Interleukin-15 in the treatment of cancer

Abstract

IL-15 is a 14–15 kDa member of the four α-helix bundle of cytokines that acts through a heterotrimeric receptor involving IL-2/IL-15R β, γc and the IL-15 specific receptor subunit IL-15R α. IL-15 stimulates the proliferation of T, B and NK cells, and induces stem, central and effector memory CD8 T cells. In rhesus macaques, continuous infusion of recombinant human IL-15 at 20 μg/kg/day was associated with approximately a 10-fold increase in the numbers of circulating NK, γ/δ cells and monocytes, and an 80- to 100-fold increase in the numbers of effector memory CD8 T cells. IL-15 has shown efficacy in murine models of malignancy. Clinical trials involving recombinant human IL-15 given by bolus infusions have been completed and by subcutaneous and continuous intravenous infusions are underway in patients with metastatic malignancy. Furthermore, clinical trials are being initiated that employ the combination of IL-15 with IL-15R α^+/− IgFc.

Over the past decade, immunotherapy has joined surgery, radiation therapy and chemotherapy as a pivotal element in the treatment of cancer [1]. Immunological approaches include monoclonal antibodies and more recently chimeric antigen receptor (CAR-T) that directly attack tumor cells [2–4]. In other strategies, the goal of cancer immunotherapy is to stimulate the host’s immune system to attack cancer cells. This approach is based on the premise that the host is making an immune response, albeit inadequate, to its cancer cells. In this arena, immunostimulatory anticheckpoint monoclonal antibodies including the anti-CTLA-4 ipilimumab and anti-PD1/PD-L1 have yielded improved patient survival [5,6]. In addition, several recombinant cytokines and hematopoietic growth factors have been approved for use as supportive agents in cancer therapy. Recombinant IL-2 is a prototypic immunotherapeutic agent approved for patients with metastatic malignancy [7]. Despite its accepted role IL-2 has negative effects. IL-2 has a dual role as an immunomodulator that stimulates proliferation of effector cells that kill cancer cells but also checkpoint cells that suppress the immune response by maintenance of inhibitory CD25⁺Foxp3⁺T regulatory cells (Tregs) and that participate in activation-induced cell death (AICD) [8,9]. Furthermore, IL-2 is associated with a serious capillary leak syndrome. These issues prompted the search for other immunotherapeutics with the benefits of IL-2 but with fewer negative side effects. It was proposed that IL-15 has favorable characteristics that could make it more effective and less toxic than IL-2 in the immunotherapy of metastatic malignancy [10–15]. In particular, IL-15 facilitates the proliferation and synthesis of immunoglobulin by activated B cells [16]. In addition, IL-15 is pivotal in mice in the generation and maintenance of NK cells; is antiapoptotic in several systems; and supports the survival of stem, central and effector CD8 memory T cells, thereby playing a critical role in the maintenance of long-lasting, high-affinity T-cell immune responses to invading pathogens [10–15,17–20]. Furthermore, in contrast to IL-2, IL-15 inhibited IL-2-mediated AICD, less consistently activated Tregs and did not cause a major capillary leak syndrome in mice or in nonhuman primates [10].

IL-15 molecular biology & expression

IL-15 is a 14–15 kDa glycoprotein encoded on chromosome 4q31 [21–23]. The human IL-15 gene comprises nine exons and four introns. There are two isoforms of IL-15 mRNA that differ in their signal peptides. One isoform consists of a 316-bp 5′-UTR, a 486-bp coding sequence and a 400-bp 3′-UTR that is translated into an IL-15 precursor protein with a long-signal peptide of 48 amino acids [24]. The other isoform of IL-15 mRNA has a short-signal peptide of 21 amino acids. Both isoforms produce the same mature protein. However, the two isoforms differ in their intracellular trafficking pattern. The IL-15 long-signal peptide is targeted to the Golgi apparatus, endosomes and the endoplasmic reticulum secretory pathway, whereas IL-15 short-signal peptide is not secreted but appears to be restricted to the cytoplasm and the nucleus [24].

IL-15 mRNA is expressed by a large number of tissues; however, IL-15 protein is largely limited to monocytes, macrophages and dendritic cells (DCs) [10,25–27]. Thus, although regulation of IL-15 protein production occurs at transcription, most of the control of its protein expression is at the level of translation [13,25–28]. Transcription of IL-15 is stimulated by type I and II interferons, CD40 ligation as well as Toll-like receptor stimuli such as lipopolysaccharide. Lucas and coworkers demonstrated that interferon type I signals by DCs and the subsequent production and trans-presentation of IL-15 by DCs to resting NK cells was necessary and sufficient for priming of NK cells [29]. Translation is impeded by multiple AUG sequences in the 5′-UTR, a long-signal peptide and a negative regulatory element in the C-terminus of the coding sequence of the mature protein that act to limit this process [25,26]. This tight regulation of IL-15 expression is required because of its potency as an inflammatory cytokine that induces TNF-α, IL-1β, IFN-γ, IL-6 and other inflammatory cytokines [30].

IL-15 receptor molecular biology

The heterotrimeric IL-15 receptor is composed of the β subunit (IL-2R/IL-15R, CD122) shared with the IL-2 receptor [10,11,22]; the common gamma (γc) subunit (CD132) shared with IL-2, IL-4, IL-7, IL-9 and IL-21; and a private IL-15 specific α subunit, IL-15R α [31–33]. The IL-15R α receptor has a 173-amino acid extracellular domain, a single 21-amino acid transmembrane region and a 37-amino acid cytoplasmic domain [10,12,13,31,32]. The human IL-15R α gene maps to chromosome 7 with over ten alternative isoforms [31,33]. It is made up of eight exons which as a result of alternative splicing can produce multiple isoforms of IL-15R α [32].

