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Biomedical subjects

D J FitzGerald

Publications and source records attributed to D J FitzGerald.

At least 55 records · Page 3Linked to original sources
PubMed1992

Antitumor effects of B3-PE and B3-LysPE40 in a nude mouse model of human breast cancer and the evaluation of B3-PE toxicity in monkeys.

B3 is a tumor-reactive monoclonal antibody (mAb) that binds to a limited number of normal tissues. Immunotoxins made with B3 coupled to either Pseudomonas exotoxin (PE) or recombinant forms of PE with a deletion of the cell-binding domain (LysPE40) have been shown to cause complete tumor regression in nude mice bearing a rapidly growing A431 (L. H. Pai et al., Proc. Natl. Acad. Sci. USA, 88: 3358-3362, 1991) human epidermoid carcinoma. In this study we show that an immunotoxin composed of mAb B3 when chemically coupled to LysPE40 (B3-LysPE40) led to complete regression of a slowly growing breast cancer, MCF-7, in nude mice when given i.v. every other day for five doses. mAb B3 coupled to native PE also produced significant regression of the MCF-7 tumor. The reactivity of mAb B3 was evaluated using an immunohistochemical method on the two responsive tumors, MCF-7 and A431, and compared with a typical human colon carcinoma specimen that has B3 antigen on its surface. The results showed that both A431 and MCF-7 xenograft tumors have similar reactivity to B3 when compared with the human colon carcinoma specimen. To evaluate the toxicity of B3-PE in primates, Cynomolgus monkeys received escalating doses of B3-PE i.v. on Days 1, 3, and 5. Based on antibody localization studies using frozen sections of normal human and monkey tissue, gastric, trachea, and bladder mucosal injury could have occurred. However, no clinical signs of injury or histological damage to these organs were seen at the doses administered. Chemical hepatitis due to PE was transient and well tolerated at doses up to 50 micrograms/kg for three doses. The lethal dose was about 100 micrograms/kg, and the cause of death was liver necrosis, as shown by necropsy. We conclude that mAb B3, when coupled to PE40 or PE, can produce strong antitumor activity in vivo. The similar level of reactivity of the B3 antibody in our tumor models with a surgical specimen of a human colon carcinoma and the toxicity study in monkeys indicate that therapeutic doses of B3-PE and B3-LysPE40 can be delivered without causing toxicity to normal organs that express B3 antigen. Although both B3-PE and B3-LysPE40 have antitumor activity in nude mice bearing a human xenograft, B3-LysPE40 is better tolerated and should be further evaluated as a therapeutic agent for cancer patients.

ADP Ribose Transferases↗
PubMed1992

Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol.

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains. Functional analysis of PE has revealed that domain I is the cell-binding domain and that domain III functions in ADP ribosylation. Domain II was originally designated as the translocation domain, mediating the transfer of domain III to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and ADP-ribosylation activities but which are not cytotoxic. However, the results do not rule out the possibility that regions of PE outside of domain II also participate in the translocation process. To investigate this problem, we have now constructed a toxin in which domain III of PE is replaced with barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens. This chimeric toxin, termed PE1-412-Bar, is cytotoxic to a murine fibroblast cell line and to a murine hybridoma resistant to the ADP-ribosylation activity of PE. A mutant form of PE1-412-Bar with an inactivating mutation in domain II at position 276 was significantly less toxic. Because the cytotoxic effect of PE1-412-Bar was due to the ribonuclease-activity of barnase molecules which had been translocated to the cytosol, we conclude that domain II of PE is not only essential but also probably sufficient to carry out the translocation process.

ADP Ribose Transferases↗
PubMed1992

Alteration of a protease-sensitive region of Pseudomonas exotoxin prolongs its survival in the circulation of mice.

