Before investigators can effectively use antibody-toxin conjugates in in vitro and in vivo protocols, it is essential that they have an understanding of the molecular features of the immunotoxin molecule essential for optimum cytotoxicity as well as the mechanism(s) by which these chimeras are internalized and processed by the target cells. Thus, the long-range goals of this work are 3-fold: (1) to determine the features of the antibody, the toxin, and the coupling reagent necessary for optimum cytotoxicity; (2) to evaluate the importance of antigen density and modulation on conjugate efficacy; and (3) to elucidate the molecular events involved in immunotoxin internalization and processing. More specifically, the immediate goals are: (1) to construct ricin (native and modified) and ricin A-/B-chain conjugates with various antibody vectors using cleavable and noncleavable crosslinking reagents; (2) to evaluate the in vivo stability of the A-chain-containing reagents; (3) to produce monoclonal antibodies to purified ricin A- and B-chain proteins; (4) to further characterize the effects of ammonium salts and proton ionopheres on conjugate internalization and processing; (5) to characterize the involvement of actin, calmodulin, and transglutaminase in conjugate translocation and processing; and (6) to determine if immunotoxins are indeed internalized via receptor-mediated endocytosis. Work to date has allowed better synthetic yields of conjugates and has resulted in a shortened protocol for production of ricin A- and B-chain polypeptides. Further, it has demonstrated the dramatic enhancement of conjugate cytotoxicity provided by ammonium salts and the proton ionophores monensin and nigericin. Finally, preclinical studies have shown that whole-ricin immunoconjugates can be safely used for the removal of target cells from human bone marrow, thus demonstrating their applicability to autologous bone marrow transplantation. (HI)
| Leonard, J E; Grothaus, C D; Taetle, R (1988) Ricin binding and protein synthesis inhibition in human hematopoietic cell lines. Blood 72:1357-63 |
| Leonard, J E; Johnson, D E; Shawler, D L et al. (1988) Inhibition of human T-cell tumor growth by T101-ricin A-chain in an athymic mouse model. Cancer Res 48:4862-7 |
| Leonard, J E; Johnson, D E; Felsen, R B et al. (1987) Establishment of a human B-cell tumor in athymic mice. Cancer Res 47:2899-902 |
| Leonard, J E; Tanney, L E; Collins, M L et al. (1987) Monoclonal antibodies to purified ricin A-chain: production and properties. Hybridoma 6:135-49 |
| Leonard, J E; Wang, Q C; Kaplan, N O et al. (1985) Kinetics of protein synthesis inactivation in human T-lymphocytes by selective monoclonal antibody-ricin conjugates. Cancer Res 45:5263-9 |
| Leonard, J E; Taetle, R; To, D et al. (1985) Preclinical studies on the use of selective antibody-ricin conjugates in autologous bone marrow transplantation. Blood 65:1149-57 |
