However, such patients are at risk of developing neutralizing antibodies, so-called inhibitors, to such exogenous factors, rendering formerly efficient therapy ineffective. Haemophilia A patients with inhibitors is a non-limiting example of a coagulopathy that is partly congenital and partly acquired. Patients that have developed inhibitors to FVIII 5 cannot be treated with conventional replacement therapy. Exogenous coagulation factors may only be administered intravenously, which is of considerable inconvenience and discomfort to patients. An inadequate FXa formation and decreased thrombin generation caused by reduced or absent FVIII activity is the reason underlying the bleeding diathesis in haemophilia A 10 patients. Proteolytic conversion of FX into its enzymatically active form FXa can be achieved by the intrinsic FX-activating complex comprising FIXa and its cofactor activated FVIII (FVIIIa). Cofactor binding increases the enzymatic activity of FIXa by about five orders of magnitude and is believed to result through multiple mechanisms as outlined by 15 Scheiflinger et al. (2008) J Thromb Haemost, 6:315-322. Notably, FVIIIa has been found to stabilize a conformation of FIXa that has increased proteolytic activity towards FX (Kolkman JA, Mertens K (2000) Biochemistry, 39:7398-7405, Zögg T, Brandstetter H (2009) Biol Chem, 390:391-400). In recent years, emicizumab (HEMLIBRA ^® ) also known as ACE910, has been approved 20 for subcutaneous prophylactic treatment of Haemophilia A with or without inhibitors against conventional replacement therapy factors. Emicizumab is a humanized, bispecific full-length anti-FIX(a)/anti-FX(a) monoclonal antibody developed by Chugai Pharmaceuticals/Roche Pharmaceuticals for the treatment of haemophilia A. Emicizumab is designed to mimic FVIII cofactor function (see Sampei et al. (2013) PLoS One, 8, 25 e57479 and WO2012/067176). Treatment with 30-50 μg of emicizumab per milliliter plasma has been speculated correspond to at least 10 to 15 IU of equivalent factor VIII activity per decilitre plasma (Shima et al., N Engl J Med 2016; 374:2044-53). However, some patients have developed inhibitors (anti-drug antibodies) against emicizumab rendering treatment with this compound ineffective. 30 Besides the generation of inhibitors as exemplified for Emicizumab, other antibody properties are also important for achieving an effective antibody-based treatment for the patient. In particular, it has been demonstrated that antibodies with high propensity for non-specific binding may lead to safety issues in the clinic. In some reports, a high level of non-specific binding caused a several-fold reduction in circulating half-life of the antibody 35 and led to ineffective and cumbersome dosing regimens for the patient (See Dobson et 2

al., Nature, volume 6, art. no.: 38644 (2016) and Avery et al., MAbs 2018, Vol.10, No.2, 244–255). WO2018/141863, WO2019/065795 (US10759870), WO2020/025672 and WO2021/152066 disclose anti-FIX(a)/anti-FX(a) bispecific full-length antibodies and their 5 use as procoagulants for use in the treatment of haemophilia by way of subcutaneous administration. One such bispecific antibody is designated Mim8 (see Østergaard H et al. Blood.2021;138:1258–68 and WO2020/025672). There are, however, still many very significant unmet medical needs in the haemophilia 10 community, in particular, in subjects with coagulopathies and in particular there is a need for reduced treatment burden and improved compounds capable of substituting for FVIII(a) for use in the treatment of a coagulopathy such as haemophilia A and related diseases. 15 SUMMARY The present invention provides procoagulant immunoglobulin single variable domain (ISVD) polypeptides derivatives, such as V _H H polypeptide derivatives, capable of binding coagulation Factor IX(a) and coagulation Factor X(a) which are highly potent and provide 20 a sufficiently long half-life such to allow for effective subcutaneous administration as well as peroral administration. Thus, in one aspect the present invention relates to procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivatives comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID 25 NO:2) or the activated form thereof, one or more protraction moiety(ies) attached to one or more surface exposed residue(s), optionally a linker (L _1-2 ) linking ISVD1 and ISVD2, and optionally one or more extension(s) (E). In one aspect the present invention relates to a procoagulant ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the 30 activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or the activated form thereof, at least one protraction moiety attached to a surface exposed residue, optionally a linker (L _1-2 ) linking ISVD1 and ISVD2, and optionally one or more extension(s) (E), wherein said first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO:1) or the activated form thereof comprising at least one of the 35 amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 (consecutive numbering), and wherein said second ISVD is capable of binding to an 3

epitope on Factor X (SEQ ID NO:2) comprising at least one of the amino acid residues N173, P174, F175, L177, L178 and D179 (consecutive numbering). The ISVD polypeptide derivative can for example be a V _H H polypeptide derivative. Another aspect the present invention relates to pharmaceutical compositions comprising 5 the ISVD polypeptides derivatives as disclosed herein. Another aspect of the invention relates to use of ISVD polypeptides derivatives disclosed herein and compositions comprising such compounds for the treatment of various forms of haemophilia and in particular haemophilia A, haemophilia A with inhibitors and acquired haemophilia A by various routes of administration including subcutaneous and peroral administration. 10 In a further aspect the invention relates to the individual component (intermediate) ISVDs or V _H H fragments that are part of an ISVD polypeptide derivative or V _H H polypeptide derivative, such as a particular anti-FIX(a) V _H H fragment or a particular anti-FX(a) V _H H fragment thereof. A further aspect of the invention relates to the manufacture of the components 15 (intermediates) of the compounds as disclosed herein including methods for modifying the isoelectric point of ISVD polypeptide derivatives capable of binding FIX(a) and FX(a) such to improve oral bioavailability of such polypeptide derivatives. BRIEF DESCRIPTION OF FIGURES 20 Figs.1a-h show non-limiting examples of anti-FX(a)/FIX(a) ISVD polypeptide derivatives. E: Extension, PM: protraction moiety, L _1-2 : linker connecting ISVD1 and ISVD2. The dashed lines indicate disulphide bonds. Fig.2a shows a non-limiting example of an anti-FX/FIX(a) V _H H polypeptide derivative25 comprising a single C18 diacid fatty acid-based protraction moiety conjugated to a C- terminal extension by way of a protraction moiety linker (L _P ), ID: L _P 1. The dashed lines indicate disulphide bonds. Fig.2b shows a non-limiting example of an anti-FX/FIX(a) V _H H polypeptide derivative 30 comprising a single tetrazole-based protraction moiety conjugated to a C-terminal extension by way of a protraction moiety linker (L _P ), ID: L _P 2. The dashed lines indicate disulphide bonds. Figs.3a-f show a detailed figurative description of compounds cmpd #20 (a), cmpd #18 35 (b), cmpd #15 (c), cmpd #13 (d), cmpd #14 (e) and cmpd #12 (f), which are non-limiting examples of anti-FX/FIX(a) V _H H polypeptide derivatives comprising a double C16 diacid 4

fatty acid-based protraction moiety conjugated to a C-terminal extension (E) by way of a protraction moiety linker (L _P ), ID L _P 1. The dashed lines indicate disulphide bonds. The extension (E) (SEQ NO:9) was used in these V _H H polypeptide derivatives. The extension comprises two cysteine residues in positions 4 and 6, respectively, which serve as 5 attachment points for the protraction moieties (PM). In a) the sequence of the V _H H1.20 - L _1-2 - V _H H2.20 - E polypeptide is represented by SEQ ID NO:634 (cmpd #20). In b) the sequence of the V _H H1.18 - L _{1-2 -} V _H H2.18 - E polypeptide is represented by SEQ ID NO:632 (cmpd #18). 10 In c) the sequence of the V _H H1.15 - L _1-2 - V _H H2.15 - E polypeptide is represented by SEQ ID NO:629 (cmpd #15). In d) the sequence of the V _H H1.13 - L _1-2 - V _H H2.13 - E polypeptide is represented by SEQ ID NO:627 (cmpd #13). In e) the sequence of the V _H H1.14 - L _1-2 - V _H H2.14 - E polypeptide is represented by SEQ 15 ID NO:628 (cmpd #14). In f) the sequence of the V _H H1.12 - L _1-2 - V _H H2.12 - E polypeptide is represented by SEQ ID NO:626 (cmpd #12). Fig.4 shows an example of titration curves (activity as result of compound concentration) 20 for cmpd #6, Mim8 and emicizumab SIA. Figs.5a and 5b show sequence alignments of anti-FIX(a) and anti-FX ISVD (V _H H fragment) sequences, respectively, wherein CDR sequences are highlighted in bold and underlined. 25 BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO:1 represents the amino acid sequence of human coagulation Factor IX. SEQ ID NO:2 represents the amino acid sequence of human coagulation Factor X. SEQ ID NOs:3-13 and 690 represent the amino acid sequences of extensions (E). SEQ ID NOs:14-26 and 691 represent the amino acid sequences of L _1-2 and L _P linkers. 30 SEQ ID NOs:27-614 represent the amino acid sequences of V _H H fragments and Complementarity Determining Regions (CDRs) thereof. SEQ ID NOs:615-691, 734 and 735 represent the amino acid sequences of V _H H polypeptides including any L _1-2 linkers and/or extensions. SEQ ID NOs:692-733 represent the sequences of peptide fragments as disclosed in 35 Example 4 herein. SEQ ID NOs:736-739 represent the amino acid sequences of potential protractors. 5

DESCRIPTION The present invention provides ISVD polypeptides derivatives capable of binding FIX(a) and FX(a) which are highly potent and provide a sufficiently long half-life such to allow for 5 effective subcutaneous as well as peroral administration. The ISVD polypeptides derivatives disclosed herein are thus suitable for treatment of various forms of haemophilia, such as haemophilia A, haemophilia A with inhibitors and acquired haemophilia A by various routes of administration including subcutaneous and peroral administration. 10 In particular, the present invention relates to bispecific protracted ISVD polypeptides, such as V _H H polypeptides, referred-to as ISVD polypeptide derivatives (or V _H H polypeptide derivatives), that can bind coagulation FIXa and coagulation FX leading to formation of activated coagulation FX (FXa) in a way that mimics the co-factor activity of coagulation factor VIIIa (FVIIIa). The V _H H polypeptide derivatives as disclosed herein show very high 15 in vitro potency, which, for example, are orders-of-magnitude higher than the bi-specific antibody emicizumab sequence identical analogue (SIA). The V _H H polypeptide derivatives also show prolonged half-life via introduced protractors, e.g. fatty acid conjugations and albumin-binder peptide fusions, in animal models such as rat, dog and pig. Non- protracted V _H H polypeptides show very rapid clearance in dog and pig. Moreover, the 20 VHH polypeptide derivatives have been engineered to enable a clinically relevant bioavailability following peroral administration via pI-lowering amino acid substitutions of surface exposed residues, and formulations using the excipients sodium N-(8-(2- hydroxybenzoyl)amino)caprylate (SNAC) and e.g. nicotinamide (NAM). Thus, upon peroral administration, the formulated and highly potent V _H H polypeptide derivatives show 25 levels of bioavailability in rat and dog animal models that are clinically relevant. Hitherto, therapeutic polypeptides such as ISVD polypeptide derivatives, such as V _H H polypeptide derivatives, having a molecular weight of more than 10 kDa have not been considered suitable for peroral administration. However, the V _H H polypeptide derivates as disclosed herein are suitable for novel oral treatment of coagulopathies, such as – but not limited to 30 – haemophilia A with or without inhibitors. The present invention thus provides procoagulant immunoglobulin single variable domain (ISVD) polypeptides derivatives, such as V _H H polypeptide derivatives, capable of binding coagulation Factor IX(a) and coagulation Factor X(a) which are highly potent and provide a sufficiently long half-life to allow for effective subcutaneous administration as well as 35 peroral administration. Thus, in one aspect the present invention relates to procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivatives comprising a first 6

ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or the activated form thereof, one or more protraction moiety(ies) attached to one or more surface exposed residue(s), optionally a linker (L _1-2 ) linking ISVD1 and ISVD2, and 5 optionally one or more extension(s) (E). Figs.1a-h show non-limiting examples of anti- FX/FIX(a) ISVD polypeptide derivatives. In one aspect the present invention relates to a procoagulant ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID 10 NO:2) or the activated form thereof, at least one protraction moiety attached to a surface exposed residue, optionally a linker (L _1-2 ) linking ISVD1 and ISVD2, and optionally one or more extension(s) (E), wherein said first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO:1) or the activated form thereof comprising at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 15 (consecutive numbering), and wherein said second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO:2) comprising at least one of the amino acid residues N173, P174, F175, L177, and L178 and D179 (consecutive numbering). In another aspect the present invention relates to a procoagulant V _H H polypeptide derivative comprising 20 a first VHH (VHH1) capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second V _H H (V _H H2) capable of binding to Factor X (SEQ ID NO:2), at least one protraction moiety attached to a surface exposed residue, optionally a linker (L _1-2 ) linking V _H H1 and V _H H2, and 25 optionally one or more extension(s) (E), wherein V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171), V _H H-2.18 (SEQ ID NO:155), V _H H-2.15 (SEQ ID NO:131), 30 V _H H-2.13 (SEQ ID NO:115), V _H H-2.14 (SEQ ID NO:123), V _H H-2.12 (SEQ ID NO:107), or V _H H-2.2 (SEQ ID NO:35), and wherein V _H H2 comprises the sequence of 35 V _H H-1.20 (SEQ ID NO:167), V _H H-1.18 (SEQ ID NO:151), 7

V _H H-1.15 (SEQ ID NO:127), V _H H-1.13 (SEQ ID NO:111), V _H H-1.14 (SEQ ID NO:119), V _H H-1.12 (SEQ ID NO:103), 5 V _H H-1.3 (SEQ ID NO:31), or V _H H-1.4 (SEQ ID NO:39). In one such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.20 (SEQ ID NO:167). 10 In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.18 (SEQ ID NO:151). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and VHH2 comprises the sequence of VHH-1.15 (SEQ ID NO:127). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID 15 NO:171) and V _H H2 comprises the sequence of V _H H-1.13 (SEQ ID NO:111). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.12 (SEQ ID NO:103). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.3 (SEQ ID NO:31). 20 In another such embodiment VHH1 comprises the sequence of VHH-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.4 (SEQ ID NO:39). In one such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.20 (SEQ ID NO:167). 25 In another such embodiment V _H H1 comprises the sequence of V _H H-2.18 (SEQ ID NO:155) and V _H H2 comprises the sequence of V _H H-1.18 (SEQ ID NO:151). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.15 (SEQ ID NO:127). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID 30 NO:171) and V _H H2 comprises the sequence of V _H H-1.13 (SEQ ID NO:111). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.12 (SEQ ID NO:103). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.3 (SEQ ID NO:31). 35 In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.4 (SEQ ID NO:39). 8

In another such embodiment V _H H1 comprises the sequence of V _H H-2.15 (SEQ ID NO:131) and V _H H2 comprises the sequence of V _H H-1.18 (SEQ ID NO:151). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID 5 NO:171) and V _H H2 comprises the sequence of V _H H-1.15 (SEQ ID NO:127). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.13 (SEQ ID NO:111). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.12 (SEQ ID NO:103). 10 In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.3 (SEQ ID NO:31). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and VHH2 comprises the sequence of VHH-1.4 (SEQ ID NO:39). 15 In another such embodiment V _H H1 comprises the sequence of V _H H-2.13 (SEQ ID NO:115) and V _H H2 comprises the sequence of V _H H-1.18 (SEQ ID NO:151). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.15 (SEQ ID NO:127). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID 20 NO:171) and VHH2 comprises the sequence of VHH-1.13 (SEQ ID NO:111). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.12 (SEQ ID NO:103). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.3 (SEQ ID NO:31). 25 In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.4 (SEQ ID NO:39). In another such embodiment V _H H1 comprises the sequence of V _H H-2.14 (SEQ ID NO:123) and V _H H2 comprises the sequence of V _H H-1.18 (SEQ ID NO:151). 30 In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.15 (SEQ ID NO:127). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.13 (SEQ ID NO:111). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID 35 NO:171) and V _H H2 comprises the sequence of V _H H-1.12 (SEQ ID NO:103). 9

In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.3 (SEQ ID NO:31). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.4 (SEQ ID NO:39). 5 In another such embodiment V _H H1 comprises the sequence of V _H H-2.12 (SEQ ID NO:107) and V _H H2 comprises the sequence of V _H H-1.18 (SEQ ID NO:151). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.15 (SEQ ID NO:127). 10 In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.13 (SEQ ID NO:111). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and VHH2 comprises the sequence of VHH-1.12 (SEQ ID NO:103). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID 15 NO:171) and V _H H2 comprises the sequence of V _H H-1.3 (SEQ ID NO:31). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.4 (SEQ ID NO:39). In another such embodiment V _H H1 comprises the sequence of V _H H-2.2 (SEQ ID NO:135) 20 and VHH2 comprises the sequence of VHH-1.18 (SEQ ID NO:151). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.15 (SEQ ID NO:127). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.13 (SEQ ID NO:111). 25 In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.12 (SEQ ID NO:103). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID NO:171) and V _H H2 comprises the sequence of V _H H-1.3 (SEQ ID NO:31). In another such embodiment V _H H1 comprises the sequence of V _H H-2.20 (SEQ ID 30 NO:171) and V _H H2 comprises the sequence of V _H H-1.4 (SEQ ID NO:39). 10

In some embodiments the protraction moiety comprises the following structure: w 5 The ISVD polypeptide derivative can for example be a V _H H polypeptide derivative. In another aspect, the present invention relates to pharmaceutical compositions comprising the ISVD polypeptides derivatives as disclosed herein. Another aspect of the invention relates to use of ISVD polypeptides derivatives disclosed herein and compositions comprising such compounds for the treatment of various forms of 10 haemophilia and in particular haemophilia A, haemophilia A with inhibitors and acquired haemophilia A by various routes of administration including but not limited to subcutaneous and peroral administration. In a further aspect the invention relates to the individual component (intermediate) ISVDs or V _H H fragments that are part of an ISVD polypeptide derivative or V _H H polypeptide 15 derivative, such as a particular anti-FIX(a) V _H H fragment or a particular anti-FX(a) V _H H fragment thereof. A further aspect of the invention relates to the manufacture of the components (intermediates) of the compounds as disclosed herein including methods for modifying the isoelectric point of ISVD polypeptide derivatives capable of binding FIX(a) and FX(a) such 20 to improve oral bioavailability of such polypeptide derivatives. DEFINITIONS In order that the present invention may be more readily understood, certain terms are defined below. Greek letters may be represented by their symbol or the corresponding written name, for 25 example: ^ = alpha; ^ = beta; ^ = epsilon; ^ = gamma; ^ = omega; etc. Also, the Greek letter of ^ may be represented by "u", e.g. in ^l=ul, or in ^M=uM. An asterisk (*) in a chemical formula designates a point of attachment. The term "a" or "an" is intended to mean "one or more." The term "comprise" and variations thereof such as "comprises" and "comprising" when preceding the recitation of 11

a step or an element, are intended to mean that the addition of further steps or elements is optional and not excluded. The term "about" is used herein to mean approximately, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by 5 extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by 10 percent, up or down (higher or lower). The term “backbone” as used herein refers to an ISVD polypeptide or V _H H polypeptide amino acid sequence including any L _1-2 linker and extension(s), but excluding a 10 protraction moiety and for the avoidance of doubt excluding any protraction moiety suitable for being fused to the backbone. The term “binding affinity” is a measure of the strength of a non-covalent interaction between two molecules, e.g. an ISVD, such as a VHH fragment and an antigen. The term is used to describe monovalent interactions. Binding affinity between two molecules, e.g. 15 an ISVD, such as a V _H H, through a monovalent interaction may be quantified by determining the equilibrium dissociation constant (K _D ). K _D can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the Surface Plasmon Resonance (SPR) method or the Isothermal Titration Calorimetry (ITC) method. The rate constants corresponding to the association and the dissociation of a monovalent 20 complex are referred to as the association rate constant ka (or kon) and dissociation rate constant k _d (or k _off ), respectively. K _D is related to k _a and k _d through the equation K _D = k _d / k _a . Following the above definition, binding affinities associated with different molecular interactions, such as the binding affinity of different ISVDs for a given antigen, may be 25 compared by comparison of the K _D values for the individual antibody/antigen complexes. The value of the dissociation constant can be determined directly by well-known methods. Standard assays to evaluate the binding ability of ligands such as ISVDs towards targets are known in the art and include, for example, ELISAs, Western blots, RIAs, and flow cytometry analysis. The binding kinetics and binding affinity of the ISVD also can be 30 assessed by standard assays known in the art, such as SPR. A competitive binding assay can be conducted in which the binding of the ISVD, such as a V _H H, to the target is compared to the binding of the target in the presence of another ligand of that target, such as another ISVD. Unless contradicted by context, the K _D is preferably determined by Surface Plasmon 35 Resonance as described herein (see example 7). 12

Preferably, the K _D value of an ISVD capable of binding to FIX(a) is 3 µM or less, such as 15 nM or less, such as 11.7 nM or less. Preferably, the K _D value of an ISVD capable of binding to FX(a) is 3 µM or less, such as 350 nM or less, such as 300 nM or less. 5 A “cross-species reactive” ISVD (or V _H H fragment) binds to e.g. FIX from all indicated species (e.g. human and cynomolgus monkey) with comparable affinity, in particular with a K _D in the range of a factor of 100, such as within a range of a factor of 50, within a range of a factor of 20, or within a range of a factor of 10. Within a K _D range of a defined factor X means that the highest affinity for a particular listed species is not more than X-times 10 higher than the lowest affinity measured for binding to a different listed species. A person skilled in the art will understand that any method for measuring affinity can be used to verify that a cross-species reactive ISVD binds to the target antigen from all listed species within a given KD factor range as described herein as long as the same conditions are applied to the K _D measurement for all listed species. Preferably, the K _D values are 15 measured using SPR, in particular at 25 °C. Preferably, the affinities are measured using the cross-species reactive ISVD, such as an V _H H. Amino acids are molecules containing an amine group and a carboxylic acid group, and, optionally, one or more additional groups, often referred to as a side chain. 20 The term "amino acid" includes canonical amino acids (which are genetically encoded), and unnatural amino acids. Non-limiting examples of unnatural amino acids are Aib ( ^- aminoisobutyric acid), deamino histidine (alternative name 3-(imidazol-4-yl)propanoic acid, abbreviated Imp (imidazopropionyl) and the d-isomers of the canonical amino acids. All amino acid residues within the polypeptide for which the optical isomer is not stated is 25 herein to be understood to mean the l-isomer, unless otherwise specified. The term “antibody” herein refers to a protein, comprising or derived from an immunoglobulin sequence, which is capable of binding to an antigen or a portion thereof An “antibody” includes - but is not limited to - full-length antibodies comprising at least four 30 polypeptide chains: two heavy chains (HC) and two light chains (LC) that are connected by disulphide bonds as well as antibodies comprising at least three polypeptide chains: two heavy chains (HC) and one light chain (LC) that are connected by disulphide bonds. One class of immunoglobulins is the IgGs. In humans, the IgG class may be divided into four sub-classes IgG1, IgG2, IgG3 and IgG4, based on the sequence of their heavy chain 35 constant regions. The light chains can be divided into two types, kappa and lambda chains, based on differences in their sequence composition. IgG molecules are composed 13

of two heavy chains, interlinked by two or more disulphide bonds, and two light chains, each attached to a heavy chain by a disulphide bond. The term “antibody” also encompasses single-domain antibodies such as V _H H fragments and V-NAR fragments. 5 The term “hypervariable region” as used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises the “complementarity determining regions” also known as the “CDRs”. “Framework” or “FR” regions are those variable domain regions other than the hypervariable region residues. Thus, an antibody or ISVD comprises from N- to C- 10 terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR3 of the heavy chain is the region which typically contributes most to antigen binding. The term “epitope” as used herein means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface elements of molecules, such 15 as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing agents capable of disrupting the structure of the protein. 20 In the context of an X-ray derived crystal structure defined by spatial coordinates of a complex between an ISVD, a V _H H fragment, and its target, the term epitope is herein, unless otherwise specified or contradicted by context, specifically defined as ISVD residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4 Å from a heavy atom in FIX/FIXa or FX/FXa. 25 The epitope for a given ISVD/antigen pair may be identified by routine methods, such as those described in the examples. For example, the ISVD and antigen may be combined and the ISVD/antigen complex may be crystallised. The crystal structure of the complex may be determined and used to identify specific sites of interaction between the ISVD and its antigen. 30 In one embodiment an ISVD polypeptide derivative as described herein comprises a first ISVD capable of binding to an epitope on FIX or the activated form thereof comprising at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 (SEQ ID NO:1) and a second ISVD is capable of binding to an epitope on FX (SEQ ID NO:2) comprising at least one of the amino acid residues N173, P174, F175, 35 L177 and L178 (consecutive numbering). 14

In one embodiment the ISVD polypeptide derivative comprises a first ISVD capable of binding to an epitope on Factor IX (SEQ ID NO:1) or the activated form thereof comprising at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 (consecutive numbering), and 5 a second ISVD capable of binding to an epitope on Factor X (SEQ ID NO:2) comprising at least one of the amino acid residues N173, P174, F175, L177, L178 (consecutive numbering) or comprising at least one of the amino acid residues N173, P174, F175, L177, L178 and D179. 10 Coagulation Factor IX (FIX) is a vitamin K-dependent coagulation factor with structural similarities to Factor VII, prothrombin, Factor X, and Protein C. FIX circulates in plasma as a single-chain zymogen (SEQ ID NO:1). The circulating zymogen form consists of 415 amino acids divided into four distinct domains comprising an N-terminal γ- carboxyglutamic acid-rich (Gla) domain, two EGF domains and a C-terminal trypsin-like 15 serine protease domain. Activation of FIX occurs by limited proteolysis at Arg145 and Arg180 to release the activation peptide (residues 146 to 180 of SEQ ID NO:1). Thus, activated FIX (FIXa) is composed of residues 1-145 of SEQ ID NO:1 (light chain) and residues 181-415 of SEQ ID NO:1 (heavy chain). Circulating FIX molecules thus comprise the FIX zymogen and the activated form of FIX 20 which are herein generally referred to as FIX and FIXa with reference to SEQ ID NO:1. Activated Factor IX is referred to as Factor IXa or FIXa. The term “FIX (SEQ ID NO:1) and/or the activated form thereof (FIXa)” may also be referred to as “FIX/FIXa” or simply “FIX(a)”. FIXa is a trypsin-like serine protease that serves a key role in haemostasis by generating, 25 as part of the tenase complex, most of the Factor Xa required to support proper thrombin formation during coagulation. FIX is herein represented by SEQ ID NO:1 corresponding to the Ala148 allelic form of human FIX (Anson et al. EMBO J.19843:1053-1060; McGraw et al., Proc Natl Acad Sci USA.198582:2847-2851; Graham et al. Am. J. Hum. Genet.198842:573-580). In the 30 present invention FIX is intended to cover all natural variants of FIX, such as the T148 variant (Uniprot ID P00740). FX is a vitamin K-dependent coagulation factor with structural similarities to Factor VII, prothrombin, FIX, and protein C. FX circulates in plasma as a two-chain zymogen 35 including residues 1-139 of SEQ ID NO:2 (light chain) and residues 143-448 of SEQ ID NO:2 (heavy chain). Human FX zymogen comprises four distinct domains comprising an 15

