Search

Background: Sickle cell disease (SCD) is an inherited red blood cell disorder with a causative substitution in the beta-globin gene that encodes beta-globin in hemoglobin. Furthermore, the ensuing vasculopathy in the microvasculature involves heightened endothelial cell adhesion, inflammation, and coagulopathy, all of which contribute to vaso-occlusive crisis (VOC) and the sequelae of SCD. In particular, dysregulation of the von Willebrand factor (VWF) and a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13) axis has been implicated in human SCD pathology., Objectives: To investigate the beneficial potential of treatment with recombinant ADAMTS13 (rADAMTS13) to alleviate VOC., Methods: Pharmacologic treatment with rADAMTS13 in vitro or in vivo was performed in a humanized mouse model of SCD that was exposed to hypoxia/reoxygenation stress as a model of VOC. Then, pharmacokinetic, pharmacodynamic, and behavioral analyses were performed., Results: Administration of rADAMTS13 to SCD mice dose-dependently increased plasma ADAMTS13 activity, reduced VWF activity/antigen ratios, and reduced baseline hemolysis (free hemoglobin and total bilirubin) within 24 hours. rADAMTS13 was administered in SCD mice, followed by hypoxia/reoxygenation stress, and reduced VWF activity/antigen ratios in parallel to significantly (p < .01) improved recovery during the reoxygenation phase. Consistent with the results in SCD mice, we demonstrate in a human in vitro system that treatment with rADAMTS13 counteracts the inhibitory activity of hemoglobin on the VWF/ADAMTS13-axis., Conclusion: Collectively, our data provide evidence that relative ADAMTS13 insufficiency in SCD mice is corrected by pharmacologic treatment with rADAMTS13 and provides an effective disease-modifying approach in a human SCD mouse model., Competing Interests: Declaration of competing interest S.C., G.S., and H. Gritsch are employees of Baxalta Innovations GmbH, a member of the Takeda group of companies, and are Takeda stock owners. P.R., H. Glantschnig, F.C., M.S., D.V., M.D., B.P., H.R., F.S., and W.H. were employees of Baxalta Innovations GmbH, a member of the Takeda group of companies, at the time of the study. L.D.F. has received grant/research support from Baxalta Innovations GmbH, a Takeda company; Roche; and Agios. E.F. and A.M. have no competing interests to declare., (Copyright © 2022 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Sickle cell disease (SCD) is an inherited red blood cell disorder that occurs worldwide. Acute vaso-occlusive crisis is the main cause of hospitalization in patients with SCD. There is growing evidence that inflammatory vasculopathy plays a key role in both acute and chronic SCD-related clinical manifestations. In a humanized mouse model of SCD, we found an increase of von Willebrand factor activity and a reduction in the ratio of a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13 (ADAMTS13) to von Willebrand factor activity similar to that observed in the human counterpart. Recombinant ADAMTS13 was administered to humanized SCD mice before they were subjected to hypoxia/reoxygenation (H/R) stress as a model of vaso-occlusive crisis. In SCD mice, recombinant ADAMTS13 reduced H/R-induced hemolysis and systemic and local inflammation in lungs and kidneys. It also diminished H/R-induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the firsttime evidence that pharmacological treatment with recombinant ADAMTS13 (TAK-755) diminished H/R-induced sickle cell-related organ damage. Thus, recombinant ADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell-related acute events.

Insufficiency of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif repeats-13) is the cause of thrombotic thrombocytopenic purpura (TTP) and contributes in microangiopathy in sickle cell disease (SCD). Recombinant ADAMTS13 effectively cleaves prothrombotic ultra-large von Willebrand factor (VWF) multimers. It is being tested as replacement therapy for TTP, and at supra-physiologic concentrations, for moderating vaso-occlusive crisis in SCD. Deficiencies of VWF, or concomitant treatment with antithrombotic drugs, could pose risks for increased bleeds in these patient populations. The purpose of the experiments was to evaluate the potential of exaggerated pharmacology and temporary bleeding risks associated with rADAMTS13 administration. We utilized safety studies in monkey and tested the effects of administering maximum-feasible doses of rADAMTS13 on nonclinical safety and spontaneous or aggressive bleeds in the rat model. Evaluation of pharmacokinetics, toxicity profiles, and challenge in a tail-tip bleeding model show that treatment with rADAMTS13 did not increase bleeding tendency, either alone, or in combination with enoxaparin or acetylsalicylic-acid. These novel findings demonstrate absence of rADAMTS13 exaggerated pharmacology without spontaneous or aggravated bleeds even at supra-physiologic (>100-fold) plasma concentrations., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608., (© 2021 by The American Society of Hematology.)

One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A., Competing Interests: A patent application has been submitted by W.X., B.D., and W.C. for the FVIII-X5 (application number W02014209942A1). W.X., B.D., and W.C. hold equity in Ivygen. J. L. is a full-time employee of Baxalta Innovations GmbH, now part of Takeda. F.H., M. K., M.d.l.R., W. H., B.M.R., F. S., and H.R. were employees of Baxalta Innovations GmbH, now part of Takeda, at the time of the study., (© 2020.)

: Accurate monitoring of coagulation, needed for optimal management of patients with haemophilia A with inhibitors, presents a challenge for treating physicians. Although global haemostatic assays may be used in this population, their utility with nonfactor therapies has yet to be established in the clinical setting. The aim of this study was to assess options for potential haemostatic activity monitoring and feasibility for factor VIII (FVIII)-equivalency measurement with a sequence identical analogue (SIA) to emicizumab using different coagulation assays. SIA was analysed using five commercial chromogenic assays and activated partial thromboplastin time (aPTT) assays including clot waveform analysis using five different triggers. Recombinant FVIII served as a comparator in all assays. Thrombin generation in haemophilia A plasma was measured using extrinsic and intrinsic trigger conditions (tissue factor or Factor XIa). Of the five chromogenic assays, a concentration-dependent increase in Factor Xa was observed with one assay, with human Factor IXa and X reagents. The SIA dose-response signal plateaued at therapeutically relevant concentrations and was nonparallel with FVIII reference, thereby not permitting FVIII-equivalence assessment. aPTT varied between reagents, with aPTT normalization occurring at low and below-therapeutic SIA concentrations. SIA [600 nmol/l (90 μg/ml)] only partially restored thrombin generation in individual haemophilia A patient plasma. FVIII-equivalence of SIA could not be determined using standard FVIII protocols and was found to be highly influenced by assay type, analytical conditions and parameters used for calculation. New and/or modified methodology and standard reagents specific for use with nonfactor therapies are required for their utilization in the clinical setting.

