key: cord-0052099-qduyo03j authors: Peyron, Ivan; Kizlik‐Masson, Claire; Dubois, Marie‐Daniéla; Atsou, Sénadé; Ferrière, Stephen; Denis, Cécile V.; Lenting, Peter J.; Casari, Caterina; Christophe, Olivier D. title: Camelid‐derived single‐chain antibodies in hemostasis: Mechanistic, diagnostic, and therapeutic applications date: 2020-09-09 journal: Res Pract Thromb Haemost DOI: 10.1002/rth2.12420 sha: 04a69e10124332730254ddeec74c8806b33a3ec9 doc_id: 52099 cord_uid: qduyo03j 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. Ever since the discovery of hybridoma technology by Kohler and Milstein in 1975, 1 monoclonal antibodies have represented very powerful tools to explore biological processes. Hemostasis, a complex biological process in which a cascade of events involving the concerted action of cellular and molecular players allows for a quick response to vessel injury, is no exception in this regard. Antibodies have been invaluable biological determinants not only to better understand hemostasis but also for diagnostics of hemostasis-related pathologies or more recently for their treatment. 2 Immunotherapy in the hemostasis field started with the use of abciximab, a humanized Fab fragment against platelet glycoprotein α 2b β 3 , widely used as antiplatelet therapy in patients undergoing percutaneous coronary intervention to avoid ischemic complications. 3 Abciximab has the capacity to block binding of α 2b β 3 to its ligands, fibrinogen and von Willebrand factor (VWF), thereby preventing platelet aggregation. 4 Following abciximab, it has taken a number of years before new antibody-based treatments could become available for hemorrhagic or thrombotic diseases. However, at least in hemophilia, therapeutic antibodies are now revolutionizing the treatment of patients. 5, 6 In particular, emicizumab, a bispecific humanized IgG4 directed against coagulation factors IX and X and marketed under the name of Hemlibra has proven efficient in treating hemophilia A patients with or without inhibitors. 7 Emicizumab mimics factor VIII cofactor activity by binding simultaneously to the enzyme factor IXa and its substrate, factor X. 7 On the thrombotic side, several antibodybased treatments are currently under development, such as ACT017, a humanized Fab fragment, directed against platelet receptor glycoprotein VI (GPVI) and intended for stroke treatment. 8 ACT017 (glenzocimab) blocks GPVI binding sites, leading to an inhibition of collagen-induced platelet aggregation. This antibody has now demonstrated its safety profile in a clinical phase I trial. 9 Antibodies targeting coagulation factors XI/XIa and XII/XIIa also hold the promise of becoming safe antithrombotic drugs since they are not expected to increase the bleeding risk. 10, 11 Although classical monoclonal antibodies and their Fab fragment or single-chain variable fragments (scFvs) derivatives remain most widely used, camelid-derived single-domain antibodies (sdAbs) have emerged as a credible next generation of antibodies offering several advantages over classic antibodies owing to their peculiar properties, including their small size, robust behavior, high affinity and specificity, and deep tissue penetration. [12] [13] [14] It is also important to mention that besides camelids, sdAbs can also be found in certain • sdAbs against coagulation proteins have helped solve biochemical research issues. • sdAbs can be used as diagnostic tools and also for therapeutics in thrombosis and hemostasis. • Protein engineering increases the versatility of sdAbs and their potential applications. sharks. 15 However, this review will focus exclusively on camelid-derived sdAbs. The first description of functional sdAbs in camelids was reported in 1993. 16 Unlike conventional antibodies, and despite their ability to bind to their target antigen with strong affinity, a fraction of antibodies derived from camelids was found to be completely devoid of light chain. From a structural point of view, sdAbs contain the CH2 and CH3 domains but lack the first classic IgG constant domain, CH1 ( Figure 1 ). The lack of CH1 domain is a necessity for their secretion in the absence of a light chain. 17 Consequently, their antigen-binding regions consist of one single VH domain termed V H H. V H Hs were reported to be the smallest intact antigen-binding single-polypeptide chains found in any natural antibody. 18 Recombinant expression of V H Hs allows for the generation of a soluble single-domain antigen-binding protein with a molecular weight of about 15 kDa. Due to their small size, these sdAbs are sometimes referred to as nanobodies. SdAbs have functional properties similar to that of conventional antibodies in terms of affinity and specificity, 19 with a binding affinity within the picomolar to nanomolar range. 20 However, distinctions also exist between sdAbs and the antigen-binding region of IgG, which is composed of two entities in two different polypeptide chains, VL and VH. Indeed, certain residues in the VH framework regions that are part of the interface with the VL are altered at the corresponding positions in sdAbs. This endows sdAbs with a more hydrophilic character, increasing their solubility. 