The laboratory diagnosis of heparin-induced thrombocytopenia (HIT) is based on an enzyme immunoassay combined with a functional test, and serotonin release assay (SRA) is the gold standard for detecting activating HIT antibodies. However, a recent atypical history of HIT prompted us to evaluate whether addition of platelet factor 4 (PF4) during SRA could improve its ability to detect pathogenic HIT antibodies. Using 5B9, a monoclonal antibody to PF4/H with a human Fc fragment, we first defined the optimal PF4 concentration for detecting low amounts of platelet-activating IgG with SRA. Plasma samples from 50 patients with suspected HIT were then studied, and SRA was positive in 17 cases (Group SRA ), with relatively high levels of PF4-specific IgG (median optical density = 2·66). SRA was also systematically performed after adding 10 μg/ml of PF4 in the reaction mixture, and significant serotonin release was measured with samples from 9 additional patients (Group PF4-SRA ). Importantly, levels of PF4-specific IgG were similar in these samples and those from the 24 persistently SRA negative patients. Moreover, the pre-test probability of HIT was intermediate/high in all 'SRA ' or 'SRA-PF4 ' patients. In conclusion, addition of exogenous PF4 might improve the detection of pathogenic HIT antibodies by SRA.

Background The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical and biological criteria, but a standard is lacking for laboratory assays. Moreover, no humanized HIT antibody is available for pathophysiological studies. Objective To characterise 5B9, a chimeric monoclonal antibody, which fully mimics the effects of human HIT antibodies. Methods/Results 5B9, a chimeric anti-platelet factor 4/heparin complexes IgG1 antibody, was obtained after immunizing specific transgenic mice. 5B9 induced heparin FcγRIIA-dependent platelet aggregation and tissue factor mRNA synthesis in monocytes. It also induced significant thrombocytopenia and thrombin generation in mice expressing human PF4 and FcγRIIA receptors. The binding of 5B9 to PF4/H complexes was inhibited by 15 of 25 HIT plasma samples and only three of 25 samples containing non-pathogenic anti-PF4/H antibodies. KKO, a murine IgG2b HIT antibody, also inhibited the binding of 5B9 to PF4/H, suggesting that epitopes recognized by both antibodies are close. A docking analysis based on V and V sequences of 5B9 showed that binding of 5B9 Fab to PF4 involved 12 and 12 residues in B and D monomers, respectively, including seven previously identified as critical to the formation of a PF4/KKO complex. Two regions (Asp-7 to Thr-15 and Ala-32 to Thr-38) therefore appeared important for the binding of 5B9 and KKO on PF4 modified by heparin. Conclusions 5B9 is the first anti-PF4/H monoclonal antibody with a human Fc fragment, which induces similar cellular activation as HIT antibodies. Moreover, 5B9 binds epitopes within PF4 that are likely to be critical for the pathogenicity of HIT antibodies.

Heparin-induced thrombocytopenia (HIT) is due to immunoglobulin G (IgG) antibodies, which bind platelet factor 4 (PF4) modified by polyanions, such as heparin (H). IgG/PF4/polyanion complexes directly activate platelets via Fc gamma type 2 receptor A (FcγRIIA) receptors. A bacterial protease, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), cleaves the hinge region of heavy-chain IgG, abolishing its ability to bind FcγR, including FcγRIIA. We evaluated whether cleavage of anti-PF4/H IgG by IdeS could suppress the pathogenicity of HIT antibodies. IdeS quickly cleaved purified 5B9, a monoclonal chimeric anti-PF4/H IgG1, which led to the formation of single cleaved 5B9 (sc5B9), without any reduction in binding ability to the PF4/H complex. However, as compared with uncleaved 5B9, the affinity of sc5B9 for platelet FcγRIIA was greatly reduced, and sc5B9 was also unable to induce heparin-dependent platelet activation. In addition, incubating IdeS in whole blood containing 5B9 or HIT plasma samples led to cleavage of anti-PF4/H antibodies, which fully abolished the ability to induce heparin-dependent platelet aggregation and tissue factor messenger RNA synthesis by monocytes. Also, when whole blood was perfused in von Willebrand factor–coated microfluidic channels, platelet aggregation and fibrin formation induced by 5B9 with heparin was strongly reduced after IdeS treatment. Finally, IdeS prevented thrombocytopenia and hypercoagulability induced by 5B9 with heparin in transgenic mice expressing human PF4 and FcγRIIA receptors. In conclusion, cleavage of anti-PF4/H IgG by IdeS abolishes heparin-dependent cellular activation induced by HIT antibodies. IdeS injection could be a potential treatment of patients with severe HIT.

