Inhibitory effect of Abrus abrin-derived peptide fraction against Dalton's lymphoma ascites model.

Peptides derived from larger molecules that are important modulators in cancer regression are becoming leads for development of therapeutic drugs. It has been reported that Abrus abrin, isolated from the seeds of Abrus precatorius, showed in vitro and in vivo antitumor properties by the induction of apoptosis. The present study was designed to evaluate the in vivo therapeutic effectiveness of abrin-derived peptide (ABP) fraction in Dalton's lymphoma (DL) mice model. The lethal dose (LD(50)) of ABP was found to be 2.25 mg/kg body weight and further the acute toxicity was determined with sublethal doses in normal mice. The acute toxicity like body weight, peripheral blood cell count, lympho-hematological and biochemical parameters remained unaffected till 200 microg/kg body weight of ABP. The sublethal doses of ABP showed very significant growth inhibitory properties in vivo DL mice model. There were 24%, 70.8% and 89.7% reductions in DL cell survival in 25, 50 and 100 microg/kg body weight of ABP, respectively. Analysis of the growth inhibitory mechanism in DL cells revealed nuclear fragmentation, and condensation with the appearance of the sub-G(0)/G(1) peak is indicative of apoptosis. Further, the Western blotting showed that apoptosis was mediated by the reduction in the ratio of Bcl-2 and Bax protein expression, and activation of caspase-3 through the release of cytochrome c in DL cells. Kaplan-Meier survival analysis showed an effective antitumor response (104.6 increase in life span (ILS) %) with a dose of 100 microg/kg body weight.

Antitumour effect of abrin on transplanted tumours in mice.

Abrin, a galactose specific lectin was purified using sepharose 4B affinity column from seeds of Abrus precatorius. It exhibited antitumour activity in mice when used at a sublethal dose of 7.5 micrograms/kg every alternate day for 10 days. Both intralesional and intraperiloneal (i.p.) administration of abrin was effective in reducing solid tumour mass development induced by Dalton's Lymphoma Ascites (DLA) and Ehrlich's Ascites Carcinoma (EAC) cells. DLA cell line was more sensitive to abrin than EAC. Abrin when injected i.p. increased the life span of ascites tumour bearing mice. Abrin when used simultaneously with tumour cells brought about maximum antitumour effect. On developed tumour masses, abrin administration brought about significant reduction in tumour volume, especially in DLA induced tumours. Prophylactic administration of abrin was found ineffective.

Molecular and biological properties of an abrin A chain immunotoxin designed for therapy of human small cell lung cancer.

An immunotoxin (IT) comprising abrin A chain attached to the mouse monoclonal antibody SWA11, recognising a cell surface antigen highly associated with human small cell lung cancer (SCLC), was synthesised using a hindered disulphide crosslinker, N-succinimidyl 3-(2-pyridyldithio) butyrate (SPDB), and purified by Blue Sepharose CL-6B affinity chromatography. The IT preparation contained monomeric conjugate, composed of one abrin A chain molecule linked to one SWA11 molecule, and was free from unconjugated A chain or antibody. The IT fully retained the cell-binding capacity of the antibody component and the ribosome-inactivating activity of the A chain. In cytotoxicity assays using the SW2 SCLC cell line in tissue culture, SWA11-SPDB-abrin A chain inhibited the incorporation of 3H-leucine by 50% at a concentration of 10 pM and by 99% at a concentration of 1 nM. The anti-tumour efficacy of the IT was tested in nude mice bearing established s.c. solid SW2 tumour xenografts. A single i.v. injection of SWA11-SPDB-abrin A chain at a non-toxic dose induced a significant 7 to 10 day growth delay that could not be matched by administration of equivalent doses of either unconjugated SWA11 or abrin A chain alone. The results of this study indicate that the antigen recognised by SWA11 is an effective target for therapy of SCLC with A chain ITs in vivo.

Pharmacokinetics in the rat of a panel of immunotoxins made with abrin A chain, ricin A chain, gelonin, and momordin.

