Cancer immune therapy with PD-1-dependent CD137 co-stimulation provides localized tumour killing without systemic toxicity.

Expression of the cell surface receptor CD137 has been shown to enhance anti-cancer T cell function via engagement with its natural ligand 4-1BBL. CD137 ligation with engineered ligands has emerged as a cancer immunotherapy strategy, yet clinical development of agonists has been hindered by either toxicity or limited efficacy. Here we show that a CD137/PD-1 bispecific antibody, IBI319, is able to overcome these limitations by coupling CD137 activation to PD-1-crosslinking. In CT26 and MC38 syngeneic mouse tumour models, IBI319 restricts T cell co-stimulation to PD-1-rich microenvironments, such as tumours and tumour-draining lymph nodes, hence systemic (liver) toxicity arising from generalised T cell activation is reduced. Besides limiting systemic T cell co-stimulation, the anti-PD-1 arm of IBI319 also exhibits checkpoint blockade functions, with an overall result of T and NK cell infiltration into tumours. Toxicology profiling in non-human primates shows that IBI319 is a well-tolerated molecule with IgG-like pharmacokinetic properties, thus a suitable candidate for further clinical development.

IBI112, a selective anti-IL23p19 monoclonal antibody, displays high efficacy in IL-23-induced psoriasiform dermatitis.

Psoriasis is a highly prevalent inflammatory skin disease. Plaque psoriasis is the most common type of psoriasis, and the interleukin (IL)-23/IL-17 axis plays a key role in disease progression. In this article, we describe IBI112, a highly potent anti-IL-23 monoclonal antibody under clinical development, which efficiently neutralizes IL23p19, a subunit of IL-23, to abrogate IL-23 binding to its receptor and block downstream signal transducer and activator of transcription 3 (STAT3) phosphorylation. Specifically, IBI112 blocked IL-23 induced downstream IL-17 production from splenocytes. In addition, IBI112 administration reduced skin thickness in a psoriasis-like epidermal hyperplasia mouse model challenged by continuous hIL-23 injection. IBI112 showed synergism with an anti-IL-1R antibody in controlling disease progression in an imiquimod (IMQ) -induced psoriasis model. Moreover, with mutations in Fc fragment of IBI112, extended half-life was observed when compared to the wild-type IgG1 version in both human-FcRn-knock-in mice and cynomolgus monkeys. IBI112 was well tolerated after high dose administration in cynomolgus monkeys. In summary, we have developed an extended half-life, anti-IL-23p19 monoclonal antibody, IBI112, which efficiently neutralized IL-23, blocked IL-23-induced IL-17 production, and alleviated disease symptoms in two mouse models of psoriasis.

Major Contribution of Caspase-9 to Honokiol-Induced Apoptotic Insults to Human Drug-Resistant Glioblastoma Cells.

Temozolomide (TMZ)-induced chemoresistance to human glioblastomas is a critical challenge now. Our previous studies showed that honokiol, a major bioactive constituent of Magnolia officinalis (Houpo), can kill human glioblastoma cells and suppresses glioblastoma growth. This study was further aimed to evaluate the effects of honokiol on human drug-resistant glioblastoma cells and the possible mechanisms. The results by data mining in the cancer genome atlas (TCGA) database and immunohistochemistry displayed that expression of caspase-9 mRNA and protein in human glioblastomas was induced. Human TMZ-resistant U87-MG-R9 glioblastoma cells were selected and prepared from human drug-sensitive U87-MG cells. Compared to human drug-sensitive U87-MG cells, TMZ did not affect viability of U87-MG-R9 glioblastoma cells. Interestingly, treatment with honokiol suppressed proliferation and survival of human drug-resistant glioblastoma cells in concentration- and time-dependent manners. Compared to caspase-8 activation, honokiol chiefly increased activity of caspase-9 in U87-MG-R9 cells. Successively, levels of cleaved caspase-3 and activities of caspase-3 and caspase-6 in human TMZ-tolerant glioblastoma cells were augmented following honokiol administration. In parallel, honokiol triggered DNA fragmentation of U87-MG-R9 cells. Accordingly, treatment of human TMZ-resistant glioblastoma cells with honokiol induced cell apoptosis but did not affect cell necrosis. Fascinatingly, suppressing caspase-9 activity using its specific inhibitors repressed honokiol-induced caspase-6 activation, DNA fragmentation, and cell apoptosis. Taken together, this study has shown the major roles of caspase-9 in transducing honokiol-induced mitochondria-dependent apoptosis in human drug-resistant glioblastoma cells. Thus, honokiol may be clinically applied as a drug candidate for treatment of glioblastoma patients with chemoresistance.

An Immunosuppressive Tick Salivary Gland Protein DsCystatin Interferes With Toll-Like Receptor Signaling by Downregulating TRAF6.

Ticks, blood-feeding arthropods, and secrete immunosuppressive molecules that inhibit host immune responses and provide survival advantages to pathogens. In this study, we characterized the immunosuppressive function of a novel tick salivary protein, DsCystatin, from Dermacentor silvarum of China. DsCystatin directly interacted with human Cathepsins L and B and inhibited their enzymatic activities. DsCystatin impaired the expression of inflammatory cytokines such as IL1ß, IFNγ, TNFα, and IL6 from mouse bone marrow-derived macrophages (BMDMs) that had been stimulated with LPS or Borrelia burgdorferi. Consistently, DsCystatin inhibited the activation of mouse BMDMs and bone marrow-derived dendritic cells by downregulating the surface expression of CD80 and CD86. Mechanically, DsCystatin inhibited LPS- or B. burgdorferi-induced NFκB activation. For the first time, we identified that DsCystatin-attenuated TLR4 signaling by targeting TRAF6. DsCystatin enhanced LPS-induced autophagy, mediated TRAF6 degradation via an autophagy dependent manner, thereby impeded the downstream phosphorylation of IκBα and the nuclear transport of NFκB. Finally, DsCystatin relieved the joint inflammation in B. burgdorferi or complete Freund's adjuvant induced mouse arthritis models. These data suggested that DsCystatin is a novel immunosuppressive protein and can potentially be used in the treatment of inflammatory diseases.

