ALBERT

Description: Background: Although complete remission rates for AML are near 70% with combination induction and consolidation chemotherapy, most patients will relapse and die from the disease or treatment complications. New agents with unique mechanisms are needed. One such class of therapeutics are fusion proteins consisting of protein synthesis inactivating peptide toxins fused to tumor cell selective ligands. DT388-IL3 is one such fusion protein. Rationale: In preclinical studies, DT388-IL3 was cytotoxic to the IL3 receptor expressing leukemia cell lines but not toxic to IL3 receptor negative cell lines. This agent was less toxic to normal progenitors and not toxic to early hematopoietic stem cells. The majority of AML progenitors overexpress IL3 receptors. Animal model work in mice bearing human leukemia cells has demonstrated anti-leukemia efficacy which is dose dependent with this agent. Toxicities in monkeys include vascular leak syndrome and pancytopenia observed only at the highest doses. The MTD in monkeys was estimated at 60mcg/kg/day. We report preliminary data on the use of DT388-IL3 fusion protein in humans from an ongoing phase I trial. Pharmacokinetics; clinical and immune response to this novel fusion protein are also being followed. Patients and Methods: Patients with refractory AML were eligible. The first dose level was qd M-W-F X six doses of DT388-IL3 at 4mcg/kg/day with dose escalation planned for subsequent patients. Patients with progression of disease or unacceptable study drug toxicity were to be removed from the study. Toxicity was graded according to NCI CTCAE version 3.0. Three patients have been treated with DT388-IL3. Serum samples were collected and will be assayed for anti-DT388-IL3 antibodies prior to and after treatment. Blood samples were obtained to measure circulating levels of active DT388-IL3 and its half life. Patient blasts were also collected prior to treatment for later analysis of expression of IL3 receptors. Result: Two patients tolerated the treatment schedule(of six doses) without any significant toxicities. Mild fever, headaches, nausea were noted. Both of these patients had progression of disease-one during treatment and one on day 15 bone marrow biospy. The above mentioned patients died secondary to disease complications at 2 weeks and 18 weeks after their last dose of the study agent respectively. DT388-IL3 levels on these two patients post infusion were below the the reliable detectable limits of the assay. The third patient became febrile and hypotensive after the first dose. The hypotension persisted and she did not receive any further doses. This patient is alive 5 weeks later with supportive care alone. DT388-IL3 levels following this patient’s dose are as follows: 2min post infusion 34.3ng/ml, 30min post infusion 1.9ng/ml, 60min post infusion 0.075ng/ml, 120min post infusion 0.003ng/ml, 240min post infusion undetectable. Conclusion: Preclinical/animal studies suggest that DT388-IL3 has anti-leukemia efficacy. Preliminary data from our ongoing phase I trial reveals minimal study agent related toxicity and no life threatening complications at this first dose level. Dose escalation is planned as per protocol.

Description: Bcl-2 acts as an important regulator of the mitochondrial pathway of apoptosis and promotes resistance of MM cells to chemotherapy. The Bcl-2 antisense oligonucleotide G3139 specifically targets Bcl-2 and may enhance the anti-tumor efficacy of Dex and Thal. In this trial G3139 was administered at 5 mg to the first 3 Pts and then 7 mg/kg/d by IVCI for 7d of 21d cycle. On day 4, Pts started Dex 40 mg daily for 4 d and Thal 100-400 mg as tolerated. After 3 cycles, responding Pts continued G3139 on a 5-week cycle with Dex 20 mg x 4d and Thal at the tolerated daily dose for up to 1 yr with an optional second yr for responding Pts. Thirty-three Pts treated to date had the following characteristics: median age 60 yrs (range: 28- 76), 22 males; 16 Pts had complex karyotypes; 14 Pts had B2M 〉 2.5 g/dl; LDH 〉1.5 normal in 7 Pts; Cr 〉1.5 mg/dl in 6 Pts; platelets

