A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models.

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model.

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Identification and characterization of a nonimmunoglobulin factor in human saliva that inhibits Streptococcus mutans glucosyltransferase.

Saliva contains an array of nonimmunoglobulin defense factors which are thought to contribute to the protection of the hard and soft tissue surfaces of the oral cavity by modulating microbial colonization and metabolism. Here we report the discovery of a putative innate defense factor in human saliva that inhibits the glucosyltransferase (GTF) of Streptococcus mutans, a virulence enzyme involved in oral colonization by this pathogen. The GTF-inhibiting factor (GIF) was initially identified as a nonimmunoglobulin salivary component that interfered with detection of antibodies to the glucan-binding region (GLU) of GTF by an enzyme-linked immunosorbent assay. This inhibitory activity was present in whole saliva and submandibular-sublingual saliva, but it was essentially absent from parotid saliva. GIF inhibited the recognition of S. mutans cell surface-associated GTF by specific antibodies but had no effect on antibodies to other cell surface antigens, suggesting that GIF specifically binds to GTF on S. mutans. GIF purified by size exclusion or affinity chromatography was used for biochemical and functional characterization. Analysis of GIF by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a high-molecular-weight glycoprotein after staining with Coomassie blue or Schiff's reagent. Heating and reduction with 2-mercaptoethanol of GIF resulted in the release of a approximately 58-kDa protein that was identified as alpha-amylase by Western blotting using anti-alpha-amylase antibodies. GLU bound blotted alpha-amylase, suggesting that the latter molecule is the GLU-binding component of the GIF complex. The ability of GTF to synthesize extracellular glucans was inhibited by GIF but not by uncomplexed alpha-amylase or an unrelated high-molecular-weight glycoprotein. In conclusion, our findings demonstrate that in human saliva, there is a high-molecular-weight glycoprotein-alpha-amylase complex which is capable of inhibiting GTF and may contribute to control of S. mutans colonization in the oral cavity.

Enhanced immunogenicity of a genetic chimeric protein consisting of two virulence antigens of Streptococcus mutans and protection against infection.

The saliva-binding region (SBR) of the cell surface antigen I/II (AgI/II) and the glucan-binding region (GLU) of the glucosyltransferase enzyme of Streptococcus mutans have been implicated in the initial adherence of S. mutans to saliva-coated tooth surfaces and the subsequent sucrose-dependent accumulation of S. mutans, respectively. Here, we describe the construction and characterization of a genetic chimeric protein consisting of the two virulence determinants SBR and GLU (SBR-GLU). The effectiveness of this construct in inducing mucosal and systemic immune responses to each virulence determinant following intranasal immunization was compared to that of each antigen alone or an equal mixture of SBR and GLU (SBR+GLU) in a mouse model. Furthermore, the ability of antibodies induced to SBR-GLU to protect against S. mutans infection was also investigated. Immunization of mice with the chimeric protein SBR-GLU resulted in significantly enhanced (P < 0.001) levels of serum immunoglobulin G (IgG) anti-SBR antibody activity compared to those in the SBR and SBR+GLU groups. The SBR-GLU-immunized mice also demonstrated a significant (P < 0.05) increase in salivary and vaginal IgA antibody responses to SBR and GLU. Analysis of the serum IgG subclass responses to SBR in mice immunized with SBR alone indicated a mixed IgG1 and IgG2a response. A preferential IgG1 response compared to an IgG2a anti-GLU response was induced in mice immunized with GLU alone. Similarly, a preferential IgG1 response was also induced to SBR when GLU was present in either a mixed or conjugated form. Finally, a significant reduction (P < 0.05) in S. mutans colonization was observed only in mice immunized with the SBR-GLU chimeric protein. Taken together, our results indicate that the chimeric protein SBR-GLU significantly enhanced mucosal immune responses to SBR and GLU and systemic immune responses to SBR. The ability of SBR-GLU to induce responses effective in protection against colonization of S. mutans suggests its potential as a vaccine antigen for dental caries.