CSL (Germany)

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3Rguidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

ObjectiveTo evaluate real‐world outcomes with rVIII‐SingleChain and other commonly used recombinant FVIII (rFVIII) products.MethodsHemophilia treatment centers in Germany (n = 21) contributed patient chart data. Inclusion criteria were prophylactic treatment with one of five rFVIII products for ≥8 weeks.ResultsMale patients (n = 225) were included: rVIII‐SingleChain (n = 40), rFVIIIFc (n = 47), octocog alfa (rFVIII; n = 58), octocog alfa (BAY 81‐8973; n = 40), or moroctocog alfa (n = 40). In patients with severe disease (n = 76), 66.6%, 70.0%, 20.0%, 7.7%, and 27.3% were dosed ≤2×/week, respectively. Irrespective of dosing frequency, mean annualized bleed rates (ABRs)/annualized spontaneous bleed rates (AsBRs) were 0.3/0.1, 0.8/0.4, 1.1/0.5, 1.5/0.8, and 1.4/0.6, and mean FVIII consumption (IU/kg/week) was 83.2, 97.2, 92.5, 104.0, and 102.1, respectively. Results for all patients were similar. Of the patients on prophylaxis with prior therapy and after switching to rVIII‐SingleChain (n = 21), mean ABR/AsBRs were 0.7/0.3 and 0.2/0.0, respectively. After switching to rVIII‐SingleChain, mean FVIII consumption reduced (109.4 vs 74.5 IU/kg/week), and percentage of patients dosed ≤2×/week increased (0% to 71.4%).ConclusionsrVIII‐SingleChain prophylaxis provides excellent bleeding protection, with potentially lowest factor consumption among the products assessed. Patients who switched to rVIII‐SingleChain prophylaxis reduced dosing frequency and consumption compared with prior treatment, with similar or potentially lower bleeding rates.

PurposePrimary (PID) and secondary immune deficiencies (SID) represent diverse groups of diagnoses, yet both can be effectively treated with intravenous immunoglobulin (IVIG) replacement therapy. Guidelines for the use of IVIG in SID vary due to the paucity of data. The objective was to analyze available IVIG Privigen® (IgPro10, CSL Behring, Bern, Switzerland) data on Efficiency Index (EI) and pharmacokinetic (PK) parameters in patients with PID and SID. MethodsThree Privigen® studies (NCT00168025, NCT00322556, and the observational study IgPro10_5001) were used to identify patients with PID and SID meeting the qualifying criteria for the PK analysis. PK properties of IVIG were estimated using a population PK model based on a standard two-compartment PK model. Immunoglobulin G (IgG) EI was calculated as the gain in serum IgG level per unit external IgG dose. ResultsA similar IVIG dose-serum IgG concentration relationship was observed in patients with PID (N = 90) and SID (N = 91). IgG EI was inversely proportional to the endogenous IgG concentration and comparable in PID (slope = −1.079) and SID (slope = −2.12). ConclusionsThese findings indicate that the disposition of Privigen® is similar during IgG replacement therapy in PID and SID. The results contribute to the understanding of IVIG treatment of SID and may support an evidence-based approach for the use of IVIG in SID in the future.

Different therapeutic approaches have been used in fetal-neonatal alloimmune thrombocytopenia, but many centers administer immunoglobulin G infusions to the pregnant woman. We studied the effect of maternal antenatal immunoglobulin infusions on fetal platelet counts in pregnancies with fetal alloimmune thrombocytopenia. We retrospectively analyzed the clinical courses of fetuses with fetal alloimmune thrombocytopenia whose mothers were treated with immunoglobulin G infusions in a single center between 1999 and 2005. In a center-specific protocol, weekly maternal immunoglobulin G infusions were given to 25 pregnant women with previously affected neonates and four women with strong platelet antibodies, but no previous history of fetal alloimmune thrombocytopenia; before each infusion diagnostic fetal blood sampling was performed to determine fetal platelet counts and immunoglobulin G levels. There were 30 fetuses with fetal alloimmune thrombocytopenia, confirmed by initial fetal blood sampling showing fetal platelet counts between 4×10(9)/L and 130×10(9)/L and antibody-coated fetal platelets using a glycoprotein specific assay. Despite weekly antenatal maternal immunoglobulin G infusions fetal platelet counts did not change significantly. Maternal and fetal immunoglobulin G levels, measured before every infusion, increased significantly with the number of maternal immunoglobulin G infusions. In this group of fetuses with fetal alloimmune thrombocytopenia no consistent increase of fetal platelets was achieved as a result of regular maternal immunoglobulin G infusions.

PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

1Emory University School of Medicine, Atlanta, Georgia, United States, 2ZLB Behring GmbH, Marburg, Germany

Immunoglobulin G (IgG) concentrates for therapeutic purposes, like passive immunotherapy, supplementation in inherited or acquired deficiencies or immunomodulation, are prepared from multidonor-derived plasma pools. They usually contain varying amounts of dimeric IgG. The essential factor influencing dimer formation is the pool size; in addition, molecular properties of IgG and a variety of production process- and formulation-specific parameters are important. Numerous experimental findings suggest that dimers are predominantly generated by interactions of idiotypic and anti-idiotypic antibodies (Ids, anti-Ids). Ab-inherent crossreactivity, frequency distribution of both the affinities for particular Id-anti-Id interactions and the corresponding dimer concentrations still have to be elucidated. All these parameters influencing molecular features and functional activity of IgG dimers hamper the assay-dependent measurement of biological efficacy and correlation of total IgG content. A more detailed understanding may help to better control the dual nature of dimer-dependent biological activity comprising both undesirable (e.g., hypotension) and desirable effects of dimeric IgG (blockade of the reticuloendothelial system, RES, in immune thrombocytopenic purpura, ITP). These effects are detectable in in vitro and in vivo models and are thought to be of relevance for humans.

Treatment satisfaction of patients with primary immunodeficiency diseases receiving hospital-based intravenous (IVIG) or home-based subcutaneous (SCIG) immunoglobulin infusions requires investigation. Evaluation of the properties and suitability of the Life Quality Index (LQI), as an instrument to assess treatment satisfaction. Patients received weekly SCIG and completed the LQI, two global treatment satisfaction questions and the CHQ-PF50 (children) or the SF-36 (adults) at baseline and 10 months. The LQI was psychometrically evaluated. The LQI comprised four scales: treatment interference (I), therapy related problems (II), therapy setting (III), treatment costs (IV). Convergent/discriminant validity for scales I, II, III was acceptable, for scale IV moderate. CHQ-PF50 scales behavior, bodily pain, global behavior, global health, mental health, parental impact-emotion significantly correlated with LQI scale II, the family activity scale with LQI scales I, III. SF-36 scale bodily pain significantly correlated with scale III. Internal consistency was good for scales I, II, III, but poor for scale IV. Score values significantly increased for scales I, III, IV in patients switching from IVIG to SCIG. Three valid LQI scales were determined. Cost-related questions should be removed due to low reliability. Patients-perceived therapy effectiveness and patient-physician/nurse interaction should be included in the instrument.