High efficiency ISVD compounds capable of replacing FVIII (A)

Abstract

The present invention provides ISVD polypeptide derivatives capable of binding to blood coagulation factor IX (a) and factor X (a), which are potent and provide sufficiently long half-lives, thereby allowing effective subcutaneous and oral administration. Thus, the ISVD polypeptide derivatives disclosed herein are suitable for treating hemophilia A, hemophilia A with inhibitors, and acquired hemophilia A by various routes of administration, including subcutaneous and oral administration.

Description

Technical Field

[0001] The present invention relates to compounds capable of binding to coagulation factor IX(a) and factor X(a) and their use in the treatment of coagulopathy such as various forms of hemophilia including hemophilia A.

[0002] Incorporation by Reference of Sequence Listing

[0003] This application is submitted with a sequence listing in electronic form. The entire contents of the sequence listing are incorporated herein by reference. Background Art

[0004] In patients with coagulopathy, such as in people with hemophilia A (HA) and hemophilia B (HB), various steps of the coagulation cascade become dysfunctional due to, for example, the absence or insufficient presence of functional coagulation factors. This dysfunction of a part of the coagulation cascade results in inadequate blood clotting and potentially life-threatening bleeding, or damage to internal organs such as joints.

[0005] Factor VIII (FVIII) deficiency, often referred to as hemophilia A, is a congenital bleeding disorder that affects approximately 420,000 people worldwide, of which approximately 105,000 are currently diagnosed. Hemophilia A has three severity levels, defined by factor FVIII plasma levels of 1% or less ("severe"), 2% to 5% ("moderate"), and 6% to 30% ("mild") (White et al. (2001) Thromb. Haemost. 85:560) or 5-<40% (according to WFH "Guidelines for the management of haemophilia" 2nd ed., Haemophilia; Epub 2012 Jul 6). Bleeding may occur spontaneously or after trauma. Approximately half of all hemophilia A patients are classified as having severe hemophilia A, experiencing severe bleeding beginning in early childhood and having frequent spontaneous or excessive bleeding episodes later in life. Bleeding usually occurs in the joints and muscles, and if not properly treated, repeated bleeding can lead to irreversible hemoarthropathy (Manco-Johnson et al. (2007) N. Engl. J. Med. 357:535-44).

[0006] Patients with hemophilia A can receive coagulation factor replacement therapy, such as exogenous FVIII. Conventional treatment consists of replacement therapy, which is provided as prophylaxis or on-demand treatment of bleeding episodes. Until recently, prophylactic management of patients with severe hemophilia A consisted of up to three intravenous injections per week of plasma-derived FVIII or recombinant FVIII or its long-acting variants.

[0007] However, such patients have the risk of generating neutralizing antibodies (so-called inhibitors) for such exogenous factors, thereby rendering previously effective therapies ineffective. Hemophilia A patients with inhibitors are non-limiting examples of partially congenital, partially acquired coagulopathy. Patients who have generated FVIII inhibitors cannot be treated with conventional replacement therapy. Exogenous coagulation factors can only be administered intravenously, which is quite inconvenient and uncomfortable for patients.

[0008] Insufficient FXa formation and decreased thrombin generation caused by reduced or absent FVIII activity are the underlying causes of the bleeding diathesis in patients with hemophilia A.

[0009] The proteolytic conversion of FX to its enzymatically active form FXa can be achieved by an intrinsic FX activation complex comprising FIXa and its cofactor-activated FVIII (FVIIIa). Cofactor binding increases the enzymatic activity of FIXa by about five orders of magnitude, and it is believed to occur by a variety of mechanisms as outlined by Scheiflinger et al. (2008) J Thromb Haemost, 6:315-322. It is noteworthy that FVIIIa has been found to stabilize the conformation of FIXa, which has increased proteolytic activity against FX (Kolkman JA, Mertens K (2000) Biochemistry, 39:7398-7405, T, Brandstetter H (2009) Biol Chem, 390:391-400).

[0010] In recent years, emicizumab Also known as ACE910, it has been approved for subcutaneous prophylactic treatment of hemophilia A with or without inhibitors to 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 hemophilia A. Emicizumab is designed to simulate FVIII cofactor function (see Sampei et al. (2013) PLoS One, 8, e57479 and WO2012 / 067176). It is speculated that the use of 30-50 μg of emicizumab per milliliter of plasma for treatment corresponds to an equivalent factor VIII activity of at least 10 to 15 IU per deciliter of plasma (Shima et al., N Engl J Med 2016; 374: 2044-53). However, some patients have developed inhibitors (anti-drug antibodies) to emicizumab, making the treatment of the compound ineffective.

[0011] In addition to generating inhibitors as exemplified for emicizumab, other antibody properties are also crucial for achieving effective antibody-based treatment for patients. In particular, it has been shown that antibodies with high nonspecific binding tendencies can cause safety issues clinically. In some reports, high levels of nonspecific binding lead to a shortened half-life of the antibody circulation, and lead to ineffective and cumbersome dosing regimens for patients (see Dobson et al., Nature, volume 6, art. no.: 38644 (2016) and Avery et al., MAbs 2018, Vol. 10, No. 2, 244-255).

[0012] WO2018 / 141863, WO2019 / 065795 (US10759870), WO2020 / 025672 and WO2021 / 152066 disclose anti-FIX(a) / anti-FX(a) bispecific full-length antibodies and their use as coagulants for treating hemophilia by subcutaneous administration. One such bispecific antibody is called Mim8 (see H et al., Blood. 2021; 138: 1258–68 and WO2020 / 025672).

[0013] However, there remain many very important unmet medical needs in the hemophilia population, particularly in patients with coagulopathy, and there is a particular need for improved compounds that reduce the treatment burden and can replace FVIII(a) for the treatment of coagulopathy such as hemophilia A and related diseases. Summary of the invention

[0014] The present invention provides procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivatives capable of binding to coagulation factor IX (a) and coagulation factor X (a), such as V H H polypeptide derivatives that are highly potent and provide a sufficiently long half-life to allow effective subcutaneous administration as well as oral administration. Thus, in one aspect, the present invention relates to 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 an activated form thereof, a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or an activated form thereof, one or more extensions attached to one or more surface exposed residues, and optionally a linker (L) connecting ISVD1 and ISVD2. 1-2 ), and optionally one or more extensions ("E").

[0015] 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 an activated form thereof, a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or an activated form thereof, at least one prolongation attached to a surface exposed residue, and optionally a linker (L) connecting ISVD1 and ISVD2. 1-2 ), and optionally one or more extensions (E), wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof, said epitope comprising at least one of amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively), and wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising at least one of amino acid residues N173, P174, F175, L177, L178 and D179 (numbered consecutively).

[0016] For example, the ISVD polypeptide derivative may be V H H polypeptide derivatives.

[0017] Another aspect of the present invention relates to pharmaceutical compositions comprising the ISVD polypeptide derivatives disclosed herein. Another aspect of the present invention relates to the use of the ISVD polypeptide derivatives disclosed herein and compositions comprising such compounds for treating various forms of hemophilia, particularly hemophilia A, hemophilia A with inhibitors, and acquired hemophilia A, by various routes of administration including subcutaneous and oral administration.

[0018] In another aspect, the present invention relates to a polypeptide as an ISVD polypeptide derivative or V H A single component (intermediate) of a portion of a H polypeptide derivative ISVD or V H H fragment, such as its specific anti-FIX(a)V H H fragment or specific anti-FX(a)V H H fragment.

[0019] Another aspect of the present invention relates to the preparation of components (intermediates) of the compounds disclosed herein, including methods for altering the isoelectric point of ISVD polypeptide derivatives capable of binding FIX(a) and FX(a) to increase the oral bioavailability of such polypeptide derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1a -h shows non-limiting examples of anti-FX(a) / FIX(a) ISVD polypeptide derivatives. E: extension, PM: prolongation, L:1-2 : The linker connecting ISVD1 and ISVD2. The dotted line indicates the disulfide bond.

[0021] Figure 2a Shows resistance to FX / FIX(a)V H Non-limiting examples of H polypeptide derivatives include: P )(ID:L P 1) A single C18 diacid fatty acid based extension conjugated to a C-terminal extension. The dotted line represents a disulfide bond.

[0022] Figure 2b Shows resistance to FX / FIX(a)V H Non-limiting examples of H polypeptide derivatives include: P )(ID:L P 2) A single tetrazole-based extension conjugated to a C-terminal extension. Dashed lines represent disulfide bonds.

[0023] Figure 3a -f shows detailed schematic descriptions of Compound #20(a), Compound #18(b), Compound #15(c), Compound #13(d), Compound #14(e) and Compound #12(f), which are compounds containing a prolonging linker (L P )(ID L P 1) Anti-FX / FIX(a)V with a C16 diacid fatty acid extension conjugated to a C-terminal extension (E) H Non-limiting examples of H polypeptide derivatives. Dashed lines represent disulfide bonds. H An extension (E) (SEQ NO: 9) is used in the H polypeptide derivatives. The extension comprises two cysteine ​​residues at positions 4 and 6, respectively, which serve as attachment points for the prolongation moiety (PM).

[0024] In a), V H H1.20-L 1-2 -V H The sequence of the H2.20-E polypeptide is represented by SEQ ID NO: 634 (Compound #20).

[0025] In b), V H H1.18-L 1-2 -V H The sequence of the H2.18-E polypeptide is represented by SEQ ID NO: 632 (Compound #18).

[0026] In c), V H H1.15-L 1-2 -V HThe sequence of the H2.15-E polypeptide is represented by SEQ ID NO: 629 (Compound #15).

[0027] In d), V H H1.13-L 1-2 -V H The sequence of the H2.13-E polypeptide is represented by SEQ ID NO: 627 (Compound #13).

[0028] In e), V H H1.14-L 1-2 -V H The sequence of the H2.14-E polypeptide is represented by SEQ ID NO: 628 (Compound #14).

[0029] In f), V H H1.12-L 1-2 -V H The sequence of the H2.12-E polypeptide is represented by SEQ ID NO: 626 (Compound #12).

[0030] Figure 4 Examples of titration curves (activity as a function of compound concentration) for Compound #6, Mim8, and Emicizumab SIA are shown.

[0031] Figure 5a and Figure 5b Anti-FIX (a) and anti-FX ISVD (V H H fragment) sequences, with the CDR sequences highlighted in bold and underlined.

[0032] Sequence Description

[0033] SEQ ID NO: 1 represents the amino acid sequence of human coagulation factor IX.

[0034] SEQ ID NO: 2 represents the amino acid sequence of human coagulation factor X.

[0035] SEQ ID NOs: 3-13 and 690 represent the amino acid sequences of the extension (E).

[0036] SEQ ID NO: 14-26 and 691 represent L 1-2 and L P Amino acid sequence of the linker.

[0037] SEQ ID NO: 27-614 represents V H The amino acid sequence of the H fragment and its complementarity determining regions (CDRs).

[0038] SEQ ID NOs: 615-691, 734 and 735 represent any L 1-2 V of the connector and / or extension H Amino acid sequence of H polypeptide.

[0039] SEQ ID NOs: 692-733 represent the sequences of the peptide fragments disclosed in Example 4 herein.

[0040] SEQ ID NOs: 736-739 represent the amino acid sequences of possible elongates. DETAILED DESCRIPTION

[0041] The present invention provides ISVD polypeptide derivatives capable of binding FIX(a) and FX(a), which are highly effective and provide a sufficiently long half-life to allow effective subcutaneous and oral administration. Therefore, the ISVD polypeptide derivatives disclosed herein are suitable for treating various forms of hemophilia, such as hemophilia A, hemophilia A with inhibitors, and acquired hemophilia A, by various routes of administration, including subcutaneous and oral administration.

[0042] In particular, the present invention relates to bispecific extended ISVD polypeptides, such as V H H polypeptide, called ISVD polypeptide derivative (or V H H polypeptide derivatives), which can bind coagulation FIXa and coagulation FX, thereby leading to the formation of activated coagulation FX (FXa) in a manner that mimics the cofactor activity of coagulation factor VIIIa (FVIIIa). H The H polypeptide derivatives showed extremely high in vitro potency, for example, several orders of magnitude higher than the bispecific antibody emicizumab sequence identical analog (SIA). H H polypeptide derivatives have also been shown to extend half-life in animal models such as rats, dogs, and pigs by introducing extensions such as fatty acid conjugation and albumin binding peptide fusion. H The H polypeptide showed very rapid clearance in dogs and pigs. In addition, V has been modified by making amino acid substitutions that lower the pI of surface exposed residues and by using excipients such as sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) and formulations such as nicotinamide (NAM). H The H polypeptide derivatives are engineered to achieve clinically meaningful bioavailability after oral administration. H H peptide derivatives have shown clinically meaningful bioavailability levels in rat and dog animal models. To date, therapeutic peptides with molecular weights exceeding 10 kDa, such as ISVD peptide derivatives, such as V HH polypeptide derivatives have not been considered suitable for oral administration. H The H polypeptide derivatives are suitable for novel oral treatments of coagulopathy such as, but not limited to, hemophilia A with or without inhibitors.

[0043] Therefore, the present invention provides procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivatives such as V H H polypeptide derivatives that are highly potent and provide a sufficiently long half-life to allow effective subcutaneous administration as well as oral administration. Thus, in one aspect, the present invention relates to 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 an activated form thereof, a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or an activated form thereof, one or more extensions attached to one or more surface exposed residues, and optionally a linker (L) connecting ISVD1 and ISVD2. 1-2 ), and optionally one or more extensions ("E"). Figure 1a -h shows non-limiting examples of anti-FX / FIX(a) ISVD polypeptide derivatives.

[0044] 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 an activated form thereof, a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or an activated form thereof, at least one prolongation attached to a surface exposed residue, and optionally a linker (L) connecting ISVD1 and ISVD2. 1-2 ), and optionally one or more extensions (E), wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof, the epitope comprising at least one of amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively), and wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising at least one of amino acid residues N173, P174, F175, L177, L178 and D179 (numbered consecutively).

[0045] In another aspect, the present invention relates to a procoagulant V H H polypeptide derivative, which comprises

[0046] A first V capable of binding to factor IX (SEQ ID NO: 1) or an activated form thereof H H(V H H1),

[0047] A second V capable of binding to Factor X (SEQ ID NO: 2) H H(V H H2),

[0048] at least one extension attached to a surface exposed residue,

[0049] Optionally, connect V H H1 and V H H2 linker (L 1-2 ),as well as

[0050] Optionally, one or more extensions (E),

[0051] Where V H H1 contains the following sequence

[0052] V H H-2.20 (SEQ ID NO: 171),

[0053] V H H-2.18 (SEQ ID NO: 155),

[0054] V H H-2.15 (SEQ ID NO: 131),

[0055] V H H-2.13 (SEQ ID NO: 115),

[0056] V H H-2.14 (SEQ ID NO: 123),

[0057] V H H-2.12 (SEQ ID NO: 107), or

[0058] V H H-2.2 (SEQ ID NO: 35),

[0059] And where V H H2 contains the following sequence

[0060] V H H-1.20 (SEQ ID NO: 167),

[0061] V H H-1.18 (SEQ ID NO: 151),

[0062] V H H-1.15 (SEQ ID NO: 127),

[0063] V H H-1.13 (SEQ ID NO: 111),

[0064] V H H-1.14 (SEQ ID NO: 119),

[0065] V H H-1.12 (SEQ ID NO: 103),

[0066] V H H-1.3 (SEQ ID NO: 31), or

[0067] V H H-1.4 (SEQ ID NO:39).

[0068] In one such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.20 (SEQ ID NO: 167).

[0069] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.18 (SEQ ID NO: 151).

[0070] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID

[0071] NO:171), and V H H2 contains V H The sequence of H-1.15 (SEQ ID NO: 127).

[0072] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.13 (SEQ ID NO: 111).

[0073] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.12 (SEQ ID NO: 103).

[0074] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.3 (SEQ ID NO:31).

[0075] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.4 (SEQ ID NO:39).

[0076] In one such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.20 (SEQ ID NO: 167).

[0077] In another such embodiment, V H H1 contains V H H-2.18 (SEQ ID NO: 155), and V H H2 contains V H The sequence of H-1.18 (SEQ ID NO: 151).

[0078] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.15 (SEQ ID NO: 127).

[0079] In another such embodiment, V H H1 contains V HH-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.13 (SEQ ID NO: 111).

[0080] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.12 (SEQ ID NO: 103).

[0081] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.3 (SEQ ID NO:31).

[0082] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.4 (SEQ ID NO:39).

[0083] In another such embodiment, V H H1 contains V H H-2.15 (SEQ ID NO: 131), and V H H2 contains V H The sequence of H-1.18 (SEQ ID NO: 151).

[0084] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.15 (SEQ ID NO: 127).

[0085] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V HThe sequence of H-1.13 (SEQ ID NO: 111).

[0086] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.12 (SEQ ID NO: 103).

[0087] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.3 (SEQ ID NO:31).

[0088] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.4 (SEQ ID NO:39).

[0089] In another such embodiment, V H H1 contains V H H-2.13 (SEQ ID NO: 115), and V H H2 contains V H The sequence of H-1.18 (SEQ ID NO: 151).

[0090] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.15 (SEQ ID NO: 127).

[0091] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.13 (SEQ ID NO: 111).

[0092] In another such embodiment, VH H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.12 (SEQ ID NO: 103).

[0093] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.3 (SEQ ID NO:31).

[0094] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.4 (SEQ ID NO:39).

[0095] In another such embodiment, V H H1 contains V H H-2.14 (SEQ ID NO: 123), and V H H2 contains V H The sequence of H-1.18 (SEQ ID NO: 151).

[0096] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.15 (SEQ ID NO: 127).

[0097] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.13 (SEQ ID NO: 111).

[0098] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains VH The sequence of H-1.12 (SEQ ID NO: 103).

[0099] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.3 (SEQ ID NO:31).

[0100] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID

[0101] NO:171), and V H H2 contains V H The sequence of H-1.4 (SEQ ID NO:39).

[0102] In another such embodiment, V H H1 contains V H H-2.12 (SEQ ID NO: 107), and V H H2 contains V H The sequence of H-1.18 (SEQ ID NO: 151).

[0103] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.15 (SEQ ID NO: 127).

[0104] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.13 (SEQ ID NO: 111).

[0105] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.12 (SEQ ID NO: 103).

[0106] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.3 (SEQ ID NO:31).

[0107] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.4 (SEQ ID NO:39).

[0108] In another such embodiment, V H H1 contains V H H-2.2 (SEQ ID NO: 135), and V H H2 contains V H The sequence of H-1.18 (SEQ ID NO: 151).

[0109] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.15 (SEQ ID NO: 127).

[0110] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.13 (SEQ ID NO: 111).

[0111] In another such embodiment, V H H1 contains V H H-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.12 (SEQ ID NO: 103).

[0112] In another such embodiment, V H H1 contains V HH-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.3 (SEQ ID NO:31).

[0113] In another such embodiment, VHH1 comprises the sequence of VHH-2.20 (SEQ ID NO: 171), and V H H2 contains V H The sequence of H-1.4 (SEQ ID NO:39).

[0114] In some embodiments, the extension portion comprises the following structure:

[0115]

[0116] Where * indicates the attachment point on the ISVD polypeptide.

[0117] For example, the ISVD polypeptide derivative may be V H H polypeptide derivatives.

[0118] In another aspect, the present invention relates to pharmaceutical compositions comprising the ISVD polypeptide derivatives disclosed herein. Another aspect of the present invention relates to the use of the ISVD polypeptide derivatives disclosed herein and compositions comprising such compounds for treating various forms of hemophilia, in particular hemophilia A, hemophilia A with inhibitors, and acquired hemophilia A, by various routes of administration including, but not limited to, subcutaneous and oral administration.

[0119] In another aspect, the present invention relates to a polypeptide as an ISVD polypeptide derivative or V H A single component (intermediate) of a portion of a H polypeptide derivative ISVD or V H H fragment, such as its specific anti-FIX(a)V H H fragment or specific anti-FX(a)V H H fragment.

[0120] Another aspect of the present invention relates to the preparation of components (intermediates) of the compounds disclosed herein, including methods for altering the isoelectric point of ISVD polypeptide derivatives capable of binding FIX(a) and FX(a) to increase the oral bioavailability of such polypeptide derivatives.

[0121] definition

[0122] In order that the present invention may be more readily understood, certain terms are defined below.

[0123] Greek letters can be represented by their symbols or corresponding written names, for example: α = alpha; β = beta; ε = epsilon; γ = gamma; ω = omega; etc. In addition, the Greek letter μ can also be represented by "u", such as in μl = ul or in μM = uM.

[0124] An asterisk (*) in a chemical formula indicates a point of attachment.

[0125] The terms “a” or “an” are intended to mean “one or more.” When the terms “comprise” and variations such as “comprising” precede recitation steps or elements, it is intended to mean that adding further steps or elements is optional and not excluded.

[0126] 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 the range by extending the boundaries above and below the numerical values. Generally, the term "about" can modify a numerical value up or down (higher or lower) to 10% above or below the stated value.

[0127] As used herein, the term "backbone" refers to an ISVD polypeptide or V H H polypeptide amino acid sequence, including any L 1-2 connectors and extensions, but not extensions, and for the avoidance of doubt, not including any extensions adapted to fuse to a framework.

[0128] The term "binding affinity" refers to the affinity between two molecules (e.g., ISVD, such as V H The strength of the non-covalent interaction between the H fragment and the antigen is measured by the equilibrium dissociation constant (K D ), two molecules (such as ISVD such as V H The K can be determined by measuring the kinetics of complex formation and dissociation, for example by surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). D The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constants k and k, respectively. a (or k 缔合 ) and the dissociation rate constant k d (or k 解离 ). Through equation K D =k d / k a , K D With k a and k d Associated.

[0129] According to the above definition, by comparing the K of each antibody / antigen complex D Values ​​allow comparison of binding affinities associated with different molecular interactions, such as the binding affinities of different ISVDs to a given antigen.

[0130] The value of the dissociation constant can be determined directly by well-known methods. Standard assays for evaluating the binding ability of ligands such as ISVDs to targets are known in the art and include, for example, ELISA, Western blotting, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of ISVDs can also be assessed by standard assays known in the art, such as SPR.

[0131] A competitive binding assay can be performed in which an ISVD such as V H Binding of H to a target is compared to binding of that target in the presence of another ligand for that target, such as another ISVD.

[0132] Unless contradicted by the context, K D Preferably it is determined by surface plasmon resonance as described herein (see Example 7).

[0133] Preferably, the K of an ISVD capable of binding to FIX(a) is D The value is 3 μM or lower, such as 15 nM or lower, such as 11.7 nM or lower.

[0134] Preferably, the K of an ISVD capable of binding to FX(a) is D The value is 3 μM or lower, such as 350 nM or lower, such as 300 nM or lower.

[0135] “Cross-species reactivity” ISVD (or V H H fragment) binds to, for example, FIX from all specified species (e.g., human and cynomolgus monkey) with comparable affinity, especially K D In the range of a factor of 100, for example, in the range of a factor of 50, in the range of a factor of 20, or in the range of a factor of 10. In the defined factor X, K D In the range, it is meant that the highest affinity for a particular listed species is no more than X times the lowest affinity measured for binding to a different listed species. One skilled in the art will appreciate that any method of measuring affinity can be used to verify that the cross-species reactive ISVD binds to the target antigen from all listed species within a given K value as described herein. D Factor range, as long as the K D The same conditions are applied to all measurements. Preferably, SPR is used to measure K D values, especially at 25°C. Preferably, a cross-species reactive ISVD such as VH H to measure affinity.

[0136] Amino acids are molecules containing an amine group and a carboxylic acid group and optionally one or more additional groups generally referred to as side chains.

[0137] The term "amino acid" includes both canonical amino acids (genetically encoded) and unnatural amino acids. Non-limiting examples of unnatural amino acids are Aib (α-aminoisobutyric acid), desaminohistidine (also known as 3-(imidazol-4-yl)propionic acid, abbreviated as Imp (imidazole propionyl) and the D-isomers of canonical amino acids. Unless otherwise specified, all amino acid residues in a polypeptide for which the optical isomer thereof is not specified are understood herein to mean the L-isomer.

