A homodimeric IL-15 superagonist F4RLI with easy preparation, improved half-life, and potent antitumor activities.

Interleukin-15 (IL-15) is a promising candidate for cancer immunotherapy due to its potent immune-activating effects. There are several IL-15 molecules currently in clinical trials but facing shortages of poor half-life, circulation instability, or complicated production and quality control processes. The aim of this study is to design a novel IL-15 superagonist to set out the above difficulties, and we constructed F4RLI consisting of the GS-linker spaced IgG4 Fc fragment, soluble IL-15 Rα (sIL-15Rα), and IL-15(N72D). Using a single plasmid transient transfection in HEK293E cells, the matured F4RLI was secreted in the form of homodimer and got purified by an easy step of protein A affinity chromatography. The F4RLI product can significantly stimulate the proliferation of human CD3+CD8+ T cells and NK cells in vitro. Meanwhile, F4RLI greatly extended the half-life and prolonged the exposure of IL-15 in mice nearly by 28- and 200-fold, respectively, in comparison with that of the IL-15 monomer. In vivo, F4RLI vastly expanded mouse splenic CD8+ T lymphocytes, illustrating its potential in tumor immunotherapy. Further studies showed that the combination of F4RLI with the immune checkpoint blocker atezolizumab played a synergistic effect in treating MC38 mouse tumor by increasing the percentage of CD8+ T cells in tumor tissue. Moreover, the combination therapy of F4RLI with the angiogenesis inhibitor bevacizumab resulted in significant tumor growth suppression in a xenograft human HT-29 mouse model. Overall, our results demonstrate a homodimeric IL-15 superagonist F4RLI with advances in manufacturing processes and biopharmaceutical applications for cancer immunotherapy. KEY POINTS: • The homodimeric structure of F4RLI facilitates its easy production processes and quality control. • The fusion with Fc and sIL-15Rα extends the plasma half-life of IL-15 by about 28-fold. • F4RLI can play synergistic antitumor activity with the PD-1/PD-L1 checkpoint inhibitor or angiogenesis inhibitor.

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.

Development of IL-15/IL-15Rα sushi domain-IgG4 Fc complexes in Pichia pastoris with potent activities and prolonged half-lives.

BACKGROUND: Interleukin-15 (IL-15) is a critical cytokine for the development, proliferation, and function of natural killer (NK) cells, NKT cells, and CD8+ memory T cells and has become one of the most promising protein molecules for the treatment of cancer and viral diseases. However, there are several limitations in applying IL-15 in therapy, such as its low yield in vitro, limited potency, and short half-life in vivo. To date, there are several recombinant IL-15 agonists based on configurational modifications that are being pursued in the treatment of cancer, such as ALT-803, which are mainly produced from mammalian cells. RESULTS: In this study, we designed two different forms of the IL-15 complex, which were formed by the noncovalent assembly of IL-15 with dimeric or monomeric sushi domain of IL-15 receptor α (SuIL-15Rα)-IgG4 Fc fusion protein and designated IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc, respectively. The two IL-15 complexes were expressed in Pichia pastoris (P. pastoris), and their activities and half-lives were evaluated and compared. Pharmacokinetic analysis showed that IL-15/SuIL-15Rα-dFc had a half-life of 14.26 h while IL-15/SuIL-15Rα-mFc had a half-life of 9.16 h in mice, which were much longer than the 0.7-h half-life of commercial recombinant human IL-15 (rhIL-15). Treatment of mice with intravenous injection of the two IL-15 complexes resulted in significant increases in NK cells, NKT cells, and memory CD8+ T cells, which were not observed after rhIL-15 treatment. Treatment of human peripheral blood mononuclear cells (PBMCs) from healthy donors with the two IL-15 complexes yielded enhanced NK and CD8+ T cell activation and proliferation, which was comparable to the effect of rhIL-15. CONCLUSIONS: These findings indicate that the IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc produced in P. pastoris exhibit potent activities and prolonged half-lives and may serve as superagonists for immunotherapy in further research and applications.

