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Background: Efgartigimod is a human IgG1 antibody Fc-fragment that reduces IgG autoantibody levels through FcRn blockade. This study reports safety of efgartigimod across IgG-mediated disorders. Methods: The safety of intravenous efgartigimod was evaluated in 204 efgartigimod-treated subjects with generalized myasthenia gravis (phase 3 ADAPT and 3-year open-label extension ADAPT+ trials), primary immune thrombocytopenia (phase 3 ADVANCE trial), or pemphigus (open-label phase 2 trial). These studies examined different efgartigimod doses (10–25 mg/kg), including cyclical dosing in generalized myasthenia gravis and continuous weekly dosing in primary immune thrombocytopenia and pemphigus. Results: Across all indications and doses studied, efgartigimod demonstrated a consistent safety profile, with treatment-emergent adverse event (TEAE) rates comparable to placebo (ADAPT, 77.4% efgartigimod/84.3% placebo; ADVANCE, 93.0% efgartigimod/95.6% placebo; and 85% in the pemphigus study). Most TEAEs were mild to moderate in severity. Discontinuation rates due to adverse events were consistently low (ADAPT, 3.6% efgartigimod/3.6% placebo; ADVANCE, 3.5% efgartigimod/2.2% placebo; and 3% of pemphigus study participants). In ADAPT+, no increases in TEAEs or infections occurred with additional efgartigimod dosing (19 cycles). Conclusions: Efgartigimod was well tolerated across indications and doses studied. Most TEAEs, including infections, were mild or moderate in severity and did not increase in frequency with recurrent dosing.

Background: Efgartigimod is a human IgG1 antibody Fc-fragment that reduces total and pathogenic IgG autoantibody levels through FcRn blockade. ADAPT was a phase 3 trial evaluating efgartigimod in patients with generalized myasthenia gravis (gMG). Patients who completed ADAPT could enroll in ADAPT+ (open-label extension). Methods: Efgartigimod (10 mg/kg intravenous) was administered in cycles of 4 weekly infusions, with subsequent cycles initiated based on clinical evaluation. ADAPT+ evaluated long-term safety and tolerability of efgartigimod in patients with gMG. Efficacy was assessed utilizing MG-ADL and QMG scores. Results: Of 167 patients from ADAPT, 151 (90%) entered ADAPT+, and 145 received ≥1 cycle as of January 2022. Over 217.55 patient-years of follow-up (mean duration per patient, 548 days), incidence of adverse events did not increase with subsequent cycles. AChR-Ab+ patients with ≥1 year of follow-up across ADAPT/ADAPT+ (n=95) received a median (range) 5.0 (0.4–7.6) cycles per year. All AChR-Ab+ patients (n=111) demonstrated consistent improvements (mean change [SE], week 3 of cycle 1) in MG-ADL (-5.0 [0.33]; up to 14 cycles) and QMG (-4.7 [0.41]; up to 7 cycles) scores during each cycle. Conclusions: These ADAPT+ analyses suggest long-term efgartigimod treatment is well tolerated and efficacious. Additional final data cut analyses will be presented at CNSF 2023.

Background: Efgartigimod is a human IgG1 antibody Fc-fragment that reduces IgG levels through FcRn blockade. A key efficacy indicator in the treatment of IgG autoantibody-mediated generalized myasthenia gravis (gMG) is improvement in MG-ADL score. Methods: The ADAPT phase 3 trial evaluated safety and efficacy of efgartigimod in patients with gMG, including reaching and maintaining of minimal symptom expression (MSE; defined as an MG-ADL total score of 0 or 1). Results: 167 patients (AChR-Ab+, n=129; AChR-Ab-, n=38) were randomized to receive treatment cycles of 4 weekly infusions of efgartigimod or placebo. Significantly more AChR-Ab+ efgartigimod-treated patients achieved MSE during cycle 1 compared to placebo-treated patients (40.0% [n=26/65] vs 11.1% [n=7/63; P<0.0001]). In cycle 2, 31.4% (n=16/51) of AChR-Ab+ patients in the efgartigimod cohort achieved MSE compared to none in the placebo cohort. MG-ADL score improved by ≥6 points in 56.9% of AChR-Ab+ efgartigimod-treated patients compared to 20.6% of placebo-treated patients in cycle 1. Most patients achieved MSE by week 4 of a cycle, paralleling early reduction in IgG levels, and MSE duration ranged from 1 to ≥10 weeks. Adverse events were predominantly mild to moderate. Conclusions: Efgartigimod treatment resulted in more patients with AChR-Ab+ gMG achieving both MSE and clinically meaningful MG-ADL improvements.