A panel of immunotoxins was constructed by chemically attaching the ribosome-inactivating proteins abrin A chain, ricin A chain, gelonin, and momordin to the monoclonal mouse IgG2a antibody Fib75 by means of a disulfide linkage. All the immunotoxins were toxic in tissue culture to the EJ human bladder carcinoma cell line expressing the antigen recognized by Fib75, inhibiting the incorporation of [3H]leucine by 50% at concentrations between 1 x 10(-10) M and 8 x 10(-10) M. The pharmacokinetics of the immunotoxins in the normal Wistar rat was determined following i.v. administration. The concentrations of intact immunotoxin in serum samples taken at various intervals after injection for up to 24 h were measured by solid-phase enzyme-linked immunosorbent assays specific for each of the four different ribosome-inactivating proteins. The Fib75 immunotoxins were cleared from the circulation with comparable, but not identical, biphasic kinetics best described by a two compartment open pharmacokinetic model. The alpha-phase half-lives of the panel, between 0.35 and 0.71 h, were similar. The beta-phase half-life of Fib75-abrin A chain, 13.3 h, was significantly longer than the beta-phase half-lives of Fib75-ricin A chain, Fib75-gelonin, and Fib75-momordin, between 7.5 and 8.6 h. Fib75-abrin A chain was found to be about 3- to 4-fold more resistant than the other immunotoxins to breakdown by reduction of the disulfide linkage between the A chain and the antibody with glutathione in vitro. This suggests that the longer serum half-life of Fib75-abrin A chain may have been due to greater stability against reduction in vivo. Analysis of serum samples obtained up to 24 h after injection of Fib75-abrin A chain revealed that the chemically intact immunotoxin present in the circulation retained full cytotoxic activity. An abrin A chain immunotoxin made with a different monoclonal mouse IgG2a antibody was also found to be more stable against reduction by glutathione in vitro than an analogous ricin A chain immunotoxin. Thus, abrin A chain may posses unique molecular properties that endow immunotoxins made with this A chain with greater stability in vivo than immunotoxins made with ricin A chain or other ribosome-inactivating proteins.

Effect of immunotoxins against Trypanosoma cruzi.

Immunotoxins were constructed with IgG antibodies against Trypanosoma cruzi surface antigens by hybridization with abrin (ITA) and ricin (ITR) A chains. The biological activity of the hybrid macromolecules was tested on the parasite forms. Motility of parasite forms was lost in vitro after incubation with ITR. In general, killing of the parasite with ITR was more efficient than with ITA. Inhibition of protein synthesis after incubation with either ITR or ITA, measured by 3H-leucine incorporation, confirmed the parasite immobilization experiments. The lethal effect was potentiated when the immunotoxins were used in the presence of 2.5 mM ammonium chloride. T. cruzi antibodies specific to cell surface antigens are excellent drug carriers that can be delivered to the target cell. However, ITR and ITA did not reduce parasitemia or increase survival of mice infected with T. cruzi.

Studies on the mechanism of action of abrin-9.2.27 immunotoxin in human melanoma cell lines.