Differential combination immunotherapy requirements for inflamed (warm) tumors versus T cell excluded (cool) tumors: engage, expand, enable, and evolve.

BACKGROUND: Different types of tumors have varying susceptibility to immunotherapy and hence require different treatment strategies; these cover a spectrum ranging from 'hot' tumors or those with high mutational burden and immune infiltrates that are more amenable to targeting to 'cold' tumors that are more difficult to treat due to the fewer targetable mutations and checkpoint markers. We hypothesized that an effective anti-tumor response requires multiple agents that would (1) engage the immune response and generate tumor-specific effector cells; (2) expand the number and breadth of the immune effector cells; (3) enable the anti-tumor activity of these immune cells in the tumor microenvironment; and (4) evolve the tumor response to widen immune effector repertoire. METHODS: A hexatherapy combination was designed and administered to MC38-CEA (warm) and 4T1 (cool) murine tumor models. The hexatherapy regimen was composed of adenovirus-based vaccine and IL-15 (interleukin-15) superagonist (N-803) to engage the immune response; anti-OX40 and anti-4-1BB to expand effector cells; anti-PD-L1 (anti-programmed death-ligand 1) to enable anti-tumor activity; and docetaxel to promote antigen spread. Primary and metastatic tumor growth inhibition were measured. The generation of anti-tumor immune effector cells was analyzed using flow cytometry, ELISpot (enzyme-linked immunospot), and RNA analysis. RESULTS: The MC38-CEA and 4T1 tumor models have differential sensitivities to the combination treatments. In the 'warm' MC38-CEA, combinations with two to five agents resulted in moderate therapeutic benefit while the hexatherapy regimen outperformed all these combinations. On the other hand, the hexatherapy regimen was required in order to decrease the primary and metastatic tumor burden in the 'cool' 4T1 model. In both models, the hexatherapy regimen promoted CD4+ and CD8+ T cell proliferation and activity. Furthermore, the hexatherapy regimen induced vaccine-specific T cells and stimulated antigen cascade. The hexatherapy regimen also limited the immunosuppressive T cell and myeloid derived suppressor cell populations, and also decreased the expression of exhaustion markers in T cells in the 4T1 model. CONCLUSION: The hexatherapy regimen is a strategic combination of immuno-oncology agents that can engage, expand, enable, and evolve the immune response and can provide therapeutic benefits in both MC38-CEA (warm) and 4T1 (cool) tumor models.

IL-15 superagonist N-803 improves IFNγ production and killing of leukemia and ovarian cancer cells by CD34+ progenitor-derived NK cells.

Allogeneic natural killer (NK) cell transfer is a potential immunotherapy to eliminate and control cancer. A promising source are CD34 + hematopoietic progenitor cells (HPCs), since large numbers of cytotoxic NK cells can be generated. Effective boosting of NK cell function can be achieved by interleukin (IL)-15. However, its in vivo half-life is short and potent trans-presentation by IL-15 receptor α (IL-15Rα) is absent. Therefore, ImmunityBio developed IL-15 superagonist N-803, which combines IL-15 with an activating mutation, an IL-15Rα sushi domain for trans-presentation, and IgG1-Fc for increased half-life. Here, we investigated whether and how N-803 improves HPC-NK cell functionality in leukemia and ovarian cancer (OC) models in vitro and in vivo in OC-bearing immunodeficient mice. We used flow cytometry-based assays, enzyme-linked immunosorbent assay, microscopy-based serial killing assays, and bioluminescence imaging, for in vitro and in vivo experiments. N-803 increased HPC-NK cell proliferation and interferon (IFN)γ production. On leukemia cells, co-culture with HPC-NK cells and N-803 increased ICAM-1 expression. Furthermore, N-803 improved HPC-NK cell-mediated (serial) leukemia killing. Treating OC spheroids with HPC-NK cells and N-803 increased IFNγ-induced CXCL10 secretion, and target killing after prolonged exposure. In immunodeficient mice bearing human OC, N-803 supported HPC-NK cell persistence in combination with total human immunoglobulins to prevent Fc-mediated HPC-NK cell depletion. Moreover, this combination treatment decreased tumor growth. In conclusion,  N-803 is a promising IL-15-based compound that boosts HPC-NK cell expansion and functionality in vitro and in vivo. Adding N-803 to HPC-NK cell therapy could improve cancer immunotherapy.