The annual “Antibody Industrial Symposium”, co organized by LabEx MAbImprove, MabDesign and Polepharma, was held in Tours, France on June 27–28, 2017. The focus was on antibody-drug-conjugates (ADCs), new entities which realize the hope of Paul Ehrlich's magic bullet. ADCs result from the bioconjugation of a highly cytotoxic drug to a selective monoclonal antibody, which acts as a vector. Building on knowledge gained during the development of three approved ADCs, brentuximab vedotin (Adcetris®), ado trastuzumab emtansine (Kadcyla®) and inotuzumab ozogamicin (Besponsa®), and the many ADCs in development, this meeting addressed strategies and the latest innovations in the field from fundamental research to manufacturing.

Hemostasis is a complex process involving the concerted action of molecular and vascular components. Its basic understanding as well as diagnostic and therapeutic aspects have greatly benefited from the use of monoclonal antibodies. Interestingly, camelid‐derived single‐domain antibodies (sdAbs), also known as V H H or nanobodies, have become available during the previous 2 decades as alternative tools in this regard. Compared to classic antibodies, sdAbs are easier to produce and their small size facilitates their engineering and functionalization. It is not surprising, therefore, that sdAbs are increasingly used in hemostasis‐related research. In addition, they have the capacity to recognize unique epitopes unavailable to full monoclonal antibodies. This property can be used to develop novel diagnostic tests identifying conformational variants of hemostatic proteins. Examples include sdAbs that bind active but not globular von Willebrand factor or free factor VIIa but not tissue factor–bound factor VIIa. Finally, sdAbs have a high therapeutic potential, exemplified by caplacizumab, a homodimeric sdAb targeting von Willebrand factor that is approved for the treatment of thrombotic thrombocytopenic purpura. In this review, the various applications of sdAbs in thrombosis and hemostasis‐related research, diagnostics, and therapeutic strategies will be discussed.

Fluorescent labelling of monoclonal antibodies (mAbs) is classically performed by chemical bioconjugation methods. The most frequent labelling technique to generate antibody–fluorophore conjugates (AFCs) involves the bioconjugation onto the mAb lysines of a dye bearing an N-hydroxysuccinimide ester or an isothiocyanate group. However, discrepancies between labelling experiments or kits can be observed, related to reproducibility issues, alteration of antigen binding, or mAb properties. The lack of information on labelling kits and the incomplete characterization of the obtained labelled mAbs largely contribute to these issues. In this work, we generated eight AFCs through either lysine or interchain cysteine cross-linking bioconjugation of green-emitting fluorophores (fluorescein or BODIPY) onto either trastuzumab or rituximab. This strategy allowed us to study the influence of fluorophore solubility, bioconjugation technology, and antibody nature on two known labelling procedures. The structures of these AFCs were thoroughly analyzed by mass spectroscopy, and their antigen binding properties were studied. We then compared these AFCs in vitro by studying their respective spectral properties and stabilities. The shelf stability profiles and sensibility to pH variation of these AFCs prove to be dye-, antibody- and labelling-technology-dependent. Fluorescence emission in AFCs was higher when lysine labelling was used, but cross-linked AFCs were revealed to be more stable. This must be taken into account for the design of any biological study involving antibody labelling.

