Biomedical subjects

D J FitzGerald

Publications and source records attributed to D J FitzGerald.

At least 91 records · Page 5Linked to original sources

PubMed1989

Cardiac allograft survival in mice treated with IL-2-PE40.

IL-2-PE40 is a chimeric protein composed of human interleukin 2 (IL-2) genetically fused to the amino terminus of a modified form of Pseudomonas exotoxin lacking its cell recognition domain. IL-2-PE40, which is extremely cytotoxic to IL-2 receptor-positive cells, was examined for its ability to prevent graft rejection in mice in which activation of T cells is prominent. We demonstrate that intraperitoneally administered IL-2-PE40 specifically and significantly prolongs the survival of vascularized heart allografts in mice. The chimeric toxin, IL-2-PE40, offers an alternative approach to the treatment of autoimmune diseases and transplant rejection in humans.

ADP Ribose Transferases↗

PubMed1988

Interleukin 2 (IL2) PE40 is cytotoxic to cells displaying either the p55 or p70 subunit of the IL2 receptor.

IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a modified form of pseudomonas exotoxin (PE). Internalization of IL2 via the individual p55 and p70 subunits of the IL2 receptor was studied using IL2-PE40 on several mouse and human cell lines expressing either the p55, the p70, or both IL2 receptor subunits. Internalization was assessed by measuring inhibition of protein synthesis caused by the toxin moiety of IL2-PE40. The results demonstrate that IL2 internalization is mediated by either the p55 receptor subunit or by the p70 subunit but is much more efficient when high affinity receptors composed of both subunits are present. IL2-PE40 is a powerful reagent for studying IL2 receptor interactions and for analyzing pathways of the immune response and its regulation.

ADP Ribose Transferases↗

PubMed1988

Selective killing of HIV-infected cells by recombinant human CD4-Pseudomonas exotoxin hybrid protein.

It is projected that in the absence of effective therapy, most individuals infected with human immunodeficiency virus (HIV) will develop acquired immune deficiency syndrome (AIDS) and ultimately succumb to a combination of opportunistic microbial infections, malignancies and direct pathogenic effects of the virus. Anti-viral agents, immunomodulators, and inhibitors of specific HIV functions are being tested as potential treatments to alleviate the high morbidity and mortality. An alternative therapeutic concept involves the development of cytotoxic agents that are targeted to kill HIV-infected cells. Here we describe the purification and characterization of a recombinant protein produced in Escherichia coli that contains the HIV-binding portion of the human CD4 molecule linked to active regions of Pseudomonas exotoxin A. This hybrid protein displays selective toxicity toward cells expressing the HIV envelope glycoprotein and thus represents a promising novel therapeutic agent for the treatment of AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Mutational analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas exotoxin which reduce cell binding and animal toxicity.

Pseudomonas exotoxin (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three structural domains. Domain I is responsible for cell recognition, II for translocation of PE across membranes and III for ADP ribosylation of elongation factor 2. Treatment of PE with reagents that react with lysine residues has been shown to lead to a reduction in cytotoxic activity apparently due to a modification of domain I (Pirker, R., FitzGerald, D. J. P., Hamilton, T. C., Ozols, R. F., Willingham, M. C., and Pastan, S. (1985) Cancer Res. 45, 751-757). To determine which lysine residues are important in cell recognition, all 12 lysines in domain I were converted to glutamates by site-directed mutagenesis. Also, two deletion mutants encompassing almost all of domain I (amino acids 4-252) or most of domain I (amino acids 4-224) were studied. The mutant proteins were produced in Escherichia coli, purified, and tested for their cytotoxic activity against Swiss 3T3 cells and in mice. The data indicate that conversion of lysine 57 to glutamate reduces cytotoxic activity towards 3T3 cells 50-100-fold and in mice about 5-fold. Deletion of amino acids 4-224 causes a similar reduction in toxicity towards cells and mice. Deletion of most of the rest of domain I (amino acids 4-252) causes a further reduction in toxicity toward cells and mice indicating this second region between amino acids 225 and 252 of domain I is also important in the toxicity of PE. Competition assays indicated that the ability of PEGlu57 to bind to 3T3 cells was greatly diminished, accounting for its diminished cytotoxic activity.

ADP Ribose Transferases↗

PubMed1988

Enhancement of the activity of immunotoxins made with either ricin A chain or Pseudomonas exotoxin in human ovarian and epidermoid carcinoma cell lines.

