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.

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.

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.

A nonpeptidyl mimic of superoxide dismutase, M40403, inhibits dose-limiting hypotension associated with interleukin-2 and increases its antitumor effects.

Interleukin-2 (IL-2) is used to treat metastatic renal cell carcinoma and malignant melanoma, but its use is limited by the severe hypotension it produces. We have shown here that M40403, a superoxide dismutase (SOD) mimetic, blocked IL-2-induced hypotension and allowed the dose of IL-2 to be increased in mice. The reversal of IL-2-mediated hypotension was associated with an increase in plasma catecholamines. In addition, M40403 increased lymphokine-activated killer (LAK) cell cytotoxicity in vitro and in vivo, through inhibition of macrophage superoxide production. Treatment of methylcholanthrene-induced (Meth A) ascites tumors with IL-2 and > or =3 mg per kg body weight M40403 induced 50% complete remissions lasting for more than 200 d, which was longer than those of untreated mice (15-d median survival) or mice treated with IL-2 alone (22-d median). Growth of subcutaneous implants of RENCA renal carcinoma was also inhibited by the combination of IL-2 and M40403. These results established that M40403 prevented IL-2 from causing dose-limiting hypotension, while enhancing its anticancer activity.

Double-Edged Sword: Interleukin-2 Promotes T Regulatory Cell Differentiation but Also Expands Interleukin-13- and Interferon-γ-Producing CD8+ T Cells via STAT6-GATA-3 Axis in Systemic Lupus Erythematosus.

Interleukin-2 (IL-2) expands the depleted T regulatory (Treg) cell population, and it has emerged as a potential therapy in systemic lupus erythematosus (SLE). However, IL-2 administration may involve the risk of expanding unwanted pro-inflammatory cells. We herein studied the effects of IL-2 on pro-inflammatory cytokine production by CD4+ and CD8+ T cells in parallel with Treg development following CD3/CD28 co-stimulation. While Treg cells are depleted in SLE patients, their CD4+ T cells were poised to receive and activate IL-2 signaling as evidenced by upregulation of CD25 and enhanced IL-2-incued STAT5 phosphorylation during Treg differentiation. In patients with SLE, however, IL-2 also expanded CD8+ T cells capable of producing interleukin-5, interkeukin-13 (IL-13), and interferon-γ (IFN-γ) that occurred with enhanced expression of GATA-3 and phosphorylation of STAT6 but not STAT5. Our data pinpoint a safety signal for systemic administration of IL-2 and challenges a long-held conceptual platform of type 1 and 2 cytokine antagonism by newly documenting the IL-2-dependent development of IL-13 and IFN-γ double-positive (IL-13+IFNγ+) CD8+ T cells in SLE.

Passive immunization against tumor necrosis factor partially abrogates interleukin 2 toxicity.

Passive immunization against TNF allowed non-tumor-bearing C3H/HEN mice and tumor-bearing C57BL/6 mice to tolerate significantly more doses of IL-2 before death (p less than 0.005 and p less than 0.001, respectively). The antitumor effect of IL-2 against both 3-d and 10-d pulmonary metastases was maintained in mice treated concurrently with neutralizing antibodies to TNF. In one experiment with 10-d pulmonary metastases, increased administration of IL-2 made possible by passive immunization against TNF significantly improved the antitumor response compared to equitoxic doses of IL-2 and control antibody. The results indicate that TNF is a mediator of IL-2 toxicity but contributes minimally to the antitumor effects of IL-2. Strategies to inhibit TNF may improve the therapeutic index of IL-2 as a neoplastic agent.

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.

Investigation of the pruritogenic effects of histamine, serotonin, tryptase, substance P and interleukin-2 in healthy dogs.

There are numerous studies of the pruritus-producing effects of histamine, serotonin, tryptase, substance P and interleukin-2 in humans and mice, but very little reported in dogs even though a common reason dogs are presented to veterinarians is pruritus. The aim of this study was to determine whether substances known to cause pruritus in humans also cause pruritus in dogs. Twenty-five clinically healthy research beagle dogs were included in the study. All dogs first received an intradermal injection of 0.05 mL saline as a control substance and were video-recorded for 20 min before and after the injection. Twenty-four hours later the dogs were randomly divided into five groups of five dogs each and randomly assigned to receive histamine, serotonin, tryptase, substance P or interleukin-2 injected intradermally each at the volume of 0.05 mL. On subsequent days, increasing concentrations of each substance were used. Before (baseline) and after the injection of each concentration of the substances, the dogs were video-recorded for 20 min. The frequency and character of pruritus episodes (scratching, licking, chewing, rubbing or rolling) were noted and these data were used for statistical analysis. The number of pruritus episodes was compared among baseline, saline and the different concentrations of each substance. The results showed that dogs did not have a significant increase in pruritic behaviour above baseline or saline after injection of any of the investigated substances (generalized linear model; P = 0.23).

99mTc-interleukin-2 scintigraphy in normal subjects and in patients with autoimmune thyroid diseases: a feasibility study.

