Characterization of B1(Fv)PE38 and B1(dsFv)PE38: single-chain and disulfide-stabilized Fv immunotoxins with increased activity that cause complete remissions of established human carcinoma xenografts in nude mice.
The mAb B1 (mouse IgG1 kappa) recognizes a carbohydrate epitope on human carcinoma cells (I. Pastan et al., Cancer Res., 51: 3781-3787, 1991). We have generated plasmids encoding immunotoxins in which the Fv domain of B1, either as a single-chain Fv or as a disulfide-stabilized Fv (dsFv), was fused to PE38, a truncated form of Pseudomonas exotoxin A. To compare the activities of the two types of recombinant immunotoxins, the proteins were prepared from cytoplasmic inclusion bodies produced in Escherichia coli. The immunotoxins were evaluated for stability, antigen binding, specific cytotoxicity, pharmacokinetics, and antitumor activity in a nude mouse model. Although the single-chain immunotoxin is relatively stable when incubated at 37 degreesC (t(1/2) approximately 4 h), the dsFv immunotoxin is much more stable, with no loss of activity after 8 h at 37 degreesC. The single-chain immunotoxin has a 2-fold better binding affinity and cytotoxicity toward antigen-positive cultured cells than the dsFv immunotoxin. The half-lives in the blood of mice of B1(Fv)PE38 (single-chain) and B1(dsFv)PE38 (disulfide-stabilized) are 23 and 27 min, respectively. Their therapeutic potential was evaluated in athymic nude mice bearing human epidermoid carcinoma xenografts. Both immunotoxins caused complete regressions of the s.c. (30-40 mm3) tumors when given i.v. in three doses of 0.025 mg/kg every other day. This is one-twentieth of the mouse LD50. Recombinant immunotoxins containing the B1(Fv) are 2-3-fold more potent antitumor agents than previously described immunotoxins containing the B3(Fv) (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 88: 8616-8620, 1991), which also target LeY and related carbohydrates in human tumors, but have a similar toxicity in mice. Thus, their therapeutic window is 2-3-fold larger. In addition, B1(dsFv)PE38 has only a 50% decrease in the apparent binding affinity of B1(Fv)PE38, whereas B3(dsFv)PE38 has a much greater loss in antigen binding.
Benhar
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Laboratory of Molecular Biology, Division of Cancer Biology Diagnosis and Centers, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Pastan
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D036002:ADP Ribose Transferases; D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D001427:Bacterial Toxins; D004354:Drug Screening Assays, Antitumor; D005098:Exotoxins / Q000737:chemistry / Q000276:immunology / Q000493:pharmacokinetics / Q000627:therapeutic use*; D006801:Humans; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins / Q000737:chemistry / Q000276:immunology / Q000493:pharmacokinetics / Q000627:therapeutic use*; D051379:Mice; D008819:Mice, Nude; D012074:Remission Induction; D014183:Transplantation, Heterologous; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A