Search

Background: International Society on Thrombosis and Haemostasis guidelines for immune-mediated thrombotic thrombocytopenic purpura (iTTP) treatment recommend concurrent therapeutic plasma exchange (TPE), immunosuppressive therapy (IST), and caplacizumab. TPE can complicate antidrug antibody (ADA) measurements by transferring pre-existing antibodies (pre-Abs) into patients via donor plasma and/or diluting treatment-emergent (TE) ADAs. Objectives: To assess the presence of ADAs in patients with iTTP who received caplacizumab. Methods: Immunogenicity data from patients with iTTP receiving caplacizumab once daily plus TPE/immunosuppressive therapy in 4 clinical trials (TITAN, HERCULES, Post-HERCULES, and a trial conducted in Japanese patients) in the clinical development program were analyzed. ADA and modified ADA assays differentiated pre-Abs from TE ADAs. A functional neutralizing antibody (NAb) assay and a neutralizing epitope characterization assay (NECA) assessed the presence of ADAs with neutralizing potential. The impact of ADAs on efficacy, pharmacokinetics/pharmacodynamics, and safety was evaluated. Results: Among 228 patients in 4 studies, prevalence of pre-Abs ranged from 17.1% (TITAN) to 56.7% (HERCULES), while TE ADA prevalence ranged from 3.1% (HERCULES) to 14.3% (Japanese study). The TE NAb-positive rate ranged from 0% (Japanese study) to 12% (Post-HERCULES) using the functional NAb assay and from 2.7% (Post- HERCULES) to 14.3% (Japanese study) using the NECA. The presence of these antibodies did not impact treatment efficacy or safety. Conclusion: A complex immunogenicity assay strategy was required to define the pre-Ab/TE ADA status of patients with iTTP treated with caplacizumab in a clinical trial setting. In addition to the wide range of pre-Abs observed, few patients had detectable TE ADAs or NAbs, neither of which affected efficacy/safety.

In cancer molecular imaging, selecting binders with high specificity and affinity for biomarkers is paramount for achieving high-contrast imaging within clinical time frames. Nanobodies have emerged as potent candidates, surpassing antibodies in pre-clinical imaging due to their convenient production, rapid renal clearance, and deeper tissue penetration. Multimerization of nanobodies is a popular strategy to enhance their affinity and pharmacokinetics; however, traditional methods are laborious and may yield heterogeneous products. In this study, we employ a Holliday junction (HJ)-like nucleic acid-based scaffold to create homogeneous nanostructures with precise multivalent and multiparatopic nanobody displays. The plug-and-play assembly allowed the screening of several nanobody multimer configurations for the detection of the breast cancer biomarker, human epidermal growth factor receptor 2 (HER2). In vitro studies demonstrated significant improvements in binding avidity, particularly with the biparatopic construct exhibiting high sensitivity, surpassing that of traditional antibody-based cell binding. Furthermore, our HJ platform allowed for adaptation from fluorescence-based to nuclear imaging, as demonstrated in xenografted mice, thereby allowing for future in vivo applications. This work highlights the potential of nucleic acid-mediated multimerization to markedly enhance nanobody binding, by exploring synergistic combinations and offering versatility for both in vitro diagnostics and cancer molecular imaging with prospects for future theranostic applications.

Radioimmunotherapy (RIT) involves combining a cytotoxic radionuclide with an antibody (Ab) targeting a tumor antigen. Compared with conventional therapies, RIT improves the therapeutic efficacy of Ab and ameliorates toxicity. This comprehensive review describes the current advancements and future prospects in RIT for treating hematologic malignancies based on recent investigations. Although β-particle RITs targeting CD20 are effective with low toxicity in patients with relapsed/refractory indolent or transformed non-Hodgkin's lymphoma, these treatments have not gained popularity because of the increasing availability of new therapies. RIT using single-domain antibodies is expected to improve tumor penetrance and reduce radiation exposure to non-target organs. To enhance RIT efficacy, α-particle RIT and pretargeted radioimmunotherapy (PRIT) are currently being developed. Alpha-particle RIT demonstrates substantial antitumor activity and reduced bystander effects due to its high linear energy transfer and short particle range. PRIT may increase the tumor-to-whole body dose ratio., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: International Society on Thrombosis and Haemostasis guidelines for immune-mediated thrombotic thrombocytopenic purpura (iTTP) treatment recommend concurrent therapeutic plasma exchange (TPE), immunosuppressive therapy (IST), and caplacizumab. TPE can complicate antidrug antibody (ADA) measurements by transferring pre-existing antibodies (pre-Abs) into patients via donor plasma and/or diluting treatment-emergent (TE) ADAs., Objectives: To assess the presence of ADAs in patients with iTTP who received caplacizumab., Methods: Immunogenicity data from patients with iTTP receiving caplacizumab once daily plus TPE/immunosuppressive therapy in 4 clinical trials (TITAN, HERCULES, Post-HERCULES, and a trial conducted in Japanese patients) in the clinical development program were analyzed. ADA and modified ADA assays differentiated pre-Abs from TE ADAs. A functional neutralizing antibody (NAb) assay and a neutralizing epitope characterization assay (NECA) assessed the presence of ADAs with neutralizing potential. The impact of ADAs on efficacy, pharmacokinetics/pharmacodynamics, and safety was evaluated., Results: Among 228 patients in 4 studies, prevalence of pre-Abs ranged from 17.1% (TITAN) to 56.7% (HERCULES), while TE ADA prevalence ranged from 3.1% (HERCULES) to 14.3% (Japanese study). The TE NAb-positive rate ranged from 0% (Japanese study) to 12% (Post-HERCULES) using the functional NAb assay and from 2.7% (Post- HERCULES) to 14.3% (Japanese study) using the NECA. The presence of these antibodies did not impact treatment efficacy or safety., Conclusion: A complex immunogenicity assay strategy was required to define the pre-Ab/TE ADA status of patients with iTTP treated with caplacizumab in a clinical trial setting. In addition to the wide range of pre-Abs observed, few patients had detectable TE ADAs or NAbs, neither of which affected efficacy/safety., (© 2024 The Authors.)

Abstract 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 VHH 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.

