Regulatory T cells control toxicity in a humanized model of IL-2 therapy.

While patient selection and clinical management have reduced high-dose IL-2 (HDIL2) immunotherapy toxicities, the immune mechanisms that underlie HDIL2-induced morbidity remain unclear. Here we show that dose-dependent morbidity and mortality of IL-2 immunotherapy can be modeled in human immune system (HIS) mice. Depletion of human T cell subsets during the HDIL2 treatment reduces toxicity, pointing to the central function of T cells. Preferential expansion of effector T cells secondary to defective suppressive capacity of regulatory T (Treg) cells after HDIL2 therapy further underscores the importance of Treg in the maintenance of immune tolerance. IL-2 toxicity is induced by selective depletion or inhibition of Treg after LDIL2 therapy, and is ameliorated in HDIL2-treated HIS mice receiving the PIM-1 kinase inhibitor, Kaempferol. Modeling IL-2 pathophysiology in HIS mice offers a means to understand the functions of effector and regulatory T cells in immune-mediated toxicities associated with cancer immunotherapy.

Ontak-like human IL-2 fusion toxin.

Ontak® is a FDA-approved diphtheria toxin-based recombinant fusion toxin for treatment of human CD25+ cutaneous T cell lymphoma (CTCL). However, it has been discontinued clinically due to the production issue related to the bacterial expression system with difficult purification. Recently we have developed monovalent and bivalent human IL-2 fusion toxins targeting human CD25+ cells using advanced unique diphtheria toxin resistant yeast Pichia Pastoris expression system. In vitro efficacy characterization using human CD25+ HUT102/6TG cells demonstrated that both monovalent and bivalent isoforms are potent and the bivalent isoform is approximately two logs more potent than the monovalent isoform. In this study, we further assessed the in vivo efficacy of the human IL-2 fusion toxins using human CD25+ HUT102/6TG tumor-bearing NSG mouse model. The data demonstrated that both monovalent and bivalent human IL-2 fusion toxins significantly prolonged the survival of the human CD25+ tumor-bearing NSG mice in a dose-dependent manner. Then we further assessed the residual tumor cells from the HUT102/6TG tumor-bearing NSG mice using the residual tumor cell bearing NSG mouse model. The results demonstrated that the residual tumor cells were still sensitive to the continual treatment with the human IL-2 fusion toxin. This yeast-expressed human IL-2 fusion toxin will be a promising candidate to replace the clinically discontinued Ontak®.

CD25 targeted therapy of chemotherapy resistant leukemic stem cells using DR5 specific TRAIL peptide.

Chemotherapy resistant leukemic stem cells (LSCs) are being targeted as a modern therapeutic approach to prevent disease relapse. LSCs isolated from methotrexate resistant side population (SP) of leukemic cell lines HL60 and MOLT4 exhibited high levels of CD25 and TRAIL R2/DR5 which are potential targets. Recombinant immunotoxin conjugating IL2α with TRAIL peptide mimetic was constructed for DR5 receptor specific targeting of LSCs and were tested in total cell population and LSCs. IL2-TRAIL peptide induced apoptosis in drug resistant SP cells from cell lines and showed potent cytotoxicity in PBMCs derived from leukemic patients with an efficacy of 81.25% in AML and 100% in CML, ALL and CLL. IL2-TRAIL peptide showed cytotoxicity in relapsed patient samples and was more effective than TRAIL or IL2-TRAIL proteins. Additionally, DR5 specific IL2-TRAIL peptide was effective in targeting and killing LSCs purified from cell lines [IC50: 952nM in HL60, 714nM in MOLT4] and relapsed patient blood samples with higher efficacy (85%) than IL2-TRAIL protein (46%). Hence, CD25 and DR5 specific targeting by IL2-TRAIL peptide may be an effective strategy for targeting drug resistant leukemic cells and LSCs.

Temporal Patterns of Novel Circulating Biomarkers in IL-2-mediated Vascular Injury in the Rat.