Adeno-associated virus serotype 8 (AAV8) gene therapy has shown efficacy in several clinical trials and is considered a highly promising technology to treat monogenic diseases such as hemophilia A and B. However, a major drawback of AAV8 gene therapy is that it can be applied only once because anti-AAV8 immunity develops after the first treatment. Readministration may be required in patients who are expected to need redosing, eg, due to organ growth, or to boost suboptimal expression levels, but no redosing protocol has been established. We have developed a preventive immune-suppressive protocol for a human factor IX (FIX) vector with an intended dose of ~5 × 10 11  vg/kg that inhibits the development of anti-AAV8 neutralizing-antibody (NAb) responses and anti-AAV8 T-cell responses using CTLA4-IgG (abatacept). In a preclinical model, transient treatment with abatacept during initial human FIX gene therapy efficiently inhibited the generation of AAV8-specific cellular and humoral responses, and thus permitted redosing of FIX. Furthermore, our data suggest that by suppression of anti-AAV8 NAb responses after the second higher dose (4 × 10 12  vg/kg) this protocol can be used to enable redosing up to such high doses. An additional advantage of CTLA4-IgG blocking CD28-mediated signals is its potential suppression of AAV8-specific cytotoxic CD8 T-cell responses, which are believed to kill transduced hepatocytes and might interfere with a successful readministration. Redosing protocols using approved drugs would be beneficial for patients because they could effortlessly be applied in clinical trials and enable safe and efficient treatment options for patients undergoing AAV8 gene therapy., (© 2020 Zurich Switzerland. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

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., (© 2020 Shire International GmbH, now part of Takeda, Zurich, Switzerland.)

Background: ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) to regulate its function. Additionally, ADAMTS13 is thought to regulate lateral association of VWF multimers to form fibrillar structures through its free thiols., Objective: The purpose of the present study is to obtain direct evidence for ADAMTS13 to engage in thiol/disulfide exchange reactions., Methods: Covalent complexes between ADAMTS13 and VWF were determined by agarose gel electrophoresis under nonreducing conditions. Free thiols in ADAMST13 were identified by a reversed phase high-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry system., Results: We demonstrate formation of covalent linkage between ADAMTS13 and VWF, which is time, concentration, temperature, and shear dependent. This interaction is independent of proteolytic activity of ADAMTS13 but depends on the C-terminal domains comprising the fifth through eighth thrombospondin type 1 repeats and C1r/C1s, Uegf, Bmp1 (CUB) domains. The interaction can be blocked by thiol-reactive agents, indicating that association is accomplished through disulfide bridge formation. Several partially reduced free thiols are identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent, although a point mutation (C1275S) in ADAMTS13 does not alter its ability to form covalent linkages with VWF. This suggests functionally relevant disulfide plasticity in ADAMTS13. Interestingly, ADAMTS13 also forms homo-oligomers under the same conditions as required for the generation of hetero-oligomeric complexes of ADAMTS13 and VWF., Conclusions: Our results suggest that a dynamic network of free thiols in ADAMTS13 undergoing intra- and inter-molecular redox reactions may add another layer of regulation to VWF function under various conditions., (© 2019 Shire International GmbH. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Extended half-life (EHL) factor therapies are needed to reduce the burden of prophylaxis and improve treatment adherence in patients with hemophilia. BAX 826 is a novel polysialylated full-length recombinant factor VIII [polysialyic acid (PSA) rFVIII] with improved pharmacokinetics (PK), prolonged pharmacology, and maintained safety attributes to enable longer-acting rFVIII therapy. In factor VIII (FVIII)-deficient hemophilic mice, PSArFVIII showed a substantially higher mean residence time (>2-fold) and exposure (>3-fold), and prolonged efficacy in tail-bleeding experiments (48 vs. 30 hours) compared with unmodified recombinant FVIII (rFVIII), as well as a potentially favorable immunogenicity profile. Reduced binding to a scavenger receptor (low-density lipoprotein receptor-related protein 1) and von Willebrand factor (VWF) as well as a largely VWF-independent circulation time in mice provide a rationale for prolonged BAX 826 activity. The significantly improved PK profile versus rFVIII was confirmed in cynomolgus monkeys [mean residence time: 23.4 vs. 10.1 hours; exposure (area under the curve from time 0 to infinity): 206 vs. 48.2 IU/ml⋅h] and is in line with results from rodent studies. Finally, safety and toxicity evaluations did not indicate increased thrombogenic potential, and repeated administration of BAX 826 to monkeys and rats was well tolerated. The favorable profile and mechanism of this novel experimental therapeutic demonstrated all of the requirements for an EHL-rFVIII candidate, and thus BAX 826 was entered into clinical assessment for the treatment of hemophilia A. SIGNIFICANCE STATEMENT: Prolongation of FVIII half-life aims to reduce the burden of prophylaxis and improve treatment outcomes in patients with hemophilia. This study shows that polysialylation of PSArFVIII resulted in prolongations of rFVIII circulation time and procoagulant activity, together with a favorable nonclinical safety profile of the experimental therapeutic., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Efficacy, safety, and manufacturability of therapeutic antibodies are influenced by their biopharmaceutical and biophysical properties. These properties can be optimized by library approaches or rationale protein design. Here, we employed a protein engineering approach to modify the variable domain of the light chain (VL) framework of an oxidized macrophage migration inhibitory factor (oxMIF)-specific antibody. The amendment of the antibody sequence was based on homology to human germline VL genes. Three regions or positions were identified in the VL domain-L1-4, L66, L79-and mutated independently or in combination to match the closest germline V gene. None of the mutations altered oxMIF specificity or affinity, but some variants improved thermal stability, aggregation propensity, and resulted in up to five-fold higher expression. Importantly, the improved biopharmaceutical properties translated into a superior pharmacokinetic profile of the antibody. Thus, optimization of the V domain framework can ameliorate the biophysical qualities of a therapeutic antibody candidate, and as result its manufacturability, and also has the potential to improve pharmacokinetics.

Preexisting immunity against adeno-associated virus (AAV) is a major challenge facing AAV gene therapy, resulting in the exclusion of patients from clinical trials. Accordingly, proper assessment of anti-AAV immunity is necessary for understanding clinical data and for product development. Previous studies on anti-AAV prevalence lack method standardization, rendering the assessment of prevalence difficult. Addressing this need, we used clinical assays that were validated according to guidelines for a comprehensive characterization of anti-AAV1, -AAV2, -AAV5, and -AAV8 immunity in large international cohorts of healthy donors and patients with hemophilia B. Here, we report a higher than expected average prevalence for anti-AAV8 (∼40%) and anti-AAV5 (∼30%) neutralizing antibodies (NAbs), which is supported by strongly correlating anti-AAV IgG antibody titers. A similar anti-AAV8 NAb prevalence was observed in hemophilia B patients. In addition, a high co-prevalence of NAbs against other serotypes makes switching to gene therapy using another serotype difficult. As anti-AAV T cell responses are believed to influence transduction, we characterized anti-AAV T cell responses using interleukin-2 (IL-2) and interferon-γ (IFN-γ) ELISpot assays, revealing a similar prevalence of IFN-γ responses (∼20%) against different serotypes that did not correlate with NAbs. These data, along with the long-term stability of NAbs, emphasize the need to develop strategies to circumvent anti-AAV immunity.