Pseudomonas exotoxin A (PE) is a single-chain 66-kDa polypeptide that kills eukaryotic cells by ADP-ribosylation of translational elongation factor 2. PE is composed of three major structural domains whose functions are binding of cells (I), translocation (II), and ADP-ribosylation (III). Here we describe a protease cleavage target that is located near arginine-490 on the surface of domain III. We made several different types of mutations near arginine-490. Deletion of arginine-490 or replacement of arginine-490 and -492 with serine and lysine or with two lysines resulted in protease-resistant molecules that were fully cytotoxic and had normal ADP-ribosylation activity. However, the half-life in mouse blood of the PE delta 490 mutant was 24 min whereas that of PE was 13 min. Furthermore, two PE mutants that were protease-hypersensitive, PEGlu246,247,249 and PEGlu57,246,247,249 (in which glutamate residues replace basic residues at the indicated positions), had very short half-lives. These data indicate that protease sensitivity is an important determinant in the half-life of PE in the circulation and suggest that the half-life of other proteins may be prolonged by removal of protease sites. Deletion of arginine-492 or the replacement of amino acids 486-491 with three glycines markedly diminished ADP-ribosylation activity and cytotoxicity, indicating that this region of domain III is also important for catalytic activity.

ADP Ribose Transferases↗
PubMed1992

Interleukin-6 fused to a mutant form of Pseudomonas exotoxin kills malignant cells from patients with multiple myeloma.

IL-6-PE4E is a recombinant protein consisting of interleukin-6 (IL-6) fused to a mutant form of Pseudomonas exotoxin in which four basic amino acids are changed to glutamate (PE4E). The chimeric toxin has been previously shown to specifically kill malignant hepatic, prostatic, epidermoid, and myeloma cell lines in vitro. To explore the possible clinical utility of IL-6-PE4E, particularly as an agent for ex vivo purging of marrow for autologous bone marrow transplantation (ABMT), we tested malignant cells from patients with multiple myeloma for sensitivity to this chimeric toxin. Ficoll-purified bone marrow cells were incubated with and without IL-6-toxin for 2 to 3 days. Eight of the 15 myeloma patients had cells that were sensitive to IL-6-toxin as measured by a decrease in the level of protein synthesis. Cells from five patients were very sensitive to IL-6-PE4E, with 50% inhibition of protein synthesis (ID50) achieved at or below 6 ng/mL (7 x 10(-11) mol/L). Cells from three additional patients showed moderate sensitivity, with ID50s between 30 and 140 ng/mL. The remaining seven samples showed little or no sensitivity, with ID50s greater than or equal to 400 ng/mL. Normal bone marrow cells or normal BFU-E and CFU-GM were resistant to the IL-6-toxin even at 1,000 ng/mL. Neither IL-6, IL-2-PE4E, nor an enzymatically deficient mutant of IL-6-PE4E was cytotoxic toward the myeloma cells, indicating that the cytotoxic effect of IL-6-PE4E required the adenosine diphosphate-ribosylation function as well as the specific ligand. Our data suggest that IL-6-toxin could be effective in ex vivo marrow purging in selected multiple myeloma patients who are candidates for ABMT, and that this toxin should also be investigated further for in vivo therapy.

ADP Ribose Transferases↗
PubMed1991

A proper amino terminus of diphtheria toxin is important for cytotoxicity.

A series of deletions and substitutions were made at the 5' end of the gene fusion between the first 388 codons of diphtheria toxin (DT) and a cDNA encoding human IL2. The chimeric protein (DT388-IL2) was expressed and purified from E. coli and found to be very cytotoxic to a human T cell line, HUT 102, that expresses a large number of IL2 receptors. Deletion of the first five amino acids of DT resulted in a non-cytotoxic chimeric protein that had both ADP-ribosylation activity and IL2 receptor binding activity. Deletion of the first two amino acids of DT had little effect on cytotoxicity, while deletion of the first four amino acids or of two acidic residues at positions 3 and 4 greatly reduced cytotoxicity. Unexpectedly, a mutant containing a single leucine in place of the first two amino acids (gly, ala) was 2-3 fold more active. The amino terminus of DT may participate in the translocation of the A chain to the cytosol in a manner similar to Pseudomonas exotoxin (PE) in which a specific C-terminal sequence has been proposed to be involved in its cytotoxicity.

Adenosine Diphosphate Ribose↗
PubMed1991

B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice.

