Bifunctional macromolecule activating both OX40 and interferon-α signaling displays potent therapeutic effects in mouse HBV and tumor models.

Combinatory enhancement of innate and adaptive immune responses is a promising strategy in immunotherapeutic drug development. Bifunctional macromolecules that simultaneously target two mechanisms may provide additional advantages over the combination of targeting two single pathways. Interferon alpha (IFNα) has been used clinically against viral infection such as the chronic infection of hepatitis B virus (CHB) as well as some types of cancers. OX40 is a costimulatory immune checkpoint molecule involved in the activation of T lymphocytes. To test whether simultaneously activating IFNα and OX40 signaling pathway could produce a synergistic therapeutic effect on CHB and tumors, we designed a bifunctional fusion protein composed of a mouse OX40 agonistic monoclonal antibody (OX86) and a mouse IFNα4, joined by a flexible (GGGGS)3 linker. This fusion protein, termed OX86-IFN, can activate both IFNα and OX40. We demonstrated that OX86-IFN could effectively activate T lymphocytes in the peripheral blood of mice. Furthermore, we showed that OX86-IFN had superior therapeutic effect to monotherapies in HBV hydrodynamic transfection and syngeneic tumor models. Collectively, our data suggests that simultaneously targeting interferon and OX40 signaling pathways by bifunctional molecule OX86-IFN elicits potent antiviral and antitumor activities, which could provide a new strategy in developing therapeutic agents against viral infection and tumors.

pH-sensitive PEG-coated hyper-branched ß-d-glucan derivative as carrier for CpG oligodeoxynucleotide delivery.

ß-d-glucan is a natural non-digestible polysaccharide that can be selectively recognized by recognition receptors such as Dectin-1 receptors, resulting in an emerging interest on exploring its capacity for carrying biological information to desired organs or cells. CpG oligodeoxynucleotide (ODN) has the potentiality to initiate an immune-stimulatory cascade via activating B cells inducing proinflammatory cytokines, which is conducive to immunotherapy and nucleic acid vaccine. Herein, we developed a pH-sensitive delivery system loading with CpG ODN by introducing poly-ethylenimine (PEI) to a hyperbranched ß-d-glucan (HBB) and coating with poly-ethylene glycol (PEG) shell via acidic liable Schiff bond. This delivery system exhibited a favorable biocompatibility and facilitated the cellular uptake of CpG ODN at pH 6.8 with the possibility of having higher accumulation in acidic cancer microenvironment. Furthermore, this carrier together with class B CpG ODN could enhance the secretion of cytokines including interleukin-6 and interferon-α as well as capable of interferon-α induction.

Targeting mitochondria by anthelmintic drug atovaquone sensitizes renal cell carcinoma to chemotherapy and immunotherapy.

Targeting mitochondria respiration is an effective therapeutic strategy in renal cell carcinoma (RCC). Atovaquone is a FDA-approved antibiotic but is also known as a mitochondrial inhibitor. We found that atovaquone inhibited proliferation and induced apoptosis of RCC cells. Mechanistically, atovaquone inhibits mitochondrial respiration in a concentration-dependent and time-dependent manner, via targeting mitochondrial respiratory complex III. Although increased glycolysis was observed in atovaquone-treated cells, atovaquone decreased ATP levels. As a consequence of mitochondrial respiration inhibition, reactive oxygen species levels were increased by atovaquone. The complete rescue of atovaquone's effects by an antioxidant suggests the important role of oxidative stress in the action of atovaquone in RCC. Importantly, atovaquone enhanced the in vitro and in vivo efficacy of 5-fluorouracil (5-FU) and interferon-α (IFN-α). Our preclinical findings suggest that atovaquone is a useful addition for RCC treatment. Our work also further demonstrates that RCC is more dependent on mitochondrial respiration than glycolysis.

Naturally Occurring Fc-Dependent Antibody From HIV-Seronegative Individuals Promotes HIV-Induced IFN-α Production.

