Antitumor effect of 5-aminolevulinic acid-mediated photodynamic therapy can be enhanced by the use of a low dose of photofrin in human tumor xenografts.

Practically all of the exogenous photosensitizers used for clinical photodynamic therapy (PDT) target mainly vasculature. Although effective in tumor destruction, they also, unavoidably, induce phototoxicity of normal tissues. Porphyrins synthesized endogenously from 5-aminolevulinic acid (ALA) accumulate within cells. Tumor eradication would be more efficient if both cellular components and vascular stroma of a tumor could be targeted. Thus, PDT with a mixture of ALA and Photofrin (Pf, a vessel-targeted sensitizer) may simultaneously destroy the two elements. Using chemical extraction assays, pharmacokinetics of ALA and ALA-induced porphyrins were studied in the plasma and tumors of nude mice bearing human WiDr and KM20L2 colonic carcinomas after an i.p. injection of 250 mg/kg body weight of ALA. Subsequently, PDT efficacy of the two tumor models with ALA, Pf, or with the two drugs in combination was evaluated. The phototoxic effects on tumor cells in vitro with the combined drugs was also determined. Moreover, histological and ultrastructural alterations of the treated tumors were investigated, and tumor cell clonogenicity was assessed as a function of time after in vivo PDT using an in vitro colony formation assay. Finally, the photosensitivity of normal skin tissue treated according to various protocols was compared. The amounts of ALA peaked at 0.5 h after administration in both plasma and WiDr tumor. The rates of ALA clearance seemed to follow a one-compartment model with half-lives of approximately 18 and 58 min in the plasma and tumor, respectively. About 100 and 60 times higher concentrations of ALA were needed to induce a given concentration of porphyrins in the plasma and tumor, respectively, although the plasma porphyrins may not only be released from blood cells but also from other organs. Similar kinetics of distribution patterns of ALA- and ALA methylester-induced porphyrins were found in the plasma and tumors, and the elimination rates were consistent with a two-compartment model. ALA induced much more porphyrins than ALA methylester in both plasma and tumors. Tumors PDT-treated with ALA plus Pf at a low dose (1 mg/kg) grew significantly more slowly than those treated with either of the drugs in both WiDr and KM20L2 models. However, the enhanced antitumor effect was not found in the tumor cells under in vitro conditions. Morphological studies demonstrated that PDT with the combined regimen resulted in necrosis of neoplastic cells and severe disruption of tumor microvasculature. This was supported by the findings obtained from the studies of in vivo PDT and in vitro clonogenic assay that showed a progressive reduction in tumor cell viability with times following PDT. Such a combined PDT protocol did not induce any phototoxicity in normal skin tissue. These data indicate that targeting both neoplastic cells and stroma with ALA and Pf (a low dose) can potentiate antitumor PDT effect with no risk of prolonged skin photosensitivity.

Determination of aluminium in liver from reindeer, moose and sheep by electrothermal atomic absorption spectrometry.

Aluminium in liver from reindeer, moose and sheep from the northeast part of Norway was determined by graphite furnace atomic absorption spectrometry following digestion of the samples with nitric acid. The concentration of aluminium in the liver was markedly higher for reindeer than for moose and sheep; the median values obtained were 0.56 microgram g-1 Al (wet wt.) for 101 reindeer, 0.06 microgram g-1 Al for 72 moose and 0.09 microgram g-1 Al for 40 sheep. The detection limit of the method was 0.01 microgram g-1 Al. The NIST SRM 1577a Bovine Liver was also analyzed.

In vitro and in vivo variation in transferrin receptor expression on a human medulloblastoma cell line.

