Production of hybrids secreting bispecific antibodies recognising CEA and doxorubicin.
A monoclonal anti-CEA secreting hybridoma (11-285-14) was made hypoxanthine, aminopterin and thymidine (HAT) sensitive 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. Since doxorubicin (Dox) is a hapten, it was conjugated to the carrier proteins keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) using 1-ethyl-3- (dimethyl-aminopropyl) carbodiimide. Dox-KLH and Dox-BSA conjugates were used to immunize mice and spleen cells from these mice were used for fusions with the HAT sensitive anti-CEA partner using standard hybridoma procedures. Enzyme linked immunosorbent assays (ELISAs) were developed to test the hybrids obtained for anti-CEA, anti-DOX, anti-BSA and bispecific monoclonal antibody (BsMab) activity. Sixteen fusions with spleen cells from DOX-KLH immunized mice yielded 621 hybrids of which 47 showed low level BsMab activity by ELISA. Eight fusions with spleen cells from DOX-BSA immunized mice yielded 297 hybrids. Fifty of these hybrids showing dual reactivity have been cloned and subcloned to yield 7 subclones with stable BsMab activity for CEA and doxorubicin.
Reddy
V S
VS
Department of Surgery, Faculty of Medicine, Memorial University, St. John's, Newfoundland, Canada.
Ford
C H
CH
D000630:Aminopterin / Q000494:pharmacology; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001375:Azaguanine / Q000633:toxicity; D002272:Carcinoembryonic Antigen / Q000032:analysis* / Q000276:immunology; D002850:Chromatography, Gel; D002999:Clone Cells / Q000187:drug effects; D004317:Doxorubicin / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas / Q000187:drug effects / Q000276:immunology; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D051379:Mice / Q000276:immunology; D011817:Rabbits / Q000276:immunology; D013936:Thymidine / Q000494:pharmacology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D007042:Hypoxanthines; D019271:Hypoxanthine; D004317:Doxorubicin; D000630:Aminopterin; D001375:Azaguanine; D013936:Thymidine