NKTR-358: A novel regulatory T-cell stimulator that selectively stimulates expansion and suppressive function of regulatory T cells for the treatment of autoimmune and inflammatory diseases.

Impaired interleukin-2 (IL-2) production and regulatory T-cell dysfunction have been implicated as immunological mechanisms central to the pathogenesis of multiple autoimmune and inflammatory diseases. NKTR-358, a novel regulatory T-cell stimulator, is an investigational therapeutic that selectively restores regulatory T-cell homeostasis in these diseases. We investigated NKTR-358's selectivity for regulatory T-cells, receptor-binding properties, ex vivo and in vivo pharmacodynamics, ability to suppress conventional T-cell proliferation in mice and non-human primates, and functional activity in a murine model of systemic lupus erythematosus. In vitro, NKTR-358 demonstrated decreased affinity for IL-2Rα, IL-2Rß, and IL-2Rαß compared with recombinant human IL-2 (rhIL-2). A single dose of NKTR-358 in cynomolgus monkeys produced a greater than 15-fold increase in regulatory T-cells, and the increase lasted until day 14, while daily rhIL-2 administration for 5 days only elicited a 3-fold increase, which lasted until day 7. Repeated dosing of NKTR-358 over 6 months in cynomolgus monkeys elicited cyclical, robust increases in regulatory T-cells with no loss in drug activity over the course of treatment. Regulatory T-cells isolated from NKTR-358-treated mice displayed a sustained, higher suppression of conventional T-cell proliferation than regulatory T-cells isolated from vehicle-treated mice. NKTR-358 treatment in a mouse model (MRL/MpJ-Faslpr) of systemic lupus erythematosus for 12 weeks maintained elevated regulatory T-cells for the treatment duration and ameliorated disease progression. Together, these results suggest that NKTR-358 has the ability to elicit sustained and preferential proliferation and activation of regulatory T-cells without corresponding effects on conventional T-cells, with improved pharmacokinetics compared with rhIL-2.

NKTR-255, a novel polymer-conjugated rhIL-15 with potent antitumor efficacy.

BACKGROUND: NKTR-255 is a novel polyethylene glycol-conjugate of recombinant human interleukin-15 (rhIL-15), which was designed to retain all known receptor binding interactions of the IL-15 molecule. We explored the biologic and pharmacologic differences between endogenous IL-15 receptor α (IL-15Rα)-dependent (NKTR-255 and rhIL-15) and IL-15Rα-independent (precomplexed rhIL-15/IL-15Rα) cytokines. METHODS: In vitro pharmacological properties of rhIL-15, NKTR-255 and precomplex cytokines (rhIL-15/IL-15Rα and rhIL-15 N72D/IL-15Rα Fc) were investigated in receptor binding, signaling and cell function. In vivo pharmacokinetic (PK) and pharmacodynamic profile of the cytokines were evaluated in normal mice. Finally, immunomodulatory effect and antitumor activity were assessed in a Daudi lymphoma model. RESULTS: NKTR-255 and rhIL-15 exhibited similar in vitro properties in receptor affinity, signaling and leukocyte degranulation, which collectively differed from precomplexed cytokines. Notably, NKTR-255 and rhIL-15 stimulated greater granzyme B secretion in human peripheral blood mononuclear cells versus precomplexed cytokines. In vivo, NKTR-255 exhibited a PK profile with reduced clearance and a longer half-life relative to rhIL-15 and demonstrated prolonged IL-15R engagement in lymphocytes compared with only transient engagement observed for rhIL-15 and precomplexed rhIL-15 N72D/IL-15Rα Fc. As a consequent, NKTR-255 provided a durable and sustained proliferation and activation of natural killer (NK) and CD8+ T cells. Importantly, NKTR-255 is more effective than the precomplexed cytokine at inducing functionally competent, cytotoxic NK cells in the tumor microenvironment and the properties of NKTR-255 translated into superior antitumor activity in a B-cell lymphoma model versus the precomplexed cytokine. CONCLUSIONS: Our results show that the novel immunotherapeutic, NKTR-255, retains the full spectrum of IL-15 biology, but with improved PK properties, over rhIL-15. These findings support the ongoing phase 1 first-in-human trial (NCT04136756) of NKTR-255 in participants with relapsed or refractory hematologic malignancies, potentially advancing rhIL-15-based immunotherapies for the treatment of cancer.

Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24Gag.

