Production of bioactive soluble interleukin-15 in complex with interleukin-15 receptor alpha from a conditionally-replicating oncolytic HSV-1.
ABSTRACT: Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the ?134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8(+) T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor ? (mIL-15R?, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15R? improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15R? was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15R? complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15R? complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units. The production of mIL-15/mIL-15R? from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15R? in various cancer models.
Project description:Limited expression and distribution of nectin-1, the major herpes simplex virus (HSV) type-1 entry-receptor, within tumors has been proposed as an impediment to oncolytic HSV (oHSV) therapy. To determine whether resistance to oHSVs in malignant peripheral nerve sheath tumors (MPNSTs) was explained by this hypothesis, nectin-1 expression and oHSV viral yields were assessed in a panel of MPNST cell lines using ?134.5-attenuated (??134.5) oHSVs and a ?134.5 wild-type (wt) virus for comparison. Although there was a correlation between nectin-1 levels and viral yields with the wt virus (R=0.75, P =0.03), there was no correlation for ??134.5 viruses (G207, R7020 or C101) and a modest trend for the second-generation oHSV C134 (R=0.62, P=0.10). Nectin-1 overexpression in resistant MPNST cell lines did not improve ??134.5 oHSV output. While multistep replication assays showed that nectin-1 overexpression improved ??134.5 oHSV cell-to-cell spread, it did not confer a sensitive phenotype to resistant cells. Finally, oHSV yields were not improved with increased nectin-1 in vivo. We conclude that nectin-1 expression is not the primary obstacle of productive infection for ??134.5 oHSVs in MPNST cell lines. In contrast, viruses that are competent in their ability to counter the antiviral response may derive benefit with higher nectin-1 expression.
Project description:Oncolytic herpes simplex virus (oHSV)-1-based vectors selectively replicate in tumor cells causing direct killing, that is, oncolysis, while sparing normal cells. The oHSVs are promising anticancer agents, but their efficacy, when used as single agents, leaves room for improvement. We hypothesized that combining the direct oncolytic and antiangiogenic activities of the interleukin (IL)-12-secreting NV1042 oHSV with microtubule disrupting agents (MDAs) would be an effective means to enhance antitumor efficacy. Vinblastine (VB) was identified among several MDAs screened, which displayed consistent and potent cytotoxic killing of both prostate cancer and endothelial cell lines. In matrigel tube-forming assays, VB was found to be highly effective at inhibiting tube formation of human umbilical vein endothelial cells. The combination of VB with NV1023 (the parental virus lacking IL-12) or NV1042 showed additive or synergistic activity against prostate cancer cell lines, and was not due to increased oHSV replication by VB. In athymic mice bearing CWR22 prostate tumors, VB in combination with NV1042 was superior to the combination of VB plus NV1023 in reducing tumor burden, appeared to be nontoxic and resulted in a statistically significant diminution in the number of CD31(+) cells as compared with other treatment groups. In human organotypic cultures using surgical samples from radical prostatectomies, both NV1023 and NV1042 were localized specifically to the epithelial cells of prostatic glands but not to the surrounding stroma. These data highlight the therapeutic advantage of combining the dual-acting antitumor and antiangiogenic activities of oHSVs and MDAs.
Project description:Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor ? (sIL-15R?). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15R? heterodimer in the medium. Purification of the IL-15 and sIL-15R? polypeptides allowed identification of the proteolytic cleavage site of IL-15R? and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15R? heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.
Project description:Interleukin (IL)-15 is an essential immune-modulator with high potential for use in cancer treatment. Natural IL-15 has a low biological potency because of its short half-life and difficulties in mass-production. IL-15R?, a member of the IL-15 receptor complex, is famous for its high affinity to IL-15 and its ability to lengthen the half-life of IL-15. We have double-transfected IL-15 and its truncated receptor IL-15R? into CT26 colon cancer cells to target them for intracellular assembly. The secreted IL-15:IL-15R? complexes were confirmed in ELISA and Co-IP experiments. IL-15:IL15R? secreting clones showed a higher anti-tumor effect than IL-15 secreting clones. Furthermore, we also evaluated the vaccine and therapeutic efficacy of the whole cancercell vaccine using mitomycin C (MMC)-treated IL-15:IL15R? secreting CT26 clones. Three sets of experiments were evaluated; (1) therapeutics, (2) vaccination, and (3) longterm protection. Wild-type CT26-bearing mice treated with a single dose of MMC-inactivated secreted IL-15:IL-15R? clones prolonged survival compared to the control group. Survival of MMC-inactivated IL-15:IL-15R? clone-vaccinated mice (without any further adjuvant) exceeded up to 100%. This protection effect even lasted for at least three months after the immunization. Secreted IL-15:IL-15R? clones challenging trigger anti-tumor response via CD4+ T, CD8+ T, and natural killer (NK) cell-dependent cytotoxicity. Our result suggested that cell-based vaccine secreting IL-15:IL-15R?, may offer the new tools for immunotherapy to treat cancer.
Project description:Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15R?). This heterodimeric IL-15:sIL-15R? complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15R? and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15R? Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly ?1-6-core-fucosylated and ?-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15R? was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed ?2-3- and ?2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and ?-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15R? of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.