Background: The catalytic domain of ADAMTS13 possesses one Zn 2+ and up to three putative Ca 2+ binding sites and can be inactivated by chelating agents. Although replenishment with an appropriate metallic cation is thought to restore the enzyme's proteolytic activity fully, ADAMTS13 stability in a metal ion-depleting environment has not been explored., Objectives: To address the stability of ADAMTS13 in citrated human plasma., Methods: ADAMTS13 activity was measured using the FRETS-VWF73 fluorogenic assay. The molar ratio of metals bound to ADAMTS13 was determined by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Higher-order structural changes were analyzed using Fourier-transformed infrared spectroscopy and dynamic light scattering., Results: ADAMTS13 was stable at room temperature for up to 24 hours irrespective of the presence of citrate (0.38%). However, at 37°C, citrate caused a time-dependent activity decrease. No ADAMTS13 activity decrease was seen in heparinized plasma, but the addition of citrate again caused ADAMTS13 instability at 37°C. Scavenging of citrate by the addition of Ca 2+ or Zn 2+ prior to but not postincubation prevented the activity decrease of the enzyme. The SEC-ICP-MS analyses showed that ADAMTS13 only bound Zn 2+ and that its reduced activity correlated with a gradual loss of bound Zn 2+ . Concomitant higher-order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less-ordered protein structures., Conclusions: Zn 2+ is required to stabilize ADAMTS13 structure at physiologic temperature, thereby preventing irreversible loss of enzyme activity. This finding is particularly important to consider when using citrated human plasma as a source of ADAMTS13 in clinical settings., (© 2019 Shire International GmbH. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Purpose: To explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation., Methods: Recombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis., Results: FL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-β sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments., Conclusions: These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.

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.

The mechanism of the efficacy of Intravenous immunoglobulins (IVIG) in autoimmune and inflammatory diseases is not well understood. This study aimed at understanding mechanisms of IVIG-mediated suppression of effector cell activities of peripheral blood mononuclear cells (PBMC) in antibody-dependent cellular cytotoxicity (ADCC). We were particularly interested in CD56 dim NK cells, the main ADCC effector cells in PBMC. Exposure of PBMC to IVIG for at least 48 h induced a caspase-3-dependent apoptotic cell death of CD56 dim NK cells without affecting CD56 bright NK cells. Induction of apoptosis in CD56 dim NK cells and concomitant suppression of ADCC effector activities of PBMC was associated with the monomer fraction of IVIG. Moreover, it was independent of IgG sialyation, did not depend on engagement of FcγRIII and could not be mimicked by IVIG (Fab') 2 or IVIG Fc preparations. The described effect could contribute to the reduction of peripheral NK cells observed during IVIG therapy in patients., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The description of hyper-functional factor IX (FIX) Padua triggered the development of BAX 335, an AAV8-based hemophilia B gene therapy vector designed to compensate for low FIX protein expression levels by expressing the FIX Padua variant, thereby reducing the exposure to viral vector. The presence of inactive FIX protein at baseline hindered conventional FIX:Ag ELISA from contributing to a more profound understanding of clinical data from the BAX 335 Phase 1/2 study (ClinicalTrials.gov: NCT01687608). By applying phage display technology, a Fab2 mini-antibody selectively binding to FIX Padua was developed and used to establish a FIX Padua-specific ELISA. The assay adequately performed, utilizing human and monkey plasma samples, and enabled the selective quantification of FIX Padua protein in human plasma samples from the BAX 335 trial. The mini-antibody also allowed the development of a chromogenic FIX Padua-specific activity assay, which adequately performed in human and mouse plasma. Collectively, the isolated FIX Padua-specific mini-antibody enabled the development of transgene-product-specific assays, which should improve the monitoring of hemophilia B gene therapies. The approach applied here for FIX Padua could be leveraged to develop variant-specific activity assays for other therapies where highly active enzymes are instrumental in achieving therapeutic levels of the transgene product.

Essentials Glycosylation heterogeneity of recombinant proteins affects pharmacokinetics and immunogenicity. N-glycomics/glycoproteomics of plasma-derived Factor VIII and 6 recombinant FVIIIs were compared. Depending on cell line, significant differences to plasma-derived FVIII were observed. Recombinant FVIIIs expressed distinct and immunologically relevant epitopes., Summary: Background/Objective Human factor VIII (FVIII) is a plasma glycoprotein, defects of which result in hemophilia A. Current substitution therapy uses FVIII products purified from human plasma or from various cell lines (recombinant FVIII) with different levels of B-domain deletion. Glycosylation is a post-translational protein modification in FVIII that has a substantial influence on its physical, functional and antigenic properties. Variation in glycosylation is likely to be the reason that FVIII products differ in their pharmacokinetics, pharmacodynamics and immunogenicity. However, the literature on FVIII glycosylation is inconsistent, preventing assembly into a coherent model. Seeking to better understand the glycosylation mechanisms underlying FVIII biology, we studied the N-glycosylation of human plasma-derived (pd)FVIII and six rFVIII products expressed in CHO, BHK or HEK cell lines. Methods FVIII samples were subjected to head-to-head detailed glycomic and glycoproteomic characterization using a combination of MALDI-MS and MS/MS, GC-MS and UPLC-UV-MS E technologies. Results/Conclusion The results of our study detail the N-glycan repertoire of pdFVIII to an unprecedented level, and for the first time, provide evidence of N-glycolylneuraminic acid (NeuGc) found on pdFVIII. Although site-specific glycosylation of rFVIII proved consistent with pdFVIII regardless of the expression system, the entire N-glycan content of each sample appeared significantly different. Although the proportion of biologically important epitopes common to all samples (i.e. sialylation and high-mannose) varied between samples, some recombinant products expressed distinct and immunologically relevant epitopes, such as LacdiNAc (LDN), fucosylated LacdiNAc (FucLDN), NeuGc, Lewis X/Y and Gal α1,3 Gal epitopes. rFVIII expressed in HEK cells showed the greatest glycomic differences to human pdFVIII., (© 2018 Shire International GmbH. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Essentials Patients with hemophilia A and inhibitors receiving emicizumab experience breakthrough bleeding. Safety concerns may exist when combining emicizumab with bypassing agents. Combined bypassing agent and bispecific antibody increased thrombin generation up to 17-fold. Thrombotic effects should be considered when combining emicizumab with plasma bypassing agent., Summary: Background Investigational non-factor products such as emicizumab offer a treatment option for patients with hemophilia and inhibitors. However, their mechanism of action raises questions regarding safety when they are combined with treatments for breakthrough bleeding. Objectives To evaluate in vitro thrombin generation (TG) and clot formation for combinations of activated prothrombin complex concentrate (aPCC), recombinant activated factor VII (rFVIIa), and a sequence-identical analog of emicizumab (SIA). Methods Therapeutic concentrations of SIA (20-600 nm) alone or with aPCC (0.05-1 U mL -1 ), isolated aPCC components or rFVIIa (0.88-5.25 μg mL -1 ) were tested for TG and compared with reference ranges for healthy donor plasma. Coagulation of FVIII-inhibited blood was determined with a widely established method, i.e. rotational thromboelastometry (ROTEM), and confirmed with the Total Thrombus-formation Analysis System. Results and conclusions SIA (600 nm) or aPCC (0.5 U mL -1 ) alone resulted in peak thrombin levels of 21.4 nm and 38.6 nm, respectively, both of which are lower than normal (83.7 ± 29.8 nm). SIA plus aPCC (0.5 U mL -1 ) increased the peak thrombin level 17-fold over SIA alone, exceeding the reference plasma value by 4.2-fold. This hypercoagulable effect occurred with 600 nmSIA combined with as little as 0.25 U mL -1 aPCC, confirmed by ROTEM. FIX was the main driver for enhanced TG. SIA plus rFVIIa (1.75 μg mL -1 ) induced a 1.8-fold increase in the peak thrombin level in platelet-rich plasma, but it did not reach the normal range. These in vitro experiments demonstrate excessive TG after administration of a combination of aPCC and SIA at clinically relevant doses. Careful judgement may be required when breakthrough bleeding is treated in patients receiving emicizumab., (© 2018 Shire International GmbH. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Essentials Aggregation is a critical quality attribute of protein therapeutics influencing immunogenicity. Aggregates and subvisible particles in 9 recombinant factor VIII (rFVIII) products were analyzed. Major differences in aggregate and particle concentrations were detected after reconstitution. rFVIII product quality determined aggregation propensity under use-relevant stress., Summary: Background Recombinant protein technologies have facilitated the development of novel factor VIII (FVIII) therapeutics with improved production efficiency, potency and half-live, and a low risk of viral transmission. The increasing number of recombinant FVIII (rFVIII) products and information on their efficacy, safety and cost allow patients and healthcare professionals to adjust treatment to individual needs. Nonetheless, 20-32% of previously untreated patients with severe hemophilia A develop inhibitory antibodies to rFVIII following treatment. The root cause of the immunogenicity of rFVIII products is not well understood. Data for human interferon and human insulin products suggest that critical quality parameters such as soluble protein aggregates (SPAs) and subvisible particles (SVPs) influence the immunogenicity of protein therapeutics. Therefore, we analyzed SPA and SVP concentrations in commercially available rFVIII products and determined how these parameters change upon exposure of rFVIII products to relevant stress conditions. Objectives Compare critical quality parameters such as SPA and SVP concentrations in rFVIII products under intended use and use-relevant stress conditions. Methods Nine rFVIII products (≥ 3 lots each) were analyzed by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) and flow cytometry-based particle analysis. Results/conclusions SPAs and SVPs were present at different concentrations in all freshly reconstituted rFVIII products: SPA concentrations ranged from 0.2% to 11.6%; SVPs were 0.7 × 10 6 to 114.0 × 10 6 / 1000 IU. Under use-relevant stress conditions (agitation and shear stress) the products formed additional SPAs and SVPs to different degrees. The collected data indicate that product quality determines its propensity to form SVPs and SPAs, and highlights differences between marketed rFVIII products., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory and tumor-promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy and diseased subjects. In this article, we report that cysteine 81 within MIF serves as a "switch cysteine" for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol reactive agents leads to significant structural rearrangements of the protein, resulting in a decreased β-sheet content and an increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adopts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF, and formation of oxMIF leads to a physiologically relevant, i.e., enzymatically active, state.