Recombinant interleukin-2 (rIL-2) administration in oncology indications is hampered by vascular toxicity, which presents as a vascular leak syndrome. We used this aspect of the toxicity of rIL-2 to evaluate candidate biomarkers of drug-induced vascular injury (DIVI) in rats given 0.36 mg/kg rIL-2 daily. Groups of rats were given either 2 or 5 doses of rIL-2 or 5 doses of rIL-2 followed by a 7-day recovery. The histomorphologic lexicon and grading scheme developed by the Vascular Injury Working Group of the Predictive Safety Testing Consortium of the Critical Path Institute were utilized to enable semiquantitative integration with circulating biomarker levels. The administration of rIL-2 was associated with time-dependent endothelial cell hyperplasia and hypertrophy and perivascular inflammation that correlated with increases in circulating angiopoietin-2, lipocalin-2, monocyte chemotactic protein-1, tissue inhibitor of metalloproteinase-1, vascular endothelial growth factor A, E-selectin, and chemokine (C-X-C motif) ligand-1, and the microRNAs miR-21, miR-132, and miR-155. The dose groups were differentially identified by panels comprising novel candidate biomarkers and traditional hematologic parameters. These results identify biomarkers of the early stages of DIVI prior to the onset of vascular smooth muscle necrosis.

Induction of vascular leak syndrome by tumor necrosis factor-alpha alone.

Although TNF-α possesses promising anticancer activity, clinical application is limited partly due to cardiovascular toxicities. TNF-α effects on vessels are likely related to vascular toxicity, but much remains poorly understood. Similarly, IL-2 is an attractive treatment option for cancers but its clinical use is limited by the side effect of vascular leak syndrome (VLS). We report here that TNF-α alone can trigger VLS. Administration of recombinant TNF-α induced VLS in normal mice and TNF-α transgenic mice exhibited VLS. Perivascular infiltrates in the lungs and specific cytokines in serum were observed in VLS-induced mice. This study shed a new light on the critical role of the TNF-α in IL-2-induced or non IL-2-induced VLS and provides important points to TNF-α therapy.

Identification of a cytotoxic form of dimeric interleukin-2 in murine tissues.

Interleukin-2 (IL-2) is a multi-faceted cytokine, known for promoting proliferation, survival, and cell death depending on the cell type and state. For example, IL-2 facilitates cell death only in activated T cells when antigen and IL-2 are abundant. The availability of IL-2 clearly impacts this process. Our laboratory recently demonstrated that IL-2 is retained in blood vessels by heparan sulfate, and that biologically active IL-2 is released from vessel tissue by heparanase. We now demonstrate that heparanase digestion also releases a dimeric form of IL-2 that is highly cytotoxic to cells expressing the IL-2 receptor. These cells include "traditional" IL-2 receptor-bearing cells such as lymphocytes, as well as those less well known for IL-2 receptor expression, such as epithelial and smooth muscle cells. The morphologic changes and rapid cell death induced by dimeric IL-2 imply that cell death is mediated by disruption of membrane permeability and subsequent necrosis. These findings suggest that IL-2 has a direct and unexpectedly broad influence on cellular homeostatic mechanisms in both immune and non-immune systems.

A four-week repeated study of intravenous toxicity of recombinant human interleukin-2 in Sprague-Dawley rats.

Interleukin-2 (IL-2) is a lymphokine with a potential role in cancer therapy. Many clinical trials of recombinant human IL-2 (rhIL-2) have been conducted to treat malignant renal carcinoma, melanoma, leukemia, lymphoma, multiple myeloma. BMI Korea has developed a method to manufacture rhIL-2 in bulk using Escherichia coli as a biosimilar. Prior to conducting human clinical trials, 4-week repeated toxicity study of rhIL-2 was conducted. In this study, rhIL-2 was administered intravenously to rats at doses of 9×10(6), 18×10(6), and 36×10(6)IU/kg/day over a period of 4 weeks. Adverse effects were observed in RBC, HGB, HCT, reticulocyte, mesenteric lymph node from middle dose, and changes of total bilirubin, femoral bone marrow, thymus, and clinical signs were observed at high dose. Local irritation was determined at low dose of female rats and at middle dose of male ones. Taken together, no observed adverse effect levels (NOAEL) was determined at dose of 9×10(6)IU/kg/day in male, and NOAEL was determined under the dose level in female rats. It suggests that present rhIL-2 is less toxic prior produced rhIL-2 and may be contribute more effective cancer-treatment strategy in human.

