Preparation Of Monoclonal Antibodies Against RON Receptor Tyrosine Kinase

Backgroud & AimReceptor tyrosine kinases (RTK) are transmembrane proteins with unique structural properties. Currently, twenty families of RTK have been identified. Abnormal RTK activation have been identified in various pathological conditions. In certain human tumors, aberrant RTK expression and activation, such as increased expression of the EGFR family members in breast and lung cancers, are hallmarks of tumor progression. Thus, RTK are potential targets for therapeutic intervention. The inhibition of RTK activities by specific inhibitors, such as Herceptin which blocks EGFR2 (Her2), significantly improves clinical outcomes in patients with breast or lung cancer.The recepteur d'origine nantais (RON) receptor tyrosine kinase belongs to the MET proto-oncogene family. Since its discovery in 1993, significant progress has been made to elucidate its biochemical and biological properties. RON has been found to be essential in embryonic development and in tumor invasive/malignant phenotypes.In this experiment, we use NIH 3T3 cell, which is transfected with wt-RON, as the antigen to prepare the monoclonal antibody against RON, then identify theproperty of the antibody. At last, we analyze the effects of the antibody to RKO cell, and then determine its mechanisms.Methods1. wt-RON NIH 3T3 wt-RON RKO RON â–³160 RKO cell were prepared withplasmids transfection.2. By immuning the mouses with wt-RON NIH 3T3 cell, hybridoma cells were prepared. Then the cells was cultured in the HAT medium.3. Hybridoma cell which secrete antibody to wt-RON were selected by modified indirect immunofluorescence.4. Limiting dilution assay was used for monoclonal culture, and those cells were selected by modified indirect immunofluorescence again.5. Ascites tumor with antibody was prepared, and the antibody was purified with caprylic acid- ammonium sulfate assay.6. Specific binding of 5 selected antibodies to 10 different cell lines was determined.7. MTT assay was used to determine the survival rate of wt-RON RKO and RON â–³ 160 RKO cell with the treatment of 5-Fu.8. MTT assay was used to determine the survival rate of wt-RON RKO and RONâ–³ 160 RKO cell with the treatment of 5 antibodies and 5-Fu.Results1. In 18 selected Hybridoma cell strains, 15 cell strains could secrete antibodies against RON steadily.2. 5 selected antibodies have different specific binding ability to 10 different cell lines.3. RONâ–³ 160 RKO cell has enhanced drug resistance to 5-Fu relative to RKO and wt-RON RKO. MSP has no dominant effect on drug resistance to 5-Fu.4. Those 5 antibodies could not inhibit the growth of RONâ–³ 160 RKO, wt-RON RKO and RKO. On the contrary, they can enhance the cell growth with treatment of 5-Fu.Conclusions1. 15 cell strains which could secrete antibodies against RON were prepared.2. The expression of RON and its variant in RKO cell could decrease apoptosis and enhance the drug resistance to 5-Fu.3. Those ant(?)bodies against RON could not neutralize RON, on the contrary, 4 of them can enhance the drug resistance to 5-Fu.