Selective antitumor effect on L10 hepatocarcinoma cells of a potent immunoconjugate composed of the A chain of abrin and a monoclonal antibody to a hepatoma-associated antigen.
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The toxic A chain of abrin was isolated by affinity chromatography and was demonstrated to be a potent inhibitor of protein synthesis in a cell-free rabbit reticulocyte system with a complete inhibitory dose at 1 X 10(-9) M. This A chain was coupled by a disulfide linkage to a purified monoclonal antibody directed against a tumor-associated antigen found on the line 10 hepatocarcinoma tumor in strain two guinea pigs. The immunoconjugate retained the functions of the individual components, i.e., antigen binding to the intact cell in vitro and inhibition of its protein synthesis. This conjugate was a selective antineoplastic agent with a cytocidal dose at 5 X 10(-9) M toward antigen-bearing cells in vitro. Several antigen-negative cells were much less susceptible to its cytotoxic effect. The cytotoxicity of the conjugate appeared to be by antibody-mediated delivery of toxic A chain into the target cell. When cells were pretreated with excess free antibody followed by a brief exposure to conjugate, there was a reversal of the cytotoxicity to antigen-positive cells but not to the antigen-negative cells. The therapeutic efficacy of the conjugate was assayed by injecting a single dose s.c. or i.v. into syngeneic guinea pigs bearing established line 10 tumors. These in vivo studies showed that (a) the conjugate was not toxic at a dosage of 60 to 1120 micrograms/guinea pig, (b) the conjugate decreased or abolished the growth of established solid tumors, (c) the conjugate delayed or inhibited tumor metastases to lymph nodes, and (d) 20 to 40% of the animals in selective groups had a long-term complete regression. (+info)
Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.
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Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells. (+info)
Phase I study of the plant protein ricin.
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A Phase I study was carried out with ricin, a plant toxin acting by inhibiting protein synthesis, on 54 cancer patients with advanced disease. Ricin was given as i.v. bolus injections every two weeks at dose levels ranging from 4.5 to 23 micrograms/sq m of estimated body surface area. Ricin was well tolerated at doses up to 18 to 20 micrograms/sq m. At these levels and at higher levels, flu-like symptoms with fatigue and muscular pain appeared and, in some patients, nausea and vomiting occurred also. No myelo-suppression was seen. Antibodies to ricin were detected in serum after two to three ricin injections. Ricin was eliminated from blood according to first order kinetics. At each dose level, the plasma concentrations, as well as the side effects, showed only minor differences between patients. The highest dose given, 23 micrograms/sq m, gave plasma concentrations twice those found previously to be therapeutically effective in tumor-bearing mice. Of 38 evaluable patients, one patient with lymphoma had a partial response. Stable disease was observed in four patients with renal cancers, in two with soft tissue sarcomas, and in one patient each with mesothelioma, thyroid, and rectal cancer. A dose of 23 micrograms/sq m is recommended for Phase II trials of ricin. (+info)
Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. I. Requirement for calcium.
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In the absence of Ca2+ cells were not sensitive to the toxic proteins abrin and modeccin and the sensitivity to ricin and diphtheria toxin was reduced. Calcium deprivation had little effect on the binding and endocytosis of abrin, modeccin, and ricin. The binding of diphtheria toxin to cells was, however, reduced, Verapamil and Co2+ inhibited 45Ca2+ uptake and protected cells against abrin and modeccin at low concentrations of Ca2+. At higher Ca2+ concentrations the protection was overcome. La3+ inhibited strongly 45Ca2+ uptake and protected well against all four toxins, even at high Ca2+ concentrations. Fe3+ also afforded protection although it did not inhibit Ca2+ uptake. The Ca2+ ionophore, A23187, which strongly increases the uptake of 45Ca2+, protected cells well against abrin and modeccin, slightly against diphtheria toxin, but not against ricin. Both Ca2+ deprivation and treatment with A23187 protected well against the hybrid toxin abrin A-chain/ricin B-chain. Such treatment afforded little protection against the hybrid ricin A-chain/abrin B-chain. Apparently the protection against abrin is associated with its A-chain. The calmodulin inhibitor, trifluoperazine, protected strongly against modeccin and diphtheria toxin. The data indicate that Ca2+ is involved in the entry mechanism for abrin, modeccin, and ricin, possibly as a Ca2+ flux together with the toxins. (+info)
Inhibitory effects of four isoabrins on the growth of sarcoma 180 cells.
