Biomedical subjects

D J FitzGerald

Publications and source records attributed to D J FitzGerald.

At least 37 records · Page 2Linked to original sources

PubMed1997

Sensitivity of human acute myeloid leukaemia to diphtheria toxin-GM-CSF fusion protein.

The potential to selectively eliminate acute myeloid leukaemia (AML) cells with the GM-CSF-diphtheria toxin fusion protein (DT-GM-CSF) was studied under conditions of autonomous proliferation in vitro with no growth factors (GFs) added and after growth stimulation with a mixture of human (hu)G-CSF, huIL-3 and huSCF. DNA synthesis was maximally inhibited after 48 h exposure to DT-GM-CSF. Cell viability and AML colony forming ability in vitro were reduced. 18/22 samples were found to be sensitive to DT-GM-CSF, with 50% inhibition of DNA synthesis (ID50) at concentrations ranging from 0.1 to 16 ng/ml, and four samples were minimally or not sensitive to DT-GM-CSF (ID50 > or = 99 ng/ml). From the 15 samples which showed autonomous proliferation, 13 were sensitive to inhibition of proliferation by DT-GM-CSF. The level of GM-CSF receptor (GM-CSFR) expression was determined by flow cytometry after labelling with specific antibodies for the alpha and beta subunits. Although the toxicity to DT-GM-CSF was specifically mediated by the GM-CSFR, no correlation was found between the level of expression of the GM-CSFR alpha or beta subunit and the sensitivity for DT-GM-CSF. These in vitro studies show that the DT-GM-CSF fusion protein can be used for specifically targeting and eliminating leukaemic cells in the majority of AML cases.

Acute Disease↗

PubMed1996

Ligand-toxin hybrids directed to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein exhibit lower toxicity than native Pseudomonas exotoxin.

Pseudomonas exotoxin (PE) binds the heavy chain of the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). To understand the significance of this interaction, novel toxin-derived gene fusions were constructed with two ligands that also bind this receptor. A 39-kDa cellular protein, termed RAP, binds LRP with high affinity and often co-purifies with it. Two RAP toxins were constructed, one with PE and one with diphtheria toxin (DT). RAP, which replaced the toxins binding domains, was combined with each of the corresponding translocating and ADP-ribosylating domains. Both RAP-toxins bound LRP with an apparent higher affinity than native PE. Despite this, RAP-PE and DT-RAP were less toxic than native PE. Apparently, RAP-toxin molecules bound and entered cells but used a pathway that afforded only low efficiency of toxin transport to the cytosol. This was evident because co-internalization with adenovirus increased the toxicity of RAP-toxins by 10-fold. We speculate that the high affinity of RAP binding may not allow the toxin's translocating and ADP-ribosylating domains to reach the cytosol but rather causes the toxin to take another pathway, possibly one that leads to lysosomes. To test this hypothesis, additional RAP-PE fusions were constructed. N-terminal or C-terminal fragments of RAP were joined to PE to produce two novel fusion proteins which were likely to have reduced affinity for LRP. Both of these shorter fusion proteins exhibited greater toxicity than full-length RAP-PE. A second ligand-toxin gene fusion was constructed between plasminogen activator inhibitor type 1 and DT. DT-plasminogen activator inhibitor type 1 formed a complex with tissue-type plasminogen activator and inhibited its proteolytic activity. However, like the RAP-toxins, this hybrid was less toxic for cells than native PE.

ADP Ribose Transferases↗

PubMed1996

8-Epi PGF2 alpha: specific analysis of an isoeicosanoid as an index of oxidant stress in vivo.

