ANALYSIS OF PRODUCTION, PURIFICATION, AND CYTOLYTIC POTENTIAL OF BI-SPECIFIC ANTIBODIES REACTIVE WITH OVARIAN-CARCINOMA-ASSOCIATED ANTIGENSAND THE T-CELL ANTIGEN CD3

OV-TL3 and MOv18 MAbs, due to their restricted specificity, have been successfully used to visualize ovarian cancer in patients and might th erefore be used to develop therapies for ovarian cancer. The bi-specif ic MAbs alphaT3/OC2 and alphaOC/TR (both being combinations of MOv18 a nd alphaCD3) have been shown to lyse ovarian tumor cells in vitro. To evaluate the relative merits of MOv18/CD3 and OV-TL 3/CD3, the present study was undertaken in which the bi-specific MAbs alphaT3/OC2 and al phaOC/TR, and a newly developed bi-specific MAb, OV-TL 3/CD3, were hig hly purified and compared for specificity, stability, purification and cytolytic potential. The dual specificity of the hybrid-hybridoma sup ernatants was analyzed by immunohistochemistry, and by testing bi-spec ific MAb-mediated cytotoxicity against relevant target cells in the pr esence of effector cells. Stability testing of bi-specific MAb-produci ng hybrid-hybridomas showed that, after sub-cloning, clones stably pro duced up to 40% bi-specific MAb even after prolonged in vitro culture. The purification of the bi-specific fractions was performed with prot ein A and by ion-exchange high-pressure liquid chromatography, dependi ng on the sub-class combination of the bi-specific MAb. The purified b i-specific MAbs were tested for their ability to mediate target-cell l ysis with the use of cytotoxic T-cell clones and activated peripheral- blood lymphocytes. The purified alphaT3/OC2, alphaOC/TR, and OV-TL 3/C D3 were all able to mediate highly specific lysis of various ovarian-c arcinoma cell lines. No correlation was found between the level of ant igen expression and bi-specific MAb-mediated cytolysis. (C) 1993 Wiley -Liss, Inc.