21, 22 In addition, the eventual presence of an extra disulfide bond between the first and third antigen-binding loops, H1 and H3, may rigidify the sdAb structure, contributing to an increased stability and activity at high temperatures and in high concentrations of denaturants, compared to classical IgGs. 23 The sdAb paratope is defined by three highly variable loops, H1, H2, and H3, which form an extended structural interface at one side of the protein (Figure 1 ). 24 Despite the smaller surface of this paratope compared to the VH-VL paratope of IgG, sdAbs can create a higher diversity by exhibiting a greater structural variation in their H1, H2, and H3 loops and by increasing the length of the H3 loop. [24] [25] [26] [27] [28] It is noteworthy that the conserved framework regions of sdAbs show a high sequence and structural homology with human VH domains of family III (VH3) 29 . This high homology not only results in a comparable immunogenicity as human VH but also increases the chances of successful humanization of these sdAbs (see also section 5 of this review). 22 Furthermore, due to their single-domain nature and lack of glycosylation, they can be produced in large amounts using standard microbial or yeast expression systems. [30] [31] [32] On the other hand, sdAbs miss some of the characteristics found in regular IgGs. For example, the absence of the Fc portion prevents these sdAbs from directly activating the complement system and immune cells, and therefore they lack cytotoxic activity. 33, 34 Furthermore, without the Fc portion, sdAbs are incapable of entering the FcRn-dependent recycling pathway. In combination with their small size, which is below the glomerular filtration capacity, this contributes to the short in vivo half-life of the sdAbs. 34, 35 Finally, with the antigen-binding paratope being restricted in size compared to classic IgG, this implies that the number of residues within this paratope that could be used to improve antigen binding are limited as well. 36 One pitfall could be that such unnatural CDR3 sequences may increase immunogenicity, a potential issue if the sdAb is selected for therapeutic purposes. The immune libraries are generally superior due to the higher affinities of the recovered sdAbs. This is likely due to the deletions/ insertions seen in the V H H genes, which occurs as clones are selected by the immune response. Such recombination events do not occur in the naïve libraries and are not naturally selected for using synthetic libraries. One important characteristic of using cDNA-based libraries (whether they may be immune, naïve or synthetic), is that they allow for a reproducible expression of the sdAbs during each round of the selection process. This is an important advantage over the use of hybridomas, in which loss of IgG expression may occur during selection. Independent of the approach underlying library engineering, numerous selection techniques including phage display, yeast display, bacterial display, mRNA/cDNA display, intracellular two-hybrid system, and ribosome display can be applied to facilitate screening and panning for a given antigen-specific sdAb. 39 The generation of a sdAb library is advantageous in the sense that it can be screened multiple times, allowing for the use of several setups for antigen presentation. This is of particular interest when the target antigen presents with complicated features, as is often the case with coagulation proteins. Indeed, coagulation factors are found in a zymogen state in the circulation and needs to be activated to exert their functions. 40 Hence, activated coagulation factors are often extremely flexible in their structure, making it sometimes difficult to identify specific binders. Following the selection protocol, sequence optimization may also be necessary to improve sdAb production efficiency, solubility, or any other desired property. The combined properties of sdAbs (eg, selection of conformationspecific binders, ease of engineering, production via routine recombinant techniques) have led to a plethora of applications for sdAbs as research tools. For instance, sdAbs have been used as crystallization chaperones for the structural investigation of diverse conformational states of flexible proteins. 39, 41 Green fluorescent protein-targeting sdAbs have been applied to optimize superresolution microscopy. 42 Since sdAbs are easily expressed in a variety of cell types, they also have been used to study intracellular processes (for which they are referred to as intrabodies). In this respect, they can be used to specifically inhibit target proteins, signal the appearance of a specific conformational state, or direct the target protein to the intracellular degradation pathway. 38, 43, 44 In addition, numerous fusion variants of sdAbs have been described. Apart from the classic fusion candidates (fluorescent proteins or IgG Fc domain), these also include horseradish peroxidase and the phospholipidbinding C1C2 domains of lactadherin. 