Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized Fv fragment.
Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. We have recently described a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin (PE38KDEL). This disulfide-stabilized immunotoxin is indistinguishable in activity and specificity from its single-chain immunotoxin counterpart (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 90: 7538-7542, 1993). We have now constructed and evaluated the stability, pharmacokinetics, and antitumor effect of a very similar disulfide-stabilized immunotoxin B3(dsFv)-PE38. This immunotoxin is specifically cytotoxic to human cancer cell lines such as A431 that express the B3 antigen on their surface. In addition, the dsFv-immunotoxin is more stable at 37 degrees C in human serum than the corresponding single-chain immunotoxin B3(Fv)-PE38. The survival of the disulfide-stabilized immunotoxin in the circulation of mice was determined by a bioassay on cultured A431 cells after administering the immunotoxin i.v. The half-life in blood was 23 min. To determine the therapeutic effects of the disulfide-stabilized immunotoxin, it was given i.v. to immunodeficient mice bearing s.c. human epidermoid carcinomas. The dsFv-immunotoxin caused complete regression of tumors with no toxic effect on mice. The antitumor effect was similar to that reported for the single-chain Fv-immunotoxin. Our data show that dsFv-immunotoxins retain full in vitro as well as in vivo activity when compared to scFv-immunotoxins. Because dsFv-immunotoxins have full activity, are more stable, and can be produced with significantly improved yields compared to scFv-immunotoxins, the dsFv-immunotoxins may be more useful for therapeutic applications than scFv-immunotoxins.
Reiter
Y
Y
Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892.
Pai
L H
LH
Brinkmann
U
U
Wang
Q C
QC
Pastan
I
I
D000818:Animals; D005260:Female; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000494:pharmacology*; D000922:Immunotoxins / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D011994:Recombinant Proteins; C055966:immunoglobulin Fv