1. Excessive free radical generation is thought to contribute to tissue injury in a broad spectrum of diseases. A particular constraint in addressing this hypothesis has been the inability to assess free radical generation in vivo and the lack of information on drugs or vitamins which act as effective antioxidants in vivo. 2. Traditional approaches have relied upon measures of substrate oxidizability or spin trapping of free radical adducts ex vivo. It is unknown how these measurements might relate, in a quantitative fashion, to the generation of reactive oxygen species in vivo. Isoeicosanoids are free radical catalyzed products of arachidonic acid. One of these compounds, 8-epi prostaglandin F2 alpha (8-epi PGF2 alpha) exhibits biological activity and may function as an autacoid. Specific analysis of this 8-epi PGF2 alpha isomer indicates that it is elevated in certain syndromes thought to be associated with oxidant stress. These include vascular reperfusion, paracetamol poisoning and liver cirrhosis. Apparently healthy individuals who smoke cigarettes or consume alcohol exhibit dose dependent increments in excretion of 8-epi PGF2 alpha. Excretion is depressed by antioxidant vitamins, although not by the nonspecific cyclooxygenase (COX) inhibitor, aspirin, even though 8-epi PGF2 alpha may be formed by either COX-1 or COX-2. 3. Specific analysis of this and other isoeicosanoids may afford an opportunity to evaluate the effects of antioxidant interventions in human diseases characterized by excessive lipid peroxidation in vivo.

Animals↗

PubMed1996

Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin.

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro, furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5-10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli, purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0-5.5. When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.

Animals↗

PubMed1996

Treatment of acute myelocytic leukemia with interleukin-6 Pseudomonas exotoxin fusion protein in a rat leukemia model.

We studied the applicability of interleukin-6 Pseudomonas exotoxin fusion protein (IL-6PE4E) for treatment of acute myelocytic leukemia (AML). Leukemic cells from five out of 10 AML patients studied expressed IL-6 receptor (IL-6R) and proliferation in vitro was inhibited in four of these cases. The potential of this approach in vivo was tested in a pre-clinical model for AML; the Brown Norway acute myelocytic leukemia (BNML). To obtain IL-6R expression levels on BNML cells comparable to the numbers expressed on human AML, human IL-6R gene transfectants of the BNML sub-line LT12 (LT12/IL-6R) were generated. IL-6PE4E is cytotoxic in vitro to LT12/IL-6R expressing 1400 high affinity IL-6R per cell with 50% inhibition of DNA synthesis at 1 ng/ml. In vivo treatment of leukemic rats carrying LT12/IL-6R leukemia indicated that the maximal tolerated dose of IL-6PE4E was 275 +/- 25 microg/kg/day, when continuously administered for 7 days and resulted in a 90% reduction in leukemic cell load. At this dose level of IL-6PE4E no reduction of normal hemopoietic progenitors was seen in non-leukemic rats. At higher dose levels (350-1050 microg/kg/day) severe systemic toxicity was encountered. On the basis of these pre-clinical studies the feasibility of growth factor-toxins for selective in vivo targeting to AML cells is evaluated.

ADP Ribose Transferases↗

PubMed1995

Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein.

The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.Z., R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. Strickland, and C.B. Saelinger. 1992. J. Biol. Chem. 267:12420-12423) [corrected] showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha 2M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor. The results of this investigation confirm that LRP mediates the internalization of PE.

ADP Ribose Transferases↗

PubMed1994

The 39-kDa receptor-associated protein regulates ligand binding by the very low density lipoprotein receptor.

