SYNTHESIS AND CHARACTERIZATION OF MONOCLONAL ANTIBODY-BETA-LACTAMASE CONJUGATES

Beta-lactamase from Enterobacter cloacae (betaL) was conjugated to the Fab'2 fragment of the monoclonal antibody L6 through a thioether link age. Although L6-Fab'2-betaL was capable of activating the antitumor p rodrug, 7-(phenylacetamido)cephalosporin mustard, it was impaired in i ts 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'-betaL and a dimeric conjuga te consisting of two individual L6-Fab' units linked to a single betaL molecule (dimeric L6-betaL) were prepared by linking L6-Fab'-SH to ma leimide-substituted betaL. Analysis of these conjugates by SDS-PAGE in dicated 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 t hat the monovalent conjugate L6-Fab'-betaL bound as well as L6-Fab'. D imeric L6-betaL displayed slightly less binding than L6-Fab'2, but bou nd substantially better than L6-Fab'2-betaL. Lower concentrations of d imeric L6-betaL compared to L6-Fab'2-betaL were required to convert th e 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 conju gate treatment, there was no significant difference in tumor concentra tion between L6-Fab'2-betaL and dimeric L6-betaL. In contrast, the blo od and normal tissue levels of dimeric L6-betaL were lower than L6-Fab '2-betaL, resulting in improved tumor to blood and tumor to normal tis sue ratios. Thus, the conjugation methodology described here may be of use for targeting strategies in which high tumor to nontumor conjugat e ratios are required in order to minimize nonspecific toxicity.