Modification of biopharmaceutical molecules by covalent conjugation of polyethylene glycol (PEG) molecules is known to enhance pharmacologic and pharmaceutical properties of proteins and other large molecules and has been used successfully in 12 approved drugs. Both linear and branched-chain PEG reagents with molecular sizes of up to 40 kDa have been used with a variety of different PEG derivatives with different linker chemistries. This review describes the properties of PEG itself, the history and evolution of PEGylation chemistry, and provides examples of PEGylated drugs with an established medical history. A trend toward the use of complex PEG architectures and larger PEG polymers, but with very pure and well-characterized PEG reagents is described. Nonclinical toxicology findings related to PEG in approved PEGylated biopharmaceuticals are summarized. The effect attributed to the PEG part of the molecules as observed in 5 of the 12 marketed products was cellular vacuolation seen microscopically mainly in phagocytic cells which is likely related to their biological function to absorb and remove particles and macromolecules from blood and tissues. Experience with marketed PEGylated products indicates that adverse effects in toxicology studies are usually related to the active part of the drug but not to the PEG moiety.

SummaryPatients with thrombotic thrombocytopenic purpura (TTP) have a deficiency of von Willebrand factor (vWF)-cleaving protease, whereas patients with hemolytic-uremic syndrome (HUS) show normal activity of this protease. Present methods for assaying vWF-cleaving protease by immunoblotting are time-intensive and cumbersome. We therefore developed a new functional assay based on the preferential binding of high-molecular-weight forms of vWF to collagen. In this assay, the diluted plasma sample to be tested is added to normal human plasma in which protease activity had been abolished. The vWF present in the protease-depleted plasma is digested by the vWF-cleaving protease in the test plasma. The proteolytic degradation leads to low-molecular-weight forms of vWF, which show impaired binding to microtiter plates coated with human collagen type III. The collagen-bound vWF is quantified using a peroxidase-conjugated rabbit antibody against human vWF. The values of vWF-cleaving protease activity in tested plasma samples are read from a calibration curve achieved by incubating the vWF-substrate with dilutions of a normal human plasma pool (NHP). Testing of plasma from patients with TTP and HUS showed that the assay can be used to distinguish between these two syndromes. The presence of an inhibitor can be detected by carrying out the test after incubation of NHP with the patient plasma sample, thus enabling differentiation of patients with familial TTP from those with non-familial TTP.

Memory B cells are responsible for the rapidly emerging antibody response after antigen reexposure. The signals required for the restimulation of memory B cells have not been fully explained. We used a murine model of anti-factor VIII (FVIII) antibody responses in hemophilia A to study the requirements for the restimula-tion of FVIII-specific memory B cells and their differentiation into anti-FVIII anti-body-producing cells. We were particularly interested in the significance of activated T cells and costimulatory interactions. Our results indicate that the re-stimulation of FVIII-specific memory B cells is strictly dependent on interactions with activated T cells. These activated T cells can be specific for either FVIII or third-party antigens. Restimulation by T cells specific for third-party antigens requires the presence of FVIII, indicating that signals induced by B-cell receptor (BCR) triggering and by interactions with activated T cells are important. The blockade of B7-1 or B7-2 as well as the blockade of CD40L inhibits the restimulation and differentiation of FVIII-specific memory B cells in vitro and in vivo. The interference with inducible costimulator-inducible costimulator ligand (ICOS-ICOSL) interactions, however, does not cause any modulation. As expected, the production of anti-FVIII antibodies by plasma cells is not dependent on any of the costimulatory interactions tested.

Human alpha1-antitrypsin (A1PI) is a well-known glycoprotein in human plasma important for the protection of tissues from proteolytic enzymes. The three N-glycosylation sites of A1PI contain diantennary N-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for A1PI. Here we present an approach to characterize A1PI isoforms from human plasma and its PTMs by LC-ESI-MS and LC-ESI-MS/MS of peptides obtained by proteolytic digestion. The single cysteine residue of A1PI formed a disulfide bridge with free cysteine. The variability of the number of antennae and hence sialic acids on glycosylation site N107, which even contained minute amounts of tetraantennary structures, emerged as a major cause for the IEF pattern of A1PI. Only negligible amounts of triantennary structures were identified attached to N70, and exclusively diantennary structures were present on site N271 in each of the isoforms analyzed. Exoglycosidase digests revealed alpha2,6-linked neuraminic acids on diantennary N-glycans, and triantennary contained additionally one single alpha2,3-neuraminic acid per N-glycan, which, together with a fucose, formed a sialyl Lewis X determinant on the beta1,4-linked N-acetylglucosamine, as shown by 2-D-HPLC of pyridylaminated asialoglycans. Fucosylation of diantennary structures was marginal and of the core alpha1,6 type.

