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Biomedical subjects

Arthur E Frankel

Publications and source records attributed to Arthur E Frankel.

At least 19 recordsLinked to original sources
PubMed2006

Systemic anthrax lethal toxin therapy produces regressions of subcutaneous human melanoma tumors in athymic nude mice.

PURPOSE: Anthrax Lethal Toxin (LeTx), composed of protective antigen and lethal factor, catalytically cleaves mitogen-activated protein kinase (MAPK) kinases and inhibits the MAPK signaling pathways. The majority of metastatic melanomas possess the V599E BRAF mutation, which constitutively activates MAPK1/2 signaling. LeTx is cytotoxic to BRAF mutant melanoma cell lines in vitro, whereas most normal cells are resistant to this toxin. In this study, we determine the in vivo potency and safety of systemically administered LeTx. EXPERIMENTAL DESIGN: A s.c. xenograft melanoma model in athymic nude mice was treated with different i.p. doses of LeTx. RESULTS: In this study, we show that in vivo systemic LeTx treatment of s.c. xenograft melanoma tumors in athymic nude mice yields partial and complete tumor regressions with minor toxicity to mice. When animal toxicity was observed, we did not find any histologic evidence of tissue damage. CONCLUSIONS: LeTx is one of the rare targeted agents to produce complete remissions of human melanomas in an animal model and thus warrants further preclinical development.

Animals↗
PubMed2006

Expression of interleukin-3 receptor subunits on defined subpopulations of acute myeloid leukemia blasts predicts the cytotoxicity of diphtheria toxin interleukin-3 fusion protein against malignant progenitors that engraft in immunodeficient mice.

The interleukin-3 receptor (IL-3R) subunits are overexpressed on acute myeloid leukemia (AML) blasts compared with normal hematopoietic cells and are thus potential targets for novel therapeutic agents. Both fluorescence-activated cell sorter (FACS) analysis and quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) were used to quantify expression of the IL-3Ralpha and beta(c) subunits on AML cells. QRT-PCR for both subunits was most predictive of killing of AML colony-forming cells (AML-CFCs) by diphtheria toxin-IL-3 fusion protein (DT(388)IL3). Among 19 patient samples, the relative level of the IL-3Ralpha was higher than the IL-3Rbeta(c) and highest in CD34(+)CD38(-)CD71(-) cells, enriched for candidate leukemia stem cells, compared with cell fractions depleted of such progenitors. Overall, the amount of IL-3Rbeta(c) subunit did not vary among sorted subpopulations. However, expression of both subunits varied by more than 10-fold among different AML samples for all subpopulations studied. The level of IL-3Rbeta(c) expression versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (set at 1000) ranged from 0.14 to 13.56 in CD34(+)CD38(-)CD71(-) cells from different samples; this value was correlated (r = .76, P = .05) with the ability of DT(388)IL3 to kill AML progenitors that engraft in beta(2)-microglobin-deficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n = 7). Thus, quantification of IL-3R subunit expression on AML blasts predicts the effectiveness IL-3R-targeted therapy in killing primitive leukemic progenitors.

Acute Disease↗
PubMed2006

Phase II clinical studies of denileukin diftitox diphtheria toxin fusion protein in patients with previously treated chronic lymphocytic leukemia.

BACKGROUND: The safety and efficacy of the interleukin-2 diphtheria toxin fusion protein (DAB(389)IL2; denileukin diftitox) directed against the IL-2 receptor (IL-2R) was tested in patients with recurrent or refractory chronic lymphocytic leukemia (CLL). METHODS: Denileukin diftitox was administered as 60-minute intravenous infusions for 5 days every 21 days at a dose of 18 mug/kg per day for up to 8 cycles. In total, 28 patients were treated in 2 multiinstitutional studies with similar eligibility criteria and treatment protocols. Twenty-two patients receive > or = 2 cycles of denileukin diftitox and were evaluable for response. RESULTS: Twelve of 22 patients achieved reductions of peripheral CLL cells, with 5 of 12 patients achieving >80% reductions. Six of 22 patients achieved reductions in the size of lymph node on examination and computed tomography scans, and all 6 of those patients met the criteria for a partial or complete response that lasted > or = 2 months. Bone marrow biopsies before and after treatment confirmed a complete remission that lasted for 1 year in 1 patient. Overall, denileukin diftitox produced complete remission in 1 of 22 patients (4%) and partial remission in 5 of 22 patients (23%) for a total remission rate of 27%. Progression-free intervals in the responders were 2 months in 2 patients and 4 months, 6 months, 7 months, and 12 months in 1 patient each. Toxicities were moderate. No infections associated with immunosuppression were seen. There was no significant correlation of response or toxicities with the numbers of denileukin diftitox cycles received or with CD25 levels. CONCLUSIONS: Follow-up studies will be required to identify predictors of response that may improve the response rate to denileukin diftitox in patients with CLL.

