Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size.

Background: The activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified.

Type 2B von Willebrand disease mutations differentially perturb autoinhibition of the A1 domain.

Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder in which a subset of point mutations in the von Willebrand factor (VWF) A1 domain and recently identified autoinhibitory module (AIM) cause spontaneous binding to glycoprotein Ibα (GPIbα) on the platelet surface. All reported type 2B VWD mutations share this enhanced binding; however, type 2B VWD manifests as variable bleeding complications and platelet levels in patients, depending on the underlying mutation. Understanding how these mutations localizing to a similar region can result in such disparate patient outcomes is essential for detailing our understanding of VWF regulatory and activation mechanisms.

Subunit Flexibility of Multimeric von Willebrand Factor/Factor VIII Complexes.

Von Willebrand factor (VWF) is a plasma glycoprotein that participates in platelet adhesion and aggregation and serves as a carrier for blood coagulation factor VIII (fVIII). Plasma VWF consists of a population of multimers that range in molecular weight from ∼ 0.55 MDa to greater than 10 MDa.

Engineering a Therapeutic Protein to Enhance the Study of Anti-Drug Immunity.

The development of anti-drug antibodies represents a significant barrier to the utilization of protein-based therapies for a wide variety of diseases. While the rate of antibody formation can vary depending on the therapeutic employed and the target patient population receiving the drug, the antigen-specific immune response underlying the development of anti-drug antibodies often remains difficult to define. This is especially true for patients with hemophilia A who, following exposure, develop antibodies against the coagulation factor, factor VIII (FVIII).

Neutralizing Antibodies Against Factor VIII Can Occur Through a Non-Germinal Center Pathway.

Humoral immunity to factor VIII (FVIII) represents a significant challenge for the treatment of patients with hemophilia A. Current paradigms indicate that neutralizing antibodies against FVIII (inhibitors) occur through a classical CD4 T cell, germinal center (GC) dependent process. However, clinical observations suggest that the nature of the immune response to FVIII may differ between patients.

Removal of single-site N-linked glycans on factor VIII alters binding of domain-specific monoclonal antibodies.

Background: A portion of individuals with hemophilia A develop neutralizing antibodies called inhibitors to glycoprotein factor VIII (FVIII). There are multiple risk factors that contribute to the risk of inhibitor formation. However, knowledge of the role of FVIII asparagine (N)-linked glycosylation in FVIII immunity is limited.

Measurement of the Translational Diffusion Coefficient and Hydrodynamic Radius of Proteins by Dynamic Light Scattering.

Diffusion is a fundamental process in biological systems that governs the molecular collisions driving biochemical reactions and membrane and transport. Measurement of the diffusion coefficient and application of the Stokes-Einstein equation produces the hydrodynamic radius, which is a commonly used gauge of particle size. Additionally, measurement of the diffusion coefficient and the sedimentation coefficient, and application of the Svedberg equation, yields the molecular weight, which is particularly useful in the characterization of very large macromolecules.

Identification of coagulation factor IX variants with enhanced activity through ancestral sequence reconstruction.

Orthologous proteins contain sequence disparity guided by natural selection. In certain cases, species-specific protein functionality predicts pharmacological enhancement, such as greater specific activity or stability. However, immunological barriers generally preclude use of nonhuman proteins as therapeutics, and difficulty exists in the identification of individual sequence determinants among the overall sequence disparity.

Conformation of the von Willebrand factor/factor VIII complex in quasi-static flow.

Von Willebrand factor (VWF) is a plasma glycoprotein that circulates noncovalently bound to blood coagulation factor VIII (fVIII). VWF is a population of multimers composed of a variable number of ∼280 kDa monomers that is activated in shear flow to bind collagen and platelet glycoprotein Ibα. Electron microscopy, atomic force microscopy, small-angle neutron scattering, and theoretical studies have produced a model in which the conformation of VWF under static conditions is a compact, globular "ball-of-yarn," implying strong, attractive forces between monomers.

The 3.2 Å structure of a bioengineered variant of blood coagulation factor VIII indicates two conformations of the C2 domain.

Background: Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost.

Preclinical Development of a Hematopoietic Stem and Progenitor Cell Bioengineered Factor VIII Lentiviral Vector Gene Therapy for Hemophilia A.

Genetically modified, autologous hematopoietic stem and progenitor cells (HSPCs) represent a new class of genetic medicine. Following this therapeutic paradigm, we are developing a product candidate, designated CD68-ET3-LV CD34, for the treatment of the severe bleeding disorder, hemophilia A. The product consists of autologous CD34 cells transduced with a human immunodeficiency virus 1-based, monocyte lineage-restricted, self-inactivating lentiviral vector (LV), termed CD68-ET3-LV, encoding a bioengineered coagulation factor VIII (fVIII) transgene, termed ET3, designed for enhanced expression.