Biological functions of IL-15

As a consequence of their sharing receptor components IL-2R/IL-15R β and γc and use of common JAK (JAK1, JAK3) STAT (STAT3, STAT5) signaling molecules, IL-2 and IL-15 share several functions. These functions include stimulation of the proliferation of activated CD4⁻CD8⁻, CD4⁺CD8⁺, CD4⁺ and CD8⁺ T cells [10,11,13,34]. Furthermore, the two cytokines facilitate the induction of cytotoxic lymphocytes and augment the proliferation and immunoglobulin synthesis by B cells that have been costimulated with an IgM-specific antibody or by ligation of CD40 [16]. IL-2 and IL-15 also stimulate the generation, proliferation and activation of NK cells [17,35]. In addition to these similarities, there are distinct differences between the functions of IL-15 and IL-2. IL-2 has a unique role in the prevention of T-cell-mediated immune responses to self. This function is explained by the fact that IL-2 is required to maintain the fitness of Fox forkhead box P3 (Foxp3) expressing Treg cells [9]. By contrast, IL-15 expression has no marked effects on Treg cells. IL-2 also has a crucial role in AICD, a process that leads to the elimination of self-reactive T cells, whereas IL-15 is an antiapoptotic agent wherein IL-15-transgenic mice have an inhibition of IL-2-induced AICD [20]. In contrast to IL-2, IL-15 has as its unique role the maintenance of a long-lasting immune response to invading pathogens. In particular, IL-15 promotes the maintenance of CD8⁺ CD44^hi memory phenotype T cells [10,11,18,19]. These observations from ex vivo functional studies are supported by an examination of mice with disrupted cytokine or cytokine receptor genes. IL-2 and IL-2R-deficient mice develop a marked enlargement of peripheral lymphoid organs that is associated with the polyclonal expansion of T and B populations [36]. These expanded populations are associated with impairments in Treg development and AICD. Furthermore, IL-2R α deficient mice develop autoimmune diseases such as hemolytic anemia and inflammatory bowel disease [36]. In contrast to these observations with IL-2, IL-15 and IL-15R α deficient mice do not develop lymphoid enlargement, increased serum immunoglobulin concentrations or autoimmune disease [37]. Rather IL-15-deficient mice have a marked reduction in the number of peripheral NK cells, NK T cells, γ/δ T cells and intestinal intraepithelial lymphocytes [37]. In contrast to the situation in mice, homeostasis of human NK cells is not IL-15 dependent although IL-15 does augment proliferation and activation of NK cells in humans [38]. In addition to its effects on T and NK cells, IL-15 also protects neutrophils from apoptosis, modulates phagocytosis and simulates the secretion of IL-8 and IL-1R antagonists by these cells [13]. IL-15 suppresses human neutrophil apoptosis through the action of several kinases including JAK2, p38 mitogen-activated protein kinase and extracellular signal-regulated kinases-1/2 [34,39–41]. In addition, IL-15 has a role in the action of the antiapoptotic Mcl-1 protein [41]. Furthermore, DCs stimulated by IL-15 demonstrate maturation and increased CD83, CD86, CD40 and MHC Class II expression [13,41].

Interaction of IL-15 with its receptor & receptor signaling

IL-15 and IL-15R α are coexpressed on monocytes and DCs following activation with interferon or CD40 ligation and Toll-like receptor activation with agents such as lipopolysaccharide that in turn induce interferon expression [42,43]. IL-15 binds to IL-15R α with high-affinity Kd >10⁻¹¹ M. Following activation-mediated coexpression in DCs, IL-15 and IL-15R α remain together on the cell surfaces, recycle through endosomal vesicles and reappear on the cell surfaces so that the combination is coexpressed for many days [43]. Dubois and coworkers and then others discovered that the IL-15R α on DCs trans-presents IL-15 to effector NK and CD8 T cells as part of an immunological synapse [43–48]. IL-15 is trans-presented to cells that express IL-2R/IL-15R β and γc but not IL-15R α. In lymphocytes, the binding of IL-15 in trans to the IL-2/IL-15R β and γc heterodimer induces JAK1 activation that subsequently phosphorylates STAT3 via the β chain and JAK3 and STAT5 via its γ chain [10–13,34]. Sathe and coworkers demonstrated a nonredundant pathway linking IL-15 via STAT5 phosphorylation and subsequent binding to the 3′ UTR of Mcl1 that is involved in the maintenance of NK cells and innate immune responses [42]. In an additional IL-15 signaling mechanism, the adapter protein Shc binds to a phosphotyrosine residue on IL-2/IL-15R β resulting in the activation of Grb2 and AKT via the Shc, Grb2, Gab2, PI3K, AKT and mTOR signaling pathway. Marcais and coworkers demonstrated that the metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells [49]. Low doses of IL-15 triggered only phosphorylation of the transcription factor STAT5, whereas high concentrations of IL-15 utilized the metabolic checkpoint kinase mTOR, stimulated the growth and nutrient uptake of NK cells and positively fed back on the receptor for IL-15 [49]. In a third signaling pathway, IL-15 trans-presentation is associated with activation of Grb2 and SOS to form a Grb2/SOS complex that in turn activates the RAS–RAF pathway that then activates the MAPK pathway involved in cellular proliferation [12,13]. Collectively these signaling pathways induce expression and activation of c-myc, c-fos, c-jun, Bcl-2, Bcl-xL and NF-kB [12,13].

In addition to its dominant trans-presentation, IL-15 can also signal in cis onto cells that express IL-15R α in addition to IL-2/IL-15R β and the common γ chain [50–56]. Cells normally bearing the β and γ chain transfected with IL-15R α were shown to efficiently signal in cis through IL-15R α and the IL-2R β/γc expressed on the surface of a single cell. With cells bearing only IL-2R/IL-15R β and γc, the addition of soluble IL-15R α facilitated IL-15 activity, whereas soluble IL-15R α inhibited the proliferative capacity of cis expressing heterotrimeric IL-15 receptors [50,55].