The genes encoding the heavy- and light-chain Fv regions of the monoclonal murine antibody B3, which recognizes a carbohydrate antigen on the surface of many human carcinomas, were cloned by PCR techniques and used to generate single-chain immunotoxins containing Pseudomonas exotoxin (PE). The light and heavy chains were connected by a flexible linker to form a single-chain antigen-binding protein, B3(Fv), which was in turn fused to truncated forms of PE lacking the cell-binding domain. The single-chain Fv and two different B3(Fv) immunotoxins, B3(Fv)-PE40 and B3(Fv)-PE38KDEL, were expressed in Escherichia coli and the single-chain immunotoxins were purified to near homogeneity. Both recombinant immunotoxins were shown to be cytotoxic specifically to carcinoma cell lines that express the B3 antigen on their surface; B3(Fv)-PE38KDEL was significantly more active. Furthermore, intravenous administration of B3(Fv)-PE38KDEL caused complete regression of human epidermoid carcinomas growing subcutaneously in immunodeficient mice.

ADP Ribose Transferases↗
PubMed1991

Analysis of sequences in domain II of Pseudomonas exotoxin A which mediate translocation.

Pseudomonas exotoxin (PE) contains 613 amino acids that are arranged into 3 structural domains. PE exerts its cell-killing effects in a series of steps initiated by binding to the cell surface and internalization into endocytic vesicles. The toxin is then cleaved within domain II near arginine-279, generating a C-terminal 37-kDa fragment that is translocated into the cytosol where it ADP-ribosylates elongation factor 2 and arrests protein synthesis. In this study, we have focused on the functions of PE which are encoded by domain II. We have used the chimeric toxin TGF alpha-PE40 to deliver the toxin's ADP-ribosylating activity to the cell cytosol. Deletion analysis revealed that sequences from 253 to 345 were essential for toxicity but sequences from 346 to 364 were dispensable. Additional point mutants were constructed which identified amino acids 339 and 343 as important residues while amino acids 344 and 345 could be altered without loss of cytotoxic activity. Our data support the idea that domain II functions by first allowing PE to be processed to a 37-kDa fragment and then key sequences such as those identified in this study mediate the translocation of ADP-ribosylation activity to the cytosol.

ADP Ribose Transferases↗
PubMed1991

Antitumor effects of interleukin 6-Pseudomonas exotoxin chimeric molecules against the human hepatocellular carcinoma, PLC/PRF/5 in mice.

IL6-PE40 and IL6-PE664Glu are chimeric molecules composed of interleukin 6 (IL6) fused to a truncated form (PE40) or a full-length mutated form (PE664Glu) of Pseudomonas exotoxin. Both forms of IL6-Pseudomonas exotoxin are cytotoxic to IL6 receptor-bearing tumor cell types in culture. In this report, we show that both IL6-PE40 and IL6-PE664Glu have antitumor activity against the hepatocellular carcinoma PLC/PRF/5 implanted s.c. in nude mice. The PLC/PRF/5 tumor contains about 2300 IL6 receptors per cell. IL6-PE664Glu showed improved therapeutic efficacy when released continuously for 7 days by an osmotic pump planted i.p. than when administered by multiple daily i.p. injections. Both forms of IL6 toxin exhibited a schedule-dependent antitumor effect. These results demonstrate that IL6-Pseudomonas exotoxin can suppress the growth of cancer which overexpresses cell surface IL6 receptors.

ADP Ribose Transferases↗
PubMed1991

Antitumor activity of a transforming growth factor alpha-Pseudomonas exotoxin fusion protein (TGF-alpha-PE40).