Background: Factor (F)XII triggers contact activation by binding to foreign surfaces, with the epidermal growth factor-like 1 (EGF-1) domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device-induced thrombosis., Objectives: To unravel and prevent EGF-1-mediated FXII surface-binding with a variable domain of heavy chain-only antibody (V H H)., Methods: FXII variants with glutamine substitutions of 2 positively charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain-specific V H Hs were raised to inhibit EGF-1-mediated FXII contact activation while preserving quiescence., Results: Two unique, positively charged patches in the EGF-1 domain were identified (upstream, 73K74K76K78H81K82H; downstream, 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in autoactivation of 94%, and prolongation of clot formation in activated partial thromboplastin time assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1-specific V H Hs were developed that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective V H H "F2" binds the positively charged patches and thereby dose-dependently extends activated partial thromboplastin time clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence., Conclusion: The 2 unique, positively charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding, making both patches crucial for contact activation. Targeting these with FXII EGF-1-specific V H Hs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable targets in preventing medical device-induced thrombosis., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Due to the serious issue of ofloxacin (OFL) abuse, there is an increasingly urgent need for accurate and rapid detection of OFL. Immunoassay has become the "golden method" for detecting OFL in complex matrix beneficial to its applicability for a large-scale screening, rapidity, and simplicity. However, traditional antibodies used in immunoassay present challenges such as time-consuming preparation, unstable sensitivity and specificity, and difficulty in directional evolution. In this paper, we successfully developed an OFL detection method based on a shark-derived single-domain antibody (ssdAb) to address these issues., Results: Using phage display technology and a heterologous expression system, OFL-specific clones 1O11, 1O13, 1O17, 1O19, 1O21, and 2O26 were successfully isolated and expressed in soluble form. Among all OFL-specific ssdAbs, the 1O17 ssdAb exhibited the highest binding affinity to OFL in a concentration-dependence manner. The limit of detection (IC 10 ) of 1O17 ssdAb was calculated as 0.34 ng/mL with a detection range of 3.40-1315.00 ng/mL, and its cross reactivity with other analogs was calculated to be less than 5.98 %, indicating high specificity and sensitivity. Molecular docking results revealed that 100Trp and 101Arg located in the CDR3 region of 1O17 ssdAb were crucial for OFL binding. In fish matrix performance tests, the 1O17 ssdAb did not demonstrate severe matrix interference in OFL-negative fish matrix, achieving satisfactory recovery rates ranging from 83.04 % to 108.82 % with high reproducibility., Significance: This research provides a new and efficient OFL detection recognition element with significant potential in immunoassay applications, broadening the application scenarios of ssdAbs. It offers valuable insights into the structure-activity relationship between ssdAbs and small molecules, laying a theoretical foundation for the further directional modification and maturation of ssdAbs in subsequent applications., Competing Interests: Declaration of Competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Necrotic enteritis is a devastating disease to poultry caused by the bacterium Clostridium perfringens. As a novel approach to combating poultry necrotic enteritis, we identified and characterized several hundred single domain antibody fragments (or nanobodies) capable of binding either the NetB toxin or the collagen-binding adhesin (CnaA) of C. perfringens. Many of the nanobodies could neutralize the in vitro functions of NetB or CnaA with inhibitory concentrations in the nanomolar range. The nanobodies were also screened for proteolytic stability in an extract derived from gastrointestinal tract fluids of chickens. A collection of 6 nanobodies (4 targeting NetB and 2 targeting CnaA) with high neutralizing activity and high gastrointestinal tract extract stability were expressed and secreted by Pichia pastoris or Bacillus subtilis. Chickens were given a feed with 1 of the 2 nanobody-containing groups: 1) nanobody-containing P. pastoris supernatants that were semi-purified, lyophilized, and enterically coated, or 2) B. subtilis spores from strains containing the nanobody genes. Compared to untreated chickens (23.75% mortality), mortality of chickens receiving feed modified with the P. pastoris and B. subtilis products decreased to 11.25 and 7.5%, respectively. These results offer a new opportunity to improve the control of poultry necrotic enteritis by incorporating highly specific nanobodies or bacteria expressing these nanobodies directly into chicken feed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Nanobodies (Nbs), which consist of only antigen-binding domains of heavy chain antibodies, have been used in a various range of applications due to their excellent properties. Nevertheless, the size of Nbs is so small that their antigen binding sites may be sterically hindered after random fixation as capture antibodies, thus leading to poor detection performance in immunoassays. To address this problem, we have focused on the multivalent modification of Nbs, wanted to retain the advantage of good stability through enlarging the size of Nbs to a certain extent, while improve its affinity and reduce its influence by spatial orientation., Results: Here, we designed homo- and heterodimeric Nbs based on Nb413 and Nb422 which recognize different epitopes of Salmonella. The affinity of engineered bivalent nanobodies for S. Enteritidis were 2 orders of magnitude higher compared to monovalent Nbs and low to sub-nM K D , as calculated by Scatchard analysis. To further explore the potential of bivalent Nbs for the detection of Salmonella, we established a sandwich ELISA based on bivalent and phage-displayed Nbs (BNb-ELISA) for multiplex Salmonella determination. Compared with monovalent Nb-based ELISA, the limit of detection (LOD) of the BNb-ELISA was shown to increase 7.5-fold to 2.364 × 10 3  CFU mL -1 for S. Enteritidis. In addition, the feasibility of this approach for S. Enteritidis detection in real samples was evaluated, with recoveries ranging from 73.0 % to 125.6 % and coefficients of variation (CV) below 7.68 %., Significance and Novelty: In this study, we developed for the first time bivalent Nbs against Salmonella and examined their improved affinity and impact on the performance of ELISA assay. It confirmed the high binding affinity and good ability of dimeric Nbs to reduce the occupation of the binding sites of immobilized antibodies. Thus, the multivalent modification of Nbs was demonstrated to be a promising means to enhance the performance of Nbs-based immunoassays for foodborne pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Human calcium sensing receptor (CaSR) senses calcium ion concentrations in vivo and is an important class of drug targets. Mutations in the receptor can lead to disorders of calcium homeostasis, including hypercalcemia and hypocalcemia. Here, 127 CaSR-targeted nanobodies were generated from camels, and four nanobodies with inhibitory function were further identified. Among these nanobodies, NB32 can effectively inhibit the mobilization of intracellular calcium ions (Ca 2+ i ) and suppress the G 12/13 and ERK 1/2 signaling pathways downstream of CaSR. Moreover, it enhanced the inhibitory effect of the calcilytics as a negative allosteric modulator (NAM). We determined the structure of complex and found NB32 bound to LB2 (Ligand-binding 2) domain of CaSR to prevent the interaction of LB2 domains of two protomers to stabilize the inactive state of CaSR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The World Health Organization advices the use of a quadrivalent vaccine as prophylaxis against influenza, to prevent severe influenza-associated disease and -mortality, and to keep up with influenza antigenic diversity. Different small molecule antivirals to treat influenza have become available. However, emergence of drug resistant influenza viruses has been observed upon use of these antivirals. An appealing alternative approach to prevent or treat influenza is the use of antibody-based antivirals, such as conventional monoclonal antibodies and single-domain antibodies (sdAbs). The surface of the influenza A and B virion is decorated with hemagglutinin molecules, which act as receptor-binding and membrane fusion proteins and represent the main target of neutralizing antibodies. SdAbs that target influenza A and B hemagglutinin have been described. In addition, sdAbs directed against the influenza A virus neuraminidase have been reported, whereas no sdAbs targeting influenza B neuraminidase have been described to date. SdAbs directed against influenza A matrix protein 2 or its ectodomain have been reported, while no sdAbs have been described targeting the influenza B matrix protein 2. Known for their high specificity, ease of production and formatting, sdAb-based antivirals could be a major leap forward in influenza control., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xavier Saelens is named as an inventor on the following patent applications, which describe single-domain antibodies directed against H5N1 influenza virus HA and NA. WO2009/147248, WO2010/081856, and WO2014/090865., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mycotoxins produced by fungi can contaminate various foods and pose significant health risks. Ensuring food safety demands rapid, highly sensitive analytical techniques. One-step Bioluminescent Enzyme Immunoassays (BLEIAs) employing nanobody-nanoluciferase fusion proteins have recently garnered attention for operational simplicity and heightened sensitivity. Nevertheless, fixed nanobody:nanoluciferase ratios in fusion proteins restrict the customization and sensitivity of traditional BLEIAs. In this study, we present a Scaffold Assembly-based BLEIA (SA-BLEIA) that overcomes these limitations through the programmable conjugation of nanobodies and luciferases onto 60-meric protein nanoscaffolds using SpyTag/SpyCatcher linkages. These nanoscaffolds facilitate the adjustable coupling of anti-aflatoxin B1 and anti-ochratoxin A nanobodies with luciferases, optimizing nanobody/luciferase ratios and diversifying specificities. Compared to conventional methods, SA-BLEIA demonstrates considerably elevated sensitivity for detecting both toxins. The elevated local concentration of luciferase significantly amplifies bioluminescence intensity, permitting reduced substrate consumption and cost-effective detection. The usage of dual-nanobody conjugates facilitates the quantification or simultaneous detection of both mycotoxins in a single test with shared reagents. The assay exhibits exceptional recovery rates in spiked cereal samples, strongly correlating with outcomes from commercial ELISA kits. Overall, this adaptable, highly sensitive, cost-effective, and multiplexed immunoassay underscores the potential of tunable scaffold assembly as a promising avenue for advancing bioanalytical diagnostic tools., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nanobodies are natural anti-SARS-CoV-2 drug candidates. Engineering multivalent nanobodies is an effective way to improve the functional binding affinity of natural nanobodies by simultaneously targeting multiple sites on viral proteins. However, multivalent nanobodies have usually been engineered by trial and error, and rational designs are still lacking. Here, we describe a structure-guided design of a self-assembled trivalent nanobody cluster targeting the SARS-CoV-2 spike protein. Using the nanobody Nb6 as a monovalent binder, we first selected a human-derived trimerization scaffold evaluated by molecular dynamics simulations, then selected an optimal linker according to the minimum distance between Nb6 and the trimerization scaffold, and finally successfully engineered a trivalent nanobody cluster called Tribody. Compared with the low-affinity monovalent counterpart (Nb6), Tribody showed much higher target binding affinity (K D  < 1 pM) and thus had a 900-fold increase in antiviral neutralization against SARS-CoV-2 pseudovirus. We determined the cryo-EM structure of the Tribody-spike complex and confirmed that all three Nb6 binders of Tribody collectively bind to the three receptor-binding domains (RBDs) of the spike and lock them in a 3-RBD-down conformation, fully consistent with our structure-guided design. This study demonstrates that synthetic nanobody clusters with human-derived self-assembled scaffolds are potential protein drugs against SARS-CoV-2 coronaviruses., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:The following authors are employes of Scinai Immunotherapeutics Ltd., a company dedicated to developing Nanobodies for clinical use. Tamar Ben-Yedidia, Oded Ovadia, Dalit Weinstein Fischer, Amir Reichman. Moreover, Matthias Dobbelstein is a member of the scientific advisory board of Scinai Immunotherapeutics Ltd.., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Staphylococcus enterotoxin B (SEB) is one of the primary virulence factors of Staphylococcus aureus but there is still a lack of targeted drugs. SEB activates immune cells via interacting with MHC-II on antigen-presenting cells, leading to the production of large amounts of pro-inflammatory cytokines. Blocking the interaction between SEB and MHC-II can avert the overactivation of immune cells. Nanobodies are the smallest functional antibodies that can bind stably to antigens. In this study, an ideal approach to obtain specific nanobodies without immunizing camelids was introduced. We constructed a library containing up to 5 × 10 8 nanobodies, and then screened those targeting SEB by using yeast surface display (YSD) technique and fluorescence-activated cell sorting (FACS). A total of 8 nanobodies with divergent complementarity-determining regions (CDRs) sequences were identified and one candidate Nb8 with high affinity to SEB was isolated. In vitro study demonstrated that Nb8 significantly inhibited SEB-induced inflammatory response. Molecular docking simulation indicated that the unique CDR3 sequence contributed to the binding of Nb8 to the MHC-II binding domain of SEB and accordingly cut off the connection between SEB and MHC-II. Our efforts contributed to the development of specific nanobodies for eliminating the threats of SEB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Deubiquitinases (DUBs) are proteolytic enzymes that catalyze the removal of ubiquitin from protein substrates. The critical role of DUBs in regulating protein ubiquitination makes them attractive drug targets in oncology, neurodegenerative disease, and antiviral development. Biochemical assays for quantifying DUB activity have enabled characterization of substrate preferences and discovery of small molecule inhibitors. However, assessing the efficacy of these inhibitors in cellular contexts to support clinical drug development has been limited by a lack of tractable cell-based assays. To address this gap, we developed a two-color flow cytometry-based assay that allows for sensitive quantification of DUB activity and inhibition in living cells. The utility of this system was demonstrated by quantifying the potency of GRL0617 against the viral DUB SARS-CoV-2 PL pro , identifying potential GRL0617 resistance mutations, and performing structure-function analysis of the vOTU domain from the recently emerged Yezo virus. In addition, the system was optimized for cellular DUBs by modifying a GFP-targeting nanobody to recruit USP7 and USP28 to benchmark a panel of reported inhibitors and assess inhibition kinetics. Together, these results demonstrate the utility of these assays for studying DUB biology in a cellular context with potential to aid in inhibitor discovery and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The third generation of genetic engineering antibodies, single-domain antibodies, have been widely reported as potential biomaterials in recognizing small molecular hazards. In this study, a shark-derived single-domain antibody was used as the recognition element for the first time to detect enrofloxacin (ENR), one of the most representative hazards in aquaculture. An ENR-specific clone named 2E6 was isolated by phage display technology. Experimental results proved that 2E6 ssdAb showed high affinity to ENR-PEI complete antigen, with the highest OD 450 value of 1.348 in binding ELISA. Through icELISA, it was determined that the IC 50 of 2E6 ssdAb to ENR was 19.230 ng/mL, while the IC 10 was 0.975 ng/mL, with rare recognition to other fluoroquinolones, which showed high sensitivity and specificity to ENR. The 2E6 ssdAb also performed excellently in fish matrix immunoassay. Results showed that the ENR-negative fish matrix did not seriously interfere with the recognition of 2E6 ssdAb to ENR-OVA, with the matrix index between 4.85% and 11.75%, while the results of icELISA in ENR-spiked fish matrix showed that 2E6 ssdAb could recognize the target ENR in different ENR-spiked concentrations of the fish matrix (10-1000 ng/mL), with the recovery between 89.30% and 126.38% and the RSD between 1.95% and 9.83%. This study broadens the application scenario of shark-derived single-domain antibodies as small molecule recognition biomaterials, providing a new recognition element on ENR detection for immunoassay., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5) is a well-characterized biomarker for the clinical diagnosis of various cancers. Nanobodies, considered the smallest antibody fragments with intact antigen-binding capacity, have gained significant attention in disease diagnosis and therapy. Due to their peculiar properties, nanobodies have become promising alternative diagnostic reagents in immunoassay. However, nanobodies-based immunoassay is still hindered by small molecular size and low antigen capture efficacy. Therefore, there is a pressing need to develop novel nanobody-based immunoassays with superior performance., Results: A novel pentameric nanobodies-based immunoassay (PNIA) was developed with enhanced sensitivity and specificity for CEACAM-5 detection. The binding epitopes of three anti-CEACAM-5 nanobodies (Nb1, Nb2 and Nb3) were analyzed. To enhance the capture and detection efficacy of CEACAM-5 in the immunoassay, we engineered bispecific nanobodies (Nb1-Nb2-rFc) as the capture antibody, and developed the FITC-labeled pentameric nanobodies (Nb3-VT1B) as the detection antibody. The binding affinities of Nb1-Nb2-rFc (1.746 × 10 -10 ) and Nb3-VT1B (1.279 × 10 -11 ) were significantly higher than those of unmodified nanobodies (Nb1-rFc, 4.063 × 10 -9 ; Nb2-rFc, 2.136 × 10 -8 ; Nb3, 3.357 × 10 -9 ). The PNIA showed a linear range of 0.625-160 ng mL -1 with a correlation coefficient R 2 of 0.9985, and a limit of detection of 0.52 ng mL -1 , which was 24-fold lower than the immunoassay using monomeric nanobody. The PNIA was validated with the spiked human serum. The average recoveries ranged from 91.8% to 102% and the coefficients of variation ranged from 0.026% to 0.082%., Significance and Novelty: The advantages of nanobodies offer a promising alternative to conventional antibodies in disease diagnosis. The novel PNIA demonstrated superior sensitivity and high specificity for the detection of CEACAM-5 antigen. This bispecific or multivalent nanobody design will provide some new insights into the design of immunoassays for clinical diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fungal keratitis (FK) is a severe infectious corneal disease. Since traditional eye drops exhibit poor dissolution and high corneal toxicity, the efficacy of current treatments for FK remains limited. It is needed to develop new approaches to control the cornea damage from FK. In this study, a nanobody (Nb) specific to β-glucan in the fungal cell wall was prepared. The conjugate of the Nb with natamycin (NAT), a traditional antifungal drug, was synthesized. Firstly, we found the Nb specific to β-glucan inhibited fungal growth by disrupting cell wall and biofilm formation.. In addition, the content of β-glucan in the fungal cell wall decreased after Nb treatment. The Nb also reduced the adhesion ability of fungal conidia to human corneal epithelial cells (HCECs). Further, we examined the difference between NAT and Nb-NAT in antifungal growth. Nb-NAT showed better antifungal effects than NAT which was caused by the interaction between Nb and β-glucan. Moreover, Nb concentration below 0.5 mg/mL did not affect the viability of HCECs. Nb-NAT had less cytotoxicity and ocular surface irritation than NAT. Nb specific to β-glucan attenuated Aspergillus fumigatus (A. fumigatus) virulence and relieved inflammatory responses in FK. Nb-NAT treatment of the cornea improved therapeutic effects compared with NAT. It decreased clinical scores and expression level of inflammatory factors. To our knowledge, this study is the first to report a Nb specific to β-glucan and Nb-NAT for the treatment of FK. These unique functions of the Nb specific to β-glucan and Nb-NAT would render it as an alternative molecule to control fungal infections including FK. STATEMENT OF SIGNIFICANCE: Fungal keratitis is a corneal disease with a high rate of blindness. Due to the poor dissolution and high corneal toxicity exhibited by traditional eye drops, the efficacy of current therapeutic treatments for fungal keratitis (FK) remains limited. To enhance the therapeutic effect of natamycin in treating fungal keratitis, this study developed an innovative approach by preparing a β-glucan-specific nanobody and loading it with the antifungal drug natamycin. The β-glucan-specific nanobody has the ability to control both fungal pathogen invasion and inflammation, which can cause damage to the cornea in FK. The conjugation with the β-glucan-specific nanobody significantly increased the antifungal capacity of natamycin and reduced its toxicity. The further application of natamycin conjugated with the β-glucan-specific nanobody could be expanded to other diseases caused by fungal pathogen infections., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare hematologic disease caused by autoantibodies against ADAMTS-13 that trigger microangiopathic hemolytic anemia. Therapeutic plasma exchange and glucocorticoids have been the mainstay of treatment for the past 30 years. In 2019, caplacizumab was approved as an addition to this regimen for the acute treatment of iTTP. Randomized controlled trials and real-world evidence have shown that caplacizumab reduces the time to platelet count normalization, refractoriness, and exacerbations of the disease, with an acceptable safety profile. In the past 5 years, there have been arguments against the upfront use of caplacizumab in all patients with iTTP, particularly related to the perceived lack of clinical benefit, safety concerns related to bleeding risk, and high costs. This perspective aimed to address these concerns in the context of the experience of expert centers that have used the drug for >5 years., Competing Interests: Declaration of competing interests L.A.V. received research funding and consulting fees from Alexion, AstraZeneca, Bayer, and Sanofi-Genzyme. C.M. received consulting fees from Takeda and Sanofi-Genzyme. P.T.B. received speaker honoraria and consultant fees from AstraZeneca, Alexion, Bayer, Novartis, Roche, Sanofi-Genzyme, Travere, Vifor CSL and participated in advisory boards for Alexion, Sanofi-Genzyme, Novartis, Travere, Takeda, Vifor CSL and Bayer. P.T.B. declares research funding from the German Research Foundation BR-2955/8. S.R.C. received consulting fees from Takeda and Sanofi-Genzyme., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Highly specific antibodies are the key reagents for developing immunoassays with a low false positive rate for environmental monitoring. Here, we provide evidence that nanobodies have the potential to achieve higher specificity than conventional antibodies and explain why from their structural features. Using sulfadimethoxine (SDM) as a model analyte, we constructed an immune phage display library and precisely isolated an ultra-specific nanobody (H1-17) by a crucial homologous antigen counter selection strategy. H1-17 showed no observable cross-reactivity (CR) with other structural analogs of 41 SDM tested, which has never been achieved by conventional antibodies. The structurally original specificity of H1-17 was illuminated and compared with that of one conventional antibody by homology modeling and site-directed mutagenesis validation. It was found that the noncanonical disulfide bond (C50-C104) of H1-17 helped CDR3 form a tailor-made binding pocket and divide it into two parts to accommodate the common structure of sulfonamides and the characteristic methoxyl group of SDM, respectively. Besides, the mutual-checking hydrogen bonds also played important roles in the specific recognition. Lastly, immunoassays with zero false positive rate were developed to screen SDM in water and milk samples, indicating that nanobodies could be reliable reagents for the accurate detection of chemical compounds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Shark variable domain of new antigen receptors (VNARs) are the smallest naturally occurring binding domains with properties of low complexity, small size, cytoplasmic expression, and ease of engineering. Green fluorescent protein (GFP) molecules have been analyzed in conventional microscopy, but their spectral characteristics preclude their use in techniques offering substantially higher resolution. Besides, the GFP molecules can be quenched in acidic environment, which makes it necessary to develop anti-GFP antibody to solve these problems. In view of the diverse applications of GFP and unique physicochemical features of VNAR, the present study aims to generate VNARs against GFP. Here, we identified 36 VNARs targeting eCGP123, an extremely stable GFP, by phage display from three immunized sharks. These VNARs bound to eCGP123 with affinity constant K D values ranging from 6.76 to 605 nM. Among them, two lead VNARs named aGFP-14 and aGFP-15 with nanomolar eCGP123-binding affinity were selected for in-depth characterization. aGFP-14 and aGFP-15 recognized similar epitopes on eCGP123. X-ray crystallography studies clarified the mechanism by which aGFP14 interacts with eCGP123. aGFP-14 also showed cross-reaction with EGFP, with K D values of 47.2 nM. Finally, immunostaining analyses demonstrated that aGFP-14 was able to bind effectively to the EGFP expressed in both cultured cells and mouse brain tissues, and can be used as a fluorescence amplifier for EGFP. Our research demonstrates a feasible idea for the screening and production of shark-derived VNARs. The two high-affinity VNARs developed in the study contribute to the diversity of GFP sdAbs and may enhance the applications of GFP., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Nanobody, a single domain antibody, has been shown a great promise for immunoassay (IA) applications. To improve the panning efficiency so as to obtain a valuable nanobody, anti-carrier protein phages in a phage display library were depleted to enhance the selection of nanobodies against the herbicide atrazine by using immunomagnetic beads conjugated with bovine serum albumin (IMB-BSA). The depletion of anti-carrier protein phages from the atrazine phage display library tripled the number of atrazine positive phage clones after four rounds of panning. One of the most sensitive phage clones Nb3 selected from the IMB-BSA depleted library was used to compare the performance with the monoclonal antibody (mAb 5D9) developed from the same immunogen. The Nb3-based IA exhibited similar specificity with the mAb 5D9-based IA, but greater thermostability and organic solvent tolerance. The half-maximum inhibition concentration (IC 50 ) of the former was 3.5-fold greater than that of the latter (36.7 ng/mL versus 10.2 ng/mL). Because the Nb3-based IA was more robust than the mAb 5D9-based IA, the method detection limit of the two assays was 7.8 ng/mL of atrazine in river samples. The depletion strategy can increase the chance to acquire high quality nanobody and can be applicable for effective development of nanobodies against other small molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: The von Willebrand factor-directed nanobody caplacizumab has greatly changed the treatment of immune thrombotic thrombocytopenic purpura (iTTP) in recent years. Data from randomized controlled trials established efficacy and safety., Objectives: This study aims to address open questions regarding patient selection, tailoring of therapy duration, obstacles in prescribing caplacizumab in iTTP, effect on adjunct treatment, and outcomes in the real-world setting., Methods: We report retrospective, observational cohorts of 113 iTTP episodes treated with caplacizumab and 119 historical control episodes treated without caplacizumab. We aggregated data from the caplacizumab phase II/III trials and real-world data from France, the United Kingdom, Germany, and Austria (846 episodes, 396 treated with caplacizumab, and 450 historical controls)., Results: Caplacizumab was efficacious in iTTP, independent of the timing of therapy initiation, but curtailed the time of active iTTP only when used in the first-line therapy within 72 hours after diagnosis and until at least partial ADAMTS13-activity remission. Aggregated data from multiple study populations showed that caplacizumab use resulted in significant absolute risk reduction of 2.87% for iTTP-related mortality (number needed to treat 35) and a relative risk reduction of 59%., Conclusion: Caplacizumab should be used in first line and until ADAMTS13-remission, lowers iTTP-related mortality and refractoriness, and decreases the number of daily plasma exchange and hospital stay. This trial is registered at www., Clinicaltrials: gov as #NCT04985318., Competing Interests: Declaration of competing interests L.A.V. received speaker honoraria and consultant fees from Sanofi-Genzyme and AstraZeneca. J.K. reports speaker honoraria and participation on advisory boards from Alexion, Sanofi, and Chiesi. W.M. reports grants from Takeda/Shire and CSL Behring and received speaker honoraria and consultant fees from Takeda/Shire and CSL Behring. P.T.B. received speaker honoraria and consultant fees from Sanofi-Genzyme, AstraZeneca, Alexion, Bayer, Travere, Pfizer, Novartis, Roche, and participated in advisory boards for Alexion, Sanofi-Genzyme, Novartis, Travere, and Bayer. He declares research funding from the German Research Foundation BR-2955/8. R.W. received speaker honoraria and consultant fees from Sanofi-Genzyme. He additionally declares research funding from the German Federal Ministry of Education and Research and the German Federal Ministry of Health. U.S. reports study fees and/or consultancy/advisory board fees from Chemocentryx/Vifor, Ablynx/Genzyme-Sanofi, Alexion, Travere, Alynlam Pharmaceuticals, and Allena Pharmaceuticals. J.M. received speaker honoraria and consultant fees from Ablynx, Alexion, and Sanofi-Genzyme. D.A.Eichenhauer has received speaker honoraria from Sanofi-Genzyme and Takeda. K.S. has received speaker honoraria from AbbVie, Merck, Pfizer, and Roche, consultant fees from AbbVie, Amgen, Bristol-Myers Squibb, Merck, Novartis, and Roche, and travel and congress fee compensation from AbbVie, Bristol-Myers Squibb, Celgene, Janssen Cilag, Lilly, and Servier. M.H. reports travel and congress fee compensation and speaker honoraria from Jazz Pharmaceuticals. P.K. reports travel support, participation in data safety monitoring boards and advisory boards, and consulting fees and/or speaker honoraria from Ablynx/Sanofi, Shire Roche, Novo Nordisk, CSL-Behring, and Biotest. F.B. reports speaker honoraria and consulting fees from Sanofi-Genzyme. A.G. reports consulting fees from Sanofi-Genzyme and Alexion and participation in advisory boards for Alexion. T.M. reports speaker honoraria from Sanofi-Aventis Deutschland GmbH. A.S. reports advisory board fees from Sanofi-Genzyme. K.S. reports speaker honoraria from AstraZeneca, Novartis, Takeda/Shire, and Vifor. M. K. reports speaker honoraria from Sanofi-Genzyme. All other authors report no conflict of interest., (Copyright © 2022 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

