The Studies Of Peptides Binding To Osteosarcoma Cells And The Mimotopes Of Surface Antigen Of Osteosarcoma Cells

It has been lenown that the treatment of osteosarcoma includes chemotherapy, operation, radiotherapy and biotherapy etc, but every of them has it's insufficiency. Target therapy only acting on tumor tissue or cells is a very promising treatment method. Monoclonal antibody(McAb) has been used as main carrier system in traditional target therapy of osteosarcoma, but it's molecule weight is high and power of pierce through tissue and cell membrane is lower and can induce human anti-mouse antibody responses (KAMA).Small molecular peptides can be used to overcome these obstacles and can be obtained from a phage display library by screening procedures. Based on the above theory, we have tried to screen 12 phage library by using live osteosarcocma cells as targets to obtain small molecular peptides binding specially to osteosarcoma cells. The peptides will help to provide the useful information for targeted chemotherapy of osteosarcoma. We have also carried studies in which c7c phage display peptide library were screened by using a 12-rner phage clone which bind to osteosarcoma cells os-732 specifically to study mimotopes of surface antigen of osteosarcoma cells.Our research can be divided into the following two parts: Part I The Study of peptides binding to osteosarcoma cells:The peptides displayed on phage binding to osteosarcoma cells os-732 were screened from 12 phage displayed library by using osteosarcoma cells as target, the eluted phage clones in every round were absorbed by normal human osteoblasts, after three rounds of biopaning, 9 of 20 phage clones were identified as positive by cell ELISA and immunohistochemistry, which could bind to osteosarcoma cells os-732, but not to normal human osteoblasts,HEK293, THP-1, MCF-7, NPC cell lines(CNE3). The DNA of positive phages was automated sequenced and their corresponding peptides were deduced according to their DNA sequences. No conserved sequence was found among these peptides. The peptides displayed on phage binding to osteosarcoma cells specifically suggested that this kind of peptide may be used for targeted chemotherapy of osteosarcoma.The different sequence of clones from screening means that phage clones can mimicry different epitope on the osteosarcoma cells surface.Part II Screening of mimotopes of osteosarcoma cell's surface antigen:For getting mimotopes on surface of osteosarcoma cells, we set up the system for screening with phage as target, and identication of specific phage clone by biotinylated phage. In briefly, mimotope sequence which could be mimics to antigenic epitope on osteosarcoma cell surface was screened from c7c phage peptide library with 12 mer peptide displayed on phage as target, and with os-732 cell lysate as elution buffer; phage clones were identified by ELISA with biotinylated 12 mer phage with target sequence. The advantages in this new system includes :(l)Taking phage with positive peptide sequence as target for screening instead of synthetic peptide which need conjugation with carrier protein; (2) prepareing biotinylated targe phage binding to mimotope phage clone, which was sensitive, specific and without biotinylated peptide, and can be used to bind to osteosarcoma cells directly.