The direct amination of glycerol with dimethylamine (DMA) was studied over catalysts based on salts of phosphomolybdic acid. The highest yield of (dimethylamino)acetone (33%) was observed over a catalyst containing 50 wt% of Cs2.5H0.5PMo12O40supported on a mesoporous silica. The corresponding sample showed good dispersion of the active phase leading to an increased number of available acid sites. With respect to the observed products, a reaction scheme was established explaining notably that the formation of 1,3-bis-dimethylamine-2-propanol and (dimethylamino)propanol was not possible due to the blocking of the required strong acid sites by DMA under reaction conditions.

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 vitroand in vivopharmacology. ALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitroassays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control. ALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration. ALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitroaffinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivoeffect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.

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).

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. Drugs were administered before, immediately after, or 15 or 60 minutes after the total occlusion of the MCA. ALX-0081 prevented MCA thrombosis and induced reperfusion when given immediately after and 15 minutes after complete occlusion and reduced brain damage without inducing hemorrhage, whereas tirofiban prevented thrombosis but did not induce reperfusion and induced striking brain hemorrhage. rtPA also induced reperfusion when given 60 minutes after occlusion but provoked brain hemorrhage. Skin bleeding time was not modified or was moderately prolonged by ALX-0081, whereas tirofiban and rtPA prolonged it. The inhibition of the GPIb–VWF axis in guinea pigs prevents cerebral artery thrombosis and induces early reperfusion without provoking intracerebral bleeding thus reducing brain infarct area.

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. In this model, acute episodes of TTP are induced by administration of an ADAMTS13-inhibiting mAb. ALX-0681 completely prevented the rapid onset of severe thrombocytopenia and schistocytic hemolytic anemia. After induction of TTP, platelet counts also rapidly recovered on administration of ALX-0681. This effect was corroborated by the full neutralization of VWF activity. The schistocytic hemolytic anemia was also halted and partially reversed by ALX-0681 treatment. Brain CT scans and post mortem analysis did not reveal any sign of bleeding, suggesting that complete neutralization of VWF by ALX-0681 under conditions of thrombocytopenia was not linked with an excessive bleeding risk. The results obtained in this study demonstrate that ALX-0681 can successfully treat and prevent the most important hallmarks of acquired TTP without evidence of a severe bleeding risk. Therefore, ALX-0681 offers an attractive new therapeutic option for acquired TTP in the clinical setting.

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 activity in vitro and in vivo. Perfusion experiments with blood from patients with acute coronary syndrome on standard antithrombotics demonstrated complete inhibition of platelet adhesion after addition of ALX-0081, while in the absence of ALX-0081 residual adhesion was observed. In a baboon efficacy and safety model measuring acute thrombosis and surgical bleeding, ALX-0081 showed a superior therapeutic window compared with marketed antithrombotics. Pharmacokinetic and biodistribution experiments demonstrated target-mediated clearance of ALX-0081, which leads to a self-regulating disposition behavior. In conclusion, these preclinical data demonstrate that ALX-0081 combines a high efficacy with an improved safety profile compared with currently marketed antithrombotics. ALX-0081 has entered clinical development.

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. Recently, other ligands for GPIb-IX-V besides the extensively studied VWF have been identified, such as : -thrombin, coagulation factor XII (FXII), high molecular weight kininogen (HMWK), factor XI (FXI), integrin Mac-1 and P-selectin. In this review, the interaction of GPIb-IX-V with its different ligands is described and the anticipated or demonstrated in vivo effects are discussed.