The present study evaluates whether the in vitro activity of immunotoxins can be enhanced by verapamil or by various antagonists of calmodulin (dansylcadaverine, trifluoperazine, chlorpromazine). The following immunotoxins made with either Pseudomonas exotoxin (PE), recombinant ricin A chain (rRTA), or ricin A chain (RTA) were used: HB21-PE and 454A12-rRTA that both recognize the human transferrin receptor; and 260F9-rRTA and 454C11-RTA that both react with human ovarian and breast cancer cells. The cytotoxicity of these immunotoxins was determined in human ovarian carcinoma cell lines and KB cells. Verapamil, that was demonstrated previously to enhance the cell-killing activity of PE immunotoxins, enhanced the activity of several ricin A chain immunotoxins, including 454A12-rRTA, 260F9-rRTA, and 454C11-RTA. Comparing 50% inhibitory dose values for inhibition of protein synthesis by 454A12-rRTA, enhancement ranged from 2- to greater than 25-fold, was dependent on the concentration of verapamil, and was greatest at short incubation times. In addition, the cytotoxicity of HB21-PE and of selected RTA immunotoxins was increased up to 30-fold by the addition of various calmodulin antagonists. The enhancing drugs did not decrease the specificity of the immunotoxins.

ADP Ribose Transferases↗

PubMed1988

Pseudomonas exotoxin--immunotoxins.

Monoclonal antibodies can be coupled with PE to make very potent ITs. Two of these ITs (PE-HB21 and OVB-3-PE) have been shown to have antitumor activity in a nude mouse model of ovarian cancer. PE ITs are at least 10-fold more active than the corresponding RTA IT. Deletion analysis of the structural gene of PE has helped assign specific functions to different portions of the molecule. Current efforts are focused on making ITs with recombinant PE.

ADP Ribose Transferases↗

PubMed1988

Cytotoxic activity of an interleukin 6-Pseudomonas exotoxin fusion protein on human myeloma cells.

A chimeric toxin composed of human interleukin 6 (IL-6) attached to a portion of Pseudomonas exotoxin (PE) devoid of its own cell recognition domain has been produced in Escherichia coli. The fusion protein (IL-6-PE40) is cytotoxic to a human myeloma cell line expressing IL-6 receptors but has no effect on IL-6 receptor-negative cells. The specificity of IL-6-PE40 cytotoxicity was demonstrated through competition with excess IL-6 and neutralization with an antibody to IL-6. IL-6-PE40 may be useful in the selective elimination of myeloma cells and other cells with high numbers of IL-6 receptors.

ADP Ribose Transferases↗

PubMed1987

Antitumor activity of an immunotoxin in a nude mouse model of human ovarian cancer.

An immunotoxin composed of an antibody to the human transferrin receptor (454A12) and ricin A chain (RTA) was shown to inhibit the growth of NIH:OVCAR-3 tumors in a nude mouse model of human ovarian cancer. Inhibition of tumor growth by 454A12-RTA was related to the dose administered. The antitumor activity of the immunotoxin was blocked by coinjection of excess antibody with immunotoxin. An immunotoxin made using 454A12 and recombinant ricin A chain (rRTA) had an activity similar to that made with native RTA. The administration of 10 micrograms or greater of the immunotoxin 454A12-RTA/rRTA had significant antitumor activity. The injection of 30 micrograms of an irrelevant immunotoxin, MOPC21-RTA, or 30 to 500 micrograms of the 454A12 antibody had no antitumor activity.

Animals↗

PubMed1987

Construction of immunotoxins using Pseudomonas exotoxin A.

The above methodology has been used to prepare a variety of PE-immunotoxins. These reagents are potent cytotoxic agents for cells in culture that bind the appropriate antibody and nontoxic when cells do not bind the antibody. PE-immunotoxins can be used to select for mutant cultured cells which lack the target of the antibody (see chapter by Gottesman [9] on drug-resistant mutants). Recently, we have also demonstrated in vivo activity when a PE-immunotoxin was used to inhibit the growth of human ovarian cancer cells in a nude mouse model of ovarian cancer. Also PE-immunotoxins are currently being evaluated in Phase 1 clinical trials for the treatment of adult T-cell leukemia.

ADP Ribose Transferases↗

PubMed1987

A monoclonal antibody-Pseudomonas toxin conjugate that specifically kills multidrug-resistant cells.

