A biomarker assay validation approach tailored to the context of use and bioanalytical platform.

It is widely acknowledged by the bioanalytical and biomarker community that biomarker assay validations should be fit-for-purpose depending on the context of use. The challenge is how to consistently apply these principles in teams responsible for measuring a disparate array of biomarkers, often on multiple analytical platforms, at various stages of the drug discovery and development pipeline and across diverse biology focus areas. To drive consistency, while maintaining the necessary flexibility to allow validations to be driven by scientific rationale and taking into consideration the context of use and associated biological and (pre)analytical factors, a framework applicable across biomarker assays was developed.

Update to the European Bioanalysis Forum recommendation on biomarkers assays; bringing context of use into practice.

In 2012, the European Bioanalysis Forum published a recommendation on biomarker method development and the bioanalysis of biomarkers in support of drug development. Since then, there has been significant discussion on how to bring the topic of context of use of biomarker assays to the forefront so that the purpose of the assay, the use of the data and the decisions being made with the data are well defined and clearly understood, not just by the bioanalytical scientist, but across all stakeholders. Therefore, it is imperative that discussions between the bioanalytical laboratory and the end users of the data happen early (and regularly) in the drug development process to enable the right assays to be developed and appropriately validated to generate the correct data and allow suitable decisions to be made.

Caplacizumab for Acquired Thrombotic Thrombocytopenic Purpura.

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is caused by aggregation of platelets on ultralarge von Willebrand factor multimers. This microvascular thrombosis causes multiorgan ischemia with potentially life-threatening complications. Daily plasma exchange and immunosuppressive therapies induce remission, but mortality and morbidity due to microthrombosis remain high.

The preclinical pharmacology of the high affinity anti-IL-6R Nanobody® ALX-0061 supports its clinical development in rheumatoid arthritis.

Introduction: The pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology.

Reperfusion of cerebral artery thrombosis by the GPIb-VWF blockade with the Nanobody ALX-0081 reduces brain infarct size in guinea pigs.

Thrombolytic therapy is the cornerstone of treatment of acute atherothrombotic ischemic stroke but is associated with brain hemorrhage; antiplatelet therapy has limited efficacy and is still associated with intracranial bleeding. Therefore, new antithrombotic approaches with a better efficacy/safety ratio are required. We have assessed the effect of ALX-0081, a Nanobody against the A1 domain of von Willebrand factor (VWF) that blocks VWF binding to GPIb, of the thrombolytic agent recombinant tissue plasminogen activator (rtPA), and of the GPIIb/IIIa antagonist tirofiban, in a middle cerebral artery (MCA) thrombosis model in guinea pigs.

Evaluation of efficacy and safety of the anti-VWF Nanobody ALX-0681 in a preclinical baboon model of acquired thrombotic thrombocytopenic purpura.

ALX-0681 is a therapeutic Nanobody targeting the A1-domain of VWF. It inhibits the interaction between ultra-large VWF and platelet GpIb-IX-V, which plays a crucial role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In the present study, we report the efficacy and safety profile of ALX-0681 in a baboon model of acquired TTP.

The in vitro effect of the new antithrombotic drug candidate ALX-0081 on blood samples of patients undergoing percutaneous coronary intervention.

Compound ALX-0081 is a bivalent humanised Nanobody® that binds the A1-domain of von Willebrand factor (VWF) with high affinity. Consequently, it can block the interaction between VWF and its platelet-receptor-glycoprotein Ib, which leads inevitably to formation of arterial thrombi. It was the objective of this study to assess the in vitro effects of ALX-0081 on platelet adhesion and aggregation in coronary artery disease (CAD) patients to determine the optimal concentration of ALX-0081 and the effect of co-medication.

Antithrombotic drug candidate ALX-0081 shows superior preclinical efficacy and safety compared with currently marketed antiplatelet drugs.

Neutralizing the interaction of the platelet receptor gpIb with VWF is an attractive strategy to treat and prevent thrombotic complications. ALX-0081 is a bivalent Nanobody which specifically targets the gpIb-binding site of VWF and interacts avidly with VWF. Nanobodies are therapeutic proteins derived from naturally occurring heavy-chain-only Abs and combine a small molecular size with a high inherent stability.

CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells.

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method.

Relation of endothelial function to residual platelet reactivity after clopidogrel in patients with stable angina pectoris undergoing percutaneous coronary intervention.