Ota and coworkers demonstrated that there is no requirement for trans-presentation of IL-15 for human CD8 T-cell proliferation [54]. Furthermore, the humanized monoclonal antibody Mik-Beta-1 directed toward IL-2/IL-15R β blocked IL-15 trans-presentation but did not inhibit IL-15 receptor action in cis [52]. In addition, in immunophysiological studies examining the ability of IL-15 expressed by a vaccine vector to influence the response induced, it was demonstrated that the presence of IL-15 during the immune-induction phase resulted in long-lasting CD8⁺ memory T cells due to an increased expression of IL-15R α [53]. The results are consistent with the ability of IL-15R α on T cells themselves to present in cis and augment the T cells’ responsiveness to IL-15 in addition to the known ability of IL-15R α on APCs to present IL-15 to T cells in trans [53]. Furthermore, Zanoni and coworkers demonstrated that IL-15 cis presentation is required for optimal NK cell activation in lipopolysaccharide-mediated inflammatory conditions [55]. Escherichia coli exposure of DCs led to IFN-β enhancement of NK-cell activation by inducing expressions of IL-15 and IL-15 receptor α not only in DCs but surprisingly also in NK cells. This process allows the transfer of IL-15 onto NK cell surfaces and its cis presentation. cis-presented NK cell-derived and trans-presented DC-derived IL-15 contributed equally to optimal NK cell activation [55].

In addition to the classical heterotrimeric IL-15 receptor JAK1, JAK3, STAT3/STAT5 pathway, there are novel receptor signaling transduction pathways for IL-15 in mast cells [57]. IL-15 signaling in mast cells does not involve IL-2/IL-15R β or the common γ chain rather it involves distinct receptors. Furthermore, mast cell IL-15 receptors recruit JAK2 and STAT5 instead of JAK1/3 and STAT3/5 that are activated in T cells. Finally, a number of groups have reported that in the absence of the common γ chain and independently of IL-2/IL-15R β and JAK/STAT signaling that IL-15 can signal via IL-15R α, JNK and NK-kB to drive RANTES production by myeloid cells [56,58,59]. Other groups have also demonstrated that IL-15R α is competent for IL-15-mediated signal transduction contrary to what was previously reported.

The efficacy of IL-15 in murine models of neoplasia

A number of studies in murine models suggested that IL-15 may prove to be of value in the therapy of neoplasia [10–14,60–67]. In particular, whereas following intravenous administration of MC38 syngeneic colon carcinoma cells wild-type B6 mice died of pulmonary metastases within 6 weeks, IL-15 transgenic mice survived for more than 8 months following infusion of the tumor cells [61]. Furthermore, Klebanoff and coworkers demonstrated that IL-15 enhanced the in vivo activity of tumor-reactive CD8⁺ T cells in the T-cell receptor transgenic mouse (pmel-1) whose CD8⁺ T cells recognized an epitope derived from the melanoma antigen gp100 [14]. Huntington and coworkers evaluated IL-15 in the HIS mouse model that involved engraftment of newborn BALB/c Rag2^−/− γc^−/− mice with human hematopoietic stem cells from fetal liver or cord blood which generates human innate and adaptive lymphocytes and DCs required for the immune response [68,69]. Trans-presented murine IL-15 inefficiently triggered human NK cells providing an explanation for the poor human NK cell reconstitution in HIS mice. This group also evaluated exogenous administration of a potent human IL-15R agonist (RLI) consisting of human IL-15 covalently linked to an extended human IL-15R α ‘sushi’ domain thus mimicking IL-15 trans-presentation. This agent was sufficient to restore human NK cell development in HIS mice. Furthermore, Bessard and coworkers demonstrated high antitumor activity of RLI, the IL-15-IL-15R α fusion protein in metastatic melanoma and colorectal cancer models in mice supporting the use of RLI as an adjuvant/therapeutic for human clinical trials [70]. In the studies of Yu [67] and Zhang [65,66], IL-15 was shown to prolong the survival of mice with established PC26, MC38 colon cancer and with TRAMP-C2 prostate cancers. On the basis of the animal and laboratory trials with IL-15, great interest was generated among leading immunotherapeutic experts participating in the NCI Immunotherapy Agent Workshop who ranked IL-15 as the most promising unavailable immunotherapeutic agent to be brought to therapeutic trials [71].

Toxicity, pharmacokinetics, immunogenicity & impact on elements of the normal immune system of recombinant human IL-15 in rhesus macaques

The safety of IL-15 was evaluated in mice and in rhesus macaques [62,72–76]. Munger et al. [62] administered 30, 60 or 180 μg of recombinant simian IL-15 by intraperitoneal injection to C57BL/6 mice three-times per day for nine doses. Mice receiving IL-15 at 180 μg demonstrated only a minimal vascular leak syndrome suggesting that this toxicity would not be dose limiting in human IL-15 trials. Mueller et al. [72] injected simian immunodeficiency virus-infected rhesus macaques with recombinant IL-15 at doses of 10 or 100 μg/kg twice weekly subcutaneously for 4 weeks [72]. IL-15 induced a nearly threefold increase in the number of peripheral CD8⁺CD3⁻ NK cells. All clinical laboratory results remained within normal limits with the exception of a nonsignificant increase in the number of circulating platelets. Berger et al. [76] administered human recombinant IL-15 to rhesus macaques at doses of 2.5–15 μg/kg daily by subcutaneous injection for up to 14 days [76]. A second set of macaques received IL-15 every 3 days at doses of 2.5, 5 or 10 μg/kg for 24 days. Daily administration of IL-15 for up to 14 days caused reversible severe neutropenia, anemia, weight loss, a generalized skin rash and a massive expansion of T cells. The bone marrow of a single neutropenic animal was found to be hypocellular.

Recombinant human IL-15 (rhIL-15) produced by the Biopharmaceutical Development Program NCI was administered at a dosing schedule of 12 daily intravenous bolus infusions at doses of 10, 20 and 50 μg/kg/day [73,74]. The only biologically meaningful laboratory abnormality was a grade 3/4 transient neutropenia. This neutropenia was shown to be secondary to a redistribution of neutrophils in that bone marrow examinations demonstrated increased marrow cellularity including cells of the neutrophil series [73]. Furthermore, neutrophils were observed in sinusoids of markedly enlarged livers and of spleens suggesting IL-15-mediated neutrophil redistribution from the circulation to the tissues. In accord with this view, Verri and coworkers demonstrated that following intraperitoneal administration of IL-15, there was a cascade of cytokines in the order of IL-18, MIP-1-α, MIP-1-β and TNF-α that was associated with a redistribution of neutrophils from the circulation to the peritoneal cavity [77].