Description: Therapeutic options for chronic lymphocytic leukemia (CLL) at relapse are limited because of myelosuppressive toxicity. Denileukin diftitox (ONTAK®, Ligand Pharmaceuticals) is a genetically engineered fusion protein comprising the enzymatically active domain of diphtheria toxin and the full length sequence of interleukin-2 (IL-2) targeting malignancies expressing the medium and high affinity IL-2 receptors. We designed a phase II study to evaluate the efficacy of ONTAK® in patients with fludarabine-refractory CLL, which is a follow-up to the previously published study (Frankel, et al, Clin. Cancer Res.2003; 9:3555). Denileukin diftitox was administered at a dose of 18μg/kg IV daily for 5 days every 3 weeks, for a maximum of 8 cycles. Thirteen patients have been treated so far, with 10 patients being evaluable for response (completed ≥ 3 cycles). Median age was 59 years (range 44–84), and 62% (8/13) were Rai stage III-IV, with a median of 3 prior therapies (range 1–6). The overall response was 40%, with 1 CR (10%, duration of response 5+ months) and 3 PR (30%, duration of response 3+, 3+ and 4+ months). Two responding patients (both PR) are still on study, while two (1 CR, 1 PR) were removed from study because of toxicities after 7 and 5 cycles, respectively. Four patients (40%) had progressive disease after cycles 3, 4, 4, and 7, respectively. One patient has completed four cycles and restaging studies are pending. Of the 3 patients not evaluable for response, two are still on study (having not completed 3 cycles), while one refused further treatment after 4 doses of cycle one. The grade 3/4 toxicities encountered were: neutropenia 4/13, thrombocytopenia 4/13, vascular leak syndrome 3/13, left ventricular cardiac dysfunction 1/13, hypotension 2/13, tachyarrhythmia 3/13, elevated PT 1/13, fatigue 1/13, rash 1/13, SIADH 1/13, constipation 1/13, vomiting 2/13, petechiae 1/13, transient elevation of GGT 1/13, transient elevation of AST/ALT 7/13, hyperglycemia 4/13, electrolyte imbalance 8/13, infection and/or febrile neutropenia 4/13, insomnia 1/13, visual disturbance 1/13, dyspnea 2/13, hypoxia 2/13. We conclude that denileukin diftitox has activity in CLL, with toxicities that can be managed with adequate premedication and close monitoring.

Description: To develop a cytotoxic drug which targets the IL-3 receptor (IL-3R) on human AML cells we previously developed a fusion protein containing a truncated form of diphtheria toxin which lacks the native binding site (DT388) fused to human IL-3. This molecule kills leukemic progenitors from many AML patients while showing little toxicity to normal hematopoietic progenitors. However, some AML samples showed little or no cell kill after exposure to this molecule. To attempt to improve the cytotoxicity of DT388IL-3 two variants of the toxin were constructed which contain alterations in the IL-3 residues that are designed to enhance binding affinity to the IL-3R. The two variants, DT388IL3[K116W] and DT388IL3[D125-133], have substitution of a hydrophobic tryptophan group at the 116 position and an eight amino acid deletion from the C-terminus of the IL-3 molecule, respectively, while the catalytic and translocation domains of DT388 remain unchanged. These variant DT388IL3 molecules and the unmodified ‘native’ fusion toxin were compared for their ability to kill AML colony forming cells (AML-CFC) from the peripheral blood of 13 newly-diagnosed AML patients and myeloid CFC from 3 normal bone marrows (NBM). AML and NBM cells were cultured for 24h with or without fusion toxin at concentrations varying between 1 and 250 ng/ml prior to plating in CFC assays. Little or no AML-CFC kill was observed for 3/13 samples. The mean % AML-CFC kill for the remaining 10 AML samples ranged from 21 – 61% for the lowest and highest concentration of native DT388IL3 tested and was significantly higher (P90% kill achieved for 6 of these at concentrations as low as 1 ng/ml. The concentration of DT388IL3[Kll6W] required to achieve ≥50% kill of AML-CFC was on average ≥5-fold lower than the concentration of native toxin required to achieve the same effect. Thus, the variant DT388IL3 molecules tested show enhanced cytotoxic activity against AML progenitors with little change in the toxicity profile against normal hematopoietic precursors. In particular, the DT388IL3[K116W] variant warrants further testing against more primitive normal and leukemic progenitors as a potentially promising new therapeutic agent for AML. Such studies are underway.

