Jack O. Egan

Novel Human Interleukin-15 Agonists

IL-15 is an immunostimulatory cytokine trans-presented with the IL-15 receptor α-chain to the shared IL-2/IL-15Rβ and common γ-chains displayed on the surface of T cells and NK cells. To further define the functionally important regions of this cytokine, activity and binding studies were conducted on human IL-15 muteins generated by site-directed mutagenesis. Amino acid substitutions of the asparagine residue at position 72, which is located at the end of helix C, were found to provide both partial agonist and superagonist activity, with various nonconservative substitutions providing enhanced activity. Particularly, the N72D substitution provided a 4–5-fold increase in biological activity of the IL-15 mutein compared with the native molecule based on proliferation assays with cells bearing human IL-15Rβ and common γ-chains. The IL-15N72D mutein exhibited superagonist activity through improved binding ability to the human IL-15Rβ-chain. However, the enhanced potency of IL-15N72D was not observed with cells expressing the mouse IL-15Rα-IL-15Rβ-γc complex, suggesting that this effect is specific to the human IL-15 receptor. The enhanced biological activity of IL-15N72D was associated with more intense phosphorylation of Jak1 and Stat5 and better anti-apoptotic activity compared with the wild-type IL-15. IL-15N72D superagonist activity was also preserved when linked to a single-chain TCR domain to generate a tumor-specific fusion protein. Thus, the human IL-15 superagonist muteins and fusions may create opportunities to construct more efficacious immunotherapeutic agents with clinical utility.

Comparison of the superagonist complex, ALT-803, to IL15 as cancer immunotherapeutics in animal models

IL15 stimulates T-cell and NK-cell responses, but not Tregs. The antitumor efficacy, biodistribution, and toxicity of an IL15-based superagonist, ALT-803, was examined in animal models and was superior, supporting its clinical development for advanced hematologic or solid tumors. IL15, a potent stimulant of CD8+ T cells and natural killer (NK) cells, is a promising cancer immunotherapeutic. ALT-803 is a complex of an IL15 superagonist mutant and a dimeric IL15 receptor αSu/Fc fusion protein that was found to exhibit enhanced biologic activity in vivo, with a substantially longer serum half-life than recombinant IL15. A single intravenous dose of ALT-803, but not IL15, eliminated well-established tumors and prolonged survival of mice bearing multiple myeloma. In this study, we extended these findings to demonstrate the superior antitumor activity of ALT-803 over IL15 in mice bearing subcutaneous B16F10 melanoma tumors and CT26 colon carcinoma metastases. Tissue biodistribution studies in mice also showed much greater retention of ALT-803 in the lymphoid organs compared with IL15, consistent with its highly potent immunostimulatory and antitumor activities in vivo. Weekly dosing with 1 mg/kg ALT-803 in C57BL/6 mice was well tolerated, yet capable of increasing peripheral blood lymphocyte, neutrophil, and monocyte counts by >8-fold. ALT-803 dose-dependent stimulation of immune cell infiltration into the lymphoid organs was also observed. Similarly, cynomolgus monkeys treated weekly with ALT-803 showed dose-dependent increases of peripheral blood lymphocyte counts, including NK, CD4+, and CD8+ memory T-cell subsets. In vitro studies demonstrated ALT-803–mediated stimulation of mouse and human immune cell proliferation and IFNγ production without inducing a broad-based release of other proinflammatory cytokines (i.e., cytokine storm). Based on these results, a weekly dosing regimen of ALT-803 has been implemented in multiple clinical studies to evaluate the dose required for effective immune cell stimulation in humans. Cancer Immunol Res; 4(1); 49–60. ©2015 AACR.

Intravesical ALT-803 and BCG Treatment Reduces Tumor Burden in a Carcinogen Induced Bladder Cancer Rat Model; a Role for Cytokine Production and NK Cell Expansion