N-terminal gamma-carboxyglutamic acid rich (Gla) domain (residues 1-45), two EGF domains, EGF1 (residues 46-82) and EGF2 (residues 85-125), respectively, and a C- terminal trypsin-like serine protease domain (residues 195-448). Activation of FX occurs by limited proteolysis at Arg194, which results in the release of the activation peptide 5 (residues 143-194). Thus, activated FX (FXa) is composed of residues 1-139 of SEQ ID NO:2 (light chain) and residues 195-448 of SEQ ID NO:2 (activated heavy chain). Circulating Factor X molecules thus comprises the FX zymogen and the activated form of FX which are herein referred to as FX and FXa, respectively, with reference to SEQ ID NO:2. In the present invention FX is intended to cover all natural variants of FX. The term 10 “FX (SEQ ID NO:2) and/or the activated form thereof (FXa)” may also be referred to as “FX/FXa” or “FX(a)”. The term “conservative substitution” as used herein refers to the case where an amino acid may be substituted to an amino acid with similar biochemical properties, for example, 15 a basic amino acid may be substituted to another basic amino acid (e.g. lysine to arginine), an acidic amino acid may be substituted to another acidic amino acid (e.g glutamate to aspartate), a neutral amino acid may be substituted to another neutral amino acid (e.g. threonine to serine), a charged amino acid may be substituted to another charged amino acid (e.g. glutamate to aspartate), a hydrophilic amino acid may be 20 substituted to another hydrophilic amino acid (e.g. asparagine to glutamine), a hydrophobic amino acid may be substituted to another hydrophobic amino acid (e.g. alanine to valine), a polar amino acid may be substituted to another polar amino acid (e.g. serine to threonine), an aromatic amino acid may be substituted to another aromatic amino acid (e.g. phenylalanine to tryptophan) and an aliphatic amino acid may be 25 substituted to another aliphatic amino acid (e.g. leucine to isoleucine). The term "excipient" as used herein refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, 30 various sugars and types of starch, L-arginine, nicotinamide, SNAC, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. The term “extension” (E) as used herein refers to a peptide or polypeptide suitable for being attached to an ISVD polypeptide. 35 The extension can be of a few amino acids, 1 to 10 amino acids, it can be longer, 10 to 30 amino acids, or it can be very long, more than 30 amino acids. An extension is preferably 16

present in the N-terminal end (N-terminal extension) or in the C-terminal end (C-terminal extension), or both, of an ISVD polypeptide, ISVD polypeptide derivative, V _H H polypeptide or V _H H polypeptide derivative. Alternatively, the extension is attached elsewhere in the ISVD polypeptide outside the CDR sequences, such as in one or more framework regions 5 or in a linker L _1-2 . The extension(s) is/are preferably recombinantly fused to the ISVD polypeptide. In other embodiments the extension(s) is/are conjugated to the ISVD polypeptide. Non-limiting examples include cpmd #22 which has a 6-amino acid C-terminal extension composed of GQACPC (SEQ ID NO:9), cpmd #6 which has a 13-amino acid C-terminal10 extension composed of GGGGCSCHHHHHH (SEQ ID NO:8), and an 11-amino acid C- terminal extension composed of GGGGSHHHHHH (SEQ ID NO:7). The purpose of the extension is to provide a point of attachment for protraction moieties and/or to provide a means for purification. Thus, the term extension does not encompass protractors and protraction moieties. 15 For example, a protractor including any protractor linker (L _P ) recombinantly fused to the N- or C-terminal end of the polypeptide is not regarded as an extension. The term “fusion” as used herein refers to in-frame joining of two or more DNA sequences which originally encode separate proteins or peptides or fragments hereof. Translation of 20 the fusion polypeptide DNA sequence will result in a single polypeptide sequence which may have functional properties derived from each of the original proteins or peptides. DNA sequences encoding fusion proteins may be created artificially by standard molecular biology methods such as overlapping PCR or DNA ligation. The resulting fusion polypeptides DNA sequence may be inserted into an appropriate expression vector that 25 supports the heterologous fusion protein expression in host organisms such as bacteria, yeast, fungus, insect cells or mammalian cells. Protraction moieties may for example be fused to the C-terminal or N-terminal of an ISVD polypeptide or V _H H polypeptide backbone. The term "host cell" as used herein covers any kind of cellular system which can be 30 engineered to generate the ISVDs disclosed herein. Host cells include – but is not limited to – cultured cells, e.g., mammalian cultured cells, such as CHO cells, HEK293T cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, fungal cells, and insect cells. 35 The term “identity” as known in the art, refers to a relationship between the sequences of two or more polypeptides, as determined by comparing the sequences. In the art, 17

"identity" also means the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical 5 model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. In the present invention similarity and identity were determined using Needleman (Needleman et al. J. Mol. Biol.1970; 48:443-453) from EMBOSS-6.6.0 using the parameters 10 and 0.5 for gaps opening and extensions, respectively (gapopen=10, gapextend=0.5). 10 The term "Immunoglobulin Single Variable Domain" or "ISVD" is used as a general term to include antigen-binding domains or fragments such as VH and VL domains, respectively. The ISVD can thus be a light chain variable domain sequence (e.g. a VL-sequence), or heavy chain variable domain sequence (e.g. a VH-sequence); for example a heavy chain 15 variable domain sequence that is derived from a conventional four-chain antibody or a light chain variable domain sequence that is derived from a conventional four-chain antibody. A particular type of ISVD is a V _H H fragment which was originally identified as a type of immunoglobulin defined as an antibody fragment consisting of a single monomeric 20 variable antibody domain. A VHH fragment is a single-domain antibody that encompasses the antigen-binding variable region of heavy-chain-only antibodies, which can be obtained from camelids. V _H H fragments have a size of around 15 kDa. They contain a single chain molecule that can bind its cognate antigen using a single domain. The antigen-binding surfaces of V _H H fragments are usually more convex (or protruding) than those of 25 conventional antibodies, which are usually flat or concave. V _H H fragments are composed of four Framework Regions (or FRs) whose sequences and structures are defined as conserved, and three Complementarity Determining Regions (or CDRs) showing high variability both in sequence content and structure conformation, which are involved in antigen binding and provide antigen specificity. 30 Another type of ISVD can be obtained from IgNARs of cartilaginous fish and a single- domain antibody thereof is designated a “V-NAR fragment”. V _H H- and V-NAR fragments do not comprise constant domains and therefore have no Fc- region which is typically part of partial/full-length and/or engineered/natural heavy-chain- 35 only antibodies [Dooley et al. (2006) Dev. Comp. Immunol.30:43–56; Muyldermans S. (2013) Annu Rev Biochem.82:775-97]. 18

A general description of camelid V _H H fragments and methods for their production and/or isolation and/or use is found inter alia in the following references WO94/04678 and WO97/49805. A general description of heavy chain immunoglobulins from cartilaginous fish and the variable regions thereof and methods for their production and/or isolation 5 and/or use is found inter alia in WO2005/118629. The total number of amino acid residues in a V _H H fragment is typically in the range of 110-140. It should, however, be noted that parts, fragments, or analogues of a V _H H are not particularly limited as to their length and/or size, if such parts, fragments, or analogues 10 meet the further requirements outlined hereinbelow and are also preferably suitable for the purposes described herein. The molecular weight of a V _H H fragment is typically in the range 12-15 kDa. pI for V _H H fragments is generally basic, as antibodies generally are, meaning that pI values are generally above 7, often between 7.5 and 8.5. A VHH fragment usually encompasses at least one disulphide bridge typically formed by conserved 15 cysteine pairs positioned in framework regions 1 and 3. Such disulphide bridge(s) ensure(s) correct folding and stability of the V _H H fragment, and it is desirable to retain such disulphide bridge(s), if, for example, side-chain modifications and/or conjugations is conducted in such a way that it targets an introduced unpaired cysteine(s) in a V _H H polypeptide. 20 In the present document, CDR sequences of ISVDs such as V _H H fragments are determined using the Kabat definition (Kontermann and Dübel, 2010, Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp.33-51). According to this method, the CDRs of the variable domain is defined as position 31-35 25 (CDR1), position 50-65 (CDR2) and position 95-102 (CDR3). However, when referring to specific amino acid residue positions in the polypeptide compounds described herein including CDRs and framework (FR) regions in the ISVD polypeptides or V _H H polypeptides consecutive numbering is used unless otherwise stated. 30 The term “ISVD polypeptide” as used herein refers to a polypeptide comprising two or more ISVDs, such as a first ISVD (ISVD1) and a second ISVD (ISVD2), e.g. connected by a linker (L _1-2 ) of any appropriate composition and length as required or without any linker at all as a direct domain fusion. 35 The term "linker" as used herein refers to at least one atom that forms a covalent bond between chemical entities. If the chemical entities are linked solely through peptide 19

bonds, the linker can be referred to as a "peptide linker". Otherwise, the linker can be referred to as a "chemical linker". An example of an ISVD polypeptide is two ISVDs connected via a linker (L _1-2 ). Another example of an ISVD polypeptide is two ISVDs connected via a linker (L _1-2 ) further 5 comprising one or more extensions. Another example of an ISVD polypeptide is two ISVDs connected without a linker (L _1-2 ) and further comprising one or more extensions, such as – but not limited to – a C-terminal and/or N-terminal extension. The linker (L _1-2 ) may for example be composed of an amino acid sequence, comprising no 10 or multiple repeats. For example, a linker may comprise 2 to 50 amino acids, 5 to 40 amino acids, or 10 to 30 amino acids. Non-limiting examples of linkers include *-GGGGS-* linker, *-GQAPGQ-* linker (SEQ ID NO:20), *-QAPGQA-* linker (SEQ ID NO:16), *-GI-* linker, *-GV-* linker, *-GT-* linker, *- 15 GL-* linker, or another amino acid composite linker. Two examples of linkers are x2 and x6 repeat composites of GGGGS, being 10 and 30 amino acid residues in length, respectively. For the avoidance of doubt, the subscript _1-2 in L _1-2 does not imply a particular direction of ISVDs being linked, i.e. a L _1-2 can - for example - link ISVD1 to ISVD2, or ISVD2 to ISVD1 20 (N- to C-terminal). SEQ ID NOs:14-24 represent non-limiting examples of L _1-2 linkers. In some embodiment the ISVD polypeptide comprises an extension (E) as outlined in any of the below formulas. 25 ISVD1 – ISVD2, or ISVD1 – ISVD2 – E, or ISVD2 – ISVD1 – E, or E - ISVD1 – ISVD2, or ISVD1 – L _1-2 – ISVD2 – E, or 30 ISVD2 – L _1-2 – ISVD1 – E, or E - ISVD1 – L _1-2 – ISVD2, The extension (E) may for example be attached to an ISVD polypeptide as outlined in any of the below formulas (N- to C-terminal): 35 ISVD1 – ISVD2, or ISVD1 – ISVD2 – E, or 20

ISVD2 – ISVD1 – E, or E - ISVD1 – ISVD2, or ISVD1 – L _1-2 – ISVD2 – E, or ISVD2 – L _1-2 – ISVD1 – E, or 5 E - ISVD1 – L _1-2 – ISVD2 An ISVD polypeptide preferably comprises a protraction moiety. In such cases the ISVD polypeptide is referred-to as an ISVD polypeptide derivative. In some embodiments the first ISVD serves as attachment point for one or more 10 protraction moieties. In some embodiments the second ISVD serves as attachment point for one or more protraction moieties. In some embodiments the L1-2 linker serves as attachment point for one or more protraction moieties. 15 For the avoidance of doubt, when a protraction moiety includes a linker (L _p ) attached to an extension (E) to an ISVD1-L _1-2 -ISVD2, the linker L _P is not considered part of the “extension”. In some aspects, ISVD polypeptides are chemically conjugated with a non-ISVD, for 20 example a small chemical non-polypeptide molecule, a carbohydrate, a fatty acid or oligopeptide or polypeptide or a protein, such as an antibody or preferably an antibody fragment. In one embodiment an ISVD polypeptide is connected to an Fc domain from an IgG antibody without use of a linker. 25 In another embodiment an ISVD polypeptide is connected to an Fc domain or fragment thereof from an IgG antibody by a linker. In preferred embodiments the extension(s) is/are fused to the ISVD polypeptide and thus not connected to the ISVD polypeptide by way of chemical conjugation. 30 In some embodiments the protraction moiety(ies) is/are fused to the ISVD polypeptide and thus not connected to the ISVD polypeptide by way of chemical conjugation. The term “isoelectric point” or “pI” as used herein refers to the pH value where the overall 35 net charge of a protein - such as an antibody - is zero. In proteins there may be many charged groups, and at the isoelectric point the sum of all these charges is zero. At a pH 21

above the isoelectric point the overall net charge of the protein will be negative, whereas at pH values below the isoelectric point the overall net charge of the protein will be positive. The pI may be either a theoretical or an experimentally determined isoelectric point. 5 The skilled person is aware of methods to determine the isoelectric point of a protein. Most commonly, the isoelectric point of a protein is computed based on the amino acid sequence of the protein. Numerous (online) tools allowing the determination of the isoelectric point of a protein are available, such as "ExPASy Compute pI/Mw"; see Protein Identification and Analysis Tools on the ExPASy Server; Gasteiger E., Hoogland C., 10 Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A.; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005), pp.571-607. Preferably, the algorithm of Skoog & Wichman, 1986. pKa of amino acid residues is used for calculating pI. The pl can also be determined experimentally and charge variants can, for example, be 15 separated using charged based-separation techniques such as isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis. In one embodiment the first and second ISVD in an ISVD polypeptide are V-NAR fragments and such compounds are designated “V-NAR polypeptides”. In another embodiment the first and second ISVD in an ISVD polypeptide are V _H H 20 fragments and such compounds are designated “VHH polypeptides”. In one such embodiments the first V _H H fragment is capable of binding FIX/FIXa and the second V _H H fragment is capable of binding FX/FXa. In a preferred embodiment the V _H H polypeptide is a bispecific V _H H polypeptide. For the avoidance of doubt, the terms multi-, tri- or bispecific are intended to reflect the 25 number of antigens bound by the ISVDs, such as V _H H fragments, i.e. not including molecules bound by a protraction moiety (if present), such as – but not limited - to albumin. In one embodiment the molecular weight of a V _H H polypeptide without a protraction moiety is in the range 27 to 29 kDa. 30 In preferred embodiments the molecular weight of a V _H H polypeptide including one or more protraction moiety(ies) (a V _H H polypeptide derivative) and optionally one or more an extension is in the range 28 to 33 kDa. A person skilled in the art will appreciate that the above embodiments are similarly applicable to V-NAR polypeptides. 35 22

The term “free cysteine” as used herein is a cysteine residue in a polypeptide chain that is available for reaction e.g. chemical conjugation and, thus, not part of a natural or an engineered internal disulphide bridge. In essence, free cysteines can be used for conjugation, albeit free cysteine residues, including recombinantly introduced free 5 cysteines, are often blocked with small thiols, such as cysteine, homocysteine, or glutathione, during recombinant expression of polypeptides in host cells. This is also observed in the recombinant production of ISVD polypeptides wherein one or more free cysteine(s) has/have been introduced. Thus, a reduction reaction using a proper reducing agent, such as bis(p-sulfonatophenyl)phenylphosphine dihydrate or tris(2- 10 carboxyethyl)phosphine hydrochloride, can be used to liberate and prepare the free cysteine for conjugation to a moiety of interest, such as – but not limited to - a protraction moiety, such as a C18 diacid gamma-Glu 2xOEG fatty acid moiety. The ISVD polypeptides, such as a V _H H polypeptide, as disclosed herein can be 15 multispecific such as – but not limited to - bispecific or trispecific. The terms “bispecific ISVD polypeptide” or “bispecific V _H H polypeptide” as used herein, refers to an ISVD polypeptide or a V _H H polypeptide, respectively, which is capable of binding to two different antigens or two different epitopes on the same antigen. The terms “trispecific ISVD polypeptide” or “trispecific V _H H polypeptide” as used herein, 20 refers to a ISVD polypeptide or a VHH polypeptide, respectively, which is capable of binding to three different antigens or three different epitopes on the same antigen or three different epitopes present on two different antigens. The terms “multispecific ISVD polypeptide” or “multispecific V _H H polypeptide” as used herein, refers to a ISVD polypeptide or a V _H H polypeptide, respectively, which is capable 25 of binding to two or more different antigens or two or more different epitopes on the same antigen. Multispecific ISVD polypeptides or multispecific V _H H polypeptides thus comprise bi- and trispecific ISVD polypeptides or V _H H polypeptides, respectively. A person skilled in the art will appreciate that the above also applies to polypeptide derivatives (i.e. including a protraction moiety). 30 The term "oral bioavailability" or "peroral bioavailability" as used herein refers to the amount of administered drug in systemic circulation after peroral administration (estimated as the area under the plasma concentration of the administrated drug versus time curve) relative to the amount of administered drug in systemic circulation after 35 intravenous administration of said drug. 23

The term “paratope” as used here in refers to the area or region on the ISVD to which an antigen specifically binds, i.e. to which it makes physical contact to the antigen. In the context of an X-ray derived crystal structure defined by spatial coordinates of a complex between an ISVD, a V _H H fragment, and its target, the term paratope is herein, 5 unless otherwise specified or contradicted by context, specifically defined as ISVD residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4 Å from a heavy atom in FIX/FIXa or FX/FXa. The paratope (and epitope) for a given ISVD/antigen pair may be identified by routine methods, such as those described in the examples. For example, the ISVD and antigen 10 may be combined and the ISVD/antigen complex may be crystallised. The crystal structure of the complex may be determined and used to identify specific sites of interaction between the ISVD and its antigen. The term "pharmaceutically acceptable excipient" means an excipient that is useful in 15 preparing a pharmaceutical composition that is generally safe, non-toxic and includes excipients that are acceptable for human pharmaceutical use. Such excipients can for example be solid, liquid or semisolid. The term "plasma half-life" as used herein refers to the time required for half the quantity 20 of a substance administered to a patient to be metabolized or eliminated from the serum or plasma of the patient by normal biological processes. The term "preferentially binds" as used herein shall be taken to mean that a binding region on the ISVD, such as a V _H H fragment, binds to one component (e.g. activated FIX) 25 in preference to, or in favour of, another component. As such, "preferential binding" does not necessarily require exclusive binding or non-detectable binding of the other component. For example, an anti-FIX(a) ISVD, such as an anti-FIX(a) V _H H fragment, may preferentially bind to activated FIX as compared to non-activated FIX. 30 The term “procoagulant antibody” refers to an antibody which potentiates blood coagulation for example by accelerating the process of blood coagulation and/or increasing the enzymatic activity of one or more coagulation factors. The term “procoagulant activity” as used herein refers to the ability of a compound, such 35 as an antibody, to potentiate blood coagulation for example by accelerating the process of blood coagulation and/or increasing the enzymatic activity of one or more coagulation 24

factors. Accordingly, the term "procoagulant activity" encompasses (but is not limited to) one or more of the activities listed below: Enhancing Factor IXa-mediated Factor X activation, as measured by an amidolytic (chromogenic or fluorogenic) assay based on FIXa-mediated FX activation. The assay 5 measures FXa through cleavage of an FXa specific peptide substrate. The substrate is produced, giving a colour that can be measured photometrically by absorbance. Shortening of clotting times, as measured by clotting assays such as Activated partial thromboplastin time (APTT) measure the activity of the intrinsic and common pathways of coagulation. Plasma is preincubated with an APTT reagent containing a contact activator, 10 e.g. ellagic acid or kaolin, and phospholipid. Calcium chloride is added to promote fibrin clot formation. Possible readouts are clotting time or clot wave form. Enhancing thrombin generation, as measured in a thrombin generation assay such as Calibrated Automated Thrombography (CAT). The thrombogram describes the concentration of thrombin in clotting plasma and is therefore a functional test of the 15 hemostatic system. The assay is based on the measurement of fluorescence that is generated by the cleavage of the fluorogenic substrate Z G G R AMC by thrombin over time. See also the method as used in Example 8 herein. Enhancing global visco-elastic properties of clot formation as measured by viscoelastic hemostatic methods e.g. in whole blood under shear stress by assays such as ROTEM 20 (Rotational thromboelastometry). In the instrument, a ball-bearing pin rotates in a stationary cup. The fibrin strands in the sample form between the wall of the cup and the pin during coagulation and the strength of the strands will affect the movement of the pin, which is detected. Shortening of the whole blood closure time (WBCT), as measured by a platelet function 25 analyzer, which is based on Von Willebrand Factor (VWF)-mediated platelet adhesion to collagen after platelet activation. High shear stress is produced which leads to platelet adhesion and platelet aggregation. The time from the beginning to end of blood flow is measured. 30 The ISVD polypeptides disclosed herein preferably comprises one, two, three or four “protraction moieties” (PM). More preferably one or two protraction moieties. The term “protraction moiety” as used herein refers to a moiety having half-life extending properties and comprising a “protractor” (P) and an optional “linker” (L _P ). The term ”protraction” thus refers to half-life extension and a protractor or protraction moiety thus 35 serves the purpose of the extending half-life of the ISVD polypeptides disclosed herein. A protraction moiety (PM) comprises a protractor “P” and an optional linker (L _P ). 25

Each protraction moiety preferably attaches to a surface exposed lysine or a cysteine residue in the polypeptide backbone of the compound. The attachment point is generally referred to as R1 (and in case of attachment of more than one protraction moiety R2, R3 and so forth wherein R1 ≠ R2 ≠ R3 and so forth). 5 The skilled person will be able to identify other surface exposed residue(s) suitable for attachment. A protraction moiety may consist of one protractor. A protraction moiety may comprise one linker (L _P ) and one protractor (P). A protraction moiety may comprise one linker and two or more protractors. 10 When the linker (L _P ) is present, the protraction moiety attaches to the ISVD polypeptide backbone via L _P . When the linker (L _P ) is absent, P attaches to the polypeptide backbone. In one embodiment a first and a second protraction moiety are present wherein said first protraction moiety comprises the structure: 15 and wherein said second protraction moiety comprises the structure 20 wherein ‘*’ (R1) represents the point of attachment on the ISVD polypeptide for the first protraction moiety, and wherein ‘**’ (R2) represents the point of attachment on the ISVD polypeptide for the second protraction moiety, wherein ‘*’ is Cys in position 257 of SEQ ID NO:629, and 25 wherein ‘**’ is Cys in position 259 of SEQ ID NO:629. 26

The ISVD polypeptide disclosed herein may comprise a single cysteine or lysine residue, to which a single protraction moiety is attached/conjugated. The ISVD polypeptide disclosed herein may comprise two lysine residues and two protraction moieties. 5 The ISVD polypeptide disclosed herein may comprise two lysine residues and two identical protraction moieties. The ISVD polypeptide disclosed herein may comprise two lysine residues and two non- identical protraction moieties. 10 The ISVD polypeptide disclosed herein may comprise three lysine residues and three protraction moieties. The ISVD polypeptide disclosed herein may comprise three lysine residues and three identical protraction moieties. The ISVD polypeptide disclosed herein may comprise three lysine residues and three 15 non-identical protraction moieties (for example, the first and second protraction moieties may be identical and the third protraction moiety different form said first and second moieties) The ISVD polypeptide disclosed herein may comprise two cysteine residues and two protraction moieties. 20 The ISVD polypeptide disclosed herein may comprise two cysteine residues and two identical protraction moieties. The ISVD polypeptide disclosed herein may comprise two cysteine residues and two non- identical protraction moieties. The ISVD polypeptide disclosed herein may comprise three cysteine residues and three 25 protraction moieties. The ISVD polypeptide disclosed herein may comprise three cysteine residues and three identical protraction moieties. The ISVD polypeptide disclosed herein may comprise three cysteine residues and three non-identical protraction moieties (for example, the first and second protraction moieties 30 may be identical and the third protraction moiety different form said first and second moieties). Where the ISVD polypeptide comprises two or three protraction moieties, the protracting moieties are preferably similar, more preferably substantially identical, or, most preferably, 35 identical. 27

In the context of chemical moieties such as the protraction moieties disclosed herein, similarity and/or identity may be determined using any suitable computer program and/or algorithm known in the art. 5 Compounds comprising a protraction moiety may be referred as “derivatives”. For example, an “ISVD polypeptide derivative” is understood to be an ISVD polypeptide comprising a protraction moiety, a “V _H H polypeptide derivative” a V _H H polypeptide comprising a protraction moiety, and a “V-NAR polypeptide derivative” a V-NAR polypeptide comprising a protraction moiety. 10 The protraction moiety may be capable of non-covalently binding to albumin, thereby promoting the circulation of the ISVD polypeptide derivative in the blood stream and prolonging its half-life. Thus, in one embodiment the protraction moiety is an albumin binding moiety. 15 The protractor (P) may comprise an acyl group. The acyl group may be branched or unbranched. The acyl group may be saturated or unsaturated. The protractor may comprise a fatty acyl group. The acyl group may be branched or unbranched. The acyl group may be saturated or unsaturated. 20 The protractor may comprise a distal carboxylic acid group. The protractor may comprise a fatty acid group. The protractor may comprise a fatty acid group and an amide group. The protractor may comprise a distal carboxylic acid group and an amide group. The protractor may comprise an alkyl group. 25 The protractor may comprise an aryl group. The protractor may comprise a tetrazole group. The protractor may comprise a sulfonic acid group. The protractor may comprise a phenoxy group. The protractor may comprise a benzoic acid group. 30 The protractor may comprise 8-30 carbon atoms. The protractor may comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms. The protractor may comprise 6-30 consecutive -CH ₂ - groups. The protractor may 35 comprise a carbon chain comprising at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive -CH ₂ - groups. 28

The protractor may comprise 12-26 carbon atoms. The “protractor” may comprise 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 carbon atoms. The protractor may comprise 10-26 consecutive -CH ₂ - groups. The protractor may comprise a carbon chain comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 5 24, 25 or 26 consecutive -CH ₂ - groups. The protractor may comprise 16-22 carbon atoms. The protractor may comprise 14-20 consecutive -CH ₂ - groups. The protractor may comprise a carbon chain comprising 14, 15, 16, 17, 18, 19 or 20 consecutive -CH ₂ - groups. 10 The protractor may comprise 16-22 consecutive carbon atoms and 14-20 consecutive - CH ₂ - groups. The protractor may comprise 16 consecutive carbon atoms and 14 consecutive -CH ₂ - groups. The protractor may comprise 18 consecutive carbon atoms and 16 consecutive -CH ₂ - 15 groups. The protractor may comprise 20 consecutive carbon atoms and 18 consecutive -CH ₂ - groups. The protractor may comprise 22 consecutive carbon atoms and 20 consecutive -CH ₂ - groups. 20 In some embodiments the protractor comprises a group defined by: Chem. a: HOOC-(CH ₂ ) _n -CO-* wherein n is an integer in the range of 8-30, which may also be referred to as a C(n+2) diacid or as Chem. a1: , wherein n is an integer in the range of 8-30. 25 The protractor may comprise an oligopeptide. In one embodiment, the protractor oligopeptide is 10 to 40 amino acids, such as 10 to 30 amino acids, such as 15 to 25 amino acids, and, preferably, 20 amino acids. The protractor oligopeptide sequence composition may for example be QRLMEDICLPRWGCLWEDDF (SEQ ID NO:736), exemplified as fusion sequence in SEQ ID NO:3 and 4 [see also WO01/45746 A2]. Cmpd 30 #71 – for example - comprises a 50-amino acid N-terminal protraction moiety composed of QRLMEDICLPRWGCLWEDDFGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4) wherein residues 1-20 is the protractor (P) and residues 21-50 is the linker (L _P ). 29

The protractor may be attached to the ISVD or ISVD polypeptide via an amino acid linker L _P . In one embodiment L _P may join the protractor (P) to the side chain of a lysine or cysteine residue in the ISVD polypeptide backbone. 5 The ISVD polypeptide derivative may comprise two protraction moieties, each of which comprises 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The ISVD polypeptide may comprise two protraction moieties, wherein each protractor (P) comprises 12, 13, 14, 15, 16, 17 or 18 consecutive -CH ₂ - groups. The ISVD polypeptide derivative may comprise two C14 diacids, two C16 diacids or two 10 C18 diacids. The ISVD polypeptide derivative may comprise three protraction moieties, each of which comprises a protractor comprising 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The ISVD polypeptide may comprise three protraction moieties, wherein the protractor comprises 10, 11, 1213, 14, 15, 16, 17 or 18 consecutive -CH ₂ - groups. 15 For the avoidance of doubt a protraction moiety even if capable of binding e.g. albumin, it is not considered a binder in terms of defining whether an ISVD polypeptide as disclosed herein is bi-, tri- or multispecific. In some embodiments an ISVD polypeptide as disclosed herein may comprise a protractor which is selected from any one of those depicted in Table 1. 20 In Table 2, LP represents the optional linker which connects the depicted protractor (P) to the ISVD polypeptide backbone. R1 represents the site of attachment in the ISVD polypeptide. 30

_, ⁷ ₁ ^# _{, ,} ^{0 6} _{4 1} ^{# #} _, ⁸ e ^h t s _t ⁿ _{e e} ^{r s} _{u e} ^{t r} _{c p} ^{u e} _{rt r} ^’ S * _‘ ⁽ ₎ P _{( s} ^r _o ^t _c ^a r _t ^o _r ^p _d ⁱ f _{c d} ⁱ _d ^{i o a} _{c i} ^a _{c i} ^a _i s ^{d d d} e ^{l 2} p ₁ ⁶ C ₁ ⁸ C ₁ C m ^a _x E ^{r :} _o ^t 1 _{c e} ^{a l} _{rt b} ^{o a} _r P ^D _I ¹ P ^{2 3 1} _{T P P 0} O W ²

o ^a r ^{p e 3} _{: o d} O ^g _e ^p i _l ⁿ _{i o} r ⁿ _e ^g _e i _l ⁿ _i ^r i ^{N o s o} _e b _{r -} ^D I ^e _{- r} ^s - _d ^{e e} _d ^{e n} _i m ^{Q n} _{i n} ^{i n} _n ^{i u} _E ^b _{c -} ^y _{i l} ^b _c ^{y g} _{- l b} ^{l S n} _i ^{n g g} _{i A} ⁿ _i m _n m ^g _{n F e} ^u _b ^o l ^{u o} l D ^{c l} _b D _{n E} ^e _{a u e} ^d _i ^{l h c} _a ^d _i ^c t _{a e} ^h _a W ^q L _e ^s _o t n ^s _o C ^s G ⁿ _{e o} ^b _{i e} ⁿ _i i _i ^r _c ^m _a ^b _i ^r _c ^m _a W _s ^{u s} _{e 0} ^{1 s} ₀ R P _f ^d L _s ^{a 3} _: ^a _e ^{d 3 a h 4} _{: d} ^{i C} _I ^d c _e ⁱ O _ti ^h _ti N ^w O ^{w a e} i _l d ^{o D} z _{E fi} ^{l D r} _e ^{N r} _e 0 ^a _p I 2 _rt ^M _L ^{m h} _t ^D I ^h R _e Q ^e _{t g} Q ^e _g C ^e _T Q ^x _e ^E S ^o _t ^E S ^o _{t 4} ^{5 1 P} _P ⁶ P ₀ O W ²

