臺灣博碩士論文加值系統

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。

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.

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
1.5.5 Others 17



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




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
Appendices 92

Arnold, J. N., M. R. Wormald, et al. (2007). "The impact of glycosylation on the biological function and structure of human immunoglobulins." Annu Rev Immunol 25: 21-50.
Asano, M., A. Yukita, et al. (1995). "Inhibition of tumor growth and metastasis by an immunoneutralizing monoclonal antibody to human vascular endothelial growth factor/vascular permeability factor121." Cancer Res 55(22): 5296-301.
Bae, D. G., Y. S. Gho, et al. (2000). "Arginine-rich anti-vascular endothelial growth factor peptides inhibit tumor growth and metastasis by blocking angiogenesis." J Biol Chem 275(18): 13588-96.
Binetruy-Tournaire, R., C. Demangel, et al. (2000). "Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis." Embo J 19(7): 1525-33.
Byzova, T. V., C. K. Goldman, et al. (2000). "A mechanism for modulation of cellular responses to VEGF: activation of the integrins." Mol Cell 6(4): 851-60.
Carmeliet, P. (2000). "Mechanisms of angiogenesis and arteriogenesis." Nat Med 6(4): 389-95.
Carmeliet, P., L. Moons, et al. (2001). "Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions." Nat Med 7(5): 575-83.
Caron, P. C., W. Laird, et al. (1992). "Engineered humanized dimeric forms of IgG are more effective antibodies." J Exp Med 176(4): 1191-5.
Carter, P. (2001). "Improving the efficacy of antibody-based cancer therapies." Nat Rev Cancer 1(2): 118-29.
Cartron, G., L. Dacheux, et al. (2002). "Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene." Blood 99(3): 754-8.
Chamow, S. M. and A. Ashkenazi (1996). "Immunoadhesins: principles and applications." Trends Biotechnol 14(2): 52-60.
Clynes, R. A., T. L. Towers, et al. (2000). "Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets." Nat Med 6(4): 443-6.
Cohen, T., D. Nahari, et al. (1996). "Interleukin 6 induces the expression of vascular endothelial growth factor." J Biol Chem 271(2): 736-41.
Cragg, M. S., R. R. French, et al. (1999). "Signaling antibodies in cancer therapy." Curr Opin Immunol 11(5): 541-7.
de Vries, C., J. A. Escobedo, et al. (1992). "The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor." Science 255(5047): 989-91.
Dias, S., K. Hattori, et al. (2000). "Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration." J Clin Invest 106(4): 511-21.
Economides, A. N., L. R. Carpenter, et al. (2003). "Cytokine traps: multi-component, high-affinity blockers of cytokine action." Nat Med 9(1): 47-52.
Eisenbeis, C. F., A. Grainger, et al. (2004). "Combination immunotherapy of B-cell non-Hodgkin's lymphoma with rituximab and interleukin-2: a preclinical and phase I study." Clin Cancer Res 10(18 Pt 1): 6101-10.
Faehling, M., J. Kroll, et al. (2002). "Essential role of calcium in vascular endothelial growth factor A-induced signaling: mechanism of the antiangiogenic effect of carboxyamidotriazole." Faseb J 16(13): 1805-7.
Ferrara, N. (2002). "VEGF and the quest for tumour angiogenesis factors." Nat Rev Cancer 2(10): 795-803.
Ferrara, N. (2004). "Vascular endothelial growth factor: basic science and clinical progress." Endocr Rev 25(4): 581-611.
Ferrara, N. and T. Davis-Smyth (1997). "The biology of vascular endothelial growth factor." Endocr Rev 18(1): 4-25.
Ferrara, N., H. P. Gerber, et al. (2003). "The biology of VEGF and its receptors." Nat Med 9(6): 669-76.
Ferrara, N. and W. J. Henzel (1989). "Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells." Biochem Biophys Res Commun 161(2): 851-8.
Ferrara, N., K. J. Hillan, et al. (2004). "Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer." Nat Rev Drug Discov 3(5): 391-400.