Description: Patients with relapsed AML over the age of 60 have a poor prognosis. Gemtuzumab ozogamicin (GO) has been approved for older pts in first relapse, although many pts who attain complete remission (CR) do not fully recover normal platelet count (so-called CRp). In vitro studies have shown that oblimersen down-regulates Bcl-2 in AML cells and enhances apoptotic cell death induced by GO. We conducted a Phase 2 study to evaluate the safety and efficacy of GO combined with oblimersen for older pts with AML. Eligibility requirements included: age ≥ 60 yrs; AML in 1st relapse; ≥ 3 mos 1st CR duration; ≥ 25% CD33-positive AML cells. Pts received oblimersen at a dose of 7 mg/kg/d for 7 days by CIV beginning on days 1 and 15; GO was given at a dose of 9 mg/m2 IV over 2 hrs on days 4 and 18. A total of 48 pts were enrolled (ITT population), all of whom received at least 1 dose of oblimersen; 9 pts failed to receive the required 2 doses of GO (per-protocol population, n=39). The median age was 67 (range, 59 to 88 yrs). Duration of 1st CR: 〈 6 mos: 7 pts; (15%); 6 to 12 mos: 29 pts (60%); 〉 12 mos: 12 pts (25%). No. of prior regimens: 1 (17 pts, 35%); 2 or 3 (26 pts, 54%); ≥ 4 (5 pts, 10%). Among treated pts, 79% completed 21 days of protocol therapy. Overall, 12 pts achieved a major response, either CR (n=5) or CRp (n=7), for an ITT response rate of 25% and a per-protocol response rate of 31%. The median time to remission was 52 days. Ten of the 12 responders survived 〉 6 mos, whereas only 6 non-responders survived ≥ 6 mos. Serious adverse events for the oblimersen/GO combination were qualitatively similar to those reported for GO alone and included among other reactions: Grade 3-4 febrile neutropenia (42%) or thrombocytopenia 33%; nausea; fever; rigors, and dyspnea. Treatment-emergent adverse reactions led to discontinuation of protocol therapy in 10 pts (21%). The most common serious adverse event was febrile neutropenia (25%). One pt (2.1%) died during treatment (sepsis) and 16 pts (33%) died within 30 days of last study medication (infection, bleeding, respiratory failure, progressive AML, and other disease-related complications). No episodes of VOD were observed. Oblimersen can be safely combined with GO; however, pts enrolled in this study appear to have had more unfavorable characteristics at entry compared with prior studies using GO alone in pts with relapsed AML. Therefore, assessment of an incremental benefit from the addition of oblimersen will require a randomized trial.

Description: Over-expression of Bcl-2 has been linked to acquired dex resistance in MM cells. Conversely, reduction of Bcl-2 using oblimersen has been shown to enhance dex-induced apoptosis in MM cell lines and in fresh MM cells in culture (Liu et al., Blood101:4105, 2003). Preliminary non-randomized clinical studies have suggested that oblimersen may potentiate the activity of VAD and dex/thalidomide in patients (pts) with resistant MM. We have conducted a randomized, multinational, Phase 3 trial of pts with advanced MM using dex given with or without oblimersen. Eligibility requirements included: relapsed or refractory MM; measurable M-protein in serum or urine; ≤ 6 prior regimens; ECOG PS ≤ 3; serum Cr ≤ 1.5 mg/dL; ANC 〉 1,000/ml; plts ≥ 50,000/ml. Exclusions: previous allogeneic transplant; other significant medical disease. Pts were stratified according to number of prior treatments (1 or 2 vs. ≥ 3 regimens), prior autologous stem cell transplant (yes/no), and primary resistance to vs. relapse from primary treatment. All pts received dex 40 mg p.o. x 4 days during weeks 1, 2 and 3. Pts randomized to oblimersen received 7 mg/kg/d x 7 days by IV infusion, beginning 3 days before dex treatment during the 1st and 3rd weeks, and during all subsequent dex cycles. Beginning on week 5, additional 4-day dex cycles were repeated in stable or responding pts every 3 weeks, for a maximum of 12 months. All pts received prophylactic H-2 antagonists, TMP/SMZ, and a bisphosphonate. Response assessments were conducted prior to each treatment cycle. The primary endpoint of the study was to compare time-to-disease progression between the 2 treatment groups. Secondary endpoint comparisons included overall survival, event-free survival at 6 months, overall response rates, response duration, clinical benefit, and safety. A total of 224 pts were randomized into the study. All pts have completed 〉 12 mos follow-up, and final results of this trial will be presented.

