Oncolytic adenovirus promotes vascular normalization and nonclassical tertiary lymphoid structure formation through STING-mediated DC activation.

Inducing a full antitumor immune response in the tumor microenvironment (TME) is essential for successful cancer immunotherapy. Here, we report that an oncolytic adenovirus carrying mIL-15 (Ad-IL15) can effectively induce antitumor immune response and inhibit tumor growth in a mouse model of cancer. We found that Ad-IL15 facilitated the activation and infiltration of immune cells, including dendritic cells (DCs), T cells and natural killer (NK) cells, in the TME. Unexpectedly, we observed that Ad-IL15 also induced vascular normalization and tertiary lymphoid structure formation in the TME. Moreover, we demonstrated these Ad-IL15-induced changes in the TME were depended on the Ad-IL15-induced activation of the STING-TBK1-IRF3 pathway in DCs. Taken together, our findings suggest that Ad-IL15 is a candidate for cancer immunotherapy that promotes immune cell activation and infiltration, tumor vascular normalization and tertiary lymphoid structure formation in the TME.

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.

Combination Cancer Immunotherapy with Dendritic Cell Vaccine and Nanoparticles Loaded with Interleukin-15 and Anti-beta-catenin siRNA Significantly Inhibits Cancer Growth and Induces Anti-Tumor Immune Response.

PURPOSE: The invention and application of new immunotherapeutic methods can compensate for the inefficiency of conventional cancer treatment approaches, partly due to the inhibitory microenvironment of the tumor. In this study, we tried to inhibit the growth of cancer cells and induce anti-tumor immune responses by silencing the expression of the ß-catenin in the tumor microenvironment and transmitting interleukin (IL)-15 cytokine to provide optimal conditions for the dendritic cell (DC) vaccine. METHODS: For this purpose, we used folic acid (FA)-conjugated SPION-carboxymethyl dextran (CMD) chitosan (C) nanoparticles (NPs) to deliver anti-ß-catenin siRNA and IL-15 to cancer cells. RESULTS: The results showed that the codelivery of ß-catenin siRNA and IL-15 significantly reduced the growth of cancer cells and increased the immune response. The treatment also considerably stimulated the performance of the DC vaccine in triggering anti-tumor immunity, which inhibited tumor development and increased survival in mice in two different cancer models. CONCLUSIONS: These findings suggest that the use of new nanocarriers such as SPION-C-CMD-FA could be an effective way to use as a novel combination therapy consisting of ß-catenin siRNA, IL-15, and DC vaccine to treat cancer.

Systemic IL-15 promotes allogeneic cell rejection in patients treated with natural killer cell adoptive therapy.

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793.

Phase I study of single agent NIZ985, a recombinant heterodimeric IL-15 agonist, in adult patients with metastatic or unresectable solid tumors.

BACKGROUND: NIZ985 is a recombinant heterodimer of physiologically active interleukin (IL-)15 and IL-15 receptor alpha. In preclinical models, NIZ985 promotes cytotoxic lymphocyte proliferation, killing function, and organ/tumor infiltration, with resultant anticancer effects. In this first-in-human study, we assessed the safety, pharmacokinetics, and immune effects of NIZ985 in patients with metastatic or unresectable solid tumors. METHODS: Single agent NIZ985 dose escalation data are reported from a phase I dose escalation/expansion study of NIZ985 as monotherapy. Adult patients (N=14) received 0.25, 0.5, 1, 2 or 4 µg/kg subcutaneous NIZ985 three times weekly (TIW) for the first 2 weeks of each 28-day cycle, in an accelerated 3+3 dose escalation trial design. IL-15 and endogenous cytokines were monitored by ELISA and multiplexed electrochemiluminescent assays. Multiparameter flow cytometry assessed the frequency, phenotype and proliferation of peripheral blood mononuclear cells. Preliminary antitumor activity was assessed by overall response rate (Response Evaluation Criteria in Solid Tumors V.1.1). RESULTS: As of March 2, 2020, median treatment duration was 7.5 weeks (range 1.1-77.1). Thirteen patients had discontinued and one (uveal melanoma) remains on treatment with stable disease. Best clinical response was stable disease (3 of 14 patients; 21%). The most frequent adverse events (AEs) were circular erythematous injection site reactions (100%), chills (71%), fatigue (57%), and fever (50%). Treatment-related grade 3/4 AEs occurred in six participants (43%); treatment-related serious AEs (SAEs) in three (21%). The per-protocol maximum tolerated dose was not reached. Pharmacokinetic accumulation of serum IL-15 in the first week was followed by significantly lower levels in week 2, likely due to more rapid cytokine consumption by an expanding lymphocyte pool. NIZ985 treatment was associated with increases in several cytokines, including interferon (IFN)-γ, IL-18, C-X-C motif chemokine ligand 10, and tumor necrosis factor-ß, plus significant induction of cytotoxic lymphocyte proliferation (including natural killer and CD8+ T cells), increased CD16+ monocytes, and increased CD163+ macrophages at injection sites. CONCLUSIONS: Subcutaneous NIZ985 TIW was generally well tolerated in patients with advanced cancer and produced immune activation paralleling preclinical observations, with induction of IFN-γ and proliferation of cytotoxic lymphocytes. Due to delayed SAEs at the two highest dose levels, administration is being changed to once-weekly in a revised protocol, as monotherapy and combined with checkpoint inhibitor spartalizumab. These alterations are expected to maximize the potential of NIZ985 as a novel immunotherapy. TRIAL REGISTRATION NUMBER: NCT02452268.