[0138] The term "antibody" as used herein refers to a protein comprising or derived from an immunoglobulin sequence, which is capable of binding to an antigen or a portion thereof.

[0139] "Antibodies" include, but are not limited to, full-length antibodies comprising at least four polypeptide chains: two heavy chains (HC) and two light chains (LC) linked by disulfide bonds, and antibodies comprising at least three polypeptide chains: two heavy chains (HC) and one light chain (LC) linked by disulfide bonds. One class of immunoglobulins is IgG. In humans, the IgG class can be divided into four subclasses: IgG1, IgG2, IgG3 and IgG4, based on the sequence of the constant region of its heavy chain. Light chains can be divided into two types: κ chains and λ chains, based on differences in their sequence composition. IgG molecules consist of two heavy chains interconnected by two or more disulfide bonds and two light chains, each of which is connected to a heavy chain by a disulfide bond. The term "antibody" also covers single domain antibodies, such as V H H fragment and V-NAR fragment.

[0140] As used herein, the term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region includes the "complementarity determining region", also referred to as "CDR".

[0141] "Framework" or "FR" regions are those variable domain regions excluding the hypervariable region residues. Thus, an antibody or ISVD comprises, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR3 of the heavy chain is the region that generally contributes most to antigen binding.

[0142] As used herein, the term "epitope" refers to a protein determinant that is capable of specific binding to an antibody. Epitopes are usually composed of surface elements of a molecule such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that when there is a denaturant that can destroy the protein structure, the binding to the former is lost, while the binding to the latter is not lost.

[0143] In ISVD, V H In the context of the X-ray crystal structure of the complex between the H fragment and its target defined by spatial coordinates, the term epitope is specifically defined herein as an ISVD residue characterized by a distance from a heavy atom in FIX / FIXa or FX / FXa, unless otherwise stated or contradicted by the context. There are heavy atoms (i.e. non-hydrogen atoms) within a distance of .

[0144] The epitope of a given ISVD / antigen pair can be identified by conventional methods such as those described in the Examples. For example, the ISVD and antigen can be combined and the ISVD / antigen complex can be crystallized. The crystal structure of the complex can be determined and used to identify specific sites of interaction between the ISVD and its antigen.

[0145] In one embodiment, the ISVD polypeptide derivative as described herein comprises a first ISVD capable of binding to an epitope on FIX or an activated form thereof comprising at least one of amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (SEQ ID NO: 1), and a second ISVD capable of binding to an epitope on FX (SEQ ID NO: 2) comprising at least one of amino acid residues N173, P174, F175, L177 and L178 (numbered consecutively).

[0146] 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 an activated form thereof, the epitope comprising at least one of amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively), and

[0147] A second ISVD capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising at least one of amino acid residues N173, P174, F175, L177, L178 (numbered consecutively), or comprising at least one of amino acid residues N173, P174, F175, L177, L178 and D179.

[0148] Coagulation factor IX (FIX) is a vitamin K-dependent coagulation factor with structural similarity 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 is composed of 415 amino acids divided into four different domains, which include a domain rich in gamma-carboxyglutamic acid (Gla) at the N-terminal, two EGF domains and a C-terminal trypsin-like serine protease domain. The activation peptide (residues 146 to 180 of SEQ ID NO:1) is released by limited proteolysis at Arg145 and Arg180, and activation of FIX occurs. Therefore, activated FIX (FIXa) is composed of residues 1-145 (light chain) of SEQ ID NO:1 and residues 181-415 (heavy chain) of SEQ ID NO:1.

[0149] Thus, circulating FIX molecules comprise the FIX zymogen and the activated form of FIX, which are generally referred to herein as FIX and FIXa, with reference to SEQ ID NO: 1.

[0150] Activated Factor IX is referred to as Factor IXa or FIXa. The term "FIX (SEQ ID NO: 1) and / or its activated form (FIXa)" may also be referred to as "FIX / FIXa", or simply "FIX(a)".

[0151] FIXa is a trypsin-like serine protease that plays a key role in hemostasis by generating the majority of factor Xa necessary to support the formation of proper thrombin during the coagulation process as part of the tenase complex.

[0152] FIX is represented herein by SEQ ID NO: 1, which corresponds to the Ala148 allele form of human FIX (Anson et al., EMBO J. 1984 3: 1053-1060;

[0153] McGraw et al., Proc Natl Acad Sci USA. 1985 82:2847-2851; Graham et al., Am. J. Hum. Genet. 1988 42:573-580). In the present invention, FIX is intended to cover all natural variants of FIX, such as the T148 variant (Uniprot ID P00740).

[0154] 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, which comprises residues 1-139 (light chain) of SEQ ID NO:2 and residues 143-448 (heavy chain) of SEQ ID NO:2. The human FX zymogen comprises four different domains, including an N-terminal domain rich in γ-carboxyglutamic acid (Gla) (residues 1-45), two EGF domains: EGF1 (residues 46-82) and EGF2 (residues 85-125), and a C-terminal trypsin-like serine protease domain (residues 195-448). FX activation occurs by limited proteolysis at Arg194, which results in the release of an activation peptide (residues 143-194). Thus, activated FX (FXa) consists of residues 1-139 (light chain) of SEQ ID NO: 2 and residues 195-448 (activated heavy chain) of SEQ ID NO: 2. Thus, circulating factor X molecules comprise FX proenzyme and activated forms of FX, which are referred to herein as FX and FXa, respectively, with reference to SEQ ID NO: 2. In the present invention, FX is intended to encompass all natural variants of FX. The term "FX (SEQ ID NO: 2) and / or its activated form (FXa)" may also be referred to as "FX / FXa" or "FX(a)".

[0155] As used herein, the term "conservative substitution" refers to situations in which an amino acid can be replaced with an amino acid having similar biochemical properties, for example, a basic amino acid can be replaced with another basic amino acid (e.g., lysine is replaced with arginine), an acidic amino acid can be replaced with another acidic amino acid (e.g., glutamic acid is replaced with aspartic acid), a neutral amino acid can be replaced with another neutral amino acid (e.g., threonine is replaced with serine), a charged amino acid can be replaced with another charged amino acid (e.g., glutamic acid is replaced with aspartic acid), a hydrophilic amino acid can be replaced with another hydrophilic amino acid (e.g., asparagine is replaced with glutamine), a hydrophobic amino acid can be replaced with another hydrophobic amino acid (e.g., alanine is replaced with valine), a polar amino acid can be replaced with another polar amino acid (e.g., serine is replaced with threonine), an aromatic amino acid can be replaced with another aromatic amino acid (e.g., phenylalanine is replaced with tryptophan), and an aliphatic amino acid can be replaced with another aliphatic amino acid (e.g., leucine is replaced with isoleucine).

[0156] The term "excipient" as used herein refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.

[0157] Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, L-arginine, niacinamide, SNAC, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0158] The term "Extension" (E) as used herein refers to a peptide or polypeptide suitable for attachment to an ISVD polypeptide.

[0159] The extension can be a few amino acids, 1 to 10 amino acids, or longer, 10 to 30 amino acids, or very long, more than 30 amino acids. The extension is preferably present in the ISVD polypeptide, ISVD polypeptide derivative, V H H polypeptide or V H The H polypeptide derivative may be present at the N-terminus (N-terminal extension) or the C-terminus (C-terminal extension), or both. Alternatively, the extension may be attached to the ISVD polypeptide at a position other than the CDR sequence, such as in one or more framework regions or at the linker L. 1-2 middle.

[0160] The extension is preferably recombinantly fused to the ISVD polypeptide. In other embodiments, the extension is conjugated to the ISVD polypeptide.

[0161] Non-limiting examples include compound #22 having a 6 amino acid C-terminal extension consisting of GQACPC (SEQ ID NO:9), compound #6 having a 13 amino acid C-terminal extension consisting of GGGGCSCHHHHHH (SEQ ID NO:8), and

[0162] The 11 amino acid C-terminal extension consists of GGGGSHHHHHH (SEQ ID NO: 7).

[0163] The purpose of the extension is to provide an attachment point for the extension portion and / or to provide a means for purification. Therefore, the term "extension" does not encompass the extension body and the extension portion.

[0164] For example, any extension linker (L) comprising a recombinant fusion to the N-terminus or C-terminus of a polypeptide P ) is not regarded as an extension.

[0165] As used herein, the term "fusion" refers to the in-frame joining of two or more DNA sequences that originally encoded separate proteins or peptides or fragments thereof. Translation of the fusion polypeptide DNA sequence will produce a single polypeptide sequence that may have functional properties derived from each of the original proteins or peptides. The DNA sequence encoding the fusion protein can be artificially created by standard molecular biology methods such as overlapping PCR or DNA ligation. The resulting fusion polypeptide DNA sequence can be inserted into an appropriate expression vector that supports heterologous fusion protein expression in a host organism such as bacteria, yeast, fungi, insect cells or mammalian cells. For example, the extension portion can be fused to an ISVD polypeptide or V H Fusion to the C-terminus or N-terminus of the H polypeptide backbone.

[0166] As used herein, the term "host cell" encompasses any type of cell system that can be engineered to produce the ISVDs disclosed herein. Host cells include, but are not limited to, cultured cells, such as cultured mammalian 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.

[0167] The term "identity" as known in the art refers to the relationship between the sequences of two or more polypeptides determined by comparing sequences. In the art, "identity" also represents the degree of sequence correlation between polypeptides, as determined by the number of matches between residue strings of two or more amino acid residues. "Identity" measures the percentage of identical matches with the smaller of the two or more sequences, with gap comparisons (if any), carried out by a specific mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptides can be easily calculated by known methods. In the present invention, Needleman (Needleman et al., J.Mol.Biol.1970; 48:443-453) from EMBOSS-6.6.0 is used to determine similarity and identity, wherein parameters 10 and 0.5 (gapopen=10, gapextend=0.5) are used for gap opening and extension, respectively.

[0168] The terms "immunoglobulin single variable domain" or "ISVD" are used as a general term to include antigen binding domains or fragments, such as VH and VL domains, respectively.

[0169] Thus, the ISVD can be a light chain variable domain sequence (e.g., a VL sequence) or a heavy chain variable domain sequence (e.g., a VH sequence); for example, a heavy chain variable domain sequence derived from a conventional four-chain antibody or a light chain variable domain sequence derived from a conventional four-chain antibody.

[0170] A specific type of ISVD is V H H fragments, which were originally identified as a type of immunoglobulin, are defined as antibody fragments consisting of a single monomeric variable antibody domain. H The H fragment is a single domain antibody that contains only the antigen-binding variable region of a heavy chain antibody obtainable from camelids. H The size of H fragments is approximately 15 kDa. They contain single-chain molecules that can bind to their cognate antigen using a single domain. H The antigen-binding surface of an H fragment is generally more convex (or protruding) than the antigen-binding surface of a conventional antibody, which is generally flat or concave. H The H fragment consists of four framework regions (or FRs), whose sequences and structures are defined as conserved, and three complementarity determining regions (or CDRs), which show high variability in both sequence content and structural conformation and are involved in antigen binding and provide antigen specificity.

[0171] Another type of ISVD can be obtained from IgNAR of cartilaginous fish, and its single domain antibody is called "V-NAR fragment".

[0172] V H H fragments and V-NAR fragments do not contain constant domains and therefore lack an Fc region, which is typically part of a partial / full-length and / or engineered / natural heavy chain-only antibody [Dooley et al. (2006) Dev. Comp. Immunol. 30:43–56; Muyldermans S. (2013) Annu Rev Biochem. 82:775-97].

[0173] About CamelidaeV H A general description of H fragments and methods for their production and / or isolation and / or use can be found in the following references: WO94 / 04678 and WO97 / 49805. A general description of heavy chain immunoglobulins and their variable regions from cartilaginous fish and methods for their production and / or isolation and / or use can be found in WO2005 / 118629.

[0174] V H The total number of amino acid residues in the H fragment is usually in the range of 110-140. However, it should be noted that H The parts, fragments or analogs of H are not particularly limited in their length and / or size, as long as such parts, fragments or analogs meet the further requirements outlined below and are also preferably suitable for the purposes described herein. H The molecular weight of the H fragment is usually in the range of 12-15 kDa. HThe pi of the H fragment is usually basic, as is usually the case with antibodies, meaning that the pi value is usually above 7, usually between 7.5 and 8.5. H The H fragment usually contains at least one disulfide bond, which is usually formed by a conserved cysteine ​​pair located in framework regions 1 and 3. Such a disulfide bond ensures that the V H The correct folding and stability of the H fragment, and if, for example, H If side chain modification and / or conjugation is performed by way of unpaired cysteines introduced into the H polypeptide, it is desirable to retain such disulfide bonds.

[0175] In this document, the Kabat definition (Kontermann and Dübel, 2010, Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51) is used to determine ISVD as V H According to this approach, the CDRs of the variable domain are defined as positions 31-35 (CDR1), positions 50-65 (CDR2), and positions 95-102 (CDR3). However, when referring to the polypeptide compounds described herein (including ISVD polypeptides or V H When referring to specific amino acid residue positions in the CDR and framework (FR) regions of an H polypeptide, consecutive numbering is used unless otherwise indicated.

[0176] As used herein, the term "ISVD polypeptide" refers to a polypeptide comprising two or more ISVDs, such as a first ISVD (ISVD1) and a second ISVD (ISVD2), which are connected as desired, for example, by a linker (L) of any appropriate composition and length. 1-2 ) connection, or as a direct domain fusion without any linker at all.

[0177] As used herein, the term "linker" refers to at least one atom that forms a covalent bond between chemical entities. If the chemical entities are connected only by peptide bonds, the linker may be referred to as a "peptide linker". Otherwise, the linker may be referred to as a "chemical linker".

[0178] An example of an ISVD polypeptide is a polypeptide connected via a linker (L 1-2 ) Two ISVDs connected.

[0179] Another example of an ISVD polypeptide is through a linker (L 1-2 ) connected to each other, further comprising one or more extensions.

[0180] Another example of an ISVD polypeptide is a polypeptide that is prepared without the use of a linker (L1-2 ), and further comprising one or more extensions, such as but not limited to C-terminal and / or N-terminal extensions.

[0181] For example, the linker (L 1-2 ) may consist of an amino acid sequence containing no repeats or containing multiple repeats.

[0182] For example, the linker can comprise 2 to 50 amino acids, 5 to 40 amino acids, or 10 to 30 amino acids.

[0183] Non-limiting examples of linkers include *-GGGGS-* linkers, *-GQAPGQ-* linkers (SEQ ID NO: 20), *-QAPGQA-* linkers (SEQ ID NO: 16), *-GI-* linkers, *-GV-* linkers, *-GT-* linkers, *-GL-* linkers, or another amino acid complex linker. Two examples of linkers are x2 and x6 repeat complexes of GGGGS, which are 10 and 30 amino acid residues in length, respectively.

[0184] For the avoidance of doubt, L 1-2 Subscript in 1-2 does not imply a specific direction of ISVD connection, i.e. 1-2 One can, for example, link ISVD1 to ISVD2, or link ISVD2 to ISVD1 (N-terminus to C-terminus).

[0185] SEQ ID NO: 14-24 represents L 1-2 Non-limiting examples of linkers.

[0186] In some embodiments, the ISVD polypeptide comprises an extension (E) as summarized by any of the following formulas:

[0187] ISVD1–ISVD2, or

[0188] ISVD1–ISVD2–E, or

[0189] ISVD2–ISVD1–E, or

[0190] E-ISVD1–ISVD2, or

[0191] ISVD1–L 1-2 –ISVD2–E, or

[0192] ISVD2–L 1-2 –ISVD1–E, or

[0193] E-ISVD1–L 1-2 –ISVD2.

[0194] For example, the extension (E) may be attached to the ISVD polypeptide as outlined by any of the following formulas (N-terminus to C-terminus):

[0195] ISVD1–ISVD2, or

[0196] ISVD1–ISVD2–E, or

[0197] ISVD2–ISVD1–E, or

[0198] E-ISVD1–ISVD2, or

[0199] ISVD1–L 1-2 –ISVD2–E, or

[0200] ISVD2–L 1-2 –ISVD1–E, or

[0201] E-ISVD1–L 1-2 –ISVD2.

[0202] The ISVD polypeptide preferably comprises an extension portion. In such a case, the ISVD polypeptide is referred to as an ISVD polypeptide derivative.

[0203] In some embodiments, the first ISVD serves as an attachment point for one or more extensions.

[0204] In some embodiments, the second ISVD serves as an attachment point for one or more extensions.

[0205] In some embodiments, L 1-2 The connector serves as an attachment point for one or more extensions.

[0206] For the avoidance of doubt, when the extended portion includes the ISVD1-L 1-2 - The extension (E) of ISVD2 is attached to the connector (L p ), the connector L p Not considered part of the “Extension”.

[0207] In some aspects, the ISVD polypeptide is chemically conjugated to a non-ISVD, such as a small chemical non-polypeptide molecule, a carbohydrate, a fatty acid, or an oligopeptide or a polypeptide or a protein, such as an antibody or preferably an antibody fragment.

[0208] In one embodiment, the ISVD polypeptide is linked to the Fc domain from an IgG antibody without the use of a linker.

[0209] In another embodiment, the ISVD polypeptide is linked to the Fc domain or fragment thereof from an IgG antibody via a linker.

[0210] In a preferred embodiment, the extension is fused to the ISVD polypeptide and is therefore not linked to the ISVD polypeptide by chemical conjugation means.

[0211] In some embodiments, the protracting moiety is fused to the ISVD polypeptide and is therefore not linked to the ISVD polypeptide by chemical conjugation means.

[0212] As used herein, the term "isoelectric point" or "pi" refers to the pH at which the total net charge of a protein, such as an antibody, is zero. There may be many charged groups in a protein, and at the isoelectric point, the sum of all these charges is zero. At pH values ​​above the isoelectric point, the total net charge of the protein is negative, while at pH values ​​below the isoelectric point, the total net charge of the protein is positive.

[0213] The pi may be a theoretical or experimentally determined isoelectric point.

[0214] Those skilled in the art are aware of methods for determining the isoelectric point of a protein. Most commonly, the isoelectric point of a protein is calculated based on its amino acid sequence. Many (online) tools are available that allow determination of the isoelectric point of a protein, such as "ExPASy Compute pI / Mw"; see Protein Identification and Analysis Tools on the ExPASy Server; Gasteiger E., Hoogland C., Gattiker A., ​​Duvaud S., Wilkins MR, Appel RD, Bairoch A.; (in) John M. Walker (ed.): The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607. Preferably, the isoelectric point of a protein is calculated using Skoog &

[0215] Algorithm of Wichman, 1986. Calculates pi using the pKa of an amino acid residue.

[0216] The pi may also be determined experimentally and the charge variants may be separated, for example, using charge-based separation techniques such as isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis.

[0217] In one embodiment, the first and second ISVDs in the ISVD polypeptide are V-NAR fragments, and such compounds are referred to as "V-NAR polypeptides."

[0218] In another embodiment, the first and second ISVDs in the ISVD polypeptide are V H H fragment, and such compounds are called "V H In one such embodiment, the first V H The H fragment can bind to FIX / FIXa, while the second V H The H fragment is capable of binding FX / FXa. In a preferred embodiment, V H H polypeptide is a bispecific V H H polypeptide.

[0219] For the avoidance of doubt, the terms multispecific, trispecific or bispecific are intended to reflect the H The number of antigens bound by the H fragment, ie excluding the molecule bound by the extending part (if present), such as but not limited to albumin.

[0220] In one embodiment, V without the extension part H The molecular weight of the H polypeptide ranges from 27 to 29 kDa.

[0221] In a preferred embodiment, one or more extensions (V H H polypeptide derivative) and optionally one or more extensions V H The molecular weight of the H polypeptide ranges from 28 to 33 kDa.

[0222] Those skilled in the art will appreciate that the above embodiments similarly apply to V-NAR polypeptides.

[0223] As used herein, the term "free cysteine" is a cysteine ​​residue in a polypeptide chain that is available for reactions such as chemical conjugation and is therefore not part of a natural or engineered internal disulfide bond. In essence, free cysteine ​​is available for conjugation, although during recombinant expression of polypeptides in host cells, free cysteine ​​residues, including recombinantly introduced free cysteine, are typically blocked with small thiols such as cysteine, homocysteine ​​or glutathione. This is also observed during recombinant production of ISVD polypeptides into which one or more free cysteines have been introduced. Therefore, reduction reactions using appropriate reducing agents such as bis(p-sulfonic acid phenyl)phenylphosphine dihydrate or tris(2-carboxyethyl)phosphine hydrochloride can be used to release and prepare free cysteine ​​for conjugation to a moiety of interest, such as, but not limited to, an extension moiety, such as a C18 diacid γ-Glu2xOEG fatty acid moiety.

[0224] The ISVD polypeptides disclosed herein, such as V H The H polypeptide may be multispecific, for example but not limited to bispecific or trispecific.

[0225] As used herein, the terms "bispecific ISVD polypeptide" or "bispecific V H "H polypeptide" refers to an ISVD polypeptide or V polypeptide that can bind to two different antigens or two different epitopes on the same antigen. H H polypeptide.

[0226] As used herein, the terms "trispecific ISVD polypeptide" or "trispecific V H "H polypeptide" refers to an ISVD polypeptide or V polypeptide that can bind to three different antigens or three different epitopes on the same antigen or three different epitopes present on two different antigens. H H polypeptide.

[0227] As used herein, the terms "multispecific ISVD polypeptide" or "multispecific V H "H polypeptide" refers to an ISVD polypeptide or V polypeptide that is capable of binding to two or more different antigens or two or more different epitopes on the same antigen. H H polypeptide. Thus, a multispecific ISVD polypeptide or a multispecific V H H polypeptides include bispecific and trispecific ISVD polypeptides or V H H polypeptide.

[0228] Those skilled in the art will appreciate that the above also applies to polypeptide derivatives (ie including extended moieties).

[0229] As used herein, the term "oral bioavailability" or "oral bioavailability" refers to the amount of an administered drug in the systemic circulation following oral administration (estimated as the area under the plasma concentration-time curve of the administered drug) relative to the amount of the administered drug in the systemic circulation following intravenous administration of the drug.

[0230] As used herein, the term "paratope" refers to the region or area on an ISVD that specifically binds to an antigen, ie, the region or area that is in physical contact with the antigen.

[0231] In ISVD, V H In the context of the X-ray crystal structure of the complex between the H fragment and its target defined by spatial coordinates, the term paratope is specifically defined herein as an ISVD residue characterized by being located at a distance from a heavy atom in FIX / FIXa or FX / FXa, unless otherwise stated or contradicted by the context. There are heavy atoms (i.e. non-hydrogen atoms) within a distance of .

[0232] The paratope (and epitope) of a given ISVD / antigen pair can be identified by conventional methods such as those described in the Examples. For example, the ISVD and antigen can be combined and the ISVD / antigen complex can be crystallized. The crystal structure of the complex can be determined and used to identify specific sites of interaction between the ISVD and its antigen.

[0233] The term "pharmaceutically acceptable excipient" means a generally safe, non-toxic excipient that can be used to prepare a pharmaceutical composition, and includes excipients that are acceptable for human pharmaceutical use. For example, such an excipient can be solid, liquid or semi-solid.

[0234] As used herein, the term "plasma half-life" refers to the time required for half the amount of a substance administered to a patient to be metabolized or eliminated from the patient's serum or plasma by normal biological processes.

[0235] As used herein, the term "preferential binding" should be understood as a binding region on an ISVD, such as V H H fragment, preferentially or favoring binding to one component (e.g., activated FIX) over another component. Therefore, "preferential binding" does not necessarily require that binding of other components is excluded or undetectable. For example, anti-FIX(a) ISVD, such as anti-FIX(a) V H The H fragment can preferentially bind to activated FIX compared to inactivated FIX.

[0236] The term "procoagulant antibody" refers to an antibody that enhances blood coagulation, for example by accelerating the blood coagulation process and / or increasing the enzymatic activity of one or more coagulation factors.