One form of multidrug resistance is due to the expression of a 170-kDa energy-dependent drug efflux pump called P-glycoprotein in the plasma membranes of human cancer cells. We have prepared conjugates of Pseudomonas toxin with the anti-P-glycoprotein monoclonal antibody MRK-16. These anti-P-glycoprotein-toxin conjugates specifically kill multidrug-resistant human KB cells. Similar conjugates could be useful in cancer therapy to reduce or eliminate multidrug-resistant tumor populations in tumors intrinsically resistant to chemotherapy or in populations that become resistant during combination chemotherapy.

ADP Ribose Transferases↗

PubMed1987

Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin.

The transforming growth factor type alpha gene has been fused to a modified Pseudomonas toxin gene from which the cell-recognition domain has been deleted. The chimeric gene has been expressed in Escherichia coli, and the chimeric protein, PE40-TGF-alpha, has been highly purified. PE40-TGF-alpha kills cells expressing epidermal growth factor receptors and has little activity against cells with few receptors. This chimeric protein might be useful in treating cancers that contain high numbers of epidermal growth factor receptors.

ADP Ribose Transferases↗

PubMed1987

Pseudomonas exotoxin coupled to a monoclonal antibody against ovarian cancer inhibits the growth of human ovarian cancer cells in a mouse model.

In an effort to devise an alternative treatment for human ovarian cancer, we have isolated a monoclonal antibody (OVB-3) that reacts with all ovarian cancers tested (10/10) but few normal tissues. An immunotoxin produced by coupling OVB-3 to Pseudomonas exotoxin kills ovarian cancer cells in tissue culture and prolongs the life of animals bearing human ovarian cancers. These data suggest that this immunotoxin should be evaluated as a treatment for ovarian cancer in women.

ADP Ribose Transferases↗

PubMed1987

Enhancement of the cytotoxic effect of anti-carcinoembryonic antigen immunotoxins by adenovirus and carboxylic ionophores.

Immunotoxins (monoclonal antibodies-ricin A-chain conjugates) directed against the tumor-associated antigen carcinoembryonic antigen (CEA) are selective in vitro cytotoxins for human adenocarcinoma cells. However, the kinetics of intoxication are relatively slow. The effects of the UV radiation-inactivated human adenovirus and the carboxylic ionophores monensin and nigericin were examined on immunotoxin cytotoxicity to the human colorectal adenocarcinoma cell line LoVo. In a 16-hour cytotoxicity assay, adenovirus reduced 33-fold the median inhibitory concentration from 3 X 10(-8) M to 9 X 10(-10) M. In timed cytotoxicity assays, 50% of control protein synthesis was reached in immunotoxin-treated cells twentyfold faster in the presence of adenovirus (0.5 hr) than in its absence (10 hr). Adenovirus produced no enhancement of immunotoxin effect on a control cell line or on a control immunotoxin on LoVo cells, demonstrating specificity of adenovirus effect. In addition, specific immunotoxin constructed with a nonreducible thioether bond, alone or with adenovirus, produced no toxicity on LoVo cells. These results suggest that both cell surface binding and presence of a reducible disulfide bond in the conjugate are necessary for adenovirus effect. Similar potentiation of the cytotoxicity of anti-CEA immunotoxins was produced by monensin and nigericin. These studies demonstrate that both adenovirus and carboxylic ionophores are potentiators of immunotoxins directed against the CEA, producing cytotoxicity equivalent to that of ricin but specific for CEA-positive adenocarcinoma cells in culture.

Adenocarcinoma↗

PubMed1986

Immunotoxins.

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Animals↗

PubMed1986

Antitumor effects of an immunotoxin made with Pseudomonas exotoxin in a nude mouse model of human ovarian cancer.

An immunotoxin composed of Pseudomonas toxin coupled to an antibody to the human transferrin receptor was evaluated for its effect on ovarian cancer. In the tumor model employed, 60 million human ovarian cancer cells were injected into the peritoneal cavity of an immunodeficient nude mouse. By day 5, cancer cells were implanted and growing in small clusters throughout the peritoneal cavity. On days 5-8, 0.3-2 micrograms of immunotoxin was injected into the peritoneal cavity. Control mice died with malignant ascites at 34-58 days after the implantation of tumor cells, whereas immunotoxin-treated mice lived to 100 days or longer. Irrelevant immunotoxins or antibody alone had no antitumor activity. These findings suggest that intraperitoneal injection of immunotoxins may have a role in the treatment of ovarian cancer.