New therapeutic agents are needed to overcome the toxicity and suboptimal efficacy observed in current treatment of glomerulonephritis (GN). BaxB01 is a fully human monoclonal antibody targeting a disease-related immunologically distinct isoform of Macrophage migration Inhibitory Factor (MIF), designated oxidized MIF (oxMIF) and locally expressed in inflammatory conditions. We report the pharmacokinetic profile of BaxB01, and its dose and exposure-related disease-modifying activity in experimentally induced rat GN. BaxB01 bound to rat oxMIF with high affinity and reduced rat macrophage migration in vitro. After intravenous administration in rats, BaxB01 demonstrated favorable pharmacokinetics, with a half-life of up to nine days. Disease modification was dose-related (≥ 10mg/kg) as demonstrated by significantly reduced proteinuria and diminished histopathological glomerular crescent formation. Importantly, a single dose was sufficient to establish an exposure-related, anti-inflammatory milieu via amelioration of glomerular cellular inflammation. Pharmacodynamic modeling corroborated these findings, consistently predicting plasma exposures that were effective in attenuating both anti-inflammatory activity and reducing loss of kidney function. This pharmacologic benefit on glomerular function and structure was sustained during established disease, while correlation analyses confirmed a link between the antibody's anti-inflammatory activity and reduced crescent formation in individual rats. Finally, safety assessment in rats showed that the experimental therapeutic was well tolerated without signs of systemic toxicity or negative impact on kidney function. These data define therapeutically relevant exposures correlated with mechanism-based activity in GN, while toxicological evaluation suggests a large therapeutic index and provides evidence for achieving safe and effective exposure to a MIF isoform-directed therapeutic in nephritis-associated disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large-scale manufacturing and raises concerns for in vivo applications, because particle components and contaminants may trigger immune responses. Here, we show that producer cell-derived polymorphic class-I major histocompatibility complexes (MHC-I) are incorporated into the LV surface and trigger allogeneic T-cell responses. By disrupting the beta-2 microglobulin gene in producer cells, we obtained MHC-free LV with substantially reduced immunogenicity. We introduce this targeted editing into a novel stable LV packaging cell line, carrying single-copy inducible vector components, which can be reproducibly converted into high-yield LV producers upon site-specific integration of the LV genome of interest. These LV efficiently transfer genes into relevant targets and are more resistant to complement-mediated inactivation, because of reduced content of the vesicular stomatitis virus envelope glycoprotein G compared to vectors produced by transient transfection. Altogether, these advances support scalable manufacturing of alloantigen-free LV with higher purity and increased complement resistance that are better suited for in vivo gene therapy., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Wall shear rates are critical for a broad variety of fluidic phenomena and are taken into account in nearly every experimental or simulation study. Generally, shear rates are not observable directly but rather derived from other parameters such as pressure and flow, often assuming somehow idealized systems. However, there is a biological system which is able to constantly measure the wall shear as a part of a regulatory circuit: The blood circulation system takes advantage of shear rate sensor (protein)molecules (multimeric forms of von Willebrand Factor, VWF), which are dissolved in the blood plasma and dramatically change their conformation under shear conditions. The conformational changes are accompanied by several functional variations and therefore interplay with the regulation of the coagulation system. In this study, we use a recombinantly produced and therefore well-defined multimeric form of VWF as a sensor which directly responds to shear rates. Shear rates, up to 32.000 s -1 , were obtained using a kind of micro-plate-to-plate rheometer capable of adsorbing shear-stretched VWF oligomeric molecules on a surface to conserve their differently stretched conformation and so allow detection of their elongation by atomic force microscopy. The laminar flow in this geometrically simple device has been characterized by adopting classical fluid dynamical models, in order to ensure well-known, stable shear rates which could be correlated quantitatively with an observed stretching of sensor molecules.

