Purification and characterization of mouse bispecific monoclonal antibodies recognizing carcinoembryonic antigen (CEA) and doxorubicin

Osborne, Perry Alonzo (1996) Purification and characterization of mouse bispecific monoclonal antibodies recognizing carcinoembryonic antigen (CEA) and doxorubicin. Masters thesis, Memorial University of Newfoundland.

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Abstract

Bispecific hybrid-hybridomas have been developed previously from an anti-carcinoembryonic antigen (CEA) hybridoma (11-285-14) and spleen cells from mice inoculated with doxorubicin-bovine serum albumin (DOX-BSA) conjugate. Seven hybrid-hybridomas were selected on the basis of binding to both CEA and DOX. The purpose of this study was to purify and characterize these antibodies. -- Each hybridoma was separately injected into Balb/c mice intraperitoneally and ascitic fluid collected. The pooled fluid was then Protein A affinity purified and then further purified using an hydroxylapatite, high performance liquid chromatography (HPLC) column. Fractions from HPLC separations were tested using various enzyme linked immunosorbent assays in order to determine the location of the bispecific antibodies. Positive fractions were pooled and used in microcytostasis assays using the high CEA expressing (SKC01) and low CEA expressing (COLO 320 DM) cell lines. One particular hybridoma (26-61-10) was selected for these in vitro assays. The bispecific antibody ⁴ DOX test was able to significantly decrease the IC₅₀ of DOX with SKCOI. The IC₅₀ with the COLO 320 DM was not altered, illustrating the specificity of the bispecific antibody for a CEA expressing cell line. The specificity of the results were confirmed when DOX was used in conjunction with a non-specific antibody (Ag8). Bispecific antibody on its own or control antibody on its own, did not have a significant effect on the viability of the cell lines. This supported the theory of the bispecific antibody delivering the drug directly to the CEA expressing cancer cells and that the increased toxicity was not due to direct antibody killing of cells. -- These results provide the first demonstration that the drug doxorubicin can be targeted to a colonic cancer cell line expressing CEA by a bispecific antibody recognising CEA and the drug.

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