• chronic lymphocytic leukemia;
  • interleukin-2 receptor;
  • progression-free interval;
  • response evaluation;
  • toxin conjugates;
  • vascular leak syndrome


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  2. Abstract


The safety and efficacy of the interleukin-2 diphtheria toxin fusion protein (DAB389IL2; denileukin diftitox) directed against the IL-2 receptor (IL-2R) was tested in patients with recurrent or refractory chronic lymphocytic leukemia (CLL).


Denileukin diftitox was administered as 60-minute intravenous infusions for 5 days every 21 days at a dose of 18 μg/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 ≥2 cycles of denileukin diftitox and were evaluable for response.


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 ≥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.


Follow-up studies will be required to identify predictors of response that may improve the response rate to denileukin diftitox in patients with CLL. Cancer 2006. © 2006 American Cancer Society.

There are 10,000 new diagnoses of chronic lymphocytic leukemia (CLL) each year in the U.S.1 The majority of patients with CLL develop progressive disease and will require therapy for adenopathy, cytopenias, organomegaly, frequent infections, and/or systemic symptoms.2 Except for a minority of patients who are appropriate candidates for allogeneic stem cell transplantation,3 most patients receive alkylating agents, adenosine analogs, and rituximab or alemtuzumab either as single agents or in combinations. Responses are frequent; however, patients develop recurrent disease almost universally within a few years.4 The recent introduction of flavopiridol and oblimersen sodium to salvage regimens has yielded some responses and occasional prolonged response durations,5, 6 but these new salvage therapies rarely are curative. Novel agents with different mechanisms of action and different toxicities (less immunosuppression) are needed. One such class of compounds is toxin conjugates composed of ligands that are directed to cell surface receptors coupled to peptide toxins.7

The interleukin-2 (IL-2) diphtheria toxin fusion protein (DAB389IL2; denileukin diftitox or ONTAK®, Seragen, Inc., Hopkinton, MA) consists of an amino-terminal methionine (the first 386 amino acid residues of mature diphtheria toxin fused to amino acid residues 1-133 of human IL-2).8 The IL-2 moiety binds to high-affinity IL-2 receptors (IL-2Rs) that are expressed on activated normal and malignant lymphocytes.9 After receptor binding, the complex is internalized into endosomes. The diphtheria toxin moiety undergoes furin cleavage and acid-mediated insertion into the endosomal membrane. The catalytic domain of diphtheria toxin is transferred to the cytosol, in which it adenosine diphosphate-ribosylates elongation factor 2, leading to inactivation of protein synthesis and cell death.10 Selective killing of IL-2R–expressing cells has been demonstrated in vitro and in vivo.11

Denileukin diftitox at a dose from 3 μg/kg daily to 31 μg/kg daily for 5 days as 5-minute to 60-minute intravenous infusions was given to 222 patients with refractory hematologic malignancies.12–15 Toxicities included asthenia, fever, nausea, malaise, vascular leak syndrome (VLS), and reversible transaminasemia. Responses were noted in 21 of 71 patients (30%) with cutaneous T-cell lymphoma patients and in 11 of 45 patients (25%) with B-cell non-Hodgkin lymphoma.

Based on these favorable clinical results and the presence of IL-2R in CLL cells,16 2 pilot studies were conducted of denileukin diftitox in patients with fludarabine-refractory CLL. In the first study, 2 of 14 evaluable patients achieved partial remissions that lasted for 14 months and ≥19 months, respectively.17 In the second study, 2 of 6 evaluable patients achieved partial remissions that lasted for ≥12 months in both patients.18 To follow up on these preliminary results, 2 multiinstitutional, Phase II studies were undertaken. Because of similarities in eligibility criteria and treatment plans and limited accrual to each study, we analyzed both of those clinical trials for the current report.


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  2. Abstract

Eligibility criteria for patient entry in the 2 studies were similar but not identical. In both studies, a diagnosis of B-cell CLL either that was refractory or intolerant to fludarabine or that recurred within 6 months after the last treatment with fludarabine was required. Patients were required to have intermediate-risk to high-risk category disease based on the modified 3-stage Rai staging system.2 Patients had to be age ≥18 years with no uncontrolled infections, no central nervous system leukemia, and a good performance status (Zubrod 1 or 2), and could not be pregnant or nursing; they also had to have an albumin level ≥3 g/dL and a bilirubin ≤1.5 times the upper limit of normal. Written informed consent was obtained for each patient in both studies.