A majority of adults without HIV infection and with a low risk of HIV-exposure have plasma IgG antibodies that enhance the rate and magnitude of HIV-induced interferon alpha (IFN-α) production. Fc-dependent IgG-HIV complexes induce IFN-α rapidly and in high titers in response to HIV concentrations that are too low to otherwise stimulate an effective IFN-α response. IFN-α promoting antibody (IPA) counters HIV-specific inhibition of IFN-α production, and compensates for the inherent delay in IFN-α production common to HIV infection and other viruses. Naturally occurring IPA has the potential to initiate a potent IFN-α response early in the course of HIV mucosal invasion in time to terminate infection prior to the creation of a pool of persistently infected cells. The current study adds IPA as a mediator of an Fc-dependent antiviral state capable of preventing HIV infection.

Interferon-alpha is involved in the luteinizing hormone-induced differentiation of rat preovulatory granulosa cells.

A surge in luteinizing hormone (LH) triggers physiological changes within the ovarian follicles, including reprogramming to induce terminal differentiation of the granulosa cells (GCs). Cytokines are members of a large regulatory network that resides in the ovaries and are involved in the regulation of steroidogenesis and gamete production. Recently we found that interferon-alpha (IFN-alpha) was overexpressed in LH-treated preovulatory GCs, as determined by a microarray analysis. In this study, we evaluated the expression of IFN-alpha and its role in the differentiation of rat preovulatory GCs. Rat GCs were treated with LH in vitro or human chorionic gonadotropin (hCG) in vivo, both of which are well-known inducers of differentiation, and IFN-alpha production and cell differentiation were determined. Stimulation of rat primary GCs with LH or hCG increased expression of IFN-alpha. LH treatment led to increased phosphorylation of PI3-K and extracellular signal-regulated kinase (ERK), and specific inhibitors for PI3-K and ERK suppressed the LH-induced IFN-alpha expression in preovulatory GCs. Furthermore, treatment with anti-rat IFN-alpha blocking antibody delayed the LH-induced differentiation of GCs and suppressed the expression of ovulation-related genes, including progesterone receptor (PR) and steroidogenic acute regulatory protein (StAR). These results indicate that LH induces IFN-alpha expression in preovulatory GCs via a PI3-K/ERK signaling pathway and that interferon-alpha production may be involved in the LH-induced differentiation of preovulatory GCs in rats.

The combination of IL-21 and IFN-alpha boosts STAT3 activation, cytotoxicity and experimental tumor therapy.

For decades cytokines such as type I interferons and IL-2 have been used in immunotherapy against cancer, viral hepatitis, and autoimmune diseases such as multiple sclerosis. However, the therapeutic use of cytokines has been hampered by their pleiotropic effects on target-cells. Thus, cytokines such as IFN-alpha and IL-2 have multiple and severe side effects. Accordingly, they are generally used at sub-optimal doses, which limit their clinical efficacy. Here we hypothesized that a combination of IFN-alpha and IL-21, a novel cytokine of the IL-2 family with anti-cancer effects, will increase the anti-cancer efficacy at sub-optimal cytokine doses. We show that the combined stimulation of target-cells with IFN-alpha and IL-21 triggers an increased STAT3 activation whereas the activation of other STATs including STAT1/2 is unaffected. In parallel, the combined stimulation with IFN-alpha and IL-21 triggers a selective increase in MHC class I expression and NK- and CD8(+) T-cell-mediated cytotoxicity. In an experimental in vivo model of renal carcinoma, the combined treatment of IFN-alpha and IL-21 also produces a significant anti-cancer effect as judged by an inhibition of tumor growth and an increased survival. Taken together our data show that the combined use of IFN-alpha and IL-21 boosts STAT3 signaling, cytotoxicity, and anti-tumor efficacy, suggesting that a combinatorial therapeutic use of these cytokines may benefit cancer patients.

Enhanced antiviral activity against foot-and-mouth disease virus by a combination of type I and II porcine interferons.