FEIBA (factor eight inhibitor bypassing activity) has a history of more than 30 years of successful use in controlling bleeding in haemophilic patients who have developed inhibitory antibodies against factor (F)VIII or FIX. Recently it was shown that FEIBA contains the proenzymes of the prothrombin complex factors, prothrombin, FVII, FIX and FX, but only very small amounts of their activation products, with the exception of FVIIa, which is contained in FEIBA in greater amounts. FEIBA controls bleeding by induction and facilitation of thrombin generation, a process for which FV is crucial. A number of biochemical in vitro and in vivo studies have shown that FXa and prothrombin play a critical role in the activity of FEIBA. Consequently, they are considered to be key components of this product. The prothrombinase complex has been found to be a major target site for FEIBA. Apart from prothrombin and FXa, FEIBA contains other proteins of the prothrombin complex, which could also facilitate haemostasis in haemophilia patients with inhibitors.

IntroductionThe development of neutralizing anti-factor VIII (FVIII) antibodies is the major complication in the treatment of patients with hemophilia A with FVIII products. 1,2 Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. [3][4][5][6] Despite clinical experience with the therapy, little is known about the immunologic mechanisms that cause the down-modulation of FVIII-specific immune responses and the induction of long-lasting immune tolerance against FVIII. We asked the question whether the restimulation of FVIII-specific memory B cells is affected by high concentrations of FVIII in vitro or high doses of FVIII in vivo. Memory B cells play an essential role in the maintenance of established antibody responses. On re-exposure to the same antigen, they are rapidly restimulated to proliferate and differentiate into antibody-secreting plasma cells (ASCs) that secrete high-affinity antibodies. 7,8 Furthermore, memory B cells have the potential to act as very efficient antigen-presenting cells and stimulators of CD4 ϩ T cells because of the expression of highaffinity antigen receptors, major histocompatibility complex (MHC) class II and costimulatory molecules. 9 It is, therefore, reasonable to believe that memory B cells have to be eradicated or functionally inactivated during a successful immune tolerance induction therapy with FVIII inhibitors in patients with hemophilia A.We used a murine model of hemophilia A that is characterized by complete deficiency of functionally active FVIII because of a targeted disruption of exon 17 of the F8 gene. 10,11 Intravenous injection of human FVIII into these mice results in high titers of anti-FVIII antibodies that have similar characteristics to those of FVIII inhibitors in patients. [12][13][14][15] Using this model, we demonstrated previously that the differentiation of FVIII-specific memory B cells into ASCs depends on the presence of activated T cells and requires CD40-CD40 ligand and CD80/CD86-CD28 costimulatory interactions. 16 Here, we show that concentrations of FVIII below the physiologic plasma concentration of 0.1 g/mL (1 U/mL) restimulate FVIII-specific memory B cells and induce their differentiation into ASCs. Concentrations above 0.1 g/mL (1 U/mL), however, inhibit memory B-cell restimulation and prevent the formation of ASCs. This inhibition is irreversible and involves the activation of caspases. Materials and methods Hemophilic E-17 miceOur colony of fully inbred hemophilic E-17 mice (characterized by a targeted disruption of exon 17 of the F8 gene) was established with a breeding pair from the original colony 10,11 and crossed into the C57BL/6J background as described. 17 All mice were male and aged 8 to 10 weeks at the beginning of the experiments. All studies were carried out in accordance with Austrian federal law (Act BG 501/1989) regulating animal experimentation and approved by the local authority in Vienna, Austria. Immunization of mice with FV...

SummaryChronic thromboembolic pulmonary hypertension (CTEPH) is an enigmatic disorder lacking signs, symptoms and classical risk factors for venous thromboembolism.The objective of the prospective case controlled study, carried out at the Pulmonary Hypertension Unit, University Hospital Vienna, Austria, was to investigate whether plasma FVIII is elevated in CTEPH patients.The study examined 122 consecutive patients diagnosed with CTEPH. Plasma FVIII was measured and compared with plasma FVIII of healthy controls (n=82) and of patients with non-thromboembolic pulmonary arterial hypertension (PAH, n=88).Results show that CTEPH patients had higher FVIII levels than controls (233±83IU/dl versus 123±40IU/dl, p<0.0001) and PAH patients (158±61IU/dl, p<0.0001). Plasma FVIII one year after surgery (212±94IU/dl) was statistically unchanged compared with preoperative values (FVIII: 226±88IU/dl, n=25). FVIII>230IU/dl was more prevalent in CTEPH patients (41%) than in controls (5%, p<0.0001) and PAH patients (22%, p=0.022).We can conclude that elevated plasma FVIII is the first prothrombotic factor identified in a large proportion of CTEPH patients.

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.