Importance of the antigen-binding valency and the nature of the cross-linking bond in ricin A-chain conjugates with antibody.
As a continuation of our work on toxin A-chain conjugates with antitumor antibodies for selective delivery of the toxin to the target cells, four ricin A-chain conjugates were prepared by linking A-chain to Fab' or F(ab')2 of rabbit IgG against L1210 with or without employing a cross-linking agent, N,N'-o-phenylenedimaleimide (PDM), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N-succinimidyl m-(N-maleimido)benzoate (SMB), and the effects of antigen-binding valency and of the nature of the cross-linking bond on their in vitro cytotoxicity were studied. The relative potencies of the conjugates in terms of IC94's were as follows: F(ab')2-SPDP-A-chain, 100; Fab'-S-S-A-chain, 21; F(ab')2-SMB-A-chain, 1.3; Fab'-PDM-A-chain 0.38. Among the four conjugates, F(ab')2-SPDP-A-chain and Fab'-S-S-A-chain can be cleaved into the homing and the cytotoxic components with 2 mM 2-mercaptoethanol. These results suggest that divalency in antigen-binding and susceptibility of the cross-linking bond to cleavage by mercapto reagent are desirable for high potency. Protein synthesis in a cell-free system of rabbit reticulocyte lysate was inhibited by Fab'-S-S-A-chain and by Fab'-PDM-A-chain as effectively as by free A-chain, indicating that the liberation of A-chain is not important, at least on ribosomes, but it is important for the A-chain to reach a ribosome after binding of the conjugates to the cell-surface.
D000818:Animals; D000912:Antibodies, Neoplasm; D000954:Antigens, Surface; D000970:Antineoplastic Agents; D001666:Binding Sites, Antibody; D002474:Cell-Free System; D003432:Cross-Linking Reagents; D007140:Immunoglobulin Fab Fragments; D007939:Leukemia L1210 / Q000276:immunology; D051379:Mice; D014176:Protein Biosynthesis; D011817:Rabbits; D012276:Ricin
D000912:Antibodies, Neoplasm; D000954:Antigens, Surface; D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D007140:Immunoglobulin Fab Fragments; D012276:Ricin