Comparative Immunogenomics of Canine Natural Killer Cells as Immunotherapy Target.

Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells have impeded translation. Outbred dogs offer an important link for studies of NK biology and immunotherapy. We analyzed gene expression of putative NK populations from healthy dogs and dogs with naturally-occurring cancers examining differential gene expression across multiple conditions, including steady-state, in vitro activation with cytokines and co-culture, and in vivo activation with inhaled IL-15 in dogs receiving IL-15 immunotherapy. We also compared dog, mouse and human CD3-NKp46+ NK cells using a novel orthologous transcriptome. Distinct transcriptional profiles between NK populations exist between conditions and in vitro versus in vivo treatments. In cross-species analysis, canine NK cells were globally more similar to human NK cells than mice. These data define canine NK cell gene expression under multiple conditions and across species, filling an important gap in translational NK studies.

Combination therapy with interleukin-15 and metformin as a synergistic treatment for pancreatic cancer.

OBJECTIVE: To investigate the efficacies and mechanisms of combination therapy with interleukin-15 (IL-15) and metformin (Met) on suppressing pancreatic cell proliferation and protecting Panc02-bearing mice. MATERIALS AND METHODS: MTT assays were applied to assess the inhibitory effects of IL-15 combined Met or Met and IL-15 alone on proliferation of normal HPDE6-C7 and Panc02 cells. After tumor grew up to 150 mm3, the Panc02-bearing xenograft model mice were randomly divided into saline group, IL-15 and Met alone group and combined treatment group (n=8) with the administration of each agent every other day for three weeks, and survival rates were recorded. The changes in tumor size, expression levels of the apoptosis, autophagy as well as Akt/mTOR/STAT3-related factors in tumor tissues were all measured. RESULTS: MTT assays demonstrated significantly inhibiting efficacy of combination therapy with IL-15 and Met on Panc02 cell proliferation compared to other groups (all p<0.05) with combination index<1 showing evident synergistic effect. Moreover, the apoptosis rate of Panc02 cell under combined treatment were 95.5±3.2% and significantly higher than those of others (all p<0.01). In addition, combined administration remarkably inhibited the growth of pancreatic carcinoma, and improved survival rate of Panc02-bearing model with less body weight loss. Furthermore, combined treatment significantly downregulated anti-apoptotic proteins as well as Akt/mTOR/STAT3 signaling pathway and upregulated autophagy related factors, respectively, compared with those of monotherapy groups in both Panc02 cells and tumor tissues. CONCLUSIONS: Combined treatment of IL-15 with Met showed synergistic anti-tumor efficacies on Panc02 cells attributing to promotion on apoptosis, autophagy and inhibition on Akt/mTOR/STAT3 signaling-transduction in Panc02-bearing model mice.

IL-15-induced lymphocytes as adjuvant cellular immunotherapy for gastric cancer.