Interleukin 15 Pharmacokinetics and Consumption by a Dynamic Cytokine Sink.

Interleukin-15 (IL-15) is crucial for the proliferation and survival of NK and CD8+ T memory cells, and of significant interest in immuno-oncology. Immune cell expansion requires continuous IL-15 exposure above a threshold concentration for an extended period. However, the short t1/2 of IL-15 makes this impossible to achieve after a single injection without a high Cmax and toxicities. The most effective way to deliver IL-15 is continuous intra-venous infusion, but this administration mode is impractical. Efforts have been devoted to developing IL-15 agonists which after a single injection maintain the cytokine in a narrow therapeutic window for a long period. Enigmatically, although the half-life extension technologies used often extend the half-life of a protein to 1 or more weeks, the modified IL-15 agonists studied usually have systemic elimination half-lives of only a few hours and rarely much longer than 1 day. These short half-lives-common to all circulating IL-15 agonists thus far reported-can be explained by a dynamic increase in clearance of the agonists that accompanies target immune cell proliferation. What is needed is an IL-15 agonist that is as effective as continuous intravenous infusion, but with the convenience and acceptance of single injections at 1-week or longer intervals.

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.

Functional and mechanistic advantage of the use of a bifunctional anti-PD-L1/IL-15 superagonist.

BACKGROUND: Anti(α)-programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) monotherapy fails to provide durable clinical benefit for most patients with carcinoma. Recent studies suggested that strategies to reduce immunosuppressive cells, promote systemic T-cell responses and lymphocyte trafficking to the tumor microenvironment (TME) may improve efficacy. N-809 is a first-in-class bifunctional agent comprising the interleukin (IL)-15 superagonist N-803 fused to two αPD-L1 domains. Thus, N-809 can potentially stimulate effector immune cells through IL-15 and block immunosuppressive PD-L1. Here, we examined the antitumor efficacy and immunomodulatory effects of N-809 versus N-803+αPD-L1 combination. METHODS: The ability of N-809 to block PD-L1 and induce IL-15-dependent immune effects was examined in vitro and in vivo. Antitumor efficacy of N-809 or N-803+αPD-L1 was evaluated in two murine carcinoma models and an extensive analysis of immune correlates was performed in the tumor and tumor-draining lymph node (dLN). RESULTS: We demonstrate that N-809 blocks PD-L1 and induces IL-15-dependent immune effects. N-809 was well-tolerated and reduced 4T1 lung metastasis, decreased MC38 tumor burden and increased survival versus N-803+αPD-L1. Compared with N-803+αPD-L1, N-809 enhanced natural killer (NK) and CD8+ T-cell activation and function in the dLN and TME, relating to increased gene expression associated with interferon and cytokine signaling, lymphoid compartment, costimulation and cytotoxicity. The higher number of TME CD8+ T cells was attributed to enhanced infiltration, not in situ expansion. Increased TME NK and CD8+ T-cell numbers correlated with augmented chemokine ligands and receptors. Moreover, in contrast to N-803+αPD-L1, N-809 reduced immunosuppressive regulatory T cells (Treg), monocytic myeloid-derived suppressor cells (M-MDSC) and M2-like macrophages in the TME. CONCLUSIONS: Our results suggest that N-809 functions by a novel immune mechanism to promote antitumor efficacy. Foremost, N-809 enhances intratumoral lymphocyte numbers by increasing trafficking via altered chemokine levels in the TME and chemokine receptor expression on CD8+ T cells and NK cells. In addition, N-809 reduces immunosuppressive and pro-tumorigenic immune cells in the TME, including Treg, M2-like macrophages and M-MDSC. Overall, these novel effects of N-809 promote an inflamed TME, leading to lower tumor burden and increased survival. These results provide mechanistic insight and rationale supporting the potential clinical study of N-809 in patients with carcinoma.

