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Targeting, Internalization, and Cytotoxicity of Methotrexate-Monoclonal Anti-Stage-specific Embryonic Antigen-1 Antibody Conjugates in Cultured F-9 Teratocarcinoma Cells¹

Department of Pathology, Boston University School of Medicine, Boston, Massachusetts 02118 [W-C. S., H.J-P. R.]; Division of Urological Surgery, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh 15261, and Veterans Administration Medical Center, Pittsburgh, Pennsylvania 15240 [B. B., T. R. H.]

Methotrexate (MTX) conjugates of a monoclonal antibody, anti-SSEA-1, containing an average of 45 mol MTX/mol of immunoglobulin M, were prepared by a carbodiimide coupling reaction. Binding experiments indicate that conjugation does not decrease the affinity of the antibody for its antigen. The conjugate strongly inhibits the growth of SSEA-1-bearing F-9 teratocarcinoma cells, with 50% inhibitory dose of 4.5 nM MTX, which makes it more active than free MTX (50% inhibitory dose of 15 nM). The drug-free antibody is not cytotoxic to F-9 cells at the concentrations used. The high efficacy of the conjugated drug may be due in part to the fact that anti-SSEA-1 antibody is an immunoglobulin M. MTX conjugated to nonspecific immunoglobulin M has little inhibitory effect (50% inhibitory dose of 150 nM). When acting on SSEA-1 negative cells, the two conjugates have only a small but identical effect. Thiamine pyrophosphate, an inhibitor of MTX transport, can prevent the cytotoxicity of the free MTX but not that of the anti-SSEA-1 conjugate. Leupeptin, an inhibitor of lysosomal protease, can partially protect F-9 cells against the antibody conjugate but not against free MTX. These results indicate that the MTX antibody conjugate binds specifically to F-9 cells, and is internalized and intracellularly degraded to release a small molecular active drug. Pretreatments of F-9 cells for 1 h with unlabeled antibody inhibits the subsequent uptake of identical concentration of labeled conjugate. The rate of internalization, however, regains almost normal values within 4 h, indicating a rapid reappearance of free antigenic sites at the cell surface.

¹ Supported by NIH Grants CA 34798, CA 14551, CA 31784, and HD 12487; and by intramural research funds from the Veterans' Administration.

² Recipient of a Cancer Research Scholar Award from American Cancer Society, Massachusetts Division. To whom requests for reprints should be addressed.

Received 10/ 4/85. Revised 2/14/86. Revised 4/28/86. Accepted 5/ 2/86.

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