Organ functions had to be maintained in both studies but different slightly. Aspartate aminotransferase and alanine aminotransferase levels had to be ≤2.5 times the upper limit of normal for Study A and ≤1.5 times the upper limit of normal for Study B. For Study A, the serum creatinine level was ≤1.5 mg/dL and creatinine clearance was ≥40 mL per minute; for Study B, the serum creatinine level was ≤1.8 mg/dL and creatinine clearance was ≥50 mL per minute. Both studies required an absolute neutrophil count ≥1000/mm3 and a platelet count ≥50,000/mm3; however, Study B permitted an absolute neutrophil count ≥500/mm3 and a platelet count ≥25,000/mm3 if CLL was the cause of cytopenia. The cardiac ejection fraction was ≥40% for Study A, whereas patients were ineligible for Class III or IV heart disease (New York Heart Association criteria) for Study B. Adequate pulmonary function (forced expiratory volume in 1 second ≥70%) and a hemoglobin level ≥8 g/dL were required only in Study A.

Pretreatment flow cytometry was performed to assess CD25 (IL-2R α) expression. Briefly, peripheral blood (100 μL) was mixed with fluorescein isothiocyanate-conjugated anti-CD19 monoclonal antibody, Cychrome (phycoerythrin [PE]-indodicarbocyanine)-conjugated anti-CD5 monoclonal antibody, and PE-conjugated anti-CD25 monoclonal antibody or PE-conjugated isotype control immunoglobulin G1 (IgG1) monoclonal antibody (10 μL each; Becton Dickinson, Mountain View, CA); incubated for 15 minutes; washed with phosphate-buffered saline (PBS); pelleted at × 600 g for 5 minutes; lyzed with l mL whole blood lysing reagent (Coulter, Hialeah, FL); fixed with 8% formaldehyde (250 μL); washed twice with PBS; resuspended in 1 mL of PBS; and analyzed on an EPICS-XL dual laser flow cytometer. Gating was performed for CD19-positive/CD5-positive lymphocytes, and the percentage of cells that demonstrated PE fluorescence intensity ≥99% of control cells stained with PE-conjugated isotype control IgG1 antibody were determined.

Denileukin diftitox produced under good manufacturing practice and aliquoted in single-use vials that contained 300 μg in 2 mL of 20 mM citrate buffer (pH 7.0), with 50 mM of ethylenediamine tetraacetic acid and 0.8% of polysorbate 20 (NDC64365-503-01; 6 vials in a package) was obtained from Ligand Pharmaceuticals, Inc. (San Diego, CA). Vials were stored at − 10°C or lower until they were used. Before treatment, vials were thawed to room temperature, and appropriate amounts of drug were dispensed in a laminar flow hood for individual daily doses by diluting with sterile saline into an empty plastic intravenous bag or plastic syringe to a final concentration of ≥15 μg/mL following the recommendations of the package insert. Patients received allopurinol at a dose of 300 mg, dexamethasone at a dose of 4 mg, acetaminophen at a dose of 650 mg, diphenhydramine at a dose of 25 mg, and 1 L of saline intravenously on treatment days. Denileukin diftitox was administered as 60-minute intravenous infusions at a dose of 18 μg/kg daily for 5 days every 21 days for a maximum of 8 cycles. All treatments were given in outpatient oncology infusion centers.

Patients were monitored with weekly histories, physical examinations, blood counts, urinalyses, and chemistries. Toxicity and toxicity grades were determined using the revised National Cancer Institute Common Toxicity Criteria (version 2.0). The Leukemia Special Adverse Event Criteria were used to grade hemoglobin, platelets, neutrophils, and fibrinogen. VLS was characterized by ≥2 of the following 3 findings: hypoalbuminemia, edema, and/or hypotension. Staging was performed pretreatment, after 2 cycles for Study B, and after 4 cycles for Study A. Lymph node disease was quantified by measurements of the area of individual lymph node masses by using the equation, area = smallest dimension × the greatest dimension determined by computerized tomography and examination. The sum of the areas of identifiable, pathologic masses was calculated before and after treatment. Responses were graded by using the criteria published by Cheson et al.2 Complete remission required the absence of constitutional symptoms, adenopathy, and organomegaly; a normal hemogram; and <30% lymphocytes in a bone marrow biopsy. Partial remission consisted of an improvement in stage, a reduction ≥50% in lymph nodes, peripheral blood values, and/or organomegaly. Evaluable patients for response were defined as those patients who received at least 2 cycles of denileukin diftitox.

The 2-sample Student t test was used to compare characteristics of the responders versus nonresponders. A chi-square test was used to assess differences between groups in the number of responders.


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Twenty eight patients were entered on the studies, including 18 patients in Study A and 10 patients in Study B. Twelve of 25 patients (48%) expressed CD25 in at least 20% of circulating CLL cells. The mean patient age was 63 years. There were 23 males and 5 females. Six patients had Rai Stage I disease, 7 patients had Rai stage II disease, 3 patients had Rai Stage III disease, and 12 patients had Rai Stage IV disease. The patients had received a mean of 3.4 prior therapies (range, 1-7 prior therapies) (Table 1).