Objectives To test the antitumor potential of lymphocytes transferred via adoptive cell therapy (ACT) in a mouse model of human gastric cancer (GC), and to evaluate the clinical efficacy and safety of combining lymphocytes as adjuvant therapy with first-line chemotherapy in patients with GC. Methods We constructed a human GC xenograft model in sublethally irradiated 6-8-week-old male NCG mice. MKN-45 cells (1 × 106 cells/mouse) were subcutaneously injected into mice's flanks. After tumors had become palpable, we randomized the mice into control, ACTIL-2, and ACTIL-15 groups. Human lymphocytes were then injected into mouse tail veins. In addition, 63 human patients with histologically or cytologically confirmed stage III-IV GC randomly received S-1 + oxaliplatin + ACTIL-15 (combination therapy group) or S-1 + oxaliplatin (chemotherapy group). Results In the mouse study, treatment with ACTIL-15 cells inhibited tumor growth on adoptive transfer, and mice that received ACTIL-15 cells had significantly longer survival rates (p < 0.05, ACTIL-15 vs. ACTIL-2). In the human study, the median survival rate of patients in the combination therapy group was 472 days (95% confidence interval [CI], 276-668 days), whereas that of patients in the chemotherapy group was 266 days (95% CI, 200-332 days; p < 0.05). Eleven percent (7/63) of patients had adverse reactions, but these reactions did not interfere with treatment. Conclusion Adoptive transfer of ACTIL-15 cells in a mouse model of GC and in patients with advanced GC treated with S1 + oxaliplatin improved survival rates in both, with an acceptable safety profile.

Co-Silencing of Tissue Transglutaminase-2 and Interleukin-15 Genes in a Celiac Disease Mimetic Mouse Model Using a Nanoparticle-in-Microsphere Oral System.

Celiac disease is a chronic inflammatory condition characterized by activation of the immune system in response to deamidation of gluten peptides brought about by tissue transglutaminase-2 (TG2). Overexpression of interleukin-15 (IL-15) in the intestinal epithelium and the lamina propria leads to the dysregulation of the immune system, leading to epithelial damage. The goal of this study was to develop an RNA interference therapeutic strategy for celiac disease using a combination of TG2 and IL-15 gene silencing in the inflamed intestine. TG2 and IL-15 silencing siRNA sequences, along with scrambled control, were encapsulated in a nanoparticle-in-microsphere oral system (NiMOS) and administered in a poly(I:C) mouse model of celiac disease. Single TG2 and IL-15 siRNA therapy and the combination showed effective gene silencing in vivo. Additionally, it was found that IL-15 gene silencing alone and combination in the NiMOS significantly reduced other proinflammatory cytokines. The tissue histopathology data also confirmed a reduction in immune cell infiltration and restoration of the mucosal architecture and barrier function in the intestine upon treatment. Overall, the results of this study show evidence that celiac disease can be potentially treated with an oral microsphere formulation using a combination of TG2 and IL-15 RNA interference therapeutic strategies.

Safety and immunological effects of recombinant canine IL-15 in dogs.

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays pivotal roles in innate and adaptive immunity. It is also a promising cytokine for treating cancer. Despite growing interest in its use as an immunotherapeutic, its safety and immunological effects in dogs have not been reported. In this study, healthy dogs were given recombinant canine IL-15 (rcIL-15) intravenously at a daily dose of 20 µg/kg for 8 days and monitored for 32 days to determine the safety and immunological effects of rcIL-15. The repeated administration of rcIL-15 was well tolerated, did not cause any serious side effects, and promoted the selective proliferation and activation of canine anti-cancer effector cells, including CD3+CD8+ cytotoxic T lymphocytes, CD3+CD5dimCD21-, and non-B/non-T NK cell populations, without stimulating Treg lymphocytes. The rcIL-15 injections also stimulated the expression of molecules and transcription factors associated with the activation and effector functions of NK cells, including CD16, NKG2D, NKp30, NKp44, NKp46, perforin, granzyme B, Ly49, T-bet, and Eomes. These results suggest that rcIL-15 might be a valuable therapeutic adjuvant to improve immunity against cancer in dogs.

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.

An unmet need: Harmonization of IL-7 and IL-15 combination for the ex vivo generation of minimally differentiated T cells.

T cell-based adoptive cell transfer therapy is now clinically used to fight cancer with CD19-targeting chimeric antigen receptor T cells. The use of other T cell-based immunotherapies relying on antigen-specific T cells, genetically modified or not, is expanding in various neoplastic diseases. T cell manufacturing has evolved through sophisticated processes to produce T cells with improved therapeutic potential. Clinical-grade manufacturing processes associated with these therapies must meet pharmaceutical requirements and therefore be standardized. Here, we focus on the use of cytokines to expand minimally differentiated T cells, as well as their standardization and harmonization in research and clinical settings.

A clinical trial of IL-15 and IL-21 combination therapy for COVID-19 is warranted.

