Von Willebrand factor (VWF) is unable to interact spontaneously with platelets because this interaction requires a conversion of the VWF A1 domain into a glycoprotein Ib␣ (GpIb␣) binding conformation. Here, we discuss a llama-derived antibody fragment (AU/VWFa-11) that specifically recognizes the GpIb␣-binding conformation. AU/VWFa-11 is unable to bind VWF in solution, but efficiently interacts with ristocetin-or botrocetin-activated VWF, VWF comprising type 2B mutation R1306Q, or immobilized VWF. These unique properties allowed us to use AU/ VWFa-11 for the detection of activated VWF in plasma of patients characterized by spontaneous VWF-platelet interactions: von Willebrand disease (VWD) type 2B and thrombotic thrombocytopenic purpura (TTP). For VWD type 2B, levels of activated VWF were increased 12-fold (P < .001) compared to levels in healthy volunteers. An inverse correlation between activated VWF levels and platelet count was observed (R 2 ‫؍‬ 0.74; P < .003). With regard to TTP, a 2-fold (P < .001) increase in activated VWF levels was found in plasma of patients with acquired TTP, whereas an 8-fold increase (P < .003) was found in congenital TTP. No overlap in levels of activated VWF could be detected between acquired and congenital TTP, suggesting that AU/VWFa-11 could be used to distinguish between both disorders. Furthermore, it could provide a tool to investigate the role of VWF in the development of thrombocytopenia in various diseases. (Blood. 2005;106: 3035-3042)

Over the last decades, advances in the knowledge of immunology have led to the identification of immune checkpoints, reinvigorating cancer immunotherapy. Although normally restricted to activated T and B cells, constitutive expression of CD70 in tumor cells has been described. Moreover, CD70 is implicated in tumor cell and regulatory T cell survival through interaction with its ligand, CD27. In this review, we summarize the targetable expression patterns of CD70 in a wide range of malignancies and the promising mechanism of anti-CD70 therapy in stimulating the anti-tumor immune response. In addition, we will discuss clinical data and future combination strategies.

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. ALX-0081 exerts potent IntroductionThe successive adhesion, activation, and aggregation of platelets are key processes in arterial thrombus formation after endothelial damage. 1,2 Both rupture of atherosclerotic plaques as well as surgical interventions to treat atherosclerosis (eg, percutaneous coronary intervention [PCI]) may cause exposure of the subendothelium and subsequent clot formation. Eventually, this can result in the occlusion of arteries, leading to ischemia, myocardial infarcts, or stroke. Given the central role of platelets in thrombosis, a substantial number of currently marketed antithrombotic drugs, such as aspirin, clopidogrel, and abciximab, target different steps involved in platelet activation and aggregation. 1,2 Thanks to their complementary mechanisms of action, the combination of these agents inhibits platelet aggregation to a greater extent than either agent alone. 3 However, the use of these antiplatelet drugs is hampered by an increased bleeding risk 1,2 and the occurrence of treatment resistance in some patients. 4 Moreover, the irreversible nature of their action can complicate the staunching of bleeding. 1,2 Inhibition of the initial adhesion of platelets to subendothelial collagen provides an alternative strategy to prevent unwanted clot formation. The plasma glycoprotein VWF plays a pivotal role in this adhesion via binding to exposed collagen on the one hand, and the interaction of its A1 domain with the gpIb-IX-V receptor complex on the surface of platelets on the other hand. [5][6][7] Interestingly, the VWF A1 domain is only exposed under high-shear conditions, 8,9 so VWF only acts as a bridging molecule between collagen and platelets in small or stenosed arteries. Therefore, it is expected that drugs inhibiting this interaction between VWF and platelets show an improved safety profile with respect to bleeding tendency. Indeed, the antithrombotic effect of several compounds targeting the gpIb-VWF-A1-axis, like aurintricarboxylic acid, 10-12 recombinant VWF fragments, 10,13-16 a recombinant gpIb chimeric protein, 17,18 anti-VWF mAbs, [19][20][21][22][23][24][25][26][27] and an anti-VWF aptamer 28 has been demonstrated in vitro and in vivo, without increasing the bleeding risk. 13,[16][17][18]21,23,25,28,29 Nevertheless, until now only 3 drug candidates have been evaluated in humans, including ALX-0081. [30][31][32][33] We developed ALX-0081, a bivalent humanized Nanobody directed against the A1 domain of VWF. Nanobodies are therapeutic proteins derived from the heavy-chain variable domains (VHH) that occur naturally in heavy-chain-only Igs of Camelidae. 34,35 Here we...

