Study On The Anti-angiogenesis Targeted On Tie2

Tie2 is an endothelial receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis. Angiopoietin is its ligand. In normal adult tissue, only the expression level of Angl/Tie2 can maintain mature vasculature, but in tumor tissue, it can achieve a high level. Angl/Tie2 has important modulating function in angiogenesis. Soluble Tie2 can be used to treat tumor, because it can block the Angl/Tie2 receptor tyrosine kinase pathway.So we have two methods to block the Angl/Tie2 receptor tyrosine kinase pathway from above:1 .expressing soluble Tie2 to neutralize Angl;2preparing antibody to block AnglTie2 binding.From the two points above, we did as follows:Ex-Tie2 gene was cloned from placenta tissue. Tie2-Fc was obtained by fusing exTie2 with Fc genes through overlapping PCR, which was inserted into MCS of B7 vector. Tie2-Fc was expressed in CHO cell and checked by use of ELISA.A semi-synthetic antibody library was constructed, from which anti-Tie2 Fab antibody was screened. Human immunoglobulin light chain genes and a part of heavy chain variable region genes were amplified by RT-PCR from fetus umbilical cord blood, light chain genes were cloned into pComb3 vector to generate light chain library; FR3-CDR3-J-CH1 segments were amplified from HBsAb's Fd fragment, VH and synthetic random CDR3 of different length were overlapped by PCR. They were inserted into light chain library to form a semi-synthetic phage antibody library. An antibody library containing 2 X 107 cfu was obtained .with more than 50% containing Fd and light chain genes.Tie2 antigen was immobilized on microplates, and specific Fabs were selected after three rounds of panning. Then the positive clones were sequenced. Germ-line gene analysis demonstrated that one of the sequenced genes belongs to heavy chain VH4 family, its variable region is derived from the rearranged Germ-line gene of DP-66. D7-27 JH3a;while the light chain is a member of vkl family, the germ-line gene is DP K 3,J K 2. The CDR3 region sequences is consensus with the random (NNK)8 design.