In one embodiment the protractor is conjugated directly onto the ISVD polypeptide, i.e. without use of a linker L _P (i.e. by way of a covalent bond). In other embodiments the protractor is conjugated directly onto the ISVD polypeptide using a linker L _P . Non-limiting examples are provided below. 5 The L _P linker - when present - may comprise Ado, Aeep or Aeeep, sulfonamide, Trx, ε- Lys, Ahx, Glu, γGlu, Gly, Ser, Ala and/or Thr. The L _P linker may comprise at least a moiety which may be represented by the following chemical formula: 10 Chem.1: *-NH-(CH ₂ ) ₂ -(O-(CH ₂ )) _k -O-(CH ₂ ) _n -CO-* or Chem.2: O H * ^N O _* , wherein k is an n integer in the range of 1-5. 15 When k=1 and n=1, the linker element may be designated Ado, or 8-amino-3,6- dioxaoctanoyl, which may be represented by the following chemical formula: Chem.3: *-NH-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -O-CH ₂ -CO-* 20 or When k=1 and n=2, the linker element may be designated Aeep, which may be 25 represented by the following chemical formula: Chem.5: *-NH-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -CO-* or Chem.6: 30 33

When k=2 and n=2, the linker element may be designated Aeeep, which may be represented by the following chemical formula: Chem.7: *-NH-(CH ₂ ) _2- O-(CH ₂ ) ₂ O-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -CO-* 5 or Chem 10 The optional linker (L _P ) may comprise an 8-amino-3,6-dioxa-octanoic acid (OEG) group having the following chemical formula Chem 8a: The optional linker (L _P ) may comprise a sulfonamide-C4 moiety. A sulfonamide-C4 group 15 is a sulfonamide group attached to a 4-butanoyl group, having the following chemical formula: Chem 9: *-NH-S(O) ₂ -CH ₂ -CH ₂ -CH ₂ -CO-* 20 or Chem 10: The optional linker L _P may comprise Trx. Trx is also referred to as Tranexamic acid, trans- 4-(aminomethyl)cyclohexanecarboxylic acid, having the following chemical formula: 25 Chem.11: *-NH-CH ₂ -(C ₆ H ₁₀ )-CO-* or 34

Chem.12: The linker L _P may comprise epsilon-lysine (ε-Lys). The linker L _P may comprise lysine (Lys). 5 The linker L _P may comprise Ahx. Ahx is also referred to as Aminocaproic acid, 6- aminohexanoic acid and is defined by Chem 13: *-NH-(CH ₂ ) ₅ -CO-* 10 or Chem 14: The linker L _P may comprise a Glu di-radical, such as Chem.15: 15 wherein the Glu di-radical may be included p times, where p is an integer in the range of 1-3. Chem.15 may also be referred to as gamma-Glu, or briefly γGlu, due to the fact that it is 20 the gamma carboxy group of the amino acid glutamic acid which is here used for connection to the epsilon-amino group of lysine. As explained above, the other linker element may, for example, be another Glu residue, or an Ado molecule. The amino group of Glu in turn forms an amide bond with the carboxy group of the protracting moiety, or with the carboxy group of, e.g., an Ado molecule, if present, or with the gamma-carboxy 25 group of, e.g., another Glu, if present. Alternatively, the ISVD polypeptide derivatives disclosed herein may comprise a linker (L _P ) which is selected from any one of those depicted in Table 2 below. R1 represents the residue in the ISVD polypeptide to which the protraction moiety is attached and P represents the protractor. 30 35

_, ⁰ ₃ ^# _{, ,} ⁸ _, ⁵ _, ⁰ _, 8 ^{6 2} e _k ² _{: 5} n ² G G G i _l O y ^N _: O G G t _e ^{N G} G i ^D I _S ^G o ^{Q D} I G _S m _E ^Q G G n ^S _{( E} G G o _i ^S ₍ ^G G t _{c S} a _{S S} ^G S _r G t G G G o G G r G G G G p _)l ^G G S ^{G G} G S ^{G a} G _S G _{S n} ^o G G G G i _{t e} G G G G p _o ^{r (} _u G G G G f ^{t o} _c - u ^s _r ^{” G -} - ” ^{G -} t _{1 e} ^{R r} _” P ₁ R _” l S _{“ o “ “} ^P _{“ p} m ^a D x ^I ₎ E P : ^L 2 ₍ r e ^l _{e b} ^{k a} _n ⁱ _{1 L} L ^P ₂ L ^P ₃ L ^P ₄ ^{P 1} _T L ₀ O W ²

Based on the disclosure herein, the skilled person will be able to determine the optimal L _P and L _1-2 linker(s) for use in a specific ISVD polypeptide derivative as disclosed herein, optionally after some limited routine experiments. For example, the linkers are preferably such that it allows each ISVD, such as a V _H H fragment, in the ISVD polypeptide to bind to 5 its target. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific ISVD polypeptide derivative as disclosed herein, optionally after some limited routine experiments. An amino acid residue in the ISVD or ISVD polypeptide backbone to which attachment 10 takes place is designated R1 herein. In case of more than one protraction moiety being attached, the further attachment site(s) may be designated R2, R3 and so forth. The protraction moiety may attach to a cysteine or lysine residue in the first ISVD portion of the ISVD polypeptide backbone. The protraction moiety may attach to a cysteine or lysine residue in the second ISVD 15 portion of the ISVD polypeptide backbone. The protraction moiety may attach to a cysteine or lysine residue in the optional linker (L _{1- 2} ) portion of the ISVD polypeptide backbone. The protraction moiety may be covalently attached to a lysine residue in the ISVD polypeptide backbone. The protraction moiety may be attached via an amide bond formed 20 between a carboxylic acid group in the protraction moiety and the epsilon amino group of the lysine residue. The protraction moiety may be covalently attached to a cysteine residue in the ISVD polypeptide backbone. The protraction moiety may be attached via a thioether bond formed between the protraction moiety and the sulphur atom of the cysteine residue in the 25 polypeptide. Thus, in some embodiments the compounds disclosed herein may comprise one, two or three lysine or cysteine residues and one, two or three protraction moieties, wherein each protraction moiety is attached to a side chain of a single lysine or cysteine residue. When attachment takes place via cysteine residue, the cysteine is preferably a free 30 cysteine. A free cysteine, in some embodiments introduced by recombinant DNA technology, may serve as a conjugation site for attaching one or more C16-, C17- or C18 diacid gamma- Glu 2xOEG fatty acid moiety. 35 In a preferred embodiment a free cysteine, optionally introduced by recombinant DNA technology, may serve as a conjugation site for attaching one, two or three C18 diacid 37

gamma-Glu 2xOEG fatty acid moiety (IUPAC name S{Beta-AA#}-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17 - carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acet yl]amino]ethoxy]ethoxy] acetyl]amino]-ethylamino]-2-oxoethyl], AA# amino acid attachment). 5 In another preferred embodiment a free cysteine, optionally introduced by recombinant DNA technology, may serve as a conjugation site for attaching one, two or three C17 diacid gamma-Glu 2xOEG fatty acid moiety (IUPAC name S{Beta-AA#}-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(16 - 10 carboxyhexadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acety l]amino]ethoxy] ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl], AA# amino acid attachment). In a most preferred embodiment a free cysteine, introduced by recombinant DNA technology, may serve as a conjugation site for attaching one, two or three C16 diacid 15 gamma-Glu 2xOEG fatty acid moiety (IUPAC name S{Beta-AA#}-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15 - carboxypentadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acet yl]amino]ethoxy] ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl], AA# amino acid attachment). 20 The term “standard chromatography” as used herein encompasses standard chromatographic methods, such as protein A, cation exchange, anion exchange, hydrophobic interaction, and hydroxyapatite chromatography. The term “surface exposed amino acid residue” as used herein refers to amino acid 25 residues whose side chain can be in contact with solvent molecules (which in general may be mostly water molecules). However, the side chain does not necessarily have to be wholly in contact with solvent molecules, and when even a portion of the side chain is in contact with the solvent molecules, the amino acid residue is defined as an "amino acid located on the surface". The amino acid residues located on the surface of a polypeptide 30 can also include amino acid residues located close to the ISVD surface and thereby can have a mutual electric charge influence from other amino acid residue(s) whose side chain, even partly, is in contact with the solvent molecules. Those of ordinary skill in the art can prepare a homology model or a machine learning based three-dimensional molecular model of a polypeptide or antibody by for example homology modelling or 35 machine learning using commercially or publicly available softwares. Alternatively, it is possible to use methods such as X-ray crystallography for three-dimensional molecular 38

model generation. The amino acid residues that may be exposed on the surface can be determined, for example, using coordinates from a three-dimensional molecular model of an antibody using a computer program such as MOE (Chemical Computing Group) or Bioluminate (Schrödinger). Surface exposed sites may be determined using algorithms 5 known in the technical field (for example, Lee and Richards (1971) J. Mol. Biol.55:379- 400; Connolly, J. Appl. Cryst. (1983) 16:548-558). Surface exposable sites can be determined using software suitable for protein modelling and analysis of three- dimensional structure information obtained from the antibody. Software available for such purposes includes, for example, the MOE (Chemical Computing Group) or Bioluminate 10 (Schrödinger). The solvent accessible surface (in Å ² ) area are calculated using a water probe with a probe radius of 1.4 Å. Furthermore, methods for determining surface exposed regions and areas using software for personal computers have been described by Pacios (Pacios, Comput. Chem.18(4):377-386 (1994); J. Mol. Model.1:46-53 (1995)). Based on such information as described above, appropriate amino acid residues located 15 on the surface of an antibody in contact with solvent can be selected. PHARMACEUTICAL COMPOSITIONS The V _H H polypeptide derivatives as disclosed herein may be prepared in pharmaceutical compositions. In some embodiments such composition comprises at least one pharmaceutically acceptable excipient. 20 The term "excipient" as used herein broadly refers to any component other than the active therapeutic ingredient (API). The excipients may serve various purposes, e.g. as a carrier, vehicle, filler, binder, lubricant, glidant, disintegrant, flow control agent, crystallization inhibitors, solubilizer, stabilizer, colouring agent, flavouring agent, surfactant, emulsifier, 25 delivery agent, hydrotrope or combinations thereof and/or to improve administration, and/or absorption of the active pharmaceutical ingredient(s). The amount of each excipient used may vary within ranges conventional in the art. Techniques and excipients which may be used to formulate oral dosage forms are 30 described in Handbook of Pharmaceutical Excipients, 8th edition, Sheskey et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2017); and Remington: the Science and Practice of Pharmacy, 22nd edition, Remington and Allen, Eds., Pharmaceutical Press (2013). 35 39

In preferred embodiments the composition comprising a V _H H polypeptide further comprises a delivery agent and a hydrotrope. A preferred delivery agent is a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid (NAC). In some embodiments the delivery agent is a salt of N-(8-(2- 5 hydroxybenzoyl)amino)caprylic acid as described in WO2007/121318. In some embodiments the delivery agent is sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (referred to as "SNAC" herein), also known as sodium 8-(salicyloylamino)octanoate. The pharmaceutical compositions preferably comprise one or more hydrotropes. 10 Hydrotropes, like a surfactant, include both a hydrophilic part and a hydrophobic and can form micelles and self-aggregate, however they solubilize solutes without micellar solubilization. In one embodiment the hydrotrope is capable of increasing the solubility of SNAC. In one embodiment the hydrotrope is nicotinamide (NAM). 15 In one embodiment the composition is a solid composition. The composition may be in a form suitable for peroral administration, such as a tablet, sachet or capsule. In one such embodiment the composition is formulated as a tablet. The solid compositions provided herein allow for an accelerated dissolution and thereby enables fast uptake of the active pharmaceutical ingredient. 20 ADMINISTRATION AND DOSAGES A compound as disclosed herein, such as a V _H H polypeptide derivative, may be administered parenterally, such as intravenously, such as intramuscularly, such as subcutaneously in an appropriate pharmaceutical composition. The compound may be administered via a non-parenteral route and preferably perorally (PO). The compound 25 may be administered prophylactically. The compound may be administered therapeutically (on demand). Subcutaneous administration The dose of the compounds to be delivered by subcutaneous administration may be from about 0.01 mg to 1 mg of the compound per day, preferably from about 0.05 mg to 5 mg 30 per day, and more preferably from about 0.1 mg to about 10 mg per day, per every second day, per every third day, per fourth day, per fifth day, per every sixth day or once weekly depending on the severity of the condition. A suitable dose may also be adjusted for a particular compound based on the properties of that compound, including its in vivo half-life or mean residence time and its biological activity. 40

In one embodiment the present invention relates to an injection device with content of said composition. Peroral (PO) administration 5 The dose of the compounds to be delivered PO may be from about 1 mg to about 300 mg of the compound per day, per every second day, per every third day depending on the severity of the condition. A suitable dose may also be adjusted for a particular compound based on the properties of that compound, including its in vivo half-life or mean residence time and its biological activity. 10 The compositions containing the compounds as disclosed herein can be administered for prophylactic and/or in some embodiments therapeutic treatments. In therapeutic applications, compositions are administered to a subject already suffering from a disease, such as any bleeding disorder as described above, in an amount sufficient to cure, 15 alleviate or partially arrest the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective amount". As will be understood by the person skilled in the art amounts effective for this purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. 20 41

EMBODIMENTS 1. A procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated 5 form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or the activated form thereof, at least one protraction moiety, optionally a linker (L _1-2 ) linking ISVD1 and ISVD2, and 10 optionally one or more extension(s) (E). 2. The ISVD polypeptide derivative according to embodiment 1 having the formula ISVD1 – ISVD2, or ISVD1 – ISVD2 – E, or 15 ISVD2 – ISVD1 – E, or E - ISVD1 – ISVD2, or ISVD1 – L1-2 – ISVD2 – E, or ISVD2 – L1-2 – ISVD1 – E, or E - ISVD1 – L1-2 – ISVD2, 20 3. The ISVD polypeptide derivative according to embodiment 1 having the formula (N- to C-terminal) ISVD1 – ISVD2, or ISVD2 – ISVD1, or 25 ISVD1 – ISVD2 – E, or ISVD2 – ISVD1 – E, or E - ISVD1 – ISVD2, or E – ISVD2 – ISVD1, or ISVD1 – L1-2 – ISVD2, or 30 ISVD2 – L1-2 – ISVD1, or ISVD1 – L1-2 – ISVD2 – E, or ISVD2 – L1-2 – ISVD1 – E, or E - ISVD1 – L1-2 – ISVD2, or E – ISVD2 – L1-2 – ISVD1. 35 42

4. The ISVD polypeptide derivative according to any of the former embodiments wherein the at least one protraction moiety(ies) is/are attached to one or more surface exposed residue(s). 5 5. The ISVD polypeptide derivative according to any of the former embodiments having the formula (N- to C-terminal) ISVD2 – L _1-2 – ISVD1 – E wherein one or more protraction moiety(ies) is/are attached to one or more surface exposed residue. 10 6. The ISVD polypeptide derivative according to any of the former embodiments having the formula (N- to C-terminal) ISVD2 – L1-2 – ISVD1 – E wherein two protraction moieties are attached to one or more surface exposed 15 residue(s) on E. 7. The ISVD polypeptide derivative according to any of the former embodiments having the formula (N- to C-terminal) ISVD2 – L _1-2 – ISVD1 – E 20 further comprising a first protraction moiety and a second protraction moiety being attached to a first surface exposed residue and a second surface exposed residue in E, respectively. 8. The ISVD polypeptide derivative according to any of the former embodiments, 25 wherein L _1-2 comprises a ratio of hydrophobic to hydrophilic amino acids of “40 to 60 %” to “60 to 40 %”. 9. The ISVD polypeptide derivative according to any of the former embodiments, wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO:1) 30 or the activated form thereof comprising at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 (consecutive numbering). 10. The ISVD polypeptide derivative according to any of the former embodiments, wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO:1) 35 or the activated form thereof comprising at least 4, 5, 6, 7 or 8 of the amino acid 43

residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 (consecutive numbering). 11. The ISVD polypeptide derivative according to any of the former embodiments, 5 wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO:1) or the activated form thereof comprising the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 (consecutive numbering). 12. The ISVD polypeptide derivative according to any of the former embodiments, 10 wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO:2) comprising at least one of the amino acid residues N173, P174, F175, L177, L178 and D179 (consecutive numbering). 13. The ISVD polypeptide derivative according to any of the former embodiments, 15 wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO:2) comprising the amino acid residues N173, P174, F175, L177 and L178 (consecutive numbering). 20 14. The ISVD polypeptide derivative according to any of the former embodiments, wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO:2) comprising at least one of the amino acid residues N173, P174, F175, L177, L178 and D179 (consecutive numbering). 25 15. The ISVD polypeptide derivative according to any of the former embodiments, wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO:2) comprising at least 3, 4, 5 or 6 of the amino acid residues N173, P174, F175, L177, L178 and D179 (consecutive numbering). 30 16. The ISVD polypeptide derivative according to any of the former embodiments, wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO:2) comprising the amino acid residues N173, P174, F175, L177, L178 and D179 (consecutive numbering). 35 17. The ISVD polypeptide derivative according to any of the former embodiments, wherein 44

the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO:1) or the activated form thereof comprising at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 (consecutive numbering), and the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO:2) 5 comprising at least one of the amino acid residues N173, P174, F175, L177, and L178 (consecutive numbering). 18. The ISVD polypeptide derivative according to any of the former embodiments, wherein 10 the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO:1) or the activated form thereof comprising the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 (consecutive numbering), and the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO:2) comprising the amino acid residues N173, P174, F175, L177, L178 and D179 15 (consecutive numbering). 19. The ISVD polypeptide derivative according to any of embodiments 1-9, wherein the first ISVD comprises a paratope comprising amino acid residues F29, N30, Y32, T54, D99, R100, S101, F102, L103, F104, Q106, A107 and N113 (SEQ ID NO:35), and 20 wherein the second ISVD comprises a paratope comprising amino acid residues a) D32, A33, M34, G35, Y37, L47, V48, A49, G50, I51, M52, N57, T58, N59, Y60, T61, K97, V99, R101 and P102 (SEQ ID NO:27), or b) A33, M34, G35, W47, V48, A49, A50, I51, S52, S57, T58, N59, Y60, A61, A97, A98, D99, G105, L107 and Y109 (SEQ ID NO:734) 25 (consecutive numbering). 20. The ISVD polypeptide derivative according to any of embodiments 1-9, wherein said first ISVD comprises 1) 30 CDR1: IYTMS (SEQ ID NO:172), optionally comprising one or two substitutions, CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:173), optionally comprising one, two or three amino acid substitutions, CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:174), optionally comprising one, two or three amino acid substitutions (Kabat definition). 35 45

21. The ISVD polypeptide derivative according to of embodiments 1-9, wherein said second ISVD comprises (A) CDR1: RYAMG (SEQ ID NO:168), optionally comprising one or two substitutions, 5 CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:169), optionally comprising one, two or three amino acid substitutions, CDR3: DDSVGDGYLDY (SEQ ID NO:170), optionally comprising one, two or three amino acid substitutions; or 10 (B) CDR1: RLAMG (SEQ ID NO:128), optionally comprising one or two amino acid substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:129), optionally comprising one, two or three substitutions, 15 CDR3: DDSVGDGYLDY (SEQ ID NO:130), optionally comprising one, two or three amino acid substitutions (Kabat definition). 22. The ISVD polypeptide derivative according to any of embodiments 1-9, wherein said 20 first ISVD comprises 1) CDR1: IYTMS (SEQ ID NO:172), optionally comprising one or two amino acid substitutions, CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:173), optionally comprising one, two 25 or three amino acid substitutions, CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:174), optionally comprising one, two or three amino acid substitutions; or and wherein said second ISVD comprises 30 (A) CDR1: RYAMG (SEQ ID NO:168), optionally comprising one or two substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:169), optionally comprising one, two or three substitutions, CDR3: DDSVGDGYLDY (SEQ ID NO:170), optionally comprising one, two or three 35 amino acid substitutions; or 46

(B) CDR1: RLAMG (SEQ ID NO:128), optionally comprising one or two substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:129), optionally comprising one, two or three substitutions, 5 CDR3: DDSVGDGYLDY (SEQ ID NO:130), optionally comprising one, two or three amino acid substitutions (Kabat numbering). 23. The ISVD polypeptide derivative according to any of embodiments 20-22, wherein 10 said substitution(s) is/are conservative substitution(s). 24. The ISVD polypeptide derivative according to any of the former embodiments, wherein the sequence of said first ISVD is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the sequence identified by SEQ ID NO:107, 115, 123, 131, 155 or 15 171, respectively. 25. The ISVD polypeptide derivative according to any of the former embodiments, wherein the sequence of said second ISVD is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the sequence identified by SEQ ID NO:103, 111, 119, 127, 151 20 or 167, respectively. 26. The ISVD polypeptide derivative according to any of the former embodiments, wherein the sequence of said first ISVD is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the sequence identified by SEQ ID NO:107, 115, 123, 131, 155 or 25 171, respectively, and wherein the sequence of said second ISVD is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the sequence identified by SEQ ID NO:103, 111, 119, 127, 151 or 167, respectively. 27. The ISVD polypeptide derivative according to any of the former embodiments, 30 wherein said first ISVD comprises the sequence of V _H H-2.20 (SEQ ID NO:171), V _H H-2.18 (SEQ ID NO:155), V _H H-2.15 (SEQ ID NO:131), V _H H-2.13 (SEQ ID NO:115), 35 V _H H-2.14 (SEQ ID NO:123), or V _H H-2.12 (SEQ ID NO:107). 47

28. The ISVD polypeptide derivative according to any of the former embodiments, wherein said second ISVD comprises the sequence of V _H H-1.20 (SEQ ID NO:167), V _H H-1.18 (SEQ ID NO:151), 5 V _H H-1.15 (SEQ ID NO:127), V _H H-1.13 (SEQ ID NO:111), V _H H-1.14 (SEQ ID NO:119), or V _H H-1.12 (SEQ ID NO:103). 29. The ISVD polypeptide derivative according to any of the former embodiments, 10 wherein said first ISVD comprises the sequence of V _H H-2.20 (SEQ ID NO:171), V _H H-2.18 (SEQ ID NO:155), VHH-2.15 (SEQ ID NO:131), V _H H-2.13 (SEQ ID NO:115), 15 V _H H-2.14 (SEQ ID NO:123), or V _H H-2.12 (SEQ ID NO:107) and wherein said second ISVD comprises the sequence of V _H H-1.20 (SEQ ID NO:167), 20 VHH-1.18 (SEQ ID NO:151), V _H H-1.15 (SEQ ID NO:127), V _H H-1.13 (SEQ ID NO:111), V _H H-1.14 (SEQ ID NO:119), or V _H H-1.12 (SEQ ID NO:103). 25 30. The ISVD polypeptide derivative according to embodiment 29, wherein said first ISVD comprises the sequence of V _H H-2.20 (SEQ ID NO:171), and wherein said second ISVD comprises the sequence of V _H H-1.20 (SEQ ID NO:167). 31. The ISVD polypeptide derivative according to embodiment 29, wherein 30 said first ISVD comprises the sequence of V _H H-2.18 (SEQ ID NO:155), and wherein said second ISVD comprises the sequence of V _H H-1.18 (SEQ ID NO:151). 32. The ISVD polypeptide derivative according to embodiments 2929, wherein said first ISVD comprises the sequence of V _H H-2.15 (SEQ ID NO:131), and wherein said second ISVD comprises the sequence of V _H H-1.15 (SEQ ID NO:127). 48

33. The ISVD polypeptide derivative according to embodiment 29, wherein said first ISVD comprises the sequence of V _H H-2.13 (SEQ ID NO:115), and wherein said second ISVD comprises the sequence of V _H H-1.13 (SEQ ID NO:111). 34. The ISVD polypeptide derivative according to embodiment 29, wherein 5 said first ISVD comprises the sequence of V _H H-2.14 (SEQ ID NO:123), and wherein said second ISVD comprises the sequence of V _H H-1.14 (SEQ ID NO:119). 35. The ISVD polypeptide derivative according to embodiment 29, wherein said first ISVD comprises the sequence of V _H H-2.12 (SEQ ID NO:107), and wherein said second ISVD comprises the sequence of V _H H-1.12 (SEQ ID NO:103). 10 36. The ISVD polypeptide derivative according to any of the former embodiments, wherein the protraction moiety(ies) comprises a protractor selected from the groups consisting of a) a C16 diacid, C17 diacid, C18 diacid, C19 diacid, C20 diacid, C21 diacid or 15 C22 diacid, b) tetrazole, c) an albumin-binder peptide, such as RLIEDICLPRWGCLWEDD (SEQ ID NO:737) d) an FcRn-binder peptide, such as QRFCTGHFGGLYPCNG (SEQ ID NO:738), 20 and e) a Fc-binder peptide, such as FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDDN (SEQ ID NO:739). 37. The ISVD polypeptide derivative according to any of the former embodiments, 25 wherein the protraction moiety(ies) comprises a protractor selected from the group consisting of C16 diacid, C17 diacid, C18 diacid, C19 diacid and C20 diacid. 38. The ISVD polypeptide derivative according to embodiment 34, wherein the protractor(s) is/are a C16 diacid protractor. 39. The ISVD polypeptide derivative according to any of the former embodiments, 30 wherein the protraction moiety comprises a linker L _P attaching the protractor to ISVD1, L _1-2 , ISVD2 or E. 40. The ISVD polypeptide derivative according to embodiment 39, wherein L _P is selected from the group consisting of 49

L _P 1 . , wherein the protraction moiety(ies) is/are attached to position 13 in ISVD2 and/or one or more of positions 12, 14, 15, 42, 44, 63, and/or 85 on ISVD1 (consecutive 5 numbering). 42. The ISVD polypeptide derivative according to any of embodiments 1-40, wherein the ISVD polypeptide derivative comprises a C-terminal extension, wherein the protraction moiety(ies) is/are attached to said C-terminal extension. 10 43. The ISVD polypeptide derivative according to any of the former embodiments, wherein the ISVD polypeptide derivative comprises a first and a second protraction moieties and a C-terminal extension, wherein the first protraction moiety and the second protraction moiety are attached to the C-terminal extension. 44. The ISVD polypeptide derivative according to embodiment 43, wherein the C-terminal 15 extension is attached to ISVD1. 45. The ISVD polypeptide derivative according to embodiment 43, wherein the C-terminal extension is attached to ISVD2. 46. The ISVD polypeptide derivative according to any of the former embodiments, wherein the protraction moieties are identical. 50

47. The ISVD polypeptide derivative according to any of the former embodiments, wherein one or more protraction moiety(ies) is/are attached to a surface exposed residue. 48. The ISVD polypeptide derivative according to any of the former embodiments, 5 wherein one or more protraction moiety(ies) is/are attached to a surface exposed residue wherein said surface exposed residue is not a residue in a CDR region. 49. The ISVD polypeptide derivative according to any of the former embodiments, wherein one or more protraction moiety(ies) is/are attached at one or more cysteine or lysine residue(s). 10 50. The ISVD polypeptide derivative according to embodiment 43 wherein said ISVD polypeptide derivative comprises SEQ ID NO:629, and wherein said first protraction moiety comprises the structure: 15 and wherein said second protraction moiety comprises the structure 20 wherein ‘*’ (R1) represents the point of attachment on the ISVD polypeptide for the first protraction moiety, and wherein ‘**’ (R2) represents the point of attachment on the ISVD polypeptide for the second protraction moiety, wherein ‘*’ is Cys in position 257 of SEQ ID NO:629, and 25 wherein ‘**’ is Cys in position 259 of SEQ ID NO:629. 51