Von Willebrand factor, an ultralarge concatemeric blood protein, must bind to platelet GPIbα during bleeding to mediate hemostasis, but not in the normal circulation to avoid thrombosis. Von Willebrand factor is proposed to be mechanically activated by flow, but the mechanism remains unclear. Using microfluidics with single-molecule imaging, we simultaneously monitored reversible Von Willebrand factor extension and binding to GPIbα under flow. We show that Von Willebrand factor is activated through a two-step conformational transition: first, elongation from compact to linear form, and subsequently, a tension-dependent local transition to a state with high affinity for GPIbα. High-affinity sites develop only in upstream regions of VWF where tension exceeds ~21 pN and depend upon electrostatic interactions. Re-compaction of Von Willebrand factor is accelerated by intramolecular interactions and increases GPIbα dissociation rate. This mechanism enables VWF to be locally activated by hydrodynamic force in hemorrhage and rapidly deactivated downstream, providing a paradigm for hierarchical mechano-regulation of receptor-ligand binding.Von Willebrand factor (VWF) is a blood protein involved in clotting and is proposed to be activated by flow, but the mechanism is unknown. Here the authors show that VWF is first converted from a compact to linear form by flow, and is subsequently activated to bind GPIbα in a tension-dependent manner.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, which was shown to be upregulated in cancers and to exhibit tumor promoting properties. Unlike other cytokines, MIF is ubiquitously present in the circulation and tissue of healthy subjects. We recently described a previously unrecognized, disease-related isoform of MIF, designated oxMIF, which is present in the circulation of patients with different inflammatory diseases. In this article, we report that oxMIF is also linked to different solid tumors as it is specifically expressed in tumor tissue from patients with colorectal, pancreatic, ovarian and lung cancer. Furthermore, oxMIF can be specifically targeted by a subset of phage display-derived fully human, monoclonal anti-MIF antibodies (mAbs) that were shown to neutralize pro-tumorigenic activities of MIF in vivo. We further demonstrate that anti-oxMIF mAbs sensitize human cancer cell lines (LNCaP, PC3, A2780 and A2780ADR) to the action of cytotoxic drugs (mitoxantrone, cisplatin and doxorubicin) in vitro and in an A2780 xenograft mouse model of ovarian cancer. We conclude that oxMIF is the disease related isoform of MIF in solid tumors and a potential new diagnostic marker and drug target in cancer.

Purpose: Recent findings demonstrated anti-PEG antibody formation in some healthy individuals and patients who have not received PEGylated biotherapeutics. Some of these findings evoked criticism because of shortcomings in the antibody assays used. To better understand this topic, we established robust antibody analytics and screened two cohorts of healthy individuals and one cohort of hemophilia patients for the expression of anti-PEG antibodies., Methods: A flow cytometry approach and a fully validated ELISA platform were established to detect specific anti-PEG antibodies. Immunohistochemistry was used to test for potential binding of anti-PEG antibodies to human tissues., Results: IgM and/or IgG anti-PEG antibodies are expressed by some healthy individuals and by some patients with hemophilia who have not received PEGylated biotherapeutics. These antibodies can be either transient or persistent and recognize PEGs of different sizes with or without terminal methoxy groups. Age and location of healthy individuals influence the prevalence of IgG but not of IgM antibodies. Anti-PEG antibodies do not cross-react with human tissues supporting the safety of the antibodies., Conclusion: We confirm that some healthy individuals and some patients with hemophilia express specific antibodies against PEG which are not associated with any pathology and do not bind to human tissues.

Unlabelled: Essentials ADAMTS-13-deficiency is a cause of thrombotic thrombocytopenic purpura (TTP). Preclinical safety of recombinant human ADAMTS-13 (BAX930) was shown in animal models. Preclinical efficacy of BAX930 was shown in a mouse model of TTP. BAX930 showed advantageous efficacy over fresh frozen plasma, the current standard of care. Click to hear Dr Cataland and Prof. Lämmle present a seminar on Thrombotic Thrombocytopenic Purpura (TTP): new Insights in Pathogenesis and Treatment Modalities., Summary: Background Thrombotic thrombocytopenic purpura (TTP) is a rare blood disorder characterized by microthrombosis in small blood vessels of the body, resulting in a low platelet count. Baxalta has developed a new recombinant ADAMTS-13 (rADAMTS-13) product (BAX930) for on-demand and prophylactic treatment of patients with hereditary TTP (hTTP). Objectives To evaluate the pharmacokinetics, efficacy and safety of BAX930 in different species, by use of an extensive preclinical program. Methods The prophylactic and therapeutic efficacies of BAX930 were tested in a previously established TTP mouse model. Pharmacokinetics were evaluated after single intravenous bolus injection in mice and rats, and after repeated dosing in cynomolgus monkeys. Toxicity was assessed in rats and monkeys, safety pharmacology in monkeys, and local tolerance in rabbits. Results BAX930 was shown to be efficacious, as demonstrated by a stabilized platelet count in ADAMTS-13 knockout mice that were thrombocytopenic when treated. Prophylactic efficacy was dose-dependent and comparable with that achieved by treatment with fresh frozen plasma, the mainstay of hTTP treatment. Therapeutic efficacy was treatment interval-dependent. Safety pharmacology evaluation did not show any deleterious effects of BAX930 on cardiovascular and respiratory functions in monkeys. The compound's pharmacokinetics were similar and dose-proportional in mice, rats, and monkeys. BAX930 was well tolerated in rats, monkeys, and rabbits, even at the highest doses tested. Conclusions These results demonstrate that BAX930 has a favorable preclinical profile, and support the clinical development of rADAMTS-13 for the treatment of hTTP., (© 2016 International Society on Thrombosis and Haemostasis.)

Neutralizing autoantibodies against factor VIII (FVIII), also called FVIII inhibitors, are the cause of acquired hemophilia A (AHA). They are quantified in the Bethesda assay or Nijmegen-modified Bethesda assay by their ability to neutralize FVIII in normal human plasma. However, FVIII inhibitors do not represent the whole spectrum of anti-FVIII autoantibodies. Here, we studied isotypes, immunoglobulin G subclasses, and apparent affinities of anti-FVIII autoantibodies to assess their prognostic value for the outcome in AHA. We analyzed baseline samples from patients enrolled in the prospective GTH-AH 01/2010 study. Our data suggest that anti-FVIII immunoglobulin A (IgA) autoantibodies are predictors of poor outcome in AHA. Anti-FVIII IgA-positive patients achieved partial remission similar to anti-FVIII IgA-negative patients but had a higher risk of subsequent recurrence. Consequently, IgA-positive patients achieved complete remission less frequently (adjusted hazard ratio [aHR], 0.35; 95% confidence interval [CI], 0.18-0.68; P < .01) and had a higher risk of death (aHR, 2.62; 95% CI, 1.11-6.22; P < .05). Anti-FVIII IgA was the strongest negative predictor of recurrence-free survival after achieving partial remission and remained significant after adjustment for baseline demographic and clinical characteristics. In conclusion, anti-FVIII IgA represents a potential novel biomarker that could be useful to predict prognosis and tailor immunosuppressive treatment of AHA., (© 2016 by The American Society of Hematology.)

