Novel synthesis and in vitro characterization of disulfide-linked ricin-monoclonal antibody conjugates devoid of galactose binding activity.
The use of tumor immunotherapy using whole ricin-antibody conjugates is complicated by the nonspecific lectin activity of the ricin B-chain which leads to toxic side effects. A novel method of coupling whole intact ricin to monoclonal antibody (MoAb) is described herein, where the nonspecific binding of the ricin B-chain is blocked. The coupling was done using the bifunctional reagents S-acetylmercaptosuccinic anhydride for antibody and succinimidyl 3-(2-pyridyldithio)propionate for ricin, and this resulted in the loss of B-chain binding activity, while impairing neither the toxic potential of the A-chain nor the activity of the MoAb. The purified immunotoxins could not bind to lactose-Sepharose and were equally cytotoxic in vitro to MoAb-reactive cell lines in the presence or absence of lactose. The coupling method was suitable for six different ricin-antibody conjugates and also using ricin deglycosylated by treatment with periodate. However, the blocking of the ricin B-chain was only effective with whole IgG molecules as F(ab')2-ricin immunotoxins could, like ricin, bind to lactose-Sepharose. Ricin-antibody conjugates reduced the [3H]leucine incorporation of appropriate target cells by 50% at a concentration of 6 to 45 ng/ml, whereas nonreactive antibody immunotoxins were not toxic to the target cells at concentrations as high as 10(4) ng/ml. The specific cytotoxicity of these immunotoxins could be inhibited by the addition of unconjugated reactive MoAb; the presence of lactose or a nonreactive MoAb did not significantly affect the observed cytotoxicity. Thus, whole ricin-antibody conjugates produced in this way do not bind nonspecifically to target cells, the most important implication being that such immunotoxins should be more potent that ricin A-chain conjugates and capable of being used in vivo.
Pietersz
G A
GA
Department of Pathology, University of Melbourne, Parkville, Victoria, Australia.
Kanellos
J
J
McKenzie
I F
IF
D000818:Animals; D000950:Antigens, Ly / Q000276:immunology; D001665:Binding Sites; D004220:Disulfides; D005690:Galactose / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D000922:Immunotoxins / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008970:Molecular Weight; D012276:Ricin / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*
D000950:Antigens, Ly; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D000922:Immunotoxins; D012276:Ricin; D005690:Galactose