[0237] As used herein, the term "procoagulant activity" refers to the ability of a compound, such as an antibody, to enhance blood coagulation, for example by accelerating the blood coagulation process and / or increasing the enzymatic activity of one or more coagulation factors. Thus, the term "procoagulant activity" encompasses (but is not limited to) one or more of the following activities:

[0238] Enhances Factor IXa-mediated Factor X activation as measured by an amide hydrolysis (chromogenic or fluorogenic) assay based on FIXa-mediated FX activation. This assay measures FXa by cleavage of a FXa-specific peptide substrate. Substrate is generated and the resulting color can be measured photometrically as absorbance.

[0239] Reduction of clotting time, as measured by coagulation tests such as the activated partial thromboplastin time (APTT), which measures the activity of the intrinsic and common coagulation pathways. Plasma is pre-incubated with an APTT reagent containing a contact activator (e.g., ellagic acid or kaolin) and a phospholipid. Calcium chloride is added to promote the formation of a fibrin clot. Possible readouts are the clotting time or the coagulation waveform.

[0240] Enhance thrombin generation, as measured by thrombin generation tests such as Calibrated Automated Thrombography (CAT). Thrombograms describe the concentration of thrombin in clotting plasma and are therefore a functional test of the hemostatic system. The test is based on the measurement of the fluorescence generated by thrombin cleaving the fluorescent substrate ZGGR AMC over time. See also the method used in Example 8 herein.

[0241] Enhances the overall viscoelastic properties of clot formation as measured by viscoelastic hemostasis methods, for example, whole blood under shear stress by tests such as ROTEM (rotational thromboelastometry). In this instrument, a ball bearing pin rotates in a stationary cup. During the clotting process, fibrin filaments in the sample form between the cup wall and the pin, and the strength of the fibrin filaments will affect the movement of the pin, which is detected.

[0242] Reduction of whole blood clotting time (WBCT), as measured by a platelet function analyzer, is based on von Willebrand factor (VWF)-mediated adhesion of platelets to collagen following platelet activation. High shear stress is generated, which leads to platelet adhesion and platelet aggregation. The time from the start to the end of blood flow is measured.

[0243] The ISVD polypeptides disclosed herein preferably comprise one, two, three or four "prolonging moieties" (PM). More preferably, one or two prolonging moieties.

[0244] As used herein, the term "prolonging moiety" refers to a prolonging moiety having the property of extending the half-life and comprising a "prolonging body" (P) and an optional "linker" (L P Thus, the term "extending" refers to half-life extension, and the extender or extending portion thus achieves the purpose of extending the half-life of the ISVD polypeptide disclosed herein.

[0245] The extension part (PM) comprises an extension body "P" and an optional linker (L P ).

[0246] Each extension is preferably attached to a surface exposed lysine or cysteine ​​residue in the polypeptide backbone of the compound. The point of attachment is generally referred to as R1 (if more than one extension is attached, then R2, R3, etc., where R1≠R2≠R3, etc.).

[0247] One skilled in the art will be able to identify other surface exposed residues suitable for attachment.

[0248] The extension portion may consist of an extension body.

[0249] The extension may include a linker (L P ) and an extension (P).

[0250] The extension portion may include a connector and two or more extensions.

[0251] When the connector (L P ) exists, the extension part is passed through L P Attached to the ISVD polypeptide backbone. P ) is absent, P is attached to the polypeptide backbone.

[0252] In one embodiment, there are first and second elongate moieties, wherein the first elongate moiety comprises the structure:

[0253]

[0254] And wherein the second extension portion comprises the following structure

[0255]

[0256] wherein '*' (R1) represents the point of attachment of the first extension moiety on the ISVD polypeptide, and

[0257] wherein '**' (R2) represents the point of attachment of the second extension moiety on the ISVD polypeptide,

[0258] wherein '*' is Cys at position 257 of SEQ ID NO:629, and

[0259] wherein '**' is Cys at position 259 of SEQ ID NO:629.

[0260] The ISVD polypeptides disclosed herein may comprise a single cysteine ​​or lysine residue to which a single prolongation moiety is attached / conjugated.

[0261] The ISVD polypeptides disclosed herein may comprise two lysine residues and two extension moieties.

[0262] The ISVD polypeptides disclosed herein may comprise two lysine residues and two identical extensions.

[0263] The ISVD polypeptides disclosed herein may comprise two lysine residues and two non-identical extensions.

[0264] The ISVD polypeptides disclosed herein may comprise three lysine residues and three extension moieties.

[0265] The ISVD polypeptides disclosed herein may comprise three lysine residues and three identical extensions.

[0266] The ISVD polypeptides disclosed herein may comprise three lysine residues and three non-identical extensions (eg, the first and second extensions may be identical, while the third extension is different from the first and second extensions).

[0267] The ISVD polypeptides disclosed herein may comprise two cysteine ​​residues and two extension moieties.

[0268] The ISVD polypeptides disclosed herein may comprise two cysteine ​​residues and two identical extensions.

[0269] The ISVD polypeptides disclosed herein may comprise two cysteine ​​residues and two non-identical extensions.

[0270] The ISVD polypeptides disclosed herein may comprise three cysteine ​​residues and three extension moieties.

[0271] The ISVD polypeptides disclosed herein may comprise three cysteine ​​residues and three identical extensions.

[0272] The ISVD polypeptides disclosed herein may comprise three cysteine ​​residues and three non-identical extensions (eg, the first and second extensions may be identical, while the third extension is different from the first and second extensions).

[0273] When the ISVD polypeptide comprises two or three extensions, these extensions are preferably similar, more preferably substantially identical, or most preferably identical.

[0274] In the case of chemical moieties such as the extended moieties disclosed herein, any suitable computer program and / or algorithm known in the art may be used to determine similarity and / or identity.

[0275] Compounds comprising an extension portion may be referred to as "derivatives". For example, an "ISVD polypeptide derivative" is understood to be an ISVD polypeptide comprising an extension portion, "V H The term "H polypeptide derivative" is understood to mean a V polypeptide containing a prolongation H H polypeptide, while "V-NAR polypeptide derivative" is understood to be a V-NAR polypeptide comprising an extended portion.

[0276] The protracting moiety may be capable of non-covalently binding to albumin, thereby promoting the circulation of the ISVD polypeptide derivative in the bloodstream and extending its half-life. Thus, in one embodiment, the protracting moiety is an albumin binding moiety.

[0277] The extension (P) may contain an acyl group. The acyl group may be branched or unbranched. The acyl group may be saturated or unsaturated. The extension may contain a fatty acyl group. The acyl group may be branched or unbranched. The acyl group may be saturated or unsaturated.

[0278] The elongate body may comprise a distal carboxylic acid group.

[0279] The elongate may comprise a fatty acid group.

[0280] The elongate may comprise fatty acid groups and amide groups.

[0281] The elongate body may comprise a distal carboxylic acid group and an amide group.

[0282] The elongate may comprise an alkyl group.

[0283] The elongate may comprise an aromatic group.

[0284] The elongate may comprise a tetrazole group.

[0285] The elongate may comprise a sulfonic acid group.

[0286] The extension may comprise a phenoxy group.

[0287] The elongate may comprise a benzoic acid group.

[0288] The elongated body may contain 8-30 carbon atoms. The elongated body may contain 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.

[0289] The extension may comprise 6-30 consecutive -CH2- groups. The extension may 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 -CH2- groups.

[0290] The elongated body may contain 12-26 carbon atoms. The "elongated body" may contain 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 carbon atoms.

[0291] The extension may comprise 10-26 consecutive -CH2- groups. The extension may comprise a carbon chain comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive -CH2- groups.

[0292] The elongated form may contain 16-22 carbon atoms.

[0293] The extension may comprise 14-20 consecutive -CH2- groups. The extension may comprise a carbon chain comprising 14, 15, 16, 17, 18, 19 or 20 consecutive -CH2- groups.

[0294] The extension may contain 16-22 consecutive carbon atoms and 14-20 consecutive -CH2- groups.

[0295] The extension may contain 16 consecutive carbon atoms and 14 consecutive -CH2- groups.

[0296] The extension may contain 18 consecutive carbon atoms and 16 consecutive -CH2- groups.

[0297] The extension may contain 20 consecutive carbon atoms and 18 consecutive -CH2- groups.

[0298] The extension may contain 22 consecutive carbon atoms and 20 consecutive -CH2- groups.

[0299] In some embodiments, the elongate comprises a group defined by the formula:

[0300] Chemical formula a: HOOC-(CH2) n -CO-*, wherein n is an integer in the range of 8-30, which may also be referred to as C(n+2) diacid or represented as

[0301] Chemical formula a1: Wherein n is an integer in the range of 8-30.

[0302] The extension body may comprise an oligopeptide. In one embodiment, the extension body 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. For example, the extension body oligopeptide sequence composition can be

[0303] QRLMEDICLPRWGCLWEDDF (SEQ ID NO: 736), which is exemplified as the fusion sequence in SEQ ID NO: 3 and 4 [see also WO01 / 45746A2]. For example, compound #71 contains a 50 amino acid N-terminal extension consisting of

[0304] QRLMEDICLPRWGCLWEDDFGGGGSGGGGSGGGGSGGGGSGGGGS GGGGS (SEQ ID NO: 4), wherein residues 1-20 are an extender (P), and residues 21-50 are a linker (L P ).

[0305] The elongated body can be connected by the amino acid linker L PAttached to an ISVD or ISVD polypeptide.

[0306] In one embodiment, L P The extension (P) may be attached to the side chain of a lysine or cysteine ​​residue in the ISVD polypeptide backbone.

[0307] The ISVD polypeptide derivative may comprise two extensions, each extension comprising 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The ISVD polypeptide may comprise two extensions, wherein each extension (P) comprises 12, 13, 14, 15, 16, 17 or 18 consecutive -CH2- groups.

[0308] The ISVD polypeptide derivative may comprise two C14 diacids, two C16 diacids, or two C18 diacids.

[0309] The ISVD polypeptide derivative may comprise three extensions, each of which comprises an extension comprising 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The ISVD polypeptide may comprise three extensions, wherein the extension comprises 10, 11, 12, 13, 14, 15, 16, 17 or 18 consecutive -CH2- groups.

[0310] For the avoidance of doubt, even if the protracted moiety is capable of binding, for example, albumin, it is not considered a binder for the purpose of defining whether an ISVD polypeptide disclosed herein is bispecific, trispecific or multispecific.

[0311] In some embodiments, the ISVD polypeptides disclosed herein may comprise an extension body selected from any of those described in Table 1.

[0312] In Table 2, L P represents an optional linker that connects the indicated extension (P) to the ISVD polypeptide backbone. R1 represents the point of attachment in the ISVD polypeptide.

[0313] Table 1: Examples of extended bodies (P) ('*' represents the same as L P or attachment point of R1)

[0314]

[0315]

[0316] In one embodiment, the elongate is conjugated directly to the ISVD polypeptide, i.e., without the use of a linker L P (i.e. via covalent bonds).

[0317] In other embodiments, a linker L is used P The elongate is conjugated directly to the ISVD polypeptide. Non-limiting examples are provided below.

[0318] L P The linker, when present, may comprise Ado, Aeep or Aeeep, sulfonamide, Trx, ε-Lys, Ahx, Glu, γGlu, Gly, Ser, Ala and / or Thr.

[0319] L P The linker may include at least a portion that can be represented by the following chemical formula:

[0320] Chemical formula 1: *-NH-(CH2)2-(O-(CH2)) k -O-(CH2) n -CO-*

[0321] or

[0322] Chemical formula 2:

[0323]

[0324] wherein k is an integer in the range of 1-5, and n is an integer in the range of 1-5.

[0325] When k=1 and n=1, the linker element may be referred to as Ado, or 8-amino-3,6-dioxaoctanoyl, which may be represented by the following chemical formula:

[0326] Chemical formula 3: *-NH-(CH2)2-O-(CH2)2-O-CH2-CO-*

[0327] or

[0328] Chemical formula 4:

[0329] When k=1 and n=2, the linker element may be referred to as Aeep, which may be represented by the following chemical formula:

[0330] Chemical formula 5: *-NH-(CH2)2-O-(CH2)2-O-(CH2)2-CO-*

[0331] or

[0332] Chemical formula 6:

[0333]

[0334] When k=2 and n=2, the linker element may be referred to as Aeeep, which may be represented by the following chemical formula:

[0335] Chemical formula 7: *-NH-(CH2) 2- O-(CH2)2O-(CH2)2-O-(CH2)2-CO-*

[0336] or

[0337] Chemical formula 8:

[0338] Optional connector (L P ) may contain an 8-amino-3,6-dioxaoctanoic acid (OEG) group having the following chemical formula:

[0339] Chemical formula 8a:

[0340] Optional connector (L P ) may contain a sulfonamide-C4 moiety. A sulfonamide-C4 group is a sulfonamide group attached to a 4-butyryl group and has the following chemical formula:

[0341] Chemical formula 9: *-NH-S(O)2-CH2-CH2-CH2-CO-*

[0342] or

[0343] Chemical formula 10:

[0344] Optional connector L P Trx may be included. Trx is also known as tranexamic acid, trans-4-(aminomethyl)cyclohexanecarboxylic acid, and has the following chemical formula:

[0345] Chemical formula 11: *-NH-CH2-(C6H 10 )-CO-*

[0346] or

[0347] Chemical formula 12:

[0348] Connector L P ε-Lysine (ε-Lys) may be included.

[0349] Connector L P Lysine (Lys) may be contained.

[0350] Connector L P Ahx may be included. Ahx is also known as aminocaproic acid, 6-aminocaproic acid, and is defined by the formula

[0351] Chemical formula 13: *-NH-(CH2)5-CO-*

[0352] or

[0353] Chemical formula 14:

[0354] Connector L P It may contain a Glu divalent group, such as

[0355] Chemical formula 15:

[0356]

[0357] The Glu divalent group may be contained p times, wherein p is an integer in the range of 1-3.

[0358] Chemical formula 15 may also be referred to as γ-Glu, or simply γGlu, because it is the γ-carboxyl group of the amino acid glutamic acid, which is used here to connect to the ε-amino group of lysine. As described above, the other linker element can be, for example, another Glu residue or an Ado molecule. The amino group of Glu in turn forms an amide bond with the carboxyl group of the extension part or with, for example, the carboxyl group of the Ado molecule (if present) or with, for example, the γ-carboxyl group of another Glu (if present).

[0359] Alternatively, the ISVD polypeptide derivatives disclosed herein may comprise a linker selected from any one of those described in Table 2 below (L P ). R1 ​​represents the residue in the ISVD polypeptide to which the extension is attached, and P represents the extension body.

[0360] Table 2: (Optional) Extension Linkers ("L P ”)

[0361]

[0362] Based on the disclosure herein, and optionally after some limited routine experimentation, one skilled in the art will be able to determine the optimal L for a particular ISVD polypeptide derivative disclosed herein. P and L 1-2 For example, the linker is preferably such that each ISVD in the ISVD polypeptide, such as V H The H fragment binds to its target. Again, based on the disclosure herein, optionally after some limited routine experimentation, one skilled in the art will be able to determine the optimal linker for a particular ISVD polypeptide derivative disclosed herein.

[0363] The amino acid residue in the ISVD or ISVD polypeptide backbone to which attachment occurs is referred to herein as R1. If more than one extension is attached, further attachment sites may be referred to as R2, R3, etc.

[0364] The extension moiety may be attached to a cysteine ​​or lysine residue in the first ISVD portion of the ISVD polypeptide backbone.

[0365] The extension moiety may be attached to a cysteine ​​or lysine residue in the second ISVD portion of the ISVD polypeptide backbone.

[0366] The extension moiety may be attached to an optional linker (L 1-2 ) portion.

[0367] The extension moiety can be covalently linked to a lysine residue in the ISVD polypeptide backbone. The extension moiety can be linked via an amide bond formed between a carboxylic acid group in the extension moiety and the epsilon amino group of the lysine residue.

[0368] The extension moiety can be covalently linked to a cysteine ​​residue in the ISVD polypeptide backbone. The extension moiety can be linked via a thioether bond formed between the extension moiety and the sulfur atom of the cysteine ​​residue in the polypeptide.

[0369] Thus, in some embodiments, the compounds disclosed herein may comprise one, two, or three lysine or cysteine ​​residues and one, two, or three prolonging moieties, wherein each prolonging moiety is attached to the side chain of a single lysine or cysteine ​​residue.

[0370] When attachment is via a cysteine ​​residue, the cysteine ​​is preferably a free cysteine.

[0371] In some embodiments a free cysteine ​​introduced by recombinant DNA technology can serve as a conjugation site for attachment of one or more C16, C17 or C18 diacid γ-Glu 2xOEG fatty acid moieties.

[0372] In a preferred embodiment, a free cysteine, optionally introduced by recombinant DNA technology, can serve as a conjugation site for attachment of one, two or three C18 diacid γ-Glu2xOEG fatty acid moieties (IUPAC name S{Beta-AA#}-[2-[2-[[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl], AA# amino acid attachment).

[0373] In another preferred embodiment, a free cysteine, optionally introduced by recombinant DNA technology, can serve as a conjugation site for attachment of one, two or three C17 diacid γ-Glu2xOEG fatty acid moieties (IUPAC name S{Beta-AA#}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(16-carboxyhexadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl], AA# amino acid attachment).

[0374] In a most preferred embodiment, a free cysteine ​​introduced by recombinant DNA technology can serve as a conjugation site for attachment of one, two or three C16 diacid γ-Glu 2xOEG fatty acid moieties (IUPAC name S{Beta-AA#}-[2-[2-[[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl], AA# amino acid attachment).

[0375] The term "standard chromatography" as used herein encompasses standard chromatography methods such as protein A, cation exchange, anion exchange, hydrophobic interaction and hydroxyapatite chromatography.

[0376] As used herein, the term "surface exposed amino acid residue" refers to an amino acid residue whose side chain can be in contact with solvent molecules (usually, it may be mainly water molecules). However, the side chain does not necessarily have to be in contact with the solvent molecules completely, and even when a part 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 the polypeptide may also include amino acid residues that are located close to the surface of the ISVD, and thus may have mutual charge influences from other amino acid residues whose side chains (even if partially) are in contact with solvent molecules. A person of ordinary skill in the art can use commercially available or publicly available software to make a homology model or a three-dimensional molecular model based on machine learning of a polypeptide or antibody, for example, by homology modeling or machine learning. Alternatively, a three-dimensional molecular model can be generated using methods such as X-ray crystallography. Amino acid residues that can be exposed on the surface can be, for example, using software such as MOE (Chemical Computing Group) or Bioluminate The surface exposed sites can be determined using computer programs such as , using coordinates from a three-dimensional molecular model of the antibody. The surface exposed sites can be determined using algorithms known in the art (e.g., Lee and Richards (1971) J. Mol. Biol. 55: 379-400; Connolly, J. Appl. Cryst. (1983) 16: 548-558). The surface exposed sites can be determined using software suitable for protein modeling and analysis of three-dimensional structural information obtained from antibodies. Software that can be used for such purposes includes, for example, MOE (Chemical Computing Group) or Bioluminate The area of ​​the solvent accessible surface (unit: )Use a probe radius of The water probe can be used to calculate. In addition, Pacios (Pacios, Comput. Chem. 18 (4): 377-386 (1994); J. Mol. Model. 1: 46-53 (1995)) has described a method for determining the region and area of ​​surface exposure using personal computer software. Based on such information as described above, appropriate amino acid residues located on the antibody surface in contact with the solvent can be selected.

[0377] Pharmaceutical composition

[0378] V disclosed in this article H The H polypeptide derivative can be prepared into a pharmaceutical composition. In some embodiments, such a composition comprises at least one pharmaceutically acceptable excipient.

[0379] As used herein, the term "excipient" broadly refers to any component other than the active therapeutic ingredient (API). Excipients can be used for a variety of purposes, such as as carriers, vehicles, fillers, binders, lubricants, glidants, disintegrants, flow control agents, crystallization inhibitors, solubilizers, stabilizers, colorants, flavoring agents, surfactants, emulsifiers, delivery agents, hydrotropes, or combinations thereof, and / or to improve the administration and / or absorption of the active pharmaceutical ingredient.

[0380] The amount of each excipient used can vary within the conventional range of the art. Techniques and excipients that can be used to prepare oral dosage forms are described in the following documents: Handbook of Pharmaceutical Excipients, 8th edition, Sheskey et al., ed., American Pharmaceuticals Association and the Pharmaceutical Press, Royal Pharmaceutical Society of Great Britain's Publishing Department (2017); and Remington: the Science and Practice of Pharmacy, 22nd edition, Remington and Allen, ed., Pharmaceutical Press (2013).

[0381] In a preferred embodiment, V H The composition of the H polypeptide further comprises a delivery agent and a hydrotrope.

[0382] A preferred delivery agent is a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid (NAC).

[0383] In some embodiments, the delivery agent is a salt of N-(8-(2-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 herein as "SNAC"), also known as sodium 8-(salicyloylamino)caprylate.

[0384] The pharmaceutical composition preferably comprises one or more hydrotropes. Hydrotropes such as surfactants contain both hydrophilic and hydrophobic portions and can form micelles and self-aggregate, yet they dissolve 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).

[0385] In one embodiment, the composition is a solid composition. The composition can be in a form suitable for oral administration, such as a tablet, a capsule or a capsule. In such an embodiment, the composition is formulated as a tablet. The solid composition provided herein allows for accelerated dissolution, thereby enabling rapid ingestion of the active pharmaceutical ingredient.

[0386] Administration and Dosage

[0387] The compounds disclosed herein, such as V H H polypeptide derivatives can be administered parenterally in appropriate pharmaceutical compositions, such as intravenous administration, intramuscular administration, and subcutaneous administration. The compound can be administered by non-parenteral routes, preferably oral (PO) administration. The compound can be administered prophylactically. The compound can be administered therapeutically (on demand).

[0388] Subcutaneous administration

[0389] The dosage of the compound delivered by subcutaneous administration may be about 0.01 mg to 1 mg per day. g Compound, preferably about 0.05 mg to 5 mg per day, more preferably about 0.1 mg to about 10 mg per day, every other day, every three days, every four days, every five days, every six days or once a week, depending on the severity of the condition. Suitable dosages can also be adjusted for specific compounds based on the properties of the compound, including its half-life or average residence time in the body and its biological activity.

[0390] In one embodiment, the present invention relates to an injection device having the composition as content.

[0391] Oral (PO) administration

[0392] The dosage of the compound delivered by PO can be from about 1 mg to about 300 mg of the compound every day, every other day, every three days, depending on the severity of the condition. The appropriate dosage can also be adjusted for a particular compound based on the properties of the compound, including its half-life or average residence time in the body and its biological activity.

[0393] Compositions containing compounds disclosed herein can be administered for preventive treatment, and / or in some embodiments for therapeutic treatment. In therapeutic applications, the composition is administered to a subject who has already suffered from a disease such as any bleeding disorder described above in an amount sufficient to cure, alleviate or partially prevent the disease and its complications. An amount sufficient to achieve this purpose is defined as a "therapeutically effective amount". It will be appreciated by those skilled in the art that the amount effective for this purpose will depend on the severity of the disease or injury and the weight and general state of the subject.

[0394] Implementation

[0395] 1. A procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivative comprising

[0396] a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO: 1) or an activated form thereof,

[0397] a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof,

[0398] at least one extension,

[0399] Optionally, a linker (L 1-2 ),as well as

[0400] Optionally, one or more extensions (E).

