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 Fv region of anti-erbB2 monoclonal antibody e23 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the inherently unstable Fv heterodimer (composed of VH and VL) is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. We have now found that e23(dsFv)-PE38KDEL is considerably more cytotoxic to antigen-positive cell lines than the corresponding single-chain immunotoxin. The basis for the enhanced cytotoxic activity is that the e23 dsFv-immunotoxin binds to erbB2 with greater affinity than the single-chain counterpart. The dsFv-immunotoxin had 4-fold increased binding compared to the scFv and almost identical to the binding affinity of e23 Fab. e23(dsFv)-PE38KDEL was also considerably more stable at 37 degrees C than the single-chain immunotoxin. The therapeutic potential of the disulfide-stabilized immunotoxin was compared with its single-chain counterpart using two animal models of immunodeficient mice bearing subcutaneous tumor xenografts of human gastric tumor N87 cells or human A431 epidermoid carcinoma cells. The antitumor effect of e23(dsFv)-PE38KDEL was significantly better than that of the single-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 same doses.
Reiter
Y
Y
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Brinkmann
U
U
Jung
S H
SH
Lee
B
B
Kasprzyk
P G
PG
King
C R
CR
Pastan
I
I
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins / Q000378:metabolism / Q000627:therapeutic use; D001667:Binding, Competitive; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000473:pathology; D002460:Cell Line; D017124:Conserved Sequence; D004220:Disulfides; D005098:Exotoxins / Q000378:metabolism* / Q000627:therapeutic use*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins / Q000378:metabolism* / Q000627:therapeutic use*; D051379:Mice; D008819:Mice, Nude; D009856:Oncogene Proteins, Viral / Q000276:immunology*; D011550:Pseudomonas aeruginosa; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins / Q000378:metabolism / Q000627:therapeutic use; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000473:pathology; D014183:Transplantation, Heterologous; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D009856:Oncogene Proteins, Viral; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A