Denileukin Diftitox Research Articles

Ribosome Inactivating Proteins: Exploiting Plant Weapons to Fight Human Cancer

Ribosomeinactivating proteins (RIPs) are potent toxins from plant or bacterial origin that work by irreversibly block ribosomes of target cells, causing apoptotic cell death. Their noteworthy activity has been exploited to therapeutic purpose in the treatment of cancer. The specificity for malignant cells has been addressed by using targeting domains such as antibodies to form immunotoxins or ligands to get chimeric fusions. These therapeutics have shown encouraging preliminary results in hematological tumors as well as in solid tumors with the most effective ones currently undergoing clinical trials. Denileukin diftitox, a fusion between the catalytic domain of the diphtheria toxin and human cytokine interleukin-2, was the first toxin-derived drug approved by the FDA. Another, more recent way to take advantage of RIPs is to utilize their DNA in the suicide gene therapy. Toxin DNA can be delivered trough a vector to or complexed and directly injected in the tumor. In the future RIPs are expected in to give a significant contribution in the treatment of the cancer, also in combination with traditional therapies. The scope of this review is to highlight advantages and drawbacks of RIP based biomedical applications, by evaluating their employment as either targeted proteins or genes as effectors.

Novel therapies for T cell lymphoma

Peripheral T-cell lymphomas (PTCL) are a heterogeneous collection of lymphomas that are associated with a very poor prognosis. Conventional therapies, historically based on protocols for aggressive B-cell lymphomas, deliver less than adequate outcomes; the majority of patients experience early relapse after frontline treatment and current 5 year overall survival is only 10–30%. Clearly, new approaches are needed. In recent years there has been a plethora of novel agents showing activity in PTCL, often in patients with advanced relapsed or refractory disease. These agents include antifolate drugs (pralatrexate), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, belinostat), nucleoside analogues (gemcitabine, forodesine, clofarabine), monoclonal antibodies (anti-CD52, anti-CD4, anti-CD2), immunotoxins (bentuximab vedotin; anti-CD30), fusion toxins (denileukin diftitox; CD25), immunomodulatory agents (IMiDs such as lenalidomide), and proteasome inhibitors (bortezomib). This is an exciting time in the treatment of PTCL, as our ever improving understanding of the distinguishing features, pathogenesis, molecular biology and progression of PTCL and the knowledge of the mechanism and efficacy of novel therapies, may see a real improvement in outcomes for patients. Biomarker co-development will be absolutely critical to advancing the field of PTCL therapy. Already, by dissecting out the biology of the disease, specific targeted approaches are being used (CD30, ALK, CCR4). Understanding the primary mechanism of action and key targets of the less specific agents such as the HDACi, lenalidome or aurora kinase inhibitors, will help us pre-select patients with the highest likelihood of benefit, reducing exposure to potential toxicities while reducing the overall health cost burden.

A Dose Finding Lead-in Study of E7777 (Diphtheria toxin fragment-Interleukin-2 Fusion Protein) in Persistent or Recurrent Cutaneous T-Cell Lymphoma (CTCL)