Cooperative Immune-Mediated Mechanisms of the HDAC Inhibitor Entinostat, an IL15 Superagonist, and a Cancer Vaccine Effectively Synergize as a Novel Cancer Therapy.

PURPOSE: Immunotherapy has demonstrated clinical efficacy in subsets of patients with solid carcinomas. Multimodal therapies using agents that can affect different arms of the immune system and/or tumor microenvironment (TME) might increase clinical responses. EXPERIMENTAL DESIGN: We demonstrate that entinostat, a class I histone deacetylase inhibitor, enhances the antitumor efficacy of the IL15 superagonist N-803 plus vaccine in 4T1 triple-negative breast and MC38-CEA colon murine carcinoma models. A comprehensive immune and gene-expression analysis was performed in the periphery and/or TME of MC38-CEA tumor-bearing mice. RESULTS: Although N-803 plus vaccine induced peripheral CD8+ T-cell activation and cytokine production, there was no reduction in tumor burden and poor tumor infiltration of CD8+ T cells with minimal levels of granzyme B. For the first time, we demonstrate that the addition of entinostat to N-803 plus vaccine promoted significant tumor control, correlating with increased expression of genes associated with tumor inflammation, enhanced infiltration of activated CD8+ T cells with maximal granzyme B, T-cell responses to multiple tumor-associated antigens, increased serum IFNγ, reduction of regulatory T cells in the TME, and decreased expression of the checkpoint V-domain Ig suppressor of T-cell activation (VISTA) on multiple immune subsets. CONCLUSIONS: Collectively, these data demonstrate that the synergistic combination of entinostat, N-803, and vaccine elicits potent antitumor activity by generating a more inflamed TME. These findings thus form the rationale for the use of this combination of agents for patients harboring poorly or noninflamed solid carcinomas.

Immunological Effects and Viral Gene Expression Determine the Efficacy of Oncolytic Measles Vaccines Encoding IL-12 or IL-15 Agonists.

Tumor-targeted immunomodulation using oncolytic viral vectors is currently being investigated as a promising strategy in cancer therapy. In a previous study, we showed that a measles virus Schwarz vaccine strain (MeVac) vector encoding an interleukin-12 fusion protein (FmIL-12) is an effective immunotherapy in the MC38cea murine colon adenocarcinoma model. We hypothesized that MeVac encoding interleukin-15 may mediate enhanced T and NK cell responses and thus increase the therapeutic efficacy, especially in NK cell-controlled tumors. Therefore, we generated MeVac vectors encoding an interleukin-15 superagonist, FmIL-15. Replication and oncolytic capacity, transgene expression, and functionality of MeVac FmIL-15 vectors were validated in vitro. Effects on the tumor immune landscape and therapeutic efficacy of both FmIL-12 and FmIL-15 vectors were studied in the MC38cea and B16hCD46 tumor models. Treatment with MeVac FmIL-15 increased T and NK cell infiltration in both models. However, MeVac FmIL-12 showed more robust viral gene expression and immune activation, resulting in superior anti-tumor efficacy. Based on these results, MeVac encoding a human IL-12 fusion protein was developed for future clinical translation.

An IL-15 Superagonist, ALT-803, Enhances Antibody-Dependent Cell-Mediated Cytotoxicity Elicited by the Monoclonal Antibody NEO-201 Against Human Carcinoma Cells.