Rapid vascular recanalization forms the basis for successful treatment of cerebral ischemia. Currently, tissue plasminogen activator (t-PA) is the only approved thrombolytic drug for ischemic stroke. However, t-PA does not always result in efficient thrombus dissolution and subsequent blood vessel recanalization. To better understand thrombus composition, we analyzed thrombi retrieved from ischemic stroke patients and found a distinct presence of von Willebrand factor (VWF) in various samples. Thrombi contained on average 20.3% ± 10.1% VWF, and this was inversely correlated with thrombus red blood cell content. We hypothesized that ADAMTS13 can exert a thrombolytic effect in VWF-containing thrombi in the setting of stroke. To test this, we generated occlusive VWF-rich thrombi in the middle cerebral artery (MCA) of mice. Infusion of t-PA did not dissolve these MCA occlusions. Interestingly, administration of ADAMTS13 5 minutes after occlusion dose-dependently dissolved these t-PA-resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes (P < .005). Delayed ADAMTS13 administration 60 minutes after occlusion was still effective but to a lesser extent (P < .05). These data show for the first time a potent thrombolytic activity of ADAMTS13 in the setting of stroke, which might become useful in treatment of acute ischemic stroke., (© 2016 by The American Society of Hematology.)

Tissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 µM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2-1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1-150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25-0.5 nM TFPI) inhibit FXa with a t1/2 between 3-15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin motif repeats 13) has antithrombotic properties because it cleaves von Willebrand factor (VWF) in smaller, less active multimers. The aim of our study was to investigate prospectively the association between ADAMTS13 activity and ischemic stroke. We included 5941 individuals ≥55 years without a history of stroke or transient ischemic attack (TIA) of the Rotterdam Study, a population-based cohort study. ADAMTS13 activity was measured at inclusion with the FRETS-VWF73 assay and VWF antigen (VWF:Ag) levels by enzyme-linked immunosorbent assay. We assessed the association among ADAMTS13 activity, VWF:Ag levels, and ischemic stroke by Cox proportional hazard analysis. The added value of ADAMTS13 activity above the traditional risk factors for ischemic stroke risk prediction was examined by the C-statistic and the net reclassification improvement index (NRI). All individuals were followed for incident stroke or TIA. Over a median follow-up time of 10.7 years (56,403 total person-years), 461 participants had a stroke, 306 of which were ischemic. After adjustment for cardiovascular risk factors, individuals with ADAMTS13 activity in the lowest quartile had a higher risk of ischemic stroke (absolute risk, 7.3%) than did those in the reference highest quartile (absolute risk, 3.8%; hazard ratio, 1.65; 95% confidence interval [CI], 1.16-2.32). Adding ADAMTS13 to the model in prediction of ischemic stroke, increased the C-statistic by 0.013 (P = .003) and provided 0.058 (95% CI, -0.002 to 0.119) NRI. Low ADAMTS13 activity is associated with the risk of ischemic stroke and improves the accuracy of risk predictions for ischemic stroke beyond traditional risk factors., (© 2015 by The American Society of Hematology.)

Objective: The metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) regulates the size of von Willebrand factor multimers. A deficiency in ADAMTS13 activity is associated with the life-threatening disease thrombotic thrombocytopenic purpura (TTP). The vast majority of patients have acquired TTP, where circulating anti-ADAMTS13 autoantibodies are causative for the decreased ADAMTS13 activity. Current treatment consists of plasma exchange, but improved therapies are highly warranted., Approach and Results: We have developed a new rat model mimicking various aspects of acquired TTP to investigate the therapeutic efficacy of human recombinant ADAMTS13. A polyclonal antibody against ADAMTS13 completely blocked endogenous rat ADAMTS13 activity in Sprague-Dawley rats. When TTP was triggered using recombinant von Willebrand factor, the animals displayed severe TTP-like symptoms, such as thrombocytopenia, hemolytic anemia, and von Willebrand factor-rich thrombi in the kidneys and brain. Subsequent injection of 400, 800, or 1600 U/kg recombinant ADAMTS13 prevented full development of these symptoms. Analysis of plasma samples confirmed that recombinant ADAMTS13 was able to override circulating anti-ADAMTS13 inhibitory antibodies, resulting in restoration of ADAMTS13 activity and degradation of ultralarge von Willebrand factor multimers., Conclusions: Recombinant ADAMTS13 was shown to be effective in averting severe acquired TTP-like symptoms in rats and holds promising value for the treatment of this severe and life-threatening disease in humans., (© 2015 American Heart Association, Inc.)

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is caused by an autoantibody-mediated deficiency of the von Willebrand factor-cleaving protease ADAMTS-13. Acute episodes of the disease are treated with a combination of immunosuppression and repeated cycles of plasma exchange to remove anti-ADAMTS-13 autoantibodies and, at the same time, replenish functional ADAMTS-13. Although this is often effective, the mortality rate has remained between 10% and 20%, highlighting the need for safer treatment options., Objectives: We previously showed that, in vitro, human recombinant ADAMTS-13 (rADAMTS-13) is able to override neutralizing antibodies and restore ADAMTS-13 activity in plasma from patients with acquired TTP. In the present study, we assessed the in vivo feasibility of this strategy by using a rat model., Methods: Wild-type rats were adjusted to an ADAMTS-13 inhibitor (inhibitor) titer of ~ 10 BU mL(-1) with goat anti-ADAMTS-13 IgG, and treated with increasing doses of rADAMTS-13. Blood samples were drawn and analyzed for ADAMTS-13-specific parameters, including FRETS-VWF73 activity, inhibitor, and ADAMTS-13-specific immune complexes (ICs). The pharmacokinetics of ADAMTS-13 activity and inhibitors were evaluated., Results: Administration of inhibitor titer-adjusted doses of rADAMTS-13 to inhibitor-treated rats predictably restored activity. Inhibitors were readily neutralized through formation of ADAMTS-13-specific ICs, which were cleared at a higher rate than the free inhibitor. Surplus protease was enzymatically active in plasma, and showed similar pharmacokinetics to ADAMTS-13 in not inhibitor-treated rats., Conclusions: Defined doses of rADAMTS-13 neutralized circulating anti-ADAMTS-13 antibodies and enabled reconstitution of ADAMTS-13 activity in plasma in our model, indicating that the protease may be a promising candidate for further exploration in treating acute episodes of acquired TTP., (© 2015 International Society on Thrombosis and Haemostasis.)

Vital functions of mammals are only possible due to the behavior of blood to coagulate most efficiently in vessels with particularly high wall shear rates. This is caused by the functional changes of the von Willebrand Factor (VWF), which mediates coagulation of blood platelets (primary hemostasis) especially when it is stretched under shear stress. Our data show that shear stretching also affects other functions of VWF: Using a customized device to simulate shear conditions and to conserve the VWF molecules in their unstable, elongated conformation, we visualize at single molecule level by AFM that VWF is preferentially cleaved by the protease ADAMTS13 at higher shear rates. In contrast to this high shear-rate-selective behavior, VWF binds FVIII more effectively only below a critical shear rate of ∼30.000 s(-1), indicating that under harsh shear conditions FVIII is released from its carrier protein. This may be required to facilitate delivery of FVIII locally to promote secondary hemostasis.