Introduction: E7777 is a recombinant cytotoxic fusion protein composed of diphtheria toxin fragments A and B and human interleukin-2. E7777 has the same amino acid sequence as denileukin diftitox (DD) (approved as ONTAK in the USA for treatment of persistent or recurrent CTCL) but with improved purity and an increased percentage of active protein monomer species. This study is a pivotal multicenter open-label single-arm study comprising a Lead-In part (approx. 20 pts) to select the E7777 dose for the Main Study (70 patients [pts]) in which primary efficacy and safety of E7777 is assessed. Dose finding is necessary since the specific bioactivity of E7777 is 1.5 – 2 times that of the prior less purified form (ONTAK). The Lead-In study is reported in this abstract.Methods: A Continual Reassessment Method (CRM) is used for dose-finding with E7777 doses ranging from 3 – 18 µg/kg to be tested. The CRM is designed to target a DLT rate of approx. 20%. Inclusion criteria were patients ≥ 18 yr old, with a diagnosis of Mycosis Fungoides (MF) or Sézary Syndrome (SS) with stage I-IV disease at study entry, measurable CD25+ tumor, and who have received at least one prior therapy for CTCL. Prior commercial DD therapy was not allowed. E7777 was administered as monotherapy by iv infusion over 60 min on 5 consecutive days every cycle of 21 days. Pts were be dosed up to 8 cycles; additional dosing is allowed if deemed beneficial by the treating physician. Required premedication included an antihistamine, anti-pyretic, and anti-emetic; in case of infusion-related reaction low-dose systemic steroid premedication was allowed. The aim of the Lead–in study was to establish the maximum tolerated dose (MTD) and the recommended Main Study dose (based on the MTD and clinical judgment), and to assess safety and tumor response. Tumor responses were assessed according to the new ISCL/EORTC criteria (Olsen 2011).Results: Seventeen pts have been treated in the Lead-In. There were 8 males and 9 females, with median age 64 (range 26-81). Thirteen pts had MF (3, 7, 1, and 2 pts with Stage I, II, III, and IV disease, respectively) and 4 had SS (Stage IV). Fourteen or more pts had at least 4 lines of prior therapy. Pts were treated for a median of 4 cycles (range 1-8 cycles) and 8 patients are ongoing. Of the 9 pts who have discontinued from the study, 5 were for disease progression, 2 for AEs, 1 completed treatment per protocol, and 1 was pt choice.Pts were treated at doses ranging from 6 to 15 µg/kg; dose levels were assigned by the CRM based on the cumulative DLT information. The dose levels for each pt are shown in chronological order in the Table below, with the corresponding DLT assessments.TableContinual Reassessment MethodPatient #Dose (µg/kg)DLT16No29No312No415Yes56No6-129No13-1512No1615Yes1712NoIn the 17 patients treated, there were two DLTs; both occurred at the 15 µg/kg dose level and were capillary leak syndrome (CLS) (one Gr 2 with hospitalization, and one Gr 4). Based on preliminary results from these 17 pts, the highest dose with a DLT rate of ≤ 20% is 12 µg/kg. The most frequent AEs occurring in ≥ 3 pts were nausea (6 pts), ALT increased and AST increased (4 pts each), and vomiting, chills, fatigue, pyrexia, myalgia, rash, staph skin infection (3 pts each). Serious AEs considered related to study drug were CLS (2 pts) and pruritus, rash, diarrhea, vomiting, hypoxemia, and tumor flare (all in 1 pt each). AEs leading to drug discontinuation were CLS (2 pts). There were no deaths on study.Five pts achieved an objective response (OR) as assessed by the investigator, including 1 pt with relapse after allogeneic stem cell transplantation who received 8 courses and achieved a complete remission with associated skin graft-vs-host disease. These responses occurred at dose levels of 6, 9, and 12 µg/kg, including in 3 pts with stage IV disease (2 MF, 1 SS).Conclusion: Dose finding of E7777 in CTCL is proceeding with the CRM method. The toxicity profile is acceptable and so far no new safety signals compared to ONTAK has been identified. Initial signs of activity have been observed. Final data from this Lead-in study will be presented. DisclosuresDuvic:Eisai Inc.: Member of safety monitoring committee for trial Other, Research Funding. Kuzel:Eisai Inc.: Research Funding. Dang:Eisai Inc.: Protocol Steering Committee Other, Research Funding. Prince:Eisai Inc.: Research Funding. Foss:Eisai Inc.: Consultancy. Guo:Eisai Inc.: Employment. Ottesen:Eisai: Employment. Ooi:Eisai Inc.: Employment. Kim:Eisai inc.: Protocol Steering Committee Other.

Abstract LB-259: Interferon-α enhances clinical benefits of regulatory T cell depletion in ovarian cancer through direct T cell effects and by inducing bystander IL-6

Abstract Tregs hinder anti-tumor immunity, and depleting them treats cancers effectively in mice. By contrast, Treg depletion in human trials has limited efficacy. In a phase I trial of advanced stage carcinomas (breast, lung, melanoma, ovarian, bladder), we showed that the IL-2/diphtheria fusion toxin denileukin diftitox (DT) significantly depleted functional CD4+CD25hiFoxp3+ Tregs in blood with improved T cell function (increased Ki-67 and interferon-γ) with minimal effects on other blood mononuclear cells. One patient with stage IV ovarian carcinoma experienced significant reduction of metastatic tumor burden with denileukin diftitox at 12 μg/kg. Additional work showed that Treg depletion effects could last up to 4 weeks, and that weekly DT at 12 μg/kg eventually depleted anti-tumor and tumor-specific CD8+ T cells. Based on these data, we conducted a phase II clinical trial of epithelial ovarian carcinomas FIGO stage III-IV failing standard treatments, using DT as a single agent at 12 μg/kg by intravenous infusion every 3-4 weeks. In this trial, DT significantly depleted blood and tumor microenvironmental Tregs with only grade I-II toxicities, but with minimal clinical efficacy in 28 consecutive patients. This trial was halted for futility according to the Simon 2-stage design. Interferon-α alone does not treat ovarian cancer, but we now show that it significantly improves clinical and immune DT-mediated Treg depletion efficacy in human ovarian cancer. In the ID8 mouse ovarian carcinoma model, DT modestly increased survival and anti-tumor immunity. Interferon-α alone did not affect Treg numbers or function, but enhanced CD8+ T cell anti-tumor immunity. Interferon-α plus DT increased mouse survival significantly over either individual drug. In type I IFNR-/- mice unable to mediate interferon-α signals, interferon-α directly increased adoptively transferred IFNR+CD8+ T cell function independent of CD4+ T cell help. When combined with DT, IFN-α reduced Treg function without further numerical Treg reduction by indirect effects on tumor microenvironmental dendritic cells. In vitro studies identified IFN-α-driven dendritic cell IL-6 as a mechanism for reducing Treg function. When three ovarian cancer patients failed DT alone in the phase II trial, two had clinical and immune benefit by adding pegylated interferon-α2a in a separate trial, with acceptable toxicities. This trial was halted due to a lack of further DT. These studies demonstrate that DT depletes Tregs in distinct human carcinomas but is unlikely to be clinically effective as a single agent. We identified novel IFN-α mechanisms that improve Treg depletion effects using FDA-approved agents that can be rapidly translated. More selective Treg depletion agents and rationale combinations with other agents could improve clinical efficacy further. Citation Format: Suzanne Thibodeaux, Vincent Hurez, Shawna Wall, Srilakshmi Pandeswara, Benjamin Daniel, Aijie Liu, Lishi Sun, Leslie Wood, Weiping Zou, Tyler Curiel. Interferon-α enhances clinical benefits of regulatory T cell depletion in ovarian cancer through direct T cell effects and by inducing bystander IL-6. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-259. doi:10.1158/1538-7445.AM2014-LB-259