51. The ISVD polypeptide derivative according to any of the former embodiments, wherein the isoelectric point of said polypeptide, as determined using isoelectric focusing, is 6.5 or less. 52. The ISVD polypeptide derivative according to any of the former embodiments wherein 5 said polypeptide derivative is a multispecific polypeptide derivative such as a bi- or trispecific polypeptide derivative. 53. The ISVD polypeptide derivative according to any of the former embodiments wherein said polypeptide derivative is a bispecific polypeptide derivative. 54. The ISVD polypeptide derivative according to any of the former embodiments wherein 10 said polypeptide derivative has a molecular weight in the range of 12-50 kDa, such as 12-48 kDa, such as 12-43 kDa, such as 12-37 kDa, such as 12-35 kDa, such as 12-32 kDa, such as 12-27 kDa, such as 12-22 kDa, such as 12-18 kDa, such as 14-50 kDa, such as 14-48 kDa, such as 14-43 kDa, such as 14-37 kDa, such as 14-35 kDa, 14-32 kDa, such as 14-27 kDa, such as 14-22 kDa, such as 14-18 kDa, such as 22-50 kDa, 15 22-48 kDa, such as 22-43 kDa, such as 22-37 kDa, such as 22-35 kDa, such as 22-32 kDa, such as 22-31 kDa, such as 22-30 kDa, such as 22-29 kDa, such as 22-28 kDa, such as 22-27 kDa, such as 24-50 kDa, 24-48 kDa, such as 24-43 kDa, such as 24-37 kDa, such as 24-35, such as 24-32 kDa, such as 24-31 kDa, 24-30 kDa, 24-29 kDa, 24-28 kDa, such as 24-27 kDa, such as 36-48 kDa, such as 36-43 kDa, such as 36-37 20 kDa, such as 28-36 kDa, such as 29-33 kDa, such as 29-30 such as about 29, such as 29, such as about 30, such as 30, such as 30-32 kDa, or such as 31 kDa. 55. The ISVD polypeptide derivative according to any of the former embodiments, which is not a membrane targeted ISVD polypeptide derivative. 25 56. The ISVD polypeptide derivative according to any of the former embodiments wherein said protraction moiety is not capable of binding components of a plasma membrane, such as aminophospholipid, such as a phosphatidylserine and/or phosphatidylethanolamine. 57. The ISVD polypeptide derivative according to any of the former embodiments, 30 wherein said protraction moiety is not capable of binding platelet surface proteins such as GPlb-lX, collagen chaperone HSP47, ephrin B1, thiol isomerase protein ERP5, Hematopoietic progenitor kinase 1-interacting protein of 55 (HIP-55), glycoprotein Vl, platelet glycoprotein 1b, platelet-derived growth factor receptor, platelet endothelial aggregation receptor I, CD31, CD36, MARKS, multimerin, integrin35 alpha lIb/beta 3, triggering receptor expressed on myeloid cells (TREM) like transcript- 1 (TLT-1), integrin-linked kinase (ILK), zyxin, collagen, P-selectin, Factor XIII, P- 52

selectin glycoprotein ligand-1, integrin alpha 6 beta 1, thrombospondin, von Willebrand factor, G6B, CD42b, syntaxin binding protein 2, phosphatidylethanolamine, fibrinogen/fibrin, filamin, stomatin, sphingolipid, CD31, CD36, CD40, CD41, CD42c, CD42, CD49b, CD61, CD62P, CD63, CD69, CD107a, CD107b, CD109, CD154, 5 PECAM-1, and/or ERPS. 58. The ISVD polypeptide derivative according to any of the former embodiments, wherein said protraction moiety is not capable of binding a membrane associated polypeptide, such as glycoproteins, GPIIb/IIIa, β2GP1, TLT-1, selectins, a coagulation factor or coagulation factor complex and/or a selectin. 10 59. The ISVD polypeptide derivative according to any of the former embodiments, wherein the mean residence time terminal half-life in blood is prolonged by at least 12 hours, at least 24 hours, at least 48 hours, at least 3, 4, 5, or 7 days as compared to the ISVD polypeptide without said protraction moiety. 60. The ISVD polypeptide derivative according to any of the former embodiments, 15 wherein said first and/or second ISVD is/are a V _H H fragment. 61. The ISVD polypeptide derivative according to any of embodiments 1-59, wherein said first and/or second ISVD is a V-NAR fragment. 62. The ISVD polypeptide derivative according to any embodiments 1-60, wherein said ISVD polypeptide derivative is a V _H H polypeptide derivative. 20 53

63. A procoagulant V _H H polypeptide derivative comprising a first V _H H capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second V _H H capable of binding to Factor X (SEQ ID NO:2), a linker (L _1-2 ) linking said first V _H H and said second V _H H, and 5 a C-terminal extension (E), and one or two protraction moiety(ies), having the formula (N- to C-terminal): “second V _H H” – L _1-2 – “first V _H H” – E wherein 10 I) said first V _H H comprises CDR1: IYTMS (SEQ ID NO:172), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:173), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:174), and 15 said second V _H H comprises CDR1: RYAMG (SEQ ID NO:168), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:169), CDR3: DDSVGDGYLDY (SEQ ID NO:170); or II) 20 said first VHH comprises CDR1: IYTMS (SEQ ID NO:132), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:133), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:134), and said second V _H H comprises 25 CDR1: RLAMG (SEQ ID NO:128), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:129), CDR3: DDSVGDGYLDY (SEQ ID NO:130); or (Kabat definition). 30 64. The procoagulant V _H H polypeptide derivative according to embodiment 63, wherein I) said first V _H H comprises the sequence of V _H H-2.20 (SEQ ID NO:171), and said second V _H H comprises the sequence of V _H H-1.20 (SEQ ID NO:167). II) 35 said first V _H H comprises the sequence of V _H H-2.18 (SEQ ID NO:155), and said second V _H H comprises the sequence of V _H H-1.18 (SEQ ID NO:151). 54

III) said first V _H H comprises the sequence of V _H H-2.15 (SEQ ID NO:131), and said second V _H H comprises the sequence of V _H H-1.15 (SEQ ID NO:127). IV) 5 said first V _H H comprises the sequence of V _H H-2.13 (SEQ ID NO:115), and said second V _H H comprises the sequence of V _H H-1.13 (SEQ ID NO:111). V) said first V _H H comprises the sequence of V _H H-2.14 (SEQ ID NO:123), and said second V _H H comprises the sequence of V _H H-1.14 (SEQ ID NO:119). 10 VI) said first V _H H comprises the sequence of V _H H-2.12 (SEQ ID NO:107), and said second V _H H comprises the sequence of V _H H-1.12 (SEQ ID NO:103). 65. The procoagulant V _H H polypeptide derivative according to any of embodiments 1-60 15 and 62-64 wherein said V _H H polypeptide derivative is a bispecific V _H H polypeptide derivative. 66. The procoagulant V _H H polypeptide derivative according to any of embodiments 63-65 wherein the linker L _1-2 comprises the amino acid residues GQAPGQ (SEQ ID NO:20). 67. The procoagulant V _H H polypeptide derivative according to any of embodiments 63-65, 20 wherein the extension (E) comprises the amino acid residues GQACPC (SEQ ID NO:9). 68. The procoagulant V _H H polypeptide derivative according to any of embodiments 63-67, wherein two protraction moieties are attached to E in positions 4 and 6 (SEQ ID NO:9), respectively. 25 69. The procoagulant V _H H polypeptide derivative according to any of embodiments 63-68, wherein said two protraction moieties are identical. 70. The procoagulant V _H H polypeptide derivative according to any of embodiments 63-69, wherein said two protraction moieties comprise the structure: 30 71. The procoagulant V _H H polypeptide derivative according to any of embodiments 63-70 comprising a first and a second protraction moiety wherein said first protraction moiety comprises the structure: 55

and wherein said second protraction moiety comprises the structure 5 wherein ‘*’ (R1) represents the point of attachment on the V _H H polypeptide derivative for the first protraction moiety, and wherein ‘**’ (R2) represents the point of attachment on the V _H H polypeptide derivative 10 for the second protraction moiety, wherein ‘*’ is Cys in position 257 of SEQ ID NO:629, and wherein ‘**’ is Cys in position 259 of SEQ ID NO:629. 72. A pharmaceutical composition comprising the ISVD polypeptide derivative or V _H H 15 polypeptide derivative according to any of embodiments 1-71. 73. A pharmaceutical composition for peroral administration comprising the ISVD polypeptide derivative or V _H H polypeptide derivative according to any of embodiments 1-7071. 74. The pharmaceutical composition according to embodiment 72 or 73, wherein the 20 ISVD polypeptide derivative is a V _H H polypeptide derivative. 75. The pharmaceutical composition according to embodiment 72 or 73, wherein the ISVD polypeptide derivative is a V-NAR polypeptide derivative. 76. The pharmaceutical composition according to any of embodiments 72-75, wherein said composition comprises a salt of N-(8-(2-hydroxybenzoyl)amino) caprylic acid. 56

77. The pharmaceutical composition according to embodiment 76, wherein said salt of N- (8-(2-hydroxybenzoyl)amino)caprylic acid is sodium N-(8-(2- hydroxybenzoyl)amino)caprylate (SNAC). 78. The pharmaceutical composition according to any of embodiments 76 to 77 further 5 comprising nicotinamide (NAM). 79. The pharmaceutical composition according to any of embodiments 74-78, wherein said composition comprises a V _H H polypeptide derivative, sodium N-(8-[2- hydroxybenzoyl] amino) caprylate (SNAC), nicotinamide (NAM) and magnesium stearate. 10 80. The pharmaceutical composition according to any of embodiments 74-78, wherein said composition comprises about 7 % w/w V _H H polypeptide derivative, about 55 % w/w sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC), about 36 % w/w nicotinamide (NAM) and about 0.5 % w/w magnesium stearate. 81. The pharmaceutical composition according to any of embodiments 73-80, wherein 15 said composition is a liquid composition. 82. The pharmaceutical composition according to any of embodiments 73-80, wherein said composition is a solid composition. 83. The pharmaceutical composition according to embodiment8282, wherein said composition is a tablet, losenge or capsule. 20 84. A method of treating a patient in need thereof comprising administering the ISVD polypeptide derivative, V _H H polypeptide derivative or pharmaceutical composition according to any of embodiments 1-83. 85. The method of treatment according to embodiment 84, wherein the patient is a patient 25 suffering from haemophilia A with or without inhibitors or acquired haemophilia A. 86. The method of treatment according to embodiment 84 or 85, wherein the treatment is a prophylactic treatment. 87. The method of treatment according to any of embodiments 84-86 wherein the ISVD polypeptide derivative, V _H H polypeptide derivative or pharmaceutical composition is 30 administered perorally. 88. The method of treatment according to any of embodiments 84-86, wherein the ISVD polypeptide derivative, V _H H polypeptide derivative or pharmaceutical composition is administered by subcutaneous injection. 35 89. The ISVD polypeptide derivative, V _H H polypeptide derivative or pharmaceutical composition according to any of embodiments 1-83 for use in medicine. 57

90. The ISVD polypeptide derivative, V _H H polypeptide derivative or composition according to any of embodiments 1-83 for use in the treatment of haemophilia, such as haemophilia A with or without inhibitors, or such as acquired haemophilia A. 91. The ISVD polypeptide derivative, V _H H polypeptide derivative or composition according 5 to any of embodiments 1-83 for use in the treatment of haemophilia A with or without inhibitors. 92. The ISVD polypeptide derivative, V _H H polypeptide derivative or composition for use according to any of embodiments 89-92, wherein the treatment is prophylactic treatment. 10 93. The ISVD polypeptide derivative, V _H H polypeptide derivative or composition for use according to any of embodiments 89-92, wherein said derivative or composition is administered perorally. 94. The ISVD polypeptide derivative, VHH polypeptide derivative or composition for use according to any of embodiments 89-92, wherein said derivative or composition is 15 administered by subcutaneous injection. 95. A method for producing a procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated 20 form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or the activated form thereof, one or more protraction moiety(ies), optionally a linker capable of linking ISVD1 and ISVD2 (“L _1-2 ”), and 25 optionally one or more extension(s) (“E”) comprising the steps of a. modifying a nucleic acid encoding the amino acid residues of a ISVD polypeptide or ISVD polypeptide derivative such that the isoelectric point of the 30 ISVD polypeptide or ISVD polypeptide derivative is reduced b. culturing host cells to express the nucleic acid encoding the ISVD polypeptide or ISVD polypeptide derivative, c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture, 35 d. purifying the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture using standard chromatography, and 58

e. attaching a protraction moiety to the ISVD polypeptide, unless such moiety is already present. 96. A method for producing a procoagulant immunoglobulin single variable domain (ISVD) 5 polypeptide or ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or the activated form thereof, a protraction moiety, optionally a linker capable of linking ISVD1 and ISVD2 (“L _1-2 ”), and optionally one or more extension(s) (“E”) comprising the steps of 10 a. modifying a nucleic acid encoding the amino acid residues of the ISVD polypeptide or ISVD polypeptide derivative such that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is 6.5 or less, b. culturing host cells to express the nucleic acid encoding ISVD polypeptide or ISVD polypeptide derivative, 15 c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture, d. purifying the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture using standard chromatography, and e. attaching a protraction moiety to the ISVD polypeptide, unless such moiety is 20 already present. 97. A method for increasing the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated 25 form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or the activated form thereof, a protraction moiety, optionally a linker capable of linking ISVD1 and ISVD2 (“L _1-2 ”), and optionally one or more extension(s) (“E”) comprising the steps of a. modifying a nucleic acid encoding the amino acid residues of the ISVD 30 polypeptide or ISVD polypeptide derivative such that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is reduced, b. culturing host cells to express the nucleic acid encoding the ISVD polypeptide or ISVD polypeptide derivative, c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host 35 cell culture, 59

d. purifying the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture using standard chromatography, and e. attaching a protraction moiety to the ISVD polypeptide, unless such moiety is already present. 5 98. A method for increasing the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or 10 the activated form thereof, a protraction moiety, optionally a linker capable of linking ISVD1 and ISVD2 (“L _1-2 ”), and optionally one or more extension(s) (“E”) comprising the steps of a. modifying a nucleic acid encoding the amino acid residues of the ISVD polypeptide or ISVD polypeptide derivative such that the isoelectric point of the 15 ISVD polypeptide or ISVD polypeptide derivative is 6.5 or less, b. culturing host cells to express the nucleic acid encoding the ISVD polypeptide or ISVD polypeptide derivative, c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture, 20 d. purifying the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture using standard chromatography, and e. attaching a protraction moiety to the ISVD polypeptide, unless such moiety is already present. 99. The method according to any of embodiments 95-98, wherein the isoelectric point is 25 reduced by at least 0.5 pH units, such as at least 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4 pH units. 100. The method according to any of embodiments 95-99, wherein step e) does not lead to an increase the isoelectric point of the ISVD polypeptide or ISVD polypeptide 30 derivative. 101. The method according to any of embodiments 95-100, wherein the isoelectric point is determined using isoelectric focusing. 102. The method according to any of embodiments 95-101, wherein the ISVD polypeptide or ISVD polypeptide derivative is encoded by a single nucleic acid. 60

103. The method according to any of embodiments 95-101, wherein the ISVD polypeptide or ISVD polypeptide derivative is a conjugate of a first and second ISVD, wherein said first and second ISVD are encoded by separate nucleic acids. 104. The method according to any of embodiments 95-103, wherein the ISVD 5 polypeptide or ISVD polypeptide derivative is a V _H H polypeptide or V _H H polypeptide derivative. 105. An ISVD polypeptide or ISVD polypeptide derivative produced using the method according to any of embodiments 95-103. 10 106. An ISVD polypeptide or ISVD polypeptide derivative obtainable using the method according to any of embodiments 95-103. 107. A V _H H polypeptide or V _H H polypeptide derivative produced using the method according to any of embodiments 95-104. 15 108. A V _H H fragment capable of binding to FIX (SEQ ID NO:1) or the activated form thereof (FIXa) wherein a. the CDR1 sequence is identified by SEQ ID NO:172, optionally comprising one or two amino acid substitutions, 20 the CDR2 sequence is identified by SEQ ID NO:173, optionally comprising one, two or three amino acid substitutions, the CDR3 sequence is identified by SEQ ID NO:174, optionally comprising one, two or three amino acid substitutions; or 25 b. the CDR1 sequence is identified by SEQ ID NO:156, optionally comprising one or two amino acid substitutions, the CDR2 sequence is identified by SEQ ID NO:157, optionally comprising one, two or three amino acid substitutions, the CDR3 sequence is identified by SEQ ID NO:158, optionally comprising 30 one, two or three amino acid substitutions; or c. the CDR1 sequence is identified by SEQ ID NO:132, optionally comprising one or two amino acid substitutions, the CDR2 sequence is identified by SEQ ID NO:133, optionally comprising 35 one, two or three amino acid substitutions, 61

the CDR3 sequence is identified by SEQ ID NO:134, optionally comprising one, two or three amino acid substitutions; or d. the CDR1 sequence is identified by SEQ ID NO:116, optionally comprising one 5 or two amino acid substitutions, the CDR2 sequence is identified by SEQ ID NO:117, optionally comprising one, two or three amino acid substitutions, the CDR3 sequence is identified by SEQ ID NO:118, optionally comprising one, two or three amino acid substitutions; or 10 e. the CDR1 sequence is identified by SEQ ID NO:124, optionally comprising one or two amino acid substitutions, the CDR2 sequence is identified by SEQ ID NO:125, optionally comprising one, two or three amino acid substitutions, 15 the CDR3 sequence is identified by SEQ ID NO:126, optionally comprising one, two or three amino acid substitutions; or f. the CDR1 sequence is identified by SEQ ID NO:108, optionally comprising one or two amino acid substitutions, 20 the CDR2 sequence is identified by SEQ ID NO:109, optionally comprising one, two or three amino acid substitutions, the CDR3 sequence is identified by SEQ ID NO:110, optionally comprising one, two or three amino acid substitutions; (Kabat definition). 25 109. A V _H H fragment capable of binding to FX (SEQ ID NO:2) wherein a. the CDR1 sequence is identified by SEQ ID NO:152, optionally comprising one or two amino acid substitutions, 30 the CDR2 sequence is identified by SEQ ID NO:153, optionally comprising one, two or three amino acid substitutions, the CDR3 sequence is identified by SEQ ID NO:154, optionally comprising one, two or three amino acid substitutions; or 35 b. the CDR1 sequence is identified by SEQ ID NO:128, optionally comprising one or two amino acid substitutions, 62

the CDR2 sequence is identified by SEQ ID NO:129, optionally comprising one, two or three amino acid substitutions, the CDR3 sequence is identified by SEQ ID NO:130, optionally comprising one, two or three amino acid substitutions. 5 110. The V _H H fragment according to embodiment 108 or 109 wherein said substitution(s) is/are conservative substitution(s). 111. The V _H H fragment according to any of embodiments 108-110 wherein said V _H H fragment is an intermediate for use in the manufacture of a V _H H polypeptide or V _H H 10 polypeptide derivative which is capable of binding to FIX (SEQ ID NO:1) or the activated form thereof (FIXa), and capable of binding to FX (SEQ ID NO:2) or the activated form thereof (FXa). 112. The VHH polypeptide according to any of embodiments 108-110 wherein said V _H H polypeptide is an intermediate for use in the manufacture of an ISVD polypeptide 15 derivative. 113. The ISVD or V _H H according to embodiment 109 or 110 wherein the activity is improved in terms of making FX more prone to proteolysis by FIXa as compared to any of the anti-FX ISVD compounds as described in WO2019/096874. 20 114. A procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or the activated 25 form thereof, at least one protraction moiety, optionally a linker (L _1-2 ) linking ISVD1 and ISVD2, and optionally one or more extension(s) (E), wherein said ISVD polypeptide is selected from the group consisting of: SEQ ID 30 NO:615-691, 734 or 735. 115. A V _H H fragment comprising the sequence of any of SEQ ID NOs:27-614. 116. A V _H H polypeptide derivative comprising the sequence of any of SEQ ID NOs:615-691 or 734-735. 35 63

117. A procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, 5 a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or the activated form thereof, at least one protraction moiety, optionally a linker (L _1-2 ) linking ISVD1 and ISVD2, and optionally one or more extension(s) (E). 10 118. The ISVD polypeptide derivative according to embodiment 117 having the formula (N- to C-terminal) ISVD2 – L _1-2 – ISVD1 – E wherein one or more protraction moiety(ies) is/are attached to one or more surface 15 exposed residue. 119. The ISVD polypeptide derivative according to embodiment 118 or 119 having the formula (N- to C-terminal) ISVD2 – L _1-2 – ISVD1 – E 20 wherein two protraction moieties are attached to one or more surface exposed residue(s) on E, and wherein the molecular weight of the derivative is in the range 20-35 kDa. 120. The ISVD polypeptide derivative according to any of embodiments 117-119, 25 wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO:1) or the activated form thereof comprising the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 (consecutive numbering), and the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO:2) or 30 the activated form thereof comprising the amino acid residues N173, P174, F175, L177, and L178 (consecutive numbering). 121. The ISVD polypeptide derivative according to any of embodiments 117-119, wherein said first ISVD comprises 64

1) CDR1: IYTMS (SEQ ID NO:172), optionally comprising one or two substitutions, CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:173), optionally comprising one, two or three substitutions, 5 CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:174), optionally comprising one, two or three substitutions; or 2) CDR1: IYTMS (SEQ ID NO:156), optionally comprising one or two substitutions, CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:157), optionally comprising one, two 10 or three substitutions, CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:158), optionally comprising one, two or three substitutions; or 3) CDR1: IYTMS (SEQ ID NO:132), optionally comprising one or two substitutions, 15 CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:133), optionally comprising one, two or three substitutions, CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:134), optionally comprising one, two or three substitutions; or 4) 20 CDR1: IYTMS (SEQ ID NO:116), optionally comprising one or two substitutions, CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:117), optionally comprising one, two or three substitutions, CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:118), optionally comprising one, two or three substitutions; or 25 5) CDR1: IYTMS (SEQ ID NO:124), optionally comprising one or two substitutions CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:125), optionally comprising one, two or three substitutions, CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:126), optionally comprising one, two 30 or three substitutions; or 6) CDR1: IYTMS (SEQ ID NO:108), optionally comprising one or two substitutions, CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:109), optionally comprising one, two or three substitutions, 35 CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:110), optionally comprising one, two or three substitutions; 65

and wherein said second ISVD comprises (A) CDR1: RYAMG (SEQ ID NO:168), optionally comprising one or two substitutions, 5 CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:169), optionally comprising one, two or three substitutions, CDR3: DDSVGDGYLDY (SEQ ID NO:170), optionally comprising one, two or three substitutions; or (B) 10 CDR1: RYAMG (SEQ ID NO:152), optionally comprising one or two substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:153), optionally comprising one, two or three substitutions, CDR3: DDSVGDGYLDY (SEQ ID NO:154), optionally comprising one, two or three substitutions; or 15 (C) CDR1: RLAMG (SEQ ID NO:128), optionally comprising one or two substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:129), optionally comprising one, two or three substitutions, CDR3: DDSVGDGYLDY (SEQ ID NO:130), optionally comprising one, two or three 20 substitutions; or (D) CDR1: RLAMG (SEQ ID NO:112), optionally comprising one or two substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:113), optionally comprising one, two or three substitutions, 25 CDR3: DDSVGDGYLDY (SEQ ID NO:114), optionally comprising one, two or three substitutions; or (E) CDR1: RLAMG (SEQ ID NO:120), optionally comprising one or two substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:121), optionally comprising one, two or 30 three substitutions, CDR3: DDSVGDGYLDY (SEQ ID NO:122), optionally comprising one, two or three substitutions; or (F) CDR1: RLAMG (SEQ ID NO:104), optionally comprising one or two substitutions, 35 CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:105), optionally comprising one, two or three substitutions, 66

CDR3: DDSVGDGYLDY (SEQ ID NO:106), optionally comprising one, two or three substitutions (Kabat definition). 5 122. The ISVD polypeptide derivative according to embodiment 121, wherein said substitution(s) is/are conservative substitution(s). 123. The ISVD polypeptide derivative according to any of embodiments 117-122, wherein said first ISVD comprises the sequence of 10 V _H H-2.20 (SEQ ID NO:171), V _H H-2.18 (SEQ ID NO:155), V _H H-2.15 (SEQ ID NO:131), VHH-2.13 (SEQ ID NO:115), V _H H-2.14 (SEQ ID NO:123), or 15 V _H H-2.12 (SEQ ID NO:107) and wherein said second ISVD comprises the sequence of V _H H-1.20 (SEQ ID NO:167), V _H H-1.18 (SEQ ID NO:151), 20 VHH-1.15 (SEQ ID NO:127), V _H H-1.13 (SEQ ID NO:111), V _H H-1.14 (SEQ ID NO:119), or V _H H-1.12 (SEQ ID NO:103). 25 124. A procoagulant V _H H polypeptide derivative comprising a first V _H H capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second V _H H capable of binding to Factor X (SEQ ID NO:2) or the activated form thereof, 30 a linker (L _1-2 ) linking said first V _H H and said second V _H H, and a C-terminal extension (E), and one or two protraction moiety(ies), having the formula (N- to C-terminal): “second V _H H” – L _1-2 – “first V _H H” – E 35 wherein I) 67

said first V _H H comprises CDR1: IYTMS (SEQ ID NO:172), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:173), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:174), and 5 said second V _H H comprises CDR1: RYAMG (SEQ ID NO:168), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:169), CDR3: DDSVGDGYLDY (SEQ ID NO:170); or 10 II) said first V _H H comprises CDR1: IYTMS (SEQ ID NO:156), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:157), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:158), and 15 said second V _H H comprises CDR1: RYAMG (SEQ ID NO:152), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:153), CDR3: DDSVGDGYLDY (SEQ ID NO:154); or 20 III) said first V _H H comprises CDR1: IYTMS (SEQ ID NO:132), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:133), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:134), and 25 said second V _H H comprises CDR1: RLAMG (SEQ ID NO:128), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:129), CDR3: DDSVGDGYLDY (SEQ ID NO:130); or 30 IV) said first V _H H comprises CDR1: IYTMS (SEQ ID NO:116), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:117), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:118), and 35 said second V _H H comprises CDR1: RLAMG (SEQ ID NO:112), 68

CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:113), CDR3: DDSVGDGYLDY (SEQ ID NO:114); or V) 5 said first V _H H comprises CDR1: IYTMS (SEQ ID NO:124), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:125), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:126), and said second V _H H comprises 10 CDR1: RLAMG (SEQ ID NO:120), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:121), CDR3: DDSVGDGYLDY (SEQ ID NO:122); or VI) 15 said first V _H H comprises CDR1: IYTMS (SEQ ID NO:108), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:109), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO:110), and said second V _H H comprises 20 CDR1: RLAMG (SEQ ID NO:104), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:105), CDR3: DDSVGDGYLDY (SEQ ID NO:106) (Kabat definition). 25 125. A pharmaceutical composition comprising the ISVD polypeptide derivative or V _H H polypeptide derivative according to any of embodiments 117-124. 126. The pharmaceutical composition according to embodiment 125, wherein said composition comprises a salt of N-(8-(2-hydroxybenzoyl)amino) caprylic acid. 30 127. The pharmaceutical composition according to embodiments 126 wherein said salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is sodium N-(8-(2- hydroxybenzoyl)amino)caprylate (SNAC). 35 128. The pharmaceutical composition according to any of embodiments 125-127 further comprising nicotinamide (NAM). 69

129. The pharmaceutical composition according to any of embodiments 125-128 wherein said composition is a solid composition. 5 130. The ISVD polypeptide derivative, V _H H polypeptide derivative or composition according to any of embodiments 117-129 for use in the treatment of haemophilia, such as haemophilia A with or without inhibitors, or such as acquired haemophilia A. 131. The ISVD polypeptide derivative, V _H H polypeptide derivative or composition for 10 use according to any of embodiments 130 wherein said polypeptide derivative or composition is administered perorally. 132. A method for increasing the oral bioavailability of a procoagulant immunoglobulin 15 single variable domain (ISVD) polypeptide or ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO:1) or the activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO:2) or the activated form thereof, a protraction moiety, optionally a linker capable of linking ISVD1 and ISVD2 (“L _1-2 ”), and optionally one or more extension(s) (“E”) comprising 20 the steps of a. modifying a nucleic acid encoding the amino acid residues of the ISVD polypeptide or ISVD polypeptide derivative such that the isoelectric point thereof is reduced, b. culturing host cells to express the nucleic acid encoding the ISVD polypeptide 25 or ISVD polypeptide derivative, c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture, d. purifying the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture using standard chromatography, and 30 e. attaching a protraction moiety to the ISVD polypeptide, unless such moiety is already present. In a preferred embodiment the compound obtained using said method for increasing the oral bioavailability of a procoagulant immunoglobulin single variable 35 domain (ISVD) polypeptide or ISVD polypeptide derivative is formulated in a pharmaceutical composition comprising SNAC and optionally also NAM. 70

In a preferred embodiment an ISVD polypeptide derivative, comprises an anti-FX ISVD connected to an anti-FIX(a) V _H H ISVD in a N-terminal to C-terminal direction (N- to C- terminal). In one such preferred embodiment an ISVD polypeptide derivative, comprises an anti-FX 5 V _H H fragment connected to an anti-FIX(a) V _H H fragment in a N-terminal to C-terminal direction (N- to C-terminal), see for example Figures 2a, 2b and 3a-f. In preferred embodiments the ISVDs or V _H H fragments capable of binding to FIX(a) preferentially binds to activated FIX (FIXa). In preferred embodiments the ISVDs or V _H H fragments capable of binding to FX 10 preferentially binds to FX zymogen, i.e. FX which has not been activated. In one embodiment the ISVD polypeptide (derivatives) or V _H H polypeptide (derivatives) of the invention is/are capable of stimulating the enzymatic activity of FIXa towards FX. In one such embodiment the stimulatory properties of said ISVD polypeptide (derivatives) or V _H H polypeptide (derivatives) is derived from the ISVD or V _H H capable of binding FIX(a). 15 Fig.5a and 5b show sequence alignments of preferred anti-FIX(a) and anti-FX ISVD (V _H H fragment) sequences, respectively, wherein CDR sequences are highlighted in bold and underline. The present invention encompasses substitution variants of the ISVDs or V _H H fragments as disclosed herein, which may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 16, 17, 18, 19 or 20 amino acid substitutions and/or deletions and/or insertions in the individual sequences disclosed herein. In some embodiments said substitutions and/or deletions and/or insertions are in one or more of the CDR sequences as disclosed herein. In some embodiments one or more of the CDR sequences is subject to amino acid substitution. In some embodiments the individual CDR sequences may comprise 0, 1, 2 25 or 3 amino acid substitutions each. For example, in the V _H H fragment according to SEQ ID NO:559, CDR1 may comprise no substitutions, CDR2 may comprise 3 substitutions and CDR3 may comprise 1 substitution. Substitution variants preferably involve the replacement of one or more amino acid(s) with the same number of amino acid(s). In some embodiments the substitution is a conservative substitution. 30 In one embodiment the ISVD polypeptide derivative, such as a V _H H polypeptide derivative comprises at least one protraction moiety which is not capable of binding components of a plasma membrane, such as aminophospholipid, such as a phosphatidylserine and/or phosphatidylethanolamine. 35 In one embodiment the protraction moiety comprises the structure: 71

wherein ‘*’ represents a point of attachment to the V _H H polypeptide. In one embodiments the ISVD polypeptide derivative, such as a V _H H polypeptide 5 derivative comprises a protraction moiety which is not capable of binding platelet surface proteins/markers, such as GPlb-lX, collagen chaperone HSP47, ephrin B1, thiol isomerase protein ERP5, Hematopoietic progenitor kinase 1-interacting protein of 55 (HIP-55), glycoprotein Vl, platelet glycoprotein 1b, platelet-derived growth factor receptor, platelet endothelial aggregation receptor I, CD31, CD36, MARKS, multimerin, integrin 10 alpha lIb/beta 3, triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1), integrin-linked kinase (ILK), zyxin, collagen, P-selectin, Factor XIII, P-selectin glycoprotein ligand-1, integrin alpha 6 beta 1, thrombospondin, von Willebrand factor, G6B, CD42b, syntaxin binding protein 2, phosphatidylethanolamine, fibrinogen/fibrin, filamin, stomatin, sphingolipid, CD31, CD36, CD40, CD41, CD42c, CD42, CD49b, CD61, 15 CD62P, CD63, CD69, CD107a, CD107b, CD109, CD154, PECAM-1, and/or ERPS. In one embodiment the ISVD polypeptide derivative, such as a V _H H polypeptide derivative comprises a protraction moiety which is not capable of binding membrane associated polypeptides, such as glycoproteins, GPIIb/IIIa, β2GP1, TLT-1, selectins, a coagulation factor or coagulation factor complex and/or a selectin. 20 In one embodiment the ISVD polypeptide derivatives, such as V _H H polypeptide derivatives, of the invention do not interfere with the effect of FVIII, such as recombinant FVIII administered to a patient suffering from haemophilia A, when said polypeptide derivatives are used in clinically relevant dosages in the treatment of haemophilia A. 25 While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. 30 72