BACKGROUND: A major mechanism of action for therapeutic antibodies is antibody-dependent cell-mediated cytotoxicity (ADCC). ALT-803 is an interleukin-15 superagonist complex that enhances ADCC against human carcinoma cells in vitro and exerts an antitumor activity in murine, rat, and human carcinomas in vivo. The authors investigated the ability of ALT-803 to modulate ADCC mediated by the humanized IgG1 monoclonal antibody (mAb) NEO-201 against human carcinoma cells. MATERIALS AND METHODS: ALT-803 modulating activity on ADCC mediated by NEO-201 was evaluated on several NEO-201 ligand-expressing human carcinoma cells. Purified human natural killer (NK) cells from multiple healthy donors were treated with ALT-803 before their use as effectors in ADCC assay. Modulation of NK cell phenotype and cytotoxic function by exposure to ALT-803 was evaluated by flow cytometry and gene expression analysis. RESULTS: ALT-803 significantly enhanced ADCC mediated by NEO-201. ALT-803 also upregulated NK activating receptors, antiapoptotic factors, and factors involved in the NK cytotoxicity, as well as downregulated gene expression of NK inhibiting receptors. CONCLUSIONS: These findings indicate that ALT-803 can enhance ADCC activity mediated by NEO-201, by modulating NK activation and cytotoxicity, suggesting a possible clinical use of ALT-803 in combination with NEO-201 for the treatment of human carcinomas.

Effectiveness of two different dose administration regimens of an IL-15 superagonist complex (ALT-803) in an orthotopic bladder cancer mouse model.

BACKGROUND: We set out to determine if the administration of subcutaneous (SQ) ALT-803 was non-inferior to standard intravesical BCG treatment in a carcinogen induced mouse (C57BL/6J) bladder cancer model. METHODS: Using this well-established carcinogen induced mouse model, we studied the effects of various dosing schemas of ALT-803 (SQ alone, SQ with intravesical BCG, intravesical alone, intravesical with intravesical BCG) compared to intravesical BCG alone (positive control) and PBS (negative control). The non-inferiority margin for the difference in bladder weight, as a surrogate for tumor mass, was defined as 7%. RESULTS: All treatment groups (i.e., ALT-803 SQ alone, ALT-803 SQ with intravesical BCG, ALT-803 intravesical alone, ALT-803 intravesical with intravesical BCG and intravesical BCG alone) demonstrated a significant reduction in tumor burden as evident by bladder weights and H&E stain (p < 0.005). Non-inferiority tests between the intravesical BCG alone group and the additional treatment groups showed that SQ ALT-803 alone (p = 0.04) and BCG plus SQ ALT-803 (p = 0.009) were non-inferior to intravesical BCG alone. In this model, we did not see an appreciable infiltration of CD4+ T, CD8+ T or CD161/KLRB1+ natural killer (NK) cells in the bladder/tumor. When assessing peripheral blood mononuclear cells, SQ ALT-803 alone resulted in a robust induction of CD8+ T cells (p < 0.01), NKG2D+ NK cells (p < 0.005) and CD3+/NKG2D+ NKT cells (p < 0.005) compared to other groups, while in splenic tissue, SQ ALT-803 alone resulted in a robust induction of CD3+/NKG2D+ NKT cells (p < 0.005) compared to other groups. CONCLUSION: Subcutaneous ALT-803 treatment alone or in combination with intravesical BCG was well tolerated and was not inferior to intravesical BCG alone. CD8+ T, NKG2D+ NK and CD3+/NKG2D+ NKT cell induction along with induction of key cytokines remain steadfast mechanisms behind ALT-803. The enhanced therapeutic index seen with BCG and ALT-803, administered SQ or intravesically, provides a powerful justification for the further development of these regimens.

Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion.

