Synthesis and characterization of monoclonal antibody-beta-lactamase conjugates

beta-Lactamase from Enterobacter cloacae (beta L) was conjugated to the Fab'2 fragment of the monoclonal antibody L6 through a thioether linkage. Although L6-Fab'2-beta L was capable of activating the antitumor prodrug, 7-(phenylacetamido)cephalosporin mustard, it was impaired in its ability to bind to antigens on the H2981 human lung adenocarcinoma cell line. As a result, studies were undertaken to prepare conjugates with preserved binding activities. L6-Fab'-beta L and a dimeric conjugate consisting of two individual L6-Fab' units linked to a single beta L molecule (dimeric L6-beta L) were prepared by linking L6-Fab'-SH to maleimide-substituted beta L. Analysis of these conjugates by SDS-PAGE indicated that the linkage involved heavy-chain thiol groups on L6 that are most likely in the hinge region and are therefore removed from the antigen binding site of the antibody. Cell binding studies revealed that the monovalent conjugate L6-Fab'-beta L bound as well as L6-Fab'. Dimeric L6-beta L displayed slightly less binding than L6-Fab'2, but bound substantially better than L6-Fab'2-beta L. Lower concentrations of dimeric L6-beta L compared to L6-Fab'2-beta L were required to convert the prodrug 7-(phenylacetamido)-cephalosporin mustard into the cytotoxic drug phenylenediamine mustard. Localization studies were performed in nude mice with H2981 subcutaneous tumor xenografts. At 96 h post conjugate treatment, there was no significant difference in tumor concentration between L6-Fab'2-beta L and dimeric L6-beta L.(ABSTRACT TRUNCATED AT 250 WORDS)