Previously, we showed that type I interferon (alpha/beta interferon [IFN-alpha/beta]) can inhibit foot-and-mouth disease virus (FMDV) replication in cell culture, and swine inoculated with 10(9) PFU of human adenovirus type 5 expressing porcine IFN-alpha (Ad5-pIFN-alpha) were protected when challenged 1 day later. In this study, we found that type II pIFN (pIFN-gamma) also has antiviral activity against FMDV in cell culture and that, in combination with pIFN-alpha, it has a synergistic antiviral effect. We also observed that while each IFN alone induced a number of IFN-stimulated genes (ISGs), the combination resulted in a synergistic induction of some ISGs. To extend these studies to susceptible animals, we inoculated groups of swine with a control Ad5, 10(8) PFU of Ad5-pIFN-alpha, low- or high-dose Ad5-pIFN-gamma, or a combination of Ad5-pIFN-alpha and low- or high-dose Ad5-pIFN-gamma and challenged all groups with FMDV 1 day later. The control group and the groups inoculated with either Ad5-pIFN-alpha or a low dose of Ad5-pIFN-gamma developed clinical disease and viremia. However, the group that received the combination of both Ad5-IFNs with the low dose of Ad5-pIFN-gamma was completely protected from challenge and had no viremia. Similarly the groups inoculated with the combination of Ad5s with the higher dose of Ad5-pIFN-gamma or with only high-dose Ad5-pIFN-gamma were protected. The protected animals did not develop antibodies against viral nonstructural (NS) proteins, while all infected animals were NS protein seropositive. No antiviral activity or significant levels of IFNs were detected in the protected groups, but there was an induction of some ISGs. The results indicate that the combination of type I and II IFNs act synergistically to inhibit FMDV replication in vitro and in vivo.

Effects of an HMG-CoA reductase inhibitor, simvastatin, on human myeloma cells.

To look for new candidates for agents to use in maintenance therapy for myeloma patients, the growth inhibitory effects of a 3-hydroxy-3-mehtylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin), simvastatin, was analyzed using human myeloma cell lines. Several investigations have indicated growth reduction in certain lineages of cancer cells including one report on myeloma, and inhibitory effects of statins on GTPases and involving MAP-kinases. Most (12 out of 13) myeloma lines examined showed growth inhibition when cultured with various concentrations (1-30 microM) of simvastatin in a dose-dependent manner. Simvastatin in combination with other biological response modifiers such as ATRA or DEX had additional effects on growth. In addition, anti-oxides prevented the simvastatin-induced growth inhibition and apoptosis. Furthermore, myeloma cells treated with simvastatin clearly showed inactivation of various MAP-kinase pathways such as ERK1/2, MEK1/2, JNK, and p38. Based on these findings, statins may be suitable for clinical usage in maintenance therapy for myeloma patients.

Interferon-mediated changes in the expression of CYP1A1 in human B lymphoblastoid (AHH-1 TK +/-) cells.

The expression of constitutive and inducible cytochrome P450s has been shown to be downregulated by interferon through an unknown pretranslational mechanism that depresses the mRNA encoding P450 apoproteins. To establish an association between gene transcription and P450 apoprotein downregulation by interferon, we studied the effect of recombinant interferon (IFN-alpha 2a) on CYP1A1 in human B lymphoblastoid cell lines. The cHoI cell line expresses inducible native CYP1A1, while the genetically engineered derivative h1A1 v2 expresses a noninducible extrachromosomal vector-derived human CYP1A1 cDNA lacking the CYP1A1 promoter region. We characterized CYP1A1 activity, apoprotein, and mRNA by ethoxyresorufin O-deethylase activity, Western immunoblotting, and Northern blot analysis, respectively. In cHoI cells, following induction with dibenz[a,h]anthracene, interferon depressed CYP1A1 apoprotein and mRNA levels by 55 and 76%, respectively, with no detectable changes in enzyme activity. In h1A1 v2, however, interferon increased CYP1A1 activity, apoprotein, and mRNA. The depression of CYP1A1 mRNA and apoprotein levels incHoI cells, in contrast with the increase observed in h1A1 v2 cells, suggests that nuclear mechanisms are essential for interferon-mediated depression of inducible P450s. From our preliminary results we propose that interferon-mediated downregulation of CYP1A1 may result from inhibition of gene transcription.