ez-ADiCon: A novel glyco-remodeling based strategy that enables preparation of homogenous antibody-drug conjugates via one-step enzymatic transglycosylation with payload-preloaded bi-antennary glycan complexes.

The conserved N-linked glycan at the Fc domain of recombinant monoclonal antibodies is an attractive target for site-specific payload conjugation for preparation of homogenous antibody-drug conjugates (ADCs). Here, we report a novel ADC constructing strategy, named "ez-ADiCon", that is achieved by one-step enzymatic transglycosylation of a payload-preloaded bi-antennary glycan oxazoline onto a deglycosylated antibody. In this method, a mixture of different glycoforms of the Fc-glycan is replaced with a pre-defined payload-linked glycan. Since two payloads are linked on each donor glycan substrate, efficient conjugation results in a highly homogenous ADC with mostly-four drug molecules per antibody, facilitating hydrophobic interaction chromatography analysis and purification. We validated this conjugation strategy using Monomethyl auristatin E (MMAE) and an anti-Human epidermal growth factor receptor 2 (anti-Her2) antibody as the model ADC components and demonstrated its target-specific in vitro cytotoxicity. Our novel conjugation strategy, ez-ADiCon, provides a new approach for the preparation of next generation ADCs.

Patient-derived monoclonal antibody neutralizes HCV infection in vitro and vivo without generating escape mutants.

In recent years, new direct-acting antivirals for hepatitis C virus (HCV) have been approved, but hepatitis C continues to pose a threat to human health. It is important to develop neutralizing anti-HCV antibodies to prevent medical and accidental infection, such as might occur via liver transplantation of chronic HCV patients and needle-stick accidents in the clinic. In this study, we sought to obtain anti-HCV antibodies using phage display screening. Phages displaying human hepatocellular carcinoma patient-derived antibodies were screened by 4 rounds of biopanning with genotype-1b and -2a HCV envelope E2 protein adsorbed to magnetic beads. The three antibodies obtained from this screen had reactivity against E2 proteins derived from both genotype-1b and -2a strains. However, in epitope analysis, these antibodies did not recognize linear peptides from an overlapping E2 epitope peptide library, and did not bind to denatured E2 protein. In addition, these antibodies showed cross-genotypic neutralizing activity against genotype-1a, -1b, -2a, and -3a cell culture-generated infectious HCV particles (HCVcc). Moreover, emergence of viral escape mutants was not observed after repeated rounds of passaging of HCV-infected cells in the presence of one such antibody, e2d066. Furthermore, injection of the e2d066 antibody into human hepatocyte-transplanted immunodeficient mice inhibited infection by J6/JFH-1 HCVcc. In conclusion, we identified conformational epitope-recognizing, cross-genotypic neutralizing antibodies using phage display screening. Notably, e2d066 antibody did not select for escape mutant emergence in vitro and demonstrated neutralizing activity in vivo. Our results suggested that these antibodies may serve as prophylactic and therapeutic agents.

Metabolites of soil microorganisms modulate amyloid ß production in Alzheimer's neurons.

Microbial flora is investigated to be related with neuropathological conditions in Alzheimer's disease (AD), and is attracting attention as a drug discovery resource. However, the relevance between the soil microbiota and the pathological condition has not been fully clarified due to the difficulty in isolation culture and the component complexity. In this study, we established a library of secondly metabolites produced in microorganism to investigate the potential effect of microorganisms on the production of amyloid ß (Aß), one of the most representative pathogens of AD. We conducted a library screening to quantify Aß and neuronal toxicity by using cortical neurons from human induced pluripotent stem cells (iPSCs) of AD patients after adding secondary metabolites. Screening results and following assessment of dose-dependency identified Verrucarin A, produced in Myrothecium spp., showed 80% decrease in Aß production. Furthermore, addition of Mer-A2026A, produced in Streptomyces pactum, showed increase in Aß42/40 ratio at the low concentration, and decrease in Aß production at the higher concentration. As a result, established library and iPSC-based phenotyping assay clarified a direct link between Aß production and soil microorganisms. These results suggest that Aß-microorganism interaction may provide insight into the AD pathophysiology with potential therapeutics.

Development of a Novel Site-Specific Pegylated Interferon Beta for Antiviral Therapy of Chronic Hepatitis B Virus.

Although nucleot(s)ide analogues and pegylated interferon alpha 2a (PEG-IFN-α2a) can suppress hepatitis B virus (HBV) replication, it is difficult to achieve complete HBV elimination from hepatocytes. A novel site-specific pegylated recombinant human IFN-ß (TRK-560) was recently developed. In the present study, we evaluated the antiviral effects of TRK-560 on HBV replication in vitro and in vivo. In vitro and in vivo HBV replication models were treated with antivirals including TRK-560, and changes in HBV markers were evaluated. To analyze antiviral mechanisms, cDNA microarray analysis and an enzyme-linked immunoassay (ELISA) were performed. TRK-560 significantly suppressed the production of intracellular HBV replication intermediates and extracellular HBV surface antigen (HBsAg) (P < 0.001 and P < 0.001, respectively), and the antiviral effects of TRK-560 were enhanced in combination with nucleot(s)ide analogues, such as entecavir and tenofovir disoproxil fumarate. The reduction in HBV DNA levels by TRK-560 treatment was significantly higher than that by PEG-IFN-α2a treatment both in vitro and in vivo (P = 0.004 and P = 0.046, respectively), and intracellular HBV covalently closed circular DNA (cccDNA) reduction by TRK-560 treatment was also significantly higher than that by PEG-IFN-α2a treatment in vivo (P = 0.0495). cDNA microarrays and ELISA for CXCL10 production revealed significant differences between TRK-560 and PEG-IFN-α2a in the induction potency of interferon-stimulated genes. TRK-560 shows a stronger antiviral potency via higher induction of interferon-stimulated genes and stronger stimulation of immune cell chemotaxis than PEG-IFN-α2a. As HBsAg loss and HBV cccDNA eradication are important clinical goals, these results suggest a potential role for TRK-560 in the development of more effective treatment for chronic hepatitis B infection.

