Interleukin-15 augments NK cell-mediated ADCC of alemtuzumab in patients with CD52+ T-cell malignancies.

Interleukin-15 (IL-15) monotherapy substantially increases the number and activity of natural killer (NK) cells and CD8+ T cells but has not produced clinical responses. In a xenograft mouse model, IL-15 enhanced the NK cell-mediated antibody-dependent cell cytotoxicity (ADCC) of the anti-CD52 antibody alemtuzumab and led to significantly more durable responses than alemtuzumab alone. To evaluate whether IL-15 potentiates ADCC in humans, we conducted a phase 1 single-center study of recombinant human IL-15 and alemtuzumab in patients with CD52-positive mature T-cell malignances. We gave IL-15 subcutaneously 5 days per week for 2 weeks in a 3 + 3 dose escalation scheme (at 0.5, 1, and 2 µg/kg), followed by standard 3 times weekly alemtuzumab IV for 4 weeks. There were no dose-limiting toxicities or severe adverse events attributable to IL-15 in the 11 patients treated. The most common adverse events were lymphopenia (100%), alemtuzumab-related infusion reactions (90%), anemia (90%), and neutropenia (72%). There were 3 partial and 2 complete responses, with an overall response rate of 45% and median duration of response 6 months. Immediately after 10 days of IL-15, there was a median 7.2-fold increase in NK cells and 2.5-fold increase in circulating CD8+ T cells, whereas the number of circulating leukemic cells decreased by a median 38% across all dose levels. Treatment with IL-15 was associated with increased expression of NKp46 and NKG2D, markers of NK-cell activation, and increased ex vivo ADCC activity of NK cells, whereas inhibitory receptors PD1 and Tim3 were decreased. This trial was registered at www.clinicaltrials.gov as #NCT02689453.

A very long-acting IL-15: implications for the immunotherapy of cancer.

BACKGROUND: Interleukin-15 (IL-15) is an important cytokine necessary for proliferation and maintenance of natural killer (NK) and CD8+ T cells, and with great promise as an immuno-oncology therapeutic. However, IL-15 has a very short half-life and a single administration does not provide the sustained exposure required for optimal stimulation of target immune cells. The purpose of this work was to develop a very long-acting prodrug that would maintain IL-15 within a narrow therapeutic window for long periods-similar to a continuous infusion. METHODS: We prepared and characterized hydrogel microspheres (MS) covalently attached to IL-15 (MS~IL-15) by a releasable linker. The pharmacokinetics and pharmacodynamics of MS~IL-15 were determined in C57BL/6J mice. The antitumor activity of MS~IL-15 as a single agent, and in combination with a suitable therapeutic antibody, was tested in a CD8+ T cell-driven bilateral transgenic adenocarcinoma mouse prostate (TRAMP)-C2 model of prostatic cancer and a NK cell-driven mouse xenograft model of human ATL (MET-1) murine model of adult T-cell leukemia. RESULTS: On subcutaneous administration to mice, the cytokine released from the depot maintained a long half-life of about 168 hours over the first 5 days, followed by an abrupt decrease to about ~30 hours in accordance with the development of a cytokine sink. A single injection of MS~IL-15 caused remarkably prolonged expansions of NK and ɣδ T cells for 2 weeks, and CD44hiCD8+ T cells for 4 weeks. In the NK cell-driven MET-1 murine model of adult T-cell leukemia, single-agent MS~IL-1550 µg or anti-CCR4 provided modest increases in survival, but a combination-through antibody-depedent cellular cytotoxicity (ADCC)-significantly extended survival. In a CD8+ T cell-driven bilateral TRAMP-C2 model of prostatic cancer, single agent subcutaneous MS~IL-15 or unilateral intratumoral agonistic anti-CD40 showed modest growth inhibition, but the combination exhibited potent, prolonged bilateral antitumor activity. CONCLUSIONS: Our results show MS~IL-15 provides a very long-acting IL-15 with low Cmax that elicits prolonged expansion of target immune cells and high anticancer activity, especially when administered in combination with a suitable immuno-oncology agent.

Effective Cytotoxicity of Dendritic Cells against Established T Cell Lymphomas in Mice.