Previously we have shown (Godal, A. et al., J. Natl. Cancer Inst., 77: 1247-1253, 1986) that the sensitivities of different melanoma cell lines to a conjugate of abrin with the anti-melanoma antibody 9.2.27 was correlated with their sensitivities to native abrin. To elucidate the underlying mechanism we have compared the binding and toxicity of the conjugate and of native abrin to two melanoma cell lines, FEMX and LOX, which differ in sensitivity to abrin. Abrin was linked by a disulfide bond to the monoclonal antibody 9.2.27, and the conjugate was purified by affinity chromatography to remove molecules with exposed galactose-binding sites on the toxin B-chain. Lactose had no effect on the binding of the immunotoxin (IT) to the cells but nevertheless reduced strongly the toxicity to the LOX cells. The differences in sensitivity to native abrin were much larger than the concurrent differences in binding. Lactose reduced the toxicity of abrin to a far greater extent than the associated reduction in binding to the cell surface. The toxicity of the immunotoxin to the FEMX cells could be prevented by pretreatment with excess 9.2.27 antibody, whereas the more abrin-sensitive LOX cells were protected only to a limited extent. Concurrent treatment of the LOX cells with antibody and lactose acted synergistically and afforded complete protection. It is suggested that the protective effect of lactose against the IT was exerted after internalization into vesicles of IT bound unspecifically to the cell surface and that the toxic moiety of the IT, the abrin A-chain, may be translocated from endocytotic vesicles to the cytosol by two alternative mechanisms, one mediated by the antibody and a second one facilitated by the B-chain and its lectin binding site. The relative significance of these mechanisms seems to differ in different target cell lines depending on their inherent sensitivities to native abrin which in turn largely reflects the ability of the cells to internalize and process surface-bound abrin.

Comparison of two anti-Thy 1.1-abrin A-chain immunotoxins prepared with different cross-linking agents: antitumor effects, in vivo fate, and tumor cell mutants.

The A-chain of the plant toxin abrin was covalently linked to monoclonal anti-Thy 1.1 antibody (OX7) with the use of either N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane hydrochloride (2IT). The SPDP reagent generates a linkage containing a disulfide bond and an amide bond, whereas the 2IT reagent generates a linkage containing a disulfide bond and an amidinium bond. The two immunotoxins were powerfully and specifically toxic to Thy 1.1-expressing murine AKR-A lymphoma cells in vitro. Both reduced the rate of protein synthesis of the cells by 50% at a concentration of 10(-11) M. However, clonogenic assays revealed that about 1% of the AKR-A cells survived treatment with high concentrations of OX7-SPDP-abrin A, whereas only about 0.1% survived treatment with similar concentrations of OX7-2IT-abrin A. Several clones of the surviving cells were isolated. Of 11 clones of cells that had survived exposure to OX7-SPDP-abrin A, 10 were resistant to further treatment with OX7-SPDP-abrin A but had normal sensitivity to OX7-2IT-abrin A. These clones expressed moderate to high levels of the Thy 1.1 antigen and were fully sensitive to abrin. In contrast, all 10 clones of cells that had survived exposure to OX7-2IT-abrin A were substantially or entirely resistant to both immunotoxins. They expressed low to high levels of the Thy 1.1 antigen and were fully sensitive to abrin. The 2IT-linked immunotoxin was much more effective than the SPDP-linked immunotoxin at protecting nu/nu mice against the growth of AKR-A lymphoma cells in the peritoneal site. A single iv injection of 0.3 nmol OX7-2IT-abrin A eradicated at least 99.99% of the tumor cells, as judged from the extension in the median survival time of the animals, whereas OX7-SPDP-abrin A eradicated only about 99% of the cells. The tumors that developed in the animals that received OX7-2IT-abrin A were Thy 1.1-negative, whereas those in the recipients of OX7-SPDP-abrin A generally expressed normal levels of the Thy 1.1 antigen. The difference in antitumor activity of the immunotoxins was not due to differences in their in vivo fate, inasmuch as they were cleared from the bloodstream at an identical rate and broke down at the same rate to release free antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Phase I study of the plant protein ricin.

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.

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.

Antibody formation against the cytotoxic proteins abrin and ricin in humans and mice.

