title: Recombinant immunotoxins containing the VH or VL domain of monoclonal antibody B3 fused to Pseudomonas exotoxin.
abstract: We prepared recombinant immunotoxins in Escherichia coli in which the VH or VL domains of mAb B3 were fused to a truncated form of Pseudomonas exotoxin (PE) (PE38KDEL). mAb B3 binds to a carbohydrate Ag found on the surfaces of many types of cancers and only a few normal tissues. PE38KDEL is a 38-kDa form of PE (66 kDa) that is missing the cell-binding domain of PE and has the carboxyl end changed from REDLK to KDEL. We show that immunotoxins in which the H chain or the L chain V region is fused to PE38KDEL bind to and kill carcinoma cells containing the B3 Ag. B3 Ag-negative cells were not affected. The cytotoxicity of these molecules is between 20- and 100-fold less than B3(Fv)-immunotoxins, containing both the H and L chain V regions. The VL-containing toxin was more active than the VH-containing toxin, indicating that the L chain of mAb B3 probably contributes more to Ag-binding than the H chain. Refolding experiments show that B3(VL)-PE38KDEL aggregates less than the VH-derivative or than a single chain immunotoxin B3(Fv)-PE38KDEL, which contains both domains in a single chain form. Furthermore, in addition to monomers, active homodimers of B3(VH)- and B3(VL)-PE38KDEL were obtained from renaturation experiments. The VL-toxin dimers, which might have their binding regions arranged in a manner similar to Bence Jones proteins (L chain homodimers), were found to have almost the same cytotoxicity as the monomers, whereas the VH-toxin dimers had decreased cytotoxic activity.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892.
authors.forename: U
authors.initials: U
authors.lastname: Brinkmann
authors.forename: B K
authors.initials: BK
authors.lastname: Lee
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics; D000918:Antibody Specificity; D001427:Bacterial Toxins; D001483:Base Sequence; D005098:Exotoxins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000922:Immunotoxins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000596:Amino Acids; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A