Deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a VWFcleaving protease, is the key factor in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy. It is well established that ADAMTS13 deficiency results in elevated plasma levels of ultralarge VWF multimers (ULVWF), which are prone to induce platelet aggregation; however, the actual trigger of TTP development remains uncertain. Here we de-

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Obizur is a highly purified recombinant porcine FVIII drug product that has been demonstrated to have a favourable safety and efficacy profile when compared with Hyate:C and can be a valuable treatment option for control of bleeding in AHA patients.

Patients with preexisting anti-adeno-associated virus serotype 8 (AAV8) neutralizing antibodies (NAbs) are currently excluded from AAV8 gene therapy trials. Therefore, the assessment of biologically relevant AAV8-NAb titers is critical for product development in gene therapy. However, standardized assays have not been routinely used to determine anti-AAV8-NAb titers, contributing to a wide range of reported anti-AAV8 prevalence rates. Using a clinical in vitro NAb assay in a separate study, a higher than expected anti-AAV8-NAb prevalence of about 50% was found in international cohorts. This comparative study has a translational character, confirming the biological relevance of anti-AAV8-antibody titers measured by this assay. The significance of low-titer anti-AAV8 NAbs is shown, along with the relevance of the in vitro assay cutoff (1:5) compared with other assays. Importantly, internally standardized reagents and purified AAV8 constructs containing 90% full capsids were used to reduce the effect of empty capsids. It was found that even very low anti-AAV8-NAb titers (<1:5) could efficiently hinder transduction in vivo, demonstrating the importance of sensitive NAb assays for clinical applications. The in vitro NAb assay was found to be more sensitive than an in vivo NAb assay and thus more suitable for patient screening. Additionally, the study showed that anti-AAV8-NAb titers <1:5 were very rare, further supporting the in vitro assay. However, assays using a lower cutoff may still be useful to explain potential variances in transgene expression. These findings support the relevance of the higher than expected prevalence of anti-AAV8 NAbs, highlighting the need for strategies to circumvent preexisting anti-AAV8 NAbs.

Baxter has developed a new recombinant factor IX (rFIX) drug product (BAX326) for treating patients with hemophilia B, or congenital FIX deficiency. An extensive preclinical program evaluated the pharmacokinetics, efficacy, and safety of BAX326 in different species. The efficacy of BAX326 was tested in three mouse models of primary pharmacodynamics: tail-tip bleeding, carotid occlusion, and thrombelastography. The pharmacokinetics was evaluated after a single intravenous bolus injection in mice, rats, and macaques. Toxicity was assessed in rats and macaques, safety pharmacology in rabbits and macaques, and immunogenicity in mice. BAX326 was shown to be efficacious in all three primary pharmacodynamic studies (P ≤ 0.0076). Hemostatic efficacy was dose related and similar for the three lots tested. Pharmacokinetic results showed that rFIX activity and rFIX antigen concentrations declined in a bi-phasic manner, similar to a previously licensed rFIX product. BAX326 was well tolerated in rabbits and macaques at all dose levels; no thrombogenic events and no adverse clinical, respiratory, or cardiovascular effects occurred. BAX326 was also shown to have a similar immunogenicity profile to the comparator rFIX product in mice. These results demonstrate that BAX326 has a favorable preclinical safety and efficacy profile, predictive of a comparable effect to that of the previously licensed rFIX in humans.

Sickle cell disease (SCD) is an inherited red blood cell disorder with a worldwide prevalence. Acute vaso-occlusive crisis (VOC) is the main cause of hospitalization in patients with SCD. Growing evidence highlights the key role of inflammatory vasculopathy in both acute and chronic SCD related clinical manifestations. In a humanized mouse model for SCD, we found an increase of vWF activity and a reduction in ADAMTS13/vWF activity ratio similar to that observed in the human counterpart. rADAMTS13 was administered to humanized SCD mice before exposure to hypoxia/reoxygenation (H/R) stress as model of VOC. In SCD mice, rADAMTS13 reduced H/R induced hemolysis and systemic and local inflammation in lungs and kidneys. rADAMTS13 also diminished H/R induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the first-time evidence that pharmacologic treatment with rADAMTS13 (TAK-755) diminished H/R induced sickle cell related organ damage. Thus, rADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell related acute events.

