Selection And Preliminary Identification Of Hepatocarcinoma-binding And Internalizing Peptides

Human hepatocellular carcinoma is one of the most common and deadly cancers in the world. Its cancer mortality rate ranks the third all over the world, and the second in our country. In recent years, with the development of Molecule Biology , Genomics and Proteomics, people know more about cancer biology and the research about human hepatocellular carcinoma makes further development. Liver cancer therapy mainly include surgery, chemo-radiotherapy, biological therapy, Chinese medicinal therapy. From 1980s, improved understanding of advances in antibody technique especial in monoclonal antibody made the targeting therapy become a potential effective choice in the curing of hepatocellular carcinoma. In 1980, Order started to use ¹³¹I anti-AFP antibody for the targeted treatment of human hepatocellular carcinoma. Many domestic and overseas scholars seriouly study liver cancer carriers, anti-liver cancer cell poison and multi-compositive treated means, which enrich the resreach about human hepatocellular carcinoma. Even though, some problems still exist in antibodies targeted therapy. High molecular weight made low penetration ability to tumor tissues, the immunogenicity of murine antibodies in humans made ill immunoreaction, which not only damage human bodies, but also made bad influence on cancer therapy. Lower molecular weight made the peptide a reasonable candidate for monoclonal antibody to lessen immunogenicity and improve the efficiency of targeted cancer therapy. Internalization of antibodies or peptides into the cell by receptor-mediated endocytosis is required for many targeted therapeutics, such as immunotoxins, immunoliposomes. A major advantage of receptor-mediated endocytosis as a drug delivery route is that therapeutic agents can be delivered specifically into target cells that overexpress the receptor and thereby increase efficacy while reducing systemic toxicity. So, internalizing antibodies or peptides have important applications.The development of Phage display peptide technology made it an efficient method to panning peptides with high affinity and specificity for almost any target. Internalizing peptides can also be isolated from Phage display peptide libraries through special selection. These targeting peptides have been successfully used in a wide range of cancer therapy.In this study, we used in vitro panning to screen peptides that can target the human hepatocellular carcinoma cell line BEL-7404 and become internalized. Their internalizing abilities were identified by in vitro and in vivo methods, which provides theoretics foundations as targeted carriers to hepatocarcinoma..This paper can be divided into three parts:A. The panning of internalizing peptides to hepatocarcinoma by in vitrophage display peptide technology.The phages from common used Ph.D.-12 Phage Display Peptide Library Kit which produced by New England Biolabs Inc were incubated with BEL-7404. After three rounds of panning, a lot of candidate internalizing phages were obtained. Accompanying with each round panning, the tittering method was carried out as the kit manual to inspect the number of phages. From the data of tittering, we were assured that the internalizing phages were enriched effectively to the tumor cell line after the three rounds of panning. Then the internalizing phage clones were picked out randomly for amplification. 20 clones were chosen for sequencing and the sequence of those phage clones were compared and analyzed. Eventually, 4 different sequences were identified. Those were DLNYFTLSSKRE( 17/20), DLNYLTLSSKRE(1/20), DLNYHARSYKRE(1/20), NLKLLDSQDWDG(1/20). 3 of 4 sequences have strong homology and the same sequence DLNY and KRE.B. Identification of hepatocarcinoma-internalizing phage display peptides both in vitro and in vivo.a. in vitro identificationThe specifically binding abilities of 4 candidate phage clones were appraised by cell-based ELISA. They all have obvious binding abilities after the calculation (p<0.05) comparing with M13. The internalizations of 4 clones were observed by fluorescence microscopy, It is obvious that 4 clones could be uptaked by BEL-7404 and BEL-7402, not HL-7702 and HEK-293. After addition of some cellular internalizing inhibitors, the internalization was obviously inhibited by examination of green staining, which meet our anticipation. The longer incubating time increased, the more phage clones internalized. Then we further used fluorescence-activated cell sorting technology to quantify the internalizing ability by the mean fluorescence intensities of cells. The FACS results indicated that 4 clones could be endocytosed by liver cancer cell lines. At the same time, different cell lines were compared about their green fluorescence. The mean fluorescence intensities in BEL-7404 or BEL-7402 were much stronger than that in HL-7702. Through the Western Blotting technology we found that the phage clone LJ01 could bind to a given 32.3kd membrane protein of hepatocarcinoma cells, not to that of normal liver cells.b. in vivo identifactionThe phage clone LJ01 was chosen for in vivo assay and the distributing situation of different tissues was identified. Obvious enrichment was identified in tumor tissues from the data of tittering. From the data of immunohistochemistry we can see brown staining in tumor tissue, which indicated the enrichment of phage clones.C. Identification of chemosynthetic-peptide LJ01 both in vitro and in vivoa. in vitro identificationThe FAM labeled LJ01 peptide was incubated with BEL-7404, BEL-7402, HL-7702, NCI-H1299, A549, NCI-H446, TCA8113, HO8910PM cells. The internalizing ability was examined using fluorescence microscopy. The specific bright staining was observed within BEL-7404, BEL-7402, a little staining in NCI-H1299 cell line, but not in the control cell line HL-7702 and other cancer cell lines.The FACS results indicated the mean fluorescence intensities in BEL-7404 or BEL-7402 were much stronger than that in HL-7702.b. in vivo identifactionThe FAM-LJ01 was injected into the tail veins of tumor-bearing mice. After blood cycle for 1h, we selected different tissues and slice up. Then the green fluorescence was observed in tumor tissue, a dim green straining in natural liver tissue, no staining in other tissues.From all the results above we can conclude that:I. 4 peptides which could specifically internalizing into hepatocarcinoma cells both in vitro and in vivo were got from 12-mer phage display library by in vitro phage display peptide technology.II. The sequence of 4 different internalizing peptides were DLNYFTLSSKRE(17/20), DLNYLTLSSKRE(l/20), DLNYHARSYKRE(1/20), NLKLLDSQDWDG(1/20). 3 of 4 peptides have the similar sequences DLNY and KRE.III. The results of in vitro and in vivo identification indicated that 4 phage display peptides have good special binding and internalizing abilities.IV. FAM labeled LJ01 peptide has good internalizing ability from the results of in vitro and in vivo identification, indicating that the chemosynthetic-peptide LJ01 without phage also sustain the special binding and internalizing abilities.