Newland: Sobi: Other: Ad Board; Novartis: Other: Ad Board, Research Funding, Speakers Bureau; Dova Pharmaeceuticals: Other: Ad Board; Grifols: Other: Ad Board; argenx: Other: Ad Board; Amgen: Other: Ad Board, Research Funding, Speakers Bureau; GSK: Research Funding. Liebman:Dova: Consultancy; Pfizer: Consultancy; Rigel: Consultancy; Novartis: Consultancy; argenx: Consultancy; Bristol-Myers: Research Funding; Janssen: Research Funding. McDonald:Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Michel:Rigel: Consultancy; Novartis: Consultancy; Amgen: Consultancy. Miyakawa:argenx: Consultancy; Kyowa Kirin: Consultancy; Zenyaku Kogyo: Consultancy; UCB: Consultancy. Parys:argenx: Current Employment. De Haard:argenx: Current Employment. Ulrichts:argenx: Current Employment. Godar:argenx: Current Employment. De Beuf:argenx: Current Employment. Ayguasanosa:argenx: Current Employment. Broome:Alexion: Honoraria; argenx: Honoraria; apellis: Honoraria; sanofi: Honoraria. Kuter:Rigel: Consultancy, Honoraria, Other, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Other: Travel Expenses, Research Funding; Actelion (Syntimmune): Consultancy, Honoraria, Other: Travel Expenses, Research Funding; Protalex: Consultancy, Honoraria, Other, Research Funding; CRICO: Consultancy, Honoraria; Immunovant: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; Kyowa-Kirin: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Principia: Consultancy, Research Funding; Protalix Biotherapeutics: Consultancy; Shionogi: Consultancy; Protalex: Consultancy, Honoraria, Research Funding; Shionogi: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Dova: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Kezar Life Sciences, Inc: Other, Research Funding; Merck Sharp Dohme: Consultancy, Honoraria; Momenta: Consultancy, Honoraria; Alnylam: Consultancy, Honoraria, Other: Travel Expenses, Research Funding; Agios: Consultancy, Honoraria, Other: Travel Expenses, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding; Zafgen: Consultancy, Honoraria; Up-To-Date: Consultancy, Honoraria, Patents & Royalties; UCB: Consultancy, Honoraria; Platelet Disorder Support Association: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Argenx: Consultancy, Honoraria, Other: Travel Expenses, Research Funding; Caremark: Consultancy, Honoraria; Immunovant: Other: Travel Expenses, Research Funding; Principia Biopharma: Consultancy, Honoraria, Other, Research Funding; Takeda (Bioverativ): Consultancy, Honoraria, Other, Research Funding; Sanofi (Genzyme): Consultancy, Honoraria.

Cardiovascular disease is the major cause of mortality in Western countries. Platelets play a crucial role in the development of arterial thrombosis and other pathophysiologies leading to clinical ischemic events. In the damaged vessel wall, platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between subendothelial collagen and the platelet receptor glycoprotein (GP) Ib/IX/V. This reversible adhesion allows platelets to roll over the damaged area, decreasing their velocity and resulting in strong platelet activation. This leads to the conformational activation of the platelet GPIIb/IIIa receptor, fibrinogen binding and finally to platelet aggregation. As each interaction (collagen-VWF, VWF-GPIb and GPIIb/IIIa-fibrinogen) plays an essential role in primary haemostasis, loss of either of these interactions results in a bleeding diathesis, implying that interfering with these interactions might result in an anti-thrombotic effect. Whereas GPIIb/IIIa antagonists indeed are effective anti-thrombotics, it has been suggested that drugs which block the initial steps of thrombus formation (collagen-VWF or VWF-GPIb interaction) might have advantages over the ones that merely inhibit platelet aggregation. In this review we will discuss and compare the development of monoclonal antibodies (moAbs) that inhibit platelet adhesion or platelet aggregation. The effect of the moAbs in in vitro experiments, in in vivo models and in clinical trials will be described. Benefits, limitations, current applications and the future perspectives in the development of antibodies for each target will be discussed.

