Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals.

Llama-derived single domain antibodies specific for Abrus agglutinin.

Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations.

Abrin induced oxidative stress mediated DNA damage in human leukemic cells and its reversal by N-acetylcysteine.

Abrin is a plant glycoprotein toxin, classified as ribosome inactivating protein (RIP) due to its property of damaging ribosomes in an irreversible manner. Many RIPs have direct DNA damaging activity. The objective of the present study was to evaluate the oxidative stress and DNA damaging potential of abrin in U937 human myeloleukemic cells. Cells were treated with abrin at IC50 of 8 ng/ml for 24h. Abrin induced a time dependent increase in reactive oxygen species and levels of antioxidant enzymes. There was significant depletion of reduced glutathione levels. DNA damage was assessed by comet assay in terms of percent head DNA, tail DNA, tail length and Olive tail moment. DNA damage was more pronounced at 4 and 8h at IC50 concentration. Abrin at 4, 8, 16 and 32 ng/ml concentration induced significant DNA damage at 4h. There was time dependent increase in levels of 8-OHdG in abrin treated cells indicating the oxidative stress mediated DNA damage. N-Acetylcysteine pretreatment at 10nM for 1h, considerably reversed the abrin induced DNA damage at 16 and 32 ng/ml. Our results clearly show oxidative DNA damage potential of abrin at low concentration.

Protection against inhalation toxicity of ricin and abrin by immunisation.

1. Abrin and ricin are highly toxic plant proteins which are very similar in structure and function and inhibit protein synthesis in eukaryotes. 2. Rats have been immunised against either toxin using formaldehyde-toxoids by three subcutaneous injections at intervals of 3 weeks. For abrin, serum titres in 14 out of 15 rats were raised to between 1:12800 and 1:51200 after two injections, 6 weeks from the start of the experiment. Titres of between 1:256 and 1:1024 were also measured in lung washes after challenge with active abrin toxin. 3. The three major antibody classes, IgG, IgM and IgA were present in the immune sera but IgG and IgA only were detected in lung washes. The proportion of IgA to IgG was higher in the lung fluid than in sera. Rats immunised by abrin toxoid were protected against 5 LCt50's of abrin by inhalation but others exposed to ricin were not. 4. For ricin, serum titres ranged from 1:800 to 1:25600 after two injections and after a third injection the titre range was the same but population samples were weighted towards the higher titres. All rats immunised with ricin toxoid survived the challenge of 5 LCt50's of ricin toxin by inhalation over the observation period of 28 days post-challenge. 5. Representative immunised rats (abrin toxoid) were taken at various times post-exposure, humanely killed and tissues were examined for pathological changes. It was concluded that an apparently severe lung lesion occurred at a later time than in non-immunised, toxin challenged rats. This damage was not lethal over the experimental observation periods. 6. Immunisation by the sub-cutaneous route therefore protects against lethality from challenge by inhalation of ricin or abrin toxins but does not prevent significant lung damage.

Immunotoxins directed against the high-molecular-weight melanoma-associated antigen. Identification of potent antibody-toxin combinations.

To study factors influencing the cytotoxicity of immunotoxins (ITs), we compared the in vitro cytotoxicity of conjugates in which the plant toxin abrin and the bacterial toxin Pseudomonas exotoxin A (PE) were coupled by 2 different procedures to 2 MAbs, 9.2.27 and NR-ML-05, which bind to different epitopes on the melanoma-associated antigen p250. The individual target cell lines differed widely in sensitivity to the different ITs, as assessed by measurement of protein synthesis inhibition. The action of the ITs was highly specific, as the toxicity of abrin and PE conjugates was respectively 20-540 and 2,200-550,000 times higher in antigen-positive cell lines (FEMX, SESX, OHS) than in the antigen-negative line KPDX. The PE conjugates prepared with the 2 different MAbs differed in potency by factors of 16-126 in the target-cell lines, but were consistently more toxic than the abrin ITs. The results demonstrate that the cytotoxicity of ITs varies with the nature of both of its moieties and that optimal results require that toxins and MAbs be matched. Moreover, the 2 coupling procedures affected differentially the binding and potency of some ITs. Each of the 2 toxins was conjugated to a sheep anti-mouse antibody (SAM) and the toxicity of these 2 conjugates was tested in an indirect approach using 9.2.27 and NR-ML-05 as primary MAbs. The results showed that the indirect procedure would have correctly predicted the most potent antibody-toxin pair, indicating that the approach may be valid for selecting suitable combinations of MAbs and toxins for use as direct ITs.