Gene therapy product release requires reliable and consistent demonstration of biopotency. In hemophilia B vectors, this is usually determined in vivo by measuring the plasma levels of the expressed human factor IX (FIX) transgene product in FIX knockout mice. To circumvent this laborious assay, we developed an in vitro method in which the HepG2 human liver cell line was infected with the vector, and the resulting FIX activity was determined in the conditioned medium using a chromogenic assay. The initial low sensitivity of the assay, particularly toward adeno-associated viral serotype 8 (AAV8), increased approximately 100-fold and allowed linear measurement in a broad range of multiplicities of infection. Statistical parameters indicated high assay repeatability (relative standard deviation (RSD) < 5%) and intra-assay reproducibility (RSD < 20%). To compare the performance of the in vitro and in vivo biopotency assay, we applied statistical analyses including regression techniques and variation decomposition to the results obtained for 25 AAV8-FIX vector lots (BAX 335). These showed a highly significant correlation, with the cell culture-based assay demonstrating less variation than the in vivo test. The in vitro assay thus constitutes a viable alternative to using animals for lot release testing.

Factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. Plasma-derived or recombinant (r) FVIII concentrates are used in patients with hemophilia A to provide a FVIII level sufficient to treat and prevent bleeding episodes. Prophylactic FVIII levels can only be maintained by administering several infusions per week. Extended FVIII circulation times would reduce the frequency of infusions, increase patient compliance, reduce the number of bleeds, and offer the possibility to achieve higher trough levels of FVIII. Prolonged circulation can be achieved by modifying the FVIII molecule with hydrophilic polymers, for example with polysialic acid (PSA). BAX 826, Baxalta's polysialylated FVIII is based on ADVATE, a full length recombinant FVIII molecule with an established extensive safety and efficacy profile. The aim of the presented studies was to assess the pharmacokinetic profile of BAX 826 in hemophilia A mice, wild-type rats, and cynomolgus monkeys. Unmodified rFVIII (ADVATE) was used as the reference compound. Test and reference compounds were administered at the same dose. Hemophilia A mice and Sprague Dawley rats were intravenously injected with BAX 826 at a target dose of 200 IU/kg rFVIII, and blood was sampled pre-dose and 5 min to 48 h after administration. Cynomolgus monkeys received a target dose of 350 IU/kg rFVIII and blood sampled 5 min to 120 h after administration. Citrated plasma was prepared and analyzed for FVIII activity (chromogenic), FVIII antigen (ELISA), and PSA-rFVIII concentration. The primary endpoint was area under the curve from administration time to the last quantifiable time point (AUC0-tlast). Mean residence time (MRT) and systemic clearance (CLs) were also assessed. Unless stated otherwise, results for FVIII activity (mice, monkeys) and FVIII antigen (rats) are presented. In mice, the AUC0-tlast for BAX 826 was 20.6 h*IU/mL, which was 2.4 times larger than for ADVATE (8.71 h*IU/mL); MRT was 10.6 h for BAX 826 and 5.8 h for ADVATE. Clearance was lower for BAX 826 (9.4 vs. 22.3 mL/h/kg). In rats, the AUC0-tlast for BAX 826 was 18.0 h*IU/mL, which was 1.8 times larger than for ADVATE (9.9 h*IU/mL). MRT was 12.5 h for BAX 826 and 4.0 h for ADVATE, and CLs was 5.3 and 28.1 mL/h/kg. In monkeys, the geometric mean of AUC0-tlast was 189.0 h*IU/mL for BAX 826 and 39.6 h*IU/mL for ADVATE. MRT was 23.4 and 10.1 h, and CLs was 2.25 and 6.72 mL/h/kg for BAX 826 and ADVATE, respectively. In monkeys, a baseline FVIII activity level was detected and adequately taken into account in calculating pharmacokinetic parameters. Nevertheless, to better follow the pharmacokinetic profile of BAX 826, the polysialylated rFVIII concentration was also assessed using a PSA specific assay. PSA-FVIII was measured in all animals after administration of BAX 826. In summary, pharmacokinetics studies in three animal species provided evidence that modification of ADVATE with PSA increases circulation time and exposure compared with the unmodified protein. Disclosures Schiviz: Baxalta Innovations GmbH: Employment. Hoebarth:Baxalta Innovations GmbH: Employment. Wolfsegger:Baxalta Innovations GmbH: Employment. Rossato:Baxalta Innovations GmbH: Employment. Weber:Baxalta Innovations GmbH: Employment. Gritsch:Baxalta Innovations GmbH: Employment. Rottensteiner:Baxalta Innovations GmbH: Employment. Turecek:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment. Hoellriegl:Baxalta Innovations GmbH: Employment. Putz:Baxalta Innovations GmbH: Employment.