Malignant cell growth within patients with B cell chronic lymphocytic leukemia (B-CLL) is largely restricted to lymphoid tissues, particularly lymph nodes. The recent in vitro finding that TLR-9 ligand (oligodeoxynucleotide [ODN]) and IL-15 exhibit strong synergy in promoting B-CLL growth may be particularly relevant to growth in these sites. This study shows IL-15-producing cells are prevalent within B-CLL-infiltrated lymph nodes and, using purified B-CLL cells from blood, investigates the mechanism for ODN and IL-15 synergy in driving B-CLL growth. ODN boosts baseline levels of phospho-RelA(S529) in B-CLL and promotes NF-κB-driven increases in IL15RA and IL2RB mRNA, followed by elevated IL-15Rα and IL-2/IL-15Rß (CD122) protein. IL-15→CD122 signaling during a critical interval, 20 to 36-48 h following initial ODN exposure, is required for optimal induction of the cycling process. Furthermore, experiments with neutralizing anti-IL-15 and anti-CD122 mAbs indicate that clonal expansion requires continued IL-15/CD122 signaling during cycling. The latter is consistent with evidence of heightened IL2RB mRNA in the fraction of recently proliferated B-CLL cells within patient peripheral blood. Compromised ODN+IL-15 growth with limited cell density is consistent with a role for upregulated IL-15Rα in facilitating homotypic trans IL-15 signaling, although there may be other explanations. Together, the findings show that ODN and IL-15 elicit temporally distinct signals that function in a coordinated manner to drive B-CLL clonal expansion.

First-in-human phase 1 clinical study of the IL-15 superagonist complex ALT-803 to treat relapse after transplantation.

New therapies for patients with hematologic malignancies who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) are needed. Interleukin 15 (IL-15) is a cytokine that stimulates CD8+ T-cell and natural killer (NK) cell antitumor responses, and we hypothesized this cytokine may augment antileukemia/antilymphoma immunity in vivo. To test this, we performed a first-in-human multicenter phase 1 trial of the IL-15 superagonist complex ALT-803 in patients who relapsed >60 days after allo-HCT. ALT-803 was administered to 33 patients via the IV or subcutaneous (SQ) routes once weekly for 4 doses (dose levels of 1, 3, 6, and 10 µg/kg). ALT-803 was well tolerated, and no dose-limiting toxicities or treatment-emergent graft-versus-host disease requiring systemic therapy was observed in this clinical setting. Adverse events following IV administration included constitutional symptoms temporally related to increased serum IL-6 and interferon-γ. To mitigate these effects, the SQ route was tested. SQ delivery resulted in self-limited injection site rashes infiltrated with lymphocytes without acute constitutional symptoms. Pharmacokinetic analysis revealed prolonged (>96 hour) serum concentrations following SQ, but not IV, injection. ALT-803 stimulated the activation, proliferation, and expansion of NK cells and CD8+ T cells without increasing regulatory T cells. Responses were observed in 19% of evaluable patients, including 1 complete remission lasting 7 months. Thus, ALT-803 is a safe, well-tolerated agent that significantly increased NK and CD8+ T cell numbers and function. This immunostimulatory IL-15 superagonist warrants further investigation to augment antitumor immunity alone and combined with other immunotherapies. This trial was registered at www.clinicaltrials.gov as #NCT01885897.

The human IL-15 superagonist ALT-803 directs SIV-specific CD8+ T cells into B-cell follicles.

Sequestering of latent HIV in follicular helper T cells within B-cell follicles that largely exclude cytotoxic T cells is a major barrier to cellular immune-based approaches to eradicate HIV. Here, we show that the clinical-grade human interleukin-15 (IL-15) superagonist ALT-803 activates and redirects simian immunodeficiency virus (SIV)-specific CD8+ T cells from the peripheral blood into B-cell follicles. In agreement with the increased trafficking of SIV-specific cytotoxic T cells to sites of cryptic viral replication, lymph nodes of elite controlling macaques contained fewer cells expressing SIV RNA or harboring SIV DNA post-ALT-803 treatment. These data establish ALT-803 as an immunotherapeutic for HIV and other chronic viral pathogens that evade host immunity by persisting in B-cell follicles.

Glycolytic requirement for NK cell cytotoxicity and cytomegalovirus control.