A 12-day bolus of intravenous administration of 20 μg/kg/day of IL-15 to rhesus macaques was associated with a 4- to 8-fold increase in the number of circulating NK and stem, central and effector memory CD8 T cells [73–75]. Subsequently, alternative routes of administration were evaluated in rhesus macaques including continuous intravenous infusion (CIV) and subcutaneous administration of IL-15 [75]. The administration of IL-15 by CIV at 20 μg/day for 10 days led to an approximately 10-fold increase in the number of circulating NK cells and a massive 80- to100-fold increase in the number of circulating effector memory CD8 T cells [75].

Subcutaneous infusions at 20 μg/kg/day for 10 days led to a more modest 10-fold expansion in the number of circulating effector memory CD8 T cells [75]. In contrast to observations with daily administration of IL-15, subcutaneous infusions of 20 μg/kg/day twice weekly were not associated with an increase in the number of total circulating lymphocytes or effector memory CD8 T cells [75]. No vascular leak syndrome, hemodynamic instability or renal failure was observed in these studies. In addition, no animal developed autoimmune disorders or antibodies to the infused IL-15.

Clinical trials using IL-15 in the treatment of cancer

Five clinical trials have been initiated using E. coli rhIL-15 in the treatment of cancer (Table 1). The primary goal of the completed trial: ‘A Phase I study of intravenous recombinant human IL-15 in adults with refractory metastatic malignant melanoma and metastatic renal cell cancer’ [78] to determine the safety, adverse event profile, dose limiting toxicity and maximum tolerated dose of rhIL-15 administered as a daily intravenous bolus infusion for 12 consecutive days in subjects with metastatic malignant melanoma or metastatic renal cell cancer [79]. This first in-human trial utilized E. coli-produced rhIL-15. The study was initially designed as a Phase I dose-escalation trial starting with an initial dose of 3 μg/kg/day for 12 days. However, after the initial patient developed grade 3 hypotension and another patient developed grade 3 thrombocytopenia the protocol was amended to add two lower doses 1.0 and 0.3 μg/kg/day. Two of four patients given the 1.0 μg/kg/day dose had persistent grade 3 alanine aminotransferase (ALT), AST elevations that were dose limiting. All nine patients with IL-15 administered at 0.3 μg/kg/day received all 12 doses without dose-limiting toxicity. The maximum tolerated dose of rhIL-15 was determined to be 0.3 μg/kg/day.

Table 1.

Clinical trials using IL-15 in cancer immunotherapy.

Clinical trial	Government clinical trials identifier	Disease + dosing strategy	Status	Ref.
A Phase I study of intravenous recombinant human IL-15 in adults with refractory metastatic malignant melanoma and metastatic renal cell cancer	NCT01021059	Phase I study with 12 daily bolus intravenous infusions in 18 patients with melanoma or renal cell cancer	This study has been completed	[78]
Recombinant interleukin-15 in treating patients with advanced melanoma, kidney cancer, non-small cell lung cancer or squamous cell head and neck cancer	NCT01727076	Melanoma, kidney, non-small cell lung cancer, squamous cell head and neck cancer. This Phase I study involves subcutaneous IL-15 daily on days 1–5 and 8–12	Currently recruiting patients	[82]
Continuous infusion of rhIL-15 for adults with advanced cancer	NCT01572493	Metastatic carcinoma. This is a Phase I study of ten daily continuous intravenous infusions of rhIL-15	Currently recruiting patients	[84]
Haploidentical donor of natural killer cell infusion with IL-15 in acute myelogenous leukemia	NCT01385423	Acute myelogenous leukemia, myelodysplastic syndrome. This is a Phase I study of rhIL-15 given after haploidentical donor NK cells	Currently recruiting patients	[85]
Use of IL-15 after chemotherapy and lymphocyte transfer in metastatic melanoma	NCT01369888	Tumor infiltrating lymphocytes in rhIL-15 in the treatment of melanoma	This study is closed	[104]

rhIL-15: Recombinant human IL-15.

There was a consistent temporal pattern of post-treatment adverse events in patients given the 3 μg/kg/day dose with fever beginning 2.5–4 h following the start of rhIL-15 infusions. Rigors occurred at approximately the 1.5–2 h time point and blood pressure dropped to a nadir of approximately 20 mm Hg below pretreatment levels. These changes were concurrent with maximum up to 50-fold elevations of circulating IL-6 and IFN-γ. In studies with chimeric antigen receptors, such symptoms could be reversed by the administration of an anti-IL-6R a monoclonal antibody [80,81]. The patients had evidence of only modest capillary leak. None of the patients produced antibodies to rhIL-15. The pharmacokinetics of rhIL-15 for 3.0, 1.0 and 0.3 μg/kg/day resulted in C_max of 47,800, 15,900 and 12,060 respectively. The survival half-lives (t_1/2) were very similar for the three dose levels 2.4, 2.7 and 2.7 h.

Flow cytometry of peripheral blood lymphocytes revealed an efflux of NK and memory CD8 T cells from the circulating blood within minutes upon IL-15 administration, followed by influx and hyperproliferation yielding 10-fold expansion of NK cells that ultimately returned to baseline [79]. Furthermore, there were significant increases in γ/δ and CD8 memory T cells. In this first in-human Phase I trial, there were no responses with stable disease as a best response. However, five patients manifested a decrease between 10 and 30% in their marker lesions and two patients had clearing of lung lesions.