Antibody formation may limit the therapeutic use of cancerostatic proteins. To study the significance of antibody formation against abrin and ricin, highly sensitive ELISA procedures for determination of anti-abrin and anti-ricin were developed. In mice treated weekly with therapeutic doses of ricin, antibodies appeared after 2-3 weeks and then rose rapidly, whereas after abrin treatment the antibody formation was slower. Ricin A-chain was found to be more immunogenic than either intact ricin or human serum albumin (HSA). Cyclophosphamide inhibited the antibody response to both abrin and ricin and a combination of cyclophosphamide and prednisolone totally inhibited both anti-abrin and anti-ricin formation during the 6-week observation period. In mice treated weekly with HSA, abrin treatment strongly reduced the anti-HSA formation, showing that abrin has an immunosuppressive effect which appeared to be stronger than that of cyclophosphamide. The existence of circulating antigen-antibody complexes could be demonstrated in the sera of toxin-treated mice by precipitation with polyethyleneglycol, whenever antibodies were detectable with ELISA. The life-span of animals given lethal ricin doses was appreciably enhanced in animals having antibody levels in excess of 10-20 ng/ml. In cancer patients treated i.v. every second week with therapeutic toxin doses, the 10-20 ng/ml levels of anti-ricin and anti-abrin were reached 6-8 weeks and 7-10 weeks after the first injection of ricin and abrin, respectively. The data indicate that the effective therapeutic use of abrin and ricin as single agents may be limited to these time frames, but that the period of effective use may be substantially prolonged if the toxins are given together with conventional cytostatic agents having immuno-suppressive activity.

Inhibitory effects of four isoabrins on the growth of sarcoma 180 cells.

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.

Comparison of two soft-agar methods for assaying chemosensitivity of human tumours in vitro: malignant melanomas.

Two soft-agar methods for assaying chemosensitivity of human cancers in vitro were compared with respect to colony morphology, plating efficiency (PE) and chemosensitivity of human melanomas. In 9 xenografts and 9 patients' biopsy specimens Method A (essentially that of Courtenay & Mills, 1978) gave considerably higher PE that Method B (essentially that of Hamburger & Salmon, 1977) and, in contrast to Method B, the number of colonies was proportional to the number of cells plated. Evidence was obtained that the observed differences in PE could be attributed to the low O2 concentration and the presence of rat red blood cells in Method A. Colony morphology was similar in the 2 assays. When cells from 4 xenografted melanomas were treated in vitro with DTIC, CCNU, vinblastine and abrin, and the inhibition of colony formation was assayed concurrently in the 2 soft-agar methods, the tumour cells appeared to be more sensitive to 3 of the drugs in Method B than in A. The results demonstrate that chemosensitivity data obtained with the 2 assays cannot be directly compared.

Lectin derivatives of methotrexate and chlorambucil as chemotherapeutic agents.

Methotrexate and chlorambucil, each covalently linked to either abrus agglutinin, abrin, ricinus agglutinin, ricin, or concanavalin A, were prepared. A single dose of the derivative injected ip into sarcoma 180-bearing noninbred N:NIH(S) white mice resulted in prolongation of the survival time and was more effective than an equivalent dose of free drug and lectin. Drug-lectin also showed a higher inhibitory effect on the DNA biosynthesis of the tumor cell than did an equivalent dose of the free drug and lectin.

In vitro sensitivity of human melanoma xenografts to cytotoxic drugs. Correlation with in vivo chemosensitivity.

Single-cell suspensions prepared from five human melanomas, grown serially as xenografts in athymic nude mice, were exposed in vitro to increasing concentrations of DTIC (Dacarbazine), CCNU (Lomustine), procarbazine, vinblastine, and the cancerostatic lectins abrin and ricin. The in vitro chemosensitivity of the cells, as measured by the drug concentrations required to inhibit colony formation in soft agar by 50%, was correlated with the growth delay of the xenografts in vivo, previously observed after treatment of the animals with maximum tolerable doses of the same drugs. It was found that for each drug the in vitro sensitivity of the different xenografts was strongly correlated with their response in vivo. The results provide evidence that the soft agar test, as carried out here, adequately reflects the relative sensitivity of the xenografts in vivo. The data indicate that human xenografts may be used to develop quantitative in vitro chemosensitivity tests.

Response to chemotherapy of human, malignant melanoma xenografts in athymic, nude mice.

