Publications by authors named "Friedrich Scheiflinger"

81 Publications

Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency.

Mol Ther Methods Clin Dev 2020 Dec 22;19:486-495. Epub 2020 Oct 22.

Herman B Wells Center for Pediatric Research, Indiana University, Indianapolis, IN 46202, USA.

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 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 nucleotide sequences were employed. A comprehensive immunogenicity assessment using stimulation assays and various 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.
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http://dx.doi.org/10.1016/j.omtm.2020.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708868PMC
December 2020

BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression.

Blood 2021 Feb;137(6):763-774

Gene Therapy Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC.

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.
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http://dx.doi.org/10.1182/blood.2019004625DOI Listing
February 2021

A bispecific antibody demonstrates limited measurability in routine coagulation assays.

Blood Coagul Fibrinolysis 2020 09;31(6):353-365

Baxalta Innovations GmbH, Vienna, Austria.

: 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.
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http://dx.doi.org/10.1097/MBC.0000000000000921DOI Listing
September 2020

Development of an Biopotency Assay for an AAV8 Hemophilia B Gene Therapy Vector Suitable for Clinical Product Release.

Mol Ther Methods Clin Dev 2020 Jun 17;17:581-588. Epub 2020 Mar 17.

Baxalta Innovations GmbH, a member of the Takeda group of companies, Uferstraße. 15, A-2304 Orth an der Donau, Austria.

Gene therapy product release requires reliable and consistent demonstration of biopotency. In hemophilia B vectors, this is usually determined 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 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 and 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 test. The assay thus constitutes a viable alternative to using animals for lot release testing.
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http://dx.doi.org/10.1016/j.omtm.2020.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139127PMC
June 2020

Blockade of the costimulatory CD28-B7 family signal axis enables repeated application of AAV8 gene vectors.

J Thromb Haemost 2020 05 2;18(5):1075-1080. Epub 2020 Mar 2.

Baxalta Innovations GmbH, A Member of the Takeda Group of Companies, Vienna, Austria.

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  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  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.
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http://dx.doi.org/10.1111/jth.14757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318590PMC
May 2020

Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics.

Antibodies (Basel) 2019 Sep 6;8(3). Epub 2019 Sep 6.

Baxalta Innovations GmbH (part of Takeda), Indusriestrasse 67, 1221 Vienna, Austria.

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.
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http://dx.doi.org/10.3390/antib8030046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784111PMC
September 2019

Identification of cysteine thiol-based linkages in ADAMTS13 in support of a non-proteolytic regulation of von Willebrand factor.

J Thromb Haemost 2019 12 3;17(12):2099-2109. Epub 2019 Sep 3.

Baxalta Innovations GmbH, a member of the Takeda group of companies, Vienna, Austria.

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.
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http://dx.doi.org/10.1111/jth.14602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916347PMC
December 2019

Evaluation of Factor VIII Polysialylation: Identification of a Longer-Acting Experimental Therapy in Mice and Monkeys.

J Pharmacol Exp Ther 2019 10 31;371(1):95-105. Epub 2019 Jul 31.

Baxalta Innovations GmbH, a Member of the Takeda Group of Companies, Vienna, Austria

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.
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http://dx.doi.org/10.1124/jpet.119.260067DOI Listing
October 2019

Prevalence of Anti-Adeno-Associated Virus Immune Responses in International Cohorts of Healthy Donors.

Mol Ther Methods Clin Dev 2019 Sep 7;14:126-133. Epub 2019 Jun 7.

Baxalta Innovations GmbH, a member of the Takeda group of companies, Vienna, Austria.

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.
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http://dx.doi.org/10.1016/j.omtm.2019.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629972PMC
September 2019

Temperature-dependent irreversible conformational change of recombinant ADAMTS13 upon metal ion chelation.

J Thromb Haemost 2019 06 21;17(6):995-1002. Epub 2019 Apr 21.

Baxalta Innovations GmbH, a member of the Takeda group of companies, Vienna, Austria.

Background: The catalytic domain of ADAMTS13 possesses one Zn and up to three putative Ca 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 or Zn prior to but not postincubation prevented the activity decrease of the enzyme. The SEC-ICP-MS analyses showed that ADAMTS13 only bound Zn and that its reduced activity correlated with a gradual loss of bound Zn . Concomitant higher-order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less-ordered protein structures.

