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

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3530-3537 ◽  
Author(s):  
Leman Yalcintepe ◽  
Arthur E. Frankel ◽  
Donna E. Hogge
Keyword(s):  
Acute Myeloid Leukemia ◽  
Fusion Protein ◽  
Diphtheria Toxin ◽  
Myeloid Leukemia ◽  
Relative Level ◽  
Immunodeficient Mice ◽  
Interleukin 3 ◽  
Qrt Pcr ◽  
Cell Fractions ◽  
Acute Myeloid

AbstractThe 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-3Rα and βc 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 (DT388IL3). Among 19 patient samples, the relative level of the IL-3Rα was higher than the IL-3Rβc 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-3Rβc 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-3Rβc 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 DT388IL3 to kill AML progenitors that engraft in β2-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.

Induction of remission in patients with acute myeloid leukemia without prolonged myelosuppression using diphtheria toxin-interleukin 3 fusion protein

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 7068-7068 ◽  
Author(s):  
A. E. Frankel ◽  
M. A. Weir ◽  
P. D. Hall ◽  
M. Holguin ◽  
C. Cable ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Fusion Protein ◽  
Diphtheria Toxin ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Cell Transplant ◽  
Interleukin 3 ◽  
History Of ◽  
Acute Myeloid

7068 The recombinant diphtheria toxin fusion protein, DT388IL3, composed of the catalytic and translocation domains of diphtheria toxin (DT388) fused to human interleukin-3 (IL3) showed selective cytotoxicity to acute myeloid leukemia (AML) stem cells both in vitro and in vivo and was prepared for a phase I clinical study (Urieto, Protein Exp Purif 33, 123, 2004). FDA approval (BB IND#11314) and IRB approvals were obtained. Seventy-five AML patients were screened and thirty-one patients treated. The median age of treated patients was 62 years (range, 25- 81 years). There were sixteen males and fifteen females. Disease was de novo in three, first relapse in ten, second relapse in eight, and refractory in ten patients. Four patients had a history of MDS, and one had a history of secondary AML. One patient each had previously received an autologous or allogeneic stem cell transplant. Cytogenetics were unfavorable in ten, intermediate in nineteen, and not done in two. Seven patients were treated with 4 μg/kg, eight patients were treated with 5.3 μg/kg, thirteen patients treated with 7.1 μg/kg, and three patients treated with 9.4 μg/kg DT388IL3. Drug-related toxicities were mild to moderate and transient including fever, chills, hypotension, hypoxemia, and hypoalbuminemia. Consistent with an absence of toxicity to normal hematopoietic progenitors, responses occurred in the absence of prolonged myelosuppression. Among thirty evaluable patients, we have observed one CR of 8 months duration, two partial remissions (PRs) lasting one and three months and three minimal responses with clearance of peripheral blasts and marrow blast cytoreductions of 89%, 90% and 93% lasting one to two months. Dose escalation is proceeding. No significant financial relationships to disclose.

scholarly journals 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

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2527-2529 ◽  
Author(s):  
Ugo Testa ◽  
Roberta Riccioni ◽  
Mauro Biffoni ◽  
Daniela Diverio ◽  
Francesco Lo-Coco ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Diphtheria Toxin ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Biochemical Parameter ◽  
Cell Killing ◽  
Receptor Expression ◽  
Interleukin 3 ◽  
Selection Of ◽  
Acute Myeloid

Abstract Leukemic blasts from patients with acute myeloid leukemia (AML) frequently express high levels of the interleukin-3 receptor α chain (IL-3Rα). 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-3Rα/IL-3Rβ 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. (Blood. 2005;106:2527-2529)

A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia

Blood ◽  
2001 ◽  
Vol 98 (4) ◽  
pp. 1166-1173 ◽  
Author(s):  
Gerald G. Wulf ◽  
Rui-Yu Wang ◽  
Ingrid Kuehnle ◽  
Douglas Weidner ◽  
Frank Marini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Side Population ◽  
Fluorescence Emission ◽  
Drug Efflux ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Acute Myeloid

The hematopoietic stem cell underlying acute myeloid leukemia (AML) is controversial. Flow cytometry and the DNA-binding dye Hoechst 33342 were previously used to identify a distinct subset of murine hematopoietic stem cells, termed the side population (SP), which rapidly expels Hoechst dye and can reconstitute the bone marrow of lethally irradiated mice. Here, the prevalence and pathogenic role of SP cells in human AML were investigated. Such cells were found in the bone marrow of more than 80% of 61 patients and had a predominant CD34low/− immunophenotype. Importantly, they carried cytogenetic markers of AML in all 11 cases of active disease examined and in 2 out of 5 cases in complete hematological remission. Comparison of daunorubicin and mitoxantrone fluorescence emission profiles revealed significantly higher drug efflux from leukemic SP cells than from non-SP cells. Three of 28 SP cell transplants generated overt AML-like disease in nonobese diabetic–severe combined immunodeficient mice. Low but persistent numbers of leukemic SP cells were detected by molecular and immunological assays in half of the remaining mice. Taken together, these findings indicate that SP cells are frequently involved in human AML and may be a target for leukemic transformation. They also suggest a mechanism by which SP cells could escape the effects of cytostatic drugs and might eventually contribute to leukemia relapse.

Targeting of a B7-1 (CD80) immunoglobulin G fusion protein to acute myeloid leukemia blasts increases their costimulatory activity for autologous remission T cells

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3138-3145 ◽  
Author(s):  
Michael Notter ◽  
Tim Willinger ◽  
Ulrike Erben ◽  
Eckhard Thiel
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Fusion Protein ◽  
Immunoglobulin G ◽  
Myeloid Leukemia ◽  
Costimulatory Molecule ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
Acute Myeloid

Abstract Transfection of tumor cells with the gene encoding the costimulatory molecule B7-1 (CD80), the ligand for CD28 and cytotoxic T lymphocye antigen-4 on T cells, has been shown to result in potent T-cell–mediated antitumor immunity. As an alternative approach, this study analyzed the costimulatory capacity of a human B7-1 immunoglobulin G (IgG) fusion protein targeted to the cell membrane of human acute myeloid leukemia (AML) blasts. Flow cytometric analysis revealed a low constitutive expression of B7-1 on human AML blasts (on average, 3.0 ± 4.3%; n = 50). In contrast, the expression of B7-2 (CD86) was highly heterogeneous and higher in AML blasts of French-American-British classification types M4 and M5 (P < .0001). The B7-1 IgG fusion protein used in this study efficiently costimulated the proliferation of resting and preactivated T cells when immobilized on plastic. After preincubation with B7-1 IgG, specific binding of the fusion protein to the high-affinity Fcγreceptor I (CD64) on leukemic cells was demonstrated and was found to increase the proliferation of both allogeneic and autologous T cells in costimulation experiments. Furthermore, targeting of B7-1 IgG to the tumor membrane resulted in increased proliferation of autologous remission T cells and had the potential to generate an enhanced redirected cytotoxic T-cell response against autologous AML blasts. In summary, the targeting of B7-1 IgG fusion protein described in this study represents a strategy alternative to gene therapy to restore the expression of the costimulatory molecule B7-1 on human AML blasts, thereby enhancing their immunogenicity for autologous T cells. This new approach may have implications for T-cell–mediated immunotherapy in AML.

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