A 39-kDa receptor associated protein (RAP) binds and inhibits ligand binding by two members of the low density lipoprotein (LDL) receptor family, gp330 and low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. To determine if additional members of the LDL receptor family may interact with RAP, Chinese hamster ovary cells were transfected with plasmids directing expression of the very low density lipoprotein (VLDL) receptor cDNA or the LDL receptor cDNA. Detergent-soluble extracts from these and normal Chinese hamster ovary cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after which the proteins were transferred to nitrocellulose membranes and incubated with RAP. When detergent extracts from normal cells were incubated with RAP, several polypeptides, including a 130-kDa protein, were observed to bind RAP. In cells transfected with the VLDL receptor cDNA, a substantial increase in RAP binding to the 130-kDa polypeptide was noted. This protein was identified as the VLDL receptor by immunoblotting. The VLDL receptor present in detergent extracts from transfected cells bound to RAP-Sepharose, and a KD of 0.7 nM for the interaction between RAP and the purified VLDL receptor was determined using enzyme-linked immunosorbent assay. The purified VLDL receptor bound 125I-labeled VLDL, but not 125I-labeled LDL, and the binding of 125I-labeled VLDL was completely inhibited by RAP. Further, RAP inhibited the uptake and degradation of 125I-VLDL by cells overexpressing the VLDL receptor. Thus the VLDL receptor represents the third member of the LDL receptor family whose ligand binding properties are antagonized by RAP. This suggests a common functional role for RAP in modulating ligand binding by members of the LDL receptor family.

Amino Acid Sequence↗

PubMed1994

Cleavage of pseudomonas exotoxin and diphtheria toxin by a furin-like enzyme prepared from beef liver.

Pseudomonas exotoxin (PE) is cleaved within mammalian cells between Arg279 and Gly280 to generate an enzymatically active COOH-terminal fragment of 37 kDa which translocates to the cytosol and ADP-ribosylates elongation factor 2. A protease, with toxin cleaving activity, was prepared from beef liver and subsequently characterized. After achieving a 500-fold enrichment in several chromatographic steps, a soluble form of this protease was identified as a furin-like enzyme. It cleaved PE on the COOH-terminal side of the sequence of RQPR (amino acids 276-279) producing the same fragments as those generated within cells. Cleavage had a pH optimum of 5.0-5.5, was inhibited by EDTA or p-hydroxymercuribenzoate but not by O-phenanthroline,N-ethylmaleimide, trans-epoxysuccinyl-L-leukcylamido-(4-guanidino)-butane, or PMSF (or other well known inhibitors of serine proteases). The beef protease cleaved PE with an apparent Km of 7 microM. A mutant form of PE, PEala281, was cleaved at the same site, with the same pH optimum, a similar Km (9 microM) but with a Vmax 150 times faster than was seen with the native toxin. Mutational analysis of the amino acids located just before the site of cleavage, confirmed the importance of arginines at P-1 and P-4. It was also noted that the introduction of a dibasic pair at 278-279 did not increase toxicity or appreciably improve the rate of cleavage. Unnicked diphtheria toxin (DT) was also cleaved by the beef protease; cleavage was on the COOH-terminal side of the sequence RVRR (amino acids 190-193), was seen at pH values ranging from 5.5 to 8.5 and had an optimum at pH 8.0. Recombinant furin cleaved PE, PEala281, and DT with the same characteristics as the beef protease. In addition, Western blot analysis revealed that anti-furin antibodies reacted specifically with components in the beef protease preparation. Immunodepletion experiments showed that all toxin-cleavage activity could be removed from the beef protease using anti-furin antibodies. The relevance of furin-mediated cleavage was further assessed by adding nicked toxins to intact cells. Nicked PE and DT both killed cells at a faster rate than their unnicked counterparts.

ADP Ribose Transferases↗

PubMed1994

Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of an interleukin-2 receptor-expressing human carcinoma.