Therapeutic options in relapsed or refractory peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) represents a relatively rare group of heterogeneous non-Hodgkin lymphomas with a very poor prognosis. Current therapies, based on historical regimens for aggressive B-cell lymphomas, have resulted in insufficient patient outcomes. The majority of patients relapse rapidly, and current 5-year overall survival rates are only 10-30%. It is evident that new approaches to treat patients with PTCL are required. In recent years, prospective studies in PTCL have been initiated, mainly in patients with relapsed/refractory disease. In some of these, selected histologic subtypes have been evaluated in detail. As a consequence, numerous new therapies have been developed and shown activity in PTCL, including: agents targeting the immune system (e.g. brentuximab vedotin, alemtuzumab, lenalidomide); histone deacetylase inhibitors (romidepsin, belinostat); antifolates (pralatrexate); fusion proteins (denileukin diftitox); nucleoside analogs (pentostatin, gemcitabine); and other agents (e.g. alisertib, plitidepsin, bendamustine, bortezomib). A variety of interesting novel combinations is also emerging. It is hoped that these innovative approaches, coupled with a greater understanding of the clinicopathologic features, pathogenesis, molecular biology, and natural history of PTCL will advance the field and improve outcomes in this challenging group of diseases. This review summarizes the currently available clinical evidence on the various approaches to treating relapsed/refractory PTCL, including the role of stem cell transplantation, with an emphasis on potential new drug therapies.

New Therapies in T-cell Lymphoma

T-cell lymphomas represent 10–12% of all patients with non-Hodgkin lymphoma (NHL) in the Western world. When cutaneous T-cell lymphoma (CTCL) is excluded, peripheral T-cell lymphoma—not otherwise specified (PTCL-NOS), anaplastic large-cell lymphoma (ALCL), and angioimmunoblastic T-cell lymphoma (AITL) represent the overwhelming majority of patients in this population. These diseases remain a great challenge to treat. They are characterized by an aggressive presentation, extranodal disease, paraneoplastic phenomena, and resistance to standard chemotherapeutics. With the rapid development of new molecular gene profiling techniques, it is highly likely that these diseases will be subclassified by molecular abnormalities in the future. An evolving understanding of the tumor molecular pathogenesis will undoubtedly lead to further novel targeted therapies. Immunoconjugates, such as brentuximab vedotin (BV), have provided outstanding responses in relapsed, refractory CD30-positive ALCL. Histone deacetylase (HDAC) inhibitors have shown activity in PTCL and are potentially synergistic with proteasome inhibitors. Denileukin diftitox is an interesting recombinant DNA fusion protein linking fragments of the diphtheria toxin to interleukin-2 (IL-2) that is tested in clinical trials. Crizotinib, a highly specific anaplastic lymphoma kinase (ALK) inhibitor, has shown great promise in small number of patients with ALK-positive ALCL; the future of patients with this disorder looks very promising. Biomarker-driven chemotherapeutics is becoming a critical part of the state-of-the-art treatment in early- and late-phase clinical trials. It is crucial that future trials include sensible biomarker-based designs when future treatments are used in these challenging disorders.