The poor prognosis associated with pediatric central nervous system tumors such as medulloblastoma has led to the development and investigation of a variety of new treatment techniques. Therapeutic agents include targeted-toxin conjugates or immunotoxins that show significant in vitro activity against many brain tumors. Transferrin receptors (TRs) are specific, cell-surface antigens that are expressed preferentially on brain tumors rather than on normal human brain tissue. This antigen has been successfully targeted in human and nonhuman brain tumors in vitro and in vivo. In this study, when TRs were used as a target in the DAOY human medulloblastoma-derived cell line in vitro, a significant level of expression was confirmed by testing the sensitivity to different immunotoxins. To ensure the relevance of the in vitro data to the in vivo situation, we also analyzed TR expression in DAOY tumors growing in athymic mice and rats. Immunocytochemistry, immunohistochemistry, immunobead binding, immunofluorescence, 125iodine-transferrin binding, and Northern blot analysis were used to compare TR expression in DAOY cells in vitro and in vivo. All in vitro assays demonstrated significant TR expression, whereas in vivo, the TR expression was negligible in the DAOY tissue. The results caution against extrapolating in vitro antigen and receptor expression data directly to the in vivo situation. Using a transferrin-toxin conjugate in a nude rat model of leptomeningeal carcinomatosis, we achieved therapeutic efficacy, despite demonstrating reduced TR expression on tumor tissue. With respect to clinical efficacy, the reduced expression of TR on DAOY medulloblastoma in vivo may be less significant than expected because of the extreme potency of immunotoxins observed in central nervous system tumors.

Ruminant uptake of nickel and other elements from industrial air pollution in the Norwegian-Russian border area.

Concentrations of the elements aluminium, arsenic, cadmium, chromium, cobalt, copper, lead, mercury, nickel, selenium and zinc in liver, and of nickel in kidneys, were studied in reindeer, moose and sheep from South Varanger in eastern Finnmark and comparable districts in western Finnmark, Norway. The study included samples from 31 reindeer, 10 moose and 10 sheep from Jarfjord (South Varanger); 31 reindeer, 27 moose and 15 sheep from Pasvik (South Varanger); and 40 reindeer, 16 moose and 15 sheep from western Finnmark. Levels of arsenic, copper, nickel and selenium were much higher in reindeer from one or both areas in South Varanger than in reindeer from western Finnmark. Levels of chromium, cobalt and zinc were also significantly higher in South Varanger reindeer than in reindeer from the reference area. Within South Varanger the highest levels of these elements were invariably found in the Jarfjord area. For the other elements studied hepatic levels in South Varanger were similar to or lower than in western Finnmark. Also in moose, higher levels of nickel and of selenium (Jarfjord only) were found in the South Varanger samples than in samples from western Finnmark. In sheep, on the other hand, levels in South Varanger samples were similar to levels in western Finnmark for all the elements studied. Comparing the results with reports on pollution of air and vegetation, it was concluded that for all the elements showing higher levels in reindeer and moose from South Varanger compared to the reference areas, the effect most probably was a result of atmospheric transport of industrial pollution from the nearby Russian towns Nikel and Zapoljarnij. The geographical and interspecies differences within the South Varanger samples support this conclusion.

In vivo efficacy of intrathecal transferrin-Pseudomonas exotoxin A immunotoxin against LOX melanoma.

Neoplastic meningitis due to the dissemination of systemic cancer or primary central nervous system tumors through the cerebrospinal fluid carries a very poor prognosis. Current treatments for this disease are ineffective, and new therapeutic modalities such as immunotoxins may be beneficial. We created an animal model of human carcinomatous meningitis with LOX melanoma-derived tissue-culture cells in athymic rats for testing the efficacy of intrathecal therapy with transferrin-Pseudomonas exotoxin A (Tfn-PE) immunotoxin. An injection of 5 x 10(5) LOX cells into the intrathecal space through an indwelling catheter resulted in the reproducible development of lower-extremity paraplegia at 9.24 +/- 1.77 days because of focal deposits of tumor growth adjacent to the thoracic and lumbar spinal cord. A dose of 2.5 or 5 micrograms of intrathecal Tfn-PE immunotoxin was neurotoxic and resulted in the deaths of 8 of 10 animals within 24 hours. Histological evidence of central nervous system damage was seen as hemorrhagic degeneration around the central canal or a pathological cleft at the level of the cervical spinal cord. Because no neurotoxicity was seen with 1 microgram of intrathecal Tfn-PE immunotoxin, this dose was administered in treatment experiments. Twenty-four hours after the intrathecal instillation of LOX cells, 10 animals received intrathecally either 1 microgram of Tfn-PE or phosphate-buffered saline with 0.1% human serum albumin (control group).(ABSTRACT TRUNCATED AT 250 WORDS)

Targeted therapy with immunotoxins in a nude rat model for leptomeningeal growth of human small cell lung cancer.

