Production of bispecific monoclonal antibodies recognising both carcinoembryonic antigen (CEA) and doxorubicin

Reddy, Vijay Subba (1993) Production of bispecific monoclonal antibodies recognising both carcinoembryonic antigen (CEA) and doxorubicin. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The objective of this project was to produce bispecific monoclonal antibodies (BsMabs) which recognise both the tumour associated carcinoembryonic antigen (CEA) and the chemotherapeutic agent doxorubicin, as a complementary approach to the use of immunoconjugates for site specific drug delivery. A monoclonal anti-CEA hybridoma (11-285-14) was made sensitive to hypoxanthine, aminopterin and thymidine (HAT) , by back selecting it in increasing concentrations of 8-azaguanine. Eight 8-azaguanine resistant fusion partners were selected based on growth characteristics and continued anti-CEA production. As doxorubicin (Dox) is a hapten, it was conjugated to carrier proteins keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) using l-ethyl-3- (dimethyl-aminopropyl) carbodiimide. Dox-KLH arid Dox-BSA conjugates were employed to immunize mice and spleen cells were used for fusions with the HAT sensitive anti-CEA 11-285-14 using standard hybridoma procedures. Enzyme linked immunosorbent assays (ELISAs) were developed to test the hybrids obtained for anti-CEA, anti-Dox, anti-BSA and dual bispecific activity. Sixteen fusions from Dox-KLH immunized mice yielded 621 hybrids of which 47 showed low level bispecificity. Eight fusions with Dox-BSA immunized mice yielded 297 hybrids. Hybrids showing dual activities were cloned and 7 out of 286 of the positive clones have been selected for expansion.

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