The Studies Of The Anti-angiogenesis Drug Directed Against Human MMP-2 And Integrin α_vβ₃

The process of angiogenesis plays an important role in many physiological and pathological conditions. Angiogenesis plays a key role in several pathological conditions such as the growth of malignant tumors and metastases, chronic inflammatory diseases and diabetic retinopathy. Therefore, the inhibition of angiogenesis could be beneficial for the treatment of cancer. The C7-1 phage clone is which we isolated from Ph.D.-C7CTM and targets to MCD protein. We improve the method of phage display in vivo and found a new control phage- a white phage clone. Then we display the C7-1 phage clone in vivo, and confirm it targets to tumor tissue.The C7-1 peptide was expressed in E.coli by a matter of C71-PEX fusion protein. In order to research the function of the fusion protein, we did some experiments such as preventing degradation, competition ELISA and the effect to endothelial cell growth. As a result, we proved the double-effect of the C71-PEX fusion protein.Integrin αvβ3 is up-regulated on cytokine - activated endothelial and smooth muscle cells as well as on vascular cells within tumors, whereas it is minimally expressed on quiescent vasculature. Thus, αvβ3 is a suitable target molecule to inhibit angiogenesis. In endothelial cells, αvβ3 induces the production of MMP-2 and subsequently interacts with the newly synthesized MMP-2 to promote vascular invasion. Without binding to β3, MMP-2 is infunctional. So we constructed a phage display single-chain variable fragment(scFv) library directed against the extracellular domain of human integrin β3 and screend for specific antibodies to it from the library. The mRNA isolated from human BT-325 cells was used to amplified the gene of the extracellular domain of human integrin β3 by RT-PCR. The expression vector of the extracellular domain of human integrin β3 was constructed and used to transforme E.coli BL21. The extracellular domain of human integrin β3 was expressed, refolded and purified, and then it was used to immunize BALB/C mice. Total splenic RNA of the immunized mice was isolated and used to amplify the heavy-chain and light-chain variable region genes of antibodies by RT-PCR. The heavy-chain and light-chain variable region genes of antibodies were then joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3 . The assembled scFv fragments were cloned into the phagemids (pCANTAB5E), and the recombinant phagemids were used to transform the competent E. coli TG1. The transformed TG1 were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The specific antibodies against the extracellular domain of human integrin β3 were enriched by four rounds of panning. The positive recombinant phages were analyzed by ELISA. As a result, the extracellular domain of human integrin β3 was overexpressed. An antibody library containing 2.6×106 different clones was obtained. The phage isolates with the binding abilities and specificity for extracellular domain of human integrin β3 were selected. This kind of scFv was expressed in E. coli . And the target protein was refolded. The work which we have done is to inhibit the tumor angiogenesis and the results could be useful for cancer therapy.