Chris Burbage

Ricin toxin contains three lectin sites which contribute to its in vivo toxicity.

Ricin intoxication of mammalian cells is initiated by B chain (RTB) binding to cell surface galactosides. Recombinant insect-derived RTB mutants with modifications in lectin-site subdomains 2 gamma, 1 alpha/2 gamma, and 1 alpha/1 beta/2 gamma were reassociated with plant RTA and tested for lethality in C57B1/6 6-8 weeks old mice. The LD50 of intraperitoneally injected castor bean ricin was 75 ng per 18 g mouse. The LD50 of single-site 2 gamma mutant heterodimer was 100 ng: the LD50 of the double-site 1 alpha/2 gamma mutant heterodimer was 500 ng, and the LD50 of the triple-site 1 alpha/1 beta,2 gamma mutant heterodimer was > 10 micrograms. Plant RTA alone had an LD50 of 300 micrograms. Animals died between 1 and 10 days post-injection. Histopathological examination of morbid animals receiving an LD50 dose of each toxin revealed only apoptosis in the thymus and spleen. The present data provide clear evidence for participation of three lectin sites in ricin in vivo toxicity. These results suggest an origin for some of the normal tissue toxicities observed with clinical trials of doubly blocked ricin conjugates and suggest modification of at least three RTB subdomains will be necessary in genetically engineered ricin fusion proteins.

Ricin fusion toxin targeted to the human granulocyte-macrophage colony stimulating factor receptor is selectively toxic to acute myeloid leukemia cells

Treatment failure of patients with acute myelogenous leukemia (AML) is frequently due to the development of multidrug resistance phenotype blasts. We have expressed a fusion protein consisting of human granulocyte-macrophage colony stimulating factor (GMCSF) fused to the N-terminus of a lectin-deficient ricin toxin B chain (RTB) in Spodoptera frugiperda insect cells. The fusion protein was purified by immunoaffinity chromatography and reassociated with chemically deglycosylated ricin toxin A chain (RTA). The resulting fusion toxin was found to react with antibodies to GMCSF, RTB and RTA and had the predicted molecular mass of 80 kDa. GMCSF-ricin bound poorly to asialofetuin (Kd = 10(6) M-1) and receptor negative cells indicating loss of lectin activity, but bound strongly to GMCSF receptor positive HL60 cells. Ligand displacement assays showed fusion toxin affinity 2.6-fold less than native GMCSF. Selective inhibition of protein synthesis was observed on receptor positive cells. Induction of apoptosis was also observed on receptor positive cells. Cells expressing multidrug resistance gene products (P-gp, Bcl2 and BclXL) were also sensitive to fusion toxin. These results suggest that GMCSF-ricin deserves further preclinical development.

IL2 fused to lectin-deficient ricin is toxic to human leukemia cells expressing the IL2 receptor

Interleukin-2 (IL2) fused to ricin B chain (RTB) with modifications of amino acid residues in each of three galactose-binding subdomains (1α, 1β and 2γ) was expressed in insect cells, purified by immunoaffinity chromatography and reassociated with ricin A chain (RTA). The fusion toxin-bound human leukemic cells with IL2 receptors and the binding was competed with IL2 but not asialofetuin. In contrast, binding was not observed with receptor negative human cell lines, and the fusion molecule very weakly bound asialofetuin (Kd = 10−6 M), indicating lectin-deficient RTB. The IL2–lectin-deficient RTB-RTA intoxicated IL2 receptor bearing cells as well as ricin or IL2–wild-type RTB-RTA. While ricin and IL2–wild-type RTB-RTA were equally toxic to receptor negative cell lines, the IL2–lectin-deficient RTB-RTA was two-two and one half logs less cytotoxic to these cell lines. The sensitivity of receptor-positive cells to the lectin-deficient fusion protein suggests that high avidity intracellular galactose binding may not be required for ricin intoxication, at least in the case of IL2 receptor-targeted molecules. Furthermore, the potent selective cytotoxicity of the fusion protein suggests that the IL2–lectin-deficient RTB-RTA and similar ricin fusion molecules directed against other leukemic cell surface receptors provide a novel class of fusion toxins for therapy of human leukemias.