Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Promotes Macrophage Activation via LDL Receptor-Independent Mechanisms.

BACKGROUND: Activated macrophages contribute to the pathogenesis of vascular disease. Vein graft failure is a major clinical problem with limited therapeutic options. PCSK9 (proprotein convertase subtilisin/kexin 9) increases low-density lipoprotein (LDL)-cholesterol levels via LDL receptor (LDLR) degradation. The role of PCSK9 in macrophage activation and vein graft failure is largely unknown, especially through LDLR-independent mechanisms. This study aimed to explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion. METHODS: We used Ldlr-/- mice to examine the LDLR-independent roles of circulating PCSK9 in experimental vein grafts. Adeno-associated virus (AAV) vector encoding a gain-of-function mutant of PCSK9 (rAAV8/D377Y-mPCSK9) induced hepatic PCSK9 overproduction. To explore novel inflammatory targets of PCSK9, we used systems biology in Ldlr-/- mouse macrophages. RESULTS: In Ldlr-/- mice, AAV-PCSK9 increased circulating PCSK9, but did not change serum cholesterol and triglyceride levels. AAV-PCSK9 promoted vein graft lesion development when compared with control AAV. In vivo molecular imaging revealed that AAV-PCSK9 increased macrophage accumulation and matrix metalloproteinase activity associated with decreased fibrillar collagen, a molecular determinant of atherosclerotic plaque stability. AAV-PCSK9 induced mRNA expression of the pro-inflammatory mediators IL-1ß (interleukin-1 beta), TNFα (tumor necrosis factor alpha), and MCP-1 (monocyte chemoattractant protein-1) in peritoneal macrophages underpinned by an in vitro analysis of Ldlr-/- mouse macrophages stimulated with endotoxin-free recombinant PCSK9. A combination of unbiased global transcriptomics and new network-based hyperedge entanglement prediction analysis identified the NF-κB (nuclear factor-kappa B) signaling molecules, lectin-like oxidized LOX-1 (LDL receptor-1), and SDC4 (syndecan-4) as potential PCSK9 targets mediating pro-inflammatory responses in macrophages. CONCLUSIONS: Circulating PCSK9 induces macrophage activation and vein graft lesion development via LDLR-independent mechanisms. PCSK9 may be a potential target for pharmacologic treatment for this unmet medical need.

Systems Approach to Discovery of Therapeutic Targets for Vein Graft Disease: PPARα Pivotally Regulates Metabolism, Activation, and Heterogeneity of Macrophages and Lesion Development.

BACKGROUND: Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discover therapeutic targets for vein graft failure. METHODS: Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The PPARs (peroxisome proliferator-activated receptors) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα small interfering RNA, or the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, quantitative polymerase chain reaction, flow cytometry, metabolic assays, and single-cell RNA sequencing on primary human and mouse macrophages. RESULTS: We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, whereas gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. CONCLUSIONS: This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacological treatment for this unmet medical need.

First-in-human phase I/Ib study of NIZ985, a recombinant heterodimer of IL-15 and IL-15Rα, as a single agent and in combination with spartalizumab in patients with advanced and metastatic solid tumors.

BACKGROUND: Preclinically, interleukin-15 (IL-15) monotherapy promotes antitumor immune responses, which are enhanced when IL-15 is used in combination with immune checkpoint inhibitors (ICIs). This first-in-human study investigated NIZ985, a recombinant heterodimer comprising physiologically active IL-15 and IL-15 receptor α, as monotherapy and in combination with spartalizumab, an anti-programmed cell death protein-1 (anti-PD-1) monoclonal antibody, in patients with advanced solid tumors. METHODS: This phase I/Ib study had two dose-escalation arms: single-agent NIZ985 administered subcutaneously thrice weekly (TIW, 2 weeks on/2 weeks off) or once weekly (QW, 3 weeks on/1 week off), and NIZ985 TIW or QW administered subcutaneously plus spartalizumab (400 mg intravenously every 4 weeks (Q4W)). The dose-expansion phase investigated NIZ985 1 µg/kg TIW/spartalizumab 400 mg Q4W in patients with anti-PD-1-sensitive or anti-PD-1-resistant tumor types stratified according to approved indications. The primary objectives were the safety, tolerability, and the maximum tolerated doses (MTDs) and/or recommended dose for expansion (RDE) of NIZ985 for the dose-expansion phase. RESULTS: As of February 17, 2020, 83 patients (median age: 63 years; range: 28-85) were treated in dose escalation (N=47; single-agent NIZ985: n=27; NIZ985/spartalizumab n=20) and dose expansion (N=36). No dose-limiting toxicities occurred nor was the MTD identified. The most common treatment-related adverse event (TRAE) was injection site reaction (primarily grades 1-2; single-agent NIZ985: 85% (23/27)); NIZ985/spartalizumab: 89% [50/56]). The most common grade 3-4 TRAE was decreased lymphocyte count (single-agent NIZ985: 7% [2/27]; NIZ985/spartalizumab: 5% [3/56]). The best overall response was stable disease in the single-agent arm (30% (8/27)) and partial response in the NIZ985/spartalizumab arm (5% [3/56]; melanoma, pancreatic cancer, gastric cancer). In dose expansion, the disease control rate was 45% (5/11) in the anti-PD-1-sensitive and 20% (5/25) in the anti-PD-1-resistant tumor type cohorts. Pharmacokinetic parameters were similar across arms. The transient increase in CD8+ T cell and natural killer cell proliferation and induction of several cytokines occurred in response to the single-agent and combination treatments. CONCLUSIONS: NIZ985 was well tolerated in the single-agent and NIZ985/spartalizumab regimens. The RDE was established at 1 µg/kg TIW. Antitumor activity of the combination was observed against tumor types known to have a poor response to ICIs. TRIAL REGISTRATION NUMBER: NCT02452268.

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.