The mixed human umbilical cord blood-derived mesenchymal stem cells show higher antitumor effect against C6 cells than the single in vitro.

OBJECTIVE: It is difficult for the treatment of neurologic tumors with current therapies, including glioma. Despite the advances in cancer therapeutics, the outcomes in these patients remain poor and, therefore, new modalities are required. Recent findings have demonstrated that human umbilical cord blood mesenchymal stem cells (UCB-MSCs) have the potential to inhibit glioma cell growth in vitro. Caspase-3 plays a critical role as an executioner of apoptosis and the level of caspase-3 expression determines the degree of apoptosis in cancer cell lines. CD133 (+) glioma displays a strong resistance to chemotherapy. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) are important cytokines in the generation of antitumor immunity. METHODS: UCB-MSCs were harvested by density gradient separation. Green fluorescence protein stable C6 cells were established. Cytotoxicity was detected by visual survival cell assay, and caspase-3 activity was assessed using immunohistochemistry staining and western blot. Flow cytometry was used to test CD133 positive C6 cells. The concentrations of IL-2 and IFN-gamma proteins secreted from UCB-MSCs were then quantified using enzyme-linked immunosorbent assay. The cytotoxicities of IL-2 alone, IFN-gamma alone, and combination of IL-2 and IFN-gamma were compared against malignant glioma cells. RESULTS: We noted a significant cytotoxicity of UCB-MSCs for malignant glioma cells. In addition, the toxicity of mixed UCB-MSCs was significantly higher than that of single UCB-MSCs for C6 glioma cells. Capase-3 levels in UCB-MSCs treatment at an effector/target (E/T) ratio of (5+5):1 were higher than those at an E/T ratio of 10:1. On the contrary, there was no change in the protein expression of capase-3 at an E/T ratio of 0:1. Immunohistochemistry staining and western blot revealed that the expression of capase-3 was higher in mixed UCB-MSCs treatment, when compared with single UCB-MSCs. We identified reductions of approximately 39 and 73% in the number of CD133 positive C6 cells treated with the single (10:1) and the mixed [(5+5):1] UCB-MSCs respectively as compared to the control group (0:1) in transwell inserts. However, the mixed UCB-MSCs secreted more immune response-related proteins (IL-2 and IFN-gamma) than the single UCB-MSCs. Combination of IL-2 and IFN-gamma might prove more cytotoxic on target cells than IL-2 alone and IFN-gamma alone. DISCUSSION: The data collected herein confirm for the first time that the mixed UCB-MSCs were shown to have more cytotoxic effects than the single UCB-MSCs through increasing the expression of caspase-3 and decreasing the expression of CD133 in C6 glioma cells. In addition, the mixed UCB-MSCs secrete more immune response-related proteins (IL-2 and IFN-gamma) than the single UCB-MSCs. Combination of IL-2 and IFN-gamma was shown to have more cytotoxic effects than IL-2 alone and IFN-gamma alone. These results demonstrate that the mixed UCB-MSCs are a potential new therapeutic agent for glioma.

A phase I/II study of chemotherapy followed by donor lymphocyte infusion plus interleukin-2 for relapsed acute leukemia after allogeneic hematopoietic cell transplantation.

