Recombinant immunotoxins containing the VH or VL domain of monoclonal antibody B3 fused to Pseudomonas exotoxin.
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.
Brinkmann
U
U
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892.
Lee
B K
BK
Pastan
I
I
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
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