Metastasis to the central nervous system in patients with small cell lung cancer is not uncommon, and a fraction of the cases have leptomeningeal disease for which no effective therapy is available. To establish an experimental model for evaluation of new therapeutic approaches for such tumor lesions, 1 x 10(6) human H-146 cells were injected directly into the cerebrospinal fluid in the cisterna magna of nude rats. Small, superficial leptomeningeal tumors developed, consistently resulting in symptoms of central nervous system involvement after a mean latency of 20 days. The model was used to study the efficacy of intrathecal targeted therapy with immunotoxins. The monoclonal anti-carcinoma antibodies MOC-31 and NrLu10 and the growth factor transferrin were conjugated to Pseudomonas exotoxin A (PE), and 1 day after tumor cell inoculation instilled in the cisterna magna as a single bolus dose of 1.5 micrograms. The antibody conjugates, which were highly cytotoxic to target cells in a protein synthesis inhibition assay in vitro, increased the symptom-free latency by 35-46%. PE had no effect, reflecting a lower in vitro cytotoxicity and possibly also a down-regulation of transferrin-receptor expression in the meningeal H-146 tumors. Delayed or repeated treatment with MOC-31-PE was less effective than day 1 administration, whereas the addition of 10% glycerol to the injection solution increased the symptom-free period to 72%. The efficacy of MOC-31-PE is superior to reported effects obtained in similar models with other therapies, and the results support the development of this immunotoxin towards clinical evaluation in small cell lung cancer patients with leptomeningeal carcinomatosis.

Comparison of two antibody-based methods for elimination of breast cancer cells from human bone marrow.

Three monoclonal antibodies reactive with antigens abundantly expressed on human carcinoma cells were used to develop and compare the efficacy of immunotoxins (ITs) and immunobeads for purging breast cancer cells from bone marrow. ITs constructed as conjugates of the monoclonal antibodies and Pseudomonas exotoxin A showed high specific cytotoxicity against three breast cancer cell lines, inhibiting protein synthesis by 50% at concentrations of 4 x 10(-13) M to 1 x 10(-10) M. Tested in a reproducible clonogenic assay, two of the ITs used at a concentration of 0.1 microgram/ml killed > 5 log units of MCF7 cells, the maximal sensitivity for assessing cytotoxic effects, and 1.5 log of T-47D tumor cells. At 1 microgram/ml, each of the three ITs eliminated > 5 log of both cell lines. The immunobead procedure removed 2.0-4.1 log of tumor cells with one purging cycle and up to 6.0 log with two cycles. The mixture of the three ITs or immunobeads was not clearly superior in efficacy, compared to the use of individual molecules, probably reflecting an overlap in expression of the respective antigens in these cell lines. For both methods, the purging efficacy was not reduced when the tumor cells were admixed with normal bone marrow cells at a ratio of 1:10. The survival of colony-forming units, granulocyte/macrophage, was 49-86% with the immunobeads and 44-75% even at high concentrations (up to 2.5 micrograms/ml x 3) of the ITs. The results indicate that each of the two immunological methods can be safely used for effective elimination of tumor cells from the graft of breast cancer patients undergoing autologous bone marrow transplantation.

Eradication of small cell lung cancer cells from human bone marrow with immunotoxins.

The potential of autologous bone marrow transplantation to improve the treatment results for patients with small cell lung cancer (SCLC) may be limited by the presence of tumor cells in the graft. We constructed immunotoxins (ITs) involving 4 monoclonal antibodies and Pseudomonas exotoxin A and investigated the cytotoxicity of the ITs to H-146 SCLC cells in the presence and absence of normal human bone marrow (BM) cells. The Pseudomonas exotoxin A conjugate with the MOC-1 antibody, which recognizes an NCAM antigen, was inactive, as tested in a reproducible soft agar assay. Conjugates involving the monoclonal antibodies MOC-31, NrLu10, and MLuC1 killed about 3.5 log tumor cells at 0.1 microgram/ml and > 5.0 log at 1 microgram/ml. In the absence of BM cells, the combination of the 3 ITs was not superior to each IT used individually. However, when H-146 cells were admixed to nucleated BM cells at the ratio of 1:10, > 5 log tumor cell kill was obtained at a concentration as low as 0.1 microgram/ml of each IT. Survival of normal BM progenitor cells was only moderately reduced by the IT treatment, even in experiments in which the 3 IT3s were used at 2.5 micrograms/ml each. Freezing and thawing of the BM, as required in a clinical setting, reduced the colony-forming unit, granulocyte-macrophage, and colony-forming unit, granulocyte-erythroid-macrophage-megakaryocyte, by 30-60% in both treated and untreated cultures. We conclude that the use of a mixture of the 3 ITs provides a safe, rapid, and effective method for eradicating SCLC cells from BM used for autologous bone marrow transplantation following high-dose chemotherapy.

