Ricin A-chain cytotoxicity depends on its presentation to the cell membrane.
Anti-ricin-A-chain monoclonal antibodies (mAbs) were raised in order to study epitopes of the A-chain involved in the initiation of its transmembrane passage. Five nonoverlapping epitopes were selected by cluster formation using a cross-inhibition assay of mAbs from 172 specific hybridomas. For each cluster, representative high affinity mAbs were selected. These mAbs were disulfide-linked to F(ab')2 fragments of a mAb directed against the CD5 antigen. The cytotoxicity of ricin A-chain bound by affinity through different epitopes to these hybrid mAbs was explored on the CD5-positive CEM cells. Without any enhancer, the hybrids displayed cytotoxicity varying in IC50 within a range of 1-10; in the presence of the enhancer NH4Cl, this variation of activity was maintained ranging from 1 to 20; with the enhancer monensin, the variation of cytotoxicity of hybrids extended over a 1-80-fold range. These differences of cytotoxicity were not correlated with mAb properties such as the inhibition of A-chain enzymatic activity, the A-chain binding affinity, the capacity for releasing A-chain at low pH, or the capacity of hybrids for delivering A-chain on the cell surface. This lack of correlation strongly suggests that the different presentations of A-chain epitopes to the cell membrane may be the essential reason for cytotoxicity variations.
Carayon
P
P
Sanofi Recherche, Centre de Montpellier, France.
Bord
A
A
Gaillard
J P
JP
Vidal
H
H
Gros
P
P
Jansen
F K
FK
D000643:Ammonium Chloride / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology; D001667:Binding, Competitive; D018956:CD5 Antigens; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects*; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D000922:Immunotoxins / Q000008:administration & dosage / Q000494:pharmacology*; D055503:Protein Multimerization; D012276:Ricin / Q000008:administration & dosage* / Q000276:immunology / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018956:CD5 Antigens; D004220:Disulfides; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D000922:Immunotoxins; D000643:Ammonium Chloride; D012276:Ricin