T cell lymphomas arise in mice that constitutively express a single TCR in the absence of NK cells. Upon TCR engagement these lymphomas are able to corrupt tumor surveillance by decreasing NK cell numbers. In this study, we investigate the outcome of interactions between these T cell lymphomas and dendritic cells. Bone marrow-derived dendritic cells mediated effective killing of T cell lymphomas after activation with IFN-γ and TLR ligands in culture. This cytotoxicity was independent of MHC compatibility. Cell lysis was reduced by the presence of the peroxynitrite inhibitors FeTTPS and L-NMMA, whereas inhibitors of apoptosis, death receptors, and degranulation were without effect, suggesting NO metabolites as the main mediators. When injected together with GM-CSF and R848 into lymphoma-bearing mice, in vitro-expanded bone marrow-derived dendritic cells caused significant survival increases. These data show that dendritic cell adaptive immunotherapy can be used as treatment against T cell lymphomas in mice.

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.

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.

Engagement of lymphoma T cell receptors causes accelerated growth and the secretion of an NK cell-inhibitory factor.

The development of T cell lymphomas in mice that constitutively express a single T cell receptor is surveilled by the action of NK cells. We investigated the effects of engaging the lymphoma TCR in this mouse model. We stimulated lymphoma cells expressing an ovalbumin-specific TCR in vivo using listeria monocytogenes as a vehicle. Infections with listeria expressing ovalbumin but not with control bacteria caused a stable change in lymphoma cells that allowed its growth in mice with normal NK cells. TCR engagement furthermore enhanced lymphoma growth in NK-cell-depleted mice suggesting a lymphoma-intrinsic change that lead to accelerated growth. The ability to grow in mice without prior NK cell depletion did not appear to be accompanied by changes in the recognition of lymphoma by NK cells. Rather, lymphoma immunization was associated with a decrease in NK cell numbers: Leukemic phases were observed for all mice starting three to eight weeks after immunizations, and leukemias were succeeded by the disappearance of NK cells from blood. We also observed strong decreases of NK cell numbers in spleens at the time of death. Co-culture experiments showed decreases in the ability of NK cells to proliferate in response to IL-15 when post-immunization lymphoma cells were present in a mechanism that did not require direct cell contact. Together these data suggest that TCR engagement caused intrinsic changes in T cell lymphoma cells resulting in both accelerated in vivo growth and in the secretion of a factor that caused NK cell disappearance.

NK cells prevent T cell lymphoma development in T cell receptor-transgenic mice.

Mice that express a single transgenic T cell receptor have a low incidence of T cell lymphoma development. We investigated whether this tumor development is restricted by surveillance mechanisms that are exerted by IL-15-dependent cells. Lymphoma incidence was increased to between 30 and 60% when TCR transgenes were expressed in IL-15-deficient mice. Mice in which NK cells had been depleted genetically or with neutralizing antibodies allowed lymphoma growth while the absence of CD8 T cells was without consequence. Half of the emerged T cell lymphomas carried Notch1 mutations. The distinct phenotype of the lymphomas involved expression of PD1, CD30, CD24, the stress receptor ligand Mult1 and MHC class I down-regulation. NK cells were able to directly lyse lymphoma cells, and neutralizations of Mult1 and class I expression prevented NK cell degranulation. Together these data support an involvement of NK cells in tumor surveillance of nascent T cell lymphomas.

IL15 Infusion of Cancer Patients Expands the Subpopulation of Cytotoxic CD56bright NK Cells and Increases NK-Cell Cytokine Release Capabilities.

The cytokine IL15 is required for survival and activation of natural killer (NK) cells as well as expansion of NK-cell populations. Here, we compare the effects of continuous IL15 infusions on NK-cell subpopulations in cancer patients. Infusions affected the CD56bright NK-cell subpopulation in that the expansion rates exceeded those of CD56dim NK-cell populations with a 350-fold increase in their total cell numbers compared with 20-fold expansion for the CD56dim subset. CD56bright NK cells responded with increased cytokine release to various stimuli, as expected given their immunoregulatory functions. Moreover, CD56bright NK cells gained the ability to kill various target cells at levels that are typical for CD56dim NK cells. Some increased cytotoxic activities were also observed for CD56dim NK cells. IL15 infusions induced expression changes on the surface of both NK-cell subsets, resulting in a previously undescribed and similar phenotype. These data suggest that IL15 infusions expand and arm CD56bright NK cells that alone or in combination with tumor-targeting antibodies may be useful in the treatment of cancer. Cancer Immunol Res; 5(10); 929-38. ©2017 AACR.