Functional characterization of two defensins, HlDFS1 and HlDFS2, from the hard tick Haemaphysalis longicornis.

BACKGROUND: Ticks are second to mosquitoes as vectors of human arthropod-borne diseases. Ticks rely heavily on antimicrobial peptides (AMPs) to defend against microbes and defensins are major components of innate immunity in ticks. RESULTS: Two novel defensin genes, named HlDFS1 and HlDFS2, were identified from a cDNA library of the hard tick Haemaphysalis longicornis collected in southeast China. The peptides encoded by both genes shares typical features of type-2 arthropod defensin superfamily. The expressions of both genes increased in ticks during blood-feeding. The synthetic minimum functional peptides HlDFS1 and HlDFS2 showed broad spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria. Moreover, HlDFS1 and HlDFS2 exhibit bactericidal activity to some drug resistant bacteria. HlDFS1, but not HlDFS2, showed inhibitory activity against fungus Candida albicans. HlDFS1 and HlDFS2 had no significant hemolysis effect on human erythrocytes at low concentrations and did not impair mammalian cell survival. Finally, HlDFS1 and HlDFS2 significantly protected mice against lethal infection by Staphylococcus aureus and Micrococcus luteus. CONCLUSIONS: HlDFS1 and HlDFS2 are two novel functional defensins from the hard tick Haemaphysalis longicornis. They showed bactericidal activity against various Gram-positive and Gram-negative bacteria and significantly protect mice against lethal bacterial infection. Thus, HlDFS1 and HlDFS2 can be introduced to the medical field as new drug candidates with antibacterial activity.

DEAD-Box Helicase DDX25 Is a Negative Regulator of Type I Interferon Pathway and Facilitates RNA Virus Infection.

Dengue is a mosquito-borne viral disease that rapidly spread in tropic and subtropic area in recent years. DEAD (Glu-Asp-Ala-Glu)-box RNA helicases have been reported to play important roles in viral infection, either as cytosolic sensors of viral nucleic acids or as essential host factors for the replication of different viruses. In this study, we reported that DDX25, a DEAD-box RNA helicase, plays a proviral role in DENV infection. The expression levels of DDX25 mRNA and protein were upregulated in DENV infected cells. During DENV infection, the intracellular viral loads were significantly lower in DDX25 silenced cells and higher in DDX25 overexpressed cells. Meanwhile, the expression level of type I interferon (IFN) was increased in DDX25 siRNA treated cells during viral infection. Consistent with the in vitro findings, the Ddx25-transgenic mice have an increased susceptibility to lethal vesicular stomatitis virus (VSV) virus challenge. The viremia was significantly higher while the anti-viral cytokine levels were lower in Ddx25-transgenic mice. Further, DDX25 modulated RIG-I signaling pathway and blocked IFNß production, by interrupting IFN regulatory factor 3 (IRF3) and NFκB activation. Thus, DDX25 is a novel negative regulator of IFN pathway and facilitates RNA virus infection.

Glycosphingolipid GM3 is Indispensable for Dengue Virus Genome Replication.

Dengue virus (DENV) causes the most prevalent arthropod-borne viral disease of humans worldwide. Glycosphingolipids (GSLs) are involved in virus infection by regulating various steps of viral-host interaction. However, the distinct role of GSLs during DENV infection remains unclear. In this study, we used mouse melanoma B16 cells and their GSL-deficient mutant counterpart GM95 cells to study the influence of GSLs on DENV infection. Surprisingly, GM95 cells were highly resistant to DENV infection compared with B16 cells. Pretreatment of B16 cells with synthetase inhibitor of GM3, the most abundant GSLs in B16 cells, or silencing GM3 synthetase T3GAL5, significantly inhibited DENV infection. DENV attachment and endocytosis were not impaired in GM95 cells, but DENV genome replication was obviously inhibited in GM95 cells compared to B16 cells. Furthermore, GM3 was colocalized with DENV viral replication complex on endoplasmic reticulum (ER) inside the B16 cells. Finally, GM3 synthetase inhibitor significantly reduced the mortality rate of suckling mice that challenged with DENV by impairing the viral replication in mouse brain. Taken together, these data indicated that GM3 was not required for DENV attachment and endocytosis, however, essential for viral genome replication. Targeting GM3 could be a novel strategy to inhibit DENV infection.

Microstructures and magnetic anisotropy of single-layered Fe-Pt alloy films by in-situ annealing.

The single-layered Fe-Pt films with thickness of 30 nm are in-situ deposited directly on Si substrate at various substrate temperatures (Ts) of 350 to 590 degrees C. As the Fe-Pt film is sputtered at substrate temperature is 350 degrees C, it shows (111) preferred orientation and tends to in-plane magnetic anisotropy. The L1(0) Fe-Pt film with (001) texture is obtained and exhibited perpendicular magnetic anisotropy as the substrate temperature is increased to 470 degrees C. The perpendicular coercivity (Hc perpendicular), saturation magnetization (Ms) and perpendicular squareness (S perpendicular) of this film are 6.9 kOe, 674 emu/cm3 and 0.89, respectively, which reveal its significant potential as perpendicular magnetic recording media.