TGF-alpha-PE40 is a chimeric protein composed of transforming growth factor alpha (TGF-alpha) linked to a modified Pseudomonas toxin from which the cell recognition domain has been deleted (PE40). TGF-alpha-PE40 has been shown to have cytotoxic effects on human cancer cell lines that express the epidermal growth factor (EGF) receptor on their surface, and when given i.p., it prolongs the survival of nude mice bearing i.p. tumors. Because several normal tissues, including liver, express EGF receptors on their surfaces, it has not been clear that this agent can be used systemically to treat EGF receptor-bearing tumors. In this study, we have delivered TGF-alpha-PE40 for 7 days by continuous infusion through a miniosmotic pump placed in the peritoneal cavity of nude immunodeficient mice. Two different human cancer cell lines that express EGF receptors on their surface were implanted s.c. One was A431, an epidermoid carcinoma; the other was DU-145, a prostate carcinoma. By using this mode of continuous i.p. delivery, we were able to achieve a constant serum level of TGF-alpha-PE40 that was nontoxic to the mice and yet delayed the growth of both tumors implanted s.c. and caused partial regression of one. We conclude that it is possible to deliver TGF-alpha-PE40 systemically and achieve a therapeutic serum level in mice without major toxicity. Although side effects may be expected, this study establishes that there is a therapeutic window for this agent in the therapy of cancers with high numbers of EGF receptors.

Animals↗
PubMed1991

Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin.

B3 is a monoclonal antibody that reacts with a carbohydrate epitope present on a variety of proteins located on the surface of many cancer cells and a limited number of normal tissues. We evaluated the cytotoxic activity of immunotoxins composed of monoclonal antibody B3 coupled to native Pseudomonas exotoxin (PE) or two recombinant forms of Pseudomonas exotoxin, PEArg57 or LysPE40, a form of PE with a deletion of the cell binding domain. All three conjugates were cytotoxic to human cell lines expressing the B3 antigen on their surface. The survival of each of the three immunotoxins in the circulation of mice was determined after administering the immunotoxin i.v. The half-life in blood of B3-PE and B3-PEArg57 was 20 hr, whereas the half-life of B3-LysPE40 was 4 hr. The short half-life of B3-LysPE40 may be due to the absence of domain I of PE. To determine the therapeutic effects of the three immunotoxins, they were given intraperitoneally to nude mice bearing subcutaneous A431 tumors. All three immunotoxins caused complete regression of 50-mm3 tumors with no toxic effects to the animals at therapeutic doses. Furthermore, substantial regression was also noted with much larger tumors. Our data indicate that the monoclonal antibody B3, when coupled to PE or recombinant forms of PE, may be useful for the treatment of tumors expressing B3 antigen. The therapeutic window was largest with B3-LysPE40, which can be administered in higher doses because it lacks sequences in domain I of PE that enable PE to bind to nontarget cells.

ADP Ribose Transferases↗
PubMed1991

Barnase toxin: a new chimeric toxin composed of pseudomonas exotoxin A and barnase.

We have constructed a chimeric toxin composed of Pseudomonas exotoxin A (PE) and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase. The chimeric protein, termed PE-Bar, reacted with both anti-PE and anti-barnase antisera and had both ADP ribosylation and ribonuclease activities. The chimeric toxin was cytotoxic to the murine fibroblast cell line L929 and to a murine hybridoma resistant to PE. A mutant form of PE-Bar lacking ADP-ribosylating activity was still cytotoxic to L929 cells. Because treatment of cells prelabeld with [3H]uridine resulted in a decrease in their RNA content, we conclude that this cytotoxic effect was due to the ribonuclease activity of barnase molecules that had been translocated to the cytosol. It is now possible to construct chimeric toxins with two or more enzymatic activities that can be delivered to the cytosol of the target cells.

ADP Ribose Transferases↗
PubMed1991

Cytotoxic activity of a recombinant fusion protein between insulin-like growth factor I and Pseudomonas exotoxin.

A chimeric toxin in which the cell binding domain of Pseudomonas exotoxin was replaced with mature human insulin-like growth factor I (IGF-I) was produced in Escherichia coli. This protein, IGF-I-PE40, was cytotoxic to human cell lines derived from a variety of tumor types, with a breast carcinoma line (MCF-7) and two hepatoma lines (HEP3B and HEPG2) showing the highest sensitivity to the toxin. The specificity of IGF-I-PE40 cytotoxicity was confirmed through competition with excess IGF-I and through blockage of toxin binding using an antibody specific to the type I IGF receptor. A potential interaction between the toxin and soluble IGF-binding proteins was also demonstrated. IGF-I-PE40 may be useful in the selective elimination of cells bearing the type I IGF receptor.