[0401] 2. The ISVD polypeptide derivative according to embodiment 1, which has the formula

[0402] ISVD1–ISVD2, or

[0403] ISVD1–ISVD2–E, or

[0404] ISVD2–ISVD1–E, or

[0405] E-ISVD1–ISVD2, or

[0406] ISVD1–L1-2–ISVD2–E, or

[0407] ISVD2–L1-2–ISVD1–E, or

[0408] E-ISVD1–L1-2–ISVD2,

[0409] 3. The ISVD polypeptide derivative according to embodiment 1, which has the following formula (N-terminus to C-terminus):

[0410] ISVD1–ISVD2, or

[0411] ISVD2–ISVD1, or

[0412] ISVD1–ISVD2–E, or

[0413] ISVD2–ISVD1–E, or

[0414] E-ISVD1–ISVD2, or

[0415] E–ISVD2–ISVD1, or

[0416] ISVD1–L1-2–ISVD2, or

[0417] ISVD2–L1-2–ISVD1, or

[0418] ISVD1–L1-2–ISVD2–E, or

[0419] ISVD2–L1-2–ISVD1–E, or

[0420] E-ISVD1–L1-2–ISVD2, or

[0421] E–ISVD2–L1-2–ISVD1.

[0422] 4. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the at least one prolonging moiety is attached to one or more surface exposed residues.

[0423] 5. The ISVD polypeptide derivative according to any one of the preceding embodiments, which has the formula (N-terminus to C-terminus)

[0424] ISVD2–L 1-2 –ISVD1–E

[0425] The one or more extension moieties are attached to the one or more surface exposed residues.

[0426] 6. The ISVD polypeptide derivative according to any one of the preceding embodiments, which has the formula (N-terminus to C-terminus)

[0427] ISVD2–L 1-2 –ISVD1–E

[0428] Two of the extensions are attached to one or more surface exposed residues on E.

[0429] 7. The ISVD polypeptide derivative according to any one of the preceding embodiments, which has the formula (N-terminus to C-terminus)

[0430] ISVD2–L 1-2 –ISVD1–E

[0431] It further includes

[0432] A first extension portion and a second extension portion, which are attached to the first surface exposed residue and the second surface exposed residue in E, respectively.

[0433] 8. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein L 1-2 The ratio of hydrophobic amino acids to hydrophilic amino acids is "40% to 60%" to "60% to 40%".

[0434] 9. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof, said epitope comprising at least one of amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively).

[0435] 10. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof comprising at least 4, 5, 6, 7 or 8 of amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively).

[0436] 11. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof, said epitope comprising amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively).

[0437] 12. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising at least one of amino acid residues N173, P174, F175, L177, L178 and D179 (numbered consecutively).

[0438] 13. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising amino acid residues N173, P174, F175, L177 and L178 (numbered consecutively).

[0439] 14. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising at least one of amino acid residues N173, P174, F175, L177, L178 and D179 (numbered consecutively).

[0440] 15. The ISVD polypeptide derivative according to any one of the preceding 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 amino acid residues N173, P174, F175, L177, L178, and D179 (numbered consecutively).

[0441] 16. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising amino acid residues N173, P174, F175, L177, L178 and D179 (numbered consecutively).

[0442] 17. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein

[0443] The first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof, the epitope comprising at least one of amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257, and N260 (numbered consecutively), and

[0444] The second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising at least one of amino acid residues N173, P174, F175, L177 and L178 (numbered consecutively).

[0445] 18. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein

[0446] The first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof, the epitope comprising amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively), and

[0447] The second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising amino acid residues N173, P174, F175, L177, L178 and D179 (numbered consecutively).

[0448] 19. The ISVD polypeptide derivative of any one 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

[0449] wherein the second ISVD comprises a paratope comprising amino acid residues

[0450] 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

[0451] 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)

[0452] (Serial numbering).

[0453] 20. The ISVD polypeptide derivative according to any one of embodiments 1-9, wherein the first ISVD comprises 1)

[0455] CDR1: IYTMS (SEQ ID NO: 172), optionally comprising one or two substitutions,

[0456] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO: 173), optionally comprising one, two or three amino acid substitutions,

[0457] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 174), optionally comprising one, two or three amino acid substitutions (Kabat definition).

[0458] 21. The ISVD polypeptide derivative according to embodiments 1-9, wherein the second ISVD comprises

[0459] (A)

[0460] CDR1: RYAMG (SEQ ID NO: 168), optionally comprising one or two substitutions,

[0461] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 169), optionally comprising one, two or three amino acid substitutions,

[0462] CDR3: DDSVGDGYLDY (SEQ ID NO: 170), optionally comprising one, two or three amino acid substitutions; or

[0463] (B)

[0464] CDR1: RLAMG (SEQ ID NO: 128), optionally comprising one or two amino acid substitutions,

[0465] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 129), optionally comprising one, two or three substitutions,

[0466] CDR3: DDSVGDGYLDY (SEQ ID NO: 130), optionally comprising one, two or three amino acid substitutions

[0467] (Kabat definition).

[0468] 22. The ISVD polypeptide derivative according to any one of embodiments 1-9, wherein the first ISVD comprises 1)

[0470] CDR1: IYTMS (SEQ ID NO: 172), optionally comprising one or two amino acid substitutions,

[0471] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO: 173), optionally comprising one, two or three amino acid substitutions,

[0472] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 174), optionally comprising one, two or three amino acid substitutions; or

[0473] and wherein the second ISVD comprises

[0474] (A)

[0475] CDR1: RYAMG (SEQ ID NO: 168), optionally comprising one or two substitutions,

[0476] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 169), optionally comprising one, two or three substitutions,

[0477] CDR3: DDSVGDGYLDY (SEQ ID NO: 170), optionally comprising one, two or three amino acid substitutions; or

[0478] (B)

[0479] CDR1: RLAMG (SEQ ID NO: 128), optionally comprising one or two substitutions,

[0480] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 129), optionally comprising one, two or three substitutions,

[0481] CDR3: DDSVGDGYLDY (SEQ ID NO: 130), optionally comprising one, two or three amino acid substitutions

[0482] (Kabat numbering).

[0483] 23. The ISVD polypeptide derivative according to any one of embodiments 20-22, wherein the substitution is a conservative substitution.

[0484] 24. The ISVD polypeptide derivative according to any of the preceding embodiments, wherein the sequence of the first ISVD is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 107, 115, 123, 131, 155 or 171, respectively.

[0485] 25. The ISVD polypeptide derivative according to any of the preceding embodiments, wherein the sequence of the second ISVD is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 103, 111, 119, 127, 151 or 167, respectively.

[0486] 26. The ISVD polypeptide derivative according to any of the preceding embodiments, wherein the sequence of the first ISVD is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 107, 115, 123, 131, 155 or 171, respectively, and wherein the sequence of the second ISVD is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 103, 111, 119, 127, 151 or 167, respectively.

[0487] 27. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the first ISVD comprises the sequence

[0488] V H H-2.20 (SEQ ID NO: 171),

[0489] V H H-2.18 (SEQ ID NO: 155),

[0490] V H H-2.15 (SEQ ID NO: 131),

[0491] V H H-2.13 (SEQ ID NO: 115),

[0492] V H H-2.14 (SEQ ID NO: 123), or

[0493] V H H-2.12 (SEQ ID NO: 107).

[0494] 28. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the second ISVD comprises the sequence

[0495] V H H-1.20 (SEQ ID NO: 167),

[0496] V HH-1.18 (SEQ ID NO: 151),

[0497] V H H-1.15 (SEQ ID NO: 127),

[0498] V H H-1.13 (SEQ ID NO: 111),

[0499] V H H-1.14 (SEQ ID NO: 119), or

[0500] V H H-1.12 (SEQ ID NO: 103).

[0501] 29. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the first ISVD comprises the sequence

[0502] V H H-2.20 (SEQ ID NO: 171),

[0503] V H H-2.18 (SEQ ID NO: 155),

[0504] V H H-2.15 (SEQ ID NO: 131),

[0505] V H H-2.13 (SEQ ID NO: 115),

[0506] V H H-2.14 (SEQ ID NO: 123), or

[0507] V H H-2.12 (SEQ ID NO: 107)

[0508] And among them

[0509] The second ISVD comprises the following sequence

[0510] V H H-1.20 (SEQ ID NO: 167),

[0511] V H H-1.18 (SEQ ID NO: 151),

[0512] V H H-1.15 (SEQ ID NO: 127),

[0513] VH H-1.13 (SEQ ID NO: 111),

[0514] V H H-1.14 (SEQ ID NO: 119), or

[0515] V H H-1.12 (SEQ ID NO: 103).

[0516] 30. The ISVD polypeptide derivative according to embodiment 29, wherein

[0517] The first ISVD includes V H H-2.20 (SEQ ID NO: 171), and wherein the second ISVD comprises V H The sequence of H-1.20 (SEQ ID NO: 167).

[0518] 31. The ISVD polypeptide derivative according to embodiment 29, wherein

[0519] The first ISVD includes V H H-2.18 (SEQ ID NO: 155), and wherein the second ISVD comprises V H The sequence of H-1.18 (SEQ ID NO: 151).

[0520] 32. The ISVD polypeptide derivative according to embodiment 29, wherein

[0521] The first ISVD includes V H H-2.15 (SEQ ID NO: 131), and wherein the second ISVD comprises V H The sequence of H-1.15 (SEQ ID NO: 127).

[0522] 33. The ISVD polypeptide derivative according to embodiment 29, wherein

[0523] The first ISVD includes V H H-2.13 (SEQ ID NO: 115), and wherein the second ISVD comprises V H The sequence of H-1.13 (SEQ ID NO: 111).

[0524] 34. The ISVD polypeptide derivative according to embodiment 29, wherein

[0525] The first ISVD includes V HH-2.14 (SEQ ID NO: 123), and wherein the second ISVD comprises V H The sequence of H-1.14 (SEQ ID NO: 119).

[0526] 35. The ISVD polypeptide derivative according to embodiment 29, wherein

[0527] The first ISVD includes V H H-2.12 (SEQ ID NO: 107), and wherein the second ISVD comprises V H The sequence of H-1.12 (SEQ ID NO: 103).

[0528] 36. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the extension portion comprises an extension selected from the group consisting of:

[0529] a) C16 diacid, C17 diacid, C18 diacid, C19 diacid, C20 diacid, C21 diacid or C22 diacid,

[0530] b) tetrazole,

[0531] c) Albumin binding peptide, such as RLIEDICLPRWGCLWEDD (SEQ ID NO: 737)

[0532] d) an FcRn binding peptide, such as QRFCTGHFGGLYPCNG (SEQ ID NO: 738), and

[0533] e) Fc binding peptides, such as

[0534] FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDDN (SEQ ID NO:739).

[0535] 37. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the extension portion comprises an extension selected from the group consisting of a C16 diacid, a C17 diacid, a C18 diacid, a C19 diacid, and a C20 diacid.

[0536] 38. The ISVD polypeptide derivative of embodiment 34, wherein the elongate is a C16 diacid elongate.

[0537] 39. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the extension portion comprises attaching the extension body to ISVD1, L 1-2 , ISVD2 or E linker L P .

[0538] 40. The ISVD polypeptide derivative according to embodiment 39, wherein L P Selected from

[0539]

[0540] 41. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the prolongation is attached to position 13 in ISVD2 and / or one or more of positions 12, 14, 15, 42, 44, 63 and / or 85 (numbered consecutively) on ISVD1.

[0541] 42. The ISVD polypeptide derivative according to any one of embodiments 1-40, wherein the ISVD polypeptide derivative comprises a C-terminal extension, wherein the prolongation moiety is attached to the C-terminal extension.

[0542] 43. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the ISVD polypeptide derivative comprises a first and a second extension moiety and a C-terminal extension, wherein the first extension moiety and the second extension moiety are attached to the C-terminal extension.

[0543] 44. The ISVD polypeptide derivative according to embodiment 43, wherein the C-terminal extension is attached to ISVD1.

[0544] 45. The ISVD polypeptide derivative according to embodiment 43, wherein the C-terminal extension is attached to ISVD2.

[0545] 46. ​​The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the extension moieties are identical.

[0546] 47. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein one or more prolonging moieties are attached to a surface exposed residue.

[0547] 48. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein one or more prolonging moieties are attached to a surface exposed residue, wherein the surface exposed residue is not a residue in a CDR region.

[0548] 49. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein one or more prolongation moieties are attached at one or more cysteine ​​or lysine residues.

[0549] 50. The ISVD polypeptide derivative according to embodiment 43, wherein

[0550] The ISVD polypeptide derivative comprises SEQ ID NO: 629, and

[0551] in

[0552] The first extension portion comprises the following structure:

[0553]

[0554] And wherein the second extension portion comprises the following structure

[0555]

[0556] wherein '*' (R1) represents the point of attachment of the first extension moiety on the ISVD polypeptide, and

[0557] wherein '**' (R2) represents the attachment point of the second extension moiety on the ISVD polypeptide,

[0558] wherein '*' is Cys at position 257 of SEQ ID NO:629, and

[0559] wherein '**' is Cys at position 259 of SEQ ID NO:629.

[0560] 51. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the isoelectric point of the polypeptide as determined using isoelectric focusing is 6.5 or lower.

[0561] 52. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the polypeptide derivative is a multispecific polypeptide derivative, such as a bispecific or trispecific polypeptide derivative.

[0562] 53. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the polypeptide derivative is a bispecific polypeptide derivative.

[0563] 54. The ISVD polypeptide derivative according to any of the preceding embodiments, wherein the molecular weight of the polypeptide derivative is 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, 22-48 kDa, such as 22-43 kDa, such as 22-37 kDa, such as 14-35 kDa, 14-32 kDa, such as 14-27 kDa, such as 14-22 kDa, such as 14-18 kDa 22-35kDa, such as 22-32kDa, such as 22-31kDa, such as 22-30kDa, such as 22-29kDa, such as 22-28kDa, such as 22-27kDa, such as 24-50kDa, 24-48kDa, such as 24-43kDa, such as 24-37kDa, such as 24-35, such as 24-32kDa, such as 24-31kDa, 24-30kDa, 24-29kDa, 24-28kDa, such as 24-27kDa, such as 36-48kDa, such as 36-43kDa, such as 36-37kDa, such as 28-36kDa, such as 29-33kDa, such as 29-30, such as about 29, such as 29, such as about 30, such as 30, such as 30-32kDa, or such as 31kDa.

[0564] 55. The ISVD polypeptide derivative according to any one of the preceding embodiments, which is not a membrane targeted ISVD polypeptide derivative.

[0565] 56. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the protracting portion is unable to bind components of the plasma membrane, such as aminophospholipids, such as phosphatidylserine and / or phosphatidylethanolamine.

[0566] 57. The ISVD polypeptide derivative according to any of the preceding embodiments, wherein the extension portion is unable to bind to platelet surface proteins such as GPlb-1X, collagen chaperone HSP47, ephrin B1, thiol isomerase protein ERP5, hematopoietic progenitor kinase 1 interacting protein 55 (HIP-55), glycoprotein V1, platelet glycoprotein 1b, platelet-derived growth factor receptor, platelet endothelial aggregation receptor I, CD31, CD36, MARKS, polyprotein, integrin α11b / β3, triggering receptor expressed on myeloid cells (TREM)-like transcript-1 (TLT-1), integrin-linked kinase (ILK), zycin, collagen, P-selectin, factor XIII, P-selectin glycoprotein ligand-1, integrin α6β1, thrombospondin, von Willebrand factor, G6B, CD42b, syntaxin binding protein 2, phosphatidylethanolamine, fibrinogen / fibrin, filamin, stomatin, sphingolipids, CD31, CD36, CD40, CD41, CD42c, CD42, CD49b, CD61, CD62P, CD63, CD69, CD107a, CD107b, CD109, CD154, PECAM-1, and / or ERPS.

[0567] 58. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the extension portion is unable to bind to a membrane-associated polypeptide, such as a glycoprotein, GPIIb / IIIa, β2GP1, TLT-1, a selectin, a coagulation factor or a coagulation factor complex and / or a selectin.

[0568] 59. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the mean residence time terminal half-life in the blood is extended by at least 12 hours, at least 24 hours, at least 48 hours, at least 3, 4, 5 or 7 days compared to an ISVD polypeptide without said extending portion.

[0569] 60. The ISVD polypeptide derivative according to any one of the preceding embodiments, wherein the first and / or second ISVD is V H H fragment.

[0570] 61. The ISVD polypeptide derivative according to any one of embodiments 1-59, wherein the first and / or second ISVD is a V-NAR fragment.

[0571] 62. The ISVD polypeptide derivative according to any one of embodiments 1-60, wherein the ISVD polypeptide derivative is V H H polypeptide derivatives.

[0572] 63. Procoagulant V HH polypeptide derivative, which comprises

[0573] A first V capable of binding to factor IX (SEQ ID NO: 1) or an activated form thereof H H,

[0574] A second V capable of binding to Factor X (SEQ ID NO: 2) H H,

[0575] Connect the first V H H and the second V H H linker (L 1-2 ),as well as

[0576] a C-terminal extension (E), and

[0577] One or two extensions,

[0578] The V H The H polypeptide derivative has the following formula (N-terminus to C-terminus):

[0579] "Second V H H”–L 1-2 – “First V H H”–E

[0580] in

[0581] I)

[0582] The first V H H contains

[0583] CDR1: IYTMS (SEQ ID NO:172),

[0584] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:173),

[0585] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 174), and

[0586] The second V H H contains

[0587] CDR1: RYAMG (SEQ ID NO:168),

[0588] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:169),

[0589] CDR3: DDSVGDGYLDY (SEQ ID NO: 170); or

[0590] II)

[0591] The first V H H contains

[0592] CDR1: IYTMS (SEQ ID NO:132),

[0593] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:133),

[0594] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 134), and

[0595] The second V H H contains

[0596] CDR1: RLAMG (SEQ ID NO:128),

[0597] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:129),

[0598] CDR3:DDSVGDGYLDY(SEQ ID NO:130);

[0599] (Kabat definition).

[0600] 64. The procoagulant V according to embodiment 63 H H polypeptide derivatives, wherein

[0601] I)

[0602] The first V H H contains V H H-2.20 (SEQ ID NO: 171), and

[0603] The second V H H contains V H The sequence of H-1.20 (SEQ ID NO: 167).

[0604] II)

[0605] The first V H H contains V H The sequence of H-2.18 (SEQ ID NO: 155), and

[0606] The second V H H contains V H The sequence of H-1.18 (SEQ ID NO: 151).

[0607] III)

[0608] The first V H H contains V H H-2.15 (SEQ ID NO: 131), and

[0609] The second V H H contains V H The sequence of H-1.15 (SEQ ID NO: 127).

[0610] IV)

[0611] The first V H H contains V H H-2.13 (SEQ ID NO: 115), and

[0612] The second V H H contains V H The sequence of H-1.13 (SEQ ID NO: 111).

[0613] V)

[0614] The first V H H contains V H The sequence of H-2.14 (SEQ ID NO: 123), and

[0615] The second V H H contains V H The sequence of H-1.14 (SEQ ID NO: 119).

[0616] VI)

[0617] The first V H H contains V H The sequence of H-2.12 (SEQ ID NO: 107), and

[0618] The second V H H contains V H The sequence of H-1.12 (SEQ ID NO: 103).

[0619] 65. The procoagulant V according to any one of embodiments 1-60 and 62-64. H H polypeptide derivative, wherein the V H H peptide derivatives are bispecific V H H polypeptide derivatives.

[0620] 66. The procoagulant V according to any one of embodiments 63-65. H H polypeptide derivative, wherein the linker L 1-2Comprising the amino acid residues GQAPGQ (SEQ ID NO: 20).

[0621] 67. The procoagulant V according to any one of embodiments 63-65. H The H polypeptide derivative, wherein the extension (E) comprises the amino acid residues GQACPC (SEQ ID NO: 9).

[0622] 68. The procoagulant V according to any one of embodiments 63-67. H The H polypeptide derivative, wherein the two extensions are attached to positions 4 and 6 of E, respectively (SEQ ID NO: 9).

[0623] 69. The procoagulant V according to any one of embodiments 63-68. H H polypeptide derivative, wherein the two extension parts are identical.

[0624] 70. The procoagulant V according to any one of embodiments 63-69. H H polypeptide derivative, wherein the two extension parts comprise the following structures:

[0625]

[0626] 71. The procoagulant V according to any one of embodiments 63-70. H H polypeptide derivative comprising a first and a second extension

[0627] The first extension portion comprises the following structure:

[0628]

[0629] And wherein the second extension portion comprises the following structure

[0630]

[0631] Wherein '*'(R1) represents the first extension part in the V H H polypeptide derivative, and

[0632] Wherein '**' (R2) represents the second extension part in the V H H attachment point on the polypeptide derivative,

[0633] wherein '*' is Cys at position 257 of SEQ ID NO:629, and

[0634] wherein '**' is Cys at position 259 of SEQ ID NO:629.

[0635] 72. A pharmaceutical composition comprising an ISVD polypeptide derivative or V according to any one of embodiments 1-71. H H polypeptide derivatives.

[0636] 73. A pharmaceutical composition for oral administration comprising an ISVD polypeptide derivative or V according to any one of embodiments 1-71. H H polypeptide derivatives.

[0637] 74. The pharmaceutical composition according to embodiment 72 or 73, wherein the ISVD polypeptide derivative is V H H polypeptide derivatives.

[0638] 75. The pharmaceutical composition of embodiment 72 or 73, wherein the ISVD polypeptide derivative is a V-NAR polypeptide derivative.

[0639] 76. A pharmaceutical composition according to any one of embodiments 72-75, wherein the composition comprises a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid.

[0640] 77. A pharmaceutical composition according to embodiment 76, wherein the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC).

[0641] 78. The pharmaceutical composition of any one of embodiments 76 to 77, further comprising nicotinamide (NAM).

[0642] 79. A pharmaceutical composition according to any one of embodiments 74-78, wherein the composition comprises V H H polypeptide derivative, sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), niacinamide (NAM) and magnesium stearate.

[0643] 80. The pharmaceutical composition of any one of embodiments 74-78, wherein the 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 niacinamide (NAM), and about 0.5% w / w magnesium stearate.

[0644] 81. The pharmaceutical composition of any one of embodiments 73-80, wherein the composition is a liquid composition.

[0645] 82. The pharmaceutical composition of any one of embodiments 73-80, wherein the composition is a solid composition.

[0646] 83. The pharmaceutical composition of embodiment 82, wherein the composition is a tablet, a lozenge or a capsule.

[0647] 84. A method of treating a patient in need thereof, comprising administering an ISVD polypeptide derivative according to any one of embodiments 1-83, V H H polypeptide derivative or pharmaceutical composition.

[0648] 85. The method of treatment according to embodiment 84, wherein the patient is a patient suffering from hemophilia A or acquired hemophilia A with or without inhibitors.

[0649] 86. The method of treatment of embodiment 84 or 85, wherein the treatment is a prophylactic treatment.

[0650] 87. The method of treatment according to any one of embodiments 84-86, wherein the ISVD polypeptide derivative, V H The H polypeptide derivative or pharmaceutical composition is administered orally.

[0651] 88. The method of treatment according to any one of embodiments 84-86, wherein the ISVD polypeptide derivative, V H The H polypeptide derivative or pharmaceutical composition is administered by subcutaneous injection.

[0652] 89. The ISVD polypeptide derivative according to any one of embodiments 1-83, V H H polypeptide derivative or pharmaceutical composition, which is used in medicine.

[0653] 90. The ISVD polypeptide derivative according to any one of embodiments 1-83, V H H polypeptide derivatives or compositions for use in treating hemophilia, such as hemophilia A with or without inhibitors, or such as acquired hemophilia A.

[0654] 91. The ISVD polypeptide derivative according to any one of embodiments 1-83, V H H polypeptide derivative or composition for use in treating hemophilia A with or without inhibitors.

[0655] 92. The ISVD polypeptide derivative for use according to any one of embodiments 89-91, V H H polypeptide derivative or composition, wherein the treatment is prophylactic treatment.

[0656] 93. An ISVD polypeptide derivative for use according to any one of embodiments 89-92, V H H polypeptide derivative or composition, wherein the derivative or composition is administered orally.

[0657] 94. The ISVD polypeptide derivative for use according to any one of embodiments 89-92, V H H polypeptide derivative or composition, wherein the derivative or composition is administered by subcutaneous injection.