Development of alternative dosing strategies

It was concluded that although IL-15 could be safely administered to patients with metastatic malignancy, rhIL-15 was not optimal when administered as an intravenous bolus due to an intense cytokine secretion and the macrophage activation syndrome that occurred in the first few hours after treatment. There were exceedingly high IL-15 C_max levels initially following bolus infusions that were sufficient to signal through the IL-2/IL-15R β and γc receptor pair that IL-15 shares with IL-2 thereby contributing to the toxicity observed [79]. To reduce the C_max obtained and to increase the period of time when IL-15 was at an optimal concentration for high-affinity IL-15 receptors, alternative receptor dosing strategies in nonhuman primate rhesus macaques were evaluated Figure 1 [75]. By administering IL-15 by CIV or subcutaneously, the exceedingly high C_max observed with bolus infusions could be avoided. With bolus intravenous infusion to rhesus macaques at 20 μg/kg/day the C_max was 720 ng/ml; with subcutaneous infusions at this dose the C_max was 50 ng/ml; whereas with CIV of this dose per day the C_max was between 2 and 4 ng/ml throughout the 10-day study [73,75]. Furthermore, the efficacy was greatest with CIV bolus infusions. In contrast to the four- to eightfold expansion of CD8 T_EM cells observed with bolus administration, CIV rhIL-15 infusions were associated with an 80- to 100-fold expansion of circulating CD8 T_EM cells in rhesus macaques [75]. Subcutaneous IL-15 at 20 μg/kg/day resulted in a more modest 10-fold expansion of CD8 T-effector memory cells [75]. To translate these preclinical trials with the goal of reducing C_max, the excess cytokine release and the macrophage activation syndrome events two additional clinical trials were initiated. These trials include the clinical trial: ‘Recombinant interleukin-15 in treating patients with advanced melanoma, kidney cancer, non-small cell lung cancer or squamous cell head and neck cancer’, [82], supported by the NCI and the Cancer Immunotherapy Trials Network, wherein at five sites recombinant IL-15 is being administered subcutaneously daily on days 1–5 and 8–12 in a study involving patients with advanced melanoma, kidney cancer, non-small cell lung cancer or squamous cell head and neck cancer [83]. Treatment repeats every 28 days for up to four courses in the absence of unacceptable toxicities are permitted. Three patients have been enrolled in each of the 0.25, 0.5, 1.0 and 2.0 μg/kg per dose cohorts [83]. Among 12 patients treated, there was only one serious adverse event, grade 2 pancreatitis in a metastatic melanoma patient that began 3 days after completing cycle one treatment at 2.0 μg/kg [83]. Flow-cytometric data indicated a consistent increase in the frequency of circulating CD3⁺CD56⁺ NK cells. In addition, in the study [84] being performed at the National Institutes of Health Clinical Center there is an evaluation of ‘continuous infusion of rhIL-15 for adults with advanced cancer.’ This study involves a dose escalation of IL-15 provided by CIV to patients with cancer for whom there is no remaining effective curative treatment available. In addition to these trials [85], ‘Haploidentical donor of natural killer cell infusion with IL-15 in acute myelogenous leukemia (AML)’ is recruiting participants [86]. This is a single-center (Masonic Cancer Center – University of Minnesota), modified standard design dose-escalation study designed to determine the maximum tolerated, minimum efficacious dose of IL-15 (intravenous recombinant human IL-15) and incidence of donor NK cell expansion by day +14 when given after haploidentical donor NK cells in patients with relapsed or refractory acute myelogenous leukemia.

Figure 1. IL-15 was administered for 10 days using four different dosing strategies.

The control group received 5% dextrose by CIV for 10 days. Each point corresponds to the mean ± SEM and indicates the fold change in the absolute count relative to the pretreatment (day −4). Gray bars above the x-axis represent the duration of IL-15 administration. The rhesus macaques received 20 μg/kg/day of IL-15 by CIV (red dots), subcutaneously daily 40 μg/kg/day (green circles), subcutaneously daily 20 μg/kg/day (gray triangles) or subcutaneously twice weekly (blue squares).

CIV: Continuous intravenous infusion; SEM: Standard error of the mean. This research was originally published in [75].

IL-15/IL-15R α

Although IL-15 administration may show efficacy in the treatment of metastatic malignancy in human clinical trials, it has not been optimal when used as a single agent for cancer therapy. A particular challenge is that there is only a low-level expression of IL-15R α on resting DC cells [87]. As noted above, for its long-term persistence and its optimal activation of NK and CD8⁺ T cells, IL-15 is predominantly expressed bound to IL-15R α on the surface of DCs where IL-15 is trans-presented to effector NK and CD8 T cells that express IL-2/IL-15R β and γc but not IL-15R α [43–48]. Indeed, the true IL-15 cytokine may not be an IL-15 monomer but rather may be considered as an IL-15R α/IL-15 heterodimeric cytokine [70,88–93]. Physiologically, IL-15 is produced as a heterodimer in association with IL-15R α. Furthermore, in mice it is the heterodimer alone that is stably produced and transported to the surface of the cell [94,95]. On cleavage, IL-15R α/IL-15 elements are associated in the serum as the sole form of circulating IL-15 [87,89,95]. The heterodimeric IL-15R α/IL-15 can be efficiently produced in a mammalian system, whereas this has not been true for monomeric IL-15. The recombinant form of IL-15R α/IL-15 has a larger overall size and when produced in a mammalian system is glycosylated in contrast to E. coli-produced monomeric IL-15. In part because of its larger size and glycosylation, the heterodimeric IL-15 has a much longer T_½ of survival in the plasma than does monomeric IL-15 [89].

To address the issue of deficient IL-15R α when IL-15 is used as a monomer, multiple agents involving IL-15 associated with IL-15R α have been generated [70,89–92]. When tested in vitro IL-15R α IgG1-Fc strongly increased the IL-15-mediated proliferation of murine NK and CD8⁺CD44^hi CD8 T cells [65,88,89]. IL-15R α/IL-15 heterodimers administered at tolerable doses to rhesus macaques led to a marked proliferation of NK, γ/δ and CD8⁺ T cells in the peripheral blood. Furthermore, IL-15 heterodimers induced the preferential expansion of both central and effector memory CD8 T cells. In addition, when mice bearing the NK sensitive syngeneic tumor B16 were treated the presence of IL-15R α linked to IgG1Fc but not IgG4Fc there was an increase in the antitumor activity [65]. Thus, a preassociation of IL-15 with IL-15R α enhanced the activities of IL-15 in vivo and in vitro.

