manner

71

Figure 19.

Effect of RBDV-IgG1 Fc on in vitro tube formation 72

Figure 20.

Statistic of HUVECs network formation 73

xiii

List of tables

Table 1. List of all chemicals used in the experiments 27 Table 2. Kits used in this study 30 Table 3. List of all primers used in this study 30 Table 4. Antibodies used in this study 31 Table 5. Cells used in this study 31 Table 6. List of all buffers and media used in this experiments 31

xiv

List of abbreviations

ADCC Antibody dependent cell-mediated cytotoxicity AMD Age-related macular degeneration

aFGF Acidic fibroblast growth factor bFGF Basic fibroblast frowth factor CDC Cell dependent cytotoxicity Cys Cysteine

GIST Gastro-intestinal stromal tumor

Flt-1 Fms-like tyrosine kinase HER-2/neu Human epiderminal growth factor receptor 2/neu HIF-1 Hypoxia induicible factor-1

IFR-α Interferon alpha

IL-6 Interleukin 6 kDa Kilo dalton

KDR Kinase domain receptor NK Natural killer cell

NO Nitric oxide NP Neuropilin

PDGF Platelet-derived growth factor

PDGFR-β Platelet-derived growth factor receptor-beta PIGF Placental growth factor

PI3 kinase Phosphotidylinositol kinase 3 kinase RTK Receptor tyrosine kinase

sFlt-1 Soluble fms-like tyrosine kinase-1 TGF-α Tranforming growth factor-alpha TGF-β Transforming growth factor-beta TNF-α Tumor necrosis factor-alpha VEGF Vascular endothelial growth factor VEGFR VEGF receptor