In vitro antiproliferative effect of interferon alpha in solid tumors: a potential predictive test.

An in vitro test for the antiproliferative effect of human leukocyte interferon (IFN-alpha) was performed in primary cultures of tumor cells obtained from 32 patients with either malignant melanoma (13), renal carcinoma (4) or bladder carcinoma (15). Our results demonstrated activity of IFN in all three groups of solid tumors. However, appreciable differences in sensitivity to antiproliferative effect of IFN between individual tumors of the same type were found. The potential of this antiproliferative test for prediction of treatment response in IFN-therapy is discussed.

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 efficacy of transferrin-toxin conjugates against glioblastoma multiforme.

The cytotoxic activity of immunotoxins constructed with human diferric transferrin (Tfn) as the carrier ligand and an abrin variant Pseudomonas exotoxin A (PE) and the diphtheria toxin mutant cross-reacting material (CRM) 107 as the toxin moieties were studied in vitro. Three malignant human cell lines, the glioblastomas multiforme SNB19 and SF295 and the LOX melanoma, and a nonhuman control murine melanoma cell line B16 were assessed. The presence of transferrin receptors on the cell lines was confirmed by direct 125I-Tfn binding assays. The 50% protein synthesis inhibitory concentration (IC50) values for all cell lines demonstrated that Tfn-abrin variant and Tfn-PE had comparable potency and were both more effective than Tfn-CRM 107. Monensin, a carboxylic ionophore, potentiated the effect of Tfn-abrin variant against glioma cells approximately 35-fold with IC50 values of 4.0 x 10(-13) M and 4.7 x 10(-12) M for SNB19 and SF295, respectively. Cytotoxic activity of Tfn-abrin variant (with or without monensin) and Tfn-PE was correlated with the degree of Tfn receptor expression measured on the cell lines. The exquisite in vitro cytotoxicity of Tfn-abrin variant and Tfn-PE immunotoxins against glioma and melanoma cells warrants further in vivo evaluation and future consideration of these agents for potential clinical application against glioblastoma multiforme and leptomeningeal neoplasia.

Kinetics of uptake and degradation of an abrin immunotoxin by melanoma cells and studies of the rates of cellular intoxication.

The mechanism of action of an abrin 9.2.27 antimelanoma antibody conjugate has been studied in 2 human melanoma cell lines, FEMX and LOX, which differ in sensitivity to the immunotoxin (IT) and to native abrin. The IT, which had been affinity purified before use to remove molecules with exposed gal-binding sites on the toxin B-chain, inhibited cellular protein synthesis at a faster rate in the LOX than in the FEMX cells despite the fact that the LOX cells express less specific antigen and bind less IT to the cell surface. Surface-bound abrin-IT, as well as surface-bound specific antibody, disappeared at equal rates from the cell surface of the 2 cell lines. After binding of labelled IT the disappearance of total cell-associated radioactivity, as well as the appearance of TCA-precipitable and TCA-soluble radioactivity in the incubation medium, occurred at faster rates in the LOX than in the FEMX cells. No free abrin or antibody B-chain complex could be detected in the medium or inside the cells. The results indicate that the different sensitivities of the melanoma cell lines reflect different abilities to process endocytosed IT and to translocate the active A-chain to the cytosol. Experiments carried out in the presence of lactose are interpreted to mean that the A-chain may be translocated to the cytosol by two mechanisms, one involving antigen-antibody interaction and one involving the B-chain, and that the lectin binding site contributes to the B-chain-facilitated mechanism.

