Structural features of the antibody-A chain linkage that influence the activity and stability of ricin A chain immunotoxins.
The importance of the various structural elements constituting a ricin A chain immunotoxin to the stability of the disulfide bond between the antibody and A chain was examined using a panel of immunoconjugates prepared with the mouse monoclonal antibody Fib75. Analogues of the standard ricin A chain immunotoxin prepared with the N-succinimidyl 3-(2-pyridyldithio)propionate disulfide cross-linker included immunoconjugates made with N-succinimidyl 4-[(iodoacetyl)amino]benzoate the thioether cross-linker; with N-succinimidyl 3-(2-pyridyldithio)butyrate, the hindered disulfide cross-linker; with a peptide spacer between the antibody and cross-linker; or with the dodecapeptide corresponding to the C-terminus of ricin A chain. The cytotoxic activities of the immunoconjugates and their susceptibility to reduction by glutathione in vitro were compared. The thioether-linked immunotoxin could not be cleaved by glutathione in vitro and had low cytotoxic potency, consistent with the requirement of a reducible disulfide linkage for activity. The hindered disulfide-linked immunotoxin was 3-fold more stable to reduction than the immunotoxin containing a standard unhindered disulfide linkage, but the cytotoxic activities of the two constructs were indistinguishable. The introduction of a flexible peptide Ala-Ala-Pro-Ala-Ala-Ala-Pro-Ala-Pro-Ala between Fib75 and the disulfide linkage introduced by SPDP had no deleterious effect on cytotoxic activity and no effect on the susceptibility of the disulfide linkage to reduction. This finding suggests that the enforced proximity of the A chain to the antibody caused by the use of a short chemical cross-linker in a conventional immunotoxin has no influence on either of these properties in this system.(ABSTRACT TRUNCATED AT 250 WORDS)
Cumber
A J
AJ
Drug Targeting Group, Institute of Cancer Research, Surrey, U.K.
Westwood
J H
JH
Henry
R V
RV
Parnell
G D
GD
Coles
B F
BF
Wawrzynczak
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism / Q000627:therapeutic use; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D046508:Culture Techniques; D004220:Disulfides / Q000737:chemistry; D004355:Drug Stability; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry* / Q000378:metabolism / Q000627:therapeutic use; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009579:Nitrobenzoates / Q000378:metabolism; D010084:Oxidation-Reduction; D012276:Ricin / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013388:Succinimides / Q000737:chemistry; D013438:Sulfhydryl Compounds; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D004220:Disulfides; D000922:Immunotoxins; D009579:Nitrobenzoates; D013388:Succinimides; D013438:Sulfhydryl Compounds; C011136:thionitrobenzoic acid; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C078238:N-succinimidyl-4-((iodoacetyl)amino)benzoate; D012276:Ricin; D005978:Glutathione