Loss of cytotoxicity and gain of cytokine production in murine tumor-activated NK cells.

NK cells are able to form a functional memory suggesting that some NK cells are surviving the activation process. We hypothesized that NK cell activation causes the development of a distinct NK cell subset and studied the fate of murine post-activation NK cells. Activation was achieved by in vivo and in vitro exposures to the melanoma tumor cell line B16 that was followed by differentiation in IL-2. When compared with control NK cells, post-activation CD25(+) NK cells expressed little granzyme B or perforin and had low lysis activity. Post-activation NK cells expressed CD27, CD90, CD127, and were low for CD11b suggesting that tumor-induced activation is restricted to an early NK cell subset. Activation of NK cells led to decreases of CD16, CD11c and increases of CD62L and the IL-18 receptor. In vivo activated but not control NK cells expressed a variety of cytokines that included IFNγ, TNFα, GM-CSF and IL-10. These data suggest that the exposure of a subset of peripheral NK cells to the B16 tumor environment caused an exhaustion of their cytolytic capacity but also a gain in their ability to produce cytokines.

Paracrine and transpresentation functions of IL-15 are mediated by diverse splice versions of IL-15Rα in human monocytes and dendritic cells.

BACKGROUND: IL-15 can either be transpresented by IL-15Rα or be secreted. RESULTS: New N- and C-terminal splice versions of human IL-15Rα determine whether IL-15 is secreted or stays bound to the cell membrane. CONCLUSION: IL-15Rα isoforms determine the mode of action of IL-15. SIGNIFICANCE: IL-15Rα isoforms may modify immune response outcomes in humans. Species-specific differences of post-translational modifications suggested the existence of human IL-15Rα isoforms. We identified eight new isoforms that are predicted to modify the intracellular C termini of IL-15Rα, and another N-terminal exon "Ex2A" that was consistently present in all but one of the C-terminal isoforms. Ex2A encodes a 49-amino acid domain that allowed the transfer of IL-15/IL-15Rα complex to the cell surface but prevented its cleavage from cell membranes and its secretion thus facilitating the transpresentation of IL-15 as part of the immunological synapse. The Ex2A domain also affected the O-glycosylation of IL-15Rα that explained the species-specific differences. The Ex2A domain appeared to be removed from major IL-15Rα species during protein maturation, but both Ex2A and IL-15Rα appeared on the surface of monocytic cells upon activation. The membrane-associated form of the only C-terminal isoform that lacked Ex2A (IC3) was retained inside the cell, but soluble IL-15/IL-15Rα complexes were readily released from cells that expressed IL-15/IL-15Rα-IC3 thus limiting this IL-15/IL-15Rα isoform to act as a secreted molecule. These data suggest that splice versions of IL-15Rα determine the range of IL-15 activities.

Selective dependence of H2-M3-restricted CD8 responses on IL-15.

We studied whether CD8 T cell responses that are mediated by unconventional MHC class Ib molecules are IL-15 dependent in mice. CD8(+) T cell responses to Listeria monocytogenes infection that are restricted by the MHC class Ib molecule H2-M3 decreased in the absence of IL-15, whereas other primary MHC class Ib- and MHC class Ia-restricted responses were IL-15 independent. This result was confirmed in MHC class Ia-deficient mice in which IL-15 deficiency also reduced H2-M3-restricted but not all CD8 T cell responses to L. monocytogenes. IL-15 deficiency did not affect proliferation or survival of responding H2-M3-restricted CD8(+) T cells, but IL-15 was necessary to detect H2-M3-restricted CD8(+) T cells in naive mice. This finding suggests that these CD8(+) T cells require IL-15 during development, but become IL-15 independent after activation. IL-15 was necessary for the survival of most class Ib-restricted CD8(+) T cells, starting at the mature thymocyte stage in naive mice, but does not affect a distinct CD44(low)/CD122(low) subpopulation. These data suggest that the nature of the selecting MHC class Ib molecule determines whether CD8(+) T cells acquire IL-15 dependence during thymic development.