NK cell activation has been shown to be metabolically regulated in vitro; however, the role of metabolism during in vivo NK cell responses to infection is unknown. We examined the role of glycolysis in NK cell function during murine cytomegalovirus (MCMV) infection and the ability of IL-15 to prime NK cells during CMV infection. The glucose metabolism inhibitor 2-deoxy-ᴅ-glucose (2DG) impaired both mouse and human NK cell cytotoxicity following priming in vitro. Similarly, MCMV-infected mice treated with 2DG had impaired clearance of NK-specific targets in vivo, which was associated with higher viral burden and susceptibility to infection on the C57BL/6 background. IL-15 priming is known to alter NK cell metabolism and metabolic requirements for activation. Treatment with the IL-15 superagonist ALT-803 rescued mice from otherwise lethal infection in an NK-dependent manner. Consistent with this, treatment of a patient with ALT-803 for recurrent CMV reactivation after hematopoietic cell transplant was associated with clearance of viremia. These studies demonstrate that NK cell-mediated control of viral infection requires glucose metabolism and that IL-15 treatment in vivo can reduce this requirement and may be effective as an antiviral therapy.

Immunobiology of the IL-15/IL-15Rα complex as an antitumor and antiviral agent.

Interleukin (IL)-15 is essential for natural killer (NK), NKT and memory (m) CD8+ T cell development and function, and is currently under investigation as an immunotherapeutic agent for the treatment of cancer. Recently, the creation of IL-15 superagonist by complexing IL-15 and its high affinity receptor alpha (IL-15 Rα) in solution, inspired by the natural trans-presentation of IL-15, advances the potential of IL-15-based tumor immunotherapy. IL-15 superagonist shows promising advantages over monomeric IL-15 such as sustaining high circulating concentrations due to prolonged half-life and more potently stimulating NK and CD8+ T effector lymphocytes. So far, there are three different forms of recombinant IL-15 superagonist fusion protein based on configurational modifications. Gene therapy using engineered cells co-expressing IL-15/IL-15 Rα complex for cancer treatment is also emerging. All forms have demonstrated efficacy in causing tumor regression in animal studies, which provides strong rationale for advancing IL-15 superagonist through clinical trials. To date, there are fourteen phase I/II IL-15 superagonist trials in cancer patients and one phase I trial in HIV patients. Information generated by ongoing trials regarding the toxicity and efficacy of IL-15 superagonist is awaited. Finally, we elaborate on immunotoxicity caused by IL-15 superagonist in preclinical studies and discuss important safety considerations.

IL-15 super-agonist (ALT-803) enhances natural killer (NK) cell function against ovarian cancer.

OBJECTIVE: Natural killer (NK) cells represent a powerful immunotherapeutic target as they lyse tumors directly, do not require differentiation, and can elicit potent inflammatory responses. The objective of these studies was to use an IL-15 super-agonist complex, ALT-803 (Altor BioScience Corporation), to enhance the function of both normal and ovarian cancer patient derived NK cells by increasing cytotoxicity and cytokine production. METHODS: NK cell function from normal donor peripheral blood mononuclear cells (PBMCs) and ovarian cancer patient ascites was assessed using flow cytometry and chromium release assays ±ALT-803 stimulation. To evaluate the ability of ALT-803 to enhance NK cell function in vivo against ovarian cancer, we used a MA148-luc ovarian cancer NOD scid gamma (NSG) xenogeneic mouse model with transferred human NK cells. RESULTS: ALT-803 potently enhanced functionality of NK cells against all ovarian cancer cell lines with significant increases seen in CD107a, IFNγ and TNFα expression depending on target cell line. Function was also rescued in NK cells derived from ovarian cancer patient ascites. Finally, only animals treated with intraperitoneal ALT-803 displayed an NK dependent significant decrease in tumor. CONCLUSIONS: ALT-803 enhances NK cell cytotoxicity against ovarian cancer in vitro and in vivo and is able to rescue functionality of NK cells derived from ovarian cancer patient ascites. These findings suggest that ALT-803 has the potential to enhance NK cell-based immunotherapeutic approaches for the treatment of ovarian cancer.