Clinical trials with heterodimeric IL-15/IL-15R α agents

As noted above, even if rhIL-15 has clinical efficacy, eventual use of monomeric IL-15 as a single agent is unlikely. Of critical importance in the present IL-15 monomer clinical trials is that there was only a very low level expression of IL-15R α on resting DCs in the serum of patients entering the IL-15 trials with a mean serum level below 2 pg/ml [87]. A number of strategies and agents are being used to circumvent this limitation. One future strategy, noted below, is to combine the optimal rhIL-15 regimen with an agonistic anti-CD40 monoclonal antibody that acts to stimulate IL-15R α expression [66]. In addition, clinical trials have been or are being initiated to evaluate IL-15 preassociated with IL-15R α or with IL-15R α IgG1-Fc [90]. Altor BioScience Corporation launched a clinical trial in patients with metastatic melanoma with the superagonist ALT-803 which represents a combination of a mutant IL-15 with a fourfold increase in biological activity given in conjunction intravenously with IL-15R α-IgG1-Fc [90]. ALT-803 is a superagonist of IL-15 consisting of two molecules of IL-15 N-to-D substituted at position 72 bound to two molecules of the ‘sushi’ domain of IL-15 receptor α and a single Fc fragment of IgG1. The Cancer Immunotherapy Trials Network has initiated a Phase I expansion trial of weekly intravenous ALT-803 in patients with advanced melanoma. Furthermore, Admune Therapeutics LLC in conjunction with the NCI is initiating a clinical trial of subcutaneously administered IL-15 associated with IL-15R α in patients with metastatic malignancy.

The agents with a combination of IL-15R α with IL-15 present a number of theoretical advantages over monomeric rhIL-15. IL-15R α/IL-15 is more physiological in that these elements are always expressed together normally and may indeed represent two elements of a heterodimeric cytokine [88–95]. In addition, the combination can be very efficiently generated in mammalian cells and are glycosylated, whereas this cannot be efficiently done with monomeric IL-15. Due to the larger size and glycosylation, the combination of IL-15R α/IL-15 has a much longer in vivo survival than the 1 h T_½ of E. coli-produced IL-15 [89]. Heterodimeric IL-15 can be given two- to three-times a week rather than the daily administration required for monomeric IL-15. The heterodimeric IL-15R μ/IL-15 is 10-fold more active in inducing the expression of NK and CD8 cells than is monomeric IL-15 [89,92]. Finally, in the B16 melanoma and TRAMP-C2 models the heterodimeric IL-15 has shown greater efficacy in the treatment of murine xenograft tumor models than monomeric IL-15 [65].

Expert commentary

IL-15 was shown to have favorable characteristics that make it more effective and less toxic than IL-2 in the treatment of cancer. Furthermore, IL-15 is pivotal in the generation and maintenance of NK cells and is effective in supporting the survival of stem, central and effector CD8 memory T cells. In addition, in contrast to IL-2, IL-15 inhibited IL-2-mediated AICD, less consistently activated Tregs and did not cause a major capillary leak syndrome in mice and in nonhuman primates. Administration of IL-15 to rhesus macaques by CIV at 20 μg/day for 10 days led to an approximately 10-fold increase in the number of circulating NK cells and an 80- to 100-fold increase in the number of circulating effector memory CD8 T cells [75]. Clinical trials utilizing E. coli-produced rhIL-15 are in a Phase I dose finding stage and are directed toward determining an appropriate dosing strategy and the maximum tolerated dose. Nevertheless, it is clear that a number of additional developments including the initiation of combination drug therapy will be required before the encouraging preclinical trials can be translated into an effective strategy utilizing IL-15 in the treatment of patients with cancer. The initial completed Phase I IL-15 bolus infusion clinical trial was associated with toxicities including fever, rigors and hypotension not predicted from the preclinical trials in nonhuman primates. These toxicities as well as hepatotoxicity led to the determination of a maximum tolerated dose of 0.3 μg/kg/day, a value much lower than that predicted from the nonhuman primate studies [79]. The toxicities observed were associated contemporaneously with up to 50-fold increases in IL-6 and IFN-γ levels. There were exceedingly high IL-15 C_max levels initially following bolus infusions that may underlie this intense cytokine secretion and macrophage activation syndrome that were observed in the first few hours following IL–15 infusions. With the goal of reducing the C_max, clinical trials have been initiated with alternative dosing strategies by administering IL-15 daily subcutaneously on days 1–5 and 8–12 or by CIV for 10 days. It is hoped that these approaches will reduce toxicity and increase the efficacy of IL-15 in increasing the number of circulating monocytes, NK cells and CD8 effector memory T cells, thereby augmenting efficacy.

Although IL-15 administration may show efficacy, it probably is not optimal when used as a single monomeric agent for cancer therapy. There is only a low level expression of IL-15R α on resting DC cells. As noted for its long-term persistence and its optimal activation of NK and CD8 T cells, IL-15 must be expressed as a heterodimer in association with IL-15R α. To address this challenge, agents with IL-15 preassociated with both IL-15R α and IL-15R α IgG1-Fc have been generated. These combinations have improved pharmacokinetics, have an impact on the mononuclear cell numbers and have greater efficacy in preclinical murine models when compared to monomeric IL-15. Trials have been initiated with both the superagonist ALT-803 (IL-15 and IL-15R α IgG1-Fc) and with IL-15/IL-15R α. It is too early in these studies to define the toxicity and efficacy that will be observed with this approach. Nevertheless, it is my view that neither IL-15 alone nor IL-15 in conjunction with IL-15R α when given alone will show major efficacy in the treatment of cancer. Preclinical studies suggest that the coadministration of IL-15 with other agents, described more extensively in the 5-year view below, will permit IL-15 to play a major role in cancer therapy. In particular, it was shown in murine models using an agonistic anti-CD40 monoclonal antibody coadministered with IL-15 that the combination was associated with an augmented IL-15R α expression and a reversal of the ‘helpless state’ of CD8 cells. This combination showed great additivity/synergy in murine syngeneic tumor models [66]. Furthermore, additivity/synergy was also observed with IL-15 given in combination with both of the checkpoint inhibitors anti-CTLA-4 and anti-PD-L1 [67]. All the strategies discussed above are based on the assumption that the host is making an immune response, albeit inadequate, to its tumor. Other approaches using IL-15 do not make this assumption. In particular, IL-15 is being incorporated in vaccines [96]. Furthermore, IL-15 is very effective in activating NK cells and monocytes suggesting that it may increase antibody-dependent cellular cytotoxicity mediated by antineoplastic monoclonal antibodies [97–99].

