Hepatocellular Carcinoma Targeting Peptide: High-Affinity Insulin Receptor Ligands Identified With Disulfide Constrained Display Random Peptide Library

BACKGROUND: Overexpression of growth factor receptors and some glycoprotein has been implicated in most of the tumors. The over expressed molecules of the tumor cell are the best sites in tumor targeting and anticancer therapies. Three methods could be employed in tumor targeting therapy. The first is to generate a monoclonal antibody against tumor high expressed molecules. The second is to select a high affinity peptide of the growth factor receptors or glycoprotein from the phage display peptide library. The third is to obtain an analogue or antilogous of the growth factors. Insulin receptors, a kind of growth factor receptors over expressed on the surface of the hepatocellular carcinoma (HCC) cells, have become potential targeted sites in HCC therapy. Phage display peptide library is a vast library of random peptide sequences expressed as fusions with coat proteins of bacteriophages. An in vitro selection process could elute the specifically-bound phages of insulin receptors. Individual phage clones are characterized by DNA sequencing. A new- peptide is synthesizedcorresponding to the DNA sequence. The new peptide will be a potential HCC-targeting molecular.AIM: The purpose of this study is to look for peptide ligands of insulin receptors by a bio-panning of a disulfide constrained phage display peptide library. We hypothesized that the peptides will play an important role in HCC diagnostic imaging and/or targeting therapy.METHODS: Rat liver insulin receptors were immobilized on a polystyrene plate by incubating in 0.1mol/L NaHCO3 overnight at 4℃. Panning was carried out by incubating a library of phage- displayed peptides with immobilized insulin receptors for 40min at room temperature. The bound phages were eluted with 0.2M glycine-HCl and amplified with 200μl Ecol.ER2738 in 20ml LB-Medium in a 250ml Erlenmeyer flask. After three round of bio panning, 12 microphage colons were picked out to amplify. 11 clones were finally sequenced according to the ELISA.A new disulfide constrained peptide CY-10 (CQSKHWRHCY) was synthesized corresponding to insert sequence. The apparent affinity constant of 125I-CY-10 binding to insulin receptors was revealed by saturation binding assay and Scachard analysis.The radioactivity of 125I-CY-10 bound to SMMC 7721 cells to the L-02 cells was compared first. Then the bio distribution of 125I-CY-10 in mice bearing human tumor was conducted following the administration of the 125I-CY-10 at 5,15,60,180,360,900min. The whole body posterior image of 131I-CY-10 in mouse bearing tumor performed 24 hr post injection. RESULTS: After three rounds, enzyme-linked immunosorbent assay (ELISA) was employed to investigate 12 individual clones bound with the insulinreceptors. All of the 12 clones were ELIS A positive. We picked out 11 clones that with a high OD value for DNA sequence. The results shows that all of the 11 colons have the same insert oligonucletide sequences ATGACGCCAATGCTTCGACTG. According to the oligonucletide sequence, a new peptide CQSKHWRHC-Y (named CY-10) was synthesized. The apparent affinity constant of 125I-CY-10 (Kd value) bind with insulin receptor is 8.909 ×10-8mol/L. The insulin receptor exhibits a 1.715-fold affinity for CY-10 than for insulin.The HCC SMMC 7721 cells bound much more 125I-CY-10 than L-02 cells. The biodistribution shows that the hepatocellular carcinoma tissue of the mice bearing human SMMC 7721 tumors has a higher uptake of 125I-CY-10 than the muscle, the bone and the brain. The 125I-CY-10 activities in tumor tissue reach maximum peak values at 3hr post-injection. The ratio of tumor to muscle at 3 hour is 2.2218. The whole body imaging of 131I-CY-10 shows a high tumor uptake.CONCLUSSION: This study generated a high-affinity peptide of insulin receptor. The uptake of peptide CY-10 in HCC tumor tissue of the naked mice uptake is high. CY-10 could be a potent targeted reagent of HCC.