Interferon-α augments the clinical efficacy of regulatory T cell depletion in ovarian cancer through direct and indirect T cell effects (VAC3P.941)

Abstract Tregs blunt anti-tumor immunity, and their depletion effectively treats tumor in mouse cancer models. However, Treg depletion in human trials is only partially effective. In our phase II ovarian cancer trial, denileukin diftitox (DT) effected significant Treg depletion in blood and the tumor microenvironment but did not produce significant clinical efficacy. Interferon-α is not an effective ovarian cancer treatment, but we show here that it significantly augments immune and clinical DT-mediated Treg depletion efficacy in human ovarian cancer. In a mouse ovarian cancer model, DT modestly improved anti-tumor immunity and survival. Interferon-α alone did not alter Treg numbers or function, but boosted CD8+ T cell anti-tumor immunity. Interferon-α plus DT significantly prolonged mouse survival over either individual agent. Using type I IFNR-/- mice that cannot mediate interferon-α signals, we showed that interferon-α directly boosted CD8+ T cell function independent of CD4+ T cell help, and in combination with DT reduced Treg function (without further numerical reduction) through indirect effects on tumor microenvironmental dendritic cells. When three ovarian cancer patients failed DT alone, two experienced immunologic and clinical benefit by adding pegylated interferon-α2a, with manageable toxicities. These studies demonstrate novel immune and clinical interferon-α anti-cancer benefits that augment Treg depletion using FDA-approved agents for rapid clinical translation.

B7-H1 blockade improves efficacy of regulatory T cell depletion as cancer immunotherapy by reducing Treg regeneration, possibly through tumor B7-H1 effects (VAC3P.950)

Abstract Immune co-signaling blockade is effective cancer immunotherapy but is not curative alone. Treg depletion has shown potential human anti-cancer efficacy, but is also not curative alone. As B7-H1 immune co-signaling facilitates Treg generation, we hypothesized that adding αB7-H1 to Treg depletion would improve treatment by hindering Treg regeneration. We show here that αB7-H1 plus denileukin diftitox (DD) to deplete Tregs is a synergistic combo that effects complete and durable (>7 mos) tumor regressions in bulky B16 melanoma, whereas neither agent alone does. αB7-H1 efficacy alone or with DD is lost in IFN-γ KO or T cell KO mice, and is mediated in part by tumor-specific CD8+ (but not CD4+) T cells. αB7-H1 is highly effective in B7-H1 KO mice, demonstrating a major role for tumor B7-H1 in treatment efficacy. Reducing tumor B7-H1-mediated Treg generation appears to be a mechanism as predicted. The αB7-H1/DD combo was also effective in ID8 ovarian cancer showing broad application. We engineered B7-H1hi/lo B16 tumors for further immune studies. Surprisingly, B7-H1hi B16 grew faster than B7-H1lo in RAG KO, suggesting B7-H1 as an adaptive immunity-independent tumor growth/survival factor. IFNγ upregulated tumor B7-H1, but αB7-H1 did not prevent IFN-γ-mediated tumor inhibition in vitro. B7-H1hi/lo tumors in B7-H1 KO are being used to define tumor vs host effects of αB7-H1 ± Treg depletion. As αB7-H1 and DD have been tested clinically, this combo is rapidly clinically translated.

Interventions for mycosis fungoides: critical commentary on a Cochrane Systematic Review.

The authors performed a systematic review of randomized controlled trials (RCTs) on interventions for any stage of typical mycosis fungoides (MF). They searched electronic databases including the Cochrane Central Register of Controlled Trials, Medline, Embase, and the Latin American and Caribbean Health Science Information database, and included reports from conference proceedings and unpublished data without language restrictions. The authors also searched trial registries affiliated with the U.S.A., Australia, the World Health Organization and the European Organisation of Research and Treatment of Cancer for studies on 'mycosis fungoides' or 'cutaneous T-cell lymphoma'. These searches were supplemented by correspondence with the groups or individuals who conducted the RCTs. The authors included RCTs with participants who were 18years of age or older, that had staging information, and in which >90% of patients had biopsy-proven typical CD4+ MF. Data on treatment and outcome of participants, including information on stage of MF, therapy, quality of life, remission or improvement, duration of remission, survival, adverse effects and toxicity were obtained from included studies. Primary outcomes were adverse effects and quality of life. Secondary outcomes were clearance of at least 90% of surface area involvement, improvement of at least 50% of surface area involvement, survival rate, relapse rate and disease-free interval. The authors also recorded potentially significant participant-related prognostic factors, such as age and sex, and tumour-related prognostic factors, such as histological subtype and systemic involvement. From 407 unique references, 14 RCTs were included with a total of 675 patients. These trials included skin-directed therapies [topical peldesine, topical imiquimod, topical hypericin, intralesional interferon (IFN)-α, psoralen ultraviolet A (PUVA) therapy, electron-beam therapy (EBT) and local radiation], systemic therapies [extracorporeal photopheresis (ECP), denileukin diftitox, bexarotene] and combination therapies (injected transfer factor with concomitant topical nitrogen mustard use). Only one meta-analysis of two studies comparing PUVA with IFN-α vs. PUVA alone could be performed, and no significant differences between the two therapies were found. Two studies on intralesional IFN-α vs. placebo were included in the review and provided opposing results, but were not examined by meta-analysis due to differences in their study design. The remainder of the Cochrane analysis reviewed outcomes of individual RCTs. There were statistically significant differences in improvement or clearance for five therapeutic regimens. One trial of topical hypericin vs. placebo found a relative benefit of hypericin, risk ratio (RR) for improvement 7·00, 95% confidence interval (CI) 1·01-48·54, P≤0·028. A trial comparing ECP with PUVA demonstrated significantly better improvement in the PUVA group (RR 0·07, 95% CI 0·00-1·00, P≤0·002). An RCT examining 'conservative', stepwise escalation from topical nitrogen mustard to 'combination therapy' with EBT and cyclophosphamide, doxorubicin, etoposide and vincristine chemotherapy found that combination therapy was superior in clearance (RR 2·18, 95% CI 1·10-4·33, P≤0·03) and improvement (RR 1·40, 95% CI 1·12-1·74, P≤0·003). However, there were no statistically significant differences in survival rates at a median follow-up of 75months. A comparison of subcutaneously injected IFN-α and acitretin vs. subcutaneously injected IFN-α and PUVA found increased clearance with IFN-α and PUVA (RR 0·54, 95% CI 0·35-0·84, P≤0·005). There were also significant reductions in grade III, severe adverse events on the World Health Organization scale; events requiring discontinuation; and neurological disorders in the IFN-α plus PUVA group. Finally, a trial comparing active vs. inactivated transfer factor found significant differences between the groups, favouring inactivated transfer factor (Fisher's exact test, P≤0·03, RR 0·09, 95% CI 0-0·61). The original study authors speculated that their results reflected a better initial prognosis for the group receiving inactivated transfer factor. None of the interventions assessed showed significant long-term benefit. Despite significantly superior clearance rates in four trials, participants in those studies had high relapse rates. This review of RCTs for MF interventions led to more questions than answers due to a dearth of adequately powered RCTs. Only one meta-analysis could be performed. The remaining review was based on single trials, many of which assessed infrequently used treatments or regimens and are not reflective of current clinical practices. Only two of the 14 RCTs assessed patient health-related quality-of-life outcomes.