Background: Platelet adhesion and aggregation at sites of vascular injury exposed to rapid blood flow require von Willebrand factor (VWF). VWF becomes immobilized by binding to subendothelial components or by a self-association at the interface of soluble and surface-bound VWF. Objectives: As this self-association has been demonstrated only under shear conditions, our first goal was to determine whether the same interaction could be observed under static conditions. Furthermore, we wanted to identify VWF domain(s) important for this self-association. Results: Biotinylated VWF (b-VWF) interacted dose-dependently and specifically with immobilized VWF in an enzyme-linked immunosorbent assay (ELISA) assay, showing that shear is not necessary to induce the VWF self-association. Whereas anti-VWF monoclonal antibodies (mAbs) had no effect on the self-association, the proteolytic VWF-fragments SpII(1366–2050) and SpIII(1–1365) inhibited the b-VWF–VWF interaction by 70 and 80%, respectively. Moreover, a specific binding of b-VWF to immobilized Sp-fragments was demonstrated. Finally, both biotinylated SpII and SpIII were able to bind specifically to both immobilized SpII and SpIII. Similar results were observed under flow conditions, which confirmed the functional relevance of our ELISA system. Conclusion: We have developed an ELISA binding assay in which a specific VWF self-association under static conditions can be demonstrated. Our results suggest a multiple domain interaction between immobilized and soluble VWF.

We have previously described a monoclonal antibody (mAb), 1C1E7, against von Willebrand factor (VWF), that increases ristocetin‐induced platelet aggregation (RIPA) and induces a preferential binding of the high‐molecular‐weight multimers of VWF to platelet GPIb. Further investigations using a rotational viscometer at a shear rate of 4000 s−1could now demonstrate that shear‐induced platelet aggregation (SIPA) is significantly increased with 1C1E7 and that this could be completely inhibited by the anti‐GPIb mAb 6D1. In contrast, platelet adhesion to a collagen surface at a shear rate of 2600 s−1, using a rectangular perfusion chamber, was significantly inhibited in the presence of 1C1E7. When citrated whole blood was incubated with 1C1E7, a spontaneous binding of VWF to the platelet GPIb could be demonstrated by flow cytometric analysis. Parallel to this, a decrease of the highest molecular weight multimers of VWF in the plasma was found. Platelets with bound VWF on their surface were able to form macroaggregates but were no longer able to adhere. These phenomena are very similar to the alterations described in von Willebrand's disease type 2B. The epitope of this mAb could be localized to the N‐terminal part of the subunit; therefore a distant conformational change in the A1 domain of VWF is suggested.

Cardiovascular diseases are one of the major causes of mortality in the western world. As platelet dependent thrombosis is of central importance in their pathophysiology, several successful strategies, targeting a specific platelet function or interaction, have been developed to prevent or treat these disorders. However, as the current antiplatelet strategies are limited in efficacy and safety, and often influence normal haemostatic functions, new compounds are being developed with improved characteristics. This review deals with the development of novel antiplatelet compounds for which evidence is available on their antithrombotic action in vivo. In a first part, these compounds, their targets and their potential applicability are discussed. The second part of this review focuses on BT tests and bleeding models and their usefulness for determination and / or prediction of the safety of novel antiplatelet compounds.

Platelet adhesion to a damaged blood vessel is the initial trigger for arterial hemostasis and thrombosis. Platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between collagen within the damaged vessel wall and the platelet receptor glycoprotein Ib / V / IX (GPIb), an interaction especially important under high shear conditions[1]. This reversible adhesion allows platelets to roll over the damaged area, which is then followed by a firm adhesion mediated by the collagen receptors (α2β1, GPVI,...) in addition[2] resulting in platelet activation. This leads to the conformational activation of the platelet αIIb α3 receptor, fibrinogen binding and finally to platelet aggregation. Over the past decades, modulation of platelet function has been a strategy for the control of cardiovascular disease. Lately, drugs have been developed that target the fibrinogen receptor αIIb α3 or the ADP receptor and many of these promising compounds have been tested in clinical trials. However the development of products that interfere with the first step of hemostasis, i.e. the platelet adhesion, has lagged behind. In this review we want to discuss (i) the in vivo studies that were performed with compounds that target proteins involved in different adhesion steps i.e. the VWF-GPIb-axis, the collagen-VWF axis and the collagen-collagen receptor axis and (ii) the possible advantages these putative new drugs could have over the current antiplatelet agents.

