Development of Anti-Idiotype Monoclonal Antibodies for the Treatment of Breast Cancer
Final rept 1 Jul 94-30 Jun 97
KENTUCKY UNIV LEXINGTON
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For the development of HER2neu mimicking anti-idiotype antibodies Ab2s, groups of female BALDc mice were immunized with three sets of monoclonal antibodies Ab1, designated 520C9, 741F8 and 454C11, directed at distinct antigenic determinants of HER2neu. Hybridomas were generated from each treatment group and one, stable Ab2 producing hybrid each of 520C9 and 741F8 were obtained, and cloned twice by limiting dilution. The isotypes of 520C9 and 741F8 Ab2s were IgG1k by ELISA. 520C9 and 741F8 Ab2 cells were used to produce mouse ascites and the Ab2 purified by affinity chromatography and confirmed by SDS-PAGE. A competitive binding assay using Ab2, Ab1 and the human breast cancer cell line SK-BR-3 which express HER2neu on their cell surface, showed that 50mu1 of each of 520C9 and 741F8 Ab2 containing supernatants inhibited the binding of Ab1 to SK-BR-3 cells by tilde 80.The specificity binding study showed that the binding of 741F8 Ab2 was to those antibodies directed against HER2neu. A competitive binding assay using 741F8 Ab2, Ab1 and SK-BR-3 cells showed that 10ng of purified 741F8 Ab2 inhibited the binding of Ab1 to SK-BR-3 cells by tilde 50. Polyclonal anti-anti-idiotype antibodies Ab3, were generated in rabbits using either purified 520C9 Ab2 or 741F8 Ab2. The Ab3 reacted with HER2neu positive tumor cells suggesting the presence of anti-tumor Ab1 antibodies.
- Medicine and Medical Research