Table 1. Clinical Characteristics of Patients with Chronic Leukocytic Leukemia Treated with Denileukin Diftitox
ParameterPercentage of Patients
Age >65 y46
Male gender82
>3 prior therapies53
Performance status 0-193
Rai Stage I21
Rai Stage II25
Lymphocyte count >100,000/μL18
Hemoglobin <10g/dL18
Platelet count <100,000/μL44
CD25-positive, >25% of cells40


Side effects were moderate in most patients. Frequent Grade 3 and 4 toxicities (including drug-related and nondrug-related toxicities) consisted of asymptomatic transient transaminasemia, neutropenia, fatigue, infections, dyspnea, VLS, emesis, rash, and thrombocytopenia (Table 2). Three patients (Patients 13, 18, and 23) died on study, including 1 patient with progressive disease, 1 patient with bacterial pneumonia, and 1 patient with Serratia sepsis and progressive disease. Two patients completed 8 cycles of denileukin diftitox. Of the remaining 23 patients, treatment was discontinued in 11 patients because of progressive disease and in 12 patients because of toxicity (3 patients had VLS, 2 patients had rash and emesis, and 1 patient each had emesis alone, rash alone, neuropathy, fatigue, fatigue and pain, pneumonia, and pleural effusion). All 4 serious infections were bacterial in origin. Three patients received 7 cycles of denileukin diftitox, 4 patients received 5 cycles, 6 patients received 4 cycles, 1 patient received 3 cycles, 6 patients received 2 cycles, and 6 patients received cycle.

Table 2. Grade 3 or Greater Toxicities (Drug Related and Unrelated) Noted in Patients with Chronic Leukocytic Leukemia Treated with Denileukin Diftitox
Side EffectPercentage of Patients
Vascular leak syndrome14
Visual floaters3
Elevation partial thromboplastin time3


Response was assessed in the blood, lymph nodes, and bone marrow, and the results are shown in Table 3 and Figures 1 and 2. Peripheral CLL cells decreased in Patients 1, 5, 9, 11, 12, 13, 14, 15, 16, 19, 22, and 24 by 76%, 71%, 83%, 88%, 60%, 81%, 44%, 97%, 44%, 44%, 80%, and 68%, respectively. Bone marrow CLL cellularity decreased (when examined) by 100% in Patients 1, 9, and 10; by 12% in Patient 11; and by 60% in Patient 15. Figure 1 shows the bone marrow biopsies before treatment and after Cycle 4 from Patient 10. Overall, denileukin diftitox produced 1 complete remission and 5 partial remissions for an overall response rate of 27% among 22 evaluable patients. Four of the responses were durable and lasted from 4 months to 12 months.

Table 3. Characteristics of Patients with Chronic Lymphocytic Leukemia who Responded to Denileukin Diftitox
Patient No.*Peripheral CLL CellsLymph Node Reduction (%)Overall ResponseLength of Response (Months)
Pretreatment/Posttreatment Count (Cells/μL)Reduction (%)
  • CLL: chronic lymphocytic leukemia; CR: complete response; PR: partial response.

  • *

    Patients 1, 9, and 10 achieved bone marrow clearance of CLL cells.

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Figure 1. Hematoxylin and eosin-stained, 6-μm paraffin sections of bone marrow biopsies that were obtained (A) before treatment and (B) after 4 treatment cycles from Patient 10 (original magnification ×400).

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Figure 2. Images of chest computed tomography scans from Patient 9 (A,C) before treatment and (B,D) after 4 treatment cycles. The arrows indicate chronic lymphocytic leukemia lymph node masses.

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Statistical Analysis

Several parameters were assessed to determine whether they predicted response. The percentage of CD25 expression was 37 ± 21% in responders and 18 ± 25% in nonresponders (2-sample Student t test; P = .11). The number of denileukin diftitox cycles was 5.0 ± 1.9 cycles for responders and 4.1 ± 2.1 cycles for nonresponders (2-sample Student t test; P = .38). Therefore, neither CD25 expression nor cycle number predicted response. Other parameters also failed to discriminate responders from nonresponders. The mean patient age was 64 ± 13 years for responders and 60 ± 10 years for nonresponders (2-sample Student t test; P = .55). The mean stage was Stage 2.5 ± 1.4 for responders and Stage 3.0 ± 1.2 for nonresponders (2-sample Student t test; P = .35). The mean hemoglobin level was 12 ± 2 g/dL for responders and 12 ± 1 g/dL for nonresponders (2-sample Student t test; P = .87).