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin composed of the variable domains of the monoclonal antibody anti-Tac, which binds to the p55 subunit of the interleukin-2 receptor (IL-2R), and a truncated form of Pseudomonas exotoxin (PE), which does not bind to the PE receptor (Chaudhary et al, Nature 339:394, 1989). Whereas its cytotoxic activity toward autoimmune and malignant target cells has been established, its efficacy in vivo remains unknown. To establish an animal model, we produced ATAC-4 cells by transfecting the gene encoding the low-affinity IL-2R (p55) into A431 epidermoid carcinoma cells. ATAC-4 cells contained low-affinity IL-2Rs (2 x 10(5)/cell) and formed tumors in nude mice. In tissue culture, protein synthesis in ATAC-4 cells was inhibited 50% (IC50) at 0.06 ng/mL (0.9 pmol/L) of anti-Tac(Fv)-PE40. IC50s for the derivatives anti-Tac(Fv)-PE38, which is missing PE amino acids 365-380, and anti-Tac(Fv)-PE38KDEL, which contains the same deletion plus the KDEL carboxyl terminus, were 0.04 and 0.025 ng/mL, respectively. All the agents produced complete tumor regressions in ATAC-4 tumor-bearing mice and anti-Tac(Fv)-PE38KDEL had significant antitumor activity at 1% of the LD50. The dose limiting toxicity of anti-Tac(Fv)-PE38KDEL was from hemorrhagic liver necrosis, which was observed at approximately 55% of the LD50.

ADP Ribose Transferases↗

PubMed1993

Mechanism of action of Pseudomonas exotoxin. Identification of a rate-limiting step.

Pseudomonas exotoxin (PE) enters cells by receptor-mediated endocytosis and is cleaved by a cellular protease between Arg279 and Gly280 to produce an NH2-terminal fragment of 28 kDa which contains the toxin's binding domain and a COOH-terminal fragment of 37 kDa which has translocating and ADP-ribosylating activity. After proteolysis, the COOH-terminal fragment reaches the endoplasmic reticulum by retrograde transport where it translocates to the cytosol and inhibits protein synthesis by ADP-ribosylating elongation factor 2. To understand how the 37-kDa fragment functions, we focused on the role of specific amino acids located near its NH2 terminus. We found that there was a 4-250-fold loss in toxic activity when tryptophan 281, leucine 284, or tyrosine 289 were changed to other residues. Mutations at these three positions did not interfere with the receptor binding, cell-mediated proteolytic cleavage, or ADP-ribosylating activity. To determine the role of these amino acids, a competition assay was devised in which the addition of excess PE delta 553, a mutant form of PE that lacks ADP-ribosylation activity, competed efficiently for the toxicity of PE. Excess PE with mutations near the NH2 terminus of the 37-kDa fragment competed poorly. This competition occurred after proteolysis since PEGly276, a mutant form of PE that is not cleaved, did not complete. We conclude that specific amino acids at the NH2 terminus of the 37-kDa fragment interact in a saturable manner with an unknown intracellular component.

ADP Ribose Transferases↗

PubMed1993

Polyethylene glycol-modified chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin.

Modification of proteins with monomethoxy-polyethylene glycol (mPEG) has been shown to prolong circulation time and to reduce immunogenicity. To make a mPEG-modified recombinant toxin that retained cytotoxic activity but had a longer residence time in circulation, we have constructed an altered form of TGF alpha-PE40, a recombinant toxin composed of human transforming growth factor alpha (TGF alpha) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell-binding domain. In the newly designed protein, termed TGF alpha R29-L2-CH2-PE38QQ delta (TCP), there are no lysine residues in the TGF alpha and PE38 portions. Human IgG4 constant region CH2 and a tetradecapeptide linker; L2, are inserted between TGF alpha and PE38. Together, L2 and CH2 contain 13 lysine residues as potential modification sites for mPEG. mPEG conjugates of TCP (PEG-TCP) were generated and the products were resolved by ion exchange chromatography. Two PEG-TCP species termed B4 and B6 retained 15 and 4% of cytotoxicity, respectively, and 26% of their receptor binding activity compared with the unmodified TCP. Both B4 and B6 had prolonged circulation times in the blood and reduced toxicity in animals. The mean residence times of B4 and B6 were 37 and 68 min, respectively, compared to 7 min for TCP. When administered i.v. to tumor bearing mice, both B4 and B6 produced marked antitumor effects whereas the unmodified TCP had none. Also, the immunogenicity of PEG-TCP was 5-10 times less than that of TCP. We suggest that the prolonged circulating time and reduced toxicity of PEG-TCP compensate for a diminished cytotoxic activity and enlarge significantly the therapeutic window of this chimeric toxin.