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The four isoabrins were shown to be capable of inhibiting the growth of tumor cells in vivo when one-fifth of their median lethal dose was used. From the in vitro experiments, the doses required for 50% inhibition of protein biosynthesis are 3.2 pg, 45 ng, 32 ng, and 10 ng/ml for abrin-a, -b, -3, and -d, respectively. Except for abrin-b, a good correlation between the inhibitory effects of abrins on the tumor growth and protein biosynthesis was observed. These isoabrins show a moderate inhibitory effect on DNA biosynthesis. (+info)
Induction of antitumor immunity by tumor cells treated with abrin.
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Abrin is known as a cytotoxic lectin. Immunization with Meth-A tumor cells which were treated in vitro with abrin induced a strong antitumor immunity in syngeneic BALB/c mice. The immunizing effect was stronger than that produced by an irradiated Meth-A tumor cell vaccine. Studies on the mechanisms of the immunizing effect with the abrin-treated tumor cells demonstrated that abrin acts as an immunoadjuvant. Furthermore, the regression of a growing Meth-A tumor was observed after abrin was injected into the tumor, while the induction of a strong antitumor immunity also occurred. It appears, therefore, that the antitumor effects of abrin are attributable to two kinds of activity: cytotoxicity and adjuvant activity. (+info)
Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. II. Effect of pH, metabolic inhibitors, and ionophores and evidence for toxin penetration from endocytotic vesicles.
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The toxicity of abrin, modeccin, and ricin to Vero cells was maximal at neutral and slightly alkaline pH, and it was strongly reduced at pH 6.0 and below. Diphtheria toxin was most toxic at low pH. Binding and endocytosis of abrin, modeccin, and ricin did not vary much within the pH range tested. High concentrations of the carboxylic ionophore Br-X-537A, protected against all four toxins. Combined treatment of cells with an inhibitor of glycolysis and an uncoupler of oxidative phosphorylation strongly inhibited endocytosis of toxins and protected against intoxication. The protective effect of Ca2+ deprivation, of pH 6.0, and of metabolic inhibitors disappeared soon after transfer of the cells to normal medium, whereas the protective effect of Br-X-537A and of trifluoperazine disappeared slowly. The decay rate of the protection by NH4Cl and by the ionophore A23187 differed with the different toxins. Cells exposed to abrin, modeccin, and ricin under protective conditions which did not inhibit endocytosis of the toxins (Ca2+ deprivation, pH 6.0, Br-X-537A), and then treated with antitoxins to inactivate extracellular toxin, were intoxicated when the protection was released. In contrast, cells exposed to toxins while endocytosis was arrested by treatment with metabolic inhibitors were not intoxicated when antitoxins were added and the metabolic inhibitors removed. Modeccin and diphtheria toxin endocytosed in the presence of trifluoperazine and NH4Cl were unable to intoxicate cells. The possibility that endocytosis is a step in the normal entry route of the toxins is discussed. (+info)
Abrogation of the non-specific toxicity of abrin conjugated to anti-lymphocyte globulin.
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A covalent conjugate of abrin and anti-human lymphocyte globulin (AHLG) was prepared in an endeavour to create a cytotoxic agent with specificity for human lymphoid cells. The AHLG--abrin conjugate was found to be around 10-fold better able to inhibit 3H-leucine uptake by the human lymphoblastoid cell line, Daudi, in tissue culture than was the control conjugate comprising abrin and normal IgG (nIgG). Both materials were less potent than native abrin. Galactose, which is known competitively to antagonize the binding of abrin to cells, strongly inhibited the toxicities of abrin and the nIgG--abrin conjugate whereas that of ALG--abrin was unimpaired. Thus, at least for Daudi cells in tissue culture, abrin can be made selectively toxic, by linkage to AHLG, towards cells bearing antigens to which the antibody moiety of the conjugate can attach. (+info)