In summary, it is clear that much work is necessary in the clinic to bring the encouraging preclinical insights and therapeutic opportunities of IL-15 to fruition. It is anticipated that with these combination agent approaches, IL-15 can play a crucial role in the treatment of cancer.

Five-year view

Dose-escalation trials of rhIL-15 with different dosing routes of administration (bolus intravenous infusion, subcutaneous or CIV) will soon be completed. However, monotherapy with IL-15 is not optimal since the physiological agent is the heterodimer IL-15R α/IL-15. Dose-escalation clinical trials will also soon be initiated and completed with the heterodimeric IL-15R α/IL-15 and with mutant IL-15/IL-15R α IgFc. When these studies have been completed, the optimal agent, dose and dosing strategy can be identified. IL-15 alone or coupled with IL-15R α can then be evaluated in a number of clinical situations including as critical elements of antitumor vaccines, cellular therapy and in association with monoclonal antibodies to augment ADCC.

Use of an agonistic anti-CD40 coadministered with IL-15 to induce (IL-15R α) & to reverse the ‘helpless state’ of CD8 T cells

Agents are available that induce IL-15R α expression on DCs that could be given in conjunction with IL-15 to circumvent the problems discussed above with IL-15 when used in monotherapy [66]. Although INF-γ induced IL-15R α on DCs it also induced Class 1 MHC expression on tumor cells and was ineffective when given in conjunction with IL-15 in CT26, MC38 and TRAMP-C2 murine tumor models [66]. In contrast, the combination of IL-15 with the agonistic anti-CD40 antibody FGK4.5 showed additivity/synergy in the MC38 murine model of colon cancer and in the TRAMP-C2 model of prostatic cancer (Figure 2) [66]. The administration of the anti-CD40 antibody was associated with an increased expression of IL-15R α on CD11⁺DCs expressing cells present in the spleens of treated mice [66]. Furthermore, the serum concentrations of murine IL-15R α as assessed by ELISA were markedly increased in anti-CD40 monoclonal antibody treated mice when compared to those in the control mouse group. In the murine syngeneic tumor model, treatment with IL-15 or with the agonistic anti-CD40 monoclonal antibody alone significantly (p < 0.01) prolonged the survival of both groups of CT26 and MC38 tumor-bearing mice when compared to mice in the PBS control group. These studies were extended to mice bearing established (80 mm³) subcutaneous TRAMP-C2 tumors and again it was demonstrated that the combination of IL-15 with anti-CD40 produced markedly additive effects when compared to either agent administered alone [66]. Using IL-15R α^−/− mice or DCs from such IL-15R α depleted mice, the IL-15-mediated induction of IL-15R α was shown to be one of the critical factors in the anti-CD40-mediated augmentation of IL-15 action in the treatment of the TRAMP-C2 model of prostatic cancer [66].

Figure 2. Combination therapy of mIL-15 with agonistic anti-CD40 mAb led to regression of established TRAMP-C2 tumors in wild-type C57BL/6 mice.

Therapy was initiated after tumors were well established with an average volume of 80 mm³. Treatment with mIL-15 alone at a dose of 2.5 μg per mouse 5 days a week for 2 weeks inhibited tumor growth slightly and prolonged survival of TRAMP-C2 tumor-bearing mice compared with mice in the PBS control group (p < 0.05), whereas treatment with an agonistic anti-CD40 mAb (200 μg per mouse on day 0 and then 100 μg per mouse on days 3, 7 and 10) provided greater inhibition of tumor growth and prolonged survival of the TRAMP-C2-bearing mice compared with mice in the PBS control group (p < 0.001). Furthermore, combination therapy with mIL-15 and anti-CD40 provided a greater therapeutic efficacy as demonstrated by the fact that all of the mice in the combination group were alive at day 60, with 80% becoming tumor free, whereas only 20% of the mice in the anti-CD40 mAb alone group and none of the mice in the PBS group or the IL-15 alone group were alive at that time.

mAb: Monoclonal antibody.

Reproduced with permission from [60]. © The American Association of Immunologists Inc. (2012).

Sckisel and coworkers [98] reported that the administration of the γc family cytokine IL-2 induced the expression of SOCS-3 in CD4 T cells that became ineffective as helper cells [98]. Although IL-2 facilitated an increase in the number of CD8 T cells, these CD8 cells induced were ‘helpless’, and inadequate as antigen-specific immune responders [98]. Furthermore, this group indicated that the ligation of CD40 relieved the ‘helpless’ CD8 T cell state so that the CD8 cells became antigen-specific immune responders. Zhang and coworkers reported related results with IL-15 [60]. As noted above, there was additivity/synergy observed with the combination of IL-15 administered with the anti-CD40 antibody FGK4.5 in an established TRAMP-C2 syngeneic tumor model [60]. Furthermore, when evaluated neither the IL-15 alone nor anti-CD40 alone increased the number of tumor-specific tetramer-positive CD8 T cells. However, the administration of the combination of IL-15 plus the agonistic anti-CD40 antibody was associated with a meaningful increase in the number of TRAMP-C2 tumor-specific SPAS-1/SNC9-H8 tetramer-positive CD8 T cells [60]. These tetramer-specific CD8 T cells were shown to be associated with protection from tumor development on rechallenge. These observations provide major support for a clinical trial to evaluate the efficacy of an agonistic optimized anti-CD40 monoclonal antibody administered in conjunction with IL-15 in the treatment of cancer.