The field of antibody development is under pressure to meet rising demands for speed, cost-effectiveness, efficacy, reliability, and large-scale production. It is costly and time-consuming to immunize animals and build a single-domain antibody (sdAb) library for each target. Using the variable domain (VHH) of heavy-chain only antibodies (HcAbs) derived from blood samples of 75 non-immunized camelid animals (51 alpacas, 13 llamas, 11 Bactrian camels), and spleens from two Bactrian camels, a naïve sdAb library with extensive megadiversity and reusability was constructed. The library was evaluated using next-generation DNA sequencing (NGS) and was found to contain hundreds of billions of unique clones. To confirm the availability of target-specific VHHs, a naive library was screened for a variety of targets. At least two VHH candidates were extracted for each target using a 20-day selection pipeline. Some binders had ultrahigh potencies, with binding affinities in the nanomolar range. This naïve library, in particular, offers the possibility of acquiring unique antibodies targeting antigens of interest with low feasible dissociation constant (kD) without the time, effort, and price associated in producing antibodies in animals via antigen injection. Overall, the study shows that the megadiverse naïve library provides a rapid, adaptable, and easy platform for antibody creation, emphasizing its therapeutic and diagnostic implications., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Factor XI (FXI) is a promising target for novel anticoagulants because it shows a strong relation to thromboembolic diseases, while fulfilling a mostly supportive role in hemostasis. Anticoagulants targeting FXI could therefore reduce the risk for thrombosis, without increasing the chance of bleeding side effects., Objectives: To generate nanobodies that can interfere with FXIa mediated activation of factor IX (FIX)., Methods: Nanobodies were selected for binding to the apple 3 domain of FXI and their effects on FXI and coagulation were measured in purified protein systems as well as in plasma-based coagulation assays. Additionally, the binding epitope of selected nanobodies was assessed by hydrogen-deuterium exchange mass spectrometry., Results: We have identified five nanobodies that inhibit FIX activation by FXI by competing with the FIX binding site on FXI. Interestingly, a sixth nanobody was found to target a different binding epitope in the apple 3 domain, resulting in competition with the FXI-high molecular weight kininogen (HK) interaction., Conclusions: We have characterized a nanobody targeting the FXI apple 3 domain that elucidates the binding orientation of HK on FXI. Moreover, we have produced five nanobodies that can inhibit the FXI-FIX interaction., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

