Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K m for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemo- IntroductionFactor IX (FIX) is a vitamin K-dependent glycoprotein and an essential protease of the hemostatic system. The domain organization of FIX is shared with factors VII, X, and protein C and comprises an N-terminal domain rich in ␥-carboxyglutamic acid (Gla), 2 epidermal growth factor-like repeats and a C-terminal trypsin-like protease domain. 1 Together they form a 55-kDa single-chain protease precursor circulating in plasma at a concentration of approximately 90nM (5 g/mL), defined as 1 IU/mL. FIX is converted to the 2-chain activated form by the tissue factor (TF)-factor VIIa (FVIIa) complex or factor XIa (FXIa). Activation occurs by limited proteolysis at Arg145 and Arg180 in the protease domain and liberates a 35-amino acid activation peptide that carries the only 2 N-linked glycans in the protein. 2,3 Subsequent assembly of FIXa with the cofactor VIIIa on the activated platelet surface greatly enhances the proteolytic activity of FIXa toward its substrate factor X (FX) and is essential for propagation of the coagulation response. 4 The importance of this activity is reflected by the occurrence of the bleeding disorder hemophilia B (HB) in individuals carrying mutations in the FIX gene. The prevalence of HB is approximately 1 in 25 000 males, and it has been estimated that approximately 84 000 people are affected worldwide. 5 The mainstay in HB treatment is substitution therapy by infusion of plasma-derived or recombinant FIX (rFIX). The therapeutic goal is to prevent bleeding episodes and to provide safe and efficacious treatment of bleedings when they occur. Because of the relatively short half-life of FIX (18-24 hours [6][7][8] ), the recommended prophylaxis regimen consists of 2 to 3 weekly infusions of 40-100 IU/kg 9 FIX to maintain trough levels above 1% and thus shifting patients from a severe to a milder phenotype. When adhered to, prophylaxis in patients without severe joint disorder is efficacious with a frequency of only 0-2 breakthrough bleeds per year in the majority of patients. 8,10 However, the need for multiple weekly infusions present challen...

Globally, the COVID-19 pandemic has had extreme consequences for the healthcare system and has led to calls for diagnostic tools to monitor and understand the transmission, pathogenesis, and epidemiology, as well as to evaluate future vaccination strategies. In this study, we have developed novel, to our knowledge, flexible ELISA-based assays for specific detection of human SARS-CoV-2 Abs against the receptor-binding domain, including an Ag sandwich ELISA relevant for large population screening and three isotype-specific assays for in-depth diagnostics. Their performance was evaluated in a cohort of 350 convalescent participants with previous COVID-19 infection, ranging from asymptomatic to critical cases. We mapped the Ab responses to different areas on protein N and S and showed that the IgM, A, and G Ab responses against receptor-binding domain are significantly correlated to the disease severity. These assays and the data generated from them are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term COVID-19 immunity.

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar K(i) values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.

Despite major advances in antibody discovery technologies, the successful development of monoclonal antibodies (mAbs) into effective therapeutic and diagnostic agents can often be impeded by developability liabilities, such as poor expression, low solubility, high viscosity and aggregation. Therefore, strategies to predict at the early phases of antibody development the risk of late-stage failure of antibody candidates are highly valuable. In this work, we employ the in silico solubility predictor CamSol to design a library of 17 variants of a humanized mAb predicted to span a broad range of solubility values, and we examine their developability potential with a battery of commonly used in vitro and in silico assays. Our results demonstrate the ability of CamSol to rationally enhance mAb developability, and provide a quantitative comparison of in vitro developability measurements with each other and with more resource-intensive solubility measurements, as well as with in silico predictors that offer a potentially faster and cheaper alternative. We observed a strong correlation between predicted and experimentally determined solubility values, as well as with measurements obtained using a panel of in vitro developability assays that probe non-specific interactions. These results indicate that computational methods have the potential to reduce or eliminate the need of carrying out laborious in vitro quality controls for large numbers of lead candidates. Overall, our study provides support to the emerging view that the implementation of in silico tools in antibody discovery campaigns can ensure rapid and early selection of antibodies with optimal developability potential.