Description: Oblimersen sodium (Ob, Genasense™) is an 18 mer phosphorothioate antisense directed against bcl-2, an antiapoptotic protein that mediates chemoresistance in malignant cells. In order to assess whether detectable intracellular concentrations (ICs) of Ob are achievable in vivo, we have developed a sensitive and specific ELISA-based assay. This assay involves Ob hybridization to a 5′-end overhang of a 3′-biotinylated capture probe ligated to a digoxigenin (Dig)-labeled probe, and detection by an anti-Dig-alkaline phosphatase system. In K562 cell extract, the assay was linear within 50–2000 pM range with a limit of quantification (LOQ) of 50 pM (equivalent to 5.0 fmol/100 μL). The withtin-run coefficients of variation (CVs) in 4 spiked concentrations were between 3–7% in 6 replicates with accuracy values between 93–109%. The between-run CVs were between 6–12% and accuracy values between 97–102%. The specificity of the assay was demonstrated by low cross reactivity with mismatched oligonucleotides and putative 3′-end metabolites shortened by 1, 2 or 3 nucleotides. Validation of IC measurement was performed in vitro in K562 cells treated with fluorescent labeled Ob conjugated to oligofectamine. At Ob concentrations between 0.1 – 10 μM, Bcl-2 mRNA downregulation measured by real-time RT-PCR occurred efficiently. Nonlinear regression analysis of a dose-response curve showed that 50% Bcl-2 downregulation (IC50) occurred at approximately 0.29 μM, corresponding to an Ob IC concentration of 37 pmole/mg protein. Cellular uptake was confirmed by microscopy and flow cytometry. To validate these results in vivo, we measured Ob IC in bone marrow samples from untreated AML pts aged 〉 60 yrs enrolled on the phase I study OSU 0164. These pts were induced with Ob 7 mg/kg/d CIVI on days 1–10, cytarabine 100 mg/m2/d CIVI on days 4–10 and daunorubicin administered iv at two dose levels (45 mg/m2/d IV and 60 mg/m2/d) on days 4–6. Among 21 pts assessable for clinical response and Bcl-2 levels, at pretreatment, Bcl-2 copy numbers (normalized to ABL) were higher among 12 pts who achieved a CR (median 85,325; range 19,120–149,100) than among 9 non-responsive (NR) pts (32,100 bcl-2 /abl copies; range 1,488–163,500) (P=.04; Mann-Whitney test). Following 72 hr Ob infusion, a decrease (−38%) in median Bcl-2/ABL mRNA copies in CR patients and an increase (+115%) in Bcl-2/ABL copies in NR pts (P=.002; Mann-Whitney test) were observed by real time RT-PCR. A trend in higher median IC of Ob was observed in CR pts (17.0 pmole/mg protein; range 1.5–30.0) as compared to NR pts (4.4 pmole/mg protein; range 0.33–28.0) (P=.06; Mann-Whitney test). Six of 7 pts with IC above the median obtained a CR. No differences were observed in the Ob plasma PKs between the CR pts [median steady state concentration (Css) 2.8 μg/mL, area-under-the-curve (AUC) 772 μg*hr/mL and clearance (Cl) 9.6 L/hr) and the NR pts (median Css 3.4 μg/mL, AUC 752 μg*hr/mL, Cl 6.4 L/hr). Although the number of samples analyzed was small, our data suggest that, despite interpatient variability of both Bcl-2 mRNA expression and Ob uptake, this antisense can be successfully delivered to pts and result in clinically relevant target downregulation. A Cancer and Leukemia Group B phase III AML study to characterize prospectively the interplay of IC levels of the Ob and Bcl-2 downregulation is in progress.

Description: 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. (Blood. 2004;104:2143-2148)

Description: Rituximab and Fludarabine are active in Waldenstrom’s macroglobulinemia (WM) producing major response rates of 40–70%. Both pre-clinical and clinical studies in related low-grade B-cell malignancies suggest that additive, and even synergistic benefit with combination Fludarabine and Rituximab may result. As such, we conducted this combination study with Rituximab and Fludarabine in WM patients. WM patients who had received 〈 2 prior therapies, and who had not previously been treated with any nucleoside analogue or Rituximab were eligible. Intended therapy was as follows: Weeks 1–4 Rituximab (375 mg/m2/week) Weeks 5, 9, 13 Fludarabine daily for 5 days (25 mg/m2) Weeks 17, 18 Rituximab (375 mg/m2/week) Weeks 19, 23, 27 Fludarabine daily for 5 days (25 mg/m2) Week 30, 31 Rituximab (375 mg/m2/week) Patients were evaluated at week 12, and if they did not progress were eligible for further therapy and were evaluable for response. 43 WM patients were enrolled with a median age of 61 (range 52–75 yrs), and median prior therapies of 1 (range 0–2). 40/43 patients continued on therapy beyond week 12. Two patients (1 PR, 1 SD) died after completing protocol therapy including one elderly patient who had an influenza pneumonia and another debilitated patient who may have had a secondary malignancy. Delays in therapy due to neutropenia were common, and 58% of patients experienced Grade III/IV neutropenia. Protocol therapy was truncated after the 4th and 5th courses of fludarabine in several patients for persistent neutropenia and/or thrombocytopenia despite delays in therapy and/or use of G-CSF support. Other complications included: Herpes zoster outbreak in 3 of the first 21 patients, before prophylactic acyclovir or equivalent was initiated; parasthesias (n=3); pneumonitis (n=2); development of bladder cancer (n=1) and a high grade lymphoma (n=1); subdural hemorrhage in a patient who had a 3-fold increase in serum viscosity following the first 4 infusions of Rituximab. On an intent to treat basis, 39/43 (90.1%) patients demonstrated a response. Response categories were as follows: CR (n=3); PR (n=32); MR (n=4). One patient remains in stable disease at 20+ months. At a median follow-up of 17 months, 36 of 39 responding patients remain in remission. Rituximab in combination with fludarabine is highly active and produces durable responses in WM.