New indirect approach to the therapeutic use of immunotoxins.

To improve the applicability of immunotoxins (ITs), we have developed a new two-step indirect procedure. The target cells to be killed are first incubated with cell-specific mouse monoclonal antibodies (MAbs). After removal of excess unbound antibody, the cells are incubated in the presence of lactose with an indirect IT, a conjugate of whole abrin and sheep anti-mouse immunoglobulin (SAM) that binds only to cells having primary mouse MAbs on their surfaces. The SAM-abrin IT is affinity purified before use to remove molecules with exposed B-chain-binding sites; it was nontoxic in the absence of the specific mouse MAbs, demonstrating the specificity of the two-step method. We compared the indirect approach, using four different primary MAbs, with the conventional method, in which abrin is coupled directly to the mouse MAbs. In three human cell lines--the melanoma line FEMX, the Burkitt cell line Rael, and the leukemia cell line KM3--the cell kill, measured by a clonogenic assay, was consistently greater with the indirect than with the direct method. In the melanoma and Rael cells, the indirect method gave a higher cell kill than even native abrin. With a mixture of two different antibodies an additive effect was observed with the indirect but not with the direct method. The new approach greatly simplifies the therapeutic application in vitro of ITs, because it permits the use of different primary antibodies, singly or in mixtures, in conjunction with only one or a few general indirect ITs. In efforts to further improve the usefulness of the indirect method, other indirect ITs containing different toxin moieties are being examined. The possibility of employing the indirect principle in vivo is being explored.

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.

Human melanoma cell lines showing striking inherent differences in sensitivity to immunotoxins containing holotoxins.

The in vitro sensitivity of human melanoma cell lines to conjugates of whole abrin or ricin linked through disulfide bonds to the monoclonal antimelanoma antibody 9.2.27 was studied. After passage of the conjugates through a Sepharose 4B column to remove molecular species with exposed binding sites on their B-chains, toxicity of the conjugates to different melanoma cell lines and nonmelanoma tumor lines was assessed by measuring their ability to inhibit cellular protein synthesis. The abrin conjugate was far more toxic to the target cells than the corresponding ricin conjugate. The 8 melanoma cell lines studied differed widely in their sensitivities to the abrin conjugate. The differences were associated with concomitant large differences in the sensitivities of the cells to the native toxins, and the significance of the level of the antigen expression became apparent only when the sensitivities of the different cell lines were normalized with respect to their sensitivity to native abrin. The observed relationship could not be accounted for by unspecific binding via the B-chain binding site of the immunotoxin. The differential sensitivity of the melanoma cell lines to the immunotoxin seems to be related to inherent differences between the cells in their ability to internalize and to process immunotoxins and toxins. The findings may have considerable practical implications.

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.

Pharmacological studies of ricin in mice and humans.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for determination of ricin in serum is presented. Using this method it was found that IV-injected ricin disappeared from the plasma of mice and cancer patients according to first-order kinetics. DBA mice were found to be more sensitive to ricin than C3H and B6D2 mice. When mice of the different strains were given the same dose of ricin, the concentrations found in liver, spleen, and kidneys were highest in the most sensitive mice. Ricin disappeared most rapidly from serum of the mice with the highest sensitivity. The inverse correlation between the rate of disappearance of ricin from serum and the tissue concentrations reached may be due to the fact that ricin is rapidly and firmly bound to cell surface receptors. Whole-body autoradiography after IV injection of 125I-labeled ricin showed the highest amount of radioactivity in liver, spleen, and adrenal cortex. Considerable amounts of radioactivity were also present in bone marrow, showing that the lack of myelosuppressive activity of ricin previously found in mice and dogs cannot be accounted for by the failure of ricin to reach the bone marrow. Part of the ricin in the tissues was present in the form of free chains, the highest fraction being present in the liver. In this tissue both the free A-chains and those present in whole ricin were found to be modified. However, the modified A-chains had retained their full capacity to inhibit protein synthesis in vitro. In cancer patients, toxicity appeared at about the same initial serum levels as in the mice, supporting the view that mouse data have a good predictive value for man. At each dose level the individual variations were modest, a finding that is important for eventual clinical use of this potent drug.

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.