Human dipeptidyl peptidase IV (DPP-IV) is a ubiquitously expressed type II transmembrane serine protease. It cleaves the penultimate positioned prolyl bonds at the N terminus of physiologically important peptides such as the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide. In this study, we have characterized different active site mutants. The Y547F mutant as well as the catalytic triad mutants S630A, D708A, and H740L showed less than 1% wild type activity. X-ray crystal structure analysis of the Y547F mutant revealed no overall changes compared with wild type apoDPP-IV, except the ablation of the hydroxyl group of Tyr 547 and a water molecule positioned in close proximity to Tyr 547 . To elucidate further the reaction mechanism, we determined the crystal structure of DPP-IV in complex with diisopropyl fluorophosphate, mimicking the tetrahedral intermediate. The kinetic and structural findings of the tyrosine residue are discussed in relation to the catalytic mechanism of DPP-IV and to the inhibitory mechanism of the 2-cyanopyrrolidine class of potent DPP-IV inhibitors, proposing an explanation for the specificity of this class of inhibitors for the S9b family among serine proteases.

Hemophilia A (HA) is a bleeding disorder resulting from deficient Factor VIII (FVIII), which normally functions as a cofactor to activated Factor IX (FIXa) that facilitates activation of Factor X (FX). To mimic this property in a bispecific antibody (biAb) format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting biAb (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant (KD) of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with KD-values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by four orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation with potencies 13 and 18 times higher than a sequence-identical analog of emicizumab, respectively. A similar potency difference was observed in a tail-vein transection model in hemophilia A mice, while reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetics of Mim8 were investigated and a half-life of 14 days demonstrated in cynomolgus monkey. In conclusion, Mim8 is a FVIIIa-mimetic with a potent and efficacious hemostatic effect based on preclinical data.

The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF⅐FVIIa complex and by FXa in the ternary TF⅐FVIIa⅐FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF⅐FVIIa-Xa complex normally but were severely impaired in binary TF⅐FVIIa⅐PAR2 signaling. The residues identified were located in the model-predicted S2 pocket of FVIIa, and complementary PAR2 P2 Leu-38 replacements demonstrated that the P2 side chain was indeed crucial for PAR2 cleavage by TF⅐FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF⅐FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactorassociated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.Coagulation factor VIIa (FVIIa) 2 in complex with its cellular receptor tissue factor (TF) mediates activation of two pathways of major physiological importance: (i) initiation of blood coagulation by activation of coagulation factors IX and X (FIX and FX), and (ii) induction of cell signaling through proteaseactivated receptors (PARs). TF⅐FVIIa-dependent signaling primarily activates PAR2, which belongs to a family of four G-protein-coupled receptors activated by specific proteolytic cleavage of their N-terminal extracellular domain (1). Cleavage unmasks a new N terminus, which serves as a tethered ligand that induces transmembrane signaling (2). Activation of PAR2 by the TF⅐FVIIa binary complex involves cellular pools of TF with low affinity for FVIIa, whereas high affinity cell surface TF mediates coagulation activation and the associated cell signaling of the ternary complex of TF⅐FVIIa⅐FXa (3). In the latter complex, FXa is the primary activator for PAR2 (4). PAR2 triggers typical G-protein-coupled as well as ␤-arrestin-dependent signaling and thereby induces cytokine, chemokine, and growth factor expression and regulates protein synthesis, cell motility, proliferation, and apoptosis (5).Although the TF pathway also induces thrombin-dependent PAR1 signaling and transcriptional respo...

The alpha/B.1.1.7 SARS-CoV-2 lineage emerged in autumn 2020 in the United Kingdom and transmitted rapidly until winter 2021 when it was responsible for most new COVID-19 cases in many European countries. The incidence domination was likely due to a fitness advantage that could be driven by the receptor-binding domain (RBD) residue change (N501Y), which also emerged independently in other variants of concern such as the beta/B.1.351 and gamma/P.1 strains. Here, we present a functional characterization of the alpha/B.1.1.7 variant and show an eightfold affinity increase towards human angiotensin-converting enzyme-2 (ACE-2). In accordance with this, transgenic hACE2 mice showed a faster disease progression and severity after infection with a low dose of B.1.1.7, compared to an early 2020 SARS-CoV-2 isolate. When challenged with sera from convalescent individuals or anti-RBD monoclonal antibodies, the N501Y variant showed a minor, but significant elevated evasion potential of ACE-2/RBD antibody neutralization. The data suggest that the single asparagine to tyrosine substitution remarkable rise in affinity may be responsible for the higher transmission rate and severity of the B.1.1.7 variant.