Ricin, the cytotoxic protein isolated from castor beans, is composed of two subunits, A-chain and B-chain. Ricin intoxicates cells by binding through its B-chain to galactose-terminated oligosaccharides found on the surface of all eukaryotic cells and then transferring its A-chain to the cytosol where it disrupts protein synthesis by inactivating ribosomes. In addition to binding, the B-chain plays an important, but not yet understood, role in the translocation of the A-chain through a cellular membrane to the cytosol. Blocking the two galactose-binding sites of native ricin by chemical modification with affinity ligands created an altered toxin, called blocked ricin, that has at least a 3500-fold lower binding affinity and is more than 1000-fold less cytotoxic than native ricin for Namalwa cells (a Burkitt's lymphoma line) but that has maintained the translocation function of the B-chain and the catalytic activity of the A-chain. Conjugation of blocked ricin to monoclonal antibodies that bind to cell surface antigens creates new cytotoxins that approach the potency of native ricin. These cytotoxins incorporate the three essential functions of natural toxins, i.e., binding to cells, transport through a membrane, and catalytic inactivation of an essential cellular process; but in addition they possess a defined cellular target specificity. Such potent immunotoxins may play an important therapeutic role in cancer treatment. Clinical trials with an anti-CD19-blocked ricin and an anti-CD33-blocked ricin conjugate against B-cell cancers and acute myeloblastic leukemia have begun.
The antibody-mediated cytotoxicity of complement can be increased by the ribosome-inactivating proteins gelonin and PAP-S. Treatment of human lymphoid cells that express CALLA with an anti-CALLA monoclonal antibody, J5, and then with rabbit complement, leaves about 6% of the cells alive. The same treatment in the presence of a sublethal concentration of gelonin or PAP-S leaves only about 0.02 and 0.3% of the cells alive respectively. This synergistic effect has potential implications for the in vitro elimination of malignant cells or of immunocompetent cells from bone marrow before its transplantation.
It has been reported previously that ammonium chloride, chloroquine, monensin, and adenovirus-2 potentiate the cytotoxicity of several protein toxins conjugated with various targeting molecules. We have tested whether these agents, and protein components of adenovirus-2, would enhance the cytotoxicity of conjugates of gelonin with J5, an antibody directed against common acute lymphoblastic leukemia-associated antigen, with 5E9, an antibody directed against human transferrin receptor, or with ricin B-chain. We found that none of these agents affected the cytotoxicity of gelonin conjugates to any significant extent. For example, monensin moderately (3-fold) enhanced the cytotoxicity of 5E9-gelonin for Namalwa cells but showed no effect when 5E9-gelonin was tested on HeLa cells. The potentiating effects of these agents for the cytotoxicity of free gelonin varied from marked to nonexistent, depending on the type of cells. In particular, adenovirus-2 potentiated the cytotoxicity of gelonin for HeLa cells but not for Namalwa cells. The three major adenoviral capsid proteins, penton, hexon, and fiber, were isolated. It was shown that penton potentiated the cytotoxicity of gelonin for HeLa cells and that hexon and fiber had no measurable effect on the cytotoxicity of gelonin. However, like the whole virus, penton was not able to affect the cytotoxicity of gelonin conjugates.
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