Project description:Interleukin-15 (IL-15) and its high affinity receptor interleukin-15 receptor alpha (IL-15R?) are widely expressed in immune cells and hepatic resident cells. IL-15 signaling has important functions in homeostasis of natural killer (NK), natural killer T (NKT) and cytotoxic T (CD8(+) T) cells, and in liver regeneration. We hypothesized that IL-15 has a protective role in liver fibrosis progression by maintaining NK cell homeostasis.Fibrosis was induced using two mechanistically distinct models. Congenic bone marrow transplantation was used to evaluate the contribution of IL-15 signaling from various compartments to NK, CD8(+) T and NKT cell homeostasis and fibrogenesis. The gene expression profile of hepatic stellate cell (HSC) from IL-15R? knockout (IL-15R?KO) mice and wild-type mice were captured using microarray analysis and validated in isolated HSC. Quantitative real-time PCR was used to assess repressors of collagen transcription.IL-15R?KO mice exhibited more fibrosis in both models. IL-15 signaling from specific types of hepatic cells had divergent roles in maintaining liver NK, CD8(+) T and NKT cells, with a direct and protective role on radio-resistant non-parenchymal cells beyond the control of NK homeostasis. HSCs isolated from IL-15R?KO mice demonstrated upregulation of collagen production. Finally, IL-15R?KO HSC with or without transforming growth factor beta (TGF-?) stimulation exhibited increased expression of fibrosis markers and decreased collagen transcription repressors expression.IL-15R? signaling has a direct anti-fibrotic effect independent of preserving NK homeostasis. These findings establish a rationale to further explore the anti-fibrotic potential of enhancing IL-15 signaling in HSCs.We investigated how a cellular protein, Interleukin-15 (IL-15), decreases the amount of scar tissue that is formed upon liver injury. We found that IL-15 and its receptor decrease the amount of scar tissue that is created by specialized liver cells (called stellate cells) and increase the number of a specific subgroup of immune cells (natural killer cells) that are known to eliminate stellate cells.GSE45612, GSE 68001 and GSE 25097.
Project description:IL-15 can either be transpresented by IL-15R? or be secreted.New N- and C-terminal splice versions of human IL-15R? determine whether IL-15 is secreted or stays bound to the cell membrane.IL-15R? isoforms determine the mode of action of IL-15.IL-15R? isoforms may modify immune response outcomes in humans. Species-specific differences of post-translational modifications suggested the existence of human IL-15R? isoforms. We identified eight new isoforms that are predicted to modify the intracellular C termini of IL-15R?, 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-15R? 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-15R? that explained the species-specific differences. The Ex2A domain appeared to be removed from major IL-15R? species during protein maturation, but both Ex2A and IL-15R? 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-15R? complexes were readily released from cells that expressed IL-15/IL-15R?-IC3 thus limiting this IL-15/IL-15R? isoform to act as a secreted molecule. These data suggest that splice versions of IL-15R? determine the range of IL-15 activities.
Project description:Interleukin (IL)-15 and its specific receptor chain, IL-15R?, support the development of various effector cells, including NK and CD8 T cells via a mechanism called trans-presentation. Whereas the dynamic of trans-presentation has been shown to involve the recycling of IL-15R? by presenting cells, the way responding cells integrate, or take advantage of this process has not been evaluated yet. To address this question, we set up a trans-presentation model using a membrane-bound IL-15.IL-15R? fusion protein, and found that IL-15 is detectable within responding cells following IL-15 trans-presentation. The role of the proteolytic cleavage of IL-15R? in this process was investigated by generating an uncleavable form of IL-15R?. We showed that IL-15 entry into responding cells necessitates the cleavage of IL-15.IL-15R? complex from the surface of IL-15 presenting cells, and observed that IL-15R? cleavage is associated with a decrease of the duration of Stat5 signaling. Once separated from presenting cells, responding cells are able to recycle IL-15.IL-15R? complexes via intracellular compartments, for residual proliferation in a time-limited manner. These studies define an unprecedented cytokine pathway in which the IL-15.IL-15R? complex cleaved from presenting cells allows responding cells to internalize, store and use IL-15.IL-15R? complex for their own proliferation and survival.
Project description:Naturally ligand independent constitutively active gp130 variants were described to be responsible for inflammatory hepatocellular adenomas. Recently, we genetically engineered a ligand-independent constitutively active gp130 variant based on homodimerization of Jun leucine zippers. Because also heterodimeric complexes within the gp130 family may have tumorigenic potential, we seek to generate ligand-independent constitutively active heterodimers for all known gp130-receptor complexes based on IL-15/IL-15R alpha-sushi fusion proteins. Ligand-independent heterodimerization of gp130 with WSX-1, LIFR, and OSMR and of OSMR with GPL led to constitutive, ligand-independent STAT1 and/or STAT3 and ERK1/2 phosphorylation. Moreover, these receptor combinations induced transcription of the STAT3 target genes c-myc and Pim-1 and factor-independent growth of stably transduced Ba/F3-gp130 cells. Here, we establish the IL-15/IL-15R alpha-sushi system as a new system to mimic constitutive and ligand-independent activation of homo- and heterodimeric receptor complexes, which might be applicable to other heterodimeric receptor families. A mutated IL-15 protein, which was still able to bind the IL-15R alpha-sushi domain, but not to beta- and gamma-receptor chains, in combination with the 2A peptide technology may be used to translate our in vitro data into the in vivo situation to assess the tumorigenic potential of gp130-heterodimeric receptor complexes.