Intravesical Bacillus Calmette-Guérin (BCG) has been shown to induce a specific immunologic response (i.e., activation of IL-2 and effector T-cells), while preclinical studies using ALT-803 (mutated IL-15 analogue combined with IL-15Rα-Fc fusion) have shown promising results by prolonging the agents half-life and stimulating CD8+ T-cells. Based on these results, we hypothesized that the intravesical administration of ALT-803 along with BCG will generate an immunologic response leading to significant bladder tumor burden reduction. Using a well-established carcinogen induced rat non-muscle invasive bladder cancer (NMIBC) model, we studied the effects of intravesical ALT-803 with and without BCG. Rat tissues were evaluated to document treatment response. Intravesical ALT-803 was safe and well tolerated alone and in combination with BCG. As a single treatment agent, ALT-803 reduced tumor burden by 35% compared to control whereas BCG alone only reduced tumor burden by 15%. However, the combination of ALT-803 plus BCG reduced tumor burden by 46% compared to control. Immune monitoring suggested that the antitumor response was linked to the production and secretion of IL-1α, IL-1β and RANTES, which in turn, induced the proliferation and activation of NK cells. Lastly, tumoral responses of the combinational treatment were associated with 76% reduction in angiogenesis, which is significantly higher than when assessed with either agent alone. The enhanced therapeutic index seen with this duplet provides justification for the development of this regimen for future clinical trials.

Inhibition of Acute Vascular Thrombosis in Chimpanzees by an Anti-Human Tissue Factor Antibody Targeting the Factor X Binding Site

Tissue factor (TF) antagonists targeting the factor VII (FVII) binding domain have been shown to interrupt acute vascular thrombus formation without impairing haemostasis in non-human primates. In this study, we evaluate whether a human/mouse chimeric monoclonal antibody (ALT-836, formerly known as Sunol-cH36) blocking the factor X/factor IX (FX/FIX) binding site of tissue factor could achieve similar clinical benefits in an arterial thrombosis model induced by surgical endarterectomy in chimpanzees. In this model, sequential surgical endarterectomies on right and left superficial femoral arteries were performed 30 days apart in five chimpanzees. A bolus (1 mg/kg) of ALT-836 was injected intravenously immediately preceding the restoration of flow in the endarterectomised femoral artery. Pre-surgical labelling of autologous platelets using (111)In-Oxine and post-surgical gamma camera imaging of (111)In-platelet deposition at endarterectomy sites was performed. The manipulated arterial segments were harvested for patency analysis 30 days following surgery. The results indicate that ALT-836 was highly effective at reducing acute vascular thrombosis, with no significant variations in surgical blood loss and template-bleeding time in the treated group compared to the control animals. These data suggest that ALT-836 is an effective and safe antithrombotic agent in preventing TF-initiated vascular thrombogenesis without compromising haemostasis.

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.

ALT-803, an IL-15 superagonist, in combination with nivolumab in patients with metastatic non-small cell lung cancer: a non-randomised, open-label, phase 1b trial

BACKGROUND Immunotherapy with PD-1 or PD-L1 blockade fails to induce a response in about 80% of patients with unselected non-small cell lung cancer (NSCLC), and many of those who do initially respond then develop resistance to treatment. Agonists that target the shared interleukin-2 (IL-2) and IL-15Rβγ pathway have induced complete and durable responses in some cancers, but no studies have been done to assess the safety or efficacy of these agonists in combination with anti-PD-1 immunotherapy. We aimed to define the safety, tolerability, and activity of this drug combination in patients with NSCLC. METHODS In this non-randomised, open-label, phase 1b trial, we enrolled patients (aged ≥18 years) with previously treated histologically or cytologically confirmed stage IIIB or IV NSCLC from three academic hospitals in the USA. Key eligibility criteria included measurable disease, eligibility to receive anti-PD-1 immunotherapy, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received the anti-PD-1 monoclonal antibody nivolumab intravenously at 3 mg/kg (then 240 mg when US Food and Drug Administration [FDA]-approved dosing changed) every 14 days (either as new treatment or continued treatment at the time of disease progression) and the IL-15 superagonist ALT-803 subcutaneously once per week on weeks 1-5 of four 6-week cycles for 6 months. ALT-803 was administered at one of four escalating dose concentrations: 6, 10, 15, or 20 μg/kg. The primary endpoint was to define safety and tolerability and to establish a recommended phase 2 dose of ALT-803 in combination with nivolumab. Analyses were per-protocol and included any patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02523469; phase 2 enrolment of patients is ongoing. FINDINGS Between Jan 18, 2016, and June 28, 2017, 23 patients were enrolled and 21 were treated at four dose levels of ALT-803 in combination with nivolumab. Two patients did not receive treatment because of the development of inter-current illness during enrolment, one patient due to leucopenia and one patient due to pulmonary dysfunction. No dose-limiting toxicities were recorded and the maximum tolerated dose was not reached. The most common adverse events were injection-site reactions (in 19 [90%] of 21 patients) and flu-like symptoms (15 [71%]). The most common grade 3 adverse events, occurring in two patients each, were lymphocytopenia and fatigue. A grade 3 myocardial infarction occurred in one patient. No grade 4 or 5 adverse events were recorded. The recommended phase 2 dose of ALT-803 is 20 μg/kg given once per week subcutaneously in combination with 240 mg intravenous nivolumab every 2 weeks. INTERPRETATION ALT-803 in combination with nivolumab can be safely administered in an outpatient setting. The promising clinical activity observed with the addition of ALT-803 to the regimen of patients with PD-1 monoclonal antibody relapsed and refractory disease shows evidence of anti-tumour activity for a new class of agents in NSCLC. FUNDING Altor BioScience (a NantWorks company), National Institutes of Health, and Medical University of South Carolina Hollings Cancer Center.