[0658] 95. A method for producing a procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivative, the ISVD polypeptide derivative comprising

[0659] a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO: 1) or an activated form thereof,

[0660] a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof,

[0661] one or more extensions,

[0662] Optionally, a linker (“L 1-2 "),as well as

[0663] Optionally, one or more extensions ("E")

[0664] The method comprises the following steps

[0665] a. modifying a nucleic acid encoding an ISVD polypeptide or an ISVD polypeptide derivative with amino acid residues such that the isoelectric point of the ISVD polypeptide or the ISVD polypeptide derivative is reduced,

[0666] b. culturing the host cell to express the nucleic acid encoding the ISVD polypeptide or ISVD polypeptide derivative,

[0667] c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture,

[0668] d. purifying said ISVD polypeptide or ISVD polypeptide derivative from said host cell culture using standard chromatography methods, and

[0669] e. Attaching a prolongation moiety to the ISVD polypeptide, unless such a moiety is already present.

[0670] 96. A method for producing a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative, the ISVD polypeptide or ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO: 1) or an activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof, a prolongation, and optionally a linker capable of linking ISVD1 and ISVD2 ("L 1-2 ”), and optionally one or more extensions (“E”), the method comprising the steps of

[0671] a. modifying the nucleic acid encoding the amino acid residues of the ISVD polypeptide or ISVD polypeptide derivative so that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is 6.5 or lower,

[0672] b. culturing the host cell to express a nucleic acid encoding an ISVD polypeptide or an ISVD polypeptide derivative,

[0673] c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture,

[0674] d. purifying said ISVD polypeptide or ISVD polypeptide derivative from said host cell culture using standard chromatography methods, and

[0675] e. Attaching a prolongation moiety to the ISVD polypeptide, unless such a moiety is already present.

[0676] 97. A method for improving the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative, the ISVD polypeptide or ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO: 1) or an activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof, a prolonging portion, and optionally a linker ("L") capable of linking ISVD1 and ISVD2. 1-2 ”), and optionally one or more extensions (“E”), the method comprising the steps of

[0677] a. modifying the nucleic acid encoding the amino acid residues of the ISVD polypeptide or ISVD polypeptide derivative so that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is reduced,

[0678] b. culturing the host cell to express the nucleic acid encoding the ISVD polypeptide or ISVD polypeptide derivative,

[0679] c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture,

[0680] d. purifying said ISVD polypeptide or ISVD polypeptide derivative from said host cell culture using standard chromatography methods, and

[0681] e. Attaching a prolongation moiety to the ISVD polypeptide, unless such a moiety is already present.

[0682] 98. A method for improving the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative, the ISVD polypeptide or ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO: 1) or an activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof, a prolonging portion, and optionally a linker ("L") capable of linking ISVD1 and ISVD2. 1-2 ”), and optionally one or more extensions (“E”), the method comprising the steps of

[0683] a. modifying the nucleic acid encoding the amino acid residues of the ISVD polypeptide or ISVD polypeptide derivative so that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is 6.5 or lower,

[0684] b. culturing the host cell to express the nucleic acid encoding the ISVD polypeptide or ISVD polypeptide derivative,

[0685] c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture,

[0686] d. purifying said ISVD polypeptide or ISVD polypeptide derivative from said host cell culture using standard chromatography methods, and

[0687] e. Attaching a prolongation moiety to the ISVD polypeptide, unless such a moiety is already present.

[0688] 99. The method of any one of embodiments 95-98, wherein the isoelectric point is lowered by at least 0.5 pH units, e.g., 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.

[0689] 100. The method according to any one of embodiments 95-99, wherein step e) does not result in an increase in the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative.

[0690] 101. The method of any one of embodiments 95-100, wherein the isoelectric point is determined using isoelectric focusing.

[0691] 102. The method according to any one of embodiments 95-101, wherein the ISVD polypeptide or ISVD polypeptide derivative is encoded by a single nucleic acid.

[0692] 103. The method according to any one of embodiments 95-101, wherein the ISVD polypeptide or ISVD polypeptide derivative is a conjugate of a first and a second ISVD, wherein the first and second ISVD are encoded by separate nucleic acids.

[0693] 104. The method according to any one of embodiments 95-103, wherein the ISVD polypeptide or ISVD polypeptide derivative is V H H polypeptide or V H H polypeptide derivatives.

[0694] 105. An ISVD polypeptide or an ISVD polypeptide derivative produced using the method according to any one of embodiments 95-103.

[0695] 106. An ISVD polypeptide or an ISVD polypeptide derivative obtainable using the method according to any one of embodiments 95-103.

[0696] 107. V produced using the method according to any one of embodiments 95-104 H H polypeptide or V H H polypeptide derivatives.

[0697] 108. V capable of binding to FIX (SEQ ID NO: 1) or its activated form (FIXa) H H-clipart

[0698] in

[0699] a. the CDR1 sequence is as shown in SEQ ID NO: 172, optionally comprising one or two amino acid substitutions,

[0700] The CDR2 sequence is as shown in SEQ ID NO: 173, optionally comprising one, two or three amino acid substitutions,

[0701] The CDR3 sequence is as shown in SEQ ID NO: 174, optionally comprising one, two or three amino acid substitutions; or

[0702] b. the CDR1 sequence is as shown in SEQ ID NO: 156, optionally comprising one or two amino acid substitutions,

[0703] The CDR2 sequence is as shown in SEQ ID NO: 157, optionally comprising one, two or three amino acid substitutions,

[0704] The CDR3 sequence is as shown in SEQ ID NO: 158, optionally comprising one, two or three amino acid substitutions; or

[0705] c. the CDR1 sequence is as shown in SEQ ID NO: 132, optionally comprising one or two amino acid substitutions,

[0706] The CDR2 sequence is as shown in SEQ ID NO: 133, optionally comprising one, two or three amino acid substitutions,

[0707] The CDR3 sequence is as shown in SEQ ID NO: 134, optionally comprising one, two or three amino acid substitutions; or

[0708] d. the CDR1 sequence is as shown in SEQ ID NO: 116, optionally comprising one or two amino acid substitutions,

[0709] The CDR2 sequence is as shown in SEQ ID NO: 117, optionally comprising one, two or three amino acid substitutions,

[0710] The CDR3 sequence is as shown in SEQ ID NO: 118, optionally comprising one, two or three amino acid substitutions; or

[0711] e. the CDR1 sequence is as shown in SEQ ID NO: 124, optionally comprising one or two amino acid substitutions,

[0712] The CDR2 sequence is as shown in SEQ ID NO: 125, optionally comprising one, two or three amino acid substitutions,

[0713] The CDR3 sequence is as shown in SEQ ID NO: 126, optionally comprising one, two or three amino acid substitutions; or

[0714] f. the CDR1 sequence is as shown in SEQ ID NO: 108, optionally comprising one or two amino acid substitutions,

[0715] The CDR2 sequence is as shown in SEQ ID NO: 109, optionally comprising one, two or three amino acid substitutions,

[0716] The CDR3 sequence is as shown in SEQ ID NO: 110, optionally comprising one, two or three amino acid substitutions;

[0717] (Kabat definition).

[0718] 109. V capable of binding to FX (SEQ ID NO: 2) H H-clipart

[0719] in

[0720] a. the CDR1 sequence is as shown in SEQ ID NO: 152, optionally comprising one or two amino acid substitutions,

[0721] The CDR2 sequence is as shown in SEQ ID NO: 153, optionally comprising one, two or three amino acid substitutions,

[0722] The CDR3 sequence is as shown in SEQ ID NO: 154, optionally comprising one, two or three amino acid substitutions; or

[0723] b. the CDR1 sequence is as shown in SEQ ID NO: 128, optionally comprising one or two amino acid substitutions,

[0724] The CDR2 sequence is as shown in SEQ ID NO: 129, optionally comprising one, two or three amino acid substitutions,

[0725] The CDR3 sequence is shown in SEQ ID NO: 130, optionally comprising one, two or three amino acid substitutions.

[0726] 110. V according to embodiment 108 or 109 H H fragment, wherein the substitution is a conservative substitution.

[0727] 111. V according to any one of embodiments 108-110 H H fragment, wherein the V H The H fragment is used to prepare a V fragment capable of binding to FIX (SEQ ID NO: 1) or its activated form (FIXa) and capable of binding to FX (SEQ ID NO: 2) or its activated form (FXa). H H polypeptide or V H Intermediates of H polypeptide derivatives.

[0728] 112. V according to any one of embodiments 108-110 H H polypeptide, wherein the V H H polypeptide is an intermediate for preparing ISVD polypeptide derivatives.

[0729] 113. The ISVD or V according to embodiment 109 or 110 H H, wherein its activity is improved in making FX more susceptible to proteolysis by FIXa compared to any anti-FX ISVD compound described in WO2019 / 096874.

[0730] 114. A procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivative comprising

[0731] a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO: 1) or an activated form thereof,

[0732] a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof,

[0733] at least one extension,

[0734] Optionally, a linker (L 1-2 ),as well as

[0735] Optionally, one or more extensions (E),

[0736] Wherein the ISVD polypeptide is selected from: SEQ ID NO: 615-691, 734 or 735.

[0737] 115. A V comprising the sequence of any one of SEQ ID NOs: 27-614 H H fragment.

[0738] 116. A V comprising the sequence of any one of SEQ ID NOs: 615-691 or 734-735 H H polypeptide derivatives.

[0739] 117. A procoagulant immunoglobulin single variable domain (ISVD) polypeptide derivative comprising

[0740] a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO: 1) or an activated form thereof,

[0741] a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof,

[0742] at least one extension,

[0743] Optionally, a linker (L 1-2 ),as well as

[0744] Optionally, one or more extensions (E).

[0745] 118. The ISVD polypeptide derivative according to embodiment 117, which has the following formula (N-terminus to C-terminus)

[0746] ISVD2–L 1-2 –ISVD1–E

[0747] The one or more extension moieties are attached to the one or more surface exposed residues.

[0748] 119. The ISVD polypeptide derivative according to embodiment 118 or 119, which has the following formula (N-terminus to C-terminus)

[0749] ISVD2–L 1-2 –ISVD1–E

[0750] wherein the two extensions are attached to one or more surface exposed residues on E, and

[0751] The molecular weight of the derivative is in the range of 20-35 kDa.

[0752] 120. The ISVD polypeptide derivative according to any one of embodiments 117-119, wherein

[0753] The first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof, the epitope comprising amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively), and

[0754] The second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) or an activated form thereof, said epitope comprising amino acid residues N173, P174, F175, L177 and L178 (numbered consecutively).

[0755] 121. The ISVD polypeptide derivative according to any one of embodiments 117-119, wherein the first ISVD comprises 1)

[0757] CDR1: IYTMS (SEQ ID NO: 172), optionally comprising one or two substitutions,

[0758] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO: 173), optionally comprising one, two or three substitutions,

[0759] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 174), optionally comprising one, two or three substitutions; or 2)

[0761] CDR1: IYTMS (SEQ ID NO: 156), optionally comprising one or two substitutions,

[0762] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO: 157), optionally comprising one, two or three substitutions,

[0763] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 158), optionally comprising one, two or three substitutions; or 3)

[0765] CDR1: IYTMS (SEQ ID NO: 132), optionally comprising one or two substitutions,

[0766] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO: 133), optionally comprising one, two or three substitutions,

[0767] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 134), optionally comprising one, two or three substitutions; or 4)

[0769] CDR1: IYTMS (SEQ ID NO: 116), optionally comprising one or two substitutions,

[0770] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO: 117), optionally comprising one, two or three substitutions,

[0771] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 118), optionally comprising one, two or three substitutions; or 5)

[0773] CDR1: IYTMS (SEQ ID NO: 124), optionally containing one or two substitutions

[0774] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO: 125), optionally comprising one, two or three substitutions,

[0775] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 126), optionally comprising one, two or three substitutions; or 6)

[0777] CDR1: IYTMS (SEQ ID NO: 108), optionally comprising one or two substitutions,

[0778] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO: 109), optionally comprising one, two or three substitutions,

[0779] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 110), optionally comprising one, two or three substitutions;

[0780] and wherein the second ISVD comprises

[0781] (A)

[0782] CDR1: RYAMG (SEQ ID NO: 168), optionally comprising one or two substitutions,

[0783] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 169), optionally comprising one, two or three substitutions,

[0784] CDR3: DDSVGDGYLDY (SEQ ID NO: 170), optionally comprising one, two or three substitutions; or

[0785] (B)

[0786] CDR1: RYAMG (SEQ ID NO: 152), optionally comprising one or two substitutions,

[0787] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 153), optionally comprising one, two or three substitutions,

[0788] CDR3: DDSVGDGYLDY (SEQ ID NO: 154), optionally comprising one, two or three substitutions; or

[0789] (C)

[0790] CDR1: RLAMG (SEQ ID NO: 128), optionally comprising one or two substitutions,

[0791] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 129), optionally comprising one, two or three substitutions,

[0792] CDR3: DDSVGDGYLDY (SEQ ID NO: 130), optionally comprising one, two or three substitutions; or

[0793] (D)

[0794] CDR1: RLAMG (SEQ ID NO: 112), optionally comprising one or two substitutions,

[0795] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 113), optionally comprising one, two or three substitutions,

[0796] CDR3: DDSVGDGYLDY (SEQ ID NO: 114), optionally comprising one, two or three substitutions; or

[0797] (E)

[0798] CDR1: RLAMG (SEQ ID NO: 120), optionally comprising one or two substitutions,

[0799] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 121), optionally comprising one, two or three substitutions,

[0800] CDR3: DDSVGDGYLDY (SEQ ID NO: 122), optionally comprising one, two or three substitutions; or

[0801] (F)

[0802] CDR1: RLAMG (SEQ ID NO: 104), optionally comprising one or two substitutions,

[0803] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 105), optionally comprising one, two or three substitutions,

[0804] CDR3: DDSVGDGYLDY (SEQ ID NO: 106), optionally comprising one, two or three substitutions

[0805] (Kabat definition).

[0806] 122. The ISVD polypeptide derivative according to embodiment 121, wherein the substitution is a conservative substitution.

[0807] 123. The ISVD polypeptide derivative according to any one of embodiments 117-122, wherein the first ISVD comprises the sequence

[0808] V H H-2.20 (SEQ ID NO: 171),

[0809] V H H-2.18 (SEQ ID NO: 155),

[0810] V H H-2.15 (SEQ ID NO: 131),

[0811] V H H-2.13 (SEQ ID NO: 115),

[0812] V H H-2.14 (SEQ ID NO: 123), or

[0813] V H H-2.12 (SEQ ID NO: 107)

[0814] And among them

[0815] The second ISVD comprises the following sequence

[0816] V H H-1.20 (SEQ ID NO: 167),

[0817] V H H-1.18 (SEQ ID NO: 151),

[0818] V H H-1.15 (SEQ ID NO: 127),

[0819] V H H-1.13 (SEQ ID NO: 111),

[0820] V H H-1.14 (SEQ ID NO: 119), or

[0821] V H H-1.12 (SEQ ID NO: 103).

[0822] 124. Procoagulant V H H polypeptide derivative, which comprises

[0823] A first V capable of binding to factor IX (SEQ ID NO: 1) or an activated form thereof HH, a second V capable of binding to factor X (SEQ ID NO: 2) or an activated form thereof H H, connect the first V H H and the second V H H linker (L 1-2 ), and a C-terminal extension (E), and

[0824] One or two extensions,

[0825] The V H The H polypeptide derivative has the following formula (N-terminus to C-terminus):

[0826] "Second V H H”–L 1-2 – “First V H H”–E

[0827] in

[0828] I)

[0829] The first V H H contains

[0830] CDR1: IYTMS (SEQ ID NO:172),

[0831] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:173),

[0832] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 174), and

[0833] The second V H H contains

[0834] CDR1: RYAMG (SEQ ID NO:168),

[0835] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:169),

[0836] CDR3: DDSVGDGYLDY (SEQ ID NO: 170); or

[0837] II)

[0838] The first V H H contains

[0839] CDR1: IYTMS (SEQ ID NO:156),

[0840] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:157),

[0841] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 158), and

[0842] The second V H H contains

[0843] CDR1: RYAMG (SEQ ID NO:152),

[0844] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:153),

[0845] CDR3: DDSVGDGYLDY (SEQ ID NO: 154); or

[0846] III)

[0847] The first V H H contains

[0848] CDR1: IYTMS (SEQ ID NO:132),

[0849] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:133),

[0850] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 134), and

[0851] The second V H H contains

[0852] CDR1: RLAMG (SEQ ID NO:128),

[0853] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:129),

[0854] CDR3: DDSVGDGYLDY (SEQ ID NO: 130); or

[0855] IV)

[0856] The first V H H contains

[0857] CDR1: IYTMS (SEQ ID NO:116),

[0858] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:117),

[0859] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 118), and

[0860] The second V H H contains

[0861] CDR1: RLAMG (SEQ ID NO:112),

[0862] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:113),

[0863] CDR3: DDSVGDGYLDY (SEQ ID NO: 114); or

[0864] V)

[0865] The first V H H contains

[0866] CDR1: IYTMS (SEQ ID NO:124),

[0867] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:125),

[0868] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 126), and

[0869] The second V H H contains

[0870] CDR1: RLAMG (SEQ ID NO:120),

[0871] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:121),

[0872] CDR3: DDSVGDGYLDY (SEQ ID NO: 122); or

[0873] VI)

[0874] The first V H H contains

[0875] CDR1: IYTMS (SEQ ID NO:108),

[0876] CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:109),

[0877] CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 110), and

[0878] The second V H H contains

[0879] CDR1: RLAMG (SEQ ID NO:104),

[0880] CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:105),

[0881] CDR3:DDSVGDGYLDY(SEQ ID NO:106)

[0882] (Kabat definition).

[0883] 125. A pharmaceutical composition comprising an ISVD polypeptide derivative or V according to any one of embodiments 117-124. H H polypeptide derivatives.

[0884] 126. A pharmaceutical composition according to embodiment 125, wherein the composition comprises a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid.

[0885] 127. A pharmaceutical composition according to embodiment 126, wherein the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC).

[0886] 128. The pharmaceutical composition of any one of embodiments 125-127, further comprising nicotinamide (NAM).

[0887] 129. The pharmaceutical composition of any one of embodiments 125-128, wherein the composition is a solid composition.

[0888] 130. The ISVD polypeptide derivative according to any one of embodiments 117-129, V H H polypeptide derivatives or compositions for use in treating hemophilia, such as hemophilia A with or without inhibitors, or such as acquired hemophilia A.

[0889] 131. The ISVD polypeptide derivative used according to embodiment 130, V H H polypeptide derivative or composition, wherein the polypeptide derivative or composition is administered orally.

[0890] 132. A method for improving the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative, the ISVD polypeptide or ISVD polypeptide derivative comprising a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO: 1) or an activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof, a prolonging portion, and optionally a linker ("L") capable of linking ISVD1 and ISVD2. 1-2 ”), and optionally one or more extensions (“E”), the method comprising the steps of

[0891] a. modifying the nucleic acid encoding the amino acid residues of the ISVD polypeptide or ISVD polypeptide derivative so that its isoelectric point is reduced,

[0892] b. culturing the host cell to express the nucleic acid encoding the ISVD polypeptide or ISVD polypeptide derivative,

[0893] c. collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture,

[0894] d. purifying said ISVD polypeptide or ISVD polypeptide derivative from said host cell culture using standard chromatography methods, and

[0895] e. Attaching a prolongation moiety to the ISVD polypeptide, unless such a moiety is already present.

[0896] In a preferred embodiment, the compound obtained using the method for improving the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative is formulated into a pharmaceutical composition comprising SNAC and optionally further comprising NAM.

[0897] In a preferred embodiment, the ISVD polypeptide derivative comprises an anti-FX ISVD and an anti-FIX(a)V linked thereto in an N-terminal to C-terminal direction (N-terminal to C-terminal). H HISVD.

[0898] In one such preferred embodiment, the ISVD polypeptide derivative comprises an anti-FX V polypeptide in an N-terminal to C-terminal direction (N-terminal to C-terminal). H H fragment and anti-FIX(a)V linked thereto H H fragment, see e.g. Figure 2a , Figure 2b and Figure 3a -f.

[0899] In a preferred embodiment, the ISVD or V that is capable of binding to FIX(a)H The H fragment preferentially binds to activated FIX (FIXa).

[0900] In a preferred embodiment, an ISVD or V that is capable of binding to FX H The H fragment preferentially binds to proenzyme of FX, i.e., FX that has not yet been activated.

[0901] In one embodiment, the ISVD polypeptide (derivative) or V H The ISVD polypeptide (derivative) or VH polypeptide (derivative) is capable of stimulating the enzymatic activity of FIXa on FX. H The stimulatory properties of the H polypeptide (derivative) are derived from the ISVD or V that can bind to FIX(a) H H.

[0902] Figure 5a and Figure 5b The preferred anti-FIX (a) and anti-FX ISVD (V H H fragment) sequences, with the CDR sequences highlighted in bold and underlined.

[0903] The present invention encompasses the ISVD or V disclosed herein. H A substitution variant of a H segment, which may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions and / or deletions and / or insertions in each sequence disclosed herein. In some embodiments, the substitutions and / or deletions and / or insertions are located in one or more CDR sequences disclosed herein. In some embodiments, one or more CDR sequences undergo amino acid substitutions. In some embodiments, each CDR sequence may each comprise 0, 1, 2 or 3 amino acid substitutions. For example, in the V according to SEQ ID NO: 559, H In the H segment, CDR1 may contain no substitutions, CDR2 may contain 3 substitutions, and CDR3 may contain 1 substitution. Substitution variants preferably involve replacing one or more amino acids with the same number of amino acids. In some embodiments, the substitution is a conservative substitution.

[0904] In one embodiment, an ISVD polypeptide derivative such as V H The H polypeptide derivative comprises at least one prolongation which is unable to bind to components of the plasma membrane, such as aminophospholipids, such as phosphatidylserine and / or phosphatidylethanolamine.

[0905] In one embodiment, the extension portion comprises the following structure:

[0906]

[0907] Where '*' represents the same as V H Attachment point for H polypeptide.

[0908] In one embodiment, an ISVD polypeptide derivative such as V H The H polypeptide derivative comprises an extension that cannot bind to platelet surface proteins / markers, such as GPlb-lX, collagen chaperone HSP47, ephrin B1, thiol isomerase protein ERP5, hematopoietic progenitor kinase 1 interacting protein 55 (HIP-55), glycoprotein Vl, platelet glycoprotein 1b, platelet-derived growth factor receptor, platelet endothelial aggregation receptor I, CD31, CD36, MARKS, polyprotein, integrin αlIb / β3, triggering receptor expressed on myeloid cells (TREM)-like transcript-1 (TLT-1), integrin-linked kinase (ILK), zoster, collagen, P-selectin, factor XIII, P-selectin glycoprotein ligand-1, integrin α6β1, thrombospondin, von Willebrand factor, G6B, CD42b, syntaxin binding protein 2, phosphatidylethanolamine, fibrinogen / fibrin, filamin, s tomati n , sphingolipids, CD31, CD36, CD40, CD41, CD42c, CD42, CD49b, CD61, CD62P, CD63, CD69, CD107a, CD107b, CD109, CD154, PECAM-1, and / or ERPS.

[0909] In one embodiment, an ISVD polypeptide derivative such as V H The H polypeptide derivative comprises an extended portion that is incapable of binding to a membrane-associated polypeptide, such as a glycoprotein, GPIIb / IIIa, β2GP1, TLT-1, selectin, a coagulation factor or a coagulation factor complex and / or a selectin.

[0910] In one embodiment, when the ISVD polypeptide derivative of the invention is H When the H polypeptide derivatives are used to treat hemophilia A at clinically relevant doses, the polypeptide derivatives do not interfere with the effects of FVIII, such as recombinant FVIII, administered to patients suffering from hemophilia A.

[0911] Although certain features of the present invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and changes that fall within the true scope of the invention.

