Monoclonal antibody-.beta.-lactamase conjugates for the activation of a cephalosporin mustard prodrug

Abstract

Cephalosporin mustard (CM) was designed as an anticancer prodrug that could be activated in a site-specific manner by monoclonal antibody-beta-lactamase conjugates targeted to antigens present on tumor cell surfaces. Purified beta-lactamases from Bacillus cereus (BC beta L) and Escherichia coli (EC beta L) catalyzed the release of phenylenediamine mustard (PDM) from CM through a fragmentation reaction which occurs after the beta-lactam ring of CM is hydrolyzed. The Km and Vmax values were 5.7 microM and 201 mumol/min per mg for BC beta L and 43 microM and 29 mumol/min per mg for EC beta L, respectively. Conjugates of BC beta L were prepared by combining the F(ab')2 fragments of the maleimide-substituted monoclonal antibodies L6 and 1F5 with thiolated BC beta L. The conjugates showed little loss in enzymatic activity and bound nearly as well as the unmodified F(ab')2 monoclonal antibodies to antigens expressed on the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). PDM was approximately 50-fold more cytotoxic than CM to H2981 cells. Treatment of the cells with L6-BC beta L followed by CM resulted in a level of cytotoxic activity that was comparable to that of PDM. This was most likely due to activation of CM by conjugate that bound to cell-surface antigens, since pretreatment of H2981 cells with BC beta L or 1F5-BC beta L enhanced the activity of CM to a lesser extent. Thus, we have shown that CM is a prodrug, and that it can be activated with immunological specificity by a monoclonal antibody-beta-lactamase conjugate.

Enzyme-Catalyzed Activation of Anticancer Prodrugs

“…7-(Phenylacetamido)-cephalosporin mustard (CM) was developed as a prodrug of the DNA-alkylating agent ENZYME-CATALYZED ACTIVATION OF PRODRUGS phenylenediamine mustard (Svensson et al, 1992). CM is activated by ␤-lactamase from Bacillus cereus and E. coli, resulting in the formation of phenylenediamine mustard with K m values of 6 and 43 M, respectively.…”

“…CM is activated by ␤-lactamase from Bacillus cereus and E. coli, resulting in the formation of phenylenediamine mustard with K m values of 6 and 43 M, respectively. CM was 50-fold less cytotoxic toward human lung adenocarcinoma cells than phenylenediamine mustard, whereas after activation by a monoclonal antibody-␤-lactamase conjugate, cytotoxicity was comparable (Svensson et al, 1992). The CM analog 7-(4-carboxybutanamido)-cephalosporin mustard (CCM) was also shown to be activated to phenylenediamine mustard by ␤-lactamases with K m values in the micromolar range (Vrudhula et al, 1993b).…”

Efficient Cancer Therapy with a Nanobody-Based Conjugate

“…After 30 min at 4°C, cells were washed with RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum. Subsequently, different concentrations of prodrug 7-(4-carboxybutanamido) cephalosporin mustard (CCM) or the drug phenylenediamine mustard (PDM) were added (19). CCM and PDM were also added to cells that were not treated with conjugates.…”

Resonance Energy Transfer Nanobiosensors Based on Affinity Binding between Apo-Enzyme and Its Substrate

“…Finally the apo-GOx was labeled with TRITC using a standard amine labeling procedure. 24 Upon analysis, the conjugated TRITC-apo-GOx (TAG) was found to have 1.67 mol of apoGOx/mol of TRITC.…”