The efficacy of donor lymphocyte infusion (DLI) for treatment of relapsed acute leukemia after allogeneic hematopoietic cell transplantation is limited. We hypothesized that interleukin-2 (IL-2) combined with DLI after chemotherapy might augment graft-versus-leukemia effects. To identify a safe and effective IL-2 regimen, a phase I/II study of DLI plus IL-2 therapy was performed for such patients. After chemotherapy, 17 patients received DLI (1 × 10(8) CD3/kg for patients with related donors, and 0.1 × 10(8) CD3/kg for those with unrelated donors) and an escalating dose of induction IL-2 (1.0, 2.0, or 3.0 × 10(6) IU/m(2)/day representing levels I [n = 7], Ia [n = 9], and II [n = 1]) for 5 days followed by maintenance (1.0 × 10(6) IU/m(2)/day) for 10 days as a continuous intravenous infusion. Unacceptable IL-2-related toxicities developed in 1 patient at level I, 2 at level Ia, and 1 at level II. Grades III-IV acute graft-versus-host disease (aGVHD) developed in 5 patients, and extensive chronic GVHD (cGVHD) developed in 8. Eight patients had a complete remission after chemotherapy prior to DLI, and 2 additional patients had a complete remission after DLI plus IL-2 therapy. In conclusion, the maximal tolerated induction dose of IL-2 combined with DLI appears to be 1.0 × 10(6) IU/m(2)/day. IL-2 administration after DLI might increase the incidence of cGVHD.

Doxycycline induces membrane expression of VE-cadherin on endothelial cells and prevents vascular hyperpermeability.

The endothelium lining blood vessels serves as a barrier against vascular hyperpermeability, and its maintenance is critical to organ health. Inflammatory mediators evoke tissue edema by disrupting the expression of membrane junctional proteins, which mediate binding between endothelial cell membranes. Endothelial cell-cell junctions form a diffusion barrier between the intravascular and interstitial space. To prevent the morbidity and mortality caused by exaggerated vascular permeability associated with pathological states (e.g., inflammatory and hypersensitivity disorders, pulmonary edema, traumatic lung injury, cerebral edema resulting from stroke, and others), it is important to develop therapeutic approaches to stabilize these interendothelial junctions. Vascular endothelial growth factor (VEGF), a potent proangiogenic cytokine, was first described as vascular permeability factor (VPF). Doxycycline, a tetracycline derivative, has been shown to inhibit angiogenesis in both humans and animal models. We now report that oral doxycycline prevents VPF/VEGF-induced vascular permeability, interleukin-2-induced pulmonary edema, and delayed-type hypersensitivity (DTH) in mice. Remarkably, doxycycline also inhibits tumor growth and tumor-associated vascular hyperpermeability. Finally, we show that doxycycline targets the adherens junction in vascular endothelial cells by inducing the total amount of VE-cadherin expression while decreasing the degree of its phosphorylation. The potential of doxycyline as a therapeutic inhibitor of vascular hyperpermeability in human clinical conditions is promising and warrants further studies.

Treg depletion-enhanced IL-2 treatment facilitates therapy of established tumors using systemically delivered oncolytic virus.

There are several roadblocks that hinder systemic delivery of oncolytic viruses to the sites of metastatic disease. These include the tumor vasculature, which provides a physical barrier to tumor-specific virus extravasation. Although interleukin-2 (IL-2) has been used in antitumor therapy, it is associated with endothelial cell injury, leading to vascular leak syndrome (VLS). Here, we demonstrate that IL-2-mediated VLS, accentuated by depletion of regulatory T cells (Treg), facilitates localization of intravenously (i.v.) delivered oncolytic virus into established tumors in immune-competent mice. IL-2, in association with Treg depletion, generates "hyperactivated" natural killer (NK) cells, possessing antitumor activity and secreting factors that facilitate virus spread/replication throughout the tumor by disrupting the tumor architecture. As a result, the combination of Treg depletion/IL-2 and systemic oncolytic virotherapy was found to be significantly more therapeutic against established disease than either treatment alone. These data demonstrate that it is possible to combine biological therapy with oncolytic virotherapy to generate systemic therapy against established tumors.