EXAMPLES LIST OF ABBREVIATIONS AIEX: Anion-exchange 5 CC: Pearson correlation coefficient CDR: Complementarity Determining Region CHO: Chinese Hamster Ovary CMK: Chloromethylketone EC ₅₀ : Half maximal effective concentration 10 EDTA: Ethylenediaminetetraacetic acid ELISA: Enzyme-Linked Immunosorbent Assay FACS: Fluorescence-Activated Cell Sorter FIX: Coagulation Factor IX FIXa: Coagulation Factor IXa 15 FX: Coagulation Factor X FXa: Coagulation Factor Xa FR: Framework Region GS: Glutamine Synthetase HA: Haemophilia A 20 HB: Haemophilia B ISVD: Immunoglobulin Single Variable Domain IV: Intravenously KO: Knock-Out MSX: L-Methionine sulfoximine 25 MRTHL: Mean residence time terminal half-life NAM: Nicotinamide OEG: 8-amino-3,6-dioxa-octanoic acid PBMC: Peripheral Blood Mononuclear Cells PCR: Polymerase Chain Reaction 30 pI: Isoelectric point PK: Pharmacokinetic PO: Perorally ROUT: Robust regression and Outlier removal (GraphPad Prism function) RMS: Root-mean-square 35 RT-PCR: Reverse Transcriptase Polymerase Chain Reaction 73

RP-UPLC: Reverse-Phase Ultra-Performance Liquid Chromatography SIA: Sequence Identical Analogue SC: Subcutaneous SNAC: Sodium N-(8-[2-hydroxybenzoyl] Amino) Caprylate 5 SPR: Surface Plasmon Resonance TGT: Thrombin Generation Test TLS: Translation-libration-screw-rotation TVT: Tail-Vein Transection UPLC: Ultra-Performance Liquid Chromatography 10 EXAMPLE 1: GENERAL METHODS & PREPARATION OF V _H H FRAGMENTS AND OTHER RECOMBINANT PROTEINS General molecular biology For general molecular biology techniques, see Molecular Cloning: A Laboratory Manual 15 (4 ^th Edition, 2014, Sambrook, Fritsch and Maniatis eds., CSHL Press, Cold Spring Harbor, NY USA). Immunizations and libraries After approval of the Ethical Committee of the Ablynx Camelid Facility (LA1400575), two 20 llama and two alpaca were immunized with human FIX and FX (Haemotologic Technologies, VT USA), respectively. Cloning of heavy chain-only antibody fragment repertoires and preparation of phage immune libraries was performed as follows. Following the final immunogen injection, blood samples were collected. From these blood25 samples, peripheral blood mononuclear cells (PBMCs) were prepared using Ficoll- Hypaque according to the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ, US). From the PBMCs, total RNA was extracted and used as starting material for RT-PCR to amplify the V _H H fragment-encoding DNA segments, essentially as described in WO2005/044858. In short, the V _H H fragment-encoding DNA fragments were 30 cloned into phagemid vector pAX212 enabling production of phage particles displaying V _H H fragments fused with His ₆ - and FLAG ₃ -tags. Subsequently, phages were prepared and stored according to standard protocols. 74

Synthetic libraries Synthetic libraries were generated by cloning synthetic V _H H gene fragments obtained and cloned from the immunizations into phagemid vector pAX190, which has the same features as the above described pAX212, but with differences in the multiple cloning site. 5 Library screening of V _H H fragments binding to FX or FIX V _H H fragment Phage Display selections were performed with the generated immune and synthetic libraries. The libraries were subjected to one to four successive rounds of enrichment against different concentrations of immobilized human FIX and FX such to 10 enrich clone screening to obtain better binders to FIX and FX (Haemotologic Technologies, VT USA) and cynomolgus FIX and FX (Novo Nordisk generated in house). In order to specifically enrich for V _H H fragments that were selective for FIXa and FX versus FIX and FXa, respectively, in certain experiments excess soluble FIX and FXa was used for competition during the incubation of the libraries with the immobilized FIXa and 15 FX. In order to specifically enrich for V _H H fragments that were selective for FIX and FX versus other structurally related coagulation factors, in certain experiments excess soluble FX and FIX, respectively, was used for competition during the incubation of the libraries with the immobilized FIX and FX. 20 Approximately 4500 individual clones from the selection outputs were screened for binding in ELISA (using periplasmic extracts from E.coli cells expressing the V _H H fragments) against human and cynomolgus FX, FXa, FIX and FIXa. Approximately 1500 clones each that showed specific binding to human FX and FIXa/FIX were identified, of which the majority showed cross-binding to cynomolgus FX and FIX. Some clones 25 showed preferential binding to FX versus FXa and FIXa versus FIX. Sequence analysis of the ELISA positive clones identified approximately 700 unique sequences of V _H H fragments binding to FIXa/FIX or FX/FXa. To optimize compounds further, intensive V _H H CDR and FR mutagenesis combined with protractor additions, linker and extension combinations were conducted with focus on enhancing the haemostatic potency via30 mutational screenings using a thrombin-generation assay, avoid target-mediated drug- disposition via screening for compounds with altered affinities, reducing immunogenicity via screening for risk sites using a MHC-associated peptide proteomics assay and enhancing in vivo oral bioavailability via pI lowering substitutions of surface-exposed residues. 35 75

Expression construct generation of V _H H fragments binding to FX or FIX Sequence analysis of V _H H fragments from phage display selection outputs was done according to commonly known procedures (Pardon et al. (2014) Nat Protoc 9: 674). V _H H fragment-containing DNA fragments, obtained by PCR with specific combinations of 5 forward FR1 and reverse FR4 primers each carrying a unique restriction site, were digested with the appropriate restriction enzymes and ligated into the matching cloning cassettes of V _H H polypeptide expression vectors as His ₆ - and/or FLAG ₃ -tagged formats for E. coli or P. pastoris expression.The ligation mixtures were then transformed to electrocompetent Escherichia coli TG1 (60502, Lucigen, Middleton, WI) or TOP10 10 (C404052, ThermoFisher Scientific, Waltham, MA) cells which were then grown under the appropriate antibiotic selection pressure (kanamycin or Zeocin). Resistant clones were verified by Sanger sequencing of a plasmid DNA (LGC Genomics, Berlin, Germany). Generic expression of V _H H fragments binding to FX or FIX in E. coli 15 V _H H fragments were expressed in E. coli TG1 from a plasmid expression vector containing the lac promoter, a resistance gene for kanamycin, an E. coli replication origin and a V _H H fragment cloning site preceded by the coding sequence for the OmpA signal peptide. In frame with the V _H H polypeptide coding sequence, the vector codes for a C- terminal FLAG ₃ and His ₆ tag. The signal peptide directs the expressed V _H H fragments to 20 the periplasmic compartment of the bacterial host. E. coli TG-1 cells containing the V _H H fragment constructs of interest were grown for 2 hours at 37°C followed by 29 hours at 30°C in baffled shaker flasks containing “5052” auto-induction medium (0.5% glycerol, 0.05% glucose, 0.2% lactose + 3 mM MgSO ₄ ). Overnight frozen cell pellets from E. coli expression cultures were then dissolved in PBS 25 (1/12.5th of the original culture volume) and incubated at 4°C for 1 hour while gently rotating. Finally, the cells were pelleted down once more and the supernatant, containing the proteins secreted into the periplasmic space, stored. Generic expression of V _H H fragments in P. pastoris 30 P. pastoris cells containing V _H H fragment constructs of interest were grown for two days (at 30°C, 200rpm) in BGCM medium. On the third day, the medium was switched to BMCM and the constructs were further grown (at 30°C, 200rpm) and induced with 0.5% v/v methanol after 8 hours. Next day the constructs were induced with 0.5% v/v methanol in the morning, at noon and in the evening. On the fifth day, the cells were spun down and 35 the supernatant (containing the secreted V _H H fragment) collected. 76

Generic expression of V _H H and antibodies binding to FX or FIX and other recombinant proteins in HEK cells Expression plasmids for transient expression in HEK293 cells were purchased from either Twist Biosciences or Thermo Fisher Scientific. Plasmids from Twist Biosciences were 5 based on the pTT vector described in Durocher, Y. et al., (2002) Nucleic Acid Res, 30: E9 while plasmids from ThermoFisherScientific were based on the pcDNA34-Topo vector (Thermo Fisher Scientific). His ₆ -tagged or non-tagged V _H H polypeptide compounds were transfected into HEK293 suspension cells in order to transiently express V _H H polypeptide compounds. Equivalent expression constructs harbouring sequences coding for anti-GLA 10 FIX and anti-GLA FX antibodies (Novo Nordisk in-house generated), emicizumab (Hoffmann-La Roche Ltd, Switzerland) sequence identical analogue (SIA) and Mim8 (Novo Nordisk, Denmark) were also expressed in HEK293 using the method described below. 15 Transient transfection of HEK293 suspension cells (Expi293 expression system, Thermo Fisher Scientific, catalogue number A14635) were performed essentially following manufacturer’s instructions. HEK293 cells were typically subcultivated every 3-4 days in Expi293 expression medium (Gibco, catalogue number A14351-01) supplemented with 1% P/S (GIBCO, catalogue number 15140-122). HEK293 cells were transfected at a cell 20 density of 2.5-3 mill/mL using Expifectamine. For each litre of HEK293 cells, the transfection was performed by diluting a total of 1 mg of plasmid DNA into 50 mL Optimem (GIBCO, catalogue number 51985-026, dilution A) and by diluting 2.7 mL Expifectamine into 50 mL Optimem (dilution B). For co-transfections (i.e. for antibodies), plasmids were used in a 1:1 ratio. Dilution A and B were mixed and incubated at room 25 temperature for 10-20 minutes. The transfection mix was hereafter added to the HEK293 cells and cells were incubated at 37 ^C in a humidified incubator with orbital rotation (85- 140 rpm). One day post-transfection, transfected cells were supplemented with 5 ml of ExpiFectamine 293 Transfection Enhancer 1 and 50 ml of ExpiFectamine 293 Transfection Enhancer 2. Cell culture supernatants were typically harvested 4-5 days 30 post-transfection by centrifugation followed by filtration. Generic expression of V _H H fragments and antibodies binding to FX and FIX(a) in CHO cells 35 Anti-FIX and anti-FX V _H H compounds and antibodies were produced in a Chinese Hamster Ovary (CHO) cells using glutamine synthetase (GS) selection. CHO cells were 77

transfected with GS expression plasmids using electroporation and hereafter subjected to selection using glutamine deprival together with MSX supplement in CD-CHO medium (Thermo Fisher Scientific, catalogue number 10743029). Stable CHO cell poolswere typically obtained after 3 weeks of culturing and pools were hereafter single-cell 5 clonedinto 384 well plates. CHO clones arising in 384 well plates were typically expanded into 96 well plates and screened for productivity. Selected producer clones were upscaled for cultivation in bioreactors at 1-L to 15-L scale using a pre-defined proprietary cell culture media (Novo Nordisk A/S). Cell viability was kept high during cultivation with a progressive decrease following this until cell culture harvest.. Here, cell supernatants 10 were cleared by centrifugation and/or depth-filter filtration using MD0HC23CL3 and MX0HC01FS1 filters (Millipore) depending on cultivation scale before proceeding with chromatography-based protein purification. Generic purification and characterization of V _H H fragments binding to FX or FIX 15 His6-tagged or non-tagged V _H H compounds were purified by MabSelectSure Protein-A resin (Cytiva) or immobilized metal affinity chromatography (IMAC) on either Ni-Excel (Cytiva) resin with Imidazole (for the latter) or acidic elution (for the former) followed by a desalting step (PD columns with Sephadex G25 resin, Cytiva) and if necessary, gel filtration chromatography (Superdex200 column, Cytiva) in PBS or HBS. Non-His6-tagged 20 VHH compounds and antibodies targeting the GLA domains of FIX and FX, respectively (generated in-house at Novo Nordisk) and emicizumab SIA (Hoffmann-La Roche Ltd, Switzerland) were purified by Protein-A resins MabSelectSure (Cytiva) or multimodal resins (Cytiva) with acidic elution followed by a desalting step (e.g. PD columns with Sephadex G25 resin, Cytiva) and if necessary, gel filtration chromatography (e.g. 25 Superdex200 column, Cytiva) in PBS or HBS. Protein integrity was analysed using a Size-Exclusion High-Performance Liquid Chromatographic (SE-HPLC) method setup on an Agilent LC 1100/1200 system and using a BIOSEP (column for separation biomolecules)-SEC-53000300×7.8 mm column (Phenomenex, cat. no. OOH-2146-K0) and a running buffer composed of 200 mM NaPhosphate pH 6.9, 300 mM NaCl and 10 % 30 isopropanol. The molecular masses of the purified V _H H polypeptide batches weres analysed using ElectroSpray Ionization Time of Flight Mass Spectrometry (ESI-TOF-MS) on a 6280 Agilent system (Agilent Technologies) with a MassPREP Desalt (Waters) column run at 0.4 ml/min in A-buffer composed of MQ-H2O/0.1% formic acid and B-buffer composed of acetonitrile/0.1% formic acid for step elution. To measure the final protein 35 concentration, a NanoDrop ^TM spectrophotometer (Thermo Scientific) was used with theoretical calculated extinction coefficients. 78

EXAMPLE 2: CONJUGATION OF PROTRACTION MOIETY TO V _H H POLYPEPTIDE CYSTEINE RESIDUE In order to protract V _H H polypeptides to improve pharmacokinetic properties, V _H H polypeptide compounds were engineered such to comprise Cysteine (Cys) substituents in 5 various backbone positions e.g. in N- or C-terminal extensions with one or two introduced Cys residues used for conjugation with one or more fatty acid protraction moiety(ies) as described further below. In order to attach the protraction moiety to the V _H H polypeptide compounds, an 10 intermediate reagent in the form of a modified protraction moiety was used. The intermediate reagent comprising the protraction moiety was prepared as described in WO2016/102562 and non-limiting examples of such intermediate reagents are shown in table 3 below: 79

_, D _I ¹ _{1 , e} ^{# v} _, ⁷ _{1 i} ^{t 0} a ₁ ^# _, ^{6 2 vi #} _{1 2 r} ^{, # # e} ₉ ^# _{, , d} ^{, 5} ₁ ¹ e ^d ₈ ^# ₂ ^{# i} _t ^# _{, ,} p ^, e ₇ ^{4 0 #} _{1 2} p ^{5 , # #} y ^l _{6 6 , , o} ^{# # 3 9 p d} ₁ ^# ₁ ^# H _p ^d _p ^{, ,} H m m _{2 8} V C C ¹ _# ¹ _# ) ₎ L ^{P ( )} r _e - _l - _l e _k m ^y _h ^{y t} _h ^t ₎ ^{t n} _i ^{a l} _{n .} t _e l _r ⁾ _{y )} ^t _e r ⁾ _{n y} ^e _{g c} ^{o x ni} _{h (} ^s _{o n (} ^{h e} B ^{x a} t _{g o} ^h _e ^r s ^e _e ^e _} ^a i ^y _{e t} r O ^e _{} r} y ^e _k ^{0 t} _e ^{m i} _{a x} ^o _r H _x ⁿ _{i 8 o} ⁿ _h ^{e t} _k N ^o _l ni _h ^t _d ⁱ m C _{e l n} A ^m _{d e c} ^a _i o _]l ⁱ H ^m _{]l i} P ^y _c ^a _i O N H ^{y d t} _c ^U _{I o} a _r ^t _d - _l m ^d O O - _o ⁶ l m ₁ C o ^{n r} _a ^y _t ^{e a} _b ^{2 y} p _e ^r _{1 t} ^{e a} _b ^r _{( d g} ^r _c ^a _a ^{C c} _l ( O _c ^a _a ^{i c} _{l c} ^{a n} _{u i} ^t _o s _c m ^{y d} h _{i o} ^y i ^{t c} m _h ^{t c} _i r ^u _{r p t} ^{o s} _r ^b _{e -} ⁾ _{a y c} ^{o i} _{r e o} O ^b - ^{) o} y _n ^{a m} _t ² ₍ ^{x n 2} ₍ ^x _{c o} ^{n c} _e - _{o 2} s _t ^{g n} _a ^[ _{ ^h _{t a} ^{c -} _{o 2} ^h _t ^e _{d -} ^e _{} e} ^{[ a} e ^e _r ² ₍ ^{y d} H N _{- ^e _{} t} y ^{n g -} _x ^o _n ^{u 2} } O ₍ - _x ^o _e ^p a ^e _t e ^a ₂ ^[ _{{ l 2} r _i ^d _{- h} ^{t y [} _{} { h} ^t _l ^{y e} _{t e} ²⁽ _{e o - - i} ^{2 m} _]l m ^a O ²⁽ _{e -} ^m _{o ]l} m ^{a a m d} _r ^[ - ^y _b ^{2[ y} _{b e} ^e _t ^{n 3} _{- o} ^r m _{a -} c ³ _{- o} ^r m _a ^c m ^r _I ^y _x ^a _{l e} ^o _b ^{y y t} _b ^r _{a p} O _x ^a _l o _b ^y _{p n} ^{i r c} _l ^o _r O _b ^{r r} _a ^c _l ^o _{r f a} ^{c o} - ^{y p} h ^] _{l a} y H N ^c - ^{y p} h ^] _l s ¹ - ^{t )} _e ⁾ _o m N H ¹ - ^{t y )} _e ⁾ _{o e} ^l _{p S} ^({ _o ⁿ _{i a} ^b O _S ^({ _o ^{m n} _{i a} ^b m - ^{1 m} _r O O - ⁵ _r a _{1 a} ^a _c H ₁ ^m _a ^a x _c E ^: _{3 e} ^l _b ^{a D} _{1 2} ¹ _I C C _{0 T} O W ²

_{, ,} 8 ₂ ⁰ _{, 4} ^{3 #} ₆ ^# _e m _c ^a _{e i} ^m _{c ]l} ^{y d} _]l ^{a y} _i ^{d -} _o ⁸ l ^y _t m ₁ - _{o l} ⁰ ₂ e ^a _b ^C ₍ ^y _t m e ^a _b ^C _{( c} ^{r a} _{a d} ^{i r d} o ^c _{l c c} a ^a _{a o} ^c _{i l} ^c m ^{y c} _i ^y _a o _{h r} ^{t o} m ^o _h ^t _c ^{i b} _{e -} ⁾ _{n r e} ⁾ _o ^{n 2} _{y (} ^{x a -} _{o c} ^b _{- y 2} ^{e 2} ₍ ^x _a - _o ^{c [} _{e {} ^h _{t d -} ^e _} ^a _{t 2} ^[ _{ ^h _t ^d - ^e _{} a} 2 ₍ ^{- y} _x ^p _e ^{h 2} ₍ ^{- y} _x ⁿ _{o 2} ^[ _{ ^o _} ^{o n} _{} - h} ^t _l ^y ₂ ^[ _{ ( _{- h} ^t _l ^{y 2} _{e o e o -} ^m _] m ²⁽ - ^m m 2 ^[ _{l -} ^{y a 2[} _]l ^{y a 3} _{- o b} ^r - ³ _{o b} ^{r y} m _{a x} ^{a c} _{l -} m _a ^c _l o _b ^{y y} b ^{r r} _{a p x} ^{a o o} _b ^y b ^r _{a p} ^o a ^{c c} _{l r -} ^{y p r ]} _{l a} ^{c c} _{l r} y ^p] _l 1 _{h -} ^{t y} - ) _e ⁾ _o ¹ _h ^{t y m} - ⁾ _e ⁾ _{o S} ^({ _{o -} ^{n 7} _{i a} ^b _{S r} ^({ _o ^m _{a -} ⁿ _i ^b _{r 1} ^m _a ^a _c ⁹ ₁ ^m _a ^a _{c 3 4} ^{1 C} C ₀ O W ²

⁸ ₆ ^{# d} _p m C o _h ^{x t} _{e e} ^{] h} _{) y l} x ^y - _o ^{5 h} - _{t l} e ^o - _z o ^a x r ^{2 o} _t ^e t _{8 -} ² - _- ^] H o ^{1 n} _{( i} - ₆ m ^{1 a} - _{l l} y ^y h ^t _{n e} ^{o ]} _f ^l o _u n _i ^s _] ^l m _y ^{t a} _{u )l} ^{b y} - _t ^{o e} _{x c} ^{o a} - _o ⁴ - ^{] m} _{o o} ^{r n} _{i b} - m 2 ^{( a} _l [- ^{y 2} _{h )} [ ^t _t ⁿ - _e ^] _e 2 _y ^g [- ^x _a 2 _o ^e [ r - ^{h 2} _t ^r [- ^e _{] e} 2[ ^{y k} _{n - x} ⁱ _l 2 ^{o [} _{- h} ^{t e} _l 2 ^[ _e ^o _{z -} - ^4[ _{o -} ^{x a} _{r o} ^t _e N - ₂ ^T _{( 5} ^{1 C} ₀ O W ²

Conjugation, purification, and analysis To the water solution containing the V _H H polypeptide with one or more introduced cysteine residue(s) for conjugation, 5 eq. BSPP (Bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt) or 1.1 eq TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) per capped 5 cysteine was added. After 1-2 h of stirring, pH was adjusted to 8.5 with aq. NaOH and 5 eq. of e.g. intermediate reagent C1 in 0.1 M NaHCO ₃ (aq.) per free cysteine in the respective V _H H polypeptide were added. The mixture was stirred gently in the dark for 1.5-16 hours. The reaction mixture was diluted with water before purification by anion exchange (AIEX) method using an Äkta system. The V _H H polypeptide with the side-chain conjugation was purified using 10 an AIEX chromatography separation method. Thus, the AIEX resin Source 30Q packed in a suitable column was used together with a sodium chloride gradient program setup on an Äkta Avant chromatography system. The buffer systems used were an equilibration buffer composed of 20 mM Tris, pH 8.5 and an elution buffer composed of 20 mM Tris, 1 M NaCl, pH 8.5. The reaction mixture was adjusted to pH 8.5 and diluted to a conductivity below 4 mS/cm using 15 MilliQ-H ₂ O or equilibration buffer. The sample was applied to the column and the column was washed after application with 5 to 10 column volumes of equilibration buffer. Separation chromatography was then performed using a shallow gradient of 30 to 50 column volumes. The gradient used was from 0 % and up to 50 % depending on pI of the V _H H polypeptide derivative that was purified. Generally, the non-conjugated parental V _H H polypeptides eluted early in the 20 gradient, VHH polypeptides with single conjugation eluted in the middle of the gradient and VHH polypeptides with multi-conjugations, meaning more than one side-chain conjugation per V _H H polypeptide molecule, eluted late in the gradient. Pooling of fractions over the main peak was conducted in a way, so high purities between 90 to 99% of V _H H polypeptide with single or double conjugate preparations were obtained. The purity analysis was performed using a 25 reverse-phase ultra-performance liquid chromatography (RP-UPLC) method based on HALO DiPhenyl column 1000 Å, 2.7 µm, 150x2.1 mm (Scantec Nordic USDPF001316) and running buffers composed of A) 0.1% v/v TFA in water and B) 0.09% v/v TFA in acetonitrile setup on a Waters Acquity UPLC system with UV and FLD detector. Column temperature was set to 60°C. The gradient program was 1) 0.0-8.0 min: 20-50% B, 2) 8.0-8.1 min: 50-80% B, 3) 8.1-9.0 min: 30 80% B, 4) 9.0-9.1 min: 80-20% B, and 5) 9.1-11.0 min: 20% B. The un-conjugated parental V _H H polypeptide eluted between 4.6-4.8 min. The V _H H polypeptide derivative with single conjugation eluted between 5.1-5.6 min with main peak at approx.5.3 min. V _H H polypeptide derivative with double conjugations eluted between 5.7-5.8 and later for offsite multi-conjugated V _H H polypeptides. Integrity of V _H H polypeptides with conjugation(s) were analysed using a SE-HPLC 35 method setup on an Agilent LC 1100/1200 system and a BIOSEP-SEC-3000300×7.8 mm column (Phenomenex, cat. no. OOH-2146-K0) and a running buffer composed of 200 mM 83

NaPhosphate pH 6.9, 300 mM NaCl and 10 % isopropanol. The molecular masses of V _H H polypeptide with conjugation(s) were analysed using ESI-TOF-MS on a 6280 Agilent system (Agilent Technologies) with a MassPREP Desalt (Waters) column run at 0.4 ml/min in A-buffer composed of MilliQ-H ₂ O/0.1% formic acid and B-buffer composed of acetonitrile/0.1% formic 5 acid for step elution. Peptide-mapping for sequence verification was performed using a combination of chymotrypsin and trypsin based digests. LC-MS system consisted of a waters Aquity UPLC combined to a Thermo Orbitrap Fusion instrument. On-line LC-MS analysis of the digests was done using a CSH C-18 column 1.7 µm, 150x2.1 mm and an acetonitril/formic acid gradient. Column temperature was 60C. Buffer A: 0,1%FA Water and Buffer B: 0,1%FA 10 Acetonitrile. The gradient program was :1) 0.0-2.0 min: 1% B, 2) 2-50 min: 1-35% B, 3) 50-51 min: 100% B and 4) 51-60 min: 100-1% B with a flow of 120 µl/min. Data were analysed using a Genedata Refiner peptide mapping workflow. Full coverage of the primary sequence was obtained. To measure protein concentration of batch preparations of VHH polypeptide derivative with conjugation(s), a NanoDrop ^TM spectrophotometer (Thermo Scientific) was used with 15 theoretical calculated extinction coefficients. Figures 1, 2 and 3 show – with increasing level of details - non-limiting examples of ISVD- and V _H H polypeptide derivatives. 84

_{n s .} ^o O m _{r i} e ^{s N t} _{- n} ^{e D} I _{5 6 6 7 7 8 9 9 9} C _t ^x Q D ^D I ^{t t} - N _n A ^Q A ^N _{( E} ^{D 1} I _. ³ _. ⁴ _. ⁵ _. ⁶ _. ⁷ _. ⁸ _. ⁹ _. S ^{S 1 e 1 1 1 1 1 1 1} H _{- n - - - - - - - E} C ^{d N} _n ^H H a V ^{H o} _n H H H ^H H H H H H H H H H H ^s _{V V V V V V V V E} U _d Q ^{n E} _{u s S} ^o F _{p . n} m ^o _i O N ^{e e e e e e e e e} O ^m _{o r} ^{e s} _n ^{D I} _n ^o _n ^o _n ^o _n ^o _{n n n n n S} ^c _t- ^e _t Q _{n n n n} ^o _n ^o _n ^o _n ^o _n ^o _n E _{f L} ^o N ^x _e ^E B w _S A ^{e T} _i : ^v _r ) 3 ^e _{e t} ) ) v ^d _{i 1} ⁿ _{t 2} ⁿ _{t 3} ⁿ _{4 5 6 7 8 9 E} ^{O t} _p ^# _e ^l _a ^# _e ^l _a ^# _e ^{l # # # # # # L} P ^: H 4 M V ^{H e} _p ^D _I ^d _p ^{v d v d} _a ^{v d} _p ^d _p ^{d d d d} m _o ⁿ _p m _o ⁿ _{p p p p p} m _o ⁿ A _e ^{l y} _l m m m m m m b ^o C _o C _o C _o C C C C C C X _p ^m ₍ ^{m m 1} _{0 E} ^a _{T ( (} O W ² ₂