In attempts to elucidate the factors determining individual differences in response of tumors to chemotherapeutic agents, the sensitivity of six human malignant melanomas growing in athymic, nude mice was studied. Six agents, viz. DTIC, CCNU, vinblastine, procarbazine, as well as the toxic lectins abrin and ricin, were administered in maximum tolerable doses to the tumor-bearing mice. The response to treatment was expressed as tumor growth delay, i.e. the number of volume double times saved by the treatment. The xenografts had very nearly retined the morphology of the parent tumors and were histologically similar. They showed different and characteristic early growth rates and also wide variations in their response to the agents tested. Unexpectedly, in the case of DTIC, CCNU and procarbazine, the response of the xenografts proved to be inversely related to the early growth rates of the tumors. For vinblastine, abrin and ricin, no correlation between response and growth rate of the tumors was apparent. Procarbazine had the best overall effect among the agents here tested. DTIC, CCNU and vinblastine had somewhat less and about equal overall efficiency. The plant toxin abrin, which acts by inhibiting protein synthesis, was at least as effective as DTIC. When abrin was given together with DTIC, the effect on the two xenografts tested was superior to that of each agent given alone. The wide variations observed in the response of the histologically similar xenografts to the different agents demonstrate that testing of the chemosensitivity of human xenografts requires the use of a panel of tumors of each histological tpye. The clear relationship found between the early growth rate of the xenografts and their sensitivity to three of the drugs most commonly used in the treatment of malignant melanomas, may have clinical implications.

Treatment of micrometastases from Lewis lung carcinoma with abrin and cyclophosphamide, given singly and in combination.

The effect of the plant toxin abrin and of cyclophosphamide given as adjuvant chemotherapy after irradiation of the primary tumor was studied in mice bearing intramuscularly growing Lewis lung carcinoma. The chemotherapy was given immediately after irradiation performed 9 days after inoculation of 10(5) tumor cells. The drugs were given in bolus injections either as single agents or concurrently. The therapeutic effect was assessed by recording the appearance of lung metastases on day 21 or the number of long-term survivors, i.e. animals alive at day 60. In the control group 21 +/- 3.4 (SD) macroscopic lung colonies was recorded on day 21. Mean survival time was 25.5 days with none of the mice alive at day 60. The optimal abrin dose (612.5 ng/kg IV) reduced the number of lung metastases to 2.6 +/- 0.8 and yielded 37% long-term survivors (11/30). The optimal dose of cyclophosphamide (150 mg/kg IP) reduced the number of lung colonies to less than 1 and yielded 61% survivors (14/23). Small to moderate doses of abrin significantly potentiated the therapeutic effect of cyclophosphamide without increasing the toxicity. The best results (18/20 or 90% long-term survivors) were obtained when the optimal dose of cyclophosphamide, 150 mg/kg, was combined with 350 ng/kg of abrin. The results obtained in this highly resistant tumor suggest that combinations of the two drugs may be useful also in other tumor types.

Effect of ricin and abrin on survival of L1210 leukemic mice and on leukemic and normal bone-marrow cells.

The effect of ricin and abrin on the survival of mice treated with L1210 leukemic cells intraperitoneally or intravenously was studied. In mice given 1 X 10(5) L1210 leukemia cells intraperitoneally a single dose of ricin (2.1 microgram/kg) intraperitoneally gave the best results, an increased life span (ILS) of 59%. Abrin also increased the life span of such animals although to a lesser extent. The effect of ricin was superior to that of 5-fluorouracil, but inferior to that of adriamycin, which gave a maximum ILS of 280%. In mice given L1210 cells intravenously no increase in life span was obtained with ricin, abrin or adiramycin, whereas 5-fluorouracil gave an ILS of 40-50%. In spleen colony assays the differential effect of ricin and abrin on the proliferative capacity of normal hematopoietic and leukemic colony-forming cells in bone marrow was studied. The differential effect of ricin was as good as that of adriamycin and considerably better than that of 5-fluorouracil. Abrin had a much smaller effect than ricin on both normal and leukemic cells. The effect of abrin on the leukemic cells was too small to be of therapeutic value. The results warrant exploration of the use of ricin in the treatment of human leukemia.