In vitro and in vivo effects of a monoclonal antibody-toxin conjugate for use in autologous bone marrow transplantation for patients with breast cancer.

We have devised a method utilizing a monoclonal antibody-toxin conjugate (LICR-LON-Fib75/abrin A-chain) for ridding bone marrow of infiltrating breast cancer cells to rescue patients with autologous bone marrow following high dose therapy. Initially we examined the activity of this conjugate in vitro. Five of seven human breast cancer cell lines were killed following exposure at 10(-8) M for 2 h; this concentration only reduced bone marrow colony formation to 83% (range, 50-100%) of control bone marrow. We then examined the pattern of bone marrow recovery after high dose melphalan (200 mg/m2) in patients with advanced breast cancer who were in remission following combination chemotherapy. To do this we compared the time of recovery of the blood count in three patients who received treated marrow and seven who received untreated marrow. Mean time to recovery of the peripheral white count (greater than 1.5 X 10(9)/liter) was 16.7 days (treated) and 18.3 days (untreated), respectively. Mean time to recovery of peripheral platelet count (greater than 50 X 10(9)/liter) was 23.7 days (treated) and 18.9 days (untreated), respectively. Patients continued in remission for 1-greater than 14 mo after high dose melphalan, and remission duration was similar in patients who received treated (6.2 mo) and untreated (7.3 mo) bone marrow. These findings indicate that treatment of bone marrow with LICR-LON-Fib75/abrin A-chain conjugate does not significantly impair bone marrow recovery, and it is, therefore, possible to rescue breast cancer patients with bone marrow that has been cleansed of infiltrating cancer cells. This may have an application in patients with poor-risk primary breast cancer who have micrometastases and who may benefit from intensive therapy, but it has minimal application in patients with more advanced disease.

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.

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.

Delivery of ricin and abrin A-chains to human carcinoma cells in culture following covalent linkage to monoclonal antibody LICR-LOND-Fib 75.

With the object of generating specific cytotoxic agents, we have prepared covalent conjugates of the A-chains of ricin and of abrin with monoclonal antibody LICR-LOND-Fib 75 and investigated their toxicity toward a human tumor cell line in culture. Both conjugates proved to be potent cytotoxins toward cells carrying the appropriate antigen. The agent containing abrin A-chain was toxic at a significantly lower concentration and exerted its maximum effect more rapidly than the one containing ricin A-chain. Inclusion of chloroquine in the incubation medium significantly enhanced the toxic action of both conjugates without loss of immunospecificity. Because of the widespread occurrence on human tumor cell lines of the antigen recognized by Fib 75, these conjugates, particularly the one containing abrin A-chain, may find application in freeing human bone marrow ex vivo of infiltrated tumor cells prior to reinfusion as an autograft.

Antitumor effects of abrin and ricin used singly and in combination with cisplatin.

Abrin, ricin, and cisplatin produced significant increases in survival times of mice inoculated with 10(6) Ehrlich ascites carcinoma or L1210 leukemia cells 24 hours prior to treatment. Combinations of abrin or ricin with cisplatin produced markedly synergistic action in prolonging survival times of mice bearing cell line A of L1210 leukemia. For example, a dosage of 1.33 micrograms per kg abrin produced a 40 percent increased length of survival (ILS) and 2.5 mg per kg of cisplatin produced a 45 percent ILS while a combination of the two agents resulted in a 229 percent ILS and produced 60-day survivors or "cures" in 20 percent of the mice treated. Similar combinations of abrin or ricin with cisplatin also produced significant additive or synergistic increases in survival times of mice bearing cell line B of L1210 leukemia or Ehrlich ascites carcinoma. Aged solutions of abrin and ricin appeared to be less toxic, but have similar antitumor effect alone or in combination with cisplatin, than freshly prepared solutions.

Studies on the toxicity and binding kinetics of abrin in normal and Epstein Barr virus-transformed lymphocyte culture-I: experimental results - 1.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Activation, stimulation and relative toxicity factor indices are studied as well as possible relationships between the DNA and protein synthesis rates, as measured by simultaneous 3H-TdR and 14C-leucine uptake.

Studies on toxicity and binding kinetics of abrin in normal and Epstein Barr virus-transformed lymphocyte culture-I: experimental results - 2.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Studies of activation and stimulation indices, as well as two new indices; the metabolic self and cross-coupling indices, lead to the prediction that there are three morphologically distinct subpopulations of EBV-transformed lymphocytes with different abrin binding site numbers. This conclusion is supported by SEM morphological differences.