Newland: Novartis: Consultancy, Honoraria, Research Funding; Angle: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Ono: Consultancy, Honoraria; Shionogi: Consultancy, Honoraria; Rigel: Consultancy, Honoraria, Research Funding; Argenx: Consultancy, Honoraria; Dova: Consultancy, Honoraria. Sánchez-González:Amgen: Consultancy, Speakers Bureau; Gilead: Speakers Bureau; Navartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Godar:Argenx: Employment. Verschueren:Argenx: Consultancy. Gandini:Argenx: Employment, Equity Ownership. Ulrichts:Argenx: Employment. Beauchamp:Argenx: Employment. Dreier:Argenx: Employment. Ward:Argenx: Equity Ownership, Honoraria, Research Funding. Michel:Rigel: Consultancy; Amgen: Consultancy; Novartis: Consultancy. Liebman:Pfizer: Consultancy; Dova: Consultancy; Bristol-Myers: Research Funding; Argenx: Consultancy; Rigel: Consultancy; Janssen: Research Funding; Novartis: Consultancy. De Haard:Argenx: Employment, Equity Ownership, Patents & Royalties. Leupin:Argenx: Employment, Equity Ownership, Patents & Royalties. Kuter:Dova: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; UCB: Consultancy, Honoraria; Momenta: Consultancy, Honoraria; Caremark: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Protalix: Consultancy, Honoraria; Principia: Consultancy, Honoraria, Research Funding; Platelet Disorder Support Association: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Principia: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Kyowa-Kirin: Consultancy, Honoraria; Kyowa-Kirin: Consultancy, Honoraria; Merck Sharp Dohme: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Merck Sharp Dohme: Consultancy, Honoraria; Protalix: Consultancy, Honoraria; Rigel: Consultancy, Honoraria, Research Funding; Rigel: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Bristol Myers Squibb (BMS): Consultancy, Honoraria, Research Funding; Alnylam: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Protalex: Consultancy, Honoraria, Research Funding; Dova: Consultancy, Honoraria; Protalex: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Up-to-Date: Consultancy, Honoraria, Patents & Royalties: 3 Up-to-Date chapters; Up-to-Date: Consultancy, Honoraria, Patents & Royalties: 3 Up-to-Date chapters; Zafgen: Consultancy, Honoraria; Takeda (Bioverativ): Consultancy, Honoraria, Research Funding; Platelet Disorder Support Association: Consultancy, Honoraria; Shinogi: Consultancy, Honoraria; Agios: Consultancy, Honoraria, Research Funding; Actelion (Syntimmune): Consultancy, Honoraria, Research Funding; Takeda (Bioverativ): Consultancy, Honoraria, Research Funding; Caremark: Consultancy, Honoraria; Kezar: Research Funding; Kezar: Research Funding; Shire: Consultancy, Honoraria; Argenx: Consultancy, Honoraria, Research Funding; Alnylam: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb (BMS): Consultancy, Honoraria, Research Funding; Argenx: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Actelion (Syntimmune): Consultancy, Honoraria, Research Funding; Shinogi: Consultancy, Honoraria; Zafgen: Consultancy, Honoraria; Momenta: Consultancy, Honoraria; UCB: Consultancy, Honoraria.

Ulrichts: argenx: Employment. Cousin:argenx: Employment. Dreier:argenx: Employment. de Haard:argenx: Employment. Leupin:argenx: Employment.

Abd-Elaziz: Ablynx NV: Consultancy. Kamphuisen:Ablynx NV: Consultancy. Lyssens:Ablynx NV: Employment. Reuvers:Ablynx NV: Consultancy. den Daas:Ablynx NV: Consultancy. Van Bockstaele:Ablynx NV: Employment. Vercruysse:Ablynx NV: Employment. Ulrichts:Ablynx NV: Employment. Baumeister:Ablynx NV: Employment. Crabbe:Ablynx NV: Employment. Compernolle:Ablynx NV: Employment. Holz:Ablynx NV: Employment.