Background: Plasma exchange (PE) is the first-line treatment for primary acquired thrombotic thrombocytopenic purpura (aTTP) with severe deficiency of ADAMTS13 activity (ADAMTS13:AC). Some patients are poor responders to PE, raising concern over multiple pathogenetic pathways., Study Design and Methods: Based on 52 aTTP patients in our national cohort study, we monitored plasma levels of ADAMTS13, clinical and laboratory findings, and outcomes. In a representative poor responder to PE, we examined an ADAMTS13 inhibitor (ADAMTS13:INH) complex in plasma milieu, by means of a large-pore isoelectric focusing (IEF) analysis., Results: Of 52 aTTP patients, 20 were good responders and 32 were poor responders. In the latter group, plasma ADAMTS13:AC levels never increased to more than 10% of normal during 14 days after PE initiation. Mean (±SD) plasma ADAMTS13:INH titers (Bethesda unit/mL) were 5.7 (±4.5) before PE, but decreased to 1.4 (±0.8) on the fourth PE day and then remarkably increased to 14.8 (±10.0) on the 10th PE day, termed "inhibitor boosting," and then slowly decreased to undetectable level over 1 month. On admission, none of the routinely available clinical and laboratory markers differentiated these two groups. However, elevated pre-PE levels of ADAMTS13:INH were correlated with a poor response. We visualized an ADAMTS13:INH (immunoglobulin G) complex in a patient plasma by an IEF analysis and found proteolytic fragment of ADAMTS13 antigen by a two-dimensional IEF and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis., Conclusion: Findings from this cohort of aTTP patients demonstrated that inhibitor boosting often occurs in aTTP patients in Japan. Poor responders could be predicted by elevated pre-PE ADAMTS13:INH levels on admission, but not by routinely collected clinical or laboratory data., (© 2015 AABB.)

Unlabelled: BACKGROUND &, Methods: Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS &, Discussion: The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures., Conclusions: From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

Purpose: Sub-visible particles were shown to facilitate unwanted immunogenicity of protein therapeutics. To understand the root cause of this phenomenon, a comprehensive analysis of these particles is required. We aimed at establishing a flow-cytometry-based technology to analyze the amount, size distribution and nature of sub-visible particles in protein solutions., Methods: We adjusted the settings of a BD FACS Canto II by tuning the forward scatter and the side scatter detectors and by using size calibration beads to facilitate the analysis of particles with sizes below 1 μM. We applied a combination of Bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and DCVJ (9-(2,2-dicyanovinyl)julolidine) to identify specific characteristics of sub-visible particles., Results: The FACS technology allows the analysis of particles between 0.75 and 10 μm in size, requiring relatively small sample volumes. Protein containing particles can be distinguished from non-protein particles and cross-β-sheet structures contained in protein particles can be identified., Conclusions: The FACS technology provides robust and reproducible results with respect to number, size distribution and specific characteristics of sub-visible particles between 0.75 and 10 μm in size. Our data for number and size distribution of particles is in good agreement with results obtained with the state-of-the-art technology micro-flow imaging.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy., (Copyright © 2015 by The American Association of Immunologists, Inc.)

FVIII is a multi-domain protein organized in a heavy and a light chain, and a B-domain whose biological function is still a matter of debate. The 3D structure of a B-domain-deleted FVIII variant has been determined by X-ray crystallography, leaving unexplained the functional nature of the flexible B-domain which could play an important role in the structure-function relationship since it is removed during the activation process. To obtain clues on the function of the B-domain, the morphology of full-length FVIII and its isolated domains was determined in the absence or presence of Ca(2+). Recombinant full-length FVIII, the purified heavy chain, light chain and B-domain as well as B-domain-deleted rFVIII were analysed in buffers of different Ca(2+) concentrations by atomic force microscopy. In the absence of Ca(2+), FVIII appeared as a globular molecule, whereas at high amounts of Ca(2+) up to 50-nm long tail structures emerged. These tails could be identified as unravelled B-domains, as images of isolated B-domains showed the same morphology and heavy chains which include the B-domain were also rich of tails, whereas the isolated light chains and B-domain-deleted FVIII lacked any deviation from a globular shape. The images further suggested that the B-domain interacts with the light chain particularly at low Ca(2+) concentrations. Our results show a Ca(2+)-regulated conformational change of the B-domain in the context of full-length rFVIII. As the B-domain tightly associated with the core of the FVIII molecule under low Ca(2+)-concentrations, a stabilizing function on FVIII under non-activating conditions may be proposed.

Recently, lower thrombin generation has been associated with excess bleeding post-cardiopulmonary bypass (CPB). Therefore, treatment to correct thrombin generation is a potentially important aspect of management of bleeding in this group of patients. The objective of the present study was to investigate the effects of fresh frozen plasma (FFP), recombinant factor VIIa (rFVIIa), prothrombin complex concentrate (PCC) and tissue factor pathway inhibitor (TFPI) inhibition on thrombin generation when added ex vivo to the plasma of patients who had undergone cardiac surgery requiring CPB. Patients undergoing elective cardiac surgery were recruited. Blood samples were collected before administration of heparin and 30 min after its reversal. Thrombin generation was measured in the presence and absence of different concentrations of FFP, rFVIIa, PCC and an anti-TFPI antibody. A total of 102 patients were recruited. Thrombin generation following CPB was lower compared with pre-CPB (median endogenous thrombin potential pre-CPB 339 nmol/l per min, post-CPB 155 nmol/l per min, P < 0.0001; median peak thrombin pre-CPB 35 nmol/l, post-CPB 11 nmol/l, P < 0.0001). Coagulation factors and anticoagulants decreased, apart from total TFPI, which increased (55-111 ng/ml, P < 0.0001), and VWF (144-170 IU/dl, P < 0.0001). Thrombin generation was corrected to pre-CPB levels by the equivalent of 15 ml/kg FFP, 45 μg/kg rFVIIa and 25 U/kg of PCC. Inhibition of TFPI resulted in an enhancement of thrombin generation significantly beyond pre-CPB levels. This study shows that FFP, rFVIIa, PCC and inhibition of TFPI correct thrombin generation in the plasma of patients who have undergone surgery requiring CPB. Inhibition of TFPI may be a further potential therapeutic strategy for managing bleeding in this group of patients.

Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nonacog gamma is a new recombinant factor IX to treat factor IX deficiency. It is indicated for control of bleeding episodes, perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with hemophilia B. Nonacog gamma was first approved in the USA in June 2013 under the trade name RIXUBIS followed by market approvals in Australia and the EU in 2014, and marketing authorization decision is pending in Japan. Nonacog gamma is derived from a recombinant Chinese hamster ovary cell line using a state of the art biotechnological manufacturing process. Recombinant factor IX is produced by Baxter's protein-free fermentation technology, which was first developed for ADVATE. The product is purified and formulated in the absence of any human or animal-derived protein. Nonacog gamma was characterized both in comprehensive in vitro and in vivo non-clinical studies as well as in an extensive clinical trial program.