Conclusions: Zn 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.
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http://dx.doi.org/10.1111/jth.14440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850365PMC
June 2019

How Full-Length FVIII Benefits from Its Heterogeneity - Insights into the Role of the B-Domain.

Pharm Res 2019 Apr 1;36(5):77. Epub 2019 Apr 1.

Research & Development, Baxalta Innovations GmbH, a Takeda company, Vienna, Austria.

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.
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http://dx.doi.org/10.1007/s11095-019-2599-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443606PMC
April 2019

Detection of Biologically Relevant Low-Titer Neutralizing Antibodies Against Adeno-Associated Virus Require Sensitive Assays.

Hum Gene Ther Methods 2019 04 29;30(2):35-43. Epub 2019 Mar 29.

Baxalta Innovations GmbH, a member of the Takeda group of companies, Vienna, Austria.

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 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 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 , demonstrating the importance of sensitive NAb assays for clinical applications. The NAb assay was found to be more sensitive than an 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 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.
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http://dx.doi.org/10.1089/hgtb.2018.263DOI Listing
April 2019

IVIG induces apoptotic cell death in CD56 NK cells resulting in inhibition of ADCC effector activity of human PBMC.

Clin Immunol 2019 01 31;198:62-70. Epub 2018 Oct 31.

Baxalta Innovations GmbH, Shire Pharmaceuticals, Vienna, Austria. Electronic address:

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 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 NK cells without affecting CD56 NK cells. Induction of apoptosis in CD56 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') or IVIG Fc preparations. The described effect could contribute to the reduction of peripheral NK cells observed during IVIG therapy in patients.
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http://dx.doi.org/10.1016/j.clim.2018.10.018DOI Listing
January 2019

Development of Methods for the Selective Measurement of the Single Amino Acid Exchange Variant Coagulation Factor IX Padua.

Mol Ther Methods Clin Dev 2018 Sep 28;10:29-37. Epub 2018 Jun 28.

Shire, Global Research, Donau-City-Str. 7, Vienna, Austria.

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.
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http://dx.doi.org/10.1016/j.omtm.2018.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039963PMC
September 2018

Role of the Cysteine 81 Residue of Macrophage Migration Inhibitory Factor as a Molecular Redox Switch.

Biochemistry 2018 03 15;57(9):1523-1532. Epub 2018 Feb 15.

Baxalta Innovations GmbH , Uferstrasse 15 , 2304 Orth an der Donau , Austria.

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.
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http://dx.doi.org/10.1021/acs.biochem.7b01156DOI Listing
March 2018

Pharmacokinetics, disease-modifying activity, and safety of an experimental therapeutic targeting an immunological isoform of macrophage migration inhibitory factor, in rat glomerulonephritis.

Eur J Pharmacol 2018 Feb 20;820:206-216. Epub 2017 Dec 20.

Research & Nonclinical Development, Shire, Industriestrasse 67, A-1220 Vienna, Austria. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ejphar.2017.12.040DOI Listing
February 2018

A bio-inspired method for direct measurement of local wall shear rates with micrometer localization using the multimeric protein von Willebrand factor as sensor molecule.

Biomicrofluidics 2017 Jul 30;11(4):044117. Epub 2017 Aug 30.

Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9, A-1060 Vienna, Austria.

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, 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.
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http://dx.doi.org/10.1063/1.5000503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577009PMC
July 2017

Genome editing for scalable production of alloantigen-free lentiviral vectors for gene therapy.

EMBO Mol Med 2017 11;9(11):1558-1573

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy

Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large-scale manufacturing and raises concerns for 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 gene therapy.
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http://dx.doi.org/10.15252/emmm.201708148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666310PMC
November 2017

Flow-induced elongation of von Willebrand factor precedes tension-dependent activation.

Nat Commun 2017 08 23;8(1):324. Epub 2017 Aug 23.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, 02115, USA.

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.
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http://dx.doi.org/10.1038/s41467-017-00230-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567343PMC
August 2017

Mining ancient proteins for next-generation drugs.

Nat Biotechnol 2017 01;35(1):28-29

Research EU, Shire, Vienna, Austria.