RhoA protein is a small GTPase that acts as a molecular switch. When bound to guanosine triphosphate (GTP), RhoA can activate several key signal pathways. Recently, nanobody Rh57 specific binding with GTP bound active RhoA was discovered and developed as a BRET biosensor without cytotoxicity. To further clarify the nanobody Rh57's mechanism of action, we co-expressed, purified, and crystallized the RhoA-Rh57 nanobody complex and solved the structure by X-ray diffraction with a resolution of 2.76 Å. The structure showed that the interaction is mainly through hydrogen bonds, salt bridges, aromatic-aromatic interactions, and hydrophobic interactions. The involved regions include CDR3 and non-hypervariable loop of Rh57, and the SWI switch loops of RhoA, respectively. The different SWI conformation of inactivated RhoA-GDP prevented the Rh57's binding. The possible explanation of Rh57 as a non-cytotoxic BRET intracellular tracer is that Rh57's binding did not overlap with downstream PRK1 and thus did not interfere with the downstream signaling pathway. Our research provides an in-depth understanding of how nanobodies recognize activated RhoA-GTP while not binding inactivated RhoA-GDP. This structural information may also provide critical information for further optimization of relevant nanobodies., Competing Interests: Declaration of competing interest We declare that we have no conflicts of interest in the authorship or publication of this contribution., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Correct elucidation of physiological and pathological processes mediated by extracellular vesicles (EV) is highly dependent on the reliability of the method used for their purification. Currently available chemical/physical protocols for sample fractionation are time-consuming, often scarcely reproducible and their yields are low. Immuno-capture based approaches could represent an effective purification alternative to obtain homogeneous EV samples. An easy-to-operate chromatography system was set-up for the purification of intact EVs based on a single domain (VHH) antibodies-copolymer matrix suitable for biological samples as different as conditioned cell culture medium and human plasma. Methacrylate-based copolymer is a porous solid support, the chemical versatility of which enables its efficient functionalization with VHHs. The combined analyses of morphological features and biomarker (CD9, CD63 and CD81) presence indicated that the recovered EVs were exosomes. The lipoprotein markers APO-A1 and APO-B were both negative in tested samples. This is the first report demonstrating the successful application of spherical porous methacrylate-based copolymer coupled with VHHs for the exosome isolation from biological fluids. This inexpensive immunoaffinity method has the potential to be applied for the isolation of EVs belonging to different morphological and physiological classes., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Protein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factors V (FVa) and VIII (FVIIIa), but also for tissue factor pathway inhibitor α (TFPIα) in the inhibition of activated factor X (FXa)., Objective: For therapeutic purposes, we aimed at generating single-domain antibodies (sdAbs) that could specifically modulate the APC-cofactor activity of PS in vivo., Methods: A llama-derived immune library of sdAbs was generated and screened on recombinant human PS by phage display. PS binders were tested in a global activated partial thromboplastin time (APTT)-based APC-cofactor activity assay., Results: A PS-specific sdAb (PS003) was found to enhance the APC-cofactor activity of PS in our APTT-based assay, and this enhancing effect was greater for a bivalent form of PS003 (PS003biv). Further characterization of PS003biv demonstrated that PS003biv also enhanced the APC-cofactor activity of PS in a tissue factor (TF)-induced thrombin generation assay and stimulated APC in the inactivation of FVa, but not FVIIIa, in plasma-based assays. Furthermore, PS003biv was directed against the sex hormone-binding globulin (SHBG)-like domain but did not inhibit the binding of PS to C4b-binding protein (C4BP) and did not interfere with the TFPIα-cofactor activity of PS. In mice, PS003biv exerted an antithrombotic effect in a FeCl 3 -induced thrombosis model, while not affecting physiological hemostasis in a tail-clip bleeding model., Discussion: Altogether, these results showed that pharmacological enhancement of the APC-cofactor activity of PS through an original anti-PS sdAb might constitute a promising and safe antithrombotic strategy., (© 2022 International Society on Thrombosis and Haemostasis.)