Platelet reactivity is greater in patients with stable angina and with more extensive peripheral vascular atherosclerosis. We sought to evaluate whether impaired peripheral microcirculatory endothelial function might correlate with platelet reactivity after clopidogrel and therefore predispose to an unfavorable outcome after percutaneous coronary intervention (PCI). In 52 consecutive patients with stable angina undergoing elective PCI, endothelial function was assessed by (1) endothelial peripheral arterial tonometry (measuring the "Endoscore"); (2) the von Willebrandt factor antigen level and ristocetin co-factor activity.

Inhibition of platelet glycoprotein Ib and its antithrombotic potential.

The platelet receptor glycoprotein (GP)Ib-IX-V complex plays a dominant role in the first steps of platelet adhesion and arterial thrombus formation. Through its interaction with the multimeric plasma protein von Willebrand factor (VWF), which is bound to the damaged subendothelial structures, GPIb-IX-V tethers the platelets from the flowing blood thereby slowing them down. This step is a prerequisite for the collagen receptors to participate in firm adhesion resulting in the formation of a first platelet layer which is the basis for further thrombus formation.

Role of glycoprotein Ibalpha in phagocytosis of platelets by macrophages.

Background: Platelet (PLT) storage at 0 to 4 degrees C suppresses bacterial multiplication, but induces clusters of glycoprotein (GP) Ibalpha that trigger their phagocytosis by macrophages and reduce their survival after transfusion. A method was sought that detects cold-induced changes in GPIbalpha involved in phagocytosis.

Study Design And Methods: Human PLTs were isolated and stored for up to 48 hours at 0 degrees C.

Platelet microparticle formation and thrombin generation under high shear are effectively suppressed by a monoclonal antibody against GPIba.

We studied the inhibition of platelet microparticle (MP) formation and thrombin generation under high shear forces. We hypothesized that an inhibitor of the GPIb a -von Willebrand factor (vWF) interaction would be more effective in suppressing MP formation and thrombin generation than GPIIb/IIIa inhibitors. Platelet-rich plasma (PRP) anticoagulated with PPACK (D-Phe-Pro-Arg chloromethyl ketone) was exposed in a cone-and-plate viscometer (shear: 5,000 s(-1) for 5 min) in the presence of antagonists to GPIb a (the monoclonal antibody [Mab] Ib-23) or to GPIIb/IIIa (abciximab, tirofiban, eptifibatide) at their IC90 determined in platelet aggregometry with ristocetin or ADP, respectively.

Shielding of the A1 domain by the D'D3 domains of von Willebrand factor modulates its interaction with platelet glycoprotein Ib-IX-V.

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D'D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbalpha.

Synergistic effect of thrombin on collagen-induced platelet procoagulant activity is mediated through protease-activated receptor-1.

Objective: In the blood coagulation process, the rate of thrombin formation is critically dependent on phosphatidylserine (PtdSer) at the surface of activated platelets. Thrombin synergistically enhances the collagen-induced platelet procoagulant response. The objective of this study is to elucidate the mechanism of this synergistic action with a focus on the intracellular Ca2+ concentration ([Ca2+]i) and the various platelet receptors for thrombin.

How does agkicetin-C bind on platelet glycoprotein Ibalpha and achieve its platelet effects?

The platelet glycoprotein (GP) Ib-IX-V receptor complex has a central role in primary haemostasis and possesses binding sites for the plasmatic adhesive protein von Willebrand Factor (VWF) and thrombin. Several snake venom components have been identified in recent years that target this receptor complex and modulate its functionality. Among them, agkicetin-C is from Deinagkistrodon acutus and proved to be a potent antagonist of GPIb-IX-V.

von Willebrand factor but not alpha-thrombin binding to platelet glycoprotein Ibalpha is influenced by the HPA-2 polymorphism.

Objective: Glycoprotein (GP) Ibalpha is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex. The N-terminal domain of the GPIbalpha chain contains binding sites for alpha-thrombin and von Willebrand factor (VWF). The human platelet alloantigen (HPA)-2 polymorphism of the GPIbalpha gene is associated with a C/T transition at nucleotide 1018, resulting in a Thr/Met dimorphism at residue 145 of GPIbalpha.

Integrin alphaIIbbeta3 and shear-dependent action of glycoprotein Ibalpha stimulate platelet-dependent thrombin formation in stirred plasma.

Under conditions of arterial-wall shear rates, platelets bind to von Willebrand factor (vWf) by way of the glycoprotein Ib (GP Ib) complex and integrin alpha(IIb)beta(3). Both adhesive receptors may also play roles in the development of procoagulant activity of platelets. Here, we investigated the effect of shear stress, as provided by a rotating cylinder, on GP Ib- and integrin alpha(IIb)beta(3)-dependent thrombin generation in coagulating platelet-rich plasma (PRP).