Nanobodies 1H9 and 1D4 were the most potent and reached complete inhibition. Open in a separate window FIGURE 1 Effect of nanobodies on factor XIa (FXIa) amidolytic activity and factor IX (FIX) activation by FXIa. apple 3 domain that elucidates the binding orientation of HK on FXI. Moreover, we have produced five nanobodies that can inhibit the FXICFIX interaction. Keywords: anticoagulants, factor XI, high molecular weight kininogen, hydrogenCdeuterium exchange mass spectrometrynanobody Essentials Factor XI (FXI) is a promising target for new anticoagulant therapy. A panel of nanobodies targeting apple 3 domain in FXI was generated. Six nanobodies showed inhibition of FXI in a variety of assays. A unique nanobody exposes a new interaction site of high molecular weight kininogen on FXI. 1.?INTRODUCTION Over the last decade factor XI (FXI) has emerged as a promising target for the development of novel anticoagulation therapy. 1 , 2 , 3 , 4 , 5 While direct oral anticoagulants (DOACs) have a lower bleeding incidence than the vitamin K antagonists they have replaced, DOACs still show a risk for bleeding due to their interference with thrombin or factor Xa, which have a crucial role in hemostasis. 3 , 4 , 6 , 7 , 8 , 9 FXI deficiency only causes a mild bleeding disorder and has been found to protect against thrombotic events such as SGI 1027 venous SGI 1027 thrombosis and ischemic stroke. 10 , 11 , 12 , 13 , 14 Accordingly, higher levels of FXI are a risk element for venous thrombosis 12 , 15 and stroke. 16 Therefore, zymogen FXI or its triggered form, FXIa, could serve as a target for safer medicines to treat thrombosis with less risk for bleeding. Numerous new treatments based on focusing on FXI are in development. These include the reduction Fst of FXI synthesis in the liver by antisense oligonucleotides, 17 , 18 small molecules that inhibit FXIa activity by obstructing the active site directly or through allosteric action, 19 , 20 anti\FXI aptamers, 21 and FXI(a)\focusing on antibodies. 22 , 23 , 24 , 25 Three FXI\focusing on antibodies (abelacimab, xisocimab 3G3, and osocimab) are either in the final stage of phase 2 or entering phase 3 medical tests. 23 , 24 , 25 , 26 , 27 While treatment with humanized antibodies seems encouraging, repeated administration of biopharmaceuticals has been known to induce adverse immune reactions through the generation of antidrug antibodies. 28 , 29 In most cases these have a negligible effect on the treatment. However, antidrug antibodies can adversely impact the pharmacokinetics and bioavailability of the drug, reducing the effectiveness of the antibody and at worst causing total neutralization of its function. Moreover, antidrug antibodies can provoke allergic reactions and even autoimmune reactions. 28 We consequently propose the development of a nanobody focusing on FXI, thought to have lower immunogenicity compared to antibodies. 30 Nanobodies are the isolated weighty chain antibody variable (VHH) domains from camelid weighty\chain antibodies. 31 These VHH domains are homologous to the VH website of a conventional antibody, and consist of three loops, so\called complementarity\determining areas (CDRs), that set up nanobody specificity. 32 , 33 Despite their small size they display a target specificity and affinity that is comparable to antibodies. 29 , 30 , 31 , 34 , 35 Moreover, nanobody size and their prolonged flexible structure of the CDR3 loop allows them to target antigenic sites that are generally not identified by antibodies. 35 While their size also causes quick renal clearance, this can be overcome by numerous protein modifications such as fusion to a human being serum albumin\binding website. 33 , SGI 1027 36 Additional favorable characteristics of nanobodies include low immunogenicity, high solubility, and good.