- _i ^{t e} n ^t V _{( -} A ^N ₍ D ₀ I ¹ _{1 .} ¹ ₂ ¹ ₃ ¹ ₄ ¹ ₅ ¹ ₆ ¹ _{7 8} H ¹ _{. . . . . .} ¹ _. ¹ _. H - ¹ H - ¹ - ¹ - ¹ - ¹ - ¹ - ^{1 1 H} H H H H H H - H - H V V ^H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V _{n s .} O m _r ^o _i ^{s N e} _n ^e _n ^e _n ^e _n ^e _n ^e _n ^e _n ^e _n ^{e e} _{t- n} ^{e D I o o o o o o o o} _n ^o N _t ^x Q _{n n n n n n n n n e} ^E S _e d _i ^{t 0} ₁ ^{1 2 3 4 5 6 7 8 #} ₁ ^# ₁ ^# ₁ ^# _{1 1 1 1 1} H V ^H _p ^{# # # # # e} _p ^{D y} _I ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p l} o m m m m m m m m m p C C C C C C C C C ¹ ₀ O W ² ₂

- _i ^{t e} V _{( n} ^t - A ^N ₍ D ₉ I ¹ _{0 .} ² ₁ ² ₂ ² ₃ ² _{4 5 6} H ¹ _{. . . .} ² _. ² _. ² _. H - ¹ H - ¹ H - ¹ H - ¹ H - ¹ H - ^{1 1} H - H - H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V _{. n s} O m _r ^o _i e ^{s N e e e e} t _{- n} ^{e D I} _n ^o _{n n n 0 n} ^o _n ^{o o 9} _{0 0 0 6} ⁹ ₆ ⁹ ₆ ⁹ ₆ N _t ^x Q _{n n e} ^E S _e d _i ^{t 9} ₁ ^{0 1 2 3 4 5 6 #} ₂ ^# ₂ ^# ₂ ^# ₂ ^# _{2 2 2} H V ^H _p ^{# # # e} _p ^{D y} _I ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p l} o m m m m m m m m C C C C C C C C ¹ _{p 0} O W ² ₂

F- ^m i _r ^{H K} _{( ( ( ( ( ( ( (} t ^e n ^t V _{( -} A ^N ₍ D ₇ I ² _{8 .} ² ₉ ² ₀ ³ ₁ ³ ₂ ³ ₃ ³ ₄ 1 _. ¹ _. ¹ _{. . . .} ³ _. H ^H - H - H - ¹ H - ¹ H - ¹ H - ¹ H - ¹ H - H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V _{s . n} O m _r ^o _i ^{N e s} _{0 0 0 0 0 0 0 t- n} ^{e D 9 9 9 9 9 9 9} ₀ ⁹ t ^I _{6 6 6 6 6 6 6 6} N ^x Q e ^E S _e d _i ^{t 7} ₂ ^{8 9 0 1 2 3 #} ₂ ^# _{2 3 3 3 3} ⁴ ₃ H V ^H _p ^{# # # # # # e} _p ^{D y} _I ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p l} o m m m m m m m m C C C C C C C C ¹ _{p 0} O W ² ₂

_. t- _{) ( I} - _i ^{t i} X F- ^m _{r a} V ^{H K} _{( ( ( ( ( ( ( ( ( i} ^{t e} n ^t - A ^N ₍ D ₅ I ³ _{6 7 8 9 .} ³ _. ³ _. ^{3 3} ₀ ⁴ ₁ ⁴ ₂ ⁴ H ^{1 H} - ^{1 1} _. ¹ _. ¹ _. ¹ _. ¹ _. ¹ H - H - H - H - H - H - H - H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V _{n s . r} ^o _i O m ^{N e e e s} t _{- n} ^{e D I} ₀ ⁹ _{0 6} ⁹ _{0 6} ⁹ _{0 0 0 n n 6} ⁹ ₆ ⁹ ₆ ⁹ ₆ ^{o o} N _t ^x Q _{n n e} ^E S _e d _i ^{t 5} ₃ ⁶ ₃ ⁷ ₃ ⁸ ₃ ⁹ ₃ ⁰ ₄ ¹ ₄ ² ₄ H V ^H _p ^{# # # # # # # # e} _p ^{D y} _I ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p l} o m m m m m m m m p C C C C C C C C ¹ ₀ O W ² ₂

- _i ^{t e} V _{( n} ^t - A ^N ₍ D ₃ I ⁴ _{4 .} ⁴ ₅ ⁴ ₆ ⁴ ₇ ⁴ ₈ ⁴ _{9 0} H ¹ _{. . . . .} ⁴ _. ⁵ _. H - ¹ H - ¹ H - ¹ H - ¹ H - ¹ H - ¹ H - ¹ H - H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V _{n s . r} ^o O m _i e ^{s N e} t _n ^e _n ^e _n ^e _n ^e _n ^e _n ^e _n ^e _{n - n} ^{e D I o o o o o o o o} N _t ^x Q _{n n n n n n n n e} ^E S _e d _i ^{t 3} ₄ ^{4 5 6 7 8 9 0 #} ₄ ^# ₄ ^# ₄ ^# _{4 4 4 5} H V ^H _p ^{# # # # e} _p ^{D y} _I ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p l} o m m m m m m m m C C C C C C C C ¹ _{p 0} O W ² ₂

F- ^m i _r ^{H K} _{( ( ( ( ( ( ( (} t ^et V _{( n -} A ^N ₍ D _{1 2} I ⁵ _. ⁵ ₃ ⁵ ₄ ⁵ ₅ ⁵ ₆ ⁵ ₇ ⁵ ₈ ^{5 1} _. ¹ _. ¹ _. ¹ _. ¹ _{. . .} H ^H - H - H - H - H - ¹ H - ¹ H - ¹ H - H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V _{n s .} O m _r ^o _i ^{s N e} _n ^{e e e e e e e e} _{t- n} ^{e D I o} _{n n} ^o _{n n} ^o _{n n n n n n} ^o _n ^o _n ^o _n ^{o o} N _t ^x Q _{n n e} ^E S _e d _i ^{t 1} ₅ ² ₅ ³ ₅ ⁴ ₅ ⁵ ₅ ⁶ ₅ ⁷ ₅ ⁸ ₅ H V ^H _p ^{# # # # # # # # e} _p ^{D y} _I ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p l} o m m m m m m m m p C C C C C C C C ¹ ₀ O W ² ₂

F- ^m i _r ^{H K} _{( ( ( ( ( ( ( (} t ^et V _{( n -} A ^N ₍ D _{9 0} I ⁵ _. ⁶ ₁ ⁶ ₂ ⁶ ₃ ⁶ ₄ ⁶ ₅ ⁶ ₆ ^{6 1} _. ¹ _. ¹ _. ¹ _{. . . .} H - H - H - H - ¹ H - ¹ H - ¹ H - ¹ H - H V ^{H H} V ^H V ^H V ^H V ^H V ^H V ^H V ^H V _{. n s} O m _r ^o _i e ^{s N e e e e e} _{0 0 0 t- n} ^{e D I} _n ^o _{n n} ^o _{n n n n} ^o _n ^o _n ^o _n ⁹ ₆ ⁹ ₆ ⁹ ₆ N _t ^x Q e ^E S _e d _i ^{t 9} ₅ ^{0 1 2 3 4 #} _{6 6 6 6 6} ⁵ ₆ ⁶ ₆ H V ^H _p ^{# # # # # # # e} _p ^{D y} _I ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p l} o m m m m m m m m p C C C C C C C C ¹ ₀ O W ² ₂

V ⁿ X _i ^{E F} - ^m _S H ^b _{a 2} ² _{2 (} ² ₍ ⁹ _{3 2} ² _{2 2 2 0 (} ^{2 2 2 6} i _r ^{H K} _{( ( ( (} t ^et V _{( n -} A ^N ₍ D ₇ I ⁶ _{8 .} ⁶ ₉ ⁶ _{0 1 2 3 4} H ¹ _{. .} ⁷ _. ⁷ _. ⁷ _. ⁷ _. ⁷ _. H - ¹ H - ¹ H - ¹ H - ¹ H - ¹ H - ¹ H - ¹ H - H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V ^H V _{. n s} O m _r ^o _i e ^{s N} _{0 0} ^e t _{- n} ^{e D I 9} _n ^e _n ^e _{0 6} ⁹ ₆ ^o _{3 4 n} ^o _{n n} ^o _n ⁹ ₆ N _t ^x Q e ^E S _e d _i ^{t 7} ₆ ^{8 #} ₆ ^{9 #} ₆ ^{0 #} ₇ ^{1 #} ₇ ² ₇ ³ ₇ ⁴ ₇ H V ^H _p ^{# # # # e} _p ^{D y} _I ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p l} o m m m m m m m m p C C C C C C C C ¹ ₀ O W ² ₂

H ^H H ^o _n ^o _n ^r _t o ^o _r ⁿ _e N N N V ^H V _r p ^p _f m g ^{o i} ₎ ^h _{c s n d} ^s ₍ ^a _t ^{t r} O ) ^{N u} _l ^e _t ⁱ _a ^t _{n -} ^p A ^N ₍ H ^t H _o ^{r D} ₅ I ⁷ _{5 3 .} ^{1 1 V 1} _{. . P -} ¹ - ^{1 )} H V ^H H H H - H _a ⁽ X _V ^H V ^H V ^I _F ^{- r} i _{o t} ^{t n} _c ^a _{e e e r )} P ^{n n n} s _a ^{/ t} _{( o} N _{o o . n} O _X F _o ^r N N m _r ^o _i ^{N e e e} - _{i P} e ^s t _{- n} ^{e D I} _n ^o _n ^o _n ^{t o} _n ^a N _t ^x Q _{n n n f e} ^E S ^{o D} _I w ^e _e d ) ) _i ^v i _{t 1 2 3 e t t r} ^{p t} _e ^{# # # d} _i ⁵ ₇ ^{6 #} ₇ ^{n #} _e ^{7 l} ₇ ^{n #} _e ^{e l} _v ^{p d} y _p ^d _p ^d _p H V ^H _p ^e _{a a} ^{O l} m m m p ^{D y} _I ^d _p ^d _p ^v _o ^{d v} _o ^: _{5 o} C C C l ⁿ _p ^{n o} m m _o m _{o e} ^{p l} H p C C ^m C ^m _b V ^{H 1} _{( (} ^a _{0 T} O W ² ₂

c ^a _r ^t _o r P r _o ^t _c ^a _{r )} P ³ P ³ P ² P ^{2 2 2 2 2 2 t} _{o ( P P P P P P} r P D _{I e} di _t p _{4 e} ^# ₅ ^# ₆ ^# ₇ ^# ₈ ^# ₉ ^{0 #} ₁ ¹ ₁ ² ₁ d ^{d d d d # # # p} _y ^l _{p p p p p} ^d _p ^d _p ^d _p ^d _{p o} m m m m m m C C C C m m m p C C C C C H VH ¹ ₀ O W ² ₂

c ^a _r ^t _o r P r _o ^t _c ^a _{r )} P ² P ² P ² P ² P ² P ^{2 2 2 2 t} _{( P P P P o} ^r P D _{I e} di _t ³ ₁ ⁴ ₁ ⁵ ₁ ⁶ ₁ ⁷ ₁ ⁸ ₁ ⁹ ₁ ^{0 1 p # # # #} _{2 2 e} ^{# # # # # p} _y ^{d l} _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p o} m m m m m m m m m p C C C C C C C C C H VH ¹ ₀ O W ² ₂

t _c ^a _r ^t _o r P r _o ^t _c ^a _{e e e e e r )} P ^{2 n} _o ^{3 n} _o ^{3 n} _o ^{3 n} _o ^{3 n 3 t} _{( P P P P P o P o} ^r N N N N N P D _{I e} di _t ² ₂ ³ ₂ ⁴ ₂ ⁵ ₂ ⁶ ₂ ^{7 8 9 0 1 2 p # #} _{2 2 2 3 3 3 e} ^{# # # # # # # # # p} _y ^{d l} _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p o} m m m m m m m m m m m p C C C C C C C C C C C H VH ¹ ₀ O W ² ₂

a _r ^t _o r P r _o ^t _c ^a _{r ) e e e e e e e e e} P ⁿ _o ^{3 n} _o ^{3 n} _o ^{3 n} _o ^{3 n} _o ⁿ _o ^{n n n t} ₍ N _P N _{P P P o o o o} ^r N N N N N N N P D _{I e} di _t ³ ₃ ⁴ ₃ ⁵ ₃ ⁶ ₃ ⁷ ₃ ^{8 9 0 1 2 3 4 5 p} _e ^{# # # # #} ₃ ^# ₃ ^# ₄ ^# ₄ ^# ₄ ^# ₄ ^# ₄ ^# ₄ ^{# p} _y ^{d l} _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p o} m m m m m m m m m m m m m p C C C C C C C C C C C C C H VH ¹ ₀ O W ² ₂

^t _c ^a _r ^t _o r P r _o ^t _c ^a _{e e e e e e e e e e e r )} P ⁿ _o ⁿ _o ⁿ _o ⁿ _o ^{n n n n n n n} _e ^{n 3 3 t} ₍ N N N N _o N _o N _o N _o N _o N _o N _o N _o N _{P P o} ^r P D _{I e} di _t ⁶ ₄ ⁷ ₄ ^{8 9 0 1 2 3 4 5 6 7 8 9 p # #} ₄ ^# ₄ ^# ₅ ^# ₅ ^# ₅ ^# ₅ ^# ₅ ^# ₅ ^# ₅ ^# _{5 5 5 e} ^{# # # p} _y ^{d l} _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p o} m m m m m m m m m m m m m m p C C C C C C C C C C C C C C HH ^{1 V} ₀ O W ² ₂

a _r ^t _o r P r _o ^t _c ^a _{r ) e} t ^{P 3} P ³ P ³ P ³ P ⁿ _{e o} ^{1 3 4 5 n} _{o o (} r N _{P P P P} N P D _{I e} di _t ⁰ ₆ ¹ ₆ ² ₆ ³ ₆ ⁴ ₆ ⁵ ₆ ⁶ ₆ ^{7 8 9 p # # # #} _{6 6 6 e} ^{# # # # # # p} _y ^{d l} _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _{p o} m m m m m m m m m m p C C C C C C C C C C H VH ¹ ₀ O W ² ₂

^s ₍ ^e _t ^{t i} _{a S 1} ⁰ _{1 r} ^e _k ni _{l )} ^” _{) )} t ₁ ^” _{” ) ” y 1} ^{P P} e R i ^“ o - R ^“ _“ - -” _{“- P} ” _P ) _{” P} L ^P _” L ^{P L“ L“} _{e e} m n ^L ₍ ^“ - _” ^“ - - ^” - ₁ ^{” n} _o ⁿ _o o ^P _{” 1 “} ^P _“ ^R _“ ^R _“ N N i ^t _{c ( ( ( (} a _{3 r} ^t L ^P ₄ L ^P ₃ L ^P ₄ L ^P o r P r _{) ) ) ” ) ” o} ^{” t} ₁ ^” c ^R ₁ R ^P _“- ^P _“- a _{r ) “-} t ^P ₍ ^” _“- ^” P ^” ₁ ^” _{1 e} ⁿ _e ^{n “} _P ^“ R ^“ R ^“ _o N _{o o} ^r ₍ 6 _{( ( (} N P _P ⁶ P ⁶ P ⁶ P D _{I e} di _t ⁰ ₇ ¹ ₇ ² ₇ ³ ₇ ^{4 5 p # # # #} ₇ ^# _{7 e} ^{# p} _y ^{d d d d d d l} _{p p p p p p o} m m m m m m p C C C C C C H VH ¹ ₀ O W ² ₂

EXAMPLE 4: EPITOPE OF ANTI-FX V _H H POLYPEPTIDES BASED ON ELISA EXPERIMENT To determine whether the binding epitope of the anti-FX V _H H polypeptide designated “cmpd#1” are located inside the activation peptide (AP) of human FX (SEQ ID NO:2), an 5 ELISA assay was setup.41 unique 12-mer peptide fragments spanning the 52 residues of the activation peptide with one amino acid spacing were immobilized in microtiter plate wells followed by incubation with the V _H H polypeptide to be tested, and detection of bound ligand was performed by addition of a secondary HRP-labelled antibody. The peptides were C-terminally conjugated to biotin and 50 µL of 1 µg/mL peptide 10 solution was used for immobilization in discrete wells of the microtiter plate which was pre-coated with 1 µg/mL streptavidin. Each well was washed with washing buffer (10mM Tris, 150mM NaCl, 2.5mM CaCl ₂ , 0.05% Tween 20, pH 8.60) followed by addition of 50 µL of 2 µg/mL FLAG-tagged anti-FX VHH polypeptide or IgG antibody to be tested. After 1 hour non-bound V _H H polypeptide was washed off using washing buffer. 15 Bound anti-FX activation peptide ligand was detected by first binding a HRP-labelled secondary antibody (for FLAG-tagged V _H H polypeptide: Anti-FLAG mAb M2-Peroxidase (HRP) (Sigma Aldrich, US) for 1 hour, and then adding 100 µL TMB-1 ELISA substrate (Kem-En-Tec Diagnostics, Denmark). The minimal epitopes were then deduced from the set of peptides giving rise to a signal 20 above baseline by identifying the common sequence covered by the set of peptides (table 6) bound by each FX binder. The first residue of the epitope was defined by the last amino acid in the first consecutive, ELISA-positive peptide, while the last residue of the epitope was defined as the first residue of the last consecutive, ELISA-positive peptide. Thus, table hereunder shows ELISA signal from consecutive peptides spanning the entire 25 FX AP for initial anti-FX V _H H polypeptides tested corresponding to Cmpd #1. ELISA signals have been normalized relative to the background signal. The grey areas mark the positive binding signals used for determining the minimal epitope, which were identified as the sequence NPFDLLDF (SEQ ID NO:692). 30 The identified epitope sequence of the FX activation peptide for cmpd#1 was used for high-resolution structure determining experiments of the epitope:paratope interactions as outlined in example 6. 102 Table 6: Epitope identification Epitope residues (SEQ ID NO) Cmpd#1 t 100

Epitope residues (SEQ ID NO) Cmpd#1 FDLLDFNQTQPE 725 438 EXAMPLE 5: CRYSTALLIZATION AND PARATOPE/EPITOPE MAPPING OF ANTI-FIX V _H H FRAGMENT CMPD#2 5 The purpose of the present study was to determine the paratope and epitope residues of the V _H H fragment designated cmpd#2. Crystallisation Crystals of the V _H H polypeptide cmpd#2 mixed in a 1:1 molar ratio with human EGR- 10 CMK-inhibited Factor IXa Gla-domain-less (wild-type) (purchased from Cambridge ProteinWorks Lot# hGDFIXAWTEGR_11) were grown using the sitting drop vapour diffusion technique at 18°C. A protein solution of 100 nl 8.4 mg/ml complex in 20mM Tris- HCl, pH 7.4, 50 mM NaCl and 2.5mM CaCl ₂ was mixed with 100 nl of 0.2 M Li sulphate, 0.1 M Tris-HCl, pH 8.5, 30% (w/v) PEG 4000 as precipitant and incubated over 60 µl 15 precipitant. Crystals appeared within two weeks. Diffraction data collection The crystal was cryo-protected by addition of 1 µl of precipitant added 20 % of ethylene glycol to the crystallisation drop prior to flash cooling in liquid nitrogen. Diffraction data 20 were collected at 100K at the Swiss Light Source beamline X06DA (1.0000 Å wavelength) using a Pilatus 2M pixel detector from Dectris. Autoindexing, integration and scaling of the data were performed with programmes from the XDS package (diffracting data statistics are summarised in Table 7). 104

Structure determination and refinement The asymmetric unit contains two cmpd#2:EGR-CMK-inhibited Factor IXa Gla-domain- less complex as judged by Matthews coefficient analysis. The structure was determined by molecular replacement using Phaser as implemented in the programme suite Phenix 5 using a structure of a predetermined V _H H:FIXa complex as search model. The correct amino acid sequence was model built using COOT and thereafter the structure was refined using steps of Phenix refinement and manual rebuilding in COOT. The refinement statistics are found in Table 7. Table 7: Data collection and refinement statistics Wavelength (Å) 1.0000 Resolution range (Å) 43.26-2.7 (2.797 - 2.7) Space group P 1 Unit cell (Å, deg) 56.5164.4764.7276.11587.44985.761 Total reflections 42366 (4277) Unique reflections 23473 (2340) Multiplicity 1.8 (1.8) Completeness (%) 96.66 (96.77) Mean I/sigma(I) 6.44 (1.40) Wilson B-factor (Å ² ) 40.75 R-merge 0.1042 (0.5247) R-meas 0.1473 (0.7416) R-pim 0.1041 (0.5241) CC1/2 0.986 (0.594) CC* 0.997 (0.863) Reflections used in refinement 23455 (2340) Reflections used for R-free 1176 (115) R-work 0.2260 (0.2951) R-free 0.2825 (0.3665) CC(work) 0.922 (0.745) CC(free) 0.829 (0.421) Number of non-hydrogen atoms 6622 macromolecules 6394 ligands 54 solvent 174 Protein residues 823 105

RMS(bonds) (Å) 0.004 RMS(angles) (deg) 0.87 Ramachandran favored (%) 94.82 Ramachandran allowed (%) 5.18 Ramachandran outliers (%) 0.00 Rotamer outliers (%) 6.39 Clashscore 9.91 Average B-factor (Å ² ) 42.93 macromolecules 43.06 ligands 42.44 Number of TLS groups 38.49 Statistics for the highest-resolution shell are shown in parentheses. Determination of epitope and paratope The epitope of cmpd#2, defined as FIX(a) residues characterized by having a heavy atom 5 (i.e. a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the V _H H polypeptide, comprises the following residues from protease domain of FIX(a): E224, T225, G226, V250, I251, R252, I253, P255, H257 and N260 according to FIX sequence (consecutive numbering) (SEQ ID NO:1). 10 The paratope of cmpd#2, defined as residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in FIX(a), comprises the following residues from cmpd#2: F29, N30, Y32, T54, D99, R100, S101, F102, L103, F104, Q106, A107 and N113 (SEQ 15 ID NO:35) (consecutive numbering). EXAMPLE 6: CRYSTALLIZATION AND PARATOPE/EPITOPE MAPPING ANTI-FX V _H H FRAGMENT CMPD#1 The purpose of the present study was to determine the paratope and epitope residues of 20 the V _H H fragment designated cmpd#1. Crystallisation Crystals of the V _H H polypeptide cmpd#1 mixed in a 1:14 molar ratio with a synthetic peptide N-term-NPFDLLD-C-term, corresponding to the activation peptide sequence 106

aa31-37 of human FX (purchased from Schafer-N ApS) as identified in example 4, were grown using the sitting drop vapour diffusion technique at 18°C. A protein solution of 150 nl 4.0 mg/ml complex in 20mM Tris-HCl, pH 7.4, 50 mM NaCl was mixed with 50 nl of 0.2 M NaCl, 2 M ammonium sulphate, 0.1 M sodium cacodylate, pH 6.5 as precipitant and 5 incubated over 60 µl precipitant. Crystals appeared within a week. Diffraction data collection The crystal was cryo-protected by addition of 1 µl of precipitant added 20% of ethylene glycol to the crystallisation drop prior to flash cooling in liquid nitrogen. Diffraction data 10 were collected at 100K at the Swiss Light Source beamline X06DA (1.0000 Å wavelength) using a Pilatus 2M pixel detector from Dectris. Autoindexing, integration and scaling of the data were performed with programmes from the XDS package (diffracting data statistics are summarised in Table 8). 15 Structure determination and refinement The asymmetric unit contains one cmpd#1:peptide complex. The structure was determined by molecular replacement using Phaser as implemented in the programme suite Phenix using the V _H H polypeptide structure designated with Protein Data Bank ID 4B41 (chain A) as search model. The correct amino acid sequence of cmpd#1 was 20 introduced and difference electron density for the synthetic peptide was identified and model built manually, all using COOT. Thereafter the structure was refined using steps of Phenix refinement and manual rebuilding in COOT. The refinement statistics are found in Table 8. Table 8: Data collection and refinement statistics Wavelength (Å) 1.0000 Resolution range (Å) 47.61-3.183 (3.297-3.183) Space group P6 ₁ 22 Unit cell (Å, deg) 83.6583.65126.329090120 Total reflections 80989 (3197) Unique reflections 4667 (353) Multiplicity 17.4 (9.1) Completeness (%) 97.57 (76.08) Mean I/sigma(I) 8.69 (1.55) Wilson B-factor (Å ² ) 50.39 R-merge 0.4146 (1.447) 107

R-meas 0.4271 (1.516) R-pim 0.1004 (0.4227) CC1/2 0.985 (0.651) CC* 0.996 (0.888) Reflections used in refinement 4664 (353) Reflections used for R-free 234 (18) R-work 0.1948 (0.2689) R-free 0.2500 (0.3981) CC(work) 0.957 (0.685) CC(free) 0.934 (0.389) Number of non-hydrogen atoms 986 macromolecules 981 ligands 5 solvent 0 Protein residues 130 RMS(bonds) (Å) 0.003 RMS(angles) (deg) 0.59 Ramachandran favored (%) 96.83 Ramachandran allowed (%) 3.17 Ramachandran outliers (%) 0.00 Rotamer outliers (%) 0.00 Clashscore 8.68 Average B-factor (Å ² ) 44.41 macromolecules 44.18 ligands 88.79 Number of TLS groups 1 Statistics for the highest-resolution shell are shown in parentheses. Determination of the epitope and paratope The epitope of the synthetic peptide, defined as the FX activation peptide residues 5 characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the V _H H polypeptide cmpd#1, comprises the following residues from the synthetic peptide N173, P174, F175, L177, and L178 according to the corresponding FX activation peptide sequence based on consecutive numbering (SEQ ID NO:2). 10 108

The paratope of V _H H polypeptide cmpd#1, defined as cmpd#1 residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the synthetic peptide, comprises the following residues from cmpd#1: D32, A33, M34, G35, Y37, L47, V48, A49, G50, I51, M52, N57, T58, N59, Y60, T61, K97, 5 V99, R101 and P102 (SEQ ID NO:27) (consecutive numbering). Construction of a homology model of the V _H H polypeptide cmpd#3 in complex with the synthetic peptide was performed, since cmpd#3 was used as the parental V _H H polypeptide sequence for rational sequence- and structure-based optimizations. 10 The crystal structure of cmpd#1 in complex with the synthetic peptide from the above described experiment was used as a starting model for the homology model of cmpd#3 in complex with the synthetic peptide. The amino acid sequences of cmpd#1 and cmpd#3 were aligned and amino acid residues differing between the two sequences were mutated in COOT to create a starting model for cmpd#3. This model, including cmpd#3, was pre- 15 processed, optimised and run through a restrained minimisation in MAESTRO from SCHRÖDINGER. The model showed the same epitope and paratope residues as described above, since sequences were identical at these positions. 20 EXAMPLE 7: SPR ANALYSIS OF ANTI-FX/ANTI-FIX(A) V _H H POLYPEPTIDES The purpose of the present study was to estimate the binding constants of selected anti- FX/anti-FIX(a) V _H H polypeptide compounds with human plasma-derived FX and Benefix ^® by Surface Plasmon Resonance (SPR) analysis. 25 Binding of purified anti-FX/FIX(a) V _H H polypeptide compounds to human plasma-derived FX (Haematologic Technologies Inc, USA) was probed by SPR. Briefly, an anti-GLA-FX, prepared recombinantly as described above, was immobilised on a CM4 sensor chip or a Xantec HLC200M using standard amine coupling chemistry at pH 5.10 nM FX (Haematologic Technologies, USA) was injected at a flow rate of 10 µL/min 30 for 30 seconds. Subsequently 1000, 100, 10, 1, 0.1 and 0 nM of V _H H polypeptide compound, according to Table 9, below were injected at a flow rate of 50 µL/min for 200 seconds to allow for binding to FX followed by a flow of 10 minutes with a running buffer (10 mM HEPES, 150 mM NaCl, 5 mM CaCl2, 0.05%(v/v) Surfactant P20, 1 mg/mL bovine serum albumin, pH 7.4) injection allowing for dissociation from FX. The running buffer was 35 also used for dilution of anti-FX V _H H polypeptide compounds. Regeneration of the chip 109

was achieved using a regeneration buffer consisting of 50 mM EDTA in running buffer, 30 seconds contact time, and a 30 µL/min flow rate. The binding data were collected at 25 °C and were analysed according to a 1:1 model using BiaEvaluation 4.1 supplied by the manufacturer (Biacore AB, Uppsala or Bruker Analyser). 5 In all cases, the binding sensograms displayed a fast on and a fast off binding kinetic profile precluding K _D determination based on kinetic analysis. Therefore, the reported K _D values are determined based on steady state analysis. Analysis resulted in the binding constants reported in Table 9 below. 10 Binding of purified anti-FX/FIX(a) V _H H polypeptide compounds to Benefix ^® (Pfizer Inc, USA) was probed by SPR. Briefly, anti-GLA-FIX, prepared recombinantly as described above, was immobilised on a CM4 sensor chip or a Xantec HLC200M using standard amine coupling chemistry at pH 5. FIX (10 nM) was injected at a flow rate of 10 µL/min for 1 minute. Subsequently 1000, 100, 10, 1, 0.1 and 0 nM of V _H H polypeptide compound 15 were injected at a flow rate of 30 µL/min for 4 minutes to allow for binding to the FIX (Benefix ^® ) followed by a flow of 5 minutes running buffer (10 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ , 0.05%(v/v) Surfactant P20, 1 mg/mL bovine serum albumin, pH 7.4) injection allowing for dissociation from FIX. The running buffer was also used for dilution of anti-FIX V _H H polypeptide compounds. Regeneration of the chip was achieved using a 20 regeneration buffer consisting of 50 mM EDTA in running buffer, 30 seconds contact time, and a 30 µL/min flow rate. The binding data were collected at 25°C and were analysed according to a 1:1 model using BiaEvaluation 4.1 supplied by the manufacturer (Biacore AB, Uppsala). In all cases, the binding sensograms displayed a fast on and a fast off binding kinetic 25 profile precluding K _D determination based on kinetic analysis. Therefore, the reported K _D values are determined based on steady state analysis. Binding constant analysis of a series of compounds are reported in Table 9. Binding affinities of FIX were generally at low-nM K _D for all compounds, while a range of 30 compounds displayed high-nM K _D for binding of FX. Target-mediated drug disposition is therefore expected to be absent for FX, while FIX interaction would be expected but limited to the required steady-state plasma concentration of the outlined compounds. Thus, the expected required plasma concentration for obtaining meaningful haemostatic coverage for the listed compounds is expected to be at low nM concentration given the 35 high potency for the listed compounds. 110