Denileukin diftitox depletes regulatory T cells without clinical benefit in advanced stage epithelial ovarian carcinoma (VAC3P.945)

Abstract Denileukin diftitox (DT) depletes regulatory T cells (Treg) that correlates with immune and clinical benefits in metastatic human melanoma and improved clinical outcomes in a renal cancer vaccine trial. We tested immune and clinical effects of Treg depletion using DT in a phase 0/I cancer trial and a phase II ovarian cancer trial. In our phase 0/I trial, we noted reductions in blood Treg prevalence and concentration (median ~18% and ~50%, respectively) 3-7 days after one intravenous DT infusion at 9 or 12 μg/kg, in 6 of 7 evaluable patients with breast, lung, and ovarian cancers, and melanoma, and increased blood IFN-γ+ and Ki-67+ T cells. Weekly DT significantly reduced metastatic tumors in one ovarian cancer patient prompting a small phase II trial in epithelial ovarian cancers. 28 patients received DT once every 3-4 weeks which significantly depleted functional Tregs from blood and the tumor microenvironment, but with variable immune outcomes and no significant clinical efficacy. Weekly DT eventually reduced effector T cells. In mouse ovarian cancer models we found that: i) DT efficacy depended on adaptive immunity, ii) its IL-2 moiety did not mediate clinical effects, and iii) its treatment mechanism appeared distinct from anti-CD25 antibody, which depleted Tregs in a human breast cancer trial. DT depletes Tregs in various carcinomas but requires more dosing and schedule studies. Treg-specific agents and combination treatments could also improve Treg depletion efficacy.

Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25+ cells

Regulatory T cells (Treg) constitute a major inhibitory cell population which suppresses immune responses. Thus, Treg have proven to be key players in the induction of transplantation tolerance, protection from autoimmune disease and prevention of the development of effective anti-tumor immune reactions. Treg express high levels of the high affinity interleukin-2 receptor (IL-2R) consisting of IL-2Rα (CD25) together with IL-2Rβ (CD122) and the common γ-chain (CD132). An effective reagent capable of depleting Treg in vivo would facilitate better cancer treatment and allow mechanistic studies of the role of Treg in transplantation tolerance and the development of autoimmune disease. In this study, we have developed a novel bivalent human IL-2 fusion toxin along with an Ontak®-like monovalent human IL-2 fusion toxin and compared the functional ability of these reagents in vitro. Here we show that genetically linking two human IL-2 domains in tandem, thereby generating a bivalent fusion toxin, results in significantly improved capacity in targeting human CD25+ cells in vitro. Binding analysis by flow cytometry showed that the bivalent human IL-2 fusion toxin has notably increased affinity for human CD25+ cells. In vitro functional analysis demonstrated that the bivalent isoform has an increased potency of approximately 2 logs in inhibiting cellular proliferation and protein synthesis in human CD25+ cells compared to the monovalent human IL-2 fusion toxin. Additionally, we performed two inhibition assays in order to verify that the fusion toxins target the cells specifically through binding of the human IL-2 domain of the fusion toxin to the human IL-2 receptor on the cell surface. These results demonstrated that 1) both monovalent and bivalent human IL-2 fusion toxins are capable of blocking the binding of biotinylated human IL-2 to human CD25 by flow cytometry; and 2) human IL-2 blocked the fusion toxins from inhibiting protein synthesis and cellular proliferation in vitro, thus confirming that the human IL-2 fusion toxins target the cells specifically through binding to the human IL-2 receptor. We believe that the bivalent human IL-2 fusion toxin will be a more potent, and therefore, more optimal agent than the current clinically-used monovalent fusion toxin (denileukin diftitox, Ontak®) for in vivo depletion of Treg.

