Short-course IL-15 given as a continuous infusion led to a massive expansion of effective NK cells: implications for combination therapy with antitumor antibodies

Abstract

Background: Full application of cytokines as oncoimmunotherapeutics requires identification of optimal regimens. Our initial effort with intravenous bolus recombinant human interleukin-15 (rhIL-15) was limited by postinfusional reactions. Subcutaneous injection and continuous intravenous infusion for 10 days (CIV-10) provided rhIL-15 with less toxicity with CIV-10 giving the best increases in CD8+ lymphocytes and natural killer (NK) cells. To ease rhIL-15 administration, we shortened time of infusion. Treatment with rhIL-15 at a dose of 3-5 µg/kg as a 5-day continuous intravenous infusion (CIV-5) had no dose-limiting toxicities while effector cell stimulation was comparable to the CIV-10 regimen.

Methods: Eleven patients with metastatic cancers were treated with rhIL-15 CIV-5, 3 µg (n=4), 4 µg (n=3), and 5 µg/kg/day (n=4) in a phase I dose-escalation study (April 6, 2012).

Results: Impressive expansions of NK cells were seen at all dose levels (mean 34-fold), including CD56bright NK cells (mean 144-fold for 4 µg/kg), as well as an increase in CD8+ T cells (mean 3.38-fold). At 5 µg/kg/day, there were no dose-limiting toxicities but pulmonary capillary leak and slower patient recovery. This led to our choice of the 4 µg/kg as CIV-5 dose for further testing. Cytolytic capacity of CD56bright and CD56dim NK cells was increased by interleukin-15 assayed by antibody-dependent cellular cytotoxicity (ADCC), natural cytotoxicity and natural killer group 2D-mediated cytotoxicity. The best response was stable disease.

Conclusions: IL-15 administered as CIV-5 substantially expanded NK cells with increased cytotoxic functions. Tumor-targeting monoclonal antibodies dependent on ADCC as their mechanism of action including alemtuzumab, obinutuzumab, avelumab, and mogamulizumab could benefit from those NK cell expansions and provide a promising therapeutic strategy.

Trial registration numbers: NCT01572493, NCT03759184, NCT03905135, NCT04185220 and NCT02689453.

Keywords: cytokines; investigational; natural killer T-cells; therapies.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Strong proliferation and phenotypical changes of NK cells induced by CIV-5. (A, B) Analyses of the two major NK-cell subsets, CD56bright and CD56dim, comparing their percentages, numbers and phenotypes before and 3 days after the end of IL-15 infusions. (A) Left panels show an example of percentages of NK cells among PBMCs (top) and percentages of both NK subsets, CD56bright and CD56dimcells (bottom), prior to and after IL-15 infusions. Middle graphs depict fold increases of each NK subset following IL-15 treatment (three patients/dose). Right graphs display percentages of the proliferation marker Ki67 expressed among each NK subset prior to and after IL-15 infusions (three patients/dose). No statistical differences have been observed among the three different doses of IL-15. (B) Phenotypical changes of NK-cell subsets following IL-15 treatment. Left: graphs show MFI of the major activating receptors expressed by NK cells. Changes in expressions of those activating markers differed between NK subsets. Expressions of NKp46, NKp30, NKG2D and CD122 were increased on CD56dimcells. Augmentations in the surface expressions of NKG2D, CD122 and DNAX accessory molecule 1 (DNAM1) were observed on CD56bright cells. Right: graphs depict percentages among each NK subset expressing surface proteins involved in inhibition or exhaustion of NK cells. IL-15 appears to have opposite effects on percentages of cells expressing TIGIT with their decreases in CD56bright cells and their increases on CD56dim cells. Decreased percentages of TIM-3+ cells were detected in both NK subsets as well as LAG3+ among the CD56dim subset. Analyses were done using nine patients (three patients/dose). *P<0.05, **P<0.01, ****P<0.0001. IL, interleukin; LAG3+, lymphocyte-activation gene 3; MFI, mean fluorescence intensity; ND, no difference; NK, natural killer; NKG2D, natural killer group 2D; PBMC, peripheral blood mononuclear cell; TIGIT, T-cell immunoreceptor with Ig and ITIM domains; TIM-3+, T-cell immunoglobulin and mucin domain containing 3.