Interleukin-15 combined with an anti-CD40 antibody provides enhanced therapeutic efficacy for murine models of colon cancer.

IL-15 has potential as an immunotherapeutic agent for cancer treatment because it is a critical factor for the proliferation and activation of natural killer (NK) and CD8(+) T cells. Administration of anti-CD40 antibodies has shown anti-tumor effects in vivo through a variety of mechanisms. Furthermore, activation of CD40 led to increased expression of IL-15 receptor-alpha by dendritic cells, an action that is critical for trans-presentation of IL-15 to NK and CD8(+) T cells. In this study, we investigated the therapeutic efficacy of the combination regimen of murine IL-15 (mIL-15) with an agonistic anti-CD40 antibody (FGK4.5) in murine lung metastasis models involving CT26 and MC38, which are murine colon cancer cell lines syngeneic to BALB/c and C57BL/6 mice, respectively. Treatment with mIL-15 or the anti-CD40 antibody alone significantly prolonged survival of both CT26 and MC38 tumor-bearing mice compared with the mice in the PBS solution control group (P < 0.01). Furthermore, combination therapy with both mIL-15 and the anti-CD40 antibody provided greater therapeutic efficacy as demonstrated by prolonged survival of the mice compared with either mIL-15 or the anti-CD40 antibody-alone groups (P < 0.001). We found that NK cells isolated from the mice that received the combination regimen expressed increased levels of intracellular granzyme B and showed stronger cytotoxic activity on the target cells. The findings from this study provide the scientific basis for clinical trials using the combination regimen of IL-15 with an anti-CD40 antibody for the treatment of patients with cancer.

Preassociation of IL-15 with IL-15R alpha-IgG1-Fc enhances its activity on proliferation of NK and CD8+/CD44high T cells and its antitumor action.

In the induction of an immune response, IL-15Ralpha on APCs transpresents IL-15 to NK and CD8(+)/CD44(high) T cells that express the IL-2/15Rbeta and gammac subunits only. In this study, we show data mimicking this transpresentation by using IL-15 preassociated with a chimeric protein that is comprised of the extracellular domain of murine IL-15Ralpha and the Fc portion of human IgG1. When tested in vitro, IL-15Ralpha-IgG1-Fc strongly increased the IL-15-mediated proliferation of murine NK and CD8(+)/CD44(high) T cells. The effect of IL-15Ralpha-IgG1-Fc was dependent on the presence of both IgG1-Fc and IL-15Ralpha. When injected into mice, IL-15Ralpha-IgG1-Fc enhanced the capacity of IL-15 to expand the number of NK and CD8(+)/CD44(high) T cells. The effect on cell numbers in vivo also depended on Fc receptor binding because reduced expansion was observed in FcRgamma(-/-) mice. NK cells cultured in IL-15/IL-15Ralpha-IgG1-Fc complex gained cytotoxic activity toward a number of NK-sensitive targets. When mice bearing the NK-sensitive syngeneic tumor B16 were treated, the presence of IL-15Ralpha-IgG1-Fc increased the antitumor activity of IL-15. Thus, a preassociation with IL-15Ralpha-IgG1-Fc enhances the activities of IL-15 in vivo and in vitro that may be useful in the treatment of tumors.

ITK and IL-15 support two distinct subsets of CD8+ T cells.