A Novel Fusion of ALT-803 (Interleukin (IL)-15 Superagonist) with an Antibody Demonstrates Antigen-specific Antitumor Responses.

IL-15 and its receptor α (IL-15Rα) are co-expressed on antigen-presenting cells, allowing transpresentation of IL-15 to immune cells bearing IL-2RßγC and stimulation of effector immune responses. We reported previously that the high-affinity interactions between an IL-15 superagonist (IL-15N72D) and the extracellular IL-15Rα sushi domain (IL-15RαSu) could be exploited to create a functional scaffold for the design of multivalent disease-targeted complexes. The IL-15N72D·IL-15RαSuFc complex, also known as ALT-803, is a multimeric complex constructed by fusing IL-15N72D·IL-15RαSu to the Fc domain of IgG1. ALT-803 is an IL-15 superagonist complex that has been developed as a potent antitumor immunotherapeutic agent and is in clinical trials. Here we describe the creation of a novel fusion molecule, 2B8T2M, using the ALT-803 scaffold fused to four single chains of the tumor-targeting monoclonal antibody rituximab. This molecule displays trispecific binding activity through its recognition of the CD20 molecule on tumor cells, stimulation via IL-2RßγC displayed on immune effector cells, and binding to Fcγ receptors on natural killer cells and macrophages. 2B8T2M activates natural killer cells to enhance antibody-dependent cellular cytotoxicity, mediates complement-dependent cytotoxicity, and induces apoptosis of B-lymphoma cells. Compared with rituximab, 2B8T2M exhibits significantly stronger antitumor activity in a xenograft SCID mouse model and depletes B cells in cynomolgus monkeys more efficiently. Thus, ALT-803 can be modified as a functional scaffold for creating multispecific, targeted IL-15-based immunotherapeutic agents and may serve as a novel platform to improve the antitumor activity and clinical efficacy of therapeutic antibodies.

Cooperative therapeutic anti-tumor effect of IL-15 agonist ALT-803 and co-targeting soluble NKG2D ligand sMIC.

Shedding of the human NKG2D ligand MIC (MHC class I-chain-related molecule) from tumor cell surfaces correlates with progression of many epithelial cancers. Shedding-derived soluble MIC (sMIC) enables tumor immune escape through multiple immune suppressive mechanisms, such as disturbing natural killer (NK) cell homeostatic maintenance, impairing NKG2D expression on NK cells and effector T cells, and facilitating the expansion of arginase I+ myeloid suppressor cells. Our recent study has demonstrated that sMIC is an effective cancer therapeutic target. Whether targeting tumor-derived sMIC would enhance current active immunotherapy is not known. Here, we determined the in vivo therapeutic effect of an antibody co-targeting sMIC with the immunostimulatory IL-15 superagonist complex, ALT-803, using genetically engineered transplantable syngeneic sMIC+ tumor models. We demonstrate that combined therapy of a nonblocking antibody neutralizing sMIC and ALT-803 improved the survival of animals bearing sMIC+ tumors in comparison to monotherapy. We further demonstrate that the enhanced therapeutic effect with combined therapy is through concurrent augmentation of NK and CD8 T cell anti-tumor responses. In particular, expression of activation-induced surface molecules and increased functional potential by cytokine secretion are improved greatly by the administration of combined therapy. Depletion of NK cells abolished the cooperative therapeutic effect. Our findings suggest that administration of the sMIC-neutralizing antibody can enhance the anti-tumor effects of ALT-803. With ALT-803 currently in clinical trials to treat progressive solid tumors, the majority of which are sMIC+, our findings provide a rationale for co-targeting sMIC to enhance the therapeutic efficacy of ALT-803 or other IL-15 agonists.