Aged↗
PubMed2006

Variant diphtheria toxin-interleukin-3 fusion proteins with increased receptor affinity have enhanced cytotoxicity against acute myeloid leukemia progenitors.

A fusion protein linking a truncated form of diphtheria toxin (DT(388)) to human interleukin-3 (DT(388)IL3) kills malignant progenitors from some patients with acute myeloid leukemia (AML) while sparing normal progenitors. This study evaluated two variants of DT(388)IL3 with increased affinity for the IL-3 receptor (IL-3R) for their cytotoxicity to AML progenitors and determined the ability of quantitative reverse transcription-PCR assessment of expression of the IL-3R subunits to predict the effectiveness of wild-type DT(388)IL3 and its variants. Both the IL-3 deletion variant (Delta125-133) and the amino acid substitution variant (K116W) showed enhanced toxicity against AML colony-forming cells (AML-CFC; but not normal CFC) compared with wild-type DT(388)IL3 with the K116W variant achieving >90% AML-CFC kill with 17 of 23 patient samples. This variant was also more effective against AML cells engrafting in nonobese diabetic severe combined immunodeficient mice. There was a significant correlation between the expression of the alpha and, particularly, the common beta subunit of the IL-3R on AML blasts detected by quantitative reverse transcription-PCR and AML-CFC kill. Thus, the combined use of IL-3R expression to select patients most likely to respond to DT(388)IL3 and the improved cytotoxicity of the K116W DT(388)IL3 variant against leukemic progenitors may enhance the clinical usefulness of these fusion proteins.

Adolescent↗
PubMed2006

Fresh frozen plasma and platelet concentrates may increase plasma anti-diphtheria toxin IgG concentrations: Implications for diphtheria fusion protein therapy.

We are developing two fusion proteins consisting of a diphtheria toxin (DT) linked to either granulocyte macrophage colony stimulating factor (DT388-GMCSF) or interleukin-3 (DT388-IL3). In trials, patients with anti-DT IgG concentrations >2.5 microg/ml had significantly lower concentrations of either fusion protein. DT389-IL2 is currently FDA approved for the treatment of cutaneous T-cell lymphomas. We noted increased concentrations of anti-DT IgG after administration of platelet concentrates (PC). Because many of these patients require transfusions, we measured the anti-DT IgG content of FFP and PC. We assayed 14 bags of FFP and 12 bags of single-donor PCs for anti-DT IgG by an enzymoimmunoassay against DT389-IL2, DT388-IL3, DT388-GMCSF. The median (percent of samples positive) of anti-DT IgG concentrations in PC against DT388-GMCSF, DT388-IL3, DT389-IL2 was 0.8 microg/ml (83%), 0.7 microg/ml (83%), and 0.3 microg/ml (58%), respectively. The median (percent of samples positive) anti-DT IgG concentration in FFP against DT388-GMCSF, DT388-IL3, and DT389-IL2 was 2.1 microg/ml (86%), 1.9 microg/ml (93%), and 1.4 microg/ml (86%), respectively. There was a strong association between anti-DT389IL2 IgG, anti-DT388IL3 IgG, and anti-DT388GMCSF IgG concentrations in both the FFP (95.6%) and PC (76.3%). Assuming a plasma volume of 3 l in a 70 kg patient, a single FFP unit would increase the plasma anti-DT389IL2 IgG, anti-DT388IL3 IgG, and anti-DT388GMCSF IgG by 0.13 microg/ml, 0.17 microg/ml, and 0.19 microg/ml, respectively. For PC, a single unit would increase plasma anti-DT389-IL2 IgG, anti-DT388-IL3 IgG, and anti-DT388-GMCSF IgG by 0.03 microg/ml, 0.06 microg/ml, and 0.07 microg/ml, respectively. In conclusion, a single FFP or PC appears to minimally increase anti-DT IgG concentrations, but multiple units may significantly do such.

Blood Platelets↗
PubMed2006

A urokinase-activated recombinant anthrax toxin is selectively cytotoxic to many human tumor cell types.

Urokinase plasminogen activator (uPA) is a tumor-specific protease highly expressed in several types of solid tumors and rarely present on normal cells under physiologic conditions. Due to its high expression on metastatic tumors, several different strategies have been used to target the urokinase system. These have mostly led to tumor growth inhibition rather than tumor regression. A different approach was adopted by replacing the furin activation site on a recombinant anthrax toxin with a urokinase activation site. The resulting toxin, PrAgU2/FP59, was highly potent against tumors both in vitro and in vivo. In this study, we show that PrAgU2/FP59 is toxic to a wide range of tumor cell lines, including non-small cell lung cancer, pancreatic cancer, and basal-like breast cancer cell lines. Of the few cell lines found to be resistant to PrAgU2/FP59, most became sensitive upon addition of exogenous pro-uPA. PrAgU2/FP59 was much less toxic to normal human cells. The potency of PrAgU2/FP59 was dependent on anthrax toxin receptor, uPA receptor, and uPA levels but not on total plasminogen activator inhibitor-1 levels. In this study, we show that PrAgU2/FP59 is a wide-range, highly potent, and highly selective toxin that is capable of specifically targeting uPA-expressing tumor cells, independently of the tissue of origin of these cells. Furthermore, we identify three molecular markers, anthrax toxin receptor, uPA, and uPA receptor, which can be used as predictors of tumor cell sensitivity to PrAgU2/FP59.

Antigens, Bacterial↗
PubMed2005

Diphtheria toxin fused to variant human interleukin-3 induces cytotoxicity of blasts from patients with acute myeloid leukemia according to the level of interleukin-3 receptor expression.

Leukemic blasts from patients with acute myeloid leukemia (AML) frequently express high levels of the interleukin-3 receptor alpha chain (IL-3Ralpha). In the present study, we have explored the sensitivity of primary leukemic blasts obtained from 34 patients with AML to a diphtheria toxin (DT) composed of the catalytic and translocation domains of DT (DT388) fused to IL-3 (DT388IL-3) and to DT388 fused to a variant IL-3 with increased binding affinity (DT388IL-3[K116W]). On a molar basis, DT388IL-3[K116W] was significantly more active than DT388IL-3 in mediating leukemic cell killing. The rate of cell killing induced by the 2 DT/IL-3 fusion proteins was significantly correlated with the level of IL-3Ralpha/IL-3Rbeta expressed on leukemic blasts. These observations support a potential use of DT388IL-3[K116W] in the treatment of refractory AMLs and provide a simple biochemical parameter for the selection of eligible patients.

Antineoplastic Agents↗
PubMed2005

CLL immunotoxins.

Immunotoxins composed of cell-selective ligands covalently linked to peptide toxins have been developed for the treatment of chemotherapy relapsed or refractory malignancies including chronic lymphocytic leukemia (CLL). A number of CLL immunotoxins have been clinically tested including T101-ricin A, H65-ricin A, DAB(486)IL2, DAB(389)IL2, RFB4 (dsFv)-PE38 and anti-Tac(Fv)-PE38. Remissions have occurred in some patients without significant myelosuppression, but novel agents continue to be needed. Kay and co-workers in this issue of Leukemia Research have targeted interleukin-4 receptors (IL4R) on CLL B cells with a recombinant IL4 pseudomonas exotoxin fusion protein (IL-4(38--37)-PE38KDEL). A fraction of patients (19%) had CLL cells that were extremely sensitive to the immunotoxin. This novel agent may provide an important new therapeutic for use in the treatment of CLL.

Antineoplastic Agents↗
PubMed2005

Interstitial diphtheria toxin-epidermal growth factor fusion protein therapy produces regressions of subcutaneous human glioblastoma multiforme tumors in athymic nude mice.

PURPOSE: The novel fusion protein, DAB389EGF, composed of the catalytic and translocation domains of diphtheria toxin (DAB389) fused with a His-Ala linker to human epidermal growth factor (EGF) was tested for antiglioma efficacy in an in vivo model of human glioma. EXPERIMENTAL DESIGN: Female athymic nude mice (ages 4-6 weeks) were inoculated s.c. with 10 million U87MG human glioma cells in the right flank. When tumor volumes reached approximately 100 mm3 (approximately 6-8 days), i.t. injections of saline, DAB389IL2, or DAB389EGF 1, 3, 5 or 10 microg in 50 microL were given every other day for three to six doses. Animals were monitored twice daily and tumor measurements were made by calipers. RESULTS: The maximal tolerated dose (MTD) of DAB389EGF was 3 microg every other day. Above the MTD, animals experienced loss of activity, reduced oral intake, and dehydration. Blood chemistries confirmed elevated blood urea nitrogen, creatinine, aspartate transaminase, and alanine transaminase. Histopathology revealed renal tubular necrosis. At the MTD, tumor regression was seen in all animals. Relapses occurred in 4 of 16 (25%) of animals after 1 month. These tumors contained EGF receptor, were sensitive in vitro to DAB389EGF, and responded to a second course of i.t. DAB389EGF. CONCLUSIONS: DAB389EGF fusion protein shows in vivo antiglioma efficacy in a s.c. tumor model and warrants further preclinical testing in an i.c. tumor model for eventual treatment of patients with recurrent or refractory EGF receptor-positive glioblastoma multiforme.