Agents to relieve checkpoints on the immune system to optimize IL-15 action

The studies above were directed toward developing strategies to increase the efficacy of externally administered IL-15 by augmenting the expression of IL-15R α on APCs. Efforts were also directed toward the coadministration of monoclonal antibodies targeting human cells and proteins that act as inhibitory checkpoints on the immune system to augment the action of coadministered IL-15. As is true with virtually all cytokines, IL-15 is associated with the expression of immunological checkpoints. IL-15 induced the inhibitory cytokine IL-10 and the expression of PD1 on CD8 T cells [67]. Furthermore, IL-15 was shown to be critical in the maintenance of CD122⁺ CD8⁺ T negative regulatory cells [100,101]. CD8⁺CD122⁺ T cells are a heterogeneous population that contains both PD1 nonexpressing memory cells as well as PD1 expressing negative regulatory cells that produce IL-10 and that negatively regulate other T cells [100,101].

To address the issue of induced checkpoints, IL-15 was administered in combination with agents to remove these inhibitory checkpoints that included antibodies to cytotoxic T-lymphocyte antigen-4 (CTLA-4) and to program death-ligand 1 (PD-L1) proteins that mediate negative T-cell signaling pathways [67,102]. Following intravenous administration of CT26 or MC38 colon carcinoma syngeneic tumors to immunologically intact mice or the establishment of subcutaneous implantation of TRAMP-C2 tumors IL-15 alone provided only a modest antitumor effect (Figure 3). Furthermore, the addition of either anti-CTLA-4 or anti-PD-L1 alone in association with IL-15 did not augment IL-15 action. However, tumor-bearing mice receiving IL-15 and the combination of both the anti-checkpoints anti-CTLA-4 and anti-PD-L1 manifested a marked prolongation of animal survival [67,102]. These observations support a clinical trial involving IL-15 or IL-15/IL-15R α with the combination of anti-CTLA-4 with anti-PD1/anti-PD-L1.

Figure 3. IL-15 showed additivity with simultaneous blockade of CTLA-4 and PD-L1 in protecting mice against TRAMP-C2 tumor growth.

Kaplan-Meier survival curves illustrate survivals of mice with treatments. IL-15 (5 mg/day 5 days a week for 2 weeks) plus IgG significantly inhibited tumor growth compared with mice receiving PBS (p = 0.037). The combination of IL-15 with either anti-CTLA-4 or anti-PD-L1 did not improve animal survival over animals receiving IL-15 alone. However, the triple combination group exhibited a significant survival advantage compared with mIL-15 alone (p = 0.022) or groups receiving mIL-15 plus anti-PD-L1 or anti-CTLA-4 alone treatment (p = 0.042 and p = 0.027, respectively). Moreover, more than 60% of triple-combination treated mice remained tumor free at study termination.

Reproduced with permission from [67].

Use of IL-15 with anticancer monoclonal antibodies to augment ADCC

The predominant approaches involving IL-15 are based on the hypothesis that the host is making an immune response, albeit inadequate, to its tumor that can be augmented by the administration of an IL-15-containing agent. However, IL-15 could also be used in systems where an additional coadministered agent provides the specificity directed toward the tumor. In particular, IL-15 could be used with anticancer vaccines, cellular therapy or with tumor-directed monoclonal antibodies [96,97,99,103]. In particular, given the capacity of IL-15 to increase the number of activated NK cells, monocytes and granulocytes, a very attractive antitumor strategy would be to use the optimal IL-15 agent and dosing strategy with antitumor monoclonal antibodies to augment ADCC cells [96,97,99,103]. Vincent and coworkers reported highly potent anti-CD20-RLI immunocytokine targeting established human B-cell lymphoma in SCID mice. In summary, since IL-15 is effective in activating NK cells, monocytes and granulocytes, trials should be initiated involving IL-15 given with cancer cell directed monoclonal antibodies. It is hoped with the diverse approaches discussed, IL-15 will take a central place in the combination treatment of cancer.

Key issues.

• IL-15 is a member of the four α-helix bundle family of cytokines. The IL-15 heterotrimeric receptor includes IL-2/IL-15R β shared with IL-2, γc shared with IL-2, IL-4, IL-7, IL-9, IL-21 and a private IL-15 receptor, IL-15R α.

• As part of an immunological synapse, on the surface of dendritic cells, IL-15R α presents IL-15 in trans to NK and memory CD8 T cells that bear the other two IL-15 receptor subunits.

• IL-15 can also function on cells that express the three cell elements of the IL-15 receptor in cis.

• IL-15 augments proliferation of T, B and NK cells and induces the expression of stem, central and effector memory CD8 T cells.

• IL-15 given by continuous intravenous infusion at 20 μg/kg/day to rhesus macaques was associated with an approximately 10-fold increase in the number of circulating NK cells and an 80- to 100-fold increase in the number of circulating effector memory CD8 T cells.

• A clinical trial of daily bolus infusions of recombinant human IL-15 for 12 days has been completed with acceptable toxicity in 18 patients with metastatic malignant melanoma and metastatic renal cell cancer. The maximum tolerated dose in this bolus infusion study was 0.3 μg/kg/day for 12 days.

• Phase I dose-escalation clinical trials with different dosing strategies that involve either subcutaneous or with continuous intravenous infusion of IL-15 have been initiated.

• Clinical trials are being initiated with IL-15 + IL-15R α or IL-15 + IL-15R α IgFc.

• Future trials are planned combining IL-15 with an agonistic anti-CD40 monoclonal antibody given to induce IL-15R α and to reverse the ‘helpless’ state of CD8 T cells.

• Subsequent trials will involve IL-15 or IL-15/IL-15R α administered in association with anticancer monoclonal antibodies to augment antibody-dependent cellular toxicity.