ADP Ribose Transferases↗

PubMed1993

Pseudomonas exotoxin-based immunotoxins containing the antibody LL2 or LL2-Fab' induce regression of subcutaneous human B-cell lymphoma in mice.

We have produced immunotoxins using LL2, a monoclonal antibody which binds to human B-cell lymphomas and which, in a radioiodinated form, induced responses in lymphoma patients (D.M. Goldberg et al., J. Clin. Oncol., 9: 548-564, 1991). We have coupled LL2 to Lys-PE38KDEL, a derivative of Pseudomonas exotoxin (PE) which does not bind to the PE receptor. LL2-PE38KDEL was cytotoxic toward several Burkitt's lymphoma lines, with 50% inhibitory concentration values ranging from 2 to 6 ng/ml (10-30 pM). Another immunotoxin, LL2-Fab'-PE38KDEL, was produced by chemically coupling the Fab' fragment of LL2 to Lys-PE38KDEL. LL2-Fab'-PE38KDEL also was cytotoxic toward the Burkitt's cells, with a 50% inhibitory concentration of 1-2 ng/ml (13-24 PM). The antibody LL2 alone had no cytotoxicity toward the malignant cells, and excess LL2 prevented the cytotoxicity of LL2-PE38KDEL and LL2-Fab'-PE38KDEL. Control immunotoxins UPC-10-PE38KDEL and Mu-9-Fab'-PE38KDEL were not cytotoxic. LL2-PE38KDEL and LL2-Fab'-PE38KDEL bound to cells with 50% and 17% of the affinity of LL2, respectively. Both immunotoxins, but not UPC-10-PE38KDEL, prevented the development of CA-46 tumors in nude mice. LL2-PE38KDEL and LL2-Fab'-PE38KDEL, but not the control immunotoxins, led to complete regressions of measurable s.c. CA-46 tumors in nude mice, when given at 50% and 35% of the 50% lethal dose, respectively. LL2 alone significantly retarded the growth of CA-46 tumors but did not cause complete tumor regressions. Immunotoxins containing derivatives of Pseudomonas exotoxin can be targeted to human B-cell lymphoma and merit further study as potential therapeutic agents.

ADP Ribose Transferases↗

PubMed1993

Immunotoxins made with a recombinant form of Pseudomonas exotoxin A that do not require proteolysis for activity.

We used recombinant DNA technology to construct a mutant form of Pseudomonas exotoxin A (PE) called cysPE35 that contains amino acids 280-364 and 381-613 of PE. cysPE35 begins at the native PE proteolytic cleavage site and contains a single cysteine residue at position 287 that can be used to conjugate the toxin to monoclonal antibodies (MAbs). Unlike immunotoxins containing larger mutant forms of PE, such as PE40 or PE38, immunotoxins containing cysPE35 linked through a disulfide bond do not require proteolysis to generate a toxin fragment able to translocate to the cytosol. cysPE35 was conjugated to several MAbs and their activities were studied in vitro and in vivo. The concentration of toxin that inhibited protein synthesis as measured by a decrease in [3H]leucine incorporation of 50% of cysPE35 conjugated through a disulfide bond to the MAb HB21, which targets the human transferrin receptor, was 1 ng/ml on A431 cells. The MAb HB21 conjugated through a thioether bond to cysPE35 was much less active (concentration of toxin that inhibited protein synthesis as measured by a decrease in [3H]leucine incorporation of 50%, 200 ng/ml). An immunotoxin containing PE38 conjugated through either a disulfide or thioether bond to the MAb HB21 had a concentration of toxin that inhibited protein synthesis as measured by a decrease in [3H]leucine incorporation of 50% of 5 ng/ml, indicating that proteolysis of PE38 may be rate limiting in the action of these immunotoxins. Two other MAbs, LL2 and B3, were also conjugated through a disulfide bond to cysPE35. Both immunotoxins were also more active against cultured cells than conjugates using PE38 or PE40, and caused complete regression of human tumor xenografts growing in nude mice. In conclusion, we have constructed a mutant form of PE which must be coupled to MAbs through a disulfide bond to produce fully active immunotoxins that do not require proteolysis to generate a toxin fragment able to reach the cell cytosol.