Figure 2

Il-15 infusions augment cytokine production and cytotoxic functions of both NK-cell subsets. (A) IL-15 treatments intensify cytokine productions by NK subsets. Top: depicts a schematic view and bottom graphs detail analyses of the major changes in cytokine productions by following intracellular detections of IFN-γ, TNF-α and GM-CSF by each NK subset when PBMCs were stimulated with IL-12 and IL-18. After treatments, two different subpopulations among CD56bright subset emerged: one subpopulation was able to secrete the three cytokines, whereas the second subpopulation coproduced IFN-γ and GM-CSF. For the CD56dim subset, only percentages of cells releasing IFN-γ increased after IL-15 infusions. (B) IL-15 treatments increase cytotoxic functions of both NK subsets. Left graphs compare lytic activities of same numbers of total NK cells prior to and after treatment at an effector: target ratio of 5:1 against three different target cells involving various activating receptors. Middle graphs (CD56bright NK cells) and right graphs (CD56dim NK cells) depict degranulation assessed via CD107 detection and intracellular IFN-γ production induced by stimulation with the same target cells at an effector: target ratio of 2:1. In the CD56bright NK subset, IL-15 treatments supported cells having abilities to degranulate and produce IFN-γ at the same time, whereas in CD56dim NK subset cells gained mainly in degranulation on target recognition. (C) IL-15 infusions increase target-induced degranulation despite TIGIT-mediated inhibition. Top histogram displays TIGIT ligand expression, CD155, on K562 and Raji target cells. Middle and bottom graphs compare percentages of CD107 upregulation among TIGIT+ and TIGIT− CD56dim NK subsets on CD155+K562 (middle graph) and CD155− aCD20Raji (bottom graph) stimulations prior and after IL-15 treatments. Although TIGIT+ CD56dim subset had lower abilities to degranulate than TIGIT− CD56dim cells when target cells express CD155, their CD107 percentages augmented after IL-15 infusions. Analyses were done using six patients (two patients/dose of IL-15 tested). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-γ, interferon gamma; IL, interleukin; ND, no difference; PBMC, peripheral blood mononuclear cell; TIGIT, T-cell immunoreceptor with Ig and ITIM domains; TNF-α, tumor necrosis factor alpha.

Figure 3

IL-15 infusions induce expansions and affect cytokine production in T-cell subsets. (A) Fold increases, proliferations with detection of Ki-67 in CD8+ cells, CD4+ cells and γδ T cells obtained after analyses of samples frozen prior and 3 days after the end of IL-15 infusions. No statistical differences were observed among the three different doses of IL-15. (B) Comparison of TNF-α, IFN-γ and IL-2 production in each T-cell subset prior and after treatments following PMA/ionomycin stimulation. No change was observed within γδ T cells. Both CD8+ and CD4+ T cells gained in their abilities to secrete TNF-α. * P<0.05, ** P<0.01, *** P<0.001. IFN-γ, interferon gamma; IL, interleukin; ND, no difference; PMA, Phorbol 12-myristate 13-acetate; TNF-α, tumor necrosis factor alpha.

Figure 4

Spider plot of CIV IL-15 treatment spider plot of 8 of 11 treated patients who had at least one restaging CT scan, showing changes from baseline in tumor burden (Y-axis) measured as the product of the longest diameters of solid metastatic target lesions of >1 cm on high-resolution CT scans (shortest diameter for lymph nodes) assessed at the end of every CIV rhIL-15 cycle (X-axis). Above the 20% increase, dashed line indicates progressive disease by RECIST criteria and below 30% reduction indicates partial response. Patients who had stable disease after their first two cycles of treatment continued to be restaged at regular intervals even though their treatment had been stopped. CIV, continuous intravenous infusion; IL, interleukin.

Figure 5

Increase in lymphocytes predominantly NK-cell count during continuous infusion of rhIL-15 IL-15 was administered at progressively increasing doses of 3, 4 and 5 µg/kg/day by 5-day CIV infusion to patients with metastatic malignancy. Three days following termination of the treatment, there was a dramatic 34-fold mean increase in the number of circulating lymphocytes, predominantly NK-cell counts, and in the 4 µg/kg/day cohort 144-fold increase in the number of circulating CD56bright NK cells. IL, interleukin; NK, natural killer; rhIL, recombinant human interleukin-15.