Previous studies of SARS-CoV-2 viral infection suggest that both the humoral and cytotoxic arms of the immune system are weak in patients with severe COVID-19 disease when compared to mild disease. A cytokine storm is also induced in severe disease. IL-15 has been shown to support the cytotoxic arm of the immune response. IL-21 has been shown to support both the cytotoxic and humoral arms of the immune response. In addition, in some settings, Il-21 has been shown to actually decrease IL-6 and TNF-alpha production, reducing the inflammatory proteins involved in the cytokine storm. Furthermore, in other settings, the combination of IL-15 and IL-21 has been shown to be more effective than either interleukin alone in promoting an effective immune response. Therefore, a clinical trial that examines the use of the combination of IL-15 and IL-21 for COVID-19 patients is warranted.

IL-15 Preconditioning Augments CAR T Cell Responses to Checkpoint Blockade for Improved Treatment of Solid Tumors.

Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8+CD62L+TCF7+IRF4- population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7+ phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.

Potent Cytolytic Activity and Specific IL15 Delivery in a Second-Generation Trispecific Killer Engager.

Natural killer (NK) cells are potent immune modulators that can quickly lyse tumor cells and elicit inflammatory responses. These characteristics make them ideal candidates for immunotherapy. However, unlike T cells, NK cells do not possess clonotypic receptors capable of specific antigen recognition and cannot expand via activating receptor signals alone. To enable NK cells with these capabilities, we created and have previously described a tri-specific killer engager (TriKE) platform capable of inducing antigen specificity and cytokine-mediated NK-cell expansion. TriKE molecules have three arms: (i) a single-chain variable fragment (scFv) against the activating receptor CD16 on NK cells to trigger NK-cell activation, (ii) an scFv against a tumor-associated antigen (CD33 here) to induce specific tumor target recognition, and (iii) an IL15 moiety to trigger NK-cell expansion and priming. Here, we demonstrate that by modifying the anti-CD16 scFv with a humanized single-domain antibody against CD16, we improved TriKE functionality. A CD33-targeting second-generation TriKE induced stronger and more specific NK-cell proliferation without T-cell stimulation, enhanced in vitro NK-cell activation and killing of CD33-expressing targets, and improved tumor control in preclinical mouse models. Given these improved functional characteristics, we propose rapid translation of second-generation TriKEs into the clinic.

IL-15 in the Combination Immunotherapy of Cancer.

We completed clinical trials of rhIL-15 by bolus, subcutaneous, and continuous intravenous infusions (CIV). IL-15 administered by CIV at 2 mcg/kg/day yielded a 38-fold increase in 10- day number of circulating NK cells, a 358-fold increase in CD56bright NK cells and a 5.8-fold increase in CD8 T cells. However, IL-15 preparations administered as monotherapy were ineffective, due to actions of immunological checkpoints and due to the lack of tumor specific targeting by NK cells. To circumvent checkpoints, trials of IL-15 in combination with other anticancer agents were initiated. Tumor-bearing mice receiving IL-15 with antibodies to CTLA-4 and PD-L1 manifested marked prolongation of survival compared to mice receiving IL-15 with either agent alone. In translation, a phase I trial was initiated involving IL-15 (rhIL-15), nivolumab and ipilimumab in patients with malignancy (NCT03388632). In rhesus macaques CIV IL-15 at 20 µg/kg/day for 10 days led to an 80-fold increase in number of circulating effector memory CD8 T cells. However, administration of γc cytokines such as IL-15 led to paralysis/depression of CD4 T-cells that was mediated through transient expression of SOCS3 that inhibited the STAT5 signaling pathway. This lost CD4 helper role could be restored alternatively by CD40 agonists. In the TRAMP-C2 prostate tumor model the combination of IL-15 with agonistic anti-CD40 produced additive effects in terms of numbers of TRAMP-C2 tumor specific Spas/SCNC/9H tetramer positive CD8 T cells expressed and tumor responses. A clinical trial is being initiated for patients with cancer using an intralesional anti-CD40 in combination with CIV rhIL-15. To translate IL-15-mediated increases in NK cells, we investigated combination therapy of IL-15 with anticancer monoclonal antibodies including rituximab in mouse models of EL-4 lymphoma transfected with human CD20 and with alemtuzumab (CAMPATH-1H) in a xenograft model of adult T cell leukemia (ATL). IL-15 enhanced the ADCC and therapeutic efficacy of both antibodies. These results provided the scientific basis for trials of IL-15 combined with alemtuzumab (anti-CD52) for patients with ATL (NCT02689453), with obinutuzumab (anti-CD20) for patients with CLL (NCT03759184), and with avelumab (anti-PD-L1) in patients with T-cell lymphoma (NCT03905135) and renal cancer (NCT04150562). In the first trial, there was elimination of circulating ATL and CLL leukemic cells in select patients.