ADP Ribose Transferases↗

PubMed1993

A recombinant form of Pseudomonas exotoxin A containing transforming growth factor alpha near its carboxyl terminus for the treatment of bladder cancer.

The epidermal growth factor receptor (EGFR) is overexpressed on the superficial layers of malignant urothelium and is suspected of playing a role in tumor progression. TP40 is a chimeric protein composed of transforming growth factor-alpha (TGF alpha) fused to a modified form of Pseudomonas exotoxin A (PE) that is selectively cytotoxic to EGFR-bearing cells and is currently undergoing clinical study for the intravesical therapy of bladder cancer. We constructed a recombinant toxin PE35/TGF alpha-KDEL as an improved agent for the local therapy of EGFR-bearing bladder cancer. PE35/TGF alpha-KDEL does not require intracellular proteolysis to generate a carboxyl-terminal fragment capable of reaching the target cell cytosol and contains a modified carboxyl-terminal sequence KDEL, that increases toxin activity. These features make PE35/TGF alpha-KDEL from 10- to 700-fold more potent than TP40 on four human bladder cancer cell lines. PE35/TGF alpha-KDEL may be a useful agent for treatment of EGFR-bearing cancers.

ADP Ribose Transferases↗

PubMed1993

Cytotoxic activities of recombinant immunotoxins composed of Pseudomonas toxin or diphtheria toxin toward lymphocytes from patients with adult T-cell leukemia.

We have previously shown that the recombinant single-chain immunotoxin anti-Tac(Fv)-PE40, composed of the variable domains of the anti-Tac monoclonal antibody in a single-chain form joined to a derivative of Pseudomonas exotoxin (PE), is cytotoxic toward malignant cells from adult T-cell leukemia (ATL) patients. Using this assay, we have now compared the activity of anti-Tac(Fv)-PE40 with that of an improved version, anti-Tac(Fv)-PE40KDEL which contains an altered carboxyl terminus, and also with two chimeric toxins made with diphtheria toxin (DT). One of these is a fusion of amino acids 1-388 of DT with anti-Tac(Fv) and is termed DT388-anti-Tac(Fv). The other, DT388-IL2, contains interleukin 2 (IL2) at the carboxyl terminus of the same DT derivative. We incubated these toxins with malignant ATL peripheral blood mononuclear cells (PBMCs) for 1-3 days and then measured [3H]leucine incorporation. We found that anti-Tac(Fv)-PE40KDEL was the most cytotoxic agent and was followed in decreasing order of activity by anti-Tac(Fv)-PE40, DT388-anti-Tac(Fv), and finally DT388-IL2. Trypan blue staining showed that inhibition of protein synthesis correlated with cell death. Time course studies showed that the recombinant toxins containing anti-Tac(Fv) were cytotoxic even if exposed to the cells for only one hour. After intravenous injection into mice, the half-life of anti-Tac(Fv)-PE40 or anti-Tac(Fv)-PE40KDEL was 30 minutes. Normal PBMCs were resistant to all four toxins. Recombinant immunotoxins made with anti-Tac merit further study as potential reagents in the treatment of ATL.

Adult↗

PubMed1992

Cell-mediated cleavage of Pseudomonas exotoxin between Arg279 and Gly280 generates the enzymatically active fragment which translocates to the cytosol.

