Preperation and identification of HER2-targeting monoclonal antibodies suppressing gastric carcinoma cells

Objective: To prepare and identify monoclonal antibodies that can specifically react with HER2 and suppress gastric tumor growth. Methods: Gastric carcinoma cell line NCI-N87 highly expressing HER2 was used to immunize 6 BABL/c mice. The spleen cells of the immunized mice were fused with SP2/0 cells and cultured with methyl cellulose. Antibodies of each clone were identified by ELISA to screen for those could positively react with HER2 antigen. Immunofluorescence of viable cells was performed to see whether the HER2-directed monoclonal antibodies could react with membrane integrated HER2. The subtypes of these monoclonal antibodies were identified by related kit purchased from Southern Biotech. The epitope of the monoclonal antibody was identified by sandwich ELISA and competition ELISA with trastuzumab. MTT cell proliferation assay was used to select functional antibody with therapeutic potential from HER2-directed monoclonal antibodies. Immunohistochemistry and Western blotting assay were performed to identify the reaction of the antibodies with HER2 antigen. Results: A total of 1 442 monoclonal antibody clones were obtained by the fusing mouse spleen cells with SP2/0 cells. Seventy-nine clones positively reacted with recombined HER2 protein in ELISA assay. Seven of these antibodies recognized membrane integrated HER2 protein in immunofluorescence detection of viable cells. The subtypes of these monoclonal antibodies were further identified. One of these 7 antibodies, 15C15, which was identified as IgG1 kapa, significantly suppressed NCI-N87 cell proliferation in MTT assay. The epitope of 15C15 was different from trastuzumab according to sandwich ELISA and competition ELISA identification. The 15C15 antibody only reacted with part of the HER2 antigen in immunohistochemistry assay and did not react with HER2 antigen (denaturated into linear structure) in Western blotting analysis. Conclusion: Using functional antibody library screening technique we have successfully obtained functional monoclonal antibodies which can suppress the gastric carcinoma cell proliferation by specially neutralizing HER2 protein. One of these antibodies, 15C15 may be applied in anti-tumor therapy of gastric carcinoma.