Salmonella are major pathogens that cause foodborne diseases. In this work, a broad-spectrum Salmonella nanobody-01 (Nb-01) was isolated and applied in the development of a streptavidin-bridged sandwich ELISA (SAB-ELISA) for simultaneously identifying five Salmonella serovars, including Salmonella Enteritidis (S. Enteritidis), Salmonella Typhimurium (S. Typhimurium), Salmonella London (S. London), Salmonella Paratyphi B (S. Paratyphi B) and Salmonella Hadar (S. Hadar). In this work, streptavidin (SA) was utilized as a scaffold to directionally immobilize biotinylated nanobody (BiNb) and Salmonella was detected by phage-displayed nanobodies. The SAB-ELISA can be accomplished within 180 min with a limit of detection (LOD) of 6.31 × 10 3 colony forming units (CFU) mL -1 , 9.15 × 10 3  CFU mL -1 , 4.23 × 10 3  CFU mL -1 , 7.31 × 10 3  CFU mL -1 and 7.25 × 10 3  CFU mL -1 towards S. Typhimurium, S. Enteritidis, S. London, S. Paratyphi B and S. Hadar, respectively. In comparison of sandwich ELISA by passive immobilization of Nb-01 on polystyrene plate, the sensitivity was increased by around 6-fold, which confirmed the enhanced immobilization efficacy of SAB-ELISA. Furthermore, the feasibility of the assay for S. Typhimurium determination in actual samples was evaluated, showing excellent recovery, inter-day and intra-day precision., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Nanobodies are the smallest antibody fragments which bind to antigens with high affinity and specificity. Due to their outstanding physicochemical stability, simplicity and cost-effective production, nanobodies have become powerful agents in therapeutic and diagnostic applications. In this work, the advantages of nanobodies were exploited to develop generic and standardized anti-human IgM reagents for serology and IgM + B-cell analysis. Selection of anti-IgM nanobodies was carried out by evaluating their yields, stability, binding kinetics and cross-reactivity with other Ig isotypes. High affinity nanobodies were selected with dissociation constants (KDs) in the nM range and high sensitivities for detection of total IgM by ELISA. The nanobodies also proved to be useful for capturing IgM in the serodiagnosis of an acute infection as demonstrated by detection of specific IgM in sera of dengue virus patients. Finally, due to the lack of an Fc region, the selected nanobodies do not require Fc receptor blocking steps, facilitating the immunophenotyping of IgM + cells by flow cytometry, an important means of diagnosis of immunodeficiencies and B-cell lymphoproliferative disorders. This work describes versatile anti-IgM nanobodies that, due to their recombinant nature and ease of reproduction at low cost, may represent an advantageous alternative to conventional anti-IgM antibodies in research and diagnosis., (Copyright © 2021. Published by Elsevier B.V.)