Pseudomonas exotoxin (PE) is a three-domain toxin which is cleaved by a cellular protease within cells and then reduced to generate two prominent fragments (Ogata, M., Chaudhary, V. K., Pastan, I., and FitzGerald, D. J. (1990) J. Biol. Chem. 265, 20678-20685). The N-terminal fragment is 28 kDa in size and contains the binding domain. The 37-kDa C-terminal fragment, which translocates to the cytosol, contains the translocation domain and the ADP-ribosylation domain. Cleavage followed by reduction is essential for toxicity since mutant forms of the toxin that cannot be cleaved by cells are nontoxic. Previous results with these mutants suggest that cleavage occurred in an arginine-rich (arginine residues are at positions 274, 276, and 279) disulfide loop near the beginning of the translocation domain, but the exact site of cleavage was not determined. Since very few molecules of the 37-kDa fragment are generated within cells it was not possible to determine the site of cleavage by performing a conventional N-terminal sequence analysis of the 37-kDa fragment. Two experimental approaches were used to overcome this limitation. First, existing amino acids near the cleavage sites were replaced with methionine residues; this was followed by the addition of [35S]methionine-labeled versions of these toxins to cells. The pattern of radioactive toxin fragments recovered from the cells indicated that the toxin was cleaved either just before or just after Arg279. Second, [3H]leucine-labeled toxin was produced and added to the cells. Sequential Edman degradations were performed on the small amount of radioactive 37-kDa fragment that could be recovered from toxin-treated cells. A peak of radioactivity in the fifth fraction indicated that leucine was the 5th amino acid on the C-terminal side of the cleavage site. This result confirmed that cleavage was between Arg279 and Gly280.

ADP Ribose Transferases↗

PubMed1992

Recombinant toxins containing the variable domains of the anti-Tac monoclonal antibody to the interleukin-2 receptor kill malignant cells from patients with chronic lymphocytic leukemia.

We have previously shown that the variable domains of the monoclonal antibody anti-Tac [anti-Tac(Fv)] can be fused to derivatives of Pseudomonas exotoxin (PE) or diphtheria toxin (DT) to produce recombinant immunotoxins that kill interleukin-2 (IL-2) receptor-bearing cells. We now report that two of these single-chain recombinant immunotoxins, anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), are cytotoxic toward peripheral blood mononuclear cells (PBMCs) from patients with chronic lymphocytic leukemia (CLL). In anti-Tac(Fv)-PE40KDEL, anti-Tac(Fv) is genetically fused to the amino terminus of PE40KDEL, a recombinant form of PE which contains amino acids 253-608 of PE and the -KDEL mutation at the carboxyl terminus. In DT388-anti-Tac(Fv), anti-Tac(Fv) is fused to the carboxyl terminus of the first 388 amino acids of DT. PBMCs from 14 patients were incubated with the recombinant toxins for 60 hours, and [3H]-leucine incorporation was measured. Anti-Tac(Fv)-PE40KDEL was cytotoxic to 7 of the 14 patient samples, with half-maximal inhibition of protein synthesis (IC50) achieved at 1.2 to 9 ng/mL (1.8 to 13 x 10(-11) mol/L). DT388-anti-Tac(Fv) was cytotoxic to 11 of the 14 samples, with IC50s ranging from less than 1 to 250 ng/mL. DT388-IL-2, in which the first 388 amino acids of DT are attached to IL-2, was marginally cytotoxic toward only 4 of 13 CLL samples tested with IC50s ranging from 100 to 550 ng/mL. Trypan blue staining of cells from several patients indicated that inhibition of protein synthesis correlated with cell death. Binding assays using [3H]-anti-Tac indicated that the CLL cells from nine of the patients contained between 400 and 2,500 sites per cell. Cells from another patient, which were resistant to both anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), had less than 100 sites per cell. We conclude that anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv) can kill CLL cells which have low numbers of IL-2 receptors, and should be investigated further for therapy of this disease.

ADP Ribose Transferases↗

PubMed1992

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor. Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.

ADP Ribose Transferases↗