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 antibo dies in which the inherently unstable V-H-V-L heterodimer is stabilize d by a disulfide bond engineered between structurally conserved framew ork positions of V-H and V-L. We have recently described a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv conne cted to a truncated form of Pseudomonas exotoxin (PE38KDEL). This disu lfide-stabilized immunotoxin is indistinguishable in activity and spec ificity from its single-chain immunotoxin counterpart (Brinkmann et al ., Proc. Natl. Acad. Sci. USA, 90: 7538-7542, 1993). We have now const ructed and evaluated the stability, pharmacokinetics, and antitumor ef fect 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 additio n, the dsFv-immunotoxin is more stable at 37 degrees C in human serum than the corresponding single-chain immunotoxin B3(Fv)-PE38. The survi val of the disulfide-stabilized immunotoxin in the circulation of mice was determined by a bioassay on cultured A431 cells after administeri ng the immunotoxin i.v. The half-life in blood was 23 min. To determin e the therapeutic effects of the disulfide-stabilized immunotoxin, it was given i.v. to immunodeficient mice bearing s.c. human epidermoid c arcinomas. The dsFv-immunotoxin caused complete regression of tumors w ith 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 com pared to scFv-immunotoxins. Because dsFv-immunotoxins have full activi ty, are more stable, and can be produced with significantly improved y ields compared to scFv-immunotoxins, the dsFv-immunotoxins may be more useful for therapeutic applications than scFv-immunotoxins.