CD8(+) T cells are commonly divided into naïve CD44(lo)CD122(lo) and "memory phenotype" CD44(hi)CD122(hi) cells. Here we show data suggesting that these two cell populations represent independent CD8(+) T cell subsets. Whereas IL-15(-/-) mice lack CD44(hi)CD122(hi) CD8(+) T cells, mice deficient in the kinase ITK lack CD44(lo)CD122(lo) cells among CD8(+) T cells. The same defects were observed during thymus development. CD44(hi)CD122(hi) cells were found among double-positive thymocytes and increased in frequency during CD8 development in wild-type mice. At the mature stage, IL-15(-/-) mice harbored virtually no CD44(hi)CD122(hi) CD8(+) thymocytes. In contrast, ITK(-/-) mice lacked CD44(lo)CD122(lo) CD8(+) cells at this stage. We generated mice with genetic deletions in both IL-15 and ITK and observed a severe reduction of all CD8(+) T cells. The two CD44(lo)CD122(lo) and CD44(hi)CD122(hi) CD8(+) T cell subsets differed in the periphery in that natural killer (NK) receptor expression was found only on CD44(hi)CD122(hi) CD8(+) T cells. This expression was paralleled by their ability to respond to both T cell receptor and NK receptor engagements. In contrast, CD44(lo)CD122(lo) CD8(+) T cells mounted stronger responses to T cell receptor stimulation but failed to recognize NK receptor ligands. Thus, whereas ITK-dependent CD44(lo)CD122(lo) CD8(+) T cells appear to represent conventional CD8(+) T cells, IL-15-dependent CD44(hi)CD122(hi) CD8(+) T cells may have functions in both adaptive and innate immunity.

Survival adjustment of mature dendritic cells by IL-15.

The survival of CD8+/CD44(hi) memory phenotype T cells depends on an IL-15 activity on nonlymphoid cells. Here, we report that IL-15 and its receptor were induced on dendritic cells (DCs) by a combination of IFN-gamma and NF-kappaB relA inducers. IL-15 conferred in an autocrine loop resistance to apoptosis that accompanied the maturation process in DCs in vitro. As an apparent result in vivo, mice deficient in IL-15 or its receptor harbor few DCs. Injecting DCs into IL-15-/- mice was associated with the appearance of CD8+/CD44(hi) T cells that depended on IL-15 expression but also correlated with the longevity of the DCs. These findings support the hypothesis that DCs mediate the effect of IL-15 on CD8+/CD44(hi) memory phenotype T cells.

Distinct pathways involving the FK506-binding proteins 12 and 12.6 underlie IL-2-versus IL-15-mediated proliferation of T cells.

The molecular basis for the different roles of IL-2 and IL-15 in lymphocyte function has been poorly defined. Searching for differences that underlie the distinct T cell responses to the two cytokines, we observed a marked susceptibility of the IL-15-induced but not of the IL-2-induced proliferation to rapamycin despite a decrease of p70S6 kinase (p70S6K) activation by the drug in response to both cytokines. Activated splenic T lymphocytes deficient in the FK506-binding protein (FKBP) 12, a target of rapamycin activity, had reduced proliferation in response to IL-15 but not to IL-2. This decreased proliferation was accompanied by reduced activation of p70S6K and of the extracellular signal-regulated kinases (ERK) after IL-15 treatment. In contrast to FKBP12-/- cells, splenic FKBP12.6-/- T cells exhibited a decreased proliferative response to IL-2 in the presence of rapamycin without affecting p70S6K or ERK activation. Thus, IL-15 induces T cell proliferation mainly via FKBP12-mediated p70S6K activation. In contrast, IL-2 signaling involves multiple pathways that include at least one additional pathway that depends on FKBP12.6.

Lymphotoxin beta receptor induces sequential activation of distinct NF-kappa B factors via separate signaling pathways.

Lymphotoxin beta receptor (LTbetaR)-induced activation of NF-kappaB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IkappaB kinase (IKK)beta- and IKKgamma-dependent degradation of IkappaBalpha and resulted in the rapid but transient activation of primarily RelA-containing NF-kappaB dimers. The alternative or second pathway proceeded via NF-kappaB-inducing kinase (NIK)-, IKKalpha-, and protein synthesis-dependent processing of the inhibitory NF-kappaB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-kappaB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKgamma regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-kappaB-induced resynthesis of IkappaBalpha, resulted in a pronounced temporal change in the nature of the NF-kappaB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-kappaB activity in mouse embryo fibroblasts in response to LTbetaR stimulation. Based on the phenotype of mice deficient in various components of the LTbetaR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures.