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Purpose: The novel fusion protein, DAB389EGF, is composed of both the catalytic and the translocation domains of diphtheria toxin that are fused to the human EGF, providing a targeting and a toxicity component. We tested DAB389EGF for antitumor activity in both in vitro and in vivo urinary bladder cancer models. Experimental Design: Human bladder cancer lines were treated with DAB389EGF and assessed for growth inhibition and clonogenic suppression. Using 6- to 8-week-old female athymic nude mice implanted orthotopically with HTB9 cells, DAB389EGF was administered intravesically twice weekly for 2 weeks. The response of the luciferase-expressing HTB9 cells was monitored via bioluminescence as the primary endpoint. Results: Treatment response with DAB389EGF was specific and robust, with an IC50 ranging from 0.5 to 15 ng/mL in eight tested bladder cancer cell lines, but greater than 50 ng/mL in the EGF receptor (EGFR)-negative H520 control cell line. Simulating short-duration intravesical therapy used clinically, a 2-hour treatment exposure of DAB389EGF (10 ng/mL) produced clonogenic suppression in three selected bladder cancer cell lines. In vivo, luciferase activity was suppressed in five of six mice treated with DAB389EGF [70 μL (1 ng/μL) per mouse], as compared with only one of six mice treated with a control diphtheria toxin (DT) fusion protein. Histologic assessment of tumor clearance correlated with the bioluminescent changes observed with DAB389EGF treatment. Immunocompetent mice treated with intravesical DAB389EGF did not show any nonspecific systemic toxicity. Conclusions: The intravesical delivery of targeted toxin fusion proteins is a novel treatment approach for non–muscle-invasive urinary bladder cancer. With appropriate targeting, the treatments are effective and well-tolerated in vivo. Clin Cancer Res; 19(1); 148–57. ©2012 AACR.

RLIP76 is a multifunctional membrane protein that transports glutathione conjugates of electrophilic compounds and other xenobiotics including chemotherapy agents out of cells. The protein is overexpressed in lung carcinomas, ovarian carcinomas, and melanomas. The protein also binds Ral and participates in mitotic spindle function, clathrin-dependent endocytosis, and triggers GTPase-activating protein activity. It is found throughout the cell, in membrane, cytosol, and the nucleus, and is known to shift between these compartments in response to stress. Loss of RLIP76 by antibody or antisense therapy is associated with increased sensitivity to radiation and chemotherapy. Conversely, liposomally delivered RLIP may treat poisoning and wounds.

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.

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.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.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.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.

Patients with relapsed or refractory chronic lymphocytic leukemia (CLL) have a poor prognosis. We tested the safety and efficacy in these patients of a diphtheria fusion protein DAB(389)IL2 (denileukin diftitox) directed against the interleukin 2 receptor that is expressed by CLL cells.DAB(389)IL2 was administered by 60 min i.v. infusions for 5 days every 21 days at 9 or 18 micro g/kg/day for up to eight cycles.Eighteen patients were treated. The mean age of the patients was 61.8 years. There were 14 males and 4 females. Two had Rai stage I, 6 had Rai stage II, and 10 had Rai stage IV. The mean number of prior treatments was 4.5 (range, 1-11). Responses were evaluated by peripheral CLL counts, computed tomography scans of all nodes and bone marrow biopsies. Twelve patients received greater than or equal to three cycles of DAB(389)IL2 and were evaluable for response. Eleven of 12 patients showed reductions of peripheral CLL cells, with 6 of 11 showing/==" BORDER="0"95% reductions. Seven of 12 patients showed reductions of node diameters on exam and computed tomography scans, and 2 of 12 showed 60 and 80% shrinkage, respectively. Pre and postbone marrow biopsies showed a reduction in CLL marrow index in 11 patients. Seven of 11 patients had50% reduction, including/==" BORDER="0"98% reduction in 3 patients. DAB(389)IL2 produced 2 of 12 (17%) partial remission and 7 of 12 (58%) minimal responses. Progression-free intervals in the responders were 1, 1, 3+, 4, 9, 10, 10+, 14 and 19+ months. Toxicities were mild to moderate and included asymptomatic, transient transaminasemia, fever, asthenia, hypoalbuminemia, nausea, vomiting, myalgias, rash, anorexia, vascular leak syndrome, and elevated creatinine kinase. Antidiphtheria toxin antibody levels were variable and ranged from 0 to 9 micro g/ml (n = 5).DAB(389)IL2 produced a rapid decrease of leukemic cells in the bone marrow and peripheral blood of most chemotherapy refractory CLL patients. Most patients also tolerated DAB(389)IL2 well, without significant myelosuppression and/or immunosuppression. The prolonged progression-free interval and subjectively observed quality-of-life in responders is intriguing. The results suggest DAB(389)IL2 has biological activity in patients with B-cell CLL. Follow-up studies of combinations or altered schedules or doses to improve the response rate are warranted.

We examined the mechanism of action of a targeted fusion toxin consisting of diphtheria toxin fused to granulocyte macrophage colony stimulating factor (GMCSF) (DT(388)-GMCSF), which was designed to selectively kill acute myeloid leukemia cells.U937 cells treated with DT(388)-GMCSF underwent apoptosis as shown by chromatin degradation and cellular and nuclear fragmentation. This apoptosis was prevented by a general caspase inhibitor. DT(388)-GMCSF treatment resulted in activation of the initiator caspases 8 and 9 and effector caspases. A selective caspase 8 inhibitor prevented activation of caspase 9, whereas a selective caspase 9 inhibitor did not prevent activation of caspase 8, indicating that caspase 8 activation is the proximal event in DT(388)-GMCSF-induced apoptosis. Caspase 8 was activated through a Fas-associated death domain protein (FADD)-dependent mechanism as demonstrated by inhibition of DT(388)-GMCSF-induced apoptosis on expression of a dominant negative FADD molecule. However, unlike most FADD-dependent apoptosis, this pathway may not involve death receptors, including Fas, tumor necrosis factor receptor 1, or tumor necrosis factor-related apoptosis-inducing ligand receptors, because inhibitors of the receptors did not prevent DT(388)-GMCSF-induced apoptosis.These data indicate that targeted toxins induce apoptosis by activating components of the death receptor pathway in a receptor-independent manner.