[0912] Example

[0913] List of abbreviations

[0914] AIEX: Ion Exchange

[0915] CC: Pearson correlation coefficient

[0916] CDR: Complementarity determining region

[0917] CHO: Chinese Hamster Ovary

[0918] CMK: Chloromethyl ketone

[0919] EC 50 : Half maximum effective concentration

[0920] EDTA: Ethylenediaminetetraacetic acid

[0921] ELISA: Enzyme-linked immunosorbent assay

[0922] FACS: Fluorescence Activated Cell Sorter

[0923] FIX: Coagulation Factor IX

[0924] FIXa: Coagulation Factor IXa

[0925] FX: Factor X

[0926] FXa: Coagulation Factor Xa

[0927] FR: Framework Region

[0928] GS: glutamine synthetase

[0929] HA: Hemophilia A

[0930] HB: Hemophilia B

[0931] ISVD: immunoglobulin single variable domain

[0932] IV: Intravenous

[0933] KO: Knockout

[0934] MSX: L-Methionine Sulfoximine

[0935] MRTHL: mean residence time terminal half-life

[0936] NAM: Niacinamide

[0937] OEG: 8-amino-3,6-dioxa-octanoic acid

[0938] PBMC: peripheral blood mononuclear cells

[0939] PCR: polymerase chain reaction

[0940] pI: isoelectric point

[0941] PK: Pharmacokinetics

[0942] PO: Oral

[0943] ROUT: Robust regression and outlier removal (GraphPad Prism function)

[0944] RMS: Root Mean Square

[0945] RT-PCR: Reverse transcriptase polymerase chain reaction

[0946] RP-UPLC: Reversed Phase Ultra-Performance Liquid Chromatography

[0947] SIA: sequence identical analogs

[0948] SC: Subcutaneous

[0949] SNAC: Sodium N-(8-[2-hydroxybenzoyl]amino)caprylate

[0950] SPR: Surface Plasmon Resonance

[0951] TGT: thrombin generation test

[0952] TLS: Translation-Vibration-Screening-Rotation

[0953] TVT: Tail vein transection

[0954] UPLC: Ultra-Performance Liquid Chromatography

[0955] Example 1: General Method and V H Preparation of H fragments and other recombinant proteins

[0956] General Molecular Biology

[0957] For general molecular biology techniques, see Molecular Cloning: A Laboratory Manual (4th edition, 2014, Sambrook, Fritsch and Maniatis eds., CSHL Press, Cold Spring Harbor, NY USA).

[0958] Immunization and libraries

[0959] Two llamas and two alpacas were immunized with human FIX and FX (Haemotologic Technologies, VT USA), respectively, after approval from the Ablynx Camelid Facility Ethics Committee (LA1400575).

[0960] Cloning of heavy chain antibody fragment repertoires only and preparation of phage immune libraries were performed as follows.

[0961] After the last immunogen injection, blood samples were collected. Peripheral blood mononuclear cells (PBMCs) were prepared from these blood samples using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, US). Total RNA was extracted from PBMCs and used as the starting material for RT-PCR to amplify the V H The DNA segment of the H fragment is essentially as described in WO2005 / 044858. H The DNA fragment of the H fragment was cloned into the phagemid vector pAX212, which allows the production of V display fused to the His6 tag and the FLAG3 tag. H Subsequently, phages were prepared and stored according to standard protocols.

[0962] Synthetic library

[0963] The synthetic library was generated by immunizing and cloning the synthetic V H The H gene fragment was cloned into the phagemid vector pAX190, which has the same features as pAX212 described above, but differs in the multiple cloning site.

[0964] V combined with FX or FIX H Library screening of H fragment

[0965] The generated immune and synthetic libraries were used to perform V H H fragment phage display selection. These libraries underwent one to four consecutive rounds of enrichment against different concentrations of immobilized human FIX and FX to enrich clones for screening for better binders to FIX and FX (Haemotologic Technologies, VT USA) and cynomolgus monkey FIX and FX (generated in-house at Novo Nordisk).

[0966] In order to specifically enrich for V that is selective for FIXa and FX over FIX and FXa, respectively HH fragments, in some experiments, were competed with excess soluble FIX and FXa during incubation of the library with immobilized FIXa and FX.

[0967] In order to specifically enrich for V that is selective for FIX and FX over other structurally related coagulation factors H H fragments, in some experiments, were competed with excess soluble FX and FIX during incubation of the library with immobilized FIX and FX, respectively.

[0968] ELISA (using H H fragment) in the periplasmic extract of E. coli cells to human and cynomolgus monkey FX, FXa, FIX and FIXa, and about 4,500 individual clones from the selection output were screened. Approximately 1500 clones that each showed specific binding to human FX and FIXa / FIX were identified, most of which showed cross-binding to cynomolgus monkey FX and FIX. Some clones showed preferential binding to FX compared to FXa and preferential binding to FIXa compared to FIX. Sequence analysis of ELISA positive clones identified V binding to FIXa / FIX or FX / FXa. H To further optimize the compounds, an in-depth V H H CDR and FR mutagenesis, combined with elongator additions, linkers, and extension combinations, focused on enhancing hemostatic potency through mutation screening using thrombin generation assays, avoiding target-mediated drug disposition by screening for affinity-altered compounds, reducing immunogenicity by screening for risk sites using MHC-associated peptide proteomics assays, and enhancing in vivo oral bioavailability through pI-lowering substitutions of surface-exposed residues.

[0969] V combined with FX or FIX H Generation of expression constructs for H fragments

[0970] V from phage display selection output H Sequence analysis of the H fragment was performed according to known procedures (Pardon et al. (2014) Nat Protoc 9:674).

[0971] Using a specific combination of forward FR1 and reverse FR4 primers, each with a unique restriction site, the V H The DNA fragment of the H fragment was digested with appropriate restriction enzymes and ligated to the V HThe H polypeptide expression vector was cloned into a matching cloning cassette as a His6-tagged form and / or a FLAG3-tagged form for expression in E. coli or P. pastoris. The ligation mixture was then transformed into electrocompetent E. coli TG1 (60502, Lucigen, Middleton, WI) or TOP10 (C404052, ThermoFisher Scientific, Waltham, MA) cells and then grown under appropriate antibiotic selection pressure (kanamycin or Zeocin). Resistant clones were verified by Sanger sequencing of plasmid DNA (LGCGenomics, Berlin, Germany).

[0972] V combined with FX or FIX H Universal Expression of H Fragments in E. coli

[0973] V was expressed in E. coli TG1 from a plasmid expression vector containing the lac promoter, kanamycin resistance gene, E. coli replication origin and VHH fragment cloning site and the preceding OmpA signal peptide coding sequence. H H fragment. H The H polypeptide coding sequence is in frame, and the vector encodes the C-terminal FLAG3 and His6 tags. The signal peptide will express the V H The H fragment is directed to the periplasmic compartment of the bacterial host.

[0974] Make it contain the target V H E. coli TG-1 cells of the H fragment construct were generated in baffled shake flasks containing "5052" autoinduction medium (0.5% glycerol, 0.05% glucose, 0.2% lactose + 3 mM MgSO4) at 37°C for 2 hours, followed by growth at 30°C for 29 hours. The frozen overnight cell pellet from the E. coli expression culture was then dissolved in PBS (1 / 12.5 of the original culture volume) and incubated at 4°C for 1 hour with gentle rotation. Finally, the cells were pelleted again and the supernatant containing the protein secreted into the periplasmic space was saved.

[0975] V H Universal Expression of H Fragment in Pichia pastoris

[0976] Make it contain the target V HThe Pichia pastoris cells of the H fragment construct were grown in BGCM medium for two days (30°C, 200rpm). On the third day, the medium was changed to BMCM, and the construct was further grown (30°C, 200rpm) and induced with 0.5% v / v methanol after 8 hours. The next day, the construct was induced with 0.5% v / v methanol in the morning, noon and evening. On the fifth day, the cells were centrifuged and the supernatant (containing secreted V H H fragment).

[0977] V combined with FX or FIX H Universal expression of H and antibodies and other recombinant proteins in HEK cells

[0978] Expression plasmids for transient expression in HEK293 cells were purchased from Twist Biosciences or ThermoFisher Scientific. The plasmid from Twist Biosciences was based on the pTT vector described in Durocher, Y. et al., (2002) Nucleic Acid Res, 30: E9, while the plasmid from ThermoFisherScientific was based on the pcDNA34-Topo vector (Thermo Fisher Scientific). H H polypeptide compound, His6-labeled or unlabeled V H H polypeptide compounds were transfected into HEK293 suspension cells. Equivalent expression constructs carrying sequences encoding anti-GLA FIX and anti-GLA FX antibodies (generated in-house at Novo Nordisk), emicizumab (Hoffmann-La Roche Ltd, Switzerland) sequence identical analog (SIA), and Mim8 (Novo Nordisk, Denmark) were also expressed in HEK293 using the methods described below.

[0979] Transient transfection of HEK293 suspension cells (Expi293 expression system, Thermo Fisher Scientific, catalog number A14635) is carried out substantially according to the manufacturer's instructions. HEK293 cells are usually subcultured every 3-4 days in Expi293 expression medium (Gibco, catalog number A14351-01) supplemented with 1% P / S (GIBCO catalog number 15140-122). HEK293 cells are transfected with a cell density of 2.5-3 million / mL using Expifectamine. For each liter of HEK293 cells, a total of 1 mg plasmid DNA is diluted into 50 mL Optimem (GIBCO, catalog number 51985-026, diluent A) and 2.7 mL Expifectamine is diluted into 50 mL Optimem (diluent B) for transfection. For cotransfection (i.e. for antibodies), plasmids are used in a 1: 1 ratio. Dilutions A and B were mixed and incubated at room temperature for 10-20 minutes. The transfection mixture was then added to HEK293 cells and the cells were incubated at 37°C in a humidified incubator with orbital rotation (85-140 rpm). One day after transfection, the 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 harvested typically 4-5 days after transfection by centrifugation and subsequent filtration. V bound to FX and FIX(a) H Universal expression of H fragments and antibodies in CHO cells

[0980] Production of anti-FIX and anti-FXV in Chinese Hamster Ovary (CHO) cells using glutamine synthetase (GS) selection HH compounds and antibodies. CHO cells were transfected with GS expression plasmids using electroporation, followed by selection using glutamine deprivation and MSX supplementation in CD-CHO medium (ThermoFisher Scientific, catalog number 10743029). Stable CHO cell pools were typically obtained after 3 weeks of culture, after which the CHO cell pools were cloned into 384-well plates in single cell form. CHO clones that appeared in 384-well plates were typically expanded into 96-well plates and screened for productivity. Selected production clones were scaled up using a predefined proprietary cell culture medium (Novo Nordisk A / S) for cultivation in bioreactors of 1-L to 15-L scales. Cell viability remained high during the culture period and then gradually declined until the cell culture was harvested. At this point, depending on the culture scale, the cell supernatant was removed by centrifugation and / or depth filter filtration using MDOHC23CL3 and MX0HC01FS1 filters (Millipore), followed by chromatography-based protein purification. V bound to FX or FIX H Universal purification and characterization of H fragments

[0981] His6-tagged or untagged V H The H compounds were purified by MabSelectSure protein A resin (Cytiva) or immobilized metal affinity chromatography (IMAC) on Ni-Excel (Cytiva) resin containing imidazole (for the latter) or by acidic elution (for the former), followed by a desalting step (PD column with Sephadex G25 resin, Cytiva) and, if necessary, gel filtration chromatography in PBS or HBS (Superdex200 column, Cytiva). Non-His6-tagged V against the GLA domain of FIX and FX, respectively HH compounds and antibodies (Novo Nordisk in-house generated) and emicizumab SIA (Hoffmann-La Roche Ltd, Switzerland) were purified by protein A resin MabSelectSure (Cytiva) or multimodal resin (Cytiva) with acidic elution followed by a desalting step (e.g., PD column with Sephadex G25 resin, Cytiva) and, if necessary, gel filtration chromatography in PBS or HBS (e.g., Superdex200 column, Cytiva). Protein integrity was analyzed using a size exclusion high performance liquid chromatography (SE-HPLC) method set up on an Agilent LC 1100 / 1200 system using a BIOSEP (column for separation of biomolecules)-SEC-53000 300×7.8 mm column (Phenomenex, catalog number OOH-2146-K0) and a running buffer consisting of 200 mM sodium phosphate pH 6.9, 300 mM NaCl and 10% isopropanol. The purified V was analyzed using electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) on a 6280 Agilent system (Agilent Technologies). H The molecular weight of the H polypeptide batch was determined using a NanoDrop TM Spectrophotometer (Thermo Scientific) and calculated theoretical extinction coefficient.

[0982] Example 2: Extension and V H Conjugation of H polypeptide cysteine ​​residues

[0983] In order to extend V H H peptide to improve pharmacokinetic properties, V H H polypeptide compounds are engineered to contain cysteine ​​(Cys) substitutions at various backbone positions, such as the introduction of one or two Cys residues in the N- or C-terminal extensions for conjugation to one or more fatty acid extensions, as further described below.

[0984] To attach the extension to the V H On the H polypeptide compound, an intermediate reagent in the form of a modified extension portion is used.

[0985] Intermediate reagents comprising an extended portion are prepared as described in WO2016 / 102562, and non-limiting examples of such intermediate reagents are shown in Table 3 below:

[0986]

[0987]

[0988] Conjugation, purification and analysis

[0989] To V containing one or more cysteine ​​residues for conjugation H 5 equivalents of BSPP (bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt) or 1.1 equivalents of TCEP (tri(2-carboxyethyl)phosphine hydrochloride) were added to the aqueous solution of H polypeptide for each blocked cysteine. After stirring for 1-2 hours, the pH was adjusted to 8.5 with aqueous NaOH solution, and the corresponding V H For each free cysteine ​​in the H polypeptide, 5 equivalents of intermediate reagent C1 in 0.1 M NaHCO3 (aq.) are added. The mixture is gently stirred in the dark for 1.5-16 hours. The reaction mixture is diluted with water and then treated with The system is purified by anion exchange (AIEX) method. AIEX chromatography is used to purify V with side chain conjugation H H polypeptide. Therefore, AIEX resin Source 30Q packed in a suitable column is similar to The sodium chloride gradient program setting on the Avant chromatography system was used. The buffer system used was an equilibrium buffer composed of 20mM Tris (pH 8.5) and an elution buffer composed of 20mM Tris, 1M NaCl (pH 8.5). The reaction mixture was adjusted to pH 8.5 and diluted to a conductivity of less than 4mS / cm using MilliQ-H2O or equilibrium buffer. The sample was added to the column and the column was washed after applying 5 to 10 column volumes of equilibrium buffer. A shallow gradient of 30 to 50 column volumes was then used for separation chromatography. The gradient used was from 0% to 50%, depending on the purified V H The pI of the H polypeptide derivative. Typically, the unconjugated parent V H The H peptide eluted early in the gradient and had a single conjugated V H The H polypeptide elutes in the middle of the gradient, while the multi-conjugated V H H polypeptide (meaning each V H The H polypeptide molecules with more than one side chain conjugated) elute at the end of the gradient. The main peak fractions are combined in a certain way to obtain 90% to 99% high purity V with single or double conjugation. H Preparation of H polypeptide. Purity analysis was performed using reverse phase ultra-high performance liquid chromatography (RP-UPLC) based on a HALO DiPhenyl column 2.7 μm, 150x2.1 mm (Scantec Nordic USDPF001316) and a running buffer consisting of A) 0.1% v / v TFA in water and B) 0.09% v / v TFA in acetonitrile, on a Waters AcquityUPLC system equipped with UV and FLD detectors. The column temperature was set at 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: 80% B, 4) 9.0-9.1 min: 80-20% B, and 5) 9.1-11.0 min: 20% B. Unconjugated parent V H The H polypeptide eluted between 4.6 and 4.8 min. H The H peptide derivative eluted between 5.1 and 5.6 min, with the main peak at about 5.3 min. H The H peptide derivative eluted between 5.7 and 5.8, followed by the offsite multi-conjugated V H The H polypeptide was purified using a SE-HPLC method setup on an Agilent LC 1100 / 1200 system and a BIOSEP-SEC-3000 300×7.8 mm column (Phenomenex, catalog number OOH-2146-K0) and a column consisting of 200 mM sodium phosphate pH

[0990] 6.9, 300 mM NaCl and 10% isopropanol running buffer was used to analyze the conjugated V H The integrity of the H polypeptide was determined using ESI-TOF-MS on a 6280 Agilent system (Agilent Technologies). HThe molecular weight of the H polypeptide was calculated using a MassPREP Desalt (Waters) column, running at 0.4 ml / min in buffer A consisting of MilliQ-H2O / 0.1% formic acid and buffer B consisting of acetonitrile / 0.1% formic acid for step elution. Peptide mapping for sequence verification was performed using a combination of chymotrypsin and trypsin-based digests. The LC-MS system consisted of a waters Aquity UPLC combined with a Thermo Orbitrap Fusion instrument. The digests were analyzed online by LC-MS using a CSH C-18 column 1.7 μm, 150x2.1 mm and an acetonitrile / formic acid gradient. The column temperature was 60°C. Buffer A: 0.1% FA water, buffer B: 0.1% FA 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 rate of 120 μl / min. Data analysis was performed using the Genedata Refiner peptide mapping workflow. Complete coverage of the primary sequence was obtained. To determine the conjugated V H Protein concentration of bulk preparations of H peptide derivatives using NanoDrop TM spectrophotometer (Thermo Scientific) and calculated theoretical extinction coefficient.

[0991] Figures 1, 2, and 3 show ISVD and V at increasing levels of detail. H Non-limiting examples of H polypeptide derivatives.

[0992]

[0993]

[0994]

[0995]

[0996]

[0997]

[0998]

[0999]

[1000]

[1001]

[1002]

[1003]

[1004]

[1005]

[1006]

[1007] Example 4: Anti-FX V based on ELISA experiment H H polypeptide epitope

[1008] To determine the anti-FX V H To determine whether the binding epitope of the H polypeptide is located within the activation peptide (AP) of human FX (SEQ ID NO: 2), an ELISA assay was established. 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 and then incubated with the V peptide to be tested. H The bound ligand was detected by adding HRP-labeled secondary antibody.

[1009] These peptides were conjugated with biotin at the C-terminus and immobilized in discrete wells of a microtiter plate pre-coated with 1 μg / mL streptavidin using 50 μL of a 1 μg / mL peptide solution. Each well was washed with wash buffer (10 mM Tris, 150 mM NaCl, 2.5 mM CaCl2, 0.05% Tween 20, pH 8.60) and then 50 μL of a 2 μg / mL FLAG-tagged anti-FXV to be tested was added. H After 1 hour, wash buffer was used to wash away unbound V H H polypeptide.

[1010] The bound anti-FX activating peptide ligand was detected as follows: first, HRP-labeled secondary antibody was bound (for FLAG-labeled V H H polypeptide: anti-FLAG mAb M2-peroxidase (HRP) (Sigma Aldrich, US)) for 1 hour, and then 100 μL of TMB-1 ELISA substrate (Kem-En-Tec Diagnostics, Denmark) was added.

[1011] The minimal epitope was then deduced from the set of peptides that produced a signal above baseline by identifying the common sequence covered by the set of peptides bound by each FX binder (Table 6). 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.

[1012] Therefore, the following table shows the initial anti-FX V corresponding to compound #1 tested. H H polypeptide, ELISA signal from a continuous peptide spanning the entire FX AP is shown. ELISA signal has been normalized to background signal.

[1013] The grey area marks the positive binding signal used to determine the minimal epitope, which was identified as the sequence NPFDLLDF (SEQ ID NO: 692).

[1014] The epitope sequence of the FX activating peptide identified for compound #1 was used in high-resolution structure determination experiments of epitope:paratope interactions as outlined in Example 6.

[1015] Table 6: Epitope identification

[1016]

[1017]

[1018] Example 5: Anti-FIX V H Crystallization and paratope / epitope mapping of H fragment compound #2

[1019] The purpose of this study was to determine the V H Paratope and epitope residues of the H fragment.

[1020] crystallization

[1021] V was mixed with human EGR-CMK-inhibited Factor IXa without Gla domain (wild type) (purchased from Cambridge Protein Works, lot number hGDFIXAWTEGR_11) at a 1:1 molar ratio. HCrystals of H polypeptide compound #2 were grown at 18°C ​​using the droplet vapor diffusion technique. 100 nl of a protein solution of 8.4 mg / ml complex in 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, and 2.5 mM CaCl2 was mixed with 100 nl of 0.2 M lithium sulfate, 0.1 M Tris-HCl (pH 8.5), 30% (w / v) PEG4000 (as a precipitant) and incubated on 60 μl of the precipitant. Crystals appeared within two weeks.

[1022] Diffraction data collection

[1023] Crystals were cryoprotected by adding 1 μl of precipitant with 20% ethylene glycol to the crystallization drop before rapid cooling in liquid nitrogen. The crystals were imaged at the Swiss Light Source beamline X06DA ( Diffraction data were collected at 100 K at 400 nm (100 nm wavelength). Automatic indexing, integration and scaling of the data were performed using programs from the XDS package (the diffraction data statistics are summarized in Table 7).

[1024] Structure determination and refinement

[1025] The asymmetric unit contains two compound #2: EGR-CMK-inhibited factor IXa Gla-free complexes as judged by Matthews coefficient analysis. H The structure of the H:FIXa complex was used as a search model and the structure was determined by molecular replacement using Phaser implemented in the program suite Phenix. The correct amino acid sequence was modeled using COOT, and the structure was then refined using Phenix refinement and manual reconstruction steps in COOT. The refinement statistics are shown in Table 7.

[1026] Table 7: Data collection and refinement statistics

[1027]

[1028]

[1029] Statistics for the highest resolution shell are shown in parentheses.

[1030] Determination of epitopes and paratopes

[1031] The epitope of compound #2 was defined as a FIX(a) residue with the following characteristics: H Heavy atoms in H polypeptides The protease domain of FIX(a) has heavy atoms (i.e., non-hydrogen atoms) within a distance of :

[1032] E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260, according to the FIX sequence (numbered consecutively) (SEQ ID NO: 1).

[1033] The paratope of compound #2 is defined as the residue with the following characteristics: a heavy atom within a distance of FIX(a) The residues from compound #2 have heavy atoms (i.e., non-hydrogen atoms) within a distance of :

[1034] F29, N30, Y32, T54, D99, R100, S101, F102, L103, F104, Q106, A107 and N113 (SEQ ID NO: 35) (numbered consecutively).

[1035] Example 6: Anti-FX V H Crystallization and paratope / epitope mapping of H fragment compound #1

[1036] The purpose of this study was to determine the V H Paratope and epitope residues of the H fragment.

[1037] crystallization

[1038] V was mixed with the synthetic peptide N-terminal-NPFDLLD-C-terminal (corresponding to the activation peptide sequence aa31-37 of human FX (purchased from Schafer-N ApS) identified in Example 4) at a molar ratio of 1:14. H Crystals of H polypeptide compound #1 were grown at 18°C ​​using the dropping vapor diffusion technique. 150 nl of a 4.0 mg / ml protein solution of the complex in 20 mM Tris-HCl (pH 7.4), 50 mM NaCl was mixed with 50 nl of 0.2 M NaCl, 2 M ammonium sulfate, 0.1 M sodium cacodylate (pH 6.5) as a precipitant and incubated on 60 μl of the precipitant. Crystals appeared within a week.

[1039] Diffraction data collection

[1040] Crystals were cryoprotected by adding 1 μl of precipitant with 20% ethylene glycol to the crystallization drop before rapid cooling in liquid nitrogen. The crystals were imaged at the Swiss Light Source beamline X06DA ( Diffraction data were collected at 100 K at 400 nm (100 nm wavelength). Automatic indexing, integration and scaling of the data were performed using programs from the XDS package (the diffraction data statistics are summarized in Table 8).

[1041] Structure determination and refinement

[1042] The asymmetric unit contains a compound #1:peptide complex. V was assigned with Protein Data Bank ID 4B41 (chain A). H The H polypeptide structure was used as a search model and the structure was determined by molecular replacement using Phaser implemented in the program suite Phenix. The correct amino acid sequence of compound #1 was introduced, and the differential electron density of the synthetic peptide was determined and modeled manually, all using COOT. The structure was then refined using Phenix refinement and a manual reconstruction step in COOT. The refinement statistics are shown in Table 8.

