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 specific for human carcinomas and beta-lactamase II from Bacillus cereus. Sequences encoding the variable regions of the L6 monoclonal antibody were assembled so as to be separated from each other by an 18-amino acid 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 export of the fusion protein to the periplasmic space of Escherichia coli. After induction, biologically active material was recovered from both culture supernatants and cell lysates. Affinity chromatography yielded about 2.5 micrograms of protein/ml of initial culture volume. The fusion protein was shown to bind to tumor cells at least as well as chemically prepared F(ab') and to maintain beta-lactamase activity at a level similar to that of the native enzyme. Tumor cells coated with the fusion 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 chain-linked variable domain-enzyme fusion proteins for the activation of anticancer prodrugs.
Goshorn
S C
SC
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Svensson
H P
HP
Kerr
D E
DE
Somerville
J E
JE
Senter
P D
PD
Fell
H P
HP
D000595:Amino Acid Sequence; D000912:Antibodies, Neoplasm / Q000008:administration & dosage / Q000737:chemistry*; D000951:Antigens, Neoplasm / Q000276:immunology*; D001483:Base Sequence; D002277:Carcinoma / Q000276:immunology*; D002470:Cell Survival / Q000187:drug effects; D002511:Cephalosporins / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009588:Nitrogen Mustard Compounds / Q000378:metabolism* / Q000494:pharmacology; D011355:Prodrugs / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage*; D014407:Tumor Cells, Cultured; D001618:beta-Lactamases / Q000008:administration & dosage / Q000737:chemistry*
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D002511:Cephalosporins; D009588:Nitrogen Mustard Compounds; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; C076378:cephalosporin mustard; D001618:beta-Lactamases