Table 9: Estimated binding constants of selected V _H H polypeptide compounds Estimated binding constants based on steady state analysis for the interaction of selected anti-FX/anti-FIX(a) V _H H polypeptide compounds with human plasma-derived FX and Benefix ^® as determined by SPR analysis. Compound ID FX binder K _D (nM) FIX binder K _D (nM) 5 EXAMPLE 8: ACTIVITY OF ANTI-FX/ANTI-FIX(A) V _H H POLYPEPTIDES IN THROMBIN GENERATION TEST (TGT) ASSAY The procoagulant activity of anti-FX/anti-FIX(a) V _H H polypeptide derivatives was determined based on their ability to promote thrombin generation in the presence of 10 procoagulant synthetic phospholipid membranes according to the principles described by Hemker et al. (Pathophysiol Haemost Thromb, 2002;32:249-253). An emicizumab sequence identical analogue (SIA) was included for comparison. Each VHH polypeptide derivative was tested in a TGT assay using normal human platelet-poor plasma (NHP) supplemented with neutralizing anti-FVIII polyclonal antibody (hereafter named HA-PPP). 15 TGTs in NHP (from healthy volunteers) supplemented with sheep anti-human FVIII polyclonal antibody (pAb, Haematologic Technologies Inc., VT, USA) were performed by standard calibrated automated thrombography using a 96-well plate fluorometer (Fluoroscan Ascent FL, Thermolabsystems, Helsinki, Finland). Reaction mixtures contained 36 μl NHP preincubated with 0.1 µg/ml anti-FVIII pAb, 4 μl test compound 20 dilution (diluted in 20 mM HEPES, 140 mM NaCl, pH 7.4, 2 % BSA), 10 μl of either 1 pM 111

tissue factor (TF, pppLow, from Thrombinoscope BV, Maastricht, The Netherlands) or 1 to 8.3 U/ml human factor XIa (Enzyme Research Laboratories, IN, USA) and 10 µl FluCa Substrate (Thrombinoscope BV, Maastricht, The Netherlands). The TGT assay was calibrated using Thrombin calibrator (Thrombinoscope BV, Maastricht, The Netherlands), 5 where 10 µl Thrombin calibrator was mixed with 36 μl NHP preincubated with 0.1 µg/ml anti-FVIII pAb, 4 μl buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA). Generally, TGT was performed at 8 concentrations of test compound (0.1, 0.3, 1, 3, 10, 30, 100, and 300 nM, final plasma concentration, or similar) or added buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) only (representing control). Normal control levels in TGT were 10 measured using NHP added buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) only. The TGT was allowed to proceed for a total of 60 minutes and the TGT parameter Peak Thrombin Height (nM) was analysed by Thrombinoscope software (Thrombinoscope BV). See figure 4 for representative titration curves for a selected test VHH polypeptide derivative, cmpd #6, and comparators emicizumab SIA and Mim8. Assessments of 15 difference of activity level were conducted as difference in EC ₅₀ relative to comparators, e.g. emicizumab SIA, or as difference in maximum thrombin peak height for a given V _H H polypeptide compound relative to maximum thrombin peak height for emicizumab SIA. The latter approach was used for screening larger numbers of V _H H polypeptide derivatives. 20 To boost activity of the V _H H polypeptide derivatives, optimal combinations of CDR mutations were identified for generating maximum activity effect. Crystal structure models such as those described in examples 5 and 6 were used to identify important paratope residues as well as for identifying surface exposed residues suited for being substituted 25 with cysteine, as conjugation sites, and pI-lowering substitutions to enhance bioavailability. Different linkers and different conjugation types were also investigated. In the following tables, activity profiles of a series of V _H H polypeptide derivatives addressing each of these parameters are outlined and will be described individually. 30 In table 10, potency EC ₅₀ values obtained from optimized V _H H polypeptide derivatives that were optimized for activity boosting mutations are shown. Principal component computational and random forest algorithms were used for mutational combinations to boost activities. The V _H H polypeptide cmpd #24 was the initial derivative without boosting mutations and compared to this V _H H polypeptide derivative and to emicizumab SIA, an 35 increase of up to 40-fold and up to 76-fold better potencies, respectively, were obtained among the best V _H H polypeptide derivatives developed. 112

Table 10: TGT activity of V _H H polypeptides derivatives, optimization of activity Compound ID Protraction moiety pI TGT activity P nd L EC t n nM) , -3) that retained activity of the V _H H polypeptides following substitution with cysteine (with and 5 without conjugation with a C18 diacid fatty acid protraction moiety P3 attached via a linker L _P 1) was performed. A total of 95 surface-exposed sites were tested with introduction of Cys substitution. Based on consecutive numbering using cmpd #23/24 as reference (representing V _H H polypeptides without any boosting mutations in CDRs), a screening for optimal site of 113 conjugation was carried out. The following residue substitutions were tested: G27C, V28C, V29C, Q30C, P31C, G32C, S34C, L35C, R36C, S38C, A40C, S42C, R55C, Q56C, A57C, P58C, G59C, K60C, E61C, R62C, Y77C, A78C, D79C, V81C, K82C, G83C, R84C, F85C, T86C, S88C, D90C, N91C, S92C, K93C, T95C, Y97C, Q99C, M100C, 5 N101C, S102C, L103C, R104C, P105C, E106C, D107C, T108C, G156C, V157C, V158C, Q159C, P160C, G161C, G162C, S163C, L164C, R165C, S167C, A169C, S171C, R184C, Q185C, A186C, P187C, G188C, K189C, E190C, R191C, Y206C, A207C, D208C, S209C, V210C, K211C, G212C, R213C, F214C, T215C, S217C, D219C, N220C, S221C, K222C, T224C, Y226C, Q228C, M229C, N230C, S231C, L232C, R233C, P234C, E235C, D236C, 10 T237C and Cys introduced into a C-terminal extension. Of these, the following 41 V _H H polypeptides could be expressed using a HEK293 expression system and successfully conjugated with reagent C3 that comprises a C18 diacid protractor moiety: Q30C, P31C, Q56C, A57C, K60C, E61C, A78C, D79C, K82C, N91C, S92C, Q99C, R104C, G156C, V158C, Q159C, P160C, G161C, G162C, S163C, 15 A169C, S171C, Q185C, A186C, P187C, G188C, K189C, E190C, A207C, D208C, S209C, V210C, T215C, S217C, D219C, N220C, S221C, K222C, S231C, R233C and Cys introduced into a C-terminal extension. These 41 V _H H polypeptides with and without conjugations were tested for activity in a TGT assay. The relative activity levels, expressed as the maximum thrombin peak height of the V _H H polypeptide derivative 20 relative to the maximum thrombin peak height of emicizumab SIA, of the VHH polypeptide (derivatives) with and without conjugations are set forward in table 11. Thus, the following 9 sites were identified as optimal for introduction of a free Cys for conjugation with a protraction moiety, e.g. using C3 as a reagent, based on retained activities before and after conjugation: Cys introduced into a C-terminal extension (Cmpd #23/24), Q30C 25 (Cmpd #25/26), V158C (Cmpd #27/28), P160C (Cmpd #29/30), G161C (Cmpd #31/32), G188C (Cmpd #33/34), E190C (Cmpd #35/36), S209C (Cmpd #37/38) and S231C (Cmpd #39/40). Table 11: TGT activity of V _H H polypeptides derivatives, protraction moiety conjugation site screening V _H H polypeptide derivatives (cmpd#23/24 TGT activity, max. peak ratio variants w/o C-term extension) C s conju ation (relative to emicizumab SIA) V _H H polypeptide_1 with C18 diacid fatty acid 1.3 n ti n

V _H H polypeptide_13, Cys substitution Q159C 1.9 VH l tid 13 ith C18 di id f tt id 14 116 V _H H polypeptide_24 with C18 diacid fatty acid 0.4 n ti n

V _H H polypeptide_36, Cys substitution S231C 1.9 V H l tid 36 ith C18 di id f tt id 19 - FX V _H H fragments was performed to optimize composition (see table 12). The relative activity levels, expressed as the maximum thrombin peak height of the V _H H polypeptide relative 5 to the maximum thrombin peak height of emicizumab SIA, of the V _H H polypeptides are set forward in table 12. From these data, V _H H polypeptides with linkers of hydrophobic composition generally displayed the highest relative activities, whereas V _H H polypeptides with linkers of acidic or hydrophilic composition displayed the lowest activities. V _H H polypeptides with linkers of mixed composition composed of QAPGQA, GQAPGQ or 10 similar showed intermediate relative activities. The optimal linker composition would preferably contain both hydrophilic and hydrophobic amino acids, thus the QAPGQA linker, for example, was investigated further for chemical stability using MS analysis showing high stability under accelerated stress conditions at 37ºC for two weeks in an isotonic buffer and neutral pH. 15 Thus, a ratio of hydrophobic to hydrophilic amino acids of “40 to 60 %” to “60 to 40 %” is preferred. The indicated VHH polypeptide all included the same set of activity boosting CDR mutations, but different set of pI-lowering mutations outside CDRs. 118

Table 12: TGT activity of V _H H polypeptides sequence with different linker L _1-2 characteristics V _H H polypeptide ID Linker (L _1-2 ) sequence TGT activity, max. peak ratio r l tiv t mi iz m b SIA 119

EXAMPLE 9: FORMULATION OF ANTI-FX/ANTI-FIX(A) V _H H POLYPEPTIDE DERIVATIVES WITH SNAC AND NAM IN DOSAGE FORM SUITABLE FOR PERORAL ADMINISTRATION To prepare for oral studies using liquid formulations with sodium N-(8-[2-hydroxybenzoyl] 5 amino) caprylate (SNAC) and nicotinamide (NAM) as excipients, the following procedure was followed. SNAC was weighed to obtain a concentration of 200 mg/ml, NAM was weighed to obtain a concentration of 1 M, HEPES buffer was weighed to obtain a final concentration of 5 mM. Powders were transferred to a glass vial and MilliQ-H ₂ O was added accordingly. The solution was stirred with a magnetic stirrer until SNAC, NAM and 10 magnesium stearate were dissolved. pH was measured and adjusted to pH 8 with 2 M NaOH. Thus, batches of purified and conjugated V _H H polypeptide derivatives of 2-5 mg/ml were liquid formulated with 200 mg/ml SNAC and with 1 M NAM final concentration. To prepare for oral studies using tablets with SNAC and NAM, the following procedure 15 was followed. Batches of purified V _H H polypeptide (derivatives) with or without fatty acid protractor conjugations were buffer exchanged into MilliQ-H ₂ O and pH adjusted to 8.0 using either 0.1 M NaOH or 0.1 M formic acid. V _H H polypeptide (derivatives) dissolved in MilliQ-H ₂ O were at concentrations from 0.5 to 4 mg/ml. Spray drying of V _H H polypeptide (derivatives) was conducted using Mini Spray Dryer B290 (BUCHI) with pump setting 5-6, 20 feed flow: 2 ml/min, inlet temp: 80-85°C, outlet temp: 45-50°C, nozzle: 1.5 mm and aspiration: 100%. To prepare tablets, powder was mixed to obtain desired compositions. The following tablet formulations were prepared. 25 Formulation #1 V _H H polypeptide (derivative): 10-20 mg SNAC: 300 mg NAM: 0 mg 30 Magnesium stearate: 5.5 mg Formulation #2 VHH polypeptide derivative: 10-20 mg SNAC: 300 mg 120

NAM: 200 mg Magnesium stearate: 5.5 mg Formulation #3 5 V _H H polypeptide derivative: 20 mg SNAC: 150 mg NAM: 100 mg Magnesium stearate: 1.25 mg 10 Powders mixed were weighed and punched for homogenic tablet using Kilian Style One (Romaco) with punch setting to simulate a rotary press. EXAMPLE 10: PERORAL AND INTRAVENOUS PHARMACOKINETIC STUDY IN RAT USING ANTI-FX/ANTI-FIX(A) V _H H POLYPEPTIDES DERIVATIVES WITH DIFFERENT 15 PI-CHANGING MUTATIONS The purpose of the present study was to investigate the effect of pI-changing mutations in anti-FX/FIX(a) V _H H polypeptide derivatives on pharmacokinetic parameters such as oral bioavailability. Liquid formulations of anti-FX/FIX(a) V _H H polypeptide derivatives were dosed 20 intravenously (IV) (IV formulation: 20 mM Hepes, 150 mM NaCl, pH 7.4) or prepared according to Example 9 and administered by oral gavage to parallel groups of Sprague Dawley rats acclimated at least one week in-house prior to study and kept in group cages with ad libitum access to standard food and water. Rats for oral dosing were fasted, with free access to water, in grid-bottomed cages from approx.2pm on the day prior to dosing 25 (for a total of 18 hours). On the day of dosing, all rats were acclimated in the procedure room for 30 minutes. Blood samples were collected immediately prior to dosing and subsequently at a number of different post-dose time points. Peroral (PO) dosed animals were kept fasted in grid- 30 bottomed cages until 4 hrs post dosing. Plasma concentrations of V _H H polypeptide derivatives were measured using a gamma-Glu-based immunoassay, in which an anti- V _H H antibody (Novo Nordisk, Denmark) and an anti-gamma-Glu (Novo Nordisk, Denmark) towards the protraction moiety of the test molecule was used. Here, 96-well MaxiSorp plates (Nunc, 439454) were coated with 2 µg/ml anti-gamma-Glu antibody, washed and 35 blocked using PBS; 0.05% tween20; 1% BSA; pH 7.4. After a wash step, compound 121

specific calibrator (0, 2.7, 8.2, 24.7, 74, 222, 667, 2000 pM) in 1% rat EDTA plasma and rat EDTA plasma samples in a minimal dilution of 100x were incubated on the plate, where the V _H H polypeptide derivative was captured via the gamma-Glu protraction moiety. After an additional wash step, a biotinylated in-house V _H H specific antibody was 5 added to the plate (0.5 nM) to make up the sandwich ELISA. After a final wash step, horseradish peroxidase (HRP)-streptavidin was added to the plate as the detection reagent. The amount of biotin labelled antibody bound to the V _H H polypeptide derivative was detected following addition of a chromogenic substrate (e.g., TMB (3,3',5,5'- tetramethylbenzidine). Optical density was measured using a Spectrometer (e.g. a 10 SpectraMax ^® M2 spectrometer (Molecular Devices)). The response was proportional to the concentration of peroxidase, which again was proportional to the concentration of the V _H H polypeptide derivative. Based on these exposure data, non-compartmental pharmacokinetic parameters were calculated using either Phoenix WinNonlin or the open source statistical analysis software R (package ‘NonCompart’). 15 The following pI-lowering substitutions T28D, K43Q, K65Q, N84D, R148Q, N159D, K172Q, K194Q and N213D (based on consecutive numbering using cmpd#4 as template) introduced at surface exposed residues outside the CDRs, were tested in different combinations (leading to V _H H polypeptide derivatives cmpd#59 to 64 and cmpd#5) and 20 compared to non-pI adjusted VHH polypeptide derivative cmpd#4. Results are given in tables 13 and 14 below for V _H H derivatives and show an effect of lowering pI on peroral bioavailability. Lowering of pI from approx.8.36 to 5.85 increased bioavailability 5- to 10-fold (see table 25 13). No effect of pI lowering on half-life was observed (see table 14). 122

m ^I _p ^O m _{3 4 5 6 6 0 6 3} ^{n i D} _o ^. ₆ ^. _{6 P} ⁿ ₍ ¹ ₂ ¹ ₂ ⁶ ₁ ⁶ ₁ ⁹ ₁ ⁶ ₁ ^d _e ^V m d ^t _{s I} ⁿ ₍ e _r ^e _{e )} ^e t p ^s _{n )} ^p ₃ ^r _e ^d _n ^{g s o} _{n n n n n n n n} ^e e ⁱ _t ^{oi a} _t ^oi _t ^oi _t ^{oi oi oi oi} g ^{a a a} _t ^a _t ^a _t ^a _t ^{a h} _ti d ⁱ _t ^{e u} _g ^u _g ^u _g ^u _g ^u _{g g g} ^w _{n n} p _{t e} ⁱ _{j j j j j} ^u _j ^u _j ^u _j ^oi _t ^oi _t p _s ⁿ _o ⁿ _o ⁿ _o ⁿ _o ⁿ _o ^{n n n s} _e ^{a a} y ^l _n ^{c c c c c} _o ^c _o ^c _o ^{c v} _{i o} ^{o e} _{g g} p i _t ^s _y ^s _y ^s _y ^s _y ^s _y ^s _y ^s _y ^s _y ^t _a ^v _t i ^{i u} _j ^u _{j r s} ⁿ _o ⁿ _o H ^{a V} H _g ^{C C C C C C C C u} _j ^m _r ^m _r ^{m e} r ^{m m m m} _r ^m _{r d n} ^{c c oi s} _y ^s _{y f} ⁿ _o ^et - ^et - ^et _{r -} ^et _{r -} ^et _{r -} ^et - ^et - ^et _{- e t} d ^{i a} _g ^{C C o a} _t ^{c a} _, ^{C C C C C C C C} _t r ^{, p u} _j ^m _r ^m _{r d o y} ^t _d ^{, i} _{c d} ^{, , , , , i} _{c d} ⁱ _{c d} ⁱ _{c d} ⁱ _{c d} ⁱ _d ^{, i} _d ⁱ _e ^p _y ⁿ _o ^e t- ^e t- t ⁱ _{c l} ^{a a} _{c c c} ^{l c C C i a} _{r i b} ^{t d} _i ^{a d} _i ^{a d} _i ^{a d} _i ^{a d} _i ^{a d} _i ^{a d} _i ^d _o ^p _, ^{r ,} _d ^{, i} _d ^{i a} _l ⁱ _o ^{r 8} P ₁ ^{8 8 8 8 8 8 8} H _o ^t _{c c c} C ₁ C _{1 1 1 1 1 1} H ^{a a} _i ^a _{i a} C C C C C C ^{V d d v} _{f r a} ^{o t} _o ^{8 8 o} _i ^{a r b} _{t P 1} C ₁ C l ^{a a} _{d r} ^D _I O _{e K :} ^{v 3} _{i 1} ^t ₄ ⁹ a ^# ₅ ^# ₅ ^{0 #} ₆ ^{1 #} ₆ ^{2 #} ₆ ^{3 4 P} _e # ₆ ^# ₆ ^# _: ^v _i ^{9 t} ₄ ^# ₅ ^{# v} _i ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^d _p ^{d 4} p _{1 a} ^v _i ^{d d 1} _e ^{l r} _{e e} ^{l r} _{p p 0 b} ^a m m m m m m m m D C _b ^a _e ^D m mO _T C C C C C C C _T D _I C C W ² ₂ ⁰ ₀ ¹

^. ₀ ^. ₀ ^. ₀ ^. ₀ ^. ₀ ^. _{0 6} ^. ₆ ^. ₆ ^. _{6 6 7 6 6 6} ^. ₆ ^. ₆ ^. ₆ ^. ₎ ² ₍ ⁿ _l ^* _T R 3 _{3 3 3 3 3} ^M ₄ s ² a _{1 d} ^e _t ^a _l 6 ₅ ^{2 .} ₄ ^{2 9 9 5 u} _c ^l 6 ^. _{4 6} ^. _{0 6} ^. _{0 6} ^. _{8 6} ^. _{5 a} ^{c s} _{i L} H _T R n ^oi _{n t} ^oi _n ^oi _n ^oi _n ^oi _n ^{oi M a} _{t . g} ^a _t u _g ^a _{t g} ^a _{t g} ^a _{t g} ^a _g ^e f _{i j} ^u _j ^u _j ^u _j ^u _j ^{u l} - n _{f o} ⁿ _o ⁿ _j ^l o ^{n n n} _a c ^{c c} _o ^c _o ^c _o ^{c hl s} _y ^s _y ^s _y ^s _y ^{s s} _a ^{n C C C C} _y C _y C _i m ^m _{r r} ^{m e} _r ^m _r ^{m m m e} _{t -} ^e _{r r r} t t C - ^e t- ^e t- ^e t- ^e t _{- e} m ^{, C, C, C C C i} _{t d} ⁱ _d ^{, , , i} _{d d d d e c c} ⁱ _c ⁱ _c ⁱ _c ⁱ _c ^{c a} _i ^a _i ^a _i ^a _i ^a _i ^a _n d ^{d d d d} _i ^{d e 8} _d ⁱ 1 ⁸ ₁ ⁸ ₁ ⁸ ₁ ⁸ ₁ ⁸ _s ^e C C C C C ₁ C _{r n} ^a _e 0 ^{1 2 3 4 M} 5 _{6 6 6 6} ^{: # # # # #} ₆ ^# _L d ^{d H 1} _{p p} ^d _p ^d _p ^d _p ^d _{p T} R ₀ m m m m m m C C MO ^C C C C _* W ² ₂ ⁰ ₀ ¹

EXAMPLE 11: INTRAVENOUS PHARMACOKINETIC STUDY IN RAT: ANTI-FX/ANTI- FIX(A) V _H H POLYPEPTIDE (DERIVATIVES) WITH DIFFERENT PROTRACTORS The purpose of the present study was to investigate the effect of different protractors on pharmacokinetic parameters such as half-life on V _H H polypeptide derivatives. 5 Liquid formulations of anti-FX/FIX(a) V _H H polypeptide derivatives prepared according to Example 9 were dosed intravenously (IV) to parallel groups of Sprague Dawley rats; acclimated at least one week in-house prior to study and kept in group cages with ad libitum access to standard food and water. Rats for oral dosing were fasted, with free access to water, in grid-bottomed cages from app.2pm on the day prior to dosing (for a 10 total of 18 hours). On the day of dosing, all rats were acclimated in the procedure room for 30 minutes. Blood samples were collected immediately prior to dosing and subsequently at a number of different post-dose time points. Plasma concentrations of V _H H polypeptide (derivatives) were measured using a His-tag- or gamma-Glu-based immunoassay, in which an anti-V _H H antibody (Novo Nordisk) towards the V _H H polypeptide derivative 15 together with an anti-His-tag antibody (R&D systems, MAB050) towards His-tag fused to the test molecule or an anti-gamma-Glu (Novo Nordisk, Denmark) towards the protraction moiety of the test molecule were used. The two immunoassay setups gave similar sensitivities, and the latter was used when the V _H H polypeptide compound had no His-tag fused to the molecule.96-well MaxiSorp plates (Nunc, 439454) were coated with the His- 20 tag or the anti-gamma-Glu antibody, washed and blocked using PBS; 0.05% tween20; 1% BSA; pH 7.4. After a wash step, derivative specific calibrator (0, 2.7, 8.2, 24.7, 74, 222, 667, 2000 pM) in 1% rat EDTA plasma and rat EDTA plasma samples in a minimal dilution of 100x were incubated on the plate, where the V _H H polypeptide derivative was captured via its His-tag or the gamma-Glu protraction moiety. After an additional wash 25 step, a biotinylated in-house V _H H specific antibody was added to the plate (0.5 nM) to make up the sandwich ELISA. After a final wash step, horseradish peroxidase (HRP)- streptavidin was added to the plate as the detection reagent. The amount of biotin labelled antibody bound to the V _H H polypeptide derivative was detected following addition of a chromogenic substrate (e.g., TMB (3,3',5,5'-tetramethylbenzidine). Optical density 30 readings were measured using a Spectrometer (e.g. a SpectraMax® M2 spectrometer (Molecular Devices)). The response was proportional to the concentration of peroxidase, which again was proportional to the concentration of the V _H H polypeptide derivative. Based on these exposure data, non-compartmental pharmacokinetic parameters were calculated using either Phoenix WinNonlin or the open source statistical analysis software 35 R (package ‘NonCompart’). 125

Results for V _H H polypeptide derivatives with different protractor types are shown in table 15 below. Results show the effect of using different fatty acids and albumin-binding peptides as protractor on median residence time terminal half-life (MRTHL). 5 Increased half-life compared to non-protracted polypeptides was observed for V _H H polypeptide derivatives having C18, C20 and tetrazole fatty acid side-chain conjugations (cmpd#66, cmpd#67 and cmpd#68: 3 to 4-fold increase), as well as for V _H H polypeptide derivatives with albumin-binding peptide protractor fusions (cmpd#70, cmpd#71, cmpd#72 and cmpd#73: 1.9 to 3.6-fold increase) with an optimal effect of using a 30 residues long 10 GlySer L _P linker fused C-terminally between the albumin-binder peptide and the V _H H polypeptide. The V _H H polypeptide derivative, cmpd #65, with a short C12 fatty acid showed the lowest half-life. This indicates that to obtain an optimal protraction of V _H H polypeptide derivatives, for prolonged half-life circulation, a fatty acid longer than C12 is preferable (such as e.g. C16, C18 or C20). 126

_L H _T ⁾ _s R ^y _a ⁹ ₁ ^{1 3 5 1 3 1 6 4 6 0 .} ₁ ^. ₆ ^. ₇ ^. ₇ ^. ₁ ^. _{5 3 3 3 7} M ^D _{0 0 0 0 0 0} ^. ₀ ^. ₀ ^. ₀ ^. ₀ ^. ₀ V _{( I c} ^a r _{n g g g} ⁿ _{g t} ^{o e e e e s} _i i ^u _j ^u _j ^u _{j o} ^u _{j k k k k} o _t ^{n n n c n n} _i ^{l n} _i ^{l n} _i ⁿ _{i L r} ^{p a} _{o t g} ^r _o ^c _o ^c _o ^{c s} _o ^{c s s s} _y C ^r _o ^s _r ^e _r ^{l e} _r ^{l e} _r ^{H e} _T n ^u _j ^t _{c y y y} ^t _{c y S} ^y _S ^y _{S S} R e ^{r n a C C C m} _r ^{a C} _{l l} ^y _l ^y _l ^M e ^f _o f _i ^c _r ^t _o d ^{d r m} _r ^m _r ^m _r ^e t _{r -} ^t _o ^m _r G- G- G- G- _. ^e f _i h _{t n p} ^et - ^et - ^et - ^{C r} i ^a _o ^{C C C ,} _p ^et _{0 d -} ¹ _{0 0 0} ^l - , ^{3, 1, 3,} _f ^{l w r} N ^{, , , i} _{o c} N ^C, _e ^d _{e e e a} i ^d i ^d i ^d i ^{hl s} _{o e} ^t _d d _c ⁱ _{c d} ⁱ _{c d} ⁱ _c ^a _i ^d _d ⁱ _{t c} ^p _t ^p _t ^p _t ^p _{a it} ^a _r ^a _i ^a _i ^a _i ^{e a} _{e e e e} ⁿ _i p ^t e _o ^{d d d} _{l i} ^{p p p p o d} _r ^{m e} _r ^e _r ^e _{r r} ^{e p r 2} ₁ ⁸ ₁ ⁰ _{2 z y} ^l _P C C C ^{a 8} r _{t 1 d d d} ^e _{d t} C ⁿ _i ⁿ _i ⁿ _i ⁿ _{i e o} ^{e p} _T ^b - ^{b b b m n} - _i ⁿ - _i ⁿ - _i ⁿ _i ⁱ _t H VH m _e u m ^u m ^u m ^c _{n f b} ^l _b ^{u o} _A ^l _{b b} ^e A ^l A ^l A _d ^{i a} _{s t} ^{e a} _{r d n K} ^D _I ^a _e P _{e :} ^{v 5} _i ^{4 5 M} 1 ^t _{6 a} ^# ₆ ^{6 #} ₆ ^{7 #} ₆ ^{8 #} ₆ ^{9 #} ₆ ^# ₄ ^{0 1 2 3 #} ₇ ^# ₇ ^# ₇ ^# ₇ ^{# :} _L ¹ _e ^{l v} _i ^{d d d d d d d d d d d H r} _{p p p p p p p p p p p T 0 b e} m RO ^a m m m m m m m m m m T D C C C C C C C C C C C ^M *W ² ₂ ⁰ ₀ ¹