Recently, we reported that distinct immunoglobulin (Ig) isotypes and IgG subclasses of factor VIII (FVIII)-specific antibodies are found in different cohorts of patients with hemophilia A and in healthy individuals. Prompted by these findings, we further investigated the distinguishing properties among the different populations of FVIII-specific antibodies. We hypothesized that the affinity of antibodies would discriminate between the neutralizing and nonneutralizing antibodies found in different study cohorts. To test this idea, we established a competition-based enzyme-linked immunosorbent assay technology to assess the apparent affinities for each isotype and IgG subclass of FVIII-specific antibodies without the need for antibody purification. We present a unique data set of apparent affinities of FVIII-specific antibodies found in healthy individuals, patients with congenital hemophilia A with and without FVIII inhibitors, and patients with acquired hemophilia A. Our data indicate that FVIII-specific antibodies found in patients with FVIII inhibitors have an up to 100-fold higher apparent affinity than that of antibodies found in patients without inhibitors and in healthy individuals. High-affinity FVIII-specific antibodies could be retrospectively detected in longitudinal samples of an individual patient with FVIII inhibitors 543 days before the first positive Bethesda assay. This finding suggests that these antibodies might serve as potential biomarkers for evolving FVIII inhibitor responses., (© 2015 by The American Society of Hematology.)

Fucoidans are complex sulfated polysaccharides extracted from brown algae. Depending on the concentration, they have been shown to stimulate and inhibit blood coagulation in vitro. Promotion of coagulation is mediated by blocking tissue factor pathway inhibitor (TFPI). We screened fucoidan extracts from four brown algae species in vitro with respect to their potential to improve coagulation in bleeding disorders. The fucoidans' pro- and anticoagulant activities were assessed by global hemostatic and standard clotting assays. Results showed that fucoidans improved coagulation parameters. Some fucoidans also activated the contact pathway of coagulation, an undesired property reported for sulfated glycosaminoglycans. Chemical evaluation of fucoidans' complex and variable structure included molecular weight (Mw), polydispersity (polyD), structural heterogeneity, and organic and inorganic impurities. Herewith, we describe a screening strategy that facilitates the identification of crude fucoidan extracts with desired biological and structural properties for improvement of compromised coagulation like in hemophilia., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Tissue factor pathway inhibitor-α (TFPIα) inhibits factor Xa by forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. TF-FVIIa forms a quaternary complex with TFPIα and FXa, which shuts off the initiation of coagulation via the extrinsic pathway., Aim: To investigate whether direct inhibition of FXa by TFPIα independently of TF plays a role in downregulating coagulation., Methods: Inhibition of FXa by TFPIα in plasma was determined by measuring thrombin generation triggered with FXa, the FX activator from Russell's viper venom (RVV-X), FXIa, or FIXa. TF-independent anticoagulant activities of TFPIα and its cofactor, protein S, were quantified: (i) after neutralization of TFPIα and protein S with anti-TFPI or anti-protein S antibodies; and (ii) in TFPI-depleted or protein S-depleted plasmas supplemented with varying amounts of TFPIα or protein S., Results: Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation in plasma triggered with RVV-X, FXa, FIXa, or FXIa. Anti-TFPI and anti-protein S antibodies decreased the lag time and increased the peak height of thrombin generation to the same extent, indicating that inhibition of FXa by TFPIα requires the presence of protein S. TFPIα and protein S titrations in TFPI-depleted or protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF also revealed TF-independent anticoagulant activity of TFPIα, which was completely dependent on the presence of protein S., Conclusion: Direct inhibition of FXa by TFPIα contributes to the downregulation of coagulation., (© 2014 International Society on Thrombosis and Haemostasis.)

Prophylaxis prevents joint and other bleeding episodes in patients with haemophilia A. Development of new factor concentrates with longer circulating half-lives may encourage patients to start, continue or resume prophylaxis. The aim of this study was to compare the pharmacodynamic effect of a PEGylated full-length recombinant factor VIII (rFVIII) concentrate with that of an unmodified rFVIII concentrate with respect to the duration of prophylactic efficacy in a murine model of haemophilic joint bleeding. Mice were pretreated with BAX 855 or unmodified rFVIII at specified times before right knee puncture to induce haemarthrosis; left knee joints served as controls. Joint bleeding was evaluated using a combination of visual and histological assessments. Administration of a single dose of unmodified rFVIII before joint puncture prevented haemarthrosis in mice up to 24 h, whereas pretreatment with BAX 855 protected the joint from bleeding up to 48 h. This pharmacodynamic study showed prolonged efficacy of BAX 855 compared to ADVATE in a haemophilia A mouse joint bleeding model. This finding supports the possibility of using BAX 855 to increase FVIII trough levels and/or extend the dosing interval in patients with haemophilia A on prophylaxis, which may potentially improve prophylactic efficacy and long-term adherence., (© 2014 John Wiley & Sons Ltd.)

Background: TFPI is a Kunitz-type protease inhibitor that downregulates the extrinsic coagulation pathway by inhibiting factor Xa (FXa) and FVIIa. All three Kunitz domains (KD1, KD2, and KD3) and protein S are required for optimal inhibition of FXa and FVIIa. There is limited information on Kunitz domain requirements of the inhibition of TF:FVIIa-catalyzed FIX and FX activation by TFPI., Aim: To investigate the role of the Kunitz domains of TFPI and protein S in the inhibition of FX and FIX activation., Methods: Inhibition of TF:FVIIa-catalyzed FX and FIX activation by full-length TFPI (TFPIFL ) and TFPI constructs was quantified from progress curves of FXa and FIXa generation measured with chromogenic substrates., Results and Conclusions: TFPIFL inhibited TF:FVIIa-catalyzed FIX activation with a Ki of 16.7 nmol L(-1) . Protein S reduced the Ki to 1.0 nmol L(-1) . TFPI1-150 and KD1-KD2 had 10-fold higher Ki values and were not stimulated by protein S. Single Kunitz domains were poor inhibitors of TF:FVIIa-catalyzed FIX activation (Ki >800 nm). FX activation was measured at limiting FVIIa and excess TF or vice versa. At both conditions, TFPIFL , TFPI1-150 , and KD1-KD2 showed similar inhibition of FX activation. However, at low phospholipid concentrations, TFPIFL was ~ 15-fold more active than TFPI1-150 or KD1-KD2. Apparently, excess phospholipids act as a kind of sink for TFPIFL , limiting its availability for TF:FVIIa inhibition. Preformed FXa:TFPIFL/1-150 complexes rapidly and stoichiometrically inhibited FIX and FX activation by TF:FVIIa, indicating that binary TFPI:FXa complex formation is the limiting step in TF:FVIIa inhibition. Protein S also enhanced inhibition of TF:FVIIa-catalyzed FX activation by TFPI., (© 2014 International Society on Thrombosis and Haemostasis.)