GENETIC CONSTRUCTION, EXPRESSION, AND CHARACTERIZATION OF A SINGLE-CHAIN ANTI-CARCINOMA ANTIBODY FUSED TO BETA-LACTAMASE

We report the genetic construction and expression of a fusion protein between an antibody single chain-linked variable domain fragment speci fic for human carcinomas and beta-lactamase II from Bacillus cereus. S equences encoding the variable regions of the L6 monoclonal antibody w ere assembled so as to be separated from each other by an 18-amino aci d linker and from the mature form of beta-lactamase by a 6-amino acid linker. The construct was placed under the transcriptional regulation of the lac promoter, and the PelB signal sequence was used to direct e xport of the fusion protein to the periplasmic space of Escherichia co li. After induction, biologically active material was recovered from b oth culture supernatants and cell lysates. Affinity chromatography yie lded about 2.5 mug of protein/ml of initial culture volume. The fusion protein was shown to bind to tumor cells at least as well as chemical ly prepared F(ab') and to maintain beta-lactamase activity at a level similar to that of the native enzyme. Tumor cells coated with the fusi on protein were sensitive to a cephalosporin mustard prodrug in a dose -dependent fashion comparable to that of enzyme chemically conjugated to F(ab'). This article demonstrates the feasibility of using single c hain-linked variable domain-enzyme fusion proteins for the activation of anticancer prodrugs.