ADP Ribose Transferases↗
PubMed1991

Biological activity of a transforming growth factor-alpha--Pseudomonas exotoxin fusion protein in vitro and in vivo.

Transforming growth factor-alpha (TGF alpha)-pseudomonas exotoxin-40 (PE40) is a chimeric protein consisting of an N-terminal TGF alpha domain fused to a C-terminal 40-kDa segment of the pseudomonas exotoxin A protein. TGF alpha-PE40 exhibits the receptor binding activity of TGF alpha and the cell killing activity of PE40. In the current study, we report that a modified TGF alpha-PE40 derivative significantly prolongs the survival of nude mice bearing tumors derived from cell lines which express the epidermal growth factor receptor (EGFR). In addition, the therapeutic benefit of this protein is mediated by specific binding to the EGF receptor. These results indicate that a therapeutic window exists in vivo for the use of some growth factor--toxin fusion proteins as anticancer agents.

ADP Ribose Transferases↗
PubMed1990

Inhibition of antibody response to Pseudomonas exotoxin and an immunotoxin containing Pseudomonas exotoxin by 15-deoxyspergualin in mice.

Immunotoxins are potent cell-killing agents that may be useful in the treatment of cancer. The early production of neutralizing antibodies to immunotoxins is one of the major limiting factors for their use in humans. 15-Deoxyspergualin (DSG), a derivative of spergualin, which is a metabolite of Bacillus laterosporus, has been found to have immunosuppressive activity in rodents, dogs, and primates. We examined the suppressive activity of DSG on the antibody response to Pseudomonas exotoxin in mice by enzyme-linked immunosorbent assay. Male BDF1 mice were immunized with a single dose of a nontoxic mutant of Pseudomonas exotoxin (40 micrograms) and then treated with i.p. injections of DSF at a dose of 10 mg/kg for 3 days. Although antibodies to Pseudomonas exotoxin were observed within 7 days in the control group, there was complete suppression of antibody production in the DSG-treated group. Immunosuppression has also been observed in animals immunized with multiple doses (10 mg x 7 d) of Pseudomonas exotoxin and treated with DSG at a dose of 5 mg/kg for 21 days. Similar immunosuppression was observed in mice given multiple doses of the immunotoxin, anti-Tac-LysPE40. We conclude that the immunosuppressive activity of DSG may be useful in increasing the duration of immunotoxin treatment.

ADP Ribose Transferases↗
PubMed1990

Expression of the interleukin 6 receptor and interleukin 6 in prostate carcinoma cells.

We have probed for the presence of interleukin 6 (IL6) receptors in prostatic carcinoma cell lines (LNCaP, DU 145, and PC3) by examining their sensitivity to the cytotoxic effects of a chimeric toxin composed of IL6 and Pseudomonas exotoxin (PE). All three cell lines were killed by IL6-PE66(4)Glu, a version of IL6-PE in which the binding domain of native PE has been mutated to debilitate PE binding to its own receptor. This cytotoxic activity confirmed the presence of IL6 receptors on prostatic carcinoma cells. We have measured the number of IL6 receptors found on these cells and have further determined that they secrete IL6. These data provide evidence that IL6 and its receptor may play an important role in human prostate cancer.

ADP Ribose Transferases↗
PubMed1990

Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol.

Pseudomonas exotoxin (PE) was incubated with cells and extracts analyzed for processed fragments. PE was proteolytically cleaved to produce a N-terminal 28-kDa and a C-terminal 37-kDa fragment, the latter being composed of a portion of domain II and all of domain III (the ADP-ribosylating domain). Cleavage was evident at 10 min after toxin addition and endosome preparations contained the processed fragments. Initially, the two fragments were linked by a disulfide bond. Subsequently, the 37-kDa fragment was reduced and translocated to the cytosol where it inactivated protein synthesis. Cytosol from toxin-treated cells was greatly enriched in the 37-kDa fragment. The 37-kDa fragment appears to be essential for toxicity since mutant PE molecules that do not produce this fragment, or cannot deliver it to the cytosol, fail to kill cells.

ADP Ribose Transferases↗