Heparin-induced thrombocytopenia (HIT) is a frequent drug-adverse event caused in the majority of patients by platelet-activating antibodies (Abs) directed against complexes of heparin (H) bound to platelet factor 4 (PF4). In most cases, HIT Abs are IgG which are potentially pathogenic as they are able to activate platelets directly in the presence of heparin via FcgRIIA receptors. The diagnosis of HIT is based on both clinical and biological criteria. However, despite recent improvements in HIT laboratory assays, a standard is always lacking for both immunological and functional assays. First, platelets from healthy donors exhibit wide variability in their response to HIT antibodies. Second, many patients who synthesize significant levels of antibodies to PF4/H while being treated with heparin do not develop HIT and the mechanisms that regulate the pathogenicity of HIT antibodies have not been fully defined. However, several studies suggested that epitope specificity of IgG antibodies is critical for the pathophysiology of HIT, especially by influencing the stability of PF4 tetramers. We therefore aimed to develop a monoclonal anti-PF4/H antibody with a human Fc fragment using transgenic mice homozygous for Cg gene of a G-class human immunoglobulin and that directly produce chimeric IgG antibodies. After immunization, several clones were found to synthesize anti-PF4 IgG antibodies but only one (5B9) produced an IgG1 that specifically bind PF4/H complexes without reactivity against PF4 alone. 5B9 was able to induce dose-dependent platelet activation and aggregation in the presence of low concentrations of UFH (0.1 and 0.5 IU/mL), and this effect was not observed without UFH or with high concentration of UFH (10 IU/ml), and was fully inhibited by IV.3 antibody. In addition, 5B9 with UFH (0.5 IU/mL) induced strong TF mRNA synthesis in isolated monocytes. Injection of 5B9 with UFH to transgenic mice expressing human PF4 and FcgRIIA receptors was followed by significant thrombocytopenia, similar to that observed with KKO, a murine IgG2b anti-PF4/H antibody. Plasma levels of thrombin/anti-thrombin (TAT) also significantly increased after injection of heparin in all mice having received 5B9 or KKO, but this effect was more pronounced on day 1 in 5B9-treated mice (mean value= 47 vs. 5 ng/mL at day 0) than in mice injected with KKO (33 vs. 11 ng/mL, respectively). Competitive immunoassays were also developed and 15 of 25 plasma samples (60%) collected in HIT patients were shown to inhibit (by at least 20%) the binding of 5B9 to PF4/H complexes compared to 3/25 (12%) samples containing non pathogenic anti-PF4/H antibodies (obtained in patients without HIT). Similar experiments were performed with KKO and its binding to PF4/H was also inhibited by 32% of HIT plasma samples (8/25), and none of the non-HIT plasmas. However, the levels of 5B9 and KKO binding inhibition to PF4/H were highly correlated (R2=0.85), thereby suggesting that the epitopes recognized by the antibodies are similar. Noticeably, KKO was also shown to inhibit in a concentration-dependent manner the binding of 5B9 to PF4/H complexes, further supporting this hypothesis. 5B9 was then sequenced and a docking model was elaborated based on VH and VL sequences of 5B9 obtained and a recently described crystal structure of KKO/PF4 tetramer complex. According to this model, 5B9 Fab likely interacts with the B and D monomers of PF4, thus possibly contributing to the stability of tetramers. In addition, the binding of 5B9 to PF4 involves 28 aa, 16 in the B monomer including Asp-7, Gln-9, Pro-34 and Pro-37 and 12 in the D monomer including Gln-9. Importantly, 13 of these 28 aa have also been identified as critical in the formation of PF4/KKO complex (Cai et al, Nat Commun. 2015;6:8277). Three regions (Asp-7 to Thr-15 and Ala-32 to Lys-41 in the B monomer and Gln-9 to Asp-18 in the D monomer) therefore appear particularly important in the binding of both 5B9 and KKO on PF4 tetramers. In conclusion, 5B9 is the first anti-PF4/H monoclonal antibody that has a human Fc fragment, and fully mimics the effect of HIT human antibodies. 5B9 could therefore be used as a standard in HIT laboratory assays. Moreover, our results support that three regions both recognized by 5B9 and KKO within PF4 tetramers are critical for the binding and pathogenicity of HIT antibodies. Disclosures No relevant conflicts of interest to declare.

Von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2-domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3-domain, the epitope of which overlaps the collagen-binding site. While KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2-domain modulates exposure of its epitope in the A3-domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact-VWF could be detected using this nanobody. By comparing plasma from volunteers to that of congenital VWD-patients, intact-VWF levels were significantly reduced for all VWD-types, and most severely in VWD-type 2A-group 2 in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD-type 1 and VWD-type 2M. A significant correlation (r=0.51, p<0.0001) between the relative amount of high molecular weight-multimers and levels of intact-VWF was observed. Reduced levels of intact-VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3-domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.