IL-15 superagonist RLI has potent immunostimulatory properties on NK cells: implications for antimetastatic treatment.

BACKGROUND: As the immune system is compromised in patients with cancer, therapeutic strategies to stimulate immunity appear promising, to avoid relapse and increase long-term overall survival. Interleukin-15 (IL-15) has similar properties to IL-2, but does not cause activation-induced cell death nor activation and proliferation of regulatory T cells (Treg), which makes it a serious candidate for anticancer immunotherapy. However, IL-15 has a short half-life and high doses are needed to achieve responses. Designed to enhance its activity, receptor-linker-IL-15 (RLI) (SO-C101) is a fusion molecule of human IL-15 covalently linked to the human IL-15Rα sushi+ domain currently assessed in a phase I/Ib clinical trial on patients with advanced/metastatic solid cancer. METHODS: We investigated the antimetastatic activity of RLI in a 4T1 mouse mammary carcinoma that spontaneously metastasizes and evaluated its immunomodulatory role in the metastatic lung microenvironment. We further characterized the proliferation, maturation and cytotoxic functions of natural killer (NK) cells in tumor-free mice treated with RLI. Finally, we explored the effect of RLI on human NK cells from healthy donors and patients with non-small cell lung cancer (NSCLC). RESULTS: RLI treatment displayed antimetastatic properties in the 4T1 mouse model. By characterizing the lung microenvironment, we observed that RLI restored the balance between NK cells and neutrophils (CD11b+ Ly6Ghigh Ly6Clow) that massively infiltrate lungs of 4T1-tumor bearing mice. In addition, the ratio between NK cells and Treg was strongly increased by RLI treatment. Further pharmacodynamic studies in tumor-free mice revealed superior proliferative and cytotoxic functions on NK cells after RLI treatment compared with IL-15 alone. Characterization of the maturation stage of NK cells demonstrated that RLI favored accumulation of CD11b+ CD27high KLRG1+ mature NK cells. Finally, RLI demonstrated potent immunostimulatory properties on human NK cells by inducing proliferation and activation of NK cells from healthy donors and enhancing cytotoxic responses to NKp30 crosslinking in NK cells from patients with NSCLC. CONCLUSIONS: Collectively, our work demonstrates superior activity of RLI compared with rhIL-15 in modulating and activating NK cells and provides additional evidences for a therapeutic strategy using RLI as antimetastatic molecule.

Combination rhIL-15 and Anti-PD-L1 (Avelumab) Enhances HIVGag-Specific CD8 T-Cell Function.

In chronic HIV infection, virus-specific cytotoxic CD8 T cells showed expression of checkpoint receptors and impaired function. Therefore, restoration of CD8 T-cell function is critical in cure strategies. Here, we show that in vitro blockade of programmed cell death ligand 1 (PD-L1) by an anti-PD-L1 antibody (avelumab) in combination with recombinant human interleukin-15 (rhIL-15) synergistically enhanced cytokine secretion by proliferating HIVGag-specific CD8 T cells. In addition, these CD8 T cells have a CXCR3+PD1-/low phenotype, suggesting a potential to traffic into peripheral tissues. In vitro, proliferating CD8 T cells express PD-L1 suggesting that anti-PD-L1 treatment also targets virus-specific CD8 T cells. Together, these data indicate that rhIL-15/avelumab combination therapy could be a useful strategy to enhance CD8 T-cell function in cure strategies.

Combined in vitro IL-12 and IL-15 stimulation promotes cellular immune response in dogs with visceral leishmaniasis.

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.

Essential role of TNF-α in gamma c cytokine aided crosstalk between dendritic cells and natural killer cells in experimental murine lymphoma.

Cooperative and cognitive interaction between the dendritic cells and natural killer cells was investigated for demonstrating the anti-tumor activity against an aggressive murine lymphoma, treated with doxorubicin. Crosstalk between the dendritic cells and the natural killer cells significantly reduced the proliferation of Dalton's lymphoma cells in a dose dependent manner. Treatment of Dalton's lymphoma cells with doxorubicin in vitro enhances the effects of crosstalk against the target cells. This crosstalk between the cells was regulated via stimulation with recombinant interleukin-15, and release of TNF-α which is critically important for the tumoricidal effects. Dendritic cells and the natural killer cells crosstalk activate both the cells and upregulate the expression of CD40, CD69 and CD86 on the dendritic cells. These findings provided new insight regarding these interactions and define a mechanism by which cellular immune response promotes tumoricidal activity against lymphoma in therapeutic setting.