IMPROVED BINDING AND ANTITUMOR-ACTIVITY OF A RECOMBINANT ANTI-ERBB2 IMMUNOTOXIN BY DISULFIDE STABILIZATION OF THE FV FRAGMENT

e23(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Pv reg ion of anti-erbB2 monoclonal antibody e23 connected to a truncated for m of Pseudomonas exotoxin (PE38KDEL), in which the inherently unstable Fv heterodimer (composed of V-H and V-L) is stabilized by a disulfide bond engineered between structurally conserved framework positions of V-H and V-L. We have now found that e23(dsFv)-PE38KDEL is considerabl y more cytotoxic to antigen-positive cell lines than the corresponding single-chain immunotoxin. The basis for the enhanced cytotoxic activi ty is that the e23 dsFv-immunotoxin binds to erbB2 with greater affini ty than the single-chain counterpart. The dsFv-immunotoxin had 4-fold increased binding compared to the scFv and almost identical to the bin ding affinity of e23 Fab. e23(dsFv)-PE38KDEL was also considerably mor e stable at 37 degrees C than the single-chain immunotoxin. The therap eutic potential of the disulfide-stabilized immunotoxin was compared w ith its single-chain counterpart using two animal models of immunodefi cient mice bearing subcutaneous tumor xenografts of human gastric tumo r N87 cells or human A431 epidermoid carcinoma cells. The antitumor ef fect of e23(dsFv)-PE38KDEL was significantly better than that of the s ingle-chain immunotoxin. e23(dsFv)-PE38KDEL caused complete regression of tumors at doses which caused no toxic effects in mice, whereas the single-chain immunotoxin did not cause complete regressions at the sa me doses.