Denileukin Diftitox plus Total Skin Electron Beam Radiation in Patients with Treatment-refractory Cutaneous T-cell Lymphoma (Mycosis Fungoides): Report of Four Cases

Denileukin Diftitox plus Total Skin Electron Beam Radiation in Patients with Treatment-refractory Cutaneous T-cell Lymphoma (Mycosis Fungoides): Report of Four Cases

HIV-1 Vpr Accelerates Viral Replication during Acute Infection by Exploitation of Proliferating CD4+ T Cells In Vivo

The precise role of viral protein R (Vpr), an HIV-1-encoded protein, during HIV-1 infection and its contribution to the development of AIDS remain unclear. Previous reports have shown that Vpr has the ability to cause G2 cell cycle arrest and apoptosis in HIV-1-infected cells in vitro. In addition, vpr is highly conserved in transmitted/founder HIV-1s and in all primate lentiviruses, which are evolutionarily related to HIV-1. Although these findings suggest an important role of Vpr in HIV-1 pathogenesis, its direct evidence in vivo has not been shown. Here, by using a human hematopoietic stem cell-transplanted humanized mouse model, we demonstrated that Vpr causes G2 cell cycle arrest and apoptosis predominantly in proliferating CCR5+ CD4+ T cells, which mainly consist of regulatory CD4+ T cells (Tregs), resulting in Treg depletion and enhanced virus production during acute infection. The Vpr-dependent enhancement of virus replication and Treg depletion is observed in CCR5-tropic but not CXCR4-tropic HIV-1-infected mice, suggesting that these effects are dependent on the coreceptor usage by HIV-1. Immune activation was observed in CCR5-tropic wild-type but not in vpr-deficient HIV-1-infected humanized mice. When humanized mice were treated with denileukin diftitox (DD), to deplete Tregs, DD-treated humanized mice showed massive activation/proliferation of memory T cells compared to the untreated group. This activation/proliferation enhanced CCR5 expression in memory CD4+ T cells and rendered them more susceptible to CCR5-tropic wild-type HIV-1 infection than to vpr-deficient virus. Taken together, these results suggest that Vpr takes advantage of proliferating CCR5+ CD4+ T cells for enhancing viremia of CCR5-tropic HIV-1. Because Tregs exist in a higher cycling state than other T cell subsets, Tregs appear to be more vulnerable to exploitation by Vpr during acute HIV-1 infection.

Cytotoxic and Gamma-Delta Subtypes Of Peripheral T-Cell Lymphoma: A Single Center Experience

BackgroundPeripheral T-cell lymphomas (TCLs) are uncommon neoplasms. TCLs in which gamma-delta T-cell receptors are expressed are extremely aggressive and rare, accounting for less than 1% of lymphoid neoplasms. The two main types of CTLs recognized in the 2008 WHO classification of lymphoid neoplasms are hepatosplenic T-cell lymphoma (HSTL) and primary cutaneous gamma-delta T-cell lymphoma (PCGD-TCL). Although rare, these diseases are associated with an aggressive clinical course, refractoriness to conventional therapies, and poor prognosis. MethodsPatients with newly diagnosed cytotoxic or gamma-delta TCLs treated from 2005-2012 were retrospectively identified. Analyses included patient characteristics, clinical presentation, pathology, treatment and outcome. ResultsTwenty-two patients (11 cutaneous gamma-delta TCL, 5 primary cutaneous aggressive epidermotropic CD8+TCL, 4 hepatosplenic gamma-delta TCL, 2 cytotoxic TCL involving the intestine) were identified. Median age was 48 (range: 18-70), 12% (12/22) were male, and 54.5% (12) presented with B symptoms. Initial therapy included CHOP based chemotherapy in 6, denileukin difititox with CHOP in 5, and single agent denileukin diftitox in 4. One patient died with hemophagocytosis prior to initiation of chemotherapy. Fifteen of 21 pts responded to initial therapy but 10 of 15 required more than one regimen containing either etoposide or gemcitabine to achieve remission. The median number of regimens was 2 (range 0-4). In the salvage setting, two patients had partial responses with romidepsin, one had a PR with pralatrexate and one had a CR after pralatrexate salvage therapy. Thirteen patients underwent stem cell transplantation in first remission: autologous in 5 pts who had no donor, and allogeneic in 8 pts. At a median follow up of 3 years, 60% relapsed, and 14 (63%) are still alive. Of the patients who underwent transplant, one auto pt died from hemophagocytosis, 2 alloBMT patients died from acute GVHD, and 2 alloBMT pts died from disease progression. One alloBMT patient with cutaneous gamma delta TCL relapsed and responded to salvage therapy. ConclusionsCytotoxic and gamma-delta TCLs are rare diseases that have traditionally responded poorly to treatment. In our experience, many patients have a suboptimal response to CHOP alone and require additional therapy to achieve remission. We report a favorable outcome after stem cell transplantation and with implementation of novel agents. Advances in understanding biology and international collaborative efforts are required to improve outcome in these rare entities. Disclosures:Off Label Use:sorafenib in t cell nhl. Foss:merck: Research Funding; spectrum: Research Funding; eisai: Membership on an entity's Board of Directors or advisory committees; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Honoraria, Research Funding; seattle genetics: Research Funding.