Animals↗
PubMed2005

Toxin conjugate therapy of cancer.

Standard approaches to the treatment of malignancies include surgery, chemotherapy, and radiation. More recently, with understanding of these processes in molecular detail, biologics have become another important aspect of treatment. Conjugate toxins are biologics that combine a potent toxin with a peptide that will target cancer cells with minimal damage to normal tissues. This review will detail examples of these conjugate toxins and their targeted malignancies.

Bacterial Toxins↗
PubMed2005

BRAF status and mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 activity indicate sensitivity of melanoma cells to anthrax lethal toxin.

Anthrax lethal toxin, composed of protective antigen and lethal factor, was tested for cytotoxicity to human melanoma cell lines and normal human cells. Eleven of 18 melanoma cell lines were sensitive to anthrax lethal toxin (IC(50) < 400 pmol/L) and 10 of these 11 sensitive cell lines carried the V599E BRAF mutation. Most normal cell types (10 of 15) were not sensitive to anthrax lethal toxin and only 5 of 15 normal human cell types were sensitive to anthrax lethal toxin (IC(50) < 400 pmol/L). These cells included monocytes and a subset of endothelial cells. In both melanoma cell lines and normal cells, anthrax toxin receptor expression levels did not correlate with anthrax lethal toxin cytotoxicity. Furthermore, an anthrax toxin receptor-deficient cell line (PR230) did not show any enhanced sensitivity to anthrax lethal toxin when transfected with anthrax toxin receptor. Anthrax lethal toxin toxicity correlated with elevated phosphorylation levels of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 in both melanoma cell lines and normal cells. Anthrax lethal toxin-sensitive melanoma cell lines and normal cells had higher phospho-MEK1/2 levels than anthrax lethal toxin-resistant melanoma cell lines and normal tissue types. U0126, a specific MEK1/2 inhibitor, was not toxic to anthrax lethal toxin-resistant melanoma cell lines but was toxic to 8 of 11 anthrax lethal toxin-sensitive cell lines. These results show that anthrax lethal toxin toxicity correlates with elevated levels of active MEK1/2 pathway but not with anthrax toxin receptor expression levels in both normal and malignant tissues. Anthrax lethal toxin may be a useful therapeutic for melanoma patients, especially those carrying the V599E BRAF mutation with constitutive activation of the mitogen-activated protein kinase pathway.

Antigens, Bacterial↗
PubMed2004

Safety evaluation of DT388IL3, a diphtheria toxin/interleukin 3 fusion protein, in the cynomolgus monkey.

We developed a fusion toxin, DT388IL3, consisting of the catalytic and translocation domains of diphtheria toxin (DT388) linked to interleukin 3 (IL3) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to estimate a range for the maximum tolerated dose (MTD) and to evaluate the dose-limiting toxicity (DLT) of DT388IL3 in cynomolgus monkeys (Macaca fasicularis), which possess cross-reactive IL3 receptors. In our previous study, we administered up to six infusions of DT388IL3 at 40, 60, or 100 microg/kg every other day to three pairs (one male monkey and one female monkey) of young adult monkeys. In five of six monkeys, results showed a dose-dependent increase in malaise and anorexia but no consistent abnormalities in serum chemistries or blood counts. There was no evidence of organ damage by blood tests or histopathology. However, the female treated at 100 microg/kg, died of moderate to severe vasculitis of multiple tissues. Based on these findings, this study repeated the 100 microg/kg group and added a group that received 150 microg/kg in an effort to confirm a dose response. Two female monkeys were treated with up to six infusions of DT388IL3 at 100 microg/kg or 150 microg/kg every other day. One additional female monkey was treated as a negative control. Monkeys in the 100 microg/kg group showed moderate malaise and anorexia, but no consistent abnormalities in blood counts or serum chemistries. Moderate elevations of liver enzymes were noted in the 150 microg/kg group in addition to severe malaise and anorexia. No significant findings were revealed at gross necropsy. The histopathological findings revealed regenerative myeloid hyperplasia and hepatic degeneration and regeneration in the 150 microg/kg group. Similar lesions of less severity were detected in the 100 microg/kg group. DT388IL3 plasma half-life was approximately 20 min with a peak concentration of approximately 2 microg/ml (30,000 pM). The IC50 for AML blasts in vitro was 6 pM. Collectively, our results suggest that DT388IL3 can be tolerated at doses up to 100 microg/kg in a nonhuman primate, which is higher than previously reported for other AML directed diphtheria toxin fusion proteins, and should in principle allow for dose escalation with reduced toxic side effects. Based on these findings a phase I clinical trial has recently been initiated with DT388IL3 for the treatment of AML.