ADP Ribose Transferases↗

PubMed1985

Characterization of immunotoxins active against ovarian cancer cell lines.

The purpose of the present study was to develop immunotoxins directed against human ovarian carcinoma cells. Four monoclonal antibodies (260F9, 454C11, 280D11, and 245E7) were chosen because they were found to bind to various ovarian carcinoma cell lines. These antibodies were covalently linked to either Pseudomonas exotoxin (PE) or ricin A chain (RTA), and the conjugates were tested against five ovarian cancer cell lines (OVCAR-2, -3, -4, -5; A1847). The ability of the immunotoxins to inhibit both protein synthesis and colony formation was evaluated. Qualitatively similar results were obtained for both types of assays. Usually, PE conjugates were more toxic than their corresponding RTA conjugates. 454C11-PE was very toxic for all ovarian carcinoma lines, whereas 454C11-RTA had low activity. Both 260F9-PE and 260F9-RTA were active in all OVCAR cell lines but not in A1847 cells. 280D11-PE was toxic for OVCAR-4; otherwise, 280D11-PE and RTA conjugates of both 280D11 and 245E7 had little activity. Specificity of immunotoxin action was shown by competition by excess antibody, nontoxicity in nontarget cells, and inactivity of an irrelevant immunotoxin. To investigate the basis of antibody-dependent differences in activity of the various immunotoxins, antibody uptake was studied in OVCAR-2 cells, and the results indicate that antibody internalization is one important factor in the activity of immunotoxins.

ADP Ribose Transferases↗

PubMed1985

Anti-transferrin receptor antibody linked to Pseudomonas exotoxin as a model immunotoxin in human ovarian carcinoma cell lines.

The present in vitro study was performed to evaluate the potential usefulness of immunotoxins in treating human ovarian carcinomas. A monoclonal antibody against the human transferrin receptor was covalently linked to Pseudomonas exotoxin. The activity of this immunotoxin (anti-TFR-PE) was studied in five ovarian carcinoma cell lines, a breast carcinoma cell line (MCF-7), and in KB cells. The ovarian carcinoma cell lines included one previously established cell line (A1847) and four recent isolates obtained from the malignant ascites of patients with metastatic ovarian carcinoma (OVCAR cell lines). While all cell lines showed inhibition of protein synthesis by anti-TFR-PE, there were quantitative differences when the level of protein synthesis was assayed after a 12-hr incubation with the immunotoxin. These differences resulted from different kinetics of anti-TFR-PE activity in the various cell lines. Higher levels of cellular binding and internalization of anti-TFR were shown to contribute to increased toxicity of anti-TFR-PE. Verapamil increased the rate of protein synthesis inhibition and thus enhanced the toxicity of anti-TFR-PE in the OVCAR cell lines.

ADP Ribose Transferases↗

PubMed1984

Pseudomonas exotoxin-anti-TAC. Cell-specific immunotoxin active against cells expressing the human T cell growth factor receptor.

An immunotoxin was constructed with an activity that discriminated between two T cell lines based on the expression of the T cell growth factor (TCGF) receptor on their cell surface. A toxic protein conjugate, designated PE-anti-TAC, was made by chemically coupling pseudomonas exotoxin (PE) to a monoclonal antibody (anti-TAC) that recognizes the human TCGF receptor. This conjugate was toxic to HUT-102 cells, a cell line that expresses the TCGF receptor, but was nontoxic for MOLT-4 cells, a receptor-negative line. The toxicity of PE-anti-TAC was enhanced 50-fold in the presence of human adenovirus type II and was reduced to control levels by adding excess anti-TAC antibody. The toxicity of PE-anti-TAC for HUT-102 cells was compared with PE-anti-transferrin receptor. To compare the route of entry for both anti-TAC and anti-TFR using electron microscopy, protein conjugates were made by coupling horseradish peroxidase (HRP) to each antibody. Anti-TFR-HRP entered HUT-102 cells by concentrative adsorptive endocytosis via coated pits, and the majority of the antibodies bound to the cell surface at 4 degrees C were seen in receptosomes by 10 min after warming to 37 degrees C. Anti-TAC-HRP was also found to enter HUT-102 cells via coated pits and receptosomes; but, in contrast to anti-TFR, anti-TAC did not selectively concentrate in coated pits, and therefore the majority of this surface-bound antibody were not internalized in HUT-102 cells by 10 min at 37 degrees C.

ADP Ribose Transferases↗