ALT-803 Transiently Reduces Simian Immunodeficiency Virus Replication in the Absence of Antiretroviral Treatment

ABSTRACT Developing biological interventions to control human immunodeficiency virus (HIV) replication in the absence of antiretroviral therapy (ART) could contribute to the development of a functional cure. As a potential alternative to ART, the interleukin-15 (IL-15) superagonist ALT-803 has been shown to boost the number and function of HIV-specific CD8+ T and NK cell populations in vitro. Four simian immunodeficiency virus (SIV)-positive rhesus macaques, three of whom possessed major histocompatibility complex alleles associated with control of SIV and all of whom had received SIV vaccine vectors that had the potential to elicit CD8+ T cell responses, were given ALT-803 in three treatment cycles. The first and second cycles of treatment were separated by 2 weeks, while the third cycle was administered after a 29-week break. ALT-803 transiently elevated the total CD8+ effector and central memory T cell and NK cell populations in peripheral blood, while viral loads transiently decreased by ∼2 logs in all animals. Virus suppression was not sustained as T cells became less responsive to ALT-803 and waned in numbers. No effect on viral loads was observed in the second cycle of ALT-803, concurrent with downregulation of the IL-2/15 common γC and β chain receptors on both CD8+ T cells and NK cells. Furthermore, populations of immunosuppressive T cells increased during the second cycle of ALT-803 treatment. During the third treatment cycle, responsiveness to ALT-803 was restored. CD8+ T cells and NK cells increased again 3- to 5-fold, and viral loads transiently decreased again by 1 to 2 logs. IMPORTANCE Overall, our data show that ALT-803 has the potential to be used as an immunomodulatory agent to elicit effective immune control of HIV/SIV replication. We identify mechanisms to explain why virus control is transient, so that this model can be used to define a clinically appropriate treatment regimen.

Novel antitumor mechanism-of-action of an IL-2 fusion protein mediated by tumor associated macrophage repolarization and innate-like CD8+ memory T cells

ALT-801, a fusion of interleukin-2 and a single-chain T cell receptor domain specific to a p53 peptide/HLA-A*0201 complex, has shown immune cell-mediated activity in human xenograft tumor SCID mouse models and in patients with metastatic malignancies. To further investigate the antitumor efficacy of this fusion protein in immunocompetent mice, we conducted mechanism-of-action studies in an orthotopic murine muscle invasive bladder cancer model. ALT-801 treatment (1.6 mg/kg i.v., day 7, 10, 14, 17 post-tumor instillation) significantly prolonged survival of C57BL/6 mice bearing orthotopic MB49luc bladder tumors when compared to equivalent treatment with either IL-2 (0.4 mg/kg) or PBS (8 mice/group; median survival: ALT-801, 48.5 days vs. IL-12, 36 days; vs.PBS, 33.5 days, p 0.05; PBS vs. ALT-801 treatment of tumor-bearing IFN-γ or IFN-γR KO mice, P > 0.05). Consistent with these results, IHC, adoptive cell transfer and expression array studies showed that ALT-801 treatment led to activation and proliferation of CD4+ and CD8+ T cells which migrate from the lymphoid tissues to the tumor site where they secrete IFN-γ. Twenty hours after ALT-801 administration, tumor associated-macrophages (TAM) in the bladder of tumor-bearing mice were also transiently repolarized from an M2 (tumor promoting) to an M1 (tumor killing) phenotype in an IFN-γ dependent manner. Additionally, in vivo ALT-801 treatment induced innate-like CD8+CD44high memory T cells to proliferate and upregulate NKG2D receptors. These cells may contribute to elevated splenocyte cytotoxic activity against mouse and human bladder tumor cells that was seen after ALT-801 administration. The results of these studies suggest that ALT-801 induces IFN-γ-dependent TAM repolarization and non-specific CD8+ memory effector T cells that promote robust and rapid antitumor activity in mice bearing orthotopic MB49luc bladder tumors. This novel immunostimulatory mechanism-of-action appears to be distinct from that of IL-2 or other T cell-based immunotherapeutics and is currently being assessed in bladder cancer patients under treatment with ALT-801.