Ferrara, N., K. Houck, et al. (1992). "Molecular and biological properties of the vascular endothelial growth factor family of proteins." Endocr Rev 13(1): 18-32.
Ferrara, N. and R. S. Kerbel (2005). "Angiogenesis as a therapeutic target." Nature 438(7070): 967-74.
Folkman, J. (1995). "Angiogenesis in cancer, vascular, rheumatoid and other disease." Nat Med 1(1): 27-31.
Folkman, J. (1996). "Fighting cancer by attacking its blood supply." Sci Am 275(3): 150-4.
Folkman, J. and M. Klagsbrun (1987). "Angiogenic factors." Science 235(4787): 442-7.
Folkman, J., E. Merler, et al. (1971). "Isolation of a tumor factor responsible for angiogenesis." J Exp Med 133(2): 275-88.
Fong, G. H., J. Rossant, et al. (1995). "Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium." Nature 376(6535): 66-70.
Fong, G. H., L. Zhang, et al. (1999). "Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice." Development 126(13): 3015-25.
Fuh, G., B. Li, et al. (1998). "Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor." J Biol Chem 273(18): 11197-204.
Fukumura, D., R. Xavier, et al. (1998). "Tumor induction of VEGF promoter activity in stromal cells." Cell 94(6): 715-25.
Gerber, H. P., V. Dixit, et al. (1998). "Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells." J Biol Chem 273(21): 13313-6.
Gerber, H. P., K. J. Hillan, et al. (1999). "VEGF is required for growth and survival in neonatal mice." Development 126(6): 1149-59.
Gerber, H. P., J. Kowalski, et al. (2000). "Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor." Cancer Res 60(22): 6253-8.
Gerber, H. P., A. McMurtrey, et al. (1998). "Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation." J Biol Chem 273(46): 30336-43.
Glennie, M. J. and J. G. van de Winkel (2003). "Renaissance of cancer therapeutic antibodies." Drug Discov Today 8(11): 503-10.
Gluck, W. L., D. Hurst, et al. (2004). "Phase I studies of interleukin (IL)-2 and rituximab in B-cell non-hodgkin's lymphoma: IL-2 mediated natural killer cell expansion correlations with clinical response." Clin Cancer Res 10(7): 2253-64.
Gorter, A. and S. Meri (1999). "Immune evasion of tumor cells using membrane-bound complement regulatory proteins." Immunol Today 20(12): 576-82.
Gospodarowicz, D., N. Ferrara, et al. (1987). "Structural characterization and biological functions of fibroblast growth factor." Endocr Rev 8(2): 95-114.
Heldin, C. H. (1995). "Dimerization of cell surface receptors in signal transduction." Cell 80(2): 213-23.
Helmlinger, G., F. Yuan, et al. (1997). "Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation." Nat Med 3(2): 177-82.
Hiratsuka, S., Y. Kataoka, et al. (2005). "Vascular endothelial growth factor A (VEGF-A) is involved in guidance of VEGF receptor-positive cells to the anterior portion of early embryos." Mol Cell Biol 25(1): 355-63.
Holash, J., S. Davis, et al. (2002). "VEGF-Trap: a VEGF blocker with potent antitumor effects." Proc Natl Acad Sci U S A 99(17): 11393-8.
Hosaka, T., K. Takase, et al. (2006). "Deletion analysis of the subunit genes of V-type Na+-ATPase from Enterococcus hirae." J Biochem (Tokyo) 139(6): 1045-52.
Katoh, O., H. Tauchi, et al. (1995). "Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation." Cancer Res 55(23): 5687-92.
Kendall, R. L. and K. A. Thomas (1993). "Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor." Proc Natl Acad Sci U S A 90(22): 10705-9.
Keyt, B. A., L. T. Berleau, et al. (1996). "The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency." J Biol Chem 271(13): 7788-95.
Keyt, B. A., H. V. Nguyen, et al. (1996). "Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis." J Biol Chem 271(10): 5638-46.
Kirschfink, M. (2001). "Targeting complement in therapy." Immunol Rev 180: 177-89.
Li, J., C. Yen, et al. (1997). "PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer." Science 275(5308): 1943-7.
Malich, G., B. Markovic, et al. (1997). "The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines." Toxicology 124(3): 179-92.