Influence of immunomodulatory drugs on the cytotoxicity induced by monoclonal antibody 17-1A and interleukin-2.

Patients treated with monoclonal antibodies and cytokines for cancer receive often co-medication, which may influence treatment efficacy. Therefore, we investigated with a flowcytometric cytotoxicity assay the effect of several immunomodulatory drugs on antibody dependent cellular cytotoxicity (ADCC), interleukin-2 (IL-2) induced cytotoxicity and IL-2-induced-ADCC. We found that dexamethasone markedly inhibited the IL-2 induced cytotoxicity and the IL-2-induced-ADCC. Ondansetron, a 5-HT-3 serotonin receptor antagonist augmented significantly ADCC. Clemastine, a histamine type-2 receptor antagonist augmented the IL-2-induced-ADCC. The TNF antagonist thalidomide suppressed ADCC whereas pentoxifylline proved to be ineffective. Other tested drugs namely ibuprofen and indomethacin, both prostaglandin E2 antagonists, cimetidine a histamine type-2 receptor antagonist, the opioid pethidine, prostaglandin E2 and histamine exerted minor effects or had no influence on the tested parameters. We conclude that glucocorticosteroids should be avoided with monoclonal antibody and cytokine treatment. According to our in vitro data the other drugs tested did not have a negative impact on cellular cytotoxicity and ADCC.

Complex combination biochemotherapy regimen in advanced metastatic melanoma in a non-intensive care unit: toxicity or benefit?

BACKGROUND: There is currently no chemotherapy or chemoimmunotherapy regimen that has shown impact on survival in patients with metastatic melanoma. Different biochemotherapy protocols showed promise with high response rates, but again without significant impact on survival. METHODS: We report the results of a retrospective analysis of a regimen consisting of dacarbazine, cisplatin, vindesine, interleukin-2 and interferon-alpha2b in 25 consecutively treated patients with regard to toxicity, efficacy and practicability. The treatment was performed on a regular dermatological ward. RESULTS: Grade III and IV toxicities were mainly haematological, with few cases of infection because of neutropenia seen. Best overall responses were CR 2/25, PR 2/25 and SD 9/25. The median progression free interval was 4 months (range 0-19) for all patients and the median survival time was 12 months (range 2-26). From a safety and practical point of view, there was no draw-back on treating patients in a non-intensive care unit. The median survival time is in the range of the one reported for monochemotherapy regimen. While there are some responding patients, the responses are short lived and go in parallel with high toxicity and impaired performance status. CONCLUSION: This complex and highly toxic chemoimmunotherapeutic regimen should not be considered as standard therapy in patients with metastatic malignant melanoma.

Doxorubicin plus interleukin-2 chemoimmunotherapy against breast cancer in mice.

As recently characterized, following s.c. implantation into syngeneic C57BL/6 mice, E0771 tumor invades locally into dermal layers and peritoneum, metastasizes to the lung, and induces a nonspecific immunosuppression in the host. Using this breast medullar adenocarcinoma model, a therapy consisting of a single moderate dose of doxorubicin followed by twice daily moderate doses of interleukin-2 for 30 days was examined for efficacy and mechanism of action when given to animals with established disease. This combination treatment, but not combinants alone, resulted in tumor-free long-term survival of 40% of the mice without significant toxicity and 83% of survivors had immune memory specific for E0771 cells. Treatment also decreased immune suppression induced by E0771 tumor. Full response to treatment required functional CD8(+) T cells, whereas depletion of natural killer cells caused only a reduction in response rate. A serum "biomarker" profile that correlated with, and seemed predictive of, response to treatment was identified by nuclear magnetic resonance-based metabonomic analysis. The efficacy of this nontoxic treatment and the potential to be able to predict which individual is responding to treatment are characteristics that make this chemoimmunotherapy attractive for clinical testing.

Histamine improves survival and protects against interleukin-2-induced pulmonary vascular leak syndrome in mice.

