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Evaluation of Monoclonal Antibody DF3 Conjugated with Ricin as a Specific Immunotoxin for in Vitro Purging of Human Bone Marrow¹

Laboratory of Clinical Pharmacology, Department of Medicine, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115

DF3 is an IgG1 monoclonal antibody (MAb) generated against a M_r 350,000–400,000 glycoprotein expressed by approximately 80% of human breast cancers. We have coupled MAb DF3 to ricin. Purification of the immunotoxin (DF3-IT) was obtained by affinity and size exclusion chromatography. DF3 antigen-positive breast cancer cell lines (ZR-75-1, BT-20, and MCF-7) and DF3 antigen-negative lung cancer cell lines (A549 and CALU6) were tested for cytotoxicity with metabolic labeling and clonogenic assays. The cells were exposed for 3 h to different concentrations of DF3-IT, MAb DF3, ricin, and a combination of unconjugated MAb DF3 and ricin. In the presence of 100 mM lactose, DF3-IT specifically inhibited protein synthesis of lines expressing DF3 antigen on their cell surface. Moreover, clonogenic survival experiments demonstrated that DF3-IT at a concentration of 1 x 10^-9 M specifically kills 2.6–2.8 log of ZR-75-1 and BT-20 cells and 1.6 log of MCF-7 cells. At the same concentration, nonspecific toxicity of DF3-IT resulted in a 30% reduction of bone marrow granulocyte and macrophage colony formation. In bone marrow-purging experiments, tumor cells were mixed with an excess of bone marrow cells and treated with DF3-IT or ricin. Tumor cell clonogenic survival assays demonstrated that the presence of bone marrow cells had no detectable effect on activity or specificity of the DF3-IT. These results thus indicate that MAb DF3 is an effective vehicle to specifically deliver toxins to cancer cells which express DF3 antigen on their surface and that DF3-IT may be useful for in vitro purging of bone marrow.

¹ This investigation was supported by PHS Grant CA-38869 awarded by the National Cancer Institute, an American-Italian Foundation for Cancer Research Grant, an NCI-EORTC grant, and a Burroughs Wellcome Award in Clinical Pharmacology (D. W. K.).

² To whom requests for reprints should be addressed.

Received 6/14/89. Revised 10/30/89. Accepted 11/ 7/89.

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