Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer

Abstract

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.

Trial registration: ClinicalTrials.gov NCT01021059.

Fig 1.

Acute lymphocyte dynamics after interleukin-15 (IL-15) administration. (A) Representation of natural killer (NK) cells among CD3− lymphocytes (top) and T-cell differentiation stages among CD8 (middle) or CD4 (bottom) cells are shown for one individual administered 3 μg/kg of IL-15 during 30-minute infusion; 10-, 20-, and 30-minute time points were taken during administration. Numbers outside red lines indicate percentage of cells inside each gate. (B) Change from baseline in proportion of NK cells is shown as mean ± SEM for four individuals. Light blue bar indicates IL-15 infusion time. (C) Changes from baseline in representation of relative proportions of CD4 and CD8 subsets are shown as mean ± SEM. Subsets were gated. (D) Proliferation (percentage of Ki-67) among NK, CD4, and CD8 subsets is shown for four individuals. T_CM, T central memory; T_EM, T effector memory; T_SCM, T stem-cell memory; T_TE, T terminal effector; T_TM, T transitional memory.

Fig 2.

Lymphocyte dynamics during and after daily interleukin-15 (IL-15) infusions. All data are shown as mean ± SEM for administration of IL-15 3 μg/kg (n = 4; all panels) or (A, B) 0.3 μg (n = 9). All fold-change values were computed by individual relative to baseline (Pre). Light blue bars indicate IL-15 infusion times. (A) Absolute count and (B) change in absolute count for total natural killer (NK) cells. (C) Representation within NK cells and (D) proliferation state of differentiation stages of NK cells. (E) Absolute CD4 and CD8 T-cell counts. Activation and proliferation states of (F) CD8 and (G) CD4 cells based on any of four markers. (H) Absolute counts of γδ T-cell lineages. P values are from two-tailed Student's t test for peak time point.

Fig 3.

Pharmacokinetic (PK) analysis after infusions of recombinant human interleukin-15 (rhIL-15) in patients with metastatic malignancy. Mean ± SEM shown for patients in each dose group. (A) Plasma concentrations of rHIL-15 after infusion. (B) Maximum serum concentration (C_max) after bolus intravenous infusions of rhIL-15 3.0, 1.0, and 0.3 μg/kg per day. (C) PK parameters for all patients. AUC, area under plasma concentration versus time curve; CL, clearance; t1/2, half-life; V1, volume of distribution into central compartment; V2, volume of distribution into peripheral compartment.

Fig 4.

Clinical and immunologic responses to recombinant human interleukin-15 (rhIL-15) infusions. For patients administered rhIL-15 (A) 3, (B) 1, or (C) 0.3 μg/kg, mean arterial pressure (MAP; upper panels) and temperature (middle panels) are shown as mean ± SEM after 12 daily infusions of rhIL-15 for one patient in each group; plasma cytokine concentrations are shown as mean ± SEM for all patients in each group after first infusion (lower panel). Fever, rigors (occurring within 2.5 to 4 hours), and hypertension followed by hypotension occurred contemporaneously with elevations in IL-6 and interferon gamma (IFN-γ). TNF-α, tumor necrosis factor alpha.

Fig 5.

Clinical activity of recombinant human interleukin-15 (rhIL-15) in patient with metastatic malignant melanoma. There were no objective remissions in 18 patients, with best response being stable disease. However, five patients had decreases of 10% to 30% in marker lesions. Two patients, including the one shown (patient No. 16), experienced clearance of lung lesions; lesion 2 at (A) baseline and (B) day 42, and lesion 3 at (C) baseline and (D) day 42. Yellow arrows indicate lung lesions; blue lines indicate area measured.

Fig 6.

Model of interleukin-15 (IL-15) –driven homeostasis. Accompanying IL-15 administration, absolute numbers of population of cells in circulating blood (eg, natural killer [NK] cells) were affected by several pathways. During and immediately after infusions, there was margination or efflux of NK populations from circulating blood so that they were nearly absent in circulation by 30 minutes. By 4 hours after infusion, influx of NK cells was detected, followed by slow normalization of cells over 2 to 3 days. After 3 days from initiation of infusions, there was hyperproliferation yielding absolute NK-cell counts > 10-fold over baseline. In the 1- to 3-week period after treatment, there was hypoproliferation until cell counts returned to baseline, after which normal homeostasis was restored. Numbers outside gold lines indicate percentage of cells inside each gate.

Fig A1.

Identification of natural killer (NK) and T-cell subsets. (A) Peripheral blood mononuclear cells from representative patient were stained with panel of monoclonal antibodies. Total NK cells were identified. Combination of CD16 and CD56 expression on total NK cells identified three subsets: CD56⁺CD16⁻, CD56⁺CD16^dim, and CD56^dimCD16^bright. Numbers outside gold lines indicate percentage of cells inside each gate. (B) Flow cytometric assay identified multiple subsets of naïve (T_N) and memory cells. CD8 T cells are shown (depicted in black in background). Naïve-like T cells (red) were first identified as CD45RO⁻CCR7⁺, further selected as CD45RA⁺CD27⁺, and finally selected as CD95⁻. T stem-cell memory (T_SCM) has same phenotype but expresses CD95. Central memory (T_CM) is CD45RO⁺CCR7⁺, transitional memory (T_TM) is CD45RO⁺CCR7⁻CD27⁺, effector memory (T_EM) is CD45RO⁺CCR7⁻CD27⁻, and terminal effector (T_TE) is CD45RO⁻CCR7⁻CD27⁻. Same strategy applied to CD4 T cells.

Fig A2.

Dose effect on lymphocyte dynamics during and after daily interleukin-15 (IL-15) infusions. All data shown as mean for administration of IL-15 3 μg/kg (n = 4) or 0.3 μg (n = 9). All fold-change values were computed by individual relative to baseline (Pre). Light blue bars indicate IL-15 infusion time. Error bars were omitted for clarity. (A) Representation within natural killer (NK) cells and (B) proliferation state of differentiation stages of NK cells. (C) Absolute CD4 and CD8 T-cell counts. (D) Absolute counts of CD8 differentiation stages. (E) Absolute counts of γδ T-cell lineages. Activation and proliferation states of (F) CD8 and (G) CD4 cells based on any of four markers. (H) Absolute counts and (I) representation (frequency within total CD4 T cells) of T regulatory cells.

Fig A3.

Comparative pharmacokinetics (PKs) for 20-μg dose at different dosing strategies in cynomolgus macaques. In our previous publications,^, we reported that with bolus intravenous (IV) infusion to rhesus macaques at 20 μg/kg per day, there was exceedingly high maximum serum concentration (C_max) of 720 ± 280 ng/mL. With identical subcutaneous (SC) dose of 20 μg/kg, C_max was 10-fold less, at 50 ± 19 ng/mL, and with continuous IV (CIV) infusion of 20 μg/kg per day, it was 2 to 4 ng/mL, maintained throughout 10-day study period. Initial high peak and rapid decline with IV bolus dosing was analogous to PKs seen in our phase I patient clinical trial. Clinical trials in patients with metastatic malignancy with interleukin-15 (IL-15) administered SC or by CIV infusions have been initiated to reduce peak IL-15 effects on cytokine release and infusional toxicities and maintain IL-15 at optimal concentration for high-affinity IL-15 receptor.