To cite this article: Hulstein JJJ, van Runnard Heimel PJ, Franx A, Lenting PJ, Bruinse HW, Silence K, de Groot PhG, Fijnheer R. Acute activation of the endothelium results in increased levels of active von Willebrand factor in hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome. 2006; 4: 2569-75. Summary. Background: HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome is a severe complication of pre-eclampsia in pregnancy, characterized by microvascular platelet thrombi. Activation of the endothelium is thought to play a key role in pre-eclampsia and HELLP syndrome. Activation of endothelial cells may lead to release of von Willebrand factor (VWF) multimers, which are highly reactive with platelets. Normally, newly released multimers are cleaved by ADAMTS13, resulting in less reactive derivatives. Objective: We hypothesized that HELLP syndrome is characterized by increased amounts of active VWF compared with healthy pregnancy and pre-eclampsia, due to acute activation of endothelial cells. This might contribute to thrombocytopenia and thrombotic microangiopathy. Methods: Active VWF and ADAMTS13 activity were measured in healthy pregnant volunteers (n ¼ 9), patients with pre-eclampsia (n ¼ 6) and patients with HELLP syndrome (n ¼ 14) at similar gestational ages. To study the role of endothelial cell activation, the propeptide/mature VWF ratio was determined, and VWF released by cultured endothelial cells was analyzed. Results: Active VWF levels were increased 2.1-fold in HELLP syndrome compared with healthy pregnant volunteers (P < 0.001) and 1.6-fold compared with patients with pre-eclampsia (P ¼ 0.001). ADAMTS13 activity was moderately decreased in patients with HELLP syndrome compared with healthy pregnant volunteers (P < 0.004), but not compared with patients with pre-eclampsia. The propeptide/mature VWF ratio was increased 1.7-fold compared with healthy pregnant volunteers (P < 0.001) and 1.5-fold compared with patients with pre-eclampsia (P < 0.05). A significant correlation was found between this ratio and the activation factor of VWF (r ¼ 0.68, P < 0.001). The amount of active VWF was increased 1.4-fold in medium of stimulated endothelial cells when compared with non-stimulated cells (P < 0.05). Conclusion: Acute endothelial cell activation in HELLP syndrome and decreased ADAMTS13 activity result in increased amounts of active VWF. This might explain the consumptive thrombocytopenia and thrombotic microangiopathy associated with HELLP syndrome. Inhibition of circulating active VWF could be a potential new approach in the treatment of patients with HELLP syndrome. J Thromb Haemost

These data suggest that targeted immunotherapy for CD70/CD27 and PD-L1/PD-1 might be promising in anaplastic thyroid cancer. However, as a low amount of tumour-infiltrating lymphocytes was observed in most lesions, combined therapy with agents enhancing the invasion of lymphocytes in the tumour region needs to be considered.

Overexpression of CD70 has been documented in a variety of solid and hematological tumors, where it is thought to play a role in tumor proliferation and evasion of immune surveillance. Here, we describe ARGX-110, a defucosylated IgG1 monoclonal antibody (mAb) that selectively targets and neutralizes CD70, the ligand of CD27.   ARGX-110 was generated by immunization of outbred llamas. The antibody was germlined to 95% human identity, and its anti-tumor efficacy was tested in several in vitro assays. ARGX-110 binds CD70 with picomolar affinity. In depletion studies, ARGX-110 lyses tumor cells with greater efficacy than its fucosylated version. In addition, ARGX-110 demonstrates strong complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis activity. ARGX-110 inhibits signaling of CD27, which results in blocking of the activation and proliferation of Tregs. In a Raji xenograft model, administration of the fucosylated version of ARGX-110 resulted in a prolonged survival at doses of 0.1 mg/kg and above. The pharmacokinetics of ARGX-110 was tested in cynomolgus monkeys; the calculated half-life is 12 days. In conclusion, ARGX-110 is a potent blocking mAb with a dual mode of action against both CD70-bearing tumor cells and CD70-dependent Tregs. This antibody is now in a Phase 1 study in patients with advanced malignancies expressing CD70 (NCT01813539).

Although normally restricted to activated T and B cells and mature dendritic cells, constitutive expression of CD70, a member of the tumor necrosis family, has been described in both hematological and solid tumors, where it increases tumor cell and regulatory T cell survival by signaling through its receptor, CD27.We have assessed the co-expression of CD70 and CD27 in non-small cell lung cancer (NSCLC) by immunohistochemistry to explore a correlation between expression of the protein and tumor histologic subtype, genetic aberrations and prognosis. Furthermore, we tested the ability of ARGX-110, a CD70-blocking antibody, to induce NK cell-mediated cytotoxicity.Our results revealed CD70 expression on the surface of both primary and metastatic NSCLC tumor cells and in the tumor microenvironment. Moreover, CD27-expressing tumor infiltrating lymphocytes were found adjacent to the tumor cells, suggesting active CD70-mediated signaling. Finally, we have shown that ARGX-110, has potent cytotoxic effects on CD70+ NSCLC cell lines.

The purpose of this study was to evaluate safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor efficacy of ARGX-110, a glyco-engineered monoclonal antibody, targeting CD70, in patients with CD70 expressing advanced malignancies. Dose escalation with a sequential 3+3 design was performed in five steps at the 0.1, 1, 2, 5, and 10 mg/kg dose levels ( = 26). ARGX-110 was administered intravenously every 3 weeks until progression or intolerable toxicity. Dose-limiting toxicity was evaluated in the 21 days following the first ARGX-110 administration (Cycle 1). Samples for pharmacokinetics and pharmacodynamics were collected. Dose-limiting toxicity was not observed and the maximum tolerated dose was not reached. ARGX-110 was generally well tolerated, with no dose-related increase in treatment-emergent adverse events (TEAE). The most common TEAE were fatigue and drug related infusion-related reactions (IRR). Of the 20 SAEs reported, five events, all IRRs, were considered related to ARGX-110. ARGX-110 demonstrates dose proportionality over the dose range 1 to 10 mg/kg, but not at 0.1 mg/kg and a terminal half-life of 10 to 13 days. The best overall response was stable disease (14/26) in all 26 evaluable patients with various malignancies and the mean duration of treatment was 15 weeks. No dose-response related antitumor activity was observed, but biomarker readouts provided signs of biological activity, particularly in patients with hematologic malignancies. This dose-escalation phase I trial provides evidence of good tolerability of ARGX-110, pharmacokinetics, and preliminary antitumor activity at all dose levels in generally heavily pretreated patients with advanced CD70-positive malignancies. .