EXAMPLE 12: PERORAL AND INTRAVENOUS PHARMACOKINETIC STUDY IN DOG: ANTI-FX/ANTI-FIX(A) V _H H POLYPEPTIDE (DERIVATIVES) FORMULATED WITH SNAC AND NAM The purpose of the present study was to investigate the effect of fatty acid protraction on 5 half-life, and if lowering of pI leads to enhanced oral bioavailability. Appropriate formulations of anti-FX/FIX(a) V _H H polypeptide (derivatives) were dosed intravenously or prepared as tablets and dosed perorally, respectively, to groups of Beagle dogs. The dogs were dosed in the morning after overnight fasting and kept fasting for 3-5 hours after a single dosing. In a subset ^§ of oral studies the dogs were given a s.c. 10 dosing of approximately 3 nmol/kg of glucagon 10 min prior to oral dosing. Blood samples were collected immediately prior to dosing and subsequently at a number of different post-dose time points. Plasma concentrations of V _H H polypeptides were measured using a His-tag-based or gamma-Glu-linker based immunoassay, in which an anti-VHH antibody (Novo Nordisk, Denmark) towards the V _H H polypeptide together with an anti-His-tag15 antibody (R&D systems, MAB050) towards His-tag fused to the test molecule or an anti- gamma-Glu (Novo Nordisk, Denmark) towards the protraction moiety of the test molecule were used. The two immunoassay setups gave similar sensitivities, and the latter was used when the V _H H polypeptide compound had no His-tag fused to the molecule.96-well MaxiSorp plates (Nunc, 439454) were coated with the antibody, washed and blocked 20 using PBS; 0.05% tween20; 1% BSA; pH 7.4. After a wash step, compound specific calibrator (0, 2.7, 8.2, 24.7, 74, 222, 667, 2000 pM) in 1% dog EDTA plasma and dog EDTA plasma samples in a minimal dilution of 100x were incubated on the plate, where the V _H H polypeptide compound was captured via its His-tag or the gamma-Glu linker motif. After an additional wash step, a biotinylated in-house V _H H specific antibody was 25 added to the plate (0.5 nM) to make up the sandwich ELISA. After a final wash step, horseradish peroxidase (HRP)-streptavidin was added to the plate as the detection reagent. The amount of biotin labelled antibody bound to the V _H H polypeptide compound was detected following addition of a chromogenic substrate (e.g., TMB (3,3',5,5'- tetramethylbenzidine). Optical density readings were measured using a Spectrometer 30 (e.g. a SpectraMax ^® M2 spectrometer (Molecular Devices)). The response was proportional to the concentration of peroxidase, which again was proportional to the concentration of the V _H H polypeptide compound. Based on these exposure data, non- compartmental pharmacokinetic parameters were calculated using either Phoenix WinNonlin or the open source statistical analysis software R (package ‘NonCompart’). 35 ^§ indicate a s.c. dosing of approximately 3 nmol/kg of glucagon 10 min prior to oral dosing. 128

Results are given in tables 16 and 17 below for three C18 diacid conjugated V _H H polypeptide derivatives and confirms a) an effect of fatty acid protraction on half-life, and 5 b) that lowering of pI leads to enhanced oral bioavailability. Thus, the observed half-lives in dog increased by 370 to 710-fold when the V _H H polypeptide derivatives were conjugated with the indicated fatty acid as compared to a non-protracted compound. The compound cmpd#6 with two C16 diacid fatty acid 10 conjugations in a C-terminal linker displayed the highest increase in half-life of approx.7.6 days. In dog, the SNAC:NAM formulation increased the oral bioavailability 2- to 8-fold compared to SNAC formulation alone. Thus, adding NAM to the oral formulation had a pronounced 15 effect enabling the oral bioavailability to and above a level that is clinically relevant i.e. approx.0.1 %. Overall considering the potency, the mean residence time and the oral bioavailability, the V _H H polypeptide derivatives as disclosed herein would be expected to be useful in the 20 treatment of haemophilia A with or without inhibitors and acquired haemophilia A, when administered perorally. 129

^d _e ^t _s ^e _y t ^{ti s} _l ⁱ n _b oi ^a t _l ^{i a} _{a )} ^{2 l} g ^v ₀ ^{5 6 3 9 4} a ^% ₍ ^. _{0 1 0} ^. ₀ ^. _{1 0} ^. _{0 0} ^. _{2 e 0} ^. ₀ ^{d * o} _i ^t _{a a a i a} m t ^{r l} _u ^l _u ^l _{a u} ^l _{a u} ^l _{a u} ^l _u ^a u _{o h} m ^F m ^r m m m m m ^{c O} _o ^r _o ^r _o ^r _o ^r _o ^r _{o -} ^e _{d )} p _e ^{a u} _j ^{u n} _{s o o o} ^{s s} _. p _y ^{l e} _g ^a _g ^a _{g t} ^u _j ^u _j ^u _j ⁿ _{j o} ⁿ _{i o} ^g _{n n} ^{c c c o} _y i ^{s s s C} _y C ^e f _i ^l - o ^{i p} _s ⁿ _o ^{n n c c e} _t ^a _y c _o ^c _o ^{c s} _y ^s _y ^h _{t g} ^C _y C _y C ^m _r ^m _f ^{l e} _r ^e _a H _n VH ^o i _t ^s _y ^s _y ^s _y ^{C C} _i w ^{u s} _j ^{m n} _r ^{m e} _r ^m _r t- t- ^hl t ^e t ^e t ^{C C} _{a f} ^{a n o} _g ^{C C C m} _r ^m _{r e o} u ^{m m m et et di c} - _, ^{C r} _{- -} ^, o ^{C C} _d ^{, i a} _{j r r r - - t c d} ⁱ _{i c} ^m t ^{a n e} t ^e t ^e t ^{C C p} _e ^r _o ^, _d ^{i t} _c ^, _d ^{, i} _d ^{i a} _i ^a _{i r} ^e _{t d o} ^c - C ^r _{- -} ^{, ,} o ^{C C} _d ⁱ _d ^{i p t} y ^l _{c c} a ^{a a} i _r ^t _{c o} ^a _{c i} ^a _i ^{d d 6 6} _{e y} ^ti _{, l} ^{r i} _o ^, _d ^{i t} _c ^, _d ^{, i} _d ⁱ _c ^a _{c i} ^a _{i o} ^p _r ^{t d r d d 8} _p ^{8 8} ₁ C ₁ ^{m C i} _{t b} ^{t a} _{l c c} ^{a i a} _i ^a _r ^t _{c c} ^{d d} _o ^r _{1 o 1 1 o} ^a _i ^a _i ^{6 6} HH _P C N C C ² _x ² _{e x} ^c _{n a r} v ^{t d r d d} a _o ^{r 8} _p ⁸ ₁ C ₁ C ^{V e} _d o _{i P 1} C _o N ₁ ⁸ C _{1 f} ⁱ C ² _x ² _x ^{o a} _s ^{e b} _{t l} ^a _{r n} a _r ^{l D} _{I d} ^D _I ^a _e O _{e :} ^d _{e §} v ^{4 1 3 §} _§ ⁵ _K P _e ^{v 4 1 3 5 M 6} _{o i 1} ^{m t} _{4 7 6 6} 6 _{1 : i} g _a ^{# # # # # # t} ₄ ^# ₇ ^# ₆ ^# ₆ ^# _{6 1} ^: _L v _i ^{d 7} p ^d _p ^d _p ^d _p ^{d d} _{1 a} ^{# # v} _i ^{d d d d d d H} _T ¹ _e ^l _o ^r _{p p e} m m m _e ^{l r} _{p p p p p p} R _{0 b} O ^{a d} m m m T ⁿ _i D C C C C C C _b ^a _e m m m m m m T D C C C C C C ^M *W ² ₂ ⁰ ₀ ¹

EXAMPLE 13: INTRAVENOUS PHARMACOKINETIC STUDY IN PIG: C18 CONJUGATED ANTI-FX/ANTI-FIX(A) V _H H POLYPEPTIDE (DERIVATIVES) The purpose of the present study was to investigate the pharmacokinetic effect of V _H H polypeptide (derivatives) with and without a C18 diacid protractor group. 5 Appropriate formulations of anti-FX/FIX(a) V _H H polypeptide (derivatives) were dosed intravenously (IV) and subcutaneously (SC), respectively, to groups of minipigs (Sus scrofa domesticus). Blood samples were collected immediately prior to dosing and subsequently at a number of different post-dose time points. Plasma concentrations of V _H H polypeptide (derivatives) were measured using a His-tag-based or gamma-Glu- 10 based immunoassay, in which an anti-V _H H antibody (Novo Nordisk) towards the V _H H polypeptide (derivative) together with an anti-His-tag antibody (R&D systems, MAB050) towards His-tag fused to the test molecule or an anti-gamma-Glu (Novo Nordisk, Denmark) towards the protraction moiety of the test molecule were used. The two immunoassay setups gave similar sensitivities, and the latter was used when the V _H H 15 polypeptide compound had no His-tag fused to the molecule.96-well MaxiSorp plates (Nunc, 439454) were coated with the His-tag or the anti-gamma-Glu antibody, washed and blocked using PBS; 0.05% tween20; 1% BSA; pH 7.4. After a wash step, derivative specific calibrator (0, 2.7, 8.2, 24.7, 74, 222, 667, 2000 pM) in 1% pig EDTA plasma and pig EDTA plasma samples in a minimal dilution of 100x were incubated on the plate, 20 where the V _H H polypeptide derivative was captured via its His-tag or the gamma-Glu protraction moiety. After an additional wash step, a biotinylated in-house V _H H specific antibody was added to the plate (0.5 nM) to make up the sandwich ELISA. After a final wash step, horseradish peroxidase (HRP)-streptavidin was added to the plate as the detection reagent. The amount of biotin labelled antibody bound to the V _H H polypeptide 25 derivative was detected following addition of a chromogenic substrate (e.g., TMB (3,3’,5,5’-tetramethylbenzidine). Optical density readings were measured using a Spectrometer (e.g. a SpectraMax® M2 spectrometer (Molecular Devices)). The response was proportional to the concentration of peroxidase, which again was proportional to the concentration of the V _H H polypeptide derivative. Non-compartmental pharmacokinetic 30 parameters were calculated using the open source statistical analysis software R (package ‘NonCompart’). Results are shown in table 18 below for V _H H polypeptides with and without a fatty acid protractor confirming an effect on half-life of fatty acid protractor conjugations for half-life extension as observed in examples 11, 12 and 13. 131

Thus, the observed half-lives in pig increased approx.15 to 20-fold when the V _H H polypeptides were conjugated with a fatty acid C18 diacid protractor compared to non- protracted V _H H compounds. 132

_t ^a _g ^u _j ^{n )} o _g c _e r ^{s k} _/ ^l o ^t _{o o} ^{0 0} ₃ ^. ₈ ^. D m _{3 3 5 4 c} ^a _r ^{n t} _{( o} ^r . p _{) d} ^{2 i} _{) ( c} ^p _u ⁿ _l a ^{o * y} _{r ) t ) ) T} t ^{g a} C ₎ R ₃ f N C d _n ^{S S} ₍ ^V I ₍ ^V I ₍ ^V I ₍ ^{M 3} r _{3 2 3 3} ^s _{a 1} a ^o _d s _n ^V I ^e _t o ₍ ^a _{l i} ^{t u} a ^t _c ^l u _a ^{c mI s 6 5} _{i p} ^{I 5} ₉ ⁵ ₉ ^. ₃ ^. ₈ ^. _{L d p} ^. ₈ ^{H e} _{8 8 5 r T} e R e ^{M n} _{i .} g ^e n f e _{i t} ^{l e} - t ⁱ _f ^{l h} _{i s a n} ^{h w l s o} e ⁱ _t ^s _y ^s _{a y} ⁿ _i d ⁱ _t ^{a C C m p} _{g e} ^u _j ^m _{r r} ^{m e} _t p ^{n e} _r t- ^e t _{- e y} ^l _{o o} ^{c r p} _, ^r _o ^{r C C mi} _t H _o ^t _o H ^t _c ^{t , ,} c ^a _{c r} ^{t a} _{d r} ⁱ _e t _{c d} ^{i a} _c ^c i ^a _{i n} V _f ^a _{r o} ^r _o ^{r d d e} _d ^{i o t a} _{o p p t} ^r P _o ^{8 8} _s ^e N _o N ₁ C ₁ C _r a _{n d} ^a K _e P _e ^{5 5 3 M} : ^{v 8} _{i 1} ^t _{7 a} ^# ₇ ^# ₄ ^# ₆ ^{: #} _L v _i ^{d d d d H} _T ¹ _e ^{l r} _{p p p p e} R _{0 b} ^a m m m m D ^D _I C MO _T C C C _* W ² ₂ ⁰ ₀ ¹ EXAMPLE 14: PHARMACODYNAMIC EFFECT OF OPTIMIZED ANTI-FX/ANTI-FIX(A) POLYPEPTIDE DERIVATIVES IN MOUSE TAIL-VEIN TRANSECTION (TVT) BLEEDING MODEL In vivo efficacy was tested in HA mouse models of moderate bleeding. In the TVT model, 5 it has previously been shown that FVIII administration reduces bleeding to the same level observed for wild-type mice. To overcome the lack of mouse cross-species reactivity of compounds testes, HA mice were supplemented with human FIX and FX before the bleeding experiments. At the end of the bleeding period, total blood loss was determined by spectrophotometric haemoglobin measurement. Plasma levels of indicated V _H H 10 polypeptide derivatives and FIX were quantified by a luminescent oxygen channelling assay using anti-V _H H (Novo Nordisk, Denmark) and anti-gammaGlu-linker antibodies (Novo Nordisk, Denmark) and anti-FIX antibodies (LS-B7226, LSBio, and FIX-2F24, in- house clone), respectively. FX levels were quantified by a commercial FX ELISA (KSP134, Nordic Biosite). From dose–response studies with the indicated V _H H 15 polypeptide derivatives, EC ₅₀ -values were determined by fitting of data to a three- parameter inverse log(dose) response equation with shared plateau values (> 0). Automatic outlier elimination with a 1% ROUT coefficient was applied and sum of squares was weighted by (blood-loss) using GraphPad Prism software (version 9.0.1). 20 Dose-response studies showed a significant reduction in blood loss, reaching blood loss levels observed in wild-type mice for the tested V _H H polypeptide derivatives compared to vehicle group and wild-type, non-haemophilic group, respectively, as shown in table 19. Estimates of EC ₅₀ effective dose ranges are presented in table 20 showing high potency, as 50% effect were observed at low nmole per kg dosage for all 3 compounds tested. 25 Table 19: Effect of anti-FX/anti-FIX(a) polypeptide derivatives in TVT bleeding mouse model Derivative # / dose Bleeding time Blood loss m l /k min AVE ± SD n = 6 nm l AVE ± SD n = 6

0.006 µmole/kg 19.7 ± 11.2 5232 ± 3698 0010 m l /k 139 ± 87 2973 ± 2973 Table 20: EC ₅₀ estimates of anti-FX/anti-FIX(a) polypeptide derivatives from TVT bleeding mouse model Derivative ID Bleeding time EC ₅₀ Blood loss EC ₅₀ m l /k m l /k 5 135 EXAMPLE 15: EX VIVO ACTIVITY DETERMINATION OF AN OPTIMIZED ANTI- FX/ANTI-FIX(A) V _H H POLYPEPTIDE DERIVATIVE POST PERORAL DOSING Following a pharmacokinetic (PK) study in wild-type dogs from example 12 to characterize PO dosing of cmpd#6, chromogenic activity was determined at two separate 5 time points at 30 minutes and 90 minutes post dosing. The measured FVIII-mimicking, chromogenic activity was compared to the plasma exposure of cmpd#6 to determine the fraction of active V _H H polypeptide derivative in serum post PO dosing. Chromogenic activity of cmpd#6 was determined using a commercial FVIII chromogenic activity assay (FVIII:C, Hyphen Biomed, France) to analyse dog serum samples. For the 10 calibration curve, cmpd#6 was spiked into 10% dog serum and serum samples were analysed with 10-times dilution using the same assay. The final activity results were corrected with 10-fold for the 10-fold dilution. Plasma exposure levels of cmpd#6 were quantified by a luminescent oxygen channelling (LOCI) assay using anti-V _H H (Novo Nordisk, Denmark) and anti-gammaGlu-linker antibodies (Novo Nordisk, Denmark). The 15 data is summarized in table 21. FVIII:C chromogenic activity was measurable in serum 30 and 90 minutes post dosing and quantification of cmpd#6 based on chromogenic activity is similar to estimation based on cmpd#6 plasma exposure measurements using LOCI assay. Any discrepancies in cmpd#6 quantification is predicted to be caused by differences in assay sensitivity. The data show that the PO administrated V _H H polypeptide 20 derivative is fully active upon oral uptake in dog, indicating that the concept of making an orally available FVIII mimetic drug to treat patients, suffering from for example haemophilia A, is possible. Table 21: Ex vivo activity of anti-FX/anti-FIX(a) polypeptide derivative post PO dosing Time post Animal FVIII:C Cmpd#6 Activity/exposure r r l d hr m ni x r R ti %

EXAMPLE 16: CRYSTALLIZATION AND PARATOPE/EPITOPE MAPPING OF ANTI- FX VHH FRAGMENT CMPD#76 The purpose of the present study was to determine the paratope and epitope residues of the V _H H fragment designated Cmpd #76 (VHH-1.15). 5 Crystallisation Crystals of the V _H H fragment Cmpd #76 produced using CHO mixed in a 1:4 molar ratio with a synthetic peptide N-term-NPFDLLD-C-term, corresponding to the activation peptide sequence aa31-37 of human FX (purchased from Apigenex) as identified in example 4, 10 were grown using the sitting drop vapour diffusion technique. A protein solution of 250 nl 5.75 mg/ml complex in 20mM Tris-HCl, pH 7.4, 50 mM NaCl was mixed with 250 nl of 4 % (v/v) Tacsimate (1.8305 M Malonic acid, 0.25 M Ammonium citrate tribasic, 0.12 M Succinic acid, 0.3 M DL-Malic acid, 0.4 M Sodium acetate trihydrate, 0.5 M Sodium formate, and 0.16 M Ammonium tartrate dibasic) pH 5.0, 12 % (w/v) Polyethylene glycol 15 3350 as precipitant and incubated over 80 µl precipitant. After 5 days of incubation at 18 °C, the crystallisation plate was transferred to 5 °C. Crystals appeared within three months. Diffraction data collection The crystal was cryo protected in precipitant added 20 % ethylene glycol prior to flash 20 cooling in liquid nitrogen. Diffraction data were collected at 100K at the Swiss Light Source beamline X10SA (1.0000 Å wavelength) using an Eiger216M pixel detector from Dectris. Autoindexing, integration and scaling of the data were performed with programmes from the XDS package (diffracting data statistics are summarised in Table 22). 25 Structure determination and refinement The asymmetric unit contains two Cmpd #76:FX AP 31-37 complexes. The structure was determined by molecular replacement using Phaser as implemented in the programme suite Phenix using a previous determined crystal structure of a related V _H H as a search model, see example 6. The correct amino acid sequence of Cmpd #76 was introduced 30 and FX AP 31-37 was model built manually in the difference electron density map, all using COOT. During rounds of refinement in Phenix and manual rebuilding in COOT, O- glycosylation on threonine residue 117 and sulphation of tyrosine residue 109 were observed and modelled. The refinement statistics are found in Table 22. 137

Table 22: Data collection and refinement statistics Wavelength (Å) 1 Resolution range (Å) 41.89 - 1.7 (1.761 - 1.7) Space group P 4 ₁ 2 ₁ 2 Unit cell (Å, deg) 66.5466.54161.74909090 Total reflections 534639 (54552) Unique reflections 40883 (4000) Multiplicity 13.1 (13.6) Completeness (%) 99.59 (99.48) Mean I/sigma(I) 17.03 (0.83) Wilson B-factor (Å ² ) 35.51 R-merge 0.07098 (3.508) R-meas 0.07398 (3.642) R-pim 0.0206 (0.9736) CC1/2 0.999 (0.72) CC* 1 (0.915) Reflections used in 40750 (3981) refinement Reflections used for R-free 1993 (193) R-work 0.1828 (0.3798) R-free 0.2077 (0.4038) CC(work) 0.963 (0.862) CC(free) 0.949 (0.702) Number of non-hydrogen 2225 atoms macromolecules 1958 ligands 84 solvent 217 Protein residues 254 RMS(bonds) 0.010 RMS(angles) 1.15 Ramachandran favored (%) 96.67 Ramachandran allowed (%) 2.92 Ramachandran outliers (%) 0.42 Rotamer outliers (%) 0.50 138

Clashscore 3.59 Average B-factor (Å ² ) 47.24 macromolecules 46.21 ligands 71.05 solvent 51.13 Number of TLS groups 1 Statistics for the highest-resolution shell are shown in parentheses. Determination of the epitope and paratope The epitope of the synthetic peptide, defined as the FX activation peptide residues 5 characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the V _H H polypeptide Cmpd #76, comprises the following residues from the synthetic peptide N173, P174, F175, L177, L178 and D179 according to the corresponding FX activation peptide sequence based on consecutive numbering (SEQ ID NO:2). 10 The paratope of V _H H fragment Cmpd #76, defined as Cmpd #76 residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the synthetic peptide, comprises the following residues from Cmpd #76: A33, M34, G35, W47, V48, A49, A50, I51, S52, S57, T58, N59, Y60, A61, A97, A98, D99, G105, L107, Y109 (SEQ ID NO:734) 15 (consecutive numbering). EXAMPLE 17: CRYSTALLIZATION AND PARATOPE/EPITOPE MAPPING OF ANTI- FX VHH FRAGMENT CMPD#77 The purpose of the present study was to determine the paratope and epitope residues of the V _H H fragment designated Cmpd #77 (V _H H1.13). 20 Crystallisation Crystals of the V _H H fragment Cmpd #77 produced from CHO mixed in a 1:4 molar ratio with a synthetic peptide N-term-NPFDLLD-C-term, corresponding to the activation peptide sequence aa31-37 of human FX (synthesized by Apigenex) were grown using the sitting 25 drop vapour diffusion technique. A protein solution of 250 nl 6.1 mg/ml complex in 20mM Tris-HCl, pH 7.4, 50 mM NaCl was mixed with 250 nl of 3 % (v/v) Tacsimate (1.8305 M Malonic acid, 0.25 M Ammonium citrate tribasic, 0.12 M Succinic acid, 0.3 M DL-Malic acid, 0.4 M Sodium acetate trihydrate, 0.5 M Sodium formate, and 0.16 M Ammonium 139

tartrate dibasic) pH 4.0, 11 % (w/v) PEG 3350 as precipitant and incubated over 80 µl precipitant. Crystal appeared 4 days of incubation at 5 °C. Diffraction data collection The crystal was cryo protected in precipitant added 20 % ethylene glycol prior to flash 5 cooling in liquid nitrogen. Diffraction data were collected at 100K at the Swiss Light Source beamline X10SA (1.0000 Å wavelength) using an Eiger216M pixel detector from Dectris. Autoindexing, integration and scaling of the data were performed with programmes from the XDS package (diffracting data statistics are summarised in Table 23). 10 Structure determination and refinement The asymmetric unit contains two Cmpd #77:FX AP 31-37 complexes. The structure was determined by molecular replacement using Phaser as implemented in the programme suite Phenix using the full asymmetric unit from the crystal structure of Cmpd #76:FX AP 31-37 in Example 16 as search model. The correct amino acid sequence of Cmpd #77 15 was introduced and rounds of refinement in Phenix and manual rebuilding in COOT were applied. The refinement statistics are found in Table 23. Table 23: Data collection and refinement statistics Wavelength (Å) 1.0000 Resolution range (Å) 32.88 - 1.7 (1.761 - 1.7) Space group P 4 ₁ 2 ₁ 2 Unit cell Å, deg) 58.9958.99160.25909090 Total reflections 418459 (41888) Unique reflections 31790 (3080) Multiplicity 13.2 (13.6) Completeness (%) 98.90 (97.90) Mean I/sigma(I) 16.44 (1.79) Wilson B-factor (Å ² ) 26.81 R-merge 0.08787 (1.558) R-meas 0.09151 (1.618) R-pim 0.02521 (0.4322) CC1/2 0.999 (0.762) CC* 1 (0.93) Reflections used in 31765 (3079) refinement 140

Reflections used for R-free 1588 (154) R-work 0.1717 (0.2765) R-free 0.2112 (0.3009) CC(work) 0.960 (0.903) CC(free) 0.935 (0.883) Number of non-hydrogen 2253 atoms macromolecules 1971 ligands 50 solvent 232 Protein residues 254 RMS(bonds) 0.011 RMS(angles) 1.03 Ramachandran favored (%) 98.33 Ramachandran allowed (%) 1.67 Ramachandran outliers (%) 0.00 Rotamer outliers (%) 0.49 Clashscore 3.83 Average B-factor (Å ² ) 35.30 macromolecules 33.83 ligands 66.50 solvent 41.00 Number of TLS groups 1 Statistics for the highest-resolution shell are shown in parentheses. Determination of the epitope and paratope The epitope of the synthetic peptide, defined as the FX activation peptide residues 5 characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the V _H H fragment Cmpd #77, comprises the following residues from the synthetic peptide N173, P174, F175, L177, L178 and D179 according to the corresponding FX activation peptide sequence based on consecutive numbering (SEQ ID NO:2). 10 The paratope of V _H H fragment Cmpd #77, defined as Cmpd #77 residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the synthetic peptide, comprises the following residues from Cmpd #77: 141

A33, M34, G35, W47, V48, A49, A50, I51, S52, S57, T58, N59, Y60, A61, A97, A98, D99, G105, L107, Y109 (SEQ ID NO:735) (consecutive numbering). EXAMPLE 18: STEADY STATE FXA GENERATION KINETICS OF BISPECIFIC V _H H 5 POLYPEPTIDE DERIVATIVES The purpose of the present study was to determine the procoagulant activity of anti- FIXa/FX bispecific V _H H polypeptide derivatives based on their ability to promote FX activation by FIXa in the presence of a procoagulant phospholipid membrane. The compounds tested are listed in Table 24 and emicizumab SIA was included for 10 comparison. The steady state FXa generation activity of each compound is reported as the parameters of Michaelis-Menten kinetics (Michaelis constant (K _M ) and the first order rate constant (k _cat /K _M )) at a given compound concentration. The compounds, the final assay concentration of which are shown in Table 24, were pre-incubation with 0.1 nM human 15 plasma-derived FIXa (Haematologic Technologies Inc, USA) and 20 µM 25:75 phosphatidyl serine:phosphatidyl choline phospholipid vesicles (Haematologic Technologies Inc, USA) in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl ₂ , 0.1% (w/v) PEG8000, pH 7.3 + 1 mg/ml BSA) for 5 min. Activation was then initiated by addition of a two-fold dilution series of human plasma-derived FX (Haematologic 20 Technologies Inc, USA) starting at 500 nM. Following activation at room temperature for 7 minutes, the reaction (50 µl) was quenched by addition of 25 µl quench buffer (50 mM HEPES, 100 mM NaCl, 60 mM EDTA, 0.1% PEG8000, pH 7.3 + 1 mg/ml BSA). The amount of FXa generated was determined by addition of 25 µl 2 mM S-2765 chromogenic substrate (Chromogenix, Sweden) and measurement of chromogenic substrate 25 conversion by absorbance measurement at 405 nm (ΔOD/min) in a microplate reader. Similarly, FX activation by free FIXa was determined at a FIXa concentration of 10 nM and a reaction time of 5 to 10 min. To covert the measured absorption at 405 nm to a corresponding FXa concentration, a FXa standard curve from 0-5 nM (50 µl) were added 25 µl quench buffer and 25 µl 2 mM S-2765 chromogenic substrate before absorption at 30 405 nm was recorded as described above. Linear regression of ΔOD/min as a function of FXa concentration produces a slope which can be used for converting measured ΔOD/min into FXa generation rate: FXa generation rate= (ΔOD/min) / (slope _{FXa standard curve} * reaction time) 35 142

Michaelis-Menten steady state kinetic parameters are determined by fitting FXa generation rate as a function of FX (substrate) concentration to the following equation: FXa generation rate = (k _cat * [FIXa] _t * [FX] _t ) / (K _M + [FX] _t ) 5 where k _cat is the enzymatic efficiency (min ^-1 ), [FIXa] _t is the total FIXa (enzyme) concentration (nM), [FX] _t is the total FX (substrate) concentration (nM) and K _M is the Michaelis constant (nM). Table 24 lists the steady state kinetic constants determined for each tested compound as well as the concentration tested. 10 Table 24: Determined steady state enzyme kinetic constants V _H H polypeptide Concentration K _M k _cat /K _M d riv tiv ID / t t d nM nM 1/min*nM The tested anti-FIXa/FX bispecific V _H H polypeptide derivatives all decrease K _M from 281.5 15 nM of free FIXa to between 3.9-9.0 nM when supplemented to a final concentration of 5 nM, and further increase the catalytic efficiency from 0.00012 min ^-1 nM ^-1 to between 0.14- 0.33 min ^-1 nM ^-1 . In comparison, emicizumab-SIA at 300 nM decrease K _M to 53.5 nM. The reduced K _M values reflect an improved assembly (spatial arrangement) of FIX(a)/FX 20 leading to a significant potency increase of the V _H H polypeptide derivatives. All compounds are tested in expected pharmacological relevant concentrations. 143