Study On Functional Monoclonal Antibody Of Gastric Carcinoma

Gastric cancer is one of the most common solid tumors worldwide.And Chinese population suffers the highest national incidence and mortality rate of gastric cancer's in the world.Presently molecular targeting therapy has shown promising anti-cancer effects in clinical use.Therefore,great efforts have been focused on the research of specific genes and proteins which play critical roles in tumor growth and metastasis for providing valuable targets for tumor target therapy.In this study,employing a new strategy of "functional monoclonal antibody(mAb) library technology",we established a mAb library and screened these mAbs which suppressed the tumor cell proliferation and metastasis so as to identify gastric cancer targets for the development of targeting therapy.BALB/c mice were immunized with NCI-N87 cell lines.Hybridomas were prepared using methyl cellulose selecting and cloning technique.First,we detected their antigen presentation on gastric cancer cell by immunofluorescence assay,and then selected the mAb which weekly or did not react with normal gastric tissue.Based on these results,monoclonal antibodies were subject to cell proliferation assay, adhesion assay,migration assay and invasion assay.Parts of the functional antibodies were also subjected for tumor treatment experiments by co-transplantation model to estimate their ability of tumor growth inhibition.Then some antibodies were purified from hybridoma supernatant and subjected for cell proliferation and tumor treatment experiments again to observe the does-dependent manner of anti-caner effects. Subsequently the subcellular location and expression profile of their antigens were determined by the immunofluorescence or immunohistochemistry.Finally we determined the molecular weight of the antigens by Western Blotting.All the data were processed in SPSS software,and the main results are as following.An antibody library containing 1,012 clones of mAb was build by fusing the spleen cells of immunized mouse with SP2/0 cells.295 clones of mAb can react with the gastric carcinoma cell.Among these mAbs,252 showed membrane reactivity.145 clones were determined weekly or barely reacted with normal gastric tissue by immunohistochemistry.Subsequent proliferation,adhesion,migration and invasion assays showed that among the 145 mAbs,22 clones could inhibit gastric tumor cell proliferation,four clones could inhibit the adhesion of tumor cell with LYEC,nine clones inhibited gastric tumor cell migration,one clone inhibited gastric tumor cell invasion.Parts of the mAbs which suppressed proliferation in vitro were screened by co-transplantation model in vivo.Among them four antibodies had a significant anti-cancer result compared to control groups.The inhibition rate of tumor growth ranged from 58%to 69%.The purified 11G5 showed does-dependent manner of anti-caner effects both in vitro and in vivo with the xenograft growth inhibitory rate of 75.73%,65.29%,53.77%for the dose of 50mg/(kg·time),25mg/(kg·time),12.5mg/(kg·time) respectively.The antigen of 11G5 mAb was determined located on the cell membrane by viable immunofluence assay and was highly expressed in gastric tumor cell but seldom expressed in normal gastric tissue.The 11G5 mAb did not react with its antigen in Western blotting,indicating this antibody recognized a conformational epitope.This study established an anti-gastric cancer antibody library,containing 1,012 clones of mAb.Among these mAbs,32 were identified as functional antibody which could inhabit proliferation,adhesion,migration and/or invasion,four clones showed the ability of tumor growth inhibition in vivo.Acquirement of this functional antibody gave an important basement for isolating and attaining functional antigen gene and protein.The antigen recognized by 11G5 mAb,which inhibited gastric cancer cell proliferation in vitro and in vivo,was located on the cell membrane and highly expressed in gastric tumor cell but seldom expressed in normal gastric tissue.This result implied its antigen might be a potential target for gastric cancer treatment.This study demonstrates that the "functional rnonoclonal antibody library technology" can screen and attain functional gene and protein related to the gastric cancer proliferation and metastasis.If further study,this technology will be an efficaciously strategy for screening and isolating the molecular targets for gastric tumor target therapy.