| Peptides directed targeted drug strategy has been proved to be a powerful method for drug specifically accumulating to the pathological site, increasing the efficiency of drug and decreasing their side effects. In order to make this strategy into application, two problems have to be solved. The one is to identify the specific molecules on target cells. The other is to find ligands which can recognize and associate those molecules.Human vascular endothelia growth factor receptor 3 (VEGFR-3) is a new promising molecular target which has been identified in 1992 as a member of III receptor tyrosine kinas family. VEGFR-3 is initially expressed in all embryonic endothelia; but its expression in the blood vessel endothelium decreases during development, and it becomes largely restricted to the lymphatic endothelium in adult tissues. Recently, more and more data showed that VEGFR-3 expression was increased in a variety of human tumours and is a good indicator for tumour metastasis and prognosis. In addition, some animal assays showed that inhibiting the signal pathway of VEGFR-3 could prevent the growth and metastasis of tumour. All these features of VEGFR-3 make it become a new promising target for tumour diagnosis and therapy. Phage display technology is a powerful tool for screening novel ligands for various target proteins, especially for identify the peptides which can be used as the targeting molecules.In our study a phage-display constrained 7 peptides library was screened with VEGFR-3. After three cycles biopanning and phage ELISA, 29 positive phages were selected. DNA sequencing revealed that one consensus peptides sequence is included in the 29 positive phages which is CSDxxHxWC. The phage, which had highest A490nm absorbance in phage ELISA was named as Phage 1, and the peptide displayed was named as P1. ELASA results indicates that Phage 1 could specifically bind to VEGFR-3 but not bind to VEGFR-1 or VEGFR-2, and what's more the binding of Phage 1 to VEGFR-3 is dose-dependent. Blast online analysis indicated that none meaningful homogeneouse sequence with P1 was found in proteins including VEGF-C and VEGF-D. Human colon cancer cell line (H29 cells) and human retinoblastoma cell line (Y79 cells) were identified as VEGFR-3 positive cells with mAb against VEGFR-3 by flow cytometry method. After constructing the nude mice bearing tumour model with HT29 cells, Phage 1 and control phages were injected into the nude mice from tail vein, Phage 1 recovered from tumor was as 2.38 times as the control phages, which indicated that Phage 1 could home to tumour tissues specifically.In order to study whether the peptide P1 still has the ability to bind the VEGFR-3 when it is departed from phage, P1 was chemically synthesized as well as the control peptide, P5. ELISA assay indicated that P1 could specifically bind to VEGFR-3 but not bind to VEGFR-1 or VEGFR-2, and the binding of P1 to VEGFR-3 is dose-dependent. In the flow cytometry assay, P1 could bind to the VEGFR-3 positive cells but not the VEGFR-3 negative cells, which is similar to the mAb to VEGFR-3. The competition assay indicates that the Phage 1 which displayed P1 could specifically inhibit the binding of P1 to VEGFR-3. What's more, the P1 could specifically inhibit the binding of FITC-P1 to VEGFR-3 positive cells. In the biodistribution assay, the FITC-P1 could home to tumour tissue compared with the control peptide, FTIC-P5.To further study whether the P1 could direct other molecules to bind to VEGFR-3, hIFN-α2a-P1 fusion gene was constructed and expressed in E.coli. The fusion protein was purified by affinity chromatography. The activity of hIFN-α2a-P1 in vitro was as same as the natural hIFN-α2a. In the ELISA assay and flow cytometry assay, the hIFN-α2a-P1 could bind to VEGFR-3 and VEGFR-3 positive cells, which indicates that P1 has the ability to direct the IFN-α2a to VEGFR-3. In the animal assay, hIFN-α2a-P1 could inhibit the growth of tumour and the inhibiting ratio is 57.1%.
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