[1043] Table 8: Data collection and refinement statistics

[1044]

[1045]

[1046] Statistics for the highest resolution shell are shown in parentheses.

[1047] Determination of epitopes and paratopes

[1048] Synthetic peptide epitopes—defined as FX activating peptide residues with the following characteristics: H Heavy atoms in H-peptide compound #1 The following residues from the synthetic peptides have heavy atoms (i.e., non-hydrogen atoms) within a distance of :

[1049] L178, according to the corresponding FX activating peptide sequence, based on consecutive numbering (SEQ ID NO: 2).

[1050] V H The paratope of H peptide compound #1 is defined as the residue of compound #1 that has the following characteristics: The residues from compound #1 have heavy atoms (i.e., non-hydrogen atoms) within a distance of :

[1051] 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) (numbered consecutively).

[1052] Conducted V H Construction of homology model of H peptide compound #3 in complex with synthetic peptide, because compound #3 is used as parent V H The H polypeptide sequence was used for rational sequence- and structure-based optimization.

[1053] The crystal structure of compound #1 in complex with a synthetic peptide from the above experiment was used as the starting model for the homology model of compound #3 in complex with a synthetic peptide. The amino acid sequences of compound #1 and compound #3 were aligned and the amino acid residues that differed between the two sequences were mutated in COOT to create a starting model for compound #3.

[1054] #3) preprocessing, optimization, and The MAESTRO was run via constrained minimization.

[1055] The model shows the same epitope and paratope residues as described above, as the sequences at these positions are identical.

[1056] Example 7: Anti-FX / Anti-FIX(a)V H SPR analysis of H peptide

[1057] The aim of this study was to estimate the activity of selected anti-FX / anti-FIX(a)V by surface plasmon resonance (SPR) analysis. H H polypeptide compounds and human plasma derived FX and The binding constant.

[1058] The binding of purified anti-FX / FIX(a) VHH polypeptide compounds to human plasma-derived FX (Haematologic Technologies Inc, USA) was probed by SPR.

[1059] Briefly, anti-GLA-FX prepared recombinantly as described above was immobilized 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 for 30 seconds. Subsequently, 1000, 100, 10, 1, 0.1 and 0 nM of V according to Table 9 below were injected at a flow rate of 50 μL / min. HThe H polypeptide compound was then injected into the 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) for 10 minutes to dissociate it from FX. The running buffer was also used to dilute the anti-FX V H H polypeptide compound. The chip was regenerated using a regeneration buffer consisting of 50 mM EDTA in the running buffer, a contact time of 30 seconds, and a flow rate of 30 μL / min. Binding data were collected at 25°C and analyzed using BiaEvaluation 4.1 provided by the manufacturer (Biacore AB, Uppsala or BrukerAnalyser) in a 1:1 model.

[1060] In all cases, the binding sensograms showed fast association and dissociation binding kinetics, ruling out K based on kinetic analysis. D Therefore, the reported K D The values ​​were determined from steady-state analysis. The analysis yielded the binding constants reported in Table 9 below.

[1061] Probing of purified anti-FX / FIX(a)V by SPR H H polypeptide compounds (Pfizer Inc, USA). Briefly, anti-GLA-FIX prepared as described above was immobilized 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 V were injected at a flow rate of 30 μL / min. H H peptide compound for 4 minutes to allow it to bind to FIX The binding was followed by injection of running buffer (5 mM HEPES, 150 mM NaCl, 5 mM CaCl2, 0.05% (v / v) surfactant P20, 1 mg / mL bovine serum albumin, pH 7.4) for 5 minutes to dissociate it from FIX. This running buffer was also used to dilute the anti-FIX V H H polypeptide compound. Regeneration of the chip was achieved using a regeneration buffer consisting of 50 mM EDTA in running buffer, a contact time of 30 seconds, and a flow rate of 30 μL / min. Binding data were collected at 25°C and analyzed using BiaEvaluation 4.1 provided by the manufacturer (Biacore AB, Uppsala) in a 1:1 model.

[1062] In all cases, the binding sensorgrams showed binding kinetics with rapid association and dissociation, thus ruling out K based on kinetic analysis. D Therefore, the reported K D The values ​​are determined based on steady-state analysis.

[1063] Binding constant analysis for a series of compounds is reported in Table 9. For all compounds, the binding affinity for FIX was generally in the low nM K D , while a series of compounds showed high nM K for FX binding D Thus, for FX, no target-mediated drug disposition is expected, whereas FIX interactions would be expected, but only at the steady-state plasma concentrations required for the compounds outlined. Thus, the plasma concentrations required to achieve meaningful hemostatic coverage expected for the listed compounds are expected to be low nM concentrations, given the high potency of the listed compounds.

[1064] Table 9: Selected V H Estimated binding constants for H peptide compounds

[1065] As determined by SPR analysis, for selected anti-FX / anti-FIX(a)V H H polypeptide compounds and human plasma derived FX and The binding constants of the interactions were estimated based on steady-state analysis.

[1066] Compound ID <![CDATA[FX conjugate K D (nM)]]> <![CDATA[FIX conjugate K D (nM)]]> Compound #4 229 11.7 Compound #5 199 2.7 Compound #6 232 4.8 Compound #7 82 2.3 Compound #8 96 2.5 Compound #9 70 1.1 Compound #10 190 5.5 Compound #11 42 6.4 Compound #12 26 7.8 Compound #13 300 2.0 Compound #14 42 1.6 Compound #15 350 3.0

[1067] Example 8: Anti-FX / Anti-FIX(a)V H Activity of H polypeptide in thrombin generation test (TGT)

[1068] Anti-FX / Anti-FIX(a)V H The procoagulant activity of the H polypeptide derivatives was determined based on their ability to promote thrombin generation in the presence of procoagulant synthetic phospholipid membranes according to the principles described by Hemker et al. (Pathophysiol Haemost Thromb, 2002; 32: 249-253). The emicizumab sequence identical analog (SIA) was included for comparison. Each V was tested in the TGT assay using normal human platelet-poor plasma (NHP) supplemented with neutralizing anti-FVIII polyclonal antibodies (hereinafter referred to as HA-PPP). H H polypeptide derivatives.

[1069] TGT in NHP (from healthy volunteers) supplemented with sheep anti-human FVIII polyclonal antibody (pAb, Haematologic Technologies Inc., VT, USA) was performed by standard calibration automatic coagulation curve method (thrombography) using a 96-well plate fluorometer (Fluoroscan Ascent FL, Thermolabsystems, Helsinki, Finland). The reaction mixture contained 36 μl NHP pre-incubated with 0.1 μg / ml anti-FVIII pAb, 4 μl test compound dilution (diluted in 20 mM HEPES, 140 mM NaCl (pH 7.4), 2% BSA), 10 μl of 1 pM tissue factor (TF, pppLow, from Thrombinoscope BV, Maastricht, the Netherlands) or 8.3 U / ml human factor XIa (Enzyme Research Laboratories, IN, USA) and 10 μl FluCa substrate (Thrombinoscope BV, Maastricht, the Netherlands). The TGT test is calibrated using thrombin calibrators (Thrombinoscope BV, Maastricht, the Netherlands), wherein 10 μl thrombin calibrators are mixed with 36 μl NHP, 4 μl buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) pre-incubated with 0.1 μg / ml anti-FVIII pAb. Typically, TGT is carried out at 8 concentrations of test compound (0.1, 0.3, 1, 3, 10, 30, 100 and 300 nM, final plasma concentration, or similar concentration) or only with buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) (representative control). The normal control level in TGT is measured using a buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) only with NHP. TGT was allowed to proceed for a total of 60 minutes and the TGT parameter Peak Thrombin Height (nM) was analyzed by Thrombinoscope software (Thrombinoscope BV). H Representative titration curves for H peptide derivatives, compound #6, and comparators emicizumab SIA and Mim8, see Figure 4 The difference in activity level was assessed relative to the EC of a comparator such as emicizumab SIA. 50 Difference or given V H The latter method was used to screen a large number of V H H polypeptide derivatives.

[1070] In order to strengthen V H The activity of H polypeptide derivatives was investigated to determine the optimal combination of CDR mutations for producing the greatest activity effect. Crystal structure models, such as those described in Examples 5 and 6, were used to identify important paratope residues and surface exposed residues suitable for cysteine ​​substitution as conjugation sites, as well as substitutions that lower the pI to enhance bioavailability. Different linkers and different conjugation types were also studied. In the table below, a series of V H The activity profiles of the H polypeptide derivatives are for each of these parameters and will be described separately.

[1071] In Table 10, the optimized V values ​​for activity enhancing mutations are shown. H The potency EC obtained by H peptide derivatives 50 Principal component calculation and random forest algorithm were used for mutation combination to enhance activity. H H polypeptide compound #24 is the original derivative without the booster mutation and is H Compared with the H peptide derivatives and emicizumab SIA, the best V H In the H polypeptide derivatives, up to 40-fold and up to 76-fold potency improvements were obtained, respectively.

[1072] Table 10: V H TGT activity of H peptide derivatives and optimization of activity

[1073]

[1074]

[1075] In Table 11, for V H The optimal conjugation sites on the H polypeptide (outside CDR1-3) were screened and these sites were replaced by cysteine ​​(with the linker L P 1 attached C18 diacid fatty acid extension P3 conjugated and unconjugated) still retains the V H Activity of H polypeptide.

[1076] A total of 95 surface exposed sites were tested by introducing Cys substitutions. HH polypeptide) were numbered consecutively to screen for the best conjugation site. 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, G8 3C, R84C, F85C, T86C, S88C, D90C, N91C, S92C, K93C, T95C, Y97C, Q99C, M100C, N101C , S102C, L103C, R104C, P105C, E106C, D107C, T108C, G156C, V157C, V158C, Q159C, P1 60C, G161C, G162C, S163C, L164C, R165C, S167C, A169C, S171C, R184C, Q185C, A186C , P187C, G188C, K189C, E190C, R191C, Y206C, A207C, D208C, S209C, V210C, K211C, G2 12C, R213C, F214C, T215C, S217C, D219C, N220C, S221C, K222C, T224C, Y226C, Q228C, M229C, N230C, S231C, L232C, R233C, P234C, E235C, D236C, T237C, and a Cys introduced into the C-terminal extension.

[1077] Among them, the following 41 types of V H The H polypeptide was expressed using the HEK293 expression system and successfully conjugated to reagent C3 containing the C18 diacid extension moiety: Q30C, P31C, Q56C, A57C, K60C, E61C, A78C, D79C, K82C, N91C, S92C, Q99C, R104C, G156C, V158C, Q159C, P160C, G161C, G1 62C, S163C, A169C, S171C, Q185C, A186C, P187C, G188C, K189C, E190C, A207C, D208C, S209C, V210C, T215C, S217C, D219C, N220C, S221C, K222C, S231C, R233C, and a Cys introduced into the C-terminal extension. These 41 V with and without conjugation were tested in TGT experiments. H The activity of H polypeptide was determined. Table 11 lists the V with and without conjugation. HThe relative activity level of H polypeptide (derivative) is expressed as V H The maximum thrombin peak height of the H polypeptide derivatives is relative to the maximum thrombin peak height of emicizumab SIA. Therefore, based on the activity retained before and after conjugation, for example, using C3 as a reagent, the following 9 sites were determined to be optimal for introducing free Cys for conjugation with the extension: Cys introduced into the C-terminal extension (compounds #23 / 24), Q30C (compounds #25 / 26), V158C (compounds #27 / 28), P160C (compounds #29 / 30), G161C (compounds #31 / 32), G188C (compounds

[1078] #33 / 34), E190C (Compound #35 / 36), S209C (Compound #37 / 38) and S231C (Compound #39 / 40).

[1079] Table 11: V H TGT activity of H peptide derivatives and screening of conjugation sites in the extended part

[1080]

[1081]

[1082]

[1083] On both anti-FIX and anti-FX V H Different linkers L between H fragments recombinant fusion 1-2 Tests were performed to optimize the composition (see Table 12). Table 12 lists V H The relative activity level of H polypeptide is expressed as V H The maximum thrombin peak height of the H polypeptide is relative to the maximum thrombin peak height of emicizumab SIA. From these data, it can be seen that the V linker with a hydrophobic composition H H peptides generally showed the highest relative activity, while V peptides with acidic or hydrophilic linkers H The H polypeptide showed the lowest activity. V with a linker consisting of a mixture of QAPGQA, GQAPGQ or similar sequences H The H polypeptide exhibited moderate relative activity. The optimal linker composition preferably contains both hydrophilic and hydrophobic amino acids, so, for example, the chemical stability of the QAPGQA linker was further investigated using MS analysis, showing high stability under accelerated stress conditions at 37°C for two weeks in isotonic buffer and neutral pH.

[1084] Therefore, the ratio of hydrophobic amino acids to hydrophilic amino acids is preferably "40% to 60%" rather than "60% to 40%". H The H polypeptides all contain the same set of activity-enhancing CDR mutations, but contain a different set of pi-lowering mutations outside the CDRs.

[1085] Table 12: Different linkers L 1-2 Characteristics of V H TGT activity of H polypeptide sequence

[1086]

[1087] Example 9: Anti-FX / Anti-FIX(a)V H The H polypeptide derivative is formulated with SNAC and NAM in a dosage form suitable for oral administration

[1088] To prepare for oral studies using a liquid formulation containing sodium N-(8-[2-hydroxybenzoyl]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, and HEPES buffer was weighed to obtain a final concentration of 5 mM. The powder was transferred to a glass vial and MilliQ-H2O was added accordingly. The solution was stirred with a magnetic stirrer until SNAC, NAM, and magnesium stearate were dissolved. The pH was measured and adjusted to pH 8 with 2M NaOH. Thus, 2-5 mg / ml of purified and conjugated V H Batches of H polypeptide derivatives were formulated as liquids with a final concentration of 200 mg / ml SNAC and 1 M NAM.

[1089] To prepare for oral studies using tablets containing SNAC and NAM, the following procedure was followed. Purified V with or without fatty acid extender conjugation was administered. H The batches of H peptide (derivative) were buffer exchanged into MilliQ-H2O and the pH was adjusted to 8.0 using 0.1 M NaOH or 0.1 M formic acid. H The concentration of H polypeptide (derivative) was 0.5 to 4 mg / ml. V was dried using a micro spray dryer B290 (BUCHI). H H polypeptide (derivative) was spray dried, pump setting was 5-6, feed flow rate: 2 ml / min, inlet temperature: 80-85°C, outlet temperature: 45-50°C, nozzle: 1.5 mm, suction rate: 100%.

[1090] To prepare tablets, the powders were blended to obtain the desired composition. The following tablet formulations were prepared.

[1091] Preparation #1

[1092]

[1093] Formulation #2

[1094]

[1095] Formulation #3

[1096]

[1097]

[1098] The blended powders were weighed and punched into homogeneous tablets using a Kilian Style One (Romaco) with a punch setting that simulates a rotary press.

[1099] Example 10: Anti-FX / anti-FIX(a)V using mutations with different altered pIs H Oral and intravenous pharmacokinetics of H polypeptide derivatives in rats

[1100] The aim of this study was to investigate the anti-FX / FIX(a)V H Effects of pI-altering mutations in H polypeptide derivatives on pharmacokinetic parameters such as oral bioavailability.

[1101] Will be anti-FX / FIX(a)V H Liquid formulations of H polypeptide derivatives were administered intravenously (IV) (IV formulation: 20 mM Hepes, 150 mM NaCl, pH 7.4) or prepared as in Example 9 and administered by oral gavage to parallel groups of Sprague Dawley rats that were acclimated in the room for at least one week prior to the study and maintained in group cages with ad libitum access to standard food and water. Rats for oral dosing were fasted in grid-bottom cages around 2 p.m. the day before dosing but had free access to water (18 hours total). On the day of dosing, all rats were acclimated in the operant chamber for 30 minutes.

[1102] Blood samples were collected just before dosing and then at various time points after dosing. Animals dosed orally (PO) were kept fasting in grid-bottom cages until 4 hours after dosing. V was measured using a γ-Glu-based immunoassay. H Plasma concentrations of H polypeptide derivatives, including anti-V HH antibody (Novo Nordisk, Denmark) and anti-γ-Glu (Novo Nordisk, Denmark) against the extended part of the test molecule. Here, 96-well MaxiSorp plates (Nunc, 439454) were coated with 2 μg / ml anti-γ-Glu antibody, washed and blocked with PBS, 0.05% Tween 20, 1% BSA (pH 7.4). After the washing steps, compound specific calibrators (0, 2.7, 8.2, 24.7, 74, 222, 667, 2000 pM) in 1% rat EDTA plasma and rat EDTA plasma samples with a minimum dilution of 100 times were incubated on the plates, where V H The H peptide derivative was captured by the γ-Glu extension. After an additional washing step, the biotinylated internal V H H-specific antibody (0.5 nM) was added to the plate to form a sandwich ELISA. After the final wash step, horseradish peroxidase (HRP)-streptavidin was added to the plate as a detection reagent. The presence of V was detected by adding a chromogenic substrate (e.g., TMB (3,3',5,5'-tetramethylbenzidine)). H The amount of biotinylated antibody bound to the H polypeptide derivative is measured using a photometer (e.g., The optical density was measured using an M2 photometer (Molecular Devices). The response was proportional to the concentration of peroxidase, which in turn was proportional to V H Based on these exposure data, non-compartmental pharmacokinetic parameters were calculated using Phoenix WinNonlin or the open source statistical analysis software R ('NonCompart' package).

[1103] The following pi-lowering substitutions were introduced at surface exposed residues outside the CDRs:

[1104] T28D, K43Q, K65Q, N84D, R148Q, N159D, K172Q, K194Q, and N213D (based on the consecutive numbering using compound #4 as template), in different combinations (generating V H H peptide derivatives, compounds #59 to 64 and compound #5), were tested and compared with V H H polypeptide derivative compound #4 was used for comparison.

[1105] The following Tables 13 and 14 give the V H H derivatives and show the effect of lowering the pI on oral bioavailability.

[1106] Lowering the pi from approximately 8.36 to 5.85 increased the bioavailability 5 to 10 fold (see Table 13).

[1107] No effect of reduced pi on half-life was observed (see Table 14).

[1108]

[1109]

[1110] Example 11: Intravenous Pharmacokinetic Study in Rats: Anti-FX / Anti-FIX(a)V with Different Elongations H H polypeptide (derivative)

[1111] The purpose of this study was to investigate the effects of different elongated bodies on V H Effect of pharmacokinetic parameters such as half-life of H polypeptide derivatives.

[1112] The anti-FX / FIX(a)V prepared according to Example 9 was H Liquid formulations of H polypeptide derivatives were administered intravenously (IV) to parallel groups of Sprague Dawley rats that were acclimated in the room for at least one week before the study and kept in group cages with free access to standard food and water. Rats for oral administration were fasted in grid-bottom cages around 2 p.m. the day before dosing, but had free access to water (18 hours total). On the day of dosing, all rats were acclimated in the operating room for 30 minutes. Blood samples were collected just before dosing and then at multiple different time points after dosing. V was measured using an immunoassay based on a His tag or γ-Glu. H Plasma concentrations of H polypeptide (derivatives) using V H Anti-V H H antibody (Novo Nordisk) and anti-His tag antibody (R&D systems, MAB050) against the His tag fused to the test molecule or anti-γ-Glu (Novo Nordisk, Denmark) against the extended part of the test molecule. Both immunoassay setups had similar sensitivity, and when V H The latter was used when the H polypeptide compound did not have a His tag fused to the molecule. 96-well MaxiSorp plates (Nunc, 439454) were coated with either the His tag or anti-γ-Glu antibody, washed and blocked using PBS, 0.05% Tween 20, 1% BSA (pH 7.4). After the washing steps, derivative-specific calibrators (0, 2.7, 8.2, 24.7, 74, 222, 667, 2000 pM) in 1% rat EDTA plasma and rat EDTA plasma samples at a minimum dilution of 100 times were incubated on the plates, with V H The H peptide derivatives were captured by their His tag or γ-Glu extension. After an additional washing step, the biotinylated internal VH H-specific antibodies were added to the plate (0.5 nM) to form a sandwich ELISA. After the final wash step, horseradish peroxidase (HRP)-streptavidin was added to the plate as a detection reagent. The presence of V was detected by adding a chromogenic substrate (e.g., TMB (3,3',5,5'-tetramethylbenzidine)). H The amount of biotinylated antibody bound to the H polypeptide derivative is measured using a spectrometer (e.g.,

[1113] Optical density readings were measured using a Spectrophotometer (Molecular Devices). The response was proportional to the concentration of peroxidase, which in turn was proportional to V H Based on these exposure data, non-compartmental pharmacokinetic parameters were calculated using Phoenix WinNonlin or the open source statistical analysis software R ('NonCompart' package).

[1114] V with different extension types H The results for the H polypeptide derivatives are shown below in Table 15. The results show the effect of using different fatty acids and albumin binding peptides as extenders on the mean residence time terminal half-life (MRTHL).

[1115] For V with C18, C20 and tetrazole fatty acid side chain conjugation H H peptide derivatives (compound #66, compound #67 and compound #68: 3- to 4-fold increase), and V with albumin binding peptide extension fused H H peptide derivatives (compound #70, compound #71, compound #72 and compound #73: 1.9 to 3.6 times increase), an extended half-life was observed compared to the non-extended peptide, where the albumin binding peptide was used in combination with V H The 30-residue GlySer L fused to the C-terminus of the H polypeptide P The linker had the best effect. V with a short C12 fatty acid H The H peptide derivative compound #65 showed the lowest half-life. This suggests that in order to obtain V H For optimal prolongation of H polypeptide derivatives, it is preferred to use fatty acids longer than C12 (such as C16, C18 or C20) in order to extend the circulation half-life.

[1116]

[1117]

[1118] Example 12: Oral and intravenous pharmacokinetic studies in dogs: anti-FX / anti-FIX(a)V formulated with SNAC and NAM H H polypeptide (derivative)

[1119] The aim of this study was to investigate the effect of fatty acid prolongation on half-life and whether a reduction in pI results in enhanced oral bioavailability.

[1120] Will be anti-FX / FIX(a)V H The appropriate formulation of H polypeptide (derivative) was administered intravenously (IV) or prepared as a tablet and administered orally to groups of beagle dogs. Dogs were dosed in the morning after an overnight fast and remained fasting for 3-5 hours after a single dose. § In the present study, approximately 3 nmol / kg of glucagon was administered subcutaneously to dogs 10 minutes prior to oral administration. Blood samples were collected immediately prior to administration and subsequently at various time points after administration. V was measured using either a His-tag-based or a γ-Glu-linker-based immunoassay. H Plasma concentrations of H polypeptide, using V H H polypeptide anti-V H H antibody (Novo Nordisk, Denmark) and anti-His tag antibody (R&Dsystems, MAB050) against the His tag fused to the test molecule or anti-γ-Glu (Novo Nordisk, Denmark) against the extended part of the test molecule. Both immunoassay setups had similar sensitivity, and when V H The latter was used when the H polypeptide compound did not have a His tag fused to the molecule. 96-well MaxiSorp plates (Nunc, 439454) were coated with antibodies, washed and blocked using PBS, 0.05% Tween 20, 1% BSA (pH 7.4). After the wash step, compound specific calibrators (0, 2.7, 8.2, 24.7, 74, 222, 667, 2000 pM) in 1% canine EDTA plasma and canine EDTA plasma samples with a minimum dilution of 100 times were incubated on the plates, where V H H polypeptide compounds are expressed by their His tag or γ -Glu linker motif to capture. After an additional wash step, the biotinylated internal V H H-specific antibodies were added to the plate (0.5 nM) to form a sandwich ELISA. After the final wash step, horseradish peroxidase (HRP)-streptavidin was added to the plate as a detection reagent. The presence of V was detected by adding a chromogenic substrate (e.g., TMB (3,3',5,5'-tetramethylbenzidine)). H The amount of biotinylated antibody bound to the H polypeptide compound can be measured using a photometer (e.g., Optical density readings were measured using a M2 photometer (Molecular Devices). The response was proportional to the concentration of peroxidase, which in turn was proportional to V H Based on these exposure data, Phoenix WinNonlin or the open source statistical analysis software R ('NonCompart'

[1121] Calculate non-compartmental pharmacokinetic parameters.