Phase I Trial of ALT-803, A Novel Recombinant IL15 Complex, in Patients with Advanced Solid Tumors

Purpose: IL15 induces the activation and proliferation of natural killer (NK) and memory CD8+ T cells and has preclinical antitumor activity. Given the superior activity and favorable kinetics of ALT-803 (IL15N72D:IL15RαSu/IgG1 Fc complex) over recombinant human IL15 (rhIL15) in animal models, we performed this first-in-human phase I trial of ALT-803 in patients with advanced solid tumors. Patients and Methods: Patients with incurable advanced melanoma, renal cell, non–small cell lung, and head and neck cancer were treated with ALT-803 0.3 to 6 μg/kg weekly intravenously or 6 to 20 μg/kg weekly subcutaneously for 4 consecutive weeks, every 6 weeks. Immune correlates included pharmacokinetics, immunogenicity, and lymphocyte expansion and function. Clinical endpoints were toxicity and antitumor activity. Results: Twenty-four patients were enrolled; 11 received intravenous and 13 received subcutaneous ALT-803. Of these patients, nine had melanoma, six renal, three head and neck, and six lung cancer. Although total lymphocyte and CD8+ T-cell expansion were modest, NK cell numbers rose significantly. Neither anti–ALT-803 antibodies nor clinical activity were observed. Overall, ALT-803 was well tolerated, with adverse effects including fatigue and nausea most commonly with intravenous administration, whereas painful injection site wheal was reported most commonly with subcutaneous ALT-803. Conclusions: Subcutaneous ALT-803 produced the expected NK cell expansion and was well tolerated with minimal cytokine toxicities and a strong local inflammatory reaction at injection sites in patients with advanced cancer. These data, together with compelling evidence of synergy in preclinical and clinical studies, provide the rationale for combining ALT-803 with other anticancer agents. Clin Cancer Res; 24(22); 5552–61. ©2018 AACR.

Tumor burden reduction in an orthotopic non-muscle-invasive bladder cancer model using IL-15 analogue (ALT-803) targeting T regulatory cells.

298 Background: A recent NCI review listed IL-15 as the most promising product candidate among twelve immunotherapy drugs that could potentially cure cancer. Preclinical studies using ALT-803 (mutated IL-15 analogue combined with IL-15Rα-Fc fusion) have shown promising results for obtaining prolonged drug half-life and stimulating CD8+ T cells and NK cells. Based on these results, we hypothesized that the administration of ALT-803 will generate an immunologic response which will reduce tumor burden in a rodent carcinogen induced orthotopic non muscle invasive bladder cancer model (NMIBC). METHODS We tested intravesical ALT-803 alone and ALT-803 in combination with Bacillus Calmette-Guérin (BCG) in a rodent carcinogen induced orthotopic NMIBC model. Rats were anesthetized then a 22-gauge Teflon transurethral catheter was placed in the bladder and urine completely drained from the bladder. Next, the saline (negative control), ALT-803 (experimental agent) or BCG (positive control) therapy was delivered by transurethral instillation and allowed to dwell in the bladder for 1 hr by occlusion of the urethra with a purse string suture. The intravesical therapy was administered weekly for a total of six weeks to mimic intravesical BCG therapy in humans. RESULTS Herein we demonstrate that ALT-803 was safe and well tolerated alone or in combination with BCG. Furthermore, ALT-803 alone reduced tumor burden by 23% whereas BCG alone reduced tumor burden by 11% compared to control. The combination of ALT-803 and BCG reduced tumor burden by 30% compared to control. Tumoral responses of the combinational treatment were associated with 76% and 80% reduction in angiogenesis and proliferation, respectively, whereas combinational therapy was associated with a 7.7-fold increase in apoptotic index compared to control. Immune monitoring suggested that the antitumor response was linked to the activation of CD8+ cells and NK cells. CONCLUSIONS The enhanced therapeutic index provided by ALT-803 plus BCG therefore provides a powerful justification for the development of this agent for future clinical trials in subjects with non-muscle invasive bladder cancer.