Denileukin diftitox (ONTAK) induces a tolerogenic phenotype in dendritic cells and stimulates survival of resting Treg

AbstractDenileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25+ cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen–specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator–dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25+ cell killing that may affect their clinical use and could be tested for novel indications. This trial was registered at www.clinicaltrials.gov, #NCT00056134.

Tumor-Specific T Cells in Human Merkel Cell Carcinomas: A Possible Role for Tregs and T-Cell Exhaustion in Reducing T-Cell Responses

Merkel cell carcinomas (MCC) are rare but highly malignant skin cancers associated with a novel polyomavirus. MCC tumors were infiltrated by T cells, including effector, central memory and regulatory T cells. Infiltrating T cells showed markedly reduced activation as evidenced by reduced expression of CD69 and CD25. Treatment of MCC tumors in vitro with IL-2 and IL-15 led to T cell activation, proliferation, enhanced cytokine production and loss of viable tumor cells from cultures. Expanded tumor-infiltrating lymphocytes showed TCR repertoire skewing and upregulation of CD137. MCC tumors implanted into immunodeficient mice failed to grow unless human T cells in the tumor grafts were depleted with denileukin diftitox, suggesting tumor-specific T cells capable of controlling tumor growth were present in MCC. Both CD4+ and CD8+ FOXP3+ regulatory T cells were frequent in MCC. 50% of non-activated T cells in MCC expressed PD-1, a marker of T-cell exhaustion, and PD-L1 and PD-L2 were expressed by a subset of tumor dendritic cells and macrophages. In summary, we observed tumor-specific T cells with suppressed activity in MCC tumors. Agents that stimulate T cell activity, block Treg function or inhibit PD-1 signaling may be effective in the treatment of this highly malignant skin cancer.

Duration of Response in Cutaneous T-Cell Lymphoma Patients Treated With Denileukin Diftitox: Results From 3 Phase III Studies

We aimed to determine duration of response in patients with CTCL treated using DD who experienced partial response, complete clinical response, or complete response. Data from 3 phase III studies were pooled. Patients received up to 8 courses of 9 or 18 μg/kg intravenous DD daily for 5 days every 21 days, or placebo. Data on DD-treated patients were analyzed by dose and CD25 status. Kaplan-Meier product limit estimates and 95% confidence intervals were calculated for duration of response and time to response. The pooled population comprised 263 DD-treated and 44 placebo-treated patients, and 100 and 7, respectively, had at least a partial response. Median duration of response using DD was 277 days vs. 81 days using placebo. Overall response vs. placebo (n = 7; 16%) as (n = 25; 31%) for DD 9 μg/kg (P= .05), (n = 56; 47%) for DD 18 μg/kg (P= .004), (n = 8; 28%) for re-treated patients (DD 18 μg/kg; P= .21), and (n = 11; 31%) for CD25 low-expression patients (DD 18 μg/kg; P= .14). Overall response rates were similar between patients who did (n = 95; 36%) and did not (n = 105; 40%) develop capillary leak syndrome (CLS); median duration of response was longer in patients who developed CLS, but was not significant (619 vs. 267 days, respectively; P= .28). Adverse events occurred in 98% of DD-treated patients; most frequent were nausea, pyrexia, fatigue, CLS, and rigors. These data indicate a durable response with DD in CTCL, even in heavily pretreated patients and those with CD25 low-expression disease.