45, 46 Besides these general applications showing (in a nonexhaustive fashion) how powerful the sdAb technology can be, more specific use of sdAbs in the thrombosis and hemostasis field will be the focus of the next few paragraphs. In the context of hemostasis, a certain number of sdAbs have been developed in the past 15-20 years, and many of those have been extremely useful, especially for biochemical studies. Indeed, many proteins relevant to maintain the hemostatic balance can exist in different forms. Best described are enzymes and cofactors that require proteolytic activation. sdAbs have the potential to differentiate small conformational differences and have therefore contributed to answer important research questions. Table 1 and Figure 3 summarize sdAbs targeting hemostasis proteins. One example of sdAb that allowed to resolve a scientific issue relates to factor VII (FVII) which can exist in a precursor (FVII) and an activated (FVIIa) state. 47 In the 1990s, FVIIa has become available for the treatment of hemophilia inhibitor patients. Its efficacy is based on the capacity of FVIIa to activate coagulation factors IX and X, thereby leading to thrombin activation. One unanswered question remained about the mechanism of action of FVIIa, based on the apparent discrepancy between the high therapeutic doses needed compared to the low physiological concentration of FVIIa. One hypothesis was that FVIIa had to compete with endogenous FVII for binding to its cofactor tissue factor (TF). 48 To assess this hypothesis, our group has recently developed a sdAb (KB-FVIIa-004) capable of selectively detecting and inhibiting proteolytic activities of FVIIa but not TF-bound FVIIa. 49 Using this unique sdAb, we have been able to demonstrate the TFindependent mode of action of FVIIa in vivo in hemophilia A mice. sdAbs have also helped decipher the sequential activation steps of factor XII (FXII), a key protein of the contact system that has recently gained a lot of attention for its potential as an antithrombotic target. 50, 51 Upon surface binding, small amounts of FXII are converted into an activated derivative, α-FXIIa. α-FXIIa is able to convert plasma prekallikrein (PPK) into plasma kallikrein (PK), and in turn PK will further amplify α-FXIIa formation. However, PK will also generate β-FXIIa, a smaller derivative of α-FXIIa that lacks the surface binding heavy chain. α-FXIIa and β-FXIIa differ in that only α-FXIIa is able to activate factor XI (FXI) into FXIa. In contrast, both variants are able to convert PPK into PK. Since PK is responsible for the release of bradykinin, a small peptide that evokes vascular leakage and tissue swelling, it is thus important to know how much α-FXIIa and β-FXIIa is present under various physiological and pathological conditions. To improve the analysis of α-FXIIa and F I G U R E 3 Targets for sdAbs in the coagulation and fibrinolysis pathways. An overview of the major activation and inactivation reactions in the coagulation and fibrinolysis pathway are shown. Red circles indicate sdAbs and their targets that have been mentioned in this review. APC, activated Protein C; α2AP, α2-antiplasmin; AT, antithrombin; F, factor; Plm, plasmin; Plg, plasminogen; PAI-1, plasminogen activator inhibitor-1; PC, protein C; PN1, Protease nexin-1; ProtS, protein S; ProtZ, protein Z; TAFI, thrombin-activatable fibrinolysis inhibitor; TM, thrombomodulin; TF, tissue factor; TFPI, tissue factor pathway inhibitor; t-PA, tissue-type plasminogen activator; u-PA, urokinase plasminogen activator; VWF, von Willebrand factor; ZPI, protein Z-dependent protease inhibitor These sdAbs thus represent an essential biochemical research tool to capture these molecules at different time points of the contact system and reveal previously unidentified agonist-dependent FXII activation pathways, which might or might not lead to activation of the coagulation cascade. 52 Another hemostatic protein which has significantly benefited from sdAbs technology for its structural and mechanistic studies is thrombin-activatable fibrinolysis inhibitor (TAFI), known for its modulatory function of the fibrinolytic process. TAFI circulates as an inactive carboxypeptidase precursor, which becomes active via proteolytic sdAbs have also been used for the analysis of other fibrinolytic proteins, such as Plasminogen Activator Inhibitor-1 and urokinase plasminogen activator (u-PA). 58-60 u-PA in particular is mainly known as a plasminogen activator, but is also involved in processes that do not require plasmin generation. 