Construction And Expression Of Single-chain Fv Antibody Against Human VEGF

The tiny blood vessels known as capillaries extend into virtually all the tissues of the body, replenishing nutrients and carrying off waste products. Under most conditions, capillaries do not increase in size or number, because the endothelial cells that line these narrow tubes do not divide. But occasionally-for example, during menstruation or when tissue is damaged-these vessels begin to grow rapidly. This proliferation of new capillaries, called angiogenesis.angiogenesis appears to be one of the crucial steps in a tumor's transition from a small, to a large malignant growth, capable of spreading to other organs throughout the body.Antiangiogenic therapy, in contrast to many other therapeutic approaches, does not aim to destroy tumors. Instead, by limiting their blood supply, it attempts to shrink tumors and prevent them from growing. Antiangiogenic drugs can stop new vessels from forming around a tumor.VEGF is a multifunctional cytokine expressed and secreted at high levels by many tumor cells. As secreted by tumor cells, VEGF is a 34-45 kDa heparin-binding, dimeric,disulfide-bonded glycoprotein that acts directly on endothelial cells (EC) by way of specific receptors to activate phospholipase C and GTP.Two high affinity VEGF receptors, both tyrosine kinases, have thus far been described. VEGF is likely to have a number of important roles in tumor biology. As a potent permeability factor, VEGF promotes extravasation of plasma fibrinogen, leading to fibrin deposition which alters the tumor extracellular matrix. This matrix promotes the ingrowth of fibroblasts and endothelial cells. Moreover, VEGF is a mitogen for endothelial cells and may play an important role in maintaining vascular endothelium and in promoting tumor angiogenesis .The murine anti-human VEGF monoclonal antibody (muMAb VEGF)was reported to potently suppress angiogenesis and growth in a variety of human tumor cells lines transplanted in nude mice.In this paper, We generated a hybridoma cell line(Ell) secreting MAb against VEGF by routine methods using synthetic peptide derived from the predicted human VEGF amino acid sequence as antigen. The MAb was proved to be the ability to specifically bind native VEGF and block the VEGF-mediated stimulation of endothelial cell growth. Then,the MAb was used to investigate VEGF expression in 20 specimens from buccal mucosal cancer patients by Immunohistochemical methods.The results showed:VEGF expression was detected in 16 (80%) out of total 20 samples. 12 specimens (high and moderate differentiation) were immunoreactive to VEGF. 4 specimens (low differentiation) were strongly stained. VEGF was mainly localized in the cytoplasm of carcinoma cell and endothelial cells of small vessels adjacent to malignant tumor cells. The intense expression of VEGF by buccal nucosal cancer provides strong evidence linking VEGF expression to the angiogenesis associated with buccal mucosal cancer. The MAb also significantly supressed the growth of cultured buccal mucosal cancer cell in BALB/c nude mice with s.c.inoculation.(datas not included here).Since the early description of monoclonal antibodies by Kohler and Milstein in 1975 , there has been considerable interest in their adoption for therapy apllication.But when the MAb administered to patients, the mouse monoclonal antibodies stimulate the production of a human antibody response - Human Anti-Mouse Antibody (HAMA). HAMA diminishes or obliterates the efficacy of the mouse antibody. One solution to the problem is the preparation of recombinant antibodies. Now,It is possible to raise small antibody fragments, such as single-chain Fvs (scFvs) against virtually any desired antigen. scFv can maintain specific binding ability of antigen as its parent MAb .These binding molecules are being used to target a wide variety of antigens for numerous diagnostic and therapeutic applications.For above reasons,so we intend to build scFv from the hybridoma cell line secreting MAb against VEGF ,total RNA was prepared and used as a template for cDNA synthesis and cloning.Reversetranscription and polymerize chain reaction(RT-PCR)was used to clone the immunoglobulin variable region gene (VH,VL) .the cloned VH and VL were linked by well documented flexible peptide bridging sequence (GGGGS)₃ in the VH-linker-VL orientation .The constructed scFv gene was inserted in pET-15S vector with a short sequence encoding a 12-amino-acid-peptide sequence(E-tag) at the 3'-end.The E-tag attachment at the C-end made it easy to test the avidity of scFv ,and expressed in E.coli. Biding activity of scfv was evaluated by immunohistochemical methods after its denature and renature treatment. The sequence analysis revealed that the full length of VH is 369bp, and VL,333bp.According to Kabat classification ,VH and VL are member of mouse Ig variable gene heavy chain subgroupII(A) and light chain subgroupIII,respectively. scFv was highly produced in the form of inclusion bodies which activity was recovered after being renatured. Immunohistochemical results showed that scFv retained almost the same antigen affinity and specificity as its parent monoclonal antibody. Our results showed that recombinant antibody scFv is potentially useful in both diagnostic and therapeutic application.Lastly, a three-demensional graphic model of scFv was constructed by homology modeling on workstation.Then a systematisc analysis was performed to determine the important residues in Fv. This study provides a method for comprehending the relationship between antibody structure and function, and benefit humanization of scFv in further research.