Platelet microparticle (MP) formation during shear-induced platelet aggregation (SIPA) contributes to a relevant part of thrombin generation. A monoclonal antibody (mAb) Ib-23 against platelet GPIb alpha (provided by B.S.) inhibits von Willebrand factor binding, ristocetin-induced platelet aggregation (RIPA), shear induced MP formation and thrombin generation. Platelet calpain affects several functions including MP-formation. One of several target substrates of calpain, filamin, binds to the cytoplasmic tail of GPIbalpha and mediates the assembly of the contractile elements during platelet activation. We therefore hypothesized that the inhibitory mechanism of our mAb Ib-23 on MP formation is a “calpeptin-like effect”, i.e. an inhibition of calpain affecting the protein tyrosine phosphorylation. PPACK-anticoagulated platelet-rich plasma or washed platelets (250′000/μl) were exposed to a shear rate of 5000 sec−1 for 5 min in a cone-plate viscometer. The mAb Ib-23 was added at the RIPA-inhibiting IC80–90. The samples were fluorescence-labelled with an anti-GPIIb-PE mAb to monitor SIPA and MP formation and analyzed by flow cytometry. Resting and sheared platelets were lysed in SDS-PAGE loading buffer for the analysis of filamin degradation and protein tyrosine phosphorylation. Results: Epitope mapping showed that mAb Ib-23 binds between residues 234 and 456 on GP Ib alpha. Exposure of platelets to high shear rates induced the activation of calpain as demonstrated by the degradation of filamin, a phenomenon blocked by the mAb Ib-23. Ib-23 did not inhibit the SIPA induced tyrosine phosphorylation of high molecular weight proteins like FAK, Syk and alpha-actinin, it even weakly enhanced the tyrosine phosphorylation of these proteins. Moreover, Ib-23 prevented the dephosphorylation of low molecular weight proteins (appr. 40 and 33kDa). Annexin V-binding was unaffected by Ib-23. In contrast, abciximab, a GP IIb/IIIa antagonist, partially inhibited SIPA and annexin binding, but not MP generation. Abciximab weakly inhibited the degradation of filamin, inhibited SIPA-induced tyrosine phosphorylation of FAK and Syk, but did not prevent the tyrosine dephosphorylation of the low molecular weight proteins. Control-experiments with calpeptin demonstrated a similar inhibition of filamin degradation as with mAb Ib-23. An isotype control antibody was without effects. The intact antibody Ib-23 was more effective than the Fab2 fragment. A mAb against the FcgammaRII receptor (IV.3, to exclude a bridging of the FcgammaRII and GpIbalpha via the antibody’s Fc part) did not affect MP-formation. We conclude that binding of mAb Ib-23 on the outside of GP Ib alpha inhibits SIPA-induced MP formation and thrombin generation possibly via a “calpeptin-like effect” which inhibits the activation of calpain and of protein tyrosine phosphatases.

Glycoprotein VI (GPVI), an essential platelet collagen receptor, belongs to the immunoglobulin (Ig) superfamily and presents two Ig-like loops in its extracellular domain (EC1, EC2). Little is known about the structures involved in GPVI binding to collagen and to its specific ligands, convulxin and collagen related peptides (CRP). Our aim was to characterize these sites using different approaches: competition assays, monoclonal antibodies (MoAb) directed against the GPVI extracellular par, phage display, molecular modelling and site-directed mutagenesis. In binding assays using soluble or cell-expressed human GPVI, we observed that: (i) collagen, CRP and convulxin, competed with one another for the binding to GPVI; (ii) GPVI interaction with either ligand is inhibited by at least two MoAb directed to different independent epitopes, and (iii) MoAb displayed selective inhibitory properties on GPVI interaction with each ligand. These observations indicate that the binding sites on GPVI for the diverse ligands seem to be distinct, exhibiting specific features but at the same time sharing common elements. MoAb 9O12 is a potent inhibitor of GPVI binding to collagen. Its epitope was mapped by screening a 15 mer linear peptide library. This allowed the identification of a motif of 7 residues of which 5 are conserved and that presented a relative homology with the GPVI 30–40 sequence. In a computer model, these residues appeared in close vicinity on a surface loop exposed at the surface of the molecule and accessible to ligands. Site-directed mutagenesis revealed that Val34 and Leu36 are critical for GPVI interaction with collagen and CRP. This study allowed the identification of residues within a loop on GPVI EC1 that might be part of a collagen/CRP-binding site.

Between Sept. 1991 and Oct. 1993, 64 patients were enrolled in this multicenter study in order to evaluate the toxicity and efficacy of a chemotherapy regimen, combining three active compounds while avoiding the pulmonary and other toxicity of mitomycin C.Vindesine 3mg/m2day 1 and 8, Ifosfamide 1200mg/m2and Platinum 30mg/m2day 1, 2 and 3 were given every 28 days. Response was evaluated each 2 courses, responders were continued until 6 courses.