61 In search for inhibitors of u-PA function, it has been observed that some of the peptide bond-based inhibitors also function as substrates. It is unclear if this dual behavior originates (i) from conformation changes within the peptide segment of the inhibitor/substrate that inserts into the active site pocket of the enzyme, (ii) from conformational changes within the enzyme structure or (iii) both. To get insight into this important question, into the u-PA active site. 58, 59 Via crystallographic analysis, they observed that the presence of such sdAbs changed the structure of u-PA, favoring option ii. Interestingly, they noticed that despite the strong inhibitory effect of the sdAb, it also was slowly proteolyzed. Following mutagenesis aiming to introduce more flexibility into the rigid H3 loop, the sdAb was proteolyzed more efficiently. This suggests that conformational changes within the peptide segment that protrudes from the active site can also contribute to whether this segment is acting as inhibitor, substrate, or both. It would be interesting to investigate whether sdAbs that penetrate the active site of other serine proteases display similar features, as this may help to develop agents that display partial or transient inhibitory properties toward their targets. Although most of the above-mentioned studies have used sdAbs as biochemical tools, they can also be essential for functional studies. One such example relates to sdAbs raised against VWF, the main molecular player in platelet adhesion to the injured vessel during primary hemostasis. 62 Although there is not any sdAb-based routine diagnostic assay available yet in clinical hematology laboratories, several sdAbs have been used for clinical research purposes, proving their usefulness in detecting specific disease-associated protein distinctions. binding assay is the ability to detect active VWF even under conditions of low VWF antigen levels. It is important to mention that under physiological conditions, extremely low levels of active VWF are present in the circulation since this platelet-binding form of VWF is considered prothrombotic. AU/VWFa-11 assays have therefore been used to detect excessive VWF activation in several pathologic conditions. 67 Best described are a bleeding-associated rare subset of von Willebrand disease (VWD) classified as VWD-type 2B and thrombotic thrombocytopenic purpura (TTP), which is related to disseminated thrombotic events in the microcirculation. VWD-type 2B is caused by gain-of-function genetic variants within or close to the VWF-A1 domain. These mutations destabilize VWF toward its platelet-binding conformation. 68 Using the AU/VWFa-11 binding assay, it was found that active VWF was raised 2-to 15-fold. 66 Moreover, an inverse correlation was observed between the amount of active VWF and platelet count, which in turn is associated with the severity of the bleeding phenotypes. 69 Similar rises in active VWF have also been measured in TTP patients, with a 2-to 12-fold increase over resting, despite a very different underlying molecular mechanism. Active VWF in TTP stems from a lack of proteolytic inactivation of VWF by its specific enzyme, ADAMTS13. 70 Interestingly, higher levels of active VWF were consistently measured in patients with congenital forms of the disease, while samples from patients suffering from the acquired forms showed smaller, albeit supraphysiologic levels of active VWF. Therefore, AU/ VWFa-11 is able to discriminate between congenital and acquired TTP forms, which is relevant to apply therapeutic decisions. 66 A number of pathologic conditions characterized by VWFdependent platelet aggregates have also been associated with elevated levels of active VWF, as quantified by AU/VWFa-11-based assays. 67 This was the case in patients suffering from HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) a severe complication of preeclampsia, 71 parasitic malaria, which is associated with thrombocytopenia and cerebral thrombotic events, 72 antiphospholipid syndrome, again characterized by low platelet counts and microthrombopathy 73 and sickle cell disease, associated with chronic hemolytic anemia and sporadic vaso-occlusion. 74 Another advantage of sdAbs is that the C-terminus is opposite to the antigen-binding surface, allowing it to be used for functionalization of the antibody. This feature has recently been used for the development of a novel diagnostic assay to measure fibrinogen levels. 76, 77 Purified sdAbs targeting fibrinogen were used in an immunobased amperometric test, allowing the rapid detection of as little as 44 μg fibrinogen/L in plasma (normal range, 1.5-4.5 g/L). In follow-up studies, the same group is now developing an agglutination-based assay, that relies on the expression of sdAbs at the Escherichia coli surface. Incubation of plasma samples containing fibrinogen with these sdAb-expressing bacteria results in a dose-dependent agglutination. 78 Given the high affinity and specificity, this type of approaches could be used for the detection of other hemostatic proteins as well. Probably owing to their relatively recent discovery, sdAbs have not yet widely found their way to the clinic. There is no doubt that this situation is likely to evolve in the next few years since given their unique properties, sdAbs have a high potential in this regard. First, despite their nonhuman origin, sdAbs are less immunogenic compared to murine-or rat-derived monoclonal IgG antibodies because of their high degree of homology to the human VH3 domain. 29 In addition, humanization of sdAbs is relatively straightforward compared to the humanization of IgG molecules. The main issue is to substitute the amino acids that correspond to those in human IgG that are involved in the VH/VL interaction, which are located in framework region 2. 