Jean M. Decker

PURPOSE: Interleukin-15 (IL-15) has significant potential in cancer immunotherapy as an activator of antitumor CD8 T and natural killer (NK) cells. The primary objectives of this trial were to determine safety, adverse event profile, dose-limiting toxicity, and maximum-tolerated dose of recombinant human IL-15 (rhIL-15) administered as a daily intravenous bolus infusion for 12 consecutive days in patients with metastatic malignancy. PATIENTS AND METHODS: We performed a first in-human trial of Escherichia coli-produced rhIL-15. Bolus infusions of 3.0, 1.0, and 0.3 μg/kg per day of IL-15 were administered for 12 consecutive days to patients with metastatic malignant melanoma or metastatic renal cell cancer. RESULTS: Flow cytometry of peripheral blood lymphocytes revealed dramatic efflux of NK and memory CD8 T cells from the circulating blood within minutes of IL-15 administration, followed by influx and hyperproliferation yielding 10-fold expansions of NK cells that ultimately returned to baseline. Up to 50-fold increases of serum levels of multiple inflammatory cytokines were observed. Dose-limiting toxicities observed in patients receiving 3.0 and 1.0 μg/kg per day were grade 3 hypotension, thrombocytopenia, and elevations of ALT and AST, resulting in 0.3 μg/kg per day being determined the maximum-tolerated dose. Indications of activity included clearance of lung lesions in two patients. CONCLUSION: IL-15 could be safely administered to patients with metastatic malignancy. IL-15 administration markedly altered homeostasis of lymphocyte subsets in blood, with NK cells and γδ cells most dramatically affected, followed by CD8 memory T cells. To reduce toxicity and increase efficacy, alternative dosing strategies have been initiated, including continuous intravenous infusions and subcutaneous IL-15 administration.

The protein portion of the immunosuppressive glycoprotein uromodulin is identical to the Tamm-Horsfall urinary glycoprotein and is synthesized in the kidney. Evidence that the glycoproteins are the same is based on amino acid sequence identity, immunologic cross-reactivity, and tissue localization to the thick ascending limb of Henle's loop. Nucleic acid sequencing of clones for uromodulin isolated from a complementary DNA bank from human kidney predicts a protein 639 amino acids in length, including a 24--amino acid leader sequence and a cysteine-rich mature protein with eight potential glycosylation sites. Uromodulin and preparations of Tamm-Horsfall glycoprotein bind to recombinant murine interleukin-1 (rIL-1) and human rIL-1 alpha, rIL-1 beta, and recombinant tumor necrosis factor (rTNF). Uromodulin isolated from urine of pregnant women by lectin adherence is more immunosuppressive than material isolated by the original salt-precipitation protocol of Tamm and Horsfall. Immunohistologic studies demonstrate that rIL-1 and rTNF bind to the same area of the human kidney that binds to antiserum specific for uromodulin. Thus, uromodulin (Tamm-Horsfall glycoprotein) may function as a unique renal regulatory glycoprotein that specifically binds to and regulates the circulating activity of a number of potent cytokines, including IL-1 and TNF.

Crude fractions of urine from pregnant women are immunosuppressive in vitro. An 85-kilodalton immunosuppressive glycoprotein purified to homogeneity from such urine inhibited in vitro assays of human T-cell and monocyte activity at concentrations of 10(-9) to 10(-11) molar. This material was nontoxic and blocked early events required for normal T-cell proliferation in vitro. On the basis of its tissue source and its in vitro activity, the name "uromodulin" is proposed for this glycoprotein.

IL-15 uses the heterotrimeric receptor IL-2/IL-15Rβ and the γ chain shared with IL-2 and the cytokine-specific IL-15Rα. Although IL-15 shares actions with IL-2 that include activation of natural killer (NK) and CD8 T cells, IL-15 is not associated with capillary leak syndrome, activation-induced cell death, or with a major effect on the number of functional regulatory T cells. To prepare for human trials to determine whether IL-15 is superior to IL-2 in cancer therapy, recombinant human IL-15 (rhIL-15) was produced under current good manufacturing practices. A safety study in rhesus macaques was performed in 4 groups of 6 animals each that received vehicle diluent control or rhIL-15 at 10, 20, or 50 μg/kg/d IV for 12 days. The major toxicity was grade 3/4 transient neutropenia. Bone marrow examinations demonstrated increased marrow cellularity, including cells of the neutrophil series. Furthermore, neutrophils were observed in sinusoids of enlarged livers and spleens, suggesting that IL-15 mediated neutrophil redistribution from the circulation to tissues. The observation that IL-15 administration was associated with increased numbers of circulating NK and CD8 central and effector-memory T cells, in conjunction with efficacy studies in murine tumor models, supports the use of multiple daily infusions of rhIL-15 in patients with metastatic malignancies.

Human peripheral blood mononuclear cells become spontaneously cytotoxic toward a broad range of erythrocyte targets after 6 to 7 days in culture. The presence of high concentrations of fetal calf serum (20%) inhibits the development of spontaneous cytotoxicity, whereas low concentrations (1 to 2%) markedly enhance cytotoxicity. Allogeneic, xenogeneic, and even syngeneic RBC are suitable targets, suggesting little, if any, target cell specificity. The addition of excess unlabeled RBC inhibits 51 Cr release from both identical and totally unrelated RBC targets; further arguing that the cytotoxic effector cell is not directed toward one and only one antigenic specificity on the target. Cell separation techniques employed at the beginning of culture demonstrate that cytotoxicity is markedly enhanced by T cell depletion by using a SRBC rosetting technique. Removal of phagocytic cells by incubation with iron particles and a magnet substantially depletes subsequent cytotoxic potential. EA rosette depletion of cells also markedly reduces subsequent cytotoxic potential. Interestingly, EA rosette depletion at the end of 7 days in culture has almost no effect on cytotoxicity, whereas E rosette depletion after 7 days continues to enhance cytotoxicity. Depletion of the cultured effector cell populations over a nylon wool column or with iron and a magnet abolished cytotoxicity. These results suggest that the cell responsible for spontaneous RBC cytotoxicity is a non-T cell which initially expresses an easily detectable Fc receptor and exhibits surface adherence properties. However, after 7 days in culture the cytotoxic cell either no longer expresses an Fc receptor, or its affinity, density, or availability are somehow altered in vitro .