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare, life-threatening disease that causes systemic platelet-rich microthrombi with multiorgan damage. The historical treatment is based on therapeutic plasma exchange (TPE) and immunosuppression. Despite survival rates exceeding 85%, unfavorable outcomes including refractoriness, death, and exacerbations of the disease during treatment still calls for a better management strategy. Caplacizumab (Cablivi) appeared recently as a new treatment in iTTP. By inhibiting binding of von Willebrand factor to platelets, caplacizumab prevents platelets aggregation and the formation of microthrombi. Two pivotal randomized controlled trials have provided positive results where the use of caplacizumab is associated with faster platelet count recovery and less unfavorable outcomes. The other strength of this agent is an impressive alleviation in the burden of care, consisting in less TPE sessions and lower volumes of plasma to achieve remission, as well as substantial shortening in the length of hospitalization. However, since the recent approval of caplacizumab for the treatment of iTTP on the basis of these studies, debates remain regarding its systematic use in this indication. Should all patients be benefited from caplacizumab? Should we reserve caplacizumab only to the more severe patients? Should caplacizumab be initiated frontline or as a salvage therapy? If applicable, how should we select patients for caplacizumab? Last, is caplacizumab treatment cost-effective? This review aims at addressing these specific questions at a time when iTTP is entering the area of targeted therapies., (© 2020 International Society on Thrombosis and Haemostasis.)

Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, life-threatening autoimmune thrombotic microangiopathy. Current standard of care is therapeutic plasma exchange, immunosuppression, and caplacizumab, an anti-von Willebrand factor nanobody, which is effective in treating aTTP episodes., Patients/methods: Here we report on seven episodes of aTTP treated without plasma exchange in six female patients in Germany and Austria. Two episodes were initial presentations of aTTP; in five instances, patients experienced a relapse. In four episodes, moderate to severe organ dysfunction was observed; three cases presented with a mild course. All patients received caplacizumab immediately once aTTP was suspected or diagnosed, and plasma exchange was omitted based on shared decision making between patient and the treating physicians., Results: We observed a rapid and robust increase of platelet counts already after the first dose of caplacizumab, leading to a doubling of platelet counts within 17 hours (median), platelet counts normalized (>150 G/L) after median 84 hours. Lactate dehydrogenase, as a surrogate parameter of organ damage, improved in parallel to the platelet counts, indicating resolving microangiopathy., Conclusions: In conclusion, in selected cases of acute bouts of aTTP, it seems feasible to delay or omit plasma exchange if platelet counts increase and organ function is stable after start of caplacizumab therapy., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Despite advances in treatment options for thrombotic thrombocytopenic purpura (TTP), there are still limited high quality data to inform clinicians regarding its management., Methods: In June 2018, the ISTH formed a multidisciplinary guideline panel to issue recommendations about treatment of TTP. The panel discussed 12 treatment questions related to both immune-mediated TTP (iTTP) and hereditary/congenital TTP (cTTP). The panel used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, including evidence-to-decision frameworks, to appraise evidence and formulate recommendations., Results: The panel agreed on eleven recommendations based on evidence ranging from very low to moderate certainty. For first episode and relapses of acute iTTP, the panel made a strong recommendation for the addition of corticosteroids to therapeutic plasma exchange (TPE), and a conditional recommendation for addition of rituximab and caplacizumab. For asymptomatic iTTP with low ADAMTS13, the panel made a conditional recommendation for rituximab outside of pregnancy, and for prophylactic TPE during pregnancy. For asymptomatic cTTP, the panel made a strong recommendation for prophylactic plasma infusion during pregnancy, but a conditional recommendation for plasma infusion or a wait and watch approach outside of pregnancy., Conclusions: The panel's recommendations are based on all the available evidence for the treatment effects of various approaches including suppressing inflammation, blocking platelet clumping, replacing the missing and/or inhibited ADAMTS13, and suppressing ADAMTS13 antibody production. There was insufficient evidence for further comparison of different treatment approaches, for which future high-quality studies in iTTP (e.g., rituximab, corticosteroids, recombinant ADAMTS13, and caplacizumab) and in cTTP (eg, recombinant ADAMTS13) are needed., (© 2020 International Society on Thrombosis and Haemostasis.)