Target-Cell-Directed Bioengineering Approaches for Gene Therapy of Hemophilia A.

Potency is a key optimization parameter for hemophilia A gene therapy product candidates. Optimization strategies include promoter engineering to increase transcription, codon optimization of mRNA to improve translation, and amino-acid substitution to promote secretion. Herein, we describe both rational and empirical design approaches to the development of a minimally sized, highly potent AAV-fVIII vector that incorporates three unique elements: a liver-directed 146-nt transcription regulatory module, a target-cell-specific codon optimization algorithm, and a high-expression bioengineered fVIII variant.

Frequency and epitope specificity of anti-factor VIII C1 domain antibodies in acquired and congenital hemophilia A.

Several studies showed that neutralizing anti-factor VIII (anti-fVIII) antibodies (inhibitors) in patients with acquired hemophilia A (AHA) and congenital hemophilia A (HA) are primarily directed to the A2 and C2 domains. In this study, the frequency and epitope specificity of anti-C1 antibodies were analyzed in acquired and congenital hemophilia inhibitor patients (n = 178). The domain specificity of antibodies was studied by homolog-scanning mutagenesis (HSM) with single human domain human/porcine fVIII proteins and antibody binding to human A2, C1, and C2 domains presented as human serum albumin (HSA) fusion proteins.

A subset of high-titer anti-factor VIII A2 domain antibodies is responsive to treatment with factor VIII.

The primary B-cell epitopes of factor VIII (fVIII) are in the A2 and C2 domains. Within the C2 domain, antibody epitope and kinetics are more important than inhibitor titer in predicting pathogenicity in a murine bleeding model. To investigate this within the A2 domain, the pathogenicity of a diverse panel of antihuman fVIII A2 domain monoclonal antibodies (MAbs) was tested in the murine model.

Tumor angiogenesis therapy using targeted delivery of Paclitaxel to the vasculature of breast cancer metastases.

Breast cancer aberrantly expresses tissue factor (TF) in cancer tissues and cancer vascular endothelial cells (VECs). TF plays a central role in cancer angiogenesis, growth, and metastasis and, as such, is a target for therapy and drug delivery. TF is the cognate receptor of factor VIIa (fVIIa).

Visualizing cancer and response to therapy in vivo using Cy5.5-labeled factor VIIa and anti-tissue factor antibody.

We have developed a specific technique for imaging cancer in vivo using Cy5.5-labeled factor VIIa (fVIIa), clotting-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibody. FVIIa is the natural ligand for TF.

The diversity of the immune response to the A2 domain of human factor VIII.

Approximately 30% of patients with severe hemophilia A develop inhibitory anti-factor VIII (fVIII) antibodies (Abs). We characterized 29 anti-human A2 monoclonal Abs (mAbs) produced in a murine hemophilia A model. A basis set of nonoverlapping mAbs was defined by competition enzyme-linked immunosorbent assay, producing 5 major groups.

A major determinant of the immunogenicity of factor VIII in a murine model is independent of its procoagulant function.

A main complication of treatment of patients with hemophilia A is the development of anti-factor VIII (fVIII) antibodies. The immunogenicity of fVIII potentially is a function of its procoagulant activity, which may result in danger signals that drive the immune response. Alternatively, intrinsic structural elements in fVIII may be particularly immunogenic.

Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein.

A point mutation leading to amino acid substitution N1922S in the A3 domain of factor VIII (fVIII) results in moderate to severe hemophilia A. A heterologous expression system comparing N1922S-fVIII and wild-type fVIII (wt-fVIII) demonstrated similar specific coagulant activities but poor secretion of N1922S-fVIII. Immunocytochemical analysis revealed that intracellular levels of N1922S-fVIII were similar to those of wt-fVIII.

Lentiviral vector platform for production of bioengineered recombinant coagulation factor VIII.

Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100 µg/l of recombinant fVIII, with a per cell production rate of 0.

The comparative immunogenicity of human and porcine factor VIII in haemophilia A mice.

Inhibitory antibodies to factor VIII (FVIII inhibitors) are the most significant complication in the management of haemophilia A. The immunogenicity of FVIII may be driven in part by structural determinants within the FVIII molecule itself. Regions of nonidentity between human and porcine FVIII possibly could drive differential immune responses.

Nonclassical anti-C2 domain antibodies are present in patients with factor VIII inhibitors.

The antihuman factor VIII (fVIII) C2 domain immune response in hemophilia A mice consists of antibodies that can be divided into 5 groups of structural epitopes and 2 groups of functional epitopes. Groups A, AB, and B consist of classical C2 antibodies that inhibit the binding of fVIII to phospholipid and von Willebrand factor. Groups BC and C contain nonclassical C2 antibodies that block the activation of fVIII by thrombin or factor Xa.