22 Since such substitutions may affect solubility or affinity, it is not uncommon that additional mutagenesis is performed to restore or even improve these parameters. A second advantage relates to the half-life of the sdAbs, which can be tailored dependent on its purpose. Due to its small size, a single unbound sdAb is rapidly eliminated from the circulation via renal clearance. This could be advantageous when used for in vivo imaging as explained in part 6.2. Alternatively, sdAbs can form a complex with their target in the circulation, and in this complex sdAbs adopt the circulatory halflife of their target. Examples hereof are caplacizumab (discussed in part 5.1), or sdAbs targeting antithrombin, which are eliminated while bound to their respective targets VWF and antithrombin. 79, 80 Finally, sdAbs can be engineered to have a prolonged half-life, for instance via fusion to proteins with a long half-life (such as the Fc region of IgG molecules or albumin), via fusion to a second sdAb that binds for example albumin, via multimerization to a size above the renal clearance limit, or via the formation of sdAb-containing nanoparticles. 81 Together, these properties make sdAbs attractive candidates for therapeutic applications whether in the area of thrombosis and hemostasis or other pathologies. So far, only one sdAb has been approved for treatment, but a number of other sdAbs are currently in clinical trials for treatment of rheumatoid arthritis, breast cancer, or other pathologies. 13 As previously mentioned, only one sdAb, caplacizumab, a homodimeric sdAb targeting the A1-domain of VWF, has been approved for therapeutic purposes. Caplacizumab interferes with the interaction between VWF and GPIbα, preventing the adhesion of platelets to VWF. 80 Originally designed to prevent thrombotic events during percutaneous coronary intervention, caplacizumab is now Although not yet in clinical development, there are also a number of other sdAbs that could be of interest for the management of thrombotic complications. In particular, sdAbs that target natural inhibitors of the fibrinolytic pathway could be useful in this regard. Inhibitory sdAbs targeting TAFI (described in section 3.3) and plasminogen activator inhibitor-1 (PAI-1), developed by the laboratory of Dr Declerck could be used to enhance thrombolysis. 54, 57 In particular, these sdAbs could be applied to enhance thrombolysis during exaggerated pulmonary thrombosis. Interestingly, a clinical trial is under consideration to improve thrombolysis via nebulization of tissue-type plasminogen activator. 85 Since sdAbs have excellent bioavailability following nebulization, 86, 87 it would be interesting to add sdAbs targeting TAFI or PAI-1 as stand-alone or adjuvant therapy. Also in hemorrhagic conditions, sdAbs could be useful, and our labo- Because sdAbs are so versatile and can be used in so many different ways, as shown in the example of the FVIII-sdAb fusion molecule, there are (almost) unlimited options in their potential applications. A few examples will be discussed below and are summarized on To date, thrombus characterization suffers from the lack of precise tools for structure and composition analysis, which is crucial to make a medical decision. This is particularly true in stroke where "time is brain" but where recanalization treatment will be more or less successful according to the etiology of the thrombus. 98 Thus, markers of thrombus composition and also of thrombus maturity are critically needed to decide whether anticoagulation, pharmacological thrombolysis, mechanical thrombolysis, or a combination of these is required. 99 The recent availability of thrombi obtained by thrombectomy has only further highlighted the differences in F I G U R E 4 Examples of potential applications of sdAbs in relation to thrombosis and hemostasis. The ease of manipulation of sdAbs makes them an ideal choice for the design of therapeutic and diagnostic tools. sdAbs can be incorporated in larger structure to participate in the gain-of-function of therapeutic compound, conjugated by cleavable or noncleavable linker. Due to their small size (associated with a high tissue penetration) and fast clearance from the bloodstream, sdAbs are suitable for the design of imaging probes. This can be achieved by the direct conjugation of target-specific sdAbs with radionucleides, or by incorporating target-specific sdAbs in nanoparticles. In the case of sdAbs with direct pro-or anticoagulant properties, increasing their half-lives requires their fusion to other proteins such as albumin or albumin adaptors, or to the Fc domain of conventional IgGs. For example, bivalent constructs bridging a target to an albumin molecule would potentially increase the half-life of the target protein to that of albumin This is why mixed imaging techniques that can combine molecular/functional/anatomic imaging are gaining increasing attention. They rely on the accuracy and precision of radiolabeled markers and sdAbs have many of the desired qualities required to constitute excellent markers for imaging. Their small molecular weight (about 15 kDa) is well below the renal elimination cutoff (~50 kDa). As a result, sdAbs size favors their tissue penetration and allows for their rapid renal clearance. This leads to a high signal-to-noise ratio and rapid imaging after administration, as demonstrated in the case of tumor imaging. 