Animals↗
PubMed2004

A urokinase-activated recombinant diphtheria toxin targeting the granulocyte-macrophage colony-stimulating factor receptor is selectively cytotoxic to human acute myeloid leukemia blasts.

Novel agents to treat acute myeloid leukemia (AML) are needed with increased efficacy and specificity. We have synthesized a dual-specificity fusion toxin DTU2GMCSF composed of the catalytic and translocation domains of diphtheria toxin (DT) fused to the granulocyte-macrophage colony-stimulating factor (GM-CSF) in which the DT furin cleavage site 163RVRRSV170 is modified to a urokinase plasminogen activator (uPA) cleavage site 163GSGRSA170, termed U2. DTU2GMCSF was highly toxic to the TF1-vRaf AML cell line (proliferation inhibition assay; IC50 = 3.14 pM), and this toxicity was greatly inhibited following pretreatment with anti-uPA and anti-GM-CSF antibodies. The activity of this toxin was then tested on a larger group of 13 human AML cell lines; 5 of the 13 cell lines were sensitive to DTU2GMCSF. An additional 5 of the 13 cell lines became sensitive when exogenous pro-uPA was added. Sensitivity to DTU2GMCSF strongly correlated with the expression levels of uPA receptors (uPARs) and GM-CSF receptors (GM-CSFRs) as well as with total uPA levels. DTU2GMCSF was less toxic to normal cells expressing uPAR or GMCSFR alone, that is, human umbilical vein endothelial cells and peripheral macrophages, respectively. These results indicate that DTU2GMCSF may be a selective and potent agent for the treatment of patients with AML.

Binding Sites↗
PubMed2004

Acute myeloid leukemia cells in G0 phase of the cell cycle that are unresponsive to conventional chemotherapy are sensitive to treatment with granulocyte-macrophage colony-stimulating factor/diphtheria toxin fusion proteins.

OBJECTIVE: Unresponsiveness to chemotherapy is a major problem in the treatment of leukemia, which can be caused by unresponsiveness of noncycling cells to cell cycle-dependent cytotoxic agents. Targeted toxins consisting of a targeting and activating cytokine (granulocyte-macrophage colony-stimulating factor [GM-CSF]) and diphtheria toxin (DT) can be used to overcome this kind of resistance of leukemic cells. In this study we manipulated the cell cycle and proliferative status of leukemic cells, explored the effect on sensitivity to DT, and determined the ability of DT388GMCSF fusion proteins to activate and subsequently kill leukemic cells. MATERIALS AND METHODS: We used the GM-CSF-dependent myeloid leukemic cell line AML-193 as a model. GM-CSF or granulocyte colony-stimulating factor (G-CSF) was used to manipulate the cell cycle and proliferative state of AML-193 cells. Cell death was quantified by 51Cr release assays. The results obtained in the AML-193 cell line model were confirmed using primary leukemic blasts. RESULTS: Similar to treatment with chemotherapy and immunotherapy, leukemic cells in resting G0 phase were relatively resistant to DT-induced cell death. Synchronized recruitment of leukemic cells into activated phases of the cell cycle by low concentrations of GM-CSF or G-CSF resulted in significant increased DT sensitivity. DT388GMCSF fusion proteins specifically targeted GM-CSF receptor-expressing cells, resulting in recruitment of leukemic cells from G0 phase of the cell cycle and subsequent kill of these cells. CONCLUSION: Leukemic cells in G0 phase, which are resistant to conventional chemotherapy, Fas-induced immunotherapy, and DT alone, can be synchronically activated and subsequently killed by DT388GMCSF fusion proteins.

Cell Division↗