The therapeutic efficacy in the treatment of metastatic cancer with high doses of interleukin-2 (IL-2) has been limited by the onset of vascular leak syndrome (VLS) and related toxicities. VLS is characterized by an increase in vascular permeability and severe hypotension resulting in interstitial edema and organ failure. This study explores the protective effects of histamine dihydrochloride (HDC) against IL-2-induced toxicities in mice. Treatment with HDC administered before or after IL-2 (1.25 x 10(6) IU, BID) was shown to protect mice from VLS-related toxicities and mortality in a dose-dependent manner. Survival rates when HDC was added were 56, 75 and 81% at doses of 0.47, 4.7 and 47.0 mg/kg, respectively, compared to 42% survival with IL-2 alone. HDC protected against IL-2-induced macroscopic pulmonary lesions, reduced edema (up to 62% reduction in lung wet/dry weight ratio) and reduced capillary leakage into the lungs as measured by a reduction in Evans Blue dye content. In addition, the systemic effect on serum cytokine levels showed that HDC only moderately lowered IL-2 induced IFN-gamma, IL-6, IL-10, IL-18 and TNF-alpha. Serum levels of IL-1beta, IL-4 and IL-12 were not measurably induced by IL-2 treatment. HDC modulates many cellular functions including regulating cytokines and blocking immune-suppression caused by reactive oxygen species (ROS) generated by the NADPH oxidase. However, the protective effect of HDC on alleviating IL-2-induced pulmonary edema was not related to ROS inhibition. Our data indicate that HDC treatment improves survival and protects against IL-2 induced VLS independent of ROS regulation in mice.

Denileukin diftitox for the treatment of steroid-resistant acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is partly mediated through activated T cells, and these cells are known to express the high-affinity receptor for interleukin 2 (IL-2R). Denileukin diftitox is composed of human IL-2 and diphtheria toxin that is cytotoxic to activated lymphocytes expressing the high-affinity IL-2R. We describe the results of a phase II study of denileukin diftitox in 22 patients with steroid-resistant aGVHD. Twenty patients were treated at dose level 1 (4.5 microg/kg daily on days 1-5 and then weekly on study days 8, 15, 22, and 29), and 2 patients were treated at dose level 2 (9.0 microg/kg delivered on the same schedule). Dose level 2 was associated with grade 3/4 renal and hepatic toxicity and vascular leak syndrome, and no further patients were treated at this level. Dose level 1 was generally well tolerated. The response of aGVHD was assessed at study days 36 and 100. Nine patients (41%) responded, all with a complete response at study day 36, and 6 patients (27%) responded at study day 100 (4 complete responses and 2 partial responses). Denileukin diftitox has promising activity in steroid-resistant aGVHD, and further study is warranted.

Phase II study of autologous transplantation with interleukin-2-incubated peripheral blood stem cells and posttransplantation interleukin-2 in relapsed or refractory non-Hodgkin lymphoma.

Previous work suggested that interleukin (IL)-2 can be used for eradicating residual disease in autologous grafts and for preventing recurrence. We report a phase II study of autologous peripheral blood stem cell transplantation with in vitro IL-2 incubation of peripheral blood stem cells and posttransplantation IL-2 in patients with recurrent or refractory non-Hodgkin lymphoma. Salvage chemotherapy consisted of ifosfamide and etoposide. Responding patients underwent autologous peripheral blood stem cell transplantation. IL-2-incubated stem cells were infused on day 0. IL-2 1 mIU/m2 was given from day 1 until day 28. Four monthly maintenance cycles of IL-2 4 mIU/m2 subcutaneously twice daily days 1 to 5 and days 8 to 11 were administered thereafter. Eighty-four evaluable patients were enrolled, and 60 proceeded to transplantation, of which 56 received IL-2-incubated stem cells. The average received dose of posttransplantation IL-2 was 30% to 50% of planned. Only 42 patients received maintenance IL-2. The average received maintenance dose of IL-2 was also approximately 30% of planned. Most dose reductions were due to toxicity or patient refusal. Three-year survival and progression-free survival for all registered patients were 43% (95% confidence interval [CI], 33%-53%) and 31% (95% CI, 21%-41%), respectively. For the 60 patients undergoing transplantation, they were 59% (95% CI, 46%-72%) and 44% (95% CI, 31%-57%), respectively. There was no relation between the dose of IL-2 received and outcome. Survival and disease-free survival of the study group were similar to those of a previous study cohort that received unmanipulated stem cells and no systemic IL-2. Administration of IL-2-incubated peripheral blood stem cells and intensive posttransplantation IL-2 was associated with considerable but rapidly reversible toxicity. No effect on long-term outcome was observed.