Novel pegylated interferon-ß as strong suppressor of the malignant ascites in a peritoneal metastasis model of human cancer.

Malignant ascites manifests as an end-stage event during the progression of a number of cancers and lacks a generally accepted standard therapy. Interferon-ß (IFN-ß) has been used to treat several cancer indications; however, little is known about the efficacy of IFN-ß on malignant ascites. In the present study, we report on the development of a novel, engineered form of human and murine IFN-ß, each conjugated with a polyethylene glycol molecule (PEG-hIFN-ß and PEG-mIFN-ß, respectively). We provide evidence that these IFN-ß molecules retain anti-viral potency comparable to unmodified IFN-ß in vitro and manifested improved pharmacokinetics in vivo. Interestingly, PEG-mIFN-ß significantly inhibited the accumulation of ascites fluid and vascular permeability of the peritoneal membrane in models of ovarian cancer and gastric cancer cell xenograft mice. We further show that PEG-hIFN-ß directly suppresses VEGF165 -induced hyperpermeability in a monolayer of human vascular endothelial cells and that PEG-mIFN-ß enhanced gene expression for a number of cell adhesion related molecules in mouse vascular endothelial cells. Taken together, these findings unveil a hitherto unrecognized potential of IFN-ß in maintaining vascular integrity, and provide proof-of-mechanism for a novel and long-acting pegylated hIFN-ß for the therapeutic treatment of malignant ascites.

Anti-allergic potential of oligomannose-coated liposome-entrapped Cry j 1 as immunotherapy for Japanese cedar pollinosis in mice.

Administration of oligomannose-coated liposomes (OMLs) in mice can induce Th1 immune responses against antigens entrapped in the OMLs. In the present study, we investigated the anti-allergic effect of treatment with oligomannose-coated liposomes (OMLs) with entrapped Cry j 1, a major allergen of Japanese cedar pollen (Cry j 1/OMLs), in Balb/c mice sensitized with Cry j 1. Pretreatment of unsensitized mice with Cry j 1/OMLs repressed the elevation of total and allergen-specific IgE levels in sera elicited in response to subsequent sensitization with Cry j 1. Cry j 1-specific IgG1 in sera also decreased in Cry j 1/OML-treated mice, while the levels of Cry j 1-specific IgG2a in these mice significantly increased after sensitization with Cry j 1. In addition, Cry j 1/OML-treated mice showed high IFN-gamma production from spleen cells and low IL-4/IFN-gamma and IL-5/IFN-gamma ratios in response to in vitro stimulation with Cry j 1. These results indicate that allergen-specific Th1 immune responses predominate over Th2 responses and control IgE elevation in Cry j 1/OML-treated mice. The anti-allergic effect of Cry j 1/OML determined by suppression of serum IgE levels was also observed in Cry j 1-presensitized mice after challenge with antigen. Thus, Cry j 1/OMLs may be useful for immunotherapeutic control of allergic reactions to Japanese cedar pollinosis.

The combination of type I interferon and ribavirin has an inhibitory effect on mouse hepatitis virus infection.

AIM: To determine the differences in efficacy between therapy using IFN-beta and ribavirin, and using IFN-alpha and ribavirin. METHODS: We studied the effect of combination therapy consisting of IFN-beta and ribavirin on mouse hepatitis virus (MHV) infection in mice. RESULTS: Combination treatment of ribavirin and IFN-alpha was more effective than IFN-alpha mono-treatment in the MHV-mouse system, and combination treatment with ribavirin and IFN-beta was more effective than IFN-beta mono-treatment in the MHV-mouse system. Furthermore, administering IFN-beta once or twice one day before combination treatment using ribavirin and IFN-alpha was more effective than administering IFN-alpha once or twice one day before the combination treatment using ribavirin and IFN-alpha. CONCLUSION: These data indicate that this MHV-infection system is a good animal model to assess anti-HCV activity for therapy using IFN and ribavirin, and suggest that administering IFN-beta before the start of combination therapy with ribavirin and IFN-alpha promotes the therapeutic effects of combination treatment with ribavirin and IFN-alpha ?in chronic hepatitis C patients.

Hemostatic effects of polymerized albumin particles bearing rGPIa/IIa in thrombocytopenic mice.

The recombinant fragment of the platelet membrane glycoprotein Ia/IIa (rGPIa/IIa) was conjugated to the polymerized albumin particles (polyAlb) with the average diameter of 180 nm. The intravenous administration of rGPIa/IIa-polyAlb to thrombocytopenic mice ([platelet] = 2.1+/-0.3 x 10(5) particles/ microL) with three doses of ca. 2.4 x 10(10), 7.2 x 10(10), and 2.4 x 10(11)particles/kg, respectively, significantly reduced their bleeding time to 426+/-71, 378+/-101, and 337+/-46 s, respectively, whereas that of the control groups (PBS) was 730+/-198 s. The injection of rGPIa/IIa-polyAlb (2.4 x 10(11)particles/kg) was approximately equal to the effect of the injection of the mouse platelets at a dose of 2.0 x 10(10) particles/kg. It was confirmed that rGPIa/IIa-polyAlb had a recognition ability against collagen and could contribute to the hemostasis in the thrombocytopenic mice as a platelet substitute.