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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.

Because of its key role in immunity, interleukin-2 (IL-2) has been studied extensively for the adoptive immunotherapy of cancer. Although systemic administration of IL-2 has been shown to stimulate antitumor responses in vivo, its efficacy in the clinic has been limited by the development of serious side effects, including the induction of vascular leak syndrome. Previously, we have identified a small peptide fragment of IL-2 that was found to contain the entire vasopermeability activity of the cytokine. The identification of the location of this potentially undesirable property of IL-2 enabled us to focus on the generation of mutant derivatives that might be lacking vasopermeability activity but that retain cytokine functionality. In addition to this discovery, our laboratory has constructed monoclonal antibody/IL-2 fusion proteins that can target this potent cytokine directly to tumor for the immunotherapy of both solid and lymphoid malignancies. Using this fusion protein technology, we have constructed a series of point mutations in the newly identified vasopermeability region of IL-2 for the purpose of deleting this activity. Fusion proteins showing reduced or deleted vasopermeability activity were then tested for their cytokine potency by several methods, including their binding to IL-2 receptors, T-cell proliferation assays, the induction of secondary cytokines, dose-escalating toxicity, and finally their ability to treat established solid tumors in syngeneic immunocompetent mice. The results of these studies clearly show that the vasopermeability activity of IL-2 can be substantially deleted by single point mutations such as Arg38Trp without grossly affecting the immune function of the cytokine.

Interleukin 18 (IL-18) was discovered as an interferon-gamma (IFN-gamma)-inducing factor and plays important roles in natural killer (NK) cell activation. IL-18 also induces proinflammatory cytokines; chemokines; helper T-cell 2 (T(H)2) cytokines (eg, IL-4, IL-13); and immunoglobulin E (Ig-E) and IgG1 production. The combination of IL-18 plus IL-2 or IL-12 up-regulates IFN-gamma gene expression and NK cytotoxicity and has synergistic antitumor activity in vivo and in vitro. Here it is reported that daily administration of IL-18 with IL-2, but not of IL-18 or IL-2 alone, induces lethal lung injury in normal mice, but not in IL-18 receptor alpha (IL-1 receptor-related protein)-deficient (IL-18 receptor alpha(-/-)) mice. Marked interstitial infiltration of lymphocytes, composed mainly of NK cells, was found in the lungs of IL-18/IL-2-treated mice. Increased cytokine and chemokine levels were observed in the sera and lungs of IL-18/IL-2-treated mice. Administration of IL-18/IL-2 was also lethal to mice treated with a metalloproteinase inhibitor, which inhibited tumor necrosis factor-alpha and Fas-ligand release. While IFN-gamma(-/-) mice were partially resistant to the treatment, IL-4(-/-), IL-13(-/-), IL-4/IL-13(-/-), and Stat6(-/-) mice were sensitive to IL-18/IL-2, indicating that these genes were not involved in the host response. The lethal effect by IL-18/IL-2 was completely eliminated in severe combined immunodeficient mice pretreated with antiasialo-GM1 antibody and normal mice pretreated with anti-NK1.1 but not with anti-CD4 or anti-CD8, monoclonal antibody. These results suggest that specific cytokines, chemokines, and NK cells are involved in the pathogenesis of interstitial pneumonia. These results suggest that the clinical use of this interleukin may result in unexpected physiological consequences.

Interleukin-12 (IL-12) is a potent immunostimulatory cytokine and an inducer of type-1 T-helper cell activity and of cytotoxic T lymphocyte and natural killer cell function. We report here the paradoxical observation that a single injection of 4,900 IU of recombinant murine IL-12 inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex (MHC) plus multiple minor antigen-mismatched bone marrow transplantation (BMT) model (A/J-->B10). The protective effect was enhanced by administration of T-cell-depleted host-type BM cells, and complete donor-type lymphohematopoietic reconstitution was observed in most animals. Treatment with a protective course of IL-12 led to increased serum interferon-gamma (IFN-gamma) levels as compared with those for GVHD controls at early time points, when IFN-gamma was produced predominantly by host-type natural killer cells, but led to almost complete inhibition of the later GVHD-associated increase in serum IFN-gamma levels, when IFN-gamma is produced predominantly by CD4+ T cells. Furthermore, IL-12 treatment was associated with marked alterations in the kinetics of donor T-cell expansion. Reductions in donor CD4+ and CD8+ T cells were observed in the spleen on day 4 post-BMT, but a marked increase in donor CD8+ cells was observed on day 7. Unlike broadly immunosuppressive methods for inhibiting GVHD, which are associated with loss of antileukemic effects, IL-12 has the potential to mediate antileukemic effects of its own; therefore, the GVHD-inhibitory effects of IL-12 described here suggest a potential application for this cytokine in clinical BMT.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TILs) causes regression of some human tumors. However, the sustained proliferation and antitumor activity of TILs requires the coadministration of potentially toxic amounts of interleukin-2 (IL-2). In an effort to overcome the requirement by T cells for IL-2, we have introduced alternative growth factor receptors that use the relatively nontoxic cytokine erythropoietin (Epo) as a ligand. In our model system, the coexpression of chimeric receptors consisting of the extracellular portion of the Epo receptor (EpoR) and the intracellular portions of the IL-2 receptor subunits, beta and gamma, conferred Epo responsiveness on a T-cell line. By contrast, cells expressing the wild-type EpoR did not proliferate in response to Epo. This suggested that Epo binding caused the activation of an IL-2 signal pathway mediated by the chimeric receptors. This approach can be used to minimize toxicity and potentially improve cancer immunotherapy with TILs.