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http://dx.doi.org/10.1038/nbt.3762DOI Listing
January 2017

Oxidized macrophage migration inhibitory factor is a potential new tissue marker and drug target in cancer.

Oncotarget 2016 11;7(45):73486-73496

Baxalta Innovations GmbH, Orth/Donau, Austria.

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.
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http://dx.doi.org/10.18632/oncotarget.11970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341993PMC
November 2016

The Mystery of Antibodies Against Polyethylene Glycol (PEG) - What do we Know?

Pharm Res 2016 09 7;33(9):2239-49. Epub 2016 Jun 7.

Baxalta Innovations GmbH, Industriestrasse 72, A-1220, Vienna, Austria.

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.
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http://dx.doi.org/10.1007/s11095-016-1961-xDOI Listing
September 2016

ADAMTS13-mediated thrombolysis of t-PA-resistant occlusions in ischemic stroke in mice.

Blood 2016 05 29;127(19):2337-45. Epub 2016 Feb 29.

Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium;

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.
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http://dx.doi.org/10.1182/blood-2015-08-662650DOI Listing
May 2016

Role of exosite binding modulators in the inhibition of Fxa by TFPI.

Thromb Haemost 2016 Mar 26;115(3):580-90. Epub 2015 Nov 26.

Stella Thomassen, Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, 6200 MD Maastricht, the Netherlands, Tel.: +31 43 388 4160, Fax: +31 43 388 4159, E-mail:

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.
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http://dx.doi.org/10.1160/TH15-04-0354DOI Listing
March 2016

Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

BMC Biotechnol 2015 Sep 18;15:87. Epub 2015 Sep 18.

Baxalta Innovations GmbH, Donau-City Straße 7, 1220, Vienna, Austria.

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.
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http://dx.doi.org/10.1186/s12896-015-0205-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574471PMC
September 2015

Shear-Dependent Interactions of von Willebrand Factor with Factor VIII and Protease ADAMTS 13 Demonstrated at a Single Molecule Level by Atomic Force Microscopy.

Anal Chem 2015 Oct 30;87(20):10299-305. Epub 2015 Sep 30.

Institute of Chemical Technologies and Analytics, Vienna University of Technology , Getreidemarkt 9/164, A-1060 Vienna, Austria.

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.
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http://dx.doi.org/10.1021/acs.analchem.5b02078DOI Listing
October 2015

Potential for Recombinant ADAMTS13 as an Effective Therapy for Acquired Thrombotic Thrombocytopenic Purpura.

Arterioscler Thromb Vasc Biol 2015 Nov 3;35(11):2336-42. Epub 2015 Sep 3.

From the Laboratory for Thrombosis Research, KU Leuven, Campus Kulak Kortrijk, Kortrijk, Belgium (C.T., S.F.D.M., K.V.); and Baxalta Innovations GmbH, Vienna, Austria (A.S., B.P., F.S., H.R.).

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.
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http://dx.doi.org/10.1161/ATVBAHA.115.306014DOI Listing
November 2015

Selective Targeting of a Disease-Related Conformational Isoform of Macrophage Migration Inhibitory Factor Ameliorates Inflammatory Conditions.

J Immunol 2015 Sep 24;195(5):2343-52. Epub 2015 Jul 24.

Baxter Biomedical Research Center, Baxter Innovations GmbH, 2304 Orth/Donau, Austria;

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.
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http://dx.doi.org/10.4049/jimmunol.1500572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4543907PMC
September 2015

Poor responder to plasma exchange therapy in acquired thrombotic thrombocytopenic purpura is associated with ADAMTS13 inhibitor boosting: visualization of an ADAMTS13 inhibitor complex and its proteolytic clearance from plasma.

Transfusion 2015 Oct 8;55(10):2321-30. Epub 2015 Jun 8.

Department of Blood Transfusion Medicine, Nara Medical University, Kashihara City, Japan.

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.
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http://dx.doi.org/10.1111/trf.13182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690831PMC
October 2015

Ca(2+) concentration-dependent conformational change of FVIII B-domain observed by atomic force microscopy.

Anal Bioanal Chem 2015 Aug 23;407(20):6051-6. Epub 2015 May 23.

Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-IAC, 1060, Vienna, Austria.

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.
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http://dx.doi.org/10.1007/s00216-015-8778-zDOI Listing
August 2015