Maniotis, A. J., R. Folberg, et al. (1999). "Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry." Am J Pathol 155(3): 739-52.
Mesiano, S., N. Ferrara, et al. (1998). "Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization." Am J Pathol 153(4): 1249-56.
Mesri, E. A., H. J. Federoff, et al. (1995). "Expression of vascular endothelial growth factor from a defective herpes simplex virus type 1 amplicon vector induces angiogenesis in mice." Circ Res 76(2): 161-7.
Muller, Y. A., B. Li, et al. (1997). "Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site." Proc Natl Acad Sci U S A 94(14): 7192-7.
Okada, F., J. W. Rak, et al. (1998). "Impact of oncogenes in tumor angiogenesis: mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells." Proc Natl Acad Sci U S A 95(7): 3609-14.
Olsson, A. K., A. Dimberg, et al. (2006). "VEGF receptor signalling - in control of vascular function." Nat Rev Mol Cell Biol 7(5): 359-71.
Pajusola, K., O. Aprelikova, et al. (1992). "FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines." Cancer Res 52(20): 5738-43.
Pertovaara, L., A. Kaipainen, et al. (1994). "Vascular endothelial growth factor is induced in response to transforming growth factor-beta in fibroblastic and epithelial cells." J Biol Chem 269(9): 6271-4.
Plate, K. H., G. Breier, et al. (1993). "Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis." Cancer Res 53(23): 5822-7.
Press, O. W., J. P. Leonard, et al. (2001). "Immunotherapy of Non-Hodgkin's lymphomas." Hematology Am Soc Hematol Educ Program: 221-40.
Prewett, M., J. Huber, et al. (1999). "Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors." Cancer Res 59(20): 5209-18.
Rosenberg, S. A., M. T. Lotze, et al. (1993). "Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer." J Natl Cancer Inst 85(8): 622-32.
Ruckman, J., L. S. Green, et al. (1998). "2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain." J Biol Chem 273(32): 20556-67.
Semenza, G. L. (2003). "Angiogenesis in ischemic and neoplastic disorders." Annu Rev Med 54: 17-28.
Shalaby, F., J. Rossant, et al. (1995). "Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice." Nature 376(6535): 62-6.
Shibusa, T., N. Shijubo, et al. (1998). "Tumor angiogenesis and vascular endothelial growth factor expression in stage I lung adenocarcinoma." Clin Cancer Res 4(6): 1483-7.
Smith, J. K., N. M. Mamoon, et al. (2004). "Emerging roles of targeted small molecule protein-tyrosine kinase inhibitors in cancer therapy." Oncol Res 14(4-5): 175-225.
Soker, S., S. Takashima, et al. (1998). "Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor." Cell 92(6): 735-45.
Tsuzuki, Y., D. Fukumura, et al. (2000). "Vascular endothelial growth factor (VEGF) modulation by targeting hypoxia-inducible factor-1alpha--> hypoxia response element--> VEGF cascade differentially regulates vascular response and growth rate in tumors." Cancer Res 60(22): 6248-52.
van Dijk, M. A. and J. G. van de Winkel (2001). "Human antibodies as next generation therapeutics." Curr Opin Chem Biol 5(4): 368-74.
Waltenberger, J., L. Claesson-Welsh, et al. (1994). "Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor." J Biol Chem 269(43): 26988-95.
Wiesmann, C., G. Fuh, et al. (1997). "Crystal structure at 1.7 A resolution of VEGF in complex with domain 2 of the Flt-1 receptor." Cell 91(5): 695-704.
Willett, C. G., Y. Boucher, et al. (2004). "Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer." Nat Med 10(2): 145-7.
Wolff, E. A., G. J. Schreiber, et al. (1993). "Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer Res 53(11): 2560-5.
Wu, Y., A. T. Hooper, et al. (2006). "The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma." Int J Cancer 119(7): 1519-29.
Yakes, F. M., B. D. Wamil, et al. (2000). "CM101 treatment overrides tumor-induced immunoprivilege leading to apoptosis." Cancer Res 60(20): 5740-6.
Yancopoulos, G. D., S. Davis, et al. (2000). "Vascular-specific growth factors and blood vessel formation." Nature 407(6801): 242-8.