Safety and efficacy of denileukin diftitox in patients with steroid-refractory acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Denileukin diftitox (Ontak), a recombinant protein composed of human interleukin 2 (IL-2) fused to diphtheria toxin, has selective cytotoxicity against activated lymphocytes expressing the high-affinity IL-2 receptor. We conducted a phase 1 study of denileukin diftitox in 30 patients with steroid refractory acute graft-versus-host disease (GVHD). Seven patients received 9 microg/kg intravenously on days 1 and 15; 18 received 9 microg/kg intravenously on days 1, 3, 5, 15, 17, and 19; and 5 received 9 microg/kg intravenously on days 1 to 5 and 15 to 19. Hepatic transaminase elevation was the dose-limiting toxicity (DLT), and dose level 2 was the maximum tolerated dose (MTD). Overall, 71% of patients responded with complete resolution (12 of 24; 50%) or partial resolution (5 of 24; 21%) of GVHD. Eight of 24 patients (33%) are alive at 6.3 to 24.6 months (median, 7.2 months). Denileukin diftitox is tolerable and has promising activity in steroid-refractory acute GVHD.

IL-2-based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial.

We determined the safety, immune activating effects, and potential efficacy of i.v. infusion of ex vivo interleukin-2 (IL-2) activated natural killer (NK) cells (part I) or IL-2 boluses (part II) during daily s.c. IL-2 administration following hematopoietic recovery from autologous transplantation. In all, 57 patients with relapsed lymphoma (n=29) or metastatic breast cancer (n=28) were enrolled. In part I of the study, 34 patients were enrolled at three dose levels of ex vivo IL-2-activated NK cells. Lymphaphereses were performed on days 28 and 42 of s.c. IL-2 administration. Following overnight ex vivo IL-2 activation of the pheresis product, the cells were reinfused the following day. In part II, 23 patients were enrolled at three dose levels of supplemental i.v. IL-2 bolus infusions, given on days 28 and 35 during s.c. IL-2 administration. Toxicities were generally mild, and no patient required hospitalization. Lytic function was markedly enhanced for fresh peripheral blood mononuclear cells (PBMNCs) obtained 1 day postinfusion of either IL-2-activated cells or IL-2 boluses. IL-2 boluses transiently increased the levels of IL-6, IFN-gamma, TNF-alpha and IL1-beta, with increases in IL-6 and IFN-gamma being dose dependent. A total of 37 patients (19 patients with lymphoma, 18 with breast cancer) treated with an optimum dose of post-transplant immunotherapy (defined as having received 1.75 x 10(6) IU/m(2)/day of s.c. IL-2 plus at least one of the planned ex vivo IL-2-activated cell infusions/IL-2 boluses) could be matched with controls from the Autologous Blood and Marrow Transplant Registry database. The matched-pairs analysis demonstrated no improvement in disease outcomes of survival and relapse. We conclude that IL-2-activated cells/IL-2 boluses can be safely administered, generate PBMNCs with enhanced cytotoxicity against NK-resistant targets, and increase cytokine levels. With this dose and schedule of administration of IL-2, no improvement in patient disease outcomes was noted. Alternative strategies will be needed to exploit the immunotherapeutic potential of IL-2-activated NK cells.