Review of the Treatment of Mycosis Fungoides and SéZary Syndrome : A Stage-Based Approach

Mycosis fungoides (MF) and Sézary Syndrome (SS) are the most common subtypes of cutaneous T-cell lymphomas. Most of patients have indolent and incurable course of disease. Therefore, treatment should be reaching the optimal benefit with minimizing the toxicity as much as possible. To achieve this aim, the management should follow a -stage-based-approach. Treatment of early-stage MF (IA-IIA) involves skin-directed therapy (SDT) including topical corticosteroids, phototherapy, topical chemotherapy, topical retinoids and radiotherapy. For aggressive/recalcitrant early-stage MF or advanced-stage MF, systemic therapy should be considered including interferone-alpha, oral retinoids including bexarotine and more recently acitretin, histone deacetylase inhibitors (HDACi), fusion toxin denileukin diftitox and chemotherapy drugs. Combined drug regimens can be considered in some situations to get the synergistic effect while lowering the individual drug's doses on the other hand. By exception of aggressive stages, chemotherapy should always come after other systemic drugs have been tried or contraindicated. Novel drugs should be considered in situations when all systemic drugs have failed.

Are there any differences in the regulations of personalized medicine among the USA, EU and Japan?

In their cautious review, Shah and Shah [1] emphasized differences in regulations of personalized medicine (PM) among the three major authorities, the US Food and Drug Administration (FDA), the European Medicines Agency (EMA) and the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan. Specific points regarding the differences, however, were not raised for the drugs they selected for discussion in their review. Given that the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) brings together the regulatory authorities and pharmaceutical industries of the USA, Europe and Japan, scientific and technical aspects of drug registration should be harmonized. To identify differences, if any, in regulations of PM, we investigated approvals of PM drugs in the three regions. As a typical example of PM, we focused on PM [2] drugs whose pharmacogenomic biomaker is required on the label. We also studied ivacaftor and pertuzumab, which were omitted in the list [2] simply because they were approved after publication of the list. The US, European and Japanese approval data on these drugs were obtained from Drugs@FDA (http://www.accessdata.fda.gov/Scripts/cder/DrugsatFDA/), European public assessment reports (http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/landing/epar_search.jsp&mid=WC0b01ac058001d125) and the PMDA website (http://www.info.pmda.go.jp/approvalSrch/PharmacySrchInit?), respectively. We defined submission/approval delay as the difference between the date of submission/approval in the USA and that in the EU or in Japan. Of 17 FDA-approved drugs and 18 indications whose biomarker is labelled as required, 13 drugs and 14 indications were approved in the EU, whereas 12 drugs and 12 indications were approved in Japan (Table 1). The median submission delay from the submission in the USA was 0 months in the EU and 21 months in Japan. The median approval delay from the approval in the USA was 6 months in the EU and 28 months in Japan. Table 1 US, EU and Japanese data on the approval of personalized medicine drugs whose pharmacogenomic biomarker is required on the label One would expect that labels would not differ significantly among countries, given that regulatory authorities evaluate the same scientific data. Both biological and nonbiological factors, however, can affect regulatory decisions. For example, a much lower incidence of cystic fibrosis [3] and melanoma [4] in Japan compared with the West could discourage the makers of ivacaftor and vemurafenib to file an application to the PMDA. Denileukin diftitox and tositumomab, which were approved for lymphoma by the FDA in 1999 and 2003, respectively, remain unavailable in both the EU and Japan, probably because better treatment modalities are available now. The approval delay in Japan was observed in other therapeutic areas [5]. The present study shows that three-quarters of the approval delay consisted of delays in submission. The approval delay without submission delay in the EU indicates that the reviews took longer for the EMA than for the FDA. The cross-sectional design of our study makes causal inference of these delays difficult. Our results show some similarities and differences in the approvals of PM drugs among the three regions of the ICH. Further studies are needed to investigate differences in postmarketing regulations of PM drugs, because such regulations are important for risk–benefit assessment of PM and are greatly affected by local factors, such as health polices, culture and financial settings.

A multicenter phase II trial to determine the safety and efficacy of combination therapy with denileukin diftitox and cyclophosphamide, doxorubicin, vincristine and prednisone in untreated peripheral T-cell lymphoma: the CONCEPT study

This phase II study to determine the safety and efficacy of denileukin diftitox (DD) and cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) enrolled patients with newly diagnosed peripheral T-cell lymphoma (PTCL). Forty-nine received DD 18 μg/kg/day (days 1, 2) with CHOP (day 3) every 21 days for ≤ 6–8 cycles. Intent-to-treat (ITT) and safety populations comprised all patients. In the ITT population, the overall response rate was 65%, median duration of response was 30 months and median progression-free survival was 12 months. Median overall survival was not attained at the end of the study, and the overall survival rate was 63.3%. The two most frequent treatment-related adverse events (AEs) were fatigue and nausea. Most frequent AEs ≥ grade 3 within the hematologic system were lymphopenia (24.5%), neutropenia (20.4%) and leukopenia (18.4%). Three treatment-related deaths occurred. DD plus CHOP was well tolerated, and progression-free and overall survival improved versus historical comparison with CHOP alone. Confirmation in larger trials is warranted.