Background: Despite advances in treatment options for thrombotic thrombocytopenic purpura (TTP), there are still limited high quality data to inform clinicians regarding its appropriate treatment., Methods: In June 2018, the ISTH formed a multidisciplinary guideline panel to issue recommendations about treatment of TTP. The panel discussed 12 treatment questions related to immune-mediated TTP (iTTP) and hereditary or congenital TTP (cTTP). The panel used the Grading of Recommendations Assessment, Development, and Evaluation approach, including evidence-to-decision frameworks, to appraise evidence and formulate recommendations., Results: The panel agreed on 11 recommendations based on evidence ranging from very low to moderate certainty. For first acute episode and relapses of iTTP, the panel made a strong recommendation for adding corticosteroids to therapeutic plasma exchange (TPE) and a conditional recommendation for adding rituximab and caplacizumab. For asymptomatic iTTP with low plasma ADAMTS13 activity, the panel made a conditional recommendation for the use of rituximab outside of pregnancy, but prophylactic TPE during pregnancy. For asymptomatic cTTP, the panel made a strong recommendation for prophylactic plasma infusion during pregnancy, and a conditional recommendation for plasma infusion or a wait and watch approach outside of pregnancy., Conclusions: The panel's recommendations are based on all the available evidence for the effects of an individual component of various treatment approaches, including suppressing inflammation, blocking platelet clumping, replacing the missing and/or inhibited ADAMTS13, and suppressing the formation of ADAMTS13 autoantibody. There was insufficient evidence for further comparing different treatment approaches (eg, TPE, corticosteroids, rituximab, and caplacizumab, etc.), for which high quality studies are needed., (© 2020 International Society on Thrombosis and Haemostasis.)

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., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Background: Protease nexin-1 (PN-1) is a member of the serine protease inhibitor (Serpin)-family, with thrombin as its main target. Current polyclonal and monoclonal antibodies against PN-1 frequently cross-react with plasminogen activator inhibitor-1 (PAI-1), a structurally and functionally homologous Serpin., Objectives: Here, we aimed to develop inhibitory single-domain antibodies (VHHs) that show specific binding to both human (hPN-1) and murine (mPN-1) PN-1., Methods: PN-1-binding VHHs were isolated via phage-display using llama-derived or synthetic VHH-libraries. Following bacterial expression, purified VHHs were analyzed in binding and activity assays., Results and Conclusions: By using a llama-derived library, 2 PN-1 specific VHHs were obtained (KB-PN1-01 and KB-PN1-02). Despite their specificity, none displayed inhibitory activity toward hPN-1 or mPN-1. From the synthetic library, 4 VHHs (H12, B11, F06, A08) could be isolated that combined efficient binding to both hPN-1 and mPN-1 with negligible binding to PAI-1. Of these, B11, F06, and A08 were able to fully restore thrombin activity by blocking PN-1. As monovalent VHH, half-maximal inhibitory concentration values for hPN-1 were 50 ± 10, 290 ± 30, and 960 ± 390 nmol/L, for B11, F06, and A08, respectively, and 1580 ± 240, 560 ± 130, and 2880 ± 770 nmol/L for mPN-1. The inhibitory potential was improved 4- to 7-fold when bivalent VHHs were engineered. Importantly, all VHHs could block PN-1 activity in plasma as well as PN-1 released from activated platelets, one of the main sources of PN-1 during hemostasis. In conclusion, we report the generation of inhibitory anti-PN-1 antibodies using a specific approach to avoid cross-reactivity with the homologous Serpin PAI-1., (© 2020 International Society on Thrombosis and Haemostasis.)

Background: Plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasminogen activators (PAs) tissue-type PA (tPA) and urokinase-type PA (uPA) plays a crucial role in many (patho)physiological processes (e.g., cardiovascular disease, tissue fibrosis) as well as in many age-related pathologies. Therefore, much effort has been put into the development of small molecule or antibody-based PAI-1 inhibitors., Objective: To elucidate the molecular mechanism of nanobody-induced PAI-1 inhibition., Methods and Results: Here we present the first crystal structures of PAI-1 in complex with two neutralizing nanobodies (Nbs). These structures, together with biochemical and biophysical characterization, reveal that Nb VHH-2g-42 (Nb42) interferes with the initial PAI-1/PA complex formation, whereas VHH-2w-64 (Nb64) redirects the PAI-1/PA interaction to PAI-1 deactivation and regeneration of active PA. Furthermore, whereas vitronectin does not have an impact on the inhibitory effect of Nb42, it strongly potentiates the inhibitory effect of Nb64, which may contribute to a strong inhibitory potential of Nb64 in vivo., Conclusions: These findings illuminate the molecular mechanisms of PAI-1 inhibition. Nb42 and Nb64 can be used as starting points to engineer further improved antibody-based PAI-1 inhibitors or guide the rational design of small molecule inhibitors to treat a wide range of PAI-1-related pathophysiological conditions., (© 2019 International Society on Thrombosis and Haemostasis.)

Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, life-threatening autoimmune thrombotic microangiopathy. Caplacizumab, an anti-von Willebrand Factor Nanobody ® , is effective for treating aTTP episodes and is well tolerated., Objectives and Methods: In the phase 3 HERCULES trial (NCT02553317), patients with aTTP received double-blind caplacizumab or placebo during daily therapeutic plasma exchange (TPE) and for ≥30 days thereafter. Patients who experienced an exacerbation while on blinded study drug treatment switched to receive open-label caplacizumab plus re-initiation of daily TPE. Exacerbations were defined as recurrence of disease occurring within 30 days after cessation of daily TPE., Results: Thirty-one patients (placebo, n = 28; caplacizumab, n = 3) had an exacerbation during double-blind treatment. Twenty-eight patients switched to open-label caplacizumab (placebo, n = 26; caplacizumab, n = 2); the three others discontinued upon exacerbation. Median time to platelet count response (≥150 × 10 9 /L) was 3.49 days upon receiving caplacizumab. There were no deaths. During open-label treatment, further exacerbation or a major thromboembolic event (vena cava thrombosis) was experienced by one patient (3.6%) each. Consistent with the double-blind phase, the most frequent treatment-emergent adverse events were catheter site hemorrhage (28.6%), headache (21.4%), and epistaxis (17.9%)., Conclusions: These results suggest that caplacizumab was efficacious and well tolerated in patients with aTTP who experienced a disease exacerbation during double-blind treatment in HERCULES., (© 2019 Sanofi. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

At the present, the identification of planktonic species in coastal water is still a time intensive process performed by highly trained personnel that relies either on qPCR or on light microscopy observation and in vitro culturing. Furthermore, the increasing danger represented by Harmful Algal Blooms (HABs) inside phytoplankton community and the recent implementation of the legislation on ballast water management to prevent the introduction of HABs and NIS (Non Indigenous Species) urge the development of faster and reliable diagnostic methods. Immuno-based approaches could fulfil this need provided that the costs for antibody selection and production will be reduced. In this work it is demonstrated for the first time the feasibility to recover nanobodies (VHHs) selective for native surface epitopes of Alexandrium minutum by direct whole cell bio-panning using a pre-immune phage display library. The recombinant nature of VHHs enabled their rapid engineering into eGFP fluorescent reagents (fluobodies) that were produced recombinantly in bacteria and are directly suitable for fluorescence microscopy and flow cytometry. Immune-detection identified also cysts and anti-Alexandrium fluobodies showed no cross-reactivity with indigenous not-toxic phytoplankton microalgae belonging to different geni. The fluobodies were able to bind selectively to the target cells in both fixed and fresh samples with minimal processing., (Copyright © 2019 Elsevier B.V. All rights reserved.)