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  2. Abstract

Patients with advanced CLL are treated for disease control rather than with curative intent with the exception of the rare patient who is eligible for allogeneic stem cell transplantation. Therefore, the objective is to achieve durable remissions with tolerable toxicities. When patients develop recurrent disease after therapy with rituximab and fludarabine, salvage regimens with alemtuzumab or combination chemotherapies often are immunosuppressive and can be associated with debilitating compromised host infections.19 There is a need for additional targeted biologics that have noncross-reactive side effects and that may be useful as single agents or in combination with other agents. Denileukin diftitox offers potential as such a reagent, and the results from current studies are encouraging. Additional work is needed to improve its tolerability and response rate.

Twenty-one percent of patients received only 1 cycle of denileukin diftitox. This off-protocol frequency was similar to the results of a pilot trial in which 28% of patients received only 1 cycle.17 The causes of protocol discontinuation included disease progression and toxicities, which included a mixture of VLS, emesis, rash, neuropathy, diffuse pain, pneumonia, pleural effusion, and fatigue. Many of these side effects are described in the package insert, and a combination of patient, nurse, and physician education; aggressive monitoring; and intervention may reduce the severity of these side effects and improve patient compliance. For 9 of the 12 patients who discontinued therapy, the reasons were VLS, rash, fatigue, emesis, and/or pain. Close communication between patients and caregivers may permit early treatment with albumin, diuretics, or fluids for VLS; topical or systemic steroids or diphenhydramine for rash; antiemetics for emesis and nausea; and narcotics for pain. Because the side effects from denileukin diftitox may be delayed in onset and may last for several weeks, early intervention may improve tolerability. Because the previous and current trials were multiinstitutional and were conducted in physician office practice settings, most caregivers had limited experience with the drug or its side effect profile. The increase in steroid premedication in the current studies did not appear to have a significant impact on the frequency or severity of toxicities.

In the current study, because the inclusion criteria differed slightly between patients in Group A and patients in Group B, we analyzed the number of cycles of denileukin diftitox that were received by these groups. There was a trend toward more cycles in Group A (4.2 ± 2.4 cycles) versus Group B (2.6 ± 1.5 cycles), although the difference had only borderline significance in a 2-sample Student t test (P = .07).

In the current study, we considered patients evaluable for response after only 2 cycles, because we observed that at least 1 patient required only 2 cycles to respond (Patient 25). Approximately 25% of patients with CLL who received ≥2 cycles of denileukin diftitox treatment responded. This matched the results from previous studies.17, 18 Considering the combined results from all denileukin diftitox-CLL trials, there have been 10 of 42 responses (in 2 of 6 patients, 6 of 22 patients, and 2 of 14 patients) for a 24% response rate and remission durations from 2 months to 20 months. The denominator was based on patients who received ≥2 cycles of denileukin diftitox. This response rate and the response duration in these heavily pretreated patients are promising, although the analysis was based on a compilation of distinct clinical trials. Can we identify the patients who are most likely to respond and, consequently, improve the response rate further in this subset?

If denileukin diftitox works by binding CLL cells and inducing cell death, then we predict an association between drug intensity and IL-2R density in CLL cells and response. It may be argued that the number of cycles does not reflect CLL cell drug exposure accurately and that the immunofluorescence measurement of a single IL-2R subunit does not correlate well with high-affinity IL-2R status, but to our knowledge these parameters are all that are currently available. Measurements of IL-2R by radioligand binding or reverse transcriptase-polymerase chain reaction analysis may yield more accurate information but were not available in the current study and would be challenging to use in clinical practice. The lack of dependence of denileukin diftitox cycle number or CD25 expression on remission induction suggests the possibility of other hypotheses for the mechanism of action of denileukin diftitox in CLL.

Beyer et al. documented an increased frequency of CTLA4-positive, FOXP3-positive, GITR-positive, CD62L-positive, transforming growth factor-β-positive, and IL-10-positive T-regulatory cells in patients who had CLL, with the greatest frequency noted among progressing patients with extensive disease.20 T-regulatory cells function to suppress antitumor immunity21 and possess high-affinity IL-2R.22 An alternative hypothesis is that denileukin diftitox depletes T-regulatory cells in CLL and permits the reemergence of natural anti-CLL immunity. To our knowledge to date, there have been no studies evaluating this mechanism. The evaluation of T-regulatory cell populations in patients with CLL who have received treatment with denileukin diftitox would be a useful beginning. Confirmation of such a mechanism also may indicate that denileukin diftitox therapy may induce antitumor immunity in other non-IL-2R-expressing tumors. The provocative responses observed with denileukin diftitox in patients with nonsmall cell lung cancer may be due to such a mechanism. With additional clinical research, denileukin diftitox may gain a niche in the armamentarium for therapy in patients with CLL and may improve the prognosis of this chronic, disabling, and ultimately fatal condition.


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  2. Abstract
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