102, 103 It is therefore possible to imagine using sdAbs fused to imaging probes for detection of fibrin/red blood cells/VWF or activated platelets in order to gain knowledge in thrombus structure. Again, the feasibility of such an approach was demonstrated using scFv. A synthetic protein-based nanoparticle containing a near infrared fluorescent molecule and functionalized with a scFv specific for activated platelets was engineered by Bonnard et al. 104 This construct exhibited a strong potential as biocompatible molecular imaging probe in an in vivo model of carotid arterial thrombosis. sdAbs have only begun to demonstrate their utility in biomedical research. The thrombosis and hemostasis community has rapidly seized their potential by developing very interesting biological tools against complex proteins and by using them to tackle unanswered biochemical issues. Notably, so far, no sdAbs against platelet receptors have been developed. Importantly, the first clinically approved sdAb is in the thrombosis and hemostasis field, demonstrating the swift embracement of this technology by our community. However, as a laboratory that has invested in sdAb technology for the past 6 years, we can testify that, although they constitute great tools, not all sdAbs are equal and working with some of them can prove challenging. Affinity maturation steps to optimize sdAbs are often necessary and can be time consuming. Only future will tell whether sdAbs will be the magic bullets of next-generation immunotherapy strategies. Continuous cultures of fused cells secreting antibody of predefined specificity Therapeutic antibodies in hemostasis. 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Determination of fibrinogen in plasma Characterization of nanobodies binding human fibrinogen selected by E. coli display Whole-cell biosensor with tunable limit of detection enables low-cost agglutination assays for medical diagnostic applications Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors Antithrombotic drug candidate ALX-0081 shows superior preclinical efficacy and safety compared with currently marketed antiplatelet drugs Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting Caplacizumab for acquired thrombotic thrombocytopenic purpura Caplacizumab treatment for acquired thrombotic thrombocytopenic purpura Reperfusion of cerebral artery thrombosis by the GPIb-VWF blockade with the Nanobody ALX-0081 reduces brain infarct size in guinea pigs Fibrinolytic abnormalities in acute respiratory distress syndrome (ARDS) and versatility of thrombolytic drugs to treat COVID-19 Delivery of ALX-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs Nanobodies(R) as inhaled biotherapeutics for lung diseases A factor VIII-nanobody fusion protein forming an ultrastable complex with VWF: effect on clearance and antibody formation Camelid single-domain antibody-fragment engineering for (pre)clinical in vivo molecular imaging applications: adjusting the bullet to its target Platelet-targeted dual pathway antithrombotic inhibits thrombosis with preserved hemostasis Novel thrombolytic drug based on thrombin cleavable microplasminogen coupled to a single-chain antibody specific for activated GPIIb/IIIa Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature Nanobodies: chemical functionalization strategies and intracellular applications Nanobody-enhanced targeting of AAV gene therapy vectors Nanobody-albumin nanoparticles (NANAPs) for the delivery of a multikinase inhibitor 17864 to EGFR overexpressing tumor cells Downregulation of EGFR by a novel multivalent nanobody-liposome platform Reprint of "Nanobodyshell functionalized thermosensitive core-crosslinked polymeric micelles for active drug targeting GpIbalpha-VWF blockade restores vessel patency by dissolving platelet aggregates formed under very high shear rate in mice Can thrombus age guide thrombolytic therapy? Cardiovasc Diagn Ther Acute ischemic stroke thrombi have an outer shell that impairs fibrinolysis Structural analysis of ischemic stroke thrombi: histological indications for therapy resistance SPECT imaging with 99mTc-labeled EGFRspecific nanobody for in vivo monitoring of EGFR expression Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer Low-fouling and biodegradable protein-based particles for thrombus imaging Generation and in vitro characterisation of inhibitory nanobodies towards plasminogen activator inhibitor 1 Development and characterization of single-domain antibodies neutralizing protease nexin-1 as tools to increase thrombin generation Identification of a novel, nanobody-induced, mechanism of TAFI inactivation and its in vivo application Camelid-derived single-chain antibodies in hemostasis: Mechanistic, diagnostic, and therapeutic applications All authors have contributed to the writing of the review and approved the final manuscript. PJL, ODC, and CVD are co-inventors on four patents related to the use of single-domain antibodies for therapeutic purposes.