PURPOSE: Radiolabelled interleukin-2 is a radiopharmaceutical used for the study of chronic inflammatory processes. (123)I-labelled interleukin-2 has successfully been used in a large number of patients affected by several immune-mediated diseases. (123)I, however, is expensive and not readily available. We have, therefore, developed a method for labelling interleukin-2 with (99m)Tc to high specific activity based on the use of an N(3)S bifunctional chelating agent. In this paper, we describe the results obtained with (99m)Tc-interleukin-2 in a series of eight normal subjects and of 12 patients with autoimmune thyroid diseases. METHODS: Biodistribution, pharmacokinetics, haematological and systemic toxicity, radiation absorbed dose and in vivo targeting were studied. RESULTS: Results showed rapid plasma clearance of (99m)Tc-interleukin-2 with retention mainly in the kidneys. Biodistribution and kinetics were similar to that observed for (123)I-interleukin-2. No acute systemic toxicity was found; a small decrease in peripheral blood lymphocytes was observed in the first hours only in patients, but it was mild and transient. (99m)Tc-interleukin-2 accumulated, to varying extents, in the thyroid of all patients affected by autoimmune thyroid diseases but not in the thyroid of normal subjects. The effective dose equivalent of a diagnostic activity of (99m)Tc-interleukin-2 (185 MBq) was 1.35 mSv. No correlation was observed between thyroid autoantibodies and uptake of (99m)Tc-interleukin-2. CONCLUSIONS: The use of (99m)Tc-interleukin-2 is safe and simple; the favourable dosimetry and biodistribution and the rapid clearance make it potentially useful for the study of chronic inflammatory diseases such as autoimmune thyroid disease.

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.

Pentoxifylline inhibits interleukin-2-induced toxicity in C57BL/6 mice but preserves antitumor efficacy.

Interleukin 2 (IL-2) mediates the regression of metastatic cancer but clinical use has been limited due to associated toxicities. Tumor necrosis factor (TNF) is an important mediator of IL-2 toxicity and may have a limited role in IL-2 antitumor efficacy. Because pentoxifylline (PTXF) inhibits TNF production, we hypothesized that PTXF would ameliorate IL-2 toxicity without compromising antitumor efficacy. Four groups of female C57BL/6 mice with pulmonary metastases from a 3-methylcholanthrene-induced fibrosarcoma (MCA-105) and four groups of nontumored mice were treated every 6 h for 4 d by intraperitoneal injections of either IL-2 alone, IL-2 and PTXF, PTXF alone, or equal volumes of saline. Upon completion of therapy, we found that PTXF suppressed many of the IL-2-induced effects including TNF production, lymphocytic infiltration of multiple organs, multiple organ edema, hepatic dysfunction, leukopenia, and thrombocytopenia. Tumor response was determined 21 d after cessation of therapy by quantitating the number and surface area of pulmonary metastases. PTXF preserved antitumor efficacy while reducing the morbidity and mortality caused by IL-2 treatment. These data strongly support the use of PTXF in extending the therapeutic index of IL-2 in the treatment of cancer.

Platelet activating factor mediates interleukin-2-induced lung injury in the rat.

Interleukin-2 was recently shown to cause acute lung injury characterized by microvascular permeability defect, interstitial edema, and leukosequestration. Similar responses can also be produced by platelet activating factor (PAF). Thus, the present study aimed to examine whether PAF plays a key role in the development of IL-2-induced lung injury in the anesthetized rat. Intravenous infusion (60 min) of recombinant human IL-2 at 10(5)-10(6) U/rat (n = 7-9) dose-dependently elevated lung water content (27 +/- 1%, P less than 0.01), myeloperoxidase activity (+84 +/- 23%, P less than 0.05), and serum thromboxane B2 (990 +/- 70%, P less than 0.01), but failed to alter blood pressure, hematocrit, serum tumor necrosis factor-alpha, and circulating leukocytes and platelets. Pretreatment (-30 min) with a potent and specific PAF antagonist, BN 50739 (10 mg/kg, intraperitoneally, n = 6) prevented the pulmonary edema (P less than 0.05) and thromboxane B2 production (P less than 0.01), and attenuated the elevation of lung myeloperoxidase activity (+18 +/- 16%, P less than 0.05) induced by IL-2. These data suggest that PAF is involved in the pathophysiological processes leading to IL-2-induced lung injury, and point to the potential therapeutic capacity of PAF antagonists in preventing pulmonary edema during IL-2 therapy.

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.

A T-cell-selective interleukin 2 mutein exhibits potent antitumor activity and is well tolerated in vivo.

Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS.

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.

Blood-brain barrier dysfunction in cats following recombinant interleukin-2 infusion.

The effects of systemic human recombinant interleukin-2 (rIL-2) infusion upon blood-brain barrier status and cerebral vascular ultrastructure were examined in cats. Each of eight animals received a single bolus i.v. infusion of rIL-2 (100,000 units/kg). Six control animals were infused with rIL-2 excipient only. Following a 1-h postinfusion survival time, the brain tissue of five rIL-2 infused and three excipient infused animals was processed and examined by light microscopy and electron microscopy for evidence of altered cerebrovascular permeability to systemically circulating horseradish peroxidase. The brain tissue of three additional rIL-2 infused animals and three excipient infused animals, sacrificed 4 h postinfusion, was examined at the light microscopic and electron microscopic levels for the presence of extravasated endogenous IgG. All animals infused with rIL-2 and four of six excipient infused animals showed increased cerebrovascular permeability to the probe used. Altered blood-brain barrier permeability, when present, was recognized in multiple loci throughout the brain, being most prominent within white matter regions. Horseradish peroxidase and IgG were observed within perivascular basal laminae and within the interstices of the brain parenchyma. Numerous endothelial lesions were observed as was flooding of endothelial cytoplasm by horseradish peroxidase or IgG. Every animal studied, regardless of permeability status, showed, within the perivascular brain parenchyma, numerous disrupted neuronal and glial processes as well as expanded intercellular spaces. This study suggests that a single systemic infusion of rIL-2 profoundly alters blood-brain barrier integrity and cerebrovascular morphological integrity. The data also suggest that some of the observed cerebrovascular effects of systemic rIL-2 infusion are due to components of the vehicle for rIL-2.