The fluorescence-linked antigen quantification (FLAQ) assay allows a fast quantification of HIV-1 p24Gag antigen. Viral supernatant are lysed and incubated with polystyrene microspheres coated with polyclonal antibodies against HIV-1 p24Gag and detector antibodies conjugated to fluorochromes (Figure 1). After washes, the fluorescence of microspheres is measured by flow cytometry and reflects the abundance of the antigen in the lysate. The speed, simplicity, and wide dynamic range of the FLAQ assay are optimum for many applications performed in HIV-1 research laboratories.

CXCL13 plus interleukin 10 is highly specific for the diagnosis of CNS lymphoma.

Establishing the diagnosis of focal brain lesions in patients with unexplained neurologic symptoms represents a challenge. The goal of this study is to provide evidence supporting functional roles for CXC chemokine ligand (CXCL)13 and interleukin (IL)-10 in central nervous system (CNS) lymphomas and to evaluate the utility of each as prognostic and diagnostic biomarkers. We demonstrate for the first time that elevated CXCL13 concentration in cerebrospinal fluid (CSF) is prognostic and that CXCL13 and CXCL12 mediate chemotaxis of lymphoma cells isolated from CNS lymphoma lesions. Expression of the activated form of Janus kinase 1 supported a role for IL-10 in prosurvival signaling. We determined the concentration of CXCL13 and IL-10 in CSF of CNS lymphoma patients and control cohorts including inflammatory and degenerative neurologic disease in a multicenter study involving 220 patients. Bivariate elevated CXCL13 plus IL-10 was 99.3% specific for primary and secondary CNS lymphoma, with sensitivity significantly greater than reference standard CSF tests. These results identify CXCL13 and IL-10 as potentially important biomarkers of CNS lymphoma that merit further evaluation and support incorporation of CXCL13 and IL-10 into diagnostic algorithms for the workup of focal brain lesions in which lymphoma is a consideration.

RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly.

The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G protein, RalB, is localized to nascent autophagosomes and is activated on nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.

qiRNA is a new type of small interfering RNA induced by DNA damage.

RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNAs) and microRNAs, several types of endogenously produced small RNAs have important roles in gene regulation, germ cell maintenance and transposon silencing. The production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases to make double-stranded RNA. The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a new class of small RNAs in the filamentous fungus Neurospora crassa. This class of small RNAs, known as qiRNAs because of their interaction with QDE-2, are about 20-21 nucleotides long (several nucleotides shorter than Neurospora siRNAs), with a strong preference for uridine at the 5' end, and originate mostly from the ribosomal DNA locus. The production of qiRNAs requires the RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3 and dicers. qiRNA biogenesis also requires DNA-damage-induced aRNAs as precursors, a process that is dependent on both QDE-1 and QDE-3. Notably, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. Furthermore, the Neurospora RNA interference mutants show increased sensitivity to DNA damage, suggesting a role for qiRNAs in the DNA-damage response by inhibiting protein translation.

A double-stranded-RNA response program important for RNA interference efficiency.

When recognized by the RNA interference (RNAi) pathway, double-stranded RNA (dsRNA) produced in eukaryotic cells results in posttranscriptional gene silencing. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that dsRNA, but not short interfering RNA (siRNA), induces the expression of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for normal gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 posttranscriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. Finally, a genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes in Neurospora. Together, our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections.

QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands.

Single-stranded small interfering RNA (siRNA) guides the cleavage of homologous mRNA by Argonaute proteins, the catalytic core of the RNA-induced silencing complex (RISC), in the conserved RNA interference (RNAi) pathway. The separation of the siRNA duplex into single strands is essential for the activation of RISC. Previous biochemical studies have suggested that Argonaute proteins cleave and remove the passenger strand of siRNA duplex from RISC, but the in vivo importance of this process and the mechanism for the removal of the nicked passenger strand are not known. Here, we show that in the filamentous fungus Neurospora, the Argonaute homolog QDE-2 and its slicer function are required for the generation of single-stranded siRNA and gene silencing in vivo. Biochemical purification of QDE-2 led to the identification of QIP, a QDE-2-interacting protein, with an exonuclease domain. The disruption of qip in Neurospora impaired gene silencing and siRNA accumulated, mostly in nicked duplex form. Furthermore, our results suggest that QIP acts as an exonuclease that cleaves and removes the nicked passenger strand from siRNA duplex in a QDE-2-dependent manner. Together, these results suggest that both the cleavage and removal of the passenger strand from the siRNA duplex are important steps in RNAi pathways.