DAB486IL-2 is a recombinant fusion toxin in which the native receptor binding domain of diphtheria toxin has been replaced with human interleukin-2 (IL-2). It selectively binds and intoxicates only cells that bear the high-affinity receptor for IL-2. In the first clinical trial of a genetically engineered ligand fusion-toxin, we have treated 18 patients with chemotherapy-resistant IL-2 receptor expressing hematologic malignancies with escalating doses of DAB486IL-2. The maximal tolerated dose of a daily intravenous bolus of DAB486IL-2 was 0.1 mg/kg per day for 10 doses, established by asymptomatic, reversible elevations of hepatic transaminases without changes in other tests of liver function. Other mild reversible side effects noted were rash, nausea, elevated creatinine, chest tightness, and fever. Pharmacokinetic analysis showed a monophasic clearance of 5.8 +/- 0.7 minutes with peak levels of 3,549 +/- 1,041 mg/mL at the 0.1 mg/kg dose. Approximately 50% of patients developed an antibody response to diphtheria toxin or DAB486IL-2. The presence of such antibodies did not preclude patients from experiencing an antitumor response as four of the six patients with antitumor effect had detectable antibody titers. Although this was a phase I trial designed to define the safety of DAB486IL-2, remissions were observed in three patients lasting from 5 to over 18 months. The ability to achieve significant tumor reductions in this group of heavily treated patients is encouraging and suggests additional trials are warranted in hematologic malignancies.

High concentrations of tumor necrosis factor (TNF) alpha have been detected in the plasma of patients undergoing immunotherapy with interleukin 2 (IL-2), suggesting that this cytokine may play a role in the fever and shocklike state induced by the administration of high-dose IL-2. Dexamethasone has been shown to inhibit the synthesis of TNF by monocytes activated in vitro by endotoxin. To determine if dexamethasone can exert a similar suppressive effect on IL-2-induced TNF synthesis in vivo, the concentration of TNF alpha was measured in plasma samples serially obtained (a) from cancer patients participating in a phase I dose escalation clinical trial with high-dose IL-2 administered in conjunction with dexamethasone (IL-2/Dex) and (b) from patients participating in concurrent studies with IL-2 alone. In contrast to the high plasma levels of TNF alpha detected in patients receiving IL-2 alone, TNF levels in most of the IL-2/Dex patients remained below the threshold of detectability of our TNF radioimmunoassay. The concurrent administration of dexamethasone also prevented the IL-2-induced increase in serum levels of C-reactive protein, a hepatic acute phase reactant whose synthesis is regulated by proinflammatory cytokines such as TNF. The steroid-treated patients also failed to develop the neutrophil chemotactic defect characteristic of IL-2 recipients. The concomitant administration of dexamethasone increased the maximum tolerated dose of IL-2 approximately threefold and markedly reduced the hypotension and organ dysfunction ordinarily observed in these patients. These results demonstrate that dexamethasone inhibits the release of TNF into the circulation of patients undergoing immunotherapy with IL-2. They further suggest that the altered spectrum and reduced severity of IL-2 side effects observed in patients receiving dexamethasone may be attributable in part to the suppressive effect of steroids on IL-2-induced TNF synthesis.

T cells from allogeneic bone marrow grafts are responsible for a graft versus leukemia effect. Use of recombinant Interleukin-2 (rIL-2) after autologous bone marrow transplantation (BMT) may enhance immune function and hopefully reproduce the allogeneic reaction. We report here the hematologic and immunologic changes observed in the first 10 patients of a phase 1 trial studying the infusion of IL-2 after autologous BMT. All patients had high-risk malignancies and received 6 days of a constant infusion of IL-2 (Eurocetus, Amsterdam, The Netherlands) at dose of 3 x 10(6) Cetus Units/m2/d, 79 +/- 12 days after autologous BMT. Clinical toxicities involving cutaneous, cholestatic, gastrointestinal, and hemodynamic effects occurred during IL-2 treatment but reversed in all cases. Completion of treatment was 91% of the scheduled dose of IL-2. Hematologic toxicity was moderate and transient with no graft failure. Increases in eosinophil and lymphocyte counts were significant (P less than .05). Stimulation of the immune system was intense and prolonged, manifested by increase numbers of CD3+, CD3+DR+, CD3+ CD25+ lymphocytes, and natural killer (NK) cells (all P less than .01), and increase of Lymphokine-activated killers (LAK) and NK activities (P less than .01 and P less than .05). This study establishes the feasibility of a 6-day administration of rIL-2 after autologous BMT leading to a major immune activation 2.5 months after BMT.