[1122] § Subcutaneous administration of approximately 3 nmol / kg of glucagon is indicated 10 minutes prior to oral administration.

[1123] The following Tables 16 and 17 give the three C18 diacid conjugated V H H peptide derivatives and confirmed

[1124] a) the effect of fatty acid extension on half-life, and

[1125] b) Reduction in pi leads to enhanced oral bioavailability.

[1126] Therefore, compared with the non-extended compound, when V H When the H polypeptide derivatives were conjugated to the indicated fatty acids, the half-life extension observed in dogs ranged from 370 to 710 fold. Compound #6, which had two C16 diacid fatty acid conjugations in the C-terminal linker, showed the highest half-life extension of approximately 7.6 days.

[1127] In dogs, the SNAC:NAM formulation increased oral bioavailability by 2- to 8-fold compared to the SNAC alone formulation. Thus, the addition of NAM to the oral formulation had a significant effect, increasing oral bioavailability to and beyond clinically meaningful levels of approximately 0.1%.

[1128] Taking into account the efficacy, average residence time and oral bioavailability, it is expected that the V disclosed herein H The H polypeptide derivatives are useful for the treatment of hemophilia A and acquired hemophilia A with or without inhibitors when administered orally.

[1129]

[1130] Example 13: Intravenous Pharmacokinetic Study in Pigs: C18-Conjugated Anti-FX / Anti-FIX(a)V H H polypeptide (derivative)

[1131] The aim of this study was to investigate the potential of V with and without a C18 diacid extender group. HPharmacokinetic effects of H polypeptide (derivatives).

[1132] Will be anti-FX / FIX(a)V H Appropriate formulations of VHH polypeptides (derivatives) were administered intravenously (IV) and subcutaneously (SC) to miniature pigs (Sus scrofa domesticus). Blood samples were collected just before administration and then at various time points after administration. Plasma concentrations of VHH polypeptides (derivatives) were measured using His-tag-based or γ-Glu-based immunoassays, wherein the VHH polypeptides (derivatives) were measured using a His-tag-based or γ-Glu-based immunoassay. H Anti-V H H antibody (Novo Nordisk) and anti-His tag antibody (R&D systems, MAB050) against the His tag fused to the test molecule or anti-γ-Glu (Novo Nordisk, Denmark) against the extended part of the test molecule. Both immunoassay setups had similar sensitivity, and when V H The latter was used when the H polypeptide compound did not have a His tag fused to the molecule. 96-well MaxiSorp plates (Nunc, 439454) were coated with either the His tag or anti-γ-Glu antibody, washed and blocked using PBS, 0.05% Tween 20, 1% BSA (pH 7.4). After the washing steps, derivative-specific calibrators (0, 2.7, 8.2, 24.7, 74, 222, 667, 2000 pM) in 1% porcine EDTA plasma and porcine EDTA plasma samples at a minimum dilution of 100-fold were incubated on the plates, with V H The H peptide derivatives were captured by their His tag or γ-Glu extension. After an additional washing step, the biotinylated internal V H H-specific antibodies were added to the plate (0.5 nM) to form a sandwich ELISA. After the final wash step, horseradish peroxidase (HRP)-streptavidin was added to the plate as a detection reagent. The presence of V was detected by adding a chromogenic substrate (e.g., TMB (3,3',5,5'-tetramethylbenzidine)). H The amount of biotinylated antibody bound to the H polypeptide derivative is measured using a photometer (e.g., Optical density readings were measured using a M2 photometer (Molecular Devices). The response was proportional to the concentration of peroxidase, which in turn was proportional to V H The non-compartmental pharmacokinetic parameters were calculated using the open source statistical analysis software R ('NonCompart' package). The following Table 18 shows the V with and without fatty acid extension. HThe results for the H polypeptide confirm the effect of conjugation of fatty acid extenders for half-life extension on half-life, as observed in Examples 11, 12 and 13.

[1133] Therefore, with the non-extended V H H compounds compared to when V H When the H polypeptide was conjugated to the fatty acid C18 diacid extension, an approximately 15- to 20-fold increase in half-life was observed in pigs.

[1134]

[1135] Example 14: Pharmacodynamic effects of optimized anti-FX / anti-FIX(a) polypeptide derivatives in the mouse tail vein transection (TVT) bleeding model

[1136] In vivo efficacy was tested in the HA mouse moderate bleeding model. In the TVT model, it has been previously shown that FVIII administration reduces bleeding to the same level as observed in wild-type mice. To overcome the problem of mice lacking cross-species reactivity of the test compounds, HA mice were supplemented with human FIX and FX prior to the bleeding experiments. At the end of the bleeding period, total blood loss was determined by spectrophotometric hemoglobin measurement. Anti-V H H (Novo Nordisk, Denmark) and anti-γGlu-linker antibody (Novo Nordisk, Denmark) and anti-FIX antibodies (LS-B7226, LSBio and FIX-2F24, in-house clones) were used to quantify the indicated V by luminescent oxygen channeling assay H Plasma levels of H polypeptide derivatives and FIX were measured. FX levels were quantified by commercial FXELISA (KSP134, Nordic Biosite). H Dose response studies of H peptide derivatives, EC was determined by fitting the data to a three-parameter antilog (dose) response equation with a shared plateau value (>0) 50 Automatic outlier elimination with a 1% ROUT coefficient was applied and sums of squares were weighted by (blood loss) using GraphPad Prism software (version 9.0.1).

[1137] Dose response studies showed significant reduction in blood loss, with the tested V H The H polypeptide derivatives achieved the levels of blood loss observed in wild-type mice, as shown in Table 19. The EC 50 The effective dose range was estimated, indicating high potency, as 50% of the effect was observed at low nmol / kg doses for all three compounds tested.

[1138] Table 19: Effects of anti-FX / anti-FIX(a) peptide derivatives in the TVT hemorrhage mouse model

[1139]

[1140] Table 20: EC of anti-FX / anti-FIX(a) peptide derivatives according to the TVT bleeding mouse model 50 estimate

[1141]

[1142] Example 15: In vitro activity assay of optimized anti-FX / anti-FIX(a) VHH polypeptide derivatives after oral administration

[1143] Following a pharmacokinetic (PK) study in wild-type dogs to characterize PO dosing of Compound #6 according to Example 12, chromogenic activity was determined at two separate time points, 30 minutes and 90 minutes post-dose. The measured chromogenic activity of simulated FVIII was compared to the plasma exposure of Compound #6 to determine the level of active FVIII in serum following PO dosing. H H fraction of peptide derivatives.

[1144] The chromogenic activity of compound #6 was determined by analyzing canine serum samples using a commercial FVIII chromogenic activity assay (FVIII:C, Hyphen Biomed, France). For the calibration curve, compound #6 was spiked into 10% canine serum and serum samples were analyzed at 10-fold dilution using the same assay. For the 10-fold dilution, the final activity results were corrected by 10-fold. H H (Novo Nordisk, Denmark) and anti-γGlu-linker antibody (Novo Nordisk, Denmark), and plasma exposure levels of compound #6 were quantified by luminescent oxygen channeling (LOCI) assay. The data are summarized in Table 21. FVIII:C chromogenic activity was detectable in serum at 30 and 90 minutes after dosing, and quantification of compound #6 based on chromogenic activity was similar to estimates based on plasma exposure measurements of compound #6 using the LOCI assay. Any differences in quantification of compound #6 are expected to be caused by differences in assay sensitivity. The data show that the PO administration of V H The H polypeptide derivative was fully active after oral ingestion in dogs, demonstrating the concept of preparing an orally available FVIII mimetic drug to treat patients with, for example, hemophilia A.

[1145] Table 21: In vitro activity of anti-FX / anti-FIX(a) polypeptide derivatives after PO administration

[1146] Example 16: Crystallization and paratope / epitope mapping of anti-FXVHH fragment compound #76

[1147] The purpose of this study was to determine the VHH-1.15 H Paratope and epitope residues of the H fragment.

[1148] crystallization

[1149] V produced using CHO was mixed with a synthetic peptide N-terminal-NPFDLLD-C-terminal (corresponding to the activation peptide sequence aa31-37 of human FX (purchased from Apigenex) identified in Example 4) at a 1:4 molar ratio. H Crystals of H fragment compound #76 were grown using the drop vapor diffusion technique. 250 nl of a 5.75 mg / ml protein solution of the complex in 20 mM 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 triammonium citrate, 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 diammonium tartrate) pH 5.0, 12% (w / v) polyethylene glycol 3350 (as a precipitant) and incubated on 80 μl of the precipitant. After incubation at 18°C ​​for 5 days, the crystallization plate was transferred to 5°C. Crystals appeared within three months.

[1150] Diffraction data collection

[1151] Crystals were cryoprotected in a precipitant with 20% ethylene glycol before rapid cooling in liquid nitrogen. The images were acquired at the Swiss Light Source beamline X10SA ( Diffraction data were collected at 100 K at 400 nm (100 nm wavelength). Automatic indexing, integration and scaling of the data were performed using programs from the XDS package (the diffraction data statistics are summarized in Table 22).

[1152] Structure determination and refinement

[1153] The asymmetric unit contains two compounds #76: FX AP 31-37 complex. HThe previously determined crystal structure of H was used as a search model and the structure was determined by molecular replacement using Phaser implemented in the program suite Phenix, see Example 6. The correct amino acid sequence of compound #76 was introduced and FX AP31-37 was manually modeled in the difference electron density map, all using COOT. During several rounds of refinement in Phenix and manual reconstruction in COOT, O-glycosylation on threonine residue 117 and sulfation of tyrosine residue 109 were observed and modeled. Refinement statistics are shown in Table 22.

[1154] Table 22: Data collection and refinement statistics

[1155]

[1156]

[1157] Statistics for the highest resolution shell are shown in parentheses.

[1158] Determination of epitopes and paratopes

[1159] Synthetic peptide epitopes—defined as FX activating peptide residues with the following characteristics: H Heavy atoms in H peptide compound #76 The peptide has heavy atoms (ie, non-hydrogen atoms) within a distance of , and includes the following residues from the synthetic peptide: N173, P174, F175, L177, L178, and D179, based on consecutive numbering according to the corresponding FX activating peptide sequence (SEQ ID NO: 2).

[1160] V H The paratope of H fragment compound #76 is defined as the residue of compound #76 that has the following characteristics: , having heavy atoms (i.e., non-hydrogen atoms) within a distance of , comprising the following residues from compound #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) (numbered consecutively).

[1161] Example 17: Crystallization and paratope / epitope mapping of anti-FX VHH fragment compound #77

[1162] The purpose of this study was to identify compound #77 (V H H1.13)V H Paratope and epitope residues of the H fragment.

[1163] crystallization

[1164] V produced from CHO cells was mixed with a synthetic peptide N-terminal-NPFDLLD-C-terminal corresponding to the activation peptide sequence aa31-37 of human FX (synthesized by Apigenex) at a 1:4 molar ratio. H Crystals of H fragment compound #77 were grown using the drop vapor diffusion technique. 250 nl of a 6.1 mg / ml protein solution of the complex in 20 mM 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 triammonium citrate, 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 diammonium tartrate) pH 4.0, 11% (w / v) PEG 3350 (as a precipitant) and incubated on 80 μl of the precipitant. Crystals appeared after incubation for 4 days at 5°C.

[1165] Diffraction data collection

[1166] Crystals were cryoprotected in a precipitant with 20% ethylene glycol before rapid cooling in liquid nitrogen. The images were acquired at the Swiss Light Source beamline X10SA ( Diffraction data were collected at 100 K at 400 nm (100 nm wavelength). Automatic indexing, integration and scaling of the data were performed using programs from the XDS package (the diffraction data statistics are summarized in Table 23).

[1167] Structure determination and refinement

[1168] The asymmetric unit contains two compound #77: FX AP 31-37 complexes. The structure was determined by molecular replacement using Phaser implemented in the program suite Phenix, using the complete asymmetric unit from the crystal structure of compound #76: FX AP 31-37 in Example 16 as a search model. The correct amino acid sequence of compound #77 was introduced and several rounds of refinement in Phenix and manual reconstruction in COOT were applied. Refinement statistics are shown in Table 23.

[1169] Table 23: Data collection and refinement statistics

[1170]

[1171]

[1172]

[1173] Statistics for the highest resolution shell are shown in parentheses.

[1174] Determination of epitopes and paratopes

[1175] Synthetic peptide epitopes—defined as FX activating peptide residues with the following characteristics: H Heavy Atoms in H Fragment Compound #77 The peptide has heavy atoms (ie, non-hydrogen atoms) within a distance of , and includes the following residues from the synthetic peptide: N173, P174, F175, L177, L178, and D179, based on consecutive numbering according to the corresponding FX activating peptide sequence (SEQ ID NO: 2).

[1176] V H The paratope of H fragment compound #77 is defined as the residue of compound #77 that has the following characteristics: The residues from compound #77 have heavy atoms (i.e., non-hydrogen atoms) within a distance of :

[1177] A33, M34, G35, W47, V48, A49, A50, I51, S52, S57, T58, N59, Y60, A61, A97, A98, D99, G105, L107, Y109 (SEQ ID NO: 735) (numbered consecutively).

[1178] Example 18: Bispecific V H Steady-state FXa generation kinetics of H-peptide derivatives

[1179] The aim of this study was to establish an anti-FIXa / FX bispecific H The procoagulant activity of the H polypeptide derivatives was determined by their ability to promote the activation of FX by FIXa in the presence of procoagulant phospholipid membranes. The compounds tested are listed in Table 24 and emicizumab SIA was included for comparison.

[1180] The steady-state FXa-generating activity of each compound was reported as the Michaelis-Menten kinetic parameter (K) at a given compound concentration. M ) and the first-order rate constant (k cat / K M)). The compounds with the final assay concentrations shown in Table 24 were pre-incubated for 5 min with 0.1 nM human plasma-derived FIXa (Haematologic Technologies Inc, USA) and 20 μM 25:75 phosphatidylserine: phosphatidylcholine phospholipid vesicles (Haematologic Technologies Inc, USA) in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w / v) PEG8000, pH 7.3+1 mg / ml BSA). Activation was then initiated by adding a two-fold dilution series of human plasma-derived FX (Haematologic Technologies Inc, USA) starting from 500 nM. After activation for 7 minutes at room temperature, the reaction (50 μl) was quenched by adding 25 μl quenching 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 adding 25 μl 2mMS-2765 chromogenic substrate (Chromogenix, Sweden) and measuring the chromogenic substrate conversion by measuring the absorbance (Δ OD / min) at 405nm in a microplate reader. Similarly, the activation of FX by free FIXa was determined with a FIXa concentration of 10 nM and a reaction time of 5 to 10 minutes. In order to convert the absorbance measured at 405nm into the corresponding FXa concentration, 25 μl quenching buffer and 25 μl 2mM S-2765 chromogenic substrate were added to the FXa standard curve of 0-5 nM (50 μl), and the absorbance at 405nm was then recorded as described above. The linear regression of Δ OD / min as a function of FXa concentration produces a slope that can be used to convert the measured Δ OD / min into FXa generation rate:

[1181] FXa generation rate = (ΔOD / min) / (slope FXa标准曲线 * Reaction time)

[1182] Michaelis-Menten steady-state kinetic parameters were determined by fitting the rate of FXa generation as a function of FX (substrate) concentration to the following equation:

[1183] FXa generation rate = (k cat *[FIXa] t *[FX] t ) / (K M +[FX] t ) where k cat is the enzyme efficiency (min -1 ), [FIXa] tis 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 test compound and concentration tested.

[1184] Table 24: Determined steady-state enzyme kinetic constants

[1185]

[1186]

[1187] in conclusion

[1188] The anti-FIXa / FX bispecific V H When the H peptide derivatives were supplemented to a final concentration of 5 nM, the K M From 281.5nM to between 3.9-9.0nM, and further increased the catalytic efficiency from 0.00012min -1 nM -1 Increased to 0.14-0.33min -1 nM -1 In contrast, 300 nM of emicizumab-SIA reduced K M Reduced to 53.5nM.

[1189] Reduced K M The value reflects the improvement in the assembly (spatial arrangement) of FIX(a) / FX, resulting in a higher V H Significant potency increase of H polypeptide derivatives. All compounds were tested at expected pharmacologically relevant concentrations.

Claims

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 an activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof, at least one extension, Optionally, a linker (L 1-2 ),as well as Optionally, one or more extensions (E), wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof, the epitope comprising at least one of amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively), and Wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2), the epitope comprising at least one of amino acid residues N173, P174, F175, L177 and L178 (numbered consecutively).

2. The ISVD polypeptide derivative according to claim 1, wherein the first ISVD is capable of binding to an epitope on Factor IX (SEQ ID NO: 1) or an activated form thereof, the epitope comprising amino acid residues E224, T225, G226, V250, 1251, R252, 1253, P255, H257 and N260 (numbered consecutively), and Wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2), the epitope comprising amino acid residues N173, P174, F175, L177 and L178 (numbered consecutively).

3. The ISVD polypeptide derivative according to claim 1 or 2, wherein the second ISVD is capable of binding to an epitope on Factor X (SEQ ID NO: 2) comprising amino acid residues N173, P174, F175, L177, L178 and D179 (numbered consecutively).

4. The ISVD polypeptide derivative of any one of claims 1 to 3, 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 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) (Serial numbering).

5. The ISVD polypeptide derivative according to any one of claims 1 to 4, wherein the at least one prolonging moiety is attached to a surface exposed amino acid residue.

6. The ISVD polypeptide derivative according to any one of claims 1 to 5, wherein the surface exposed residues are not residues in the CDR regions.

7. The ISVD polypeptide derivative according to any one of claims 1 to 6, having the following formula (N-terminus to C-terminus): ISVD2–L 1-2 –ISVD1–E wherein the two extensions are attached to one or more surface exposed amino acid residues on E, and The molecular weight of the ISVD polypeptide derivative is in the range of 20-35 kDa.

8. The ISVD polypeptide derivative according to any one of claims 1 to 7, Wherein the 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 or three amino acid substitutions, CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 174), optionally comprising one, two or three amino acid substitutions (Kabat definition), and Wherein the second ISVD comprises (A) CDR1: RYAMG (SEQ ID NO: 152), optionally comprising one or two amino acid substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 153), optionally comprising one, two or three amino acid substitutions, CDR3: DDSVGDGYLDY (SEQ ID NO: 154), optionally comprising one, two or three amino acid substitutions (Kabat definition); or (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 amino acid substitutions, CDR3: DDSVGDGYLDY (SEQ ID NO: 130), optionally comprising one, two or three amino acid substitutions (Kabat definition); or (C) CDR1: RYAMG (SEQ ID NO: 32), optionally comprising one or two amino acid substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 33), optionally comprising one, two or three amino acid substitutions, CDR3: DYSSGDGYLDY (SEQ ID NO: 34), optionally comprising one, two or three amino acid substitutions (Kabat definition); or (D) CDR1: RYAMG (SEQ ID NO: 40), optionally comprising one or two amino acid substitutions, CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 41), optionally comprising one, two or three amino acid substitutions, CDR3: DDSSGDGYLDY (SEQ ID NO: 42), optionally comprising one, two or three amino acid substitutions (Kabat definition).

9. The ISVD polypeptide derivative according to claim 8, wherein the substitution is a conservative substitution.

10. 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 an activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof, at least one extension attached to a surface exposed amino acid residue, Optionally, a linker (L 1-2 ),as well as Optionally, one or more extensions (E), The first ISVD comprises the following sequence 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), 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 the second ISVD comprises the sequence V H H-1.20(SEQ ID NO:167), V H H-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), V H H-1.12(SEQ ID NO:103), V H H-1.3 (SEQ ID NO: 31), or V H H-1.4(SEQ ID NO:39)。 11. The ISVD polypeptide derivative according to any one of the preceding claims, wherein The first ISVD includes V H H-2.15 (SEQ ID NO: 131), and wherein the second ISVD comprises V H The sequence of H-1.15 (SEQ ID NO: 127).

12. The ISVD polypeptide derivative according to claim 10 or 11, which has the following formula (N-terminus to C-terminus): ISVD2–L 1-2 –ISVD1–E wherein the two extensions are attached to one or more surface exposed amino acid residues on E, and The molecular weight of the ISVD polypeptide derivative is in the range of 20-35 kDa.

13. The ISVD polypeptide derivative according to any one of claims 10 to 12, wherein The ISVD polypeptide derivative comprises SEQ ID NO: 629, and in The first extension portion comprises the following structure: And wherein the second extension portion comprises the following structure wherein '*' is Cys at position 257 of SEQ ID NO:629, and wherein '**' is Cys at position 259 of SEQ ID NO:

629.

14. The ISVD polypeptide derivative according to any one of the preceding claims, wherein the ISVD polypeptide derivative is V H H polypeptide derivatives.

15. Procoagulant V H H polypeptide derivative, which comprises A first V capable of binding to factor IX (SEQ ID NO: 1) or an activated form thereof H H, A second V capable of binding to factor X (SEQ ID NO: 2) or an activated form thereof H H, Connect the first V H H and the second V H H linker (L 1-2 ),as well as having a C-terminal extension (E) of SEQ ID NO: 9, The V H The H polypeptide derivative has the following formula (N-terminus to C-terminus): "Second V H H”–L 1-2 - "First V H H”–E It further comprises first and second extensions attached to E, in 1) The first V H H contains CDR1: IYTMS (SEQ ID NO:172), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:173), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 174) (Kabat definition), and The second V H H contains CDR1: RYAMG (SEQ ID NO:152), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:153), CDR3: DDSVGDGYLDY (SEQ ID NO: 154) (Kabat definition); or 2) The first V H H contains CDR1: IYTMS (SEQ ID NO:132), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:133), CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 134) (Kabat definition), and The second V H H contains CDR1: RLAMG (SEQ ID NO:128), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO:129), CDR3: DDSVGDGYLDY (SEQ ID NO: 130) (Kabat definition), The first extension portion comprises the following structure: And wherein the second extension portion comprises the following structure wherein '*' is Cys (C) at position 257 of SEQ ID NO: 629, and wherein '**' is Cys (C) at position 259 of SEQ ID NO:

629.

16. V according to claim 15 H H polypeptide derivative, wherein the V H The H polypeptide derivative comprises SEQ ID NO:

629.

17. An ISVD polypeptide derivative or V according to any one of the preceding claims. H H polypeptide derivative, wherein the ISVD polypeptide derivative or V H The H polypeptide derivative is a bispecific ISVD polypeptide derivative or V H H polypeptide derivatives.

18. A pharmaceutical composition comprising an ISVD polypeptide derivative or V according to any one of the preceding claims. H H polypeptide derivative, and one or more pharmaceutically acceptable excipients.

19. The pharmaceutical composition of claim 18, wherein the composition comprises a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid.

20. The pharmaceutical composition of claim 19, wherein the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC), and wherein the composition further comprises nicotinamide (NAM).

21. An ISVD polypeptide derivative according to any one of the preceding claims, V H H polypeptide derivative or composition for use in treating hemophilia A or acquired hemophilia A with or without inhibitors.

22. An ISVD polypeptide derivative for use according to any one of the preceding claims, V H H polypeptide derivative or composition, wherein the polypeptide derivative or composition is administered orally.

23. A V comprising a CDR selected from the group consisting of H H fragment: CDR1: IYTMS (SEQ ID NO:172), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:173), and CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 174) (Kabat definition); CDR1: RYAMG (SEQ ID NO:152), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 153), and CDR3: DDSVGDGYLDY (SEQ ID NO: 154) (Kabat definition); CDR1: IYTMS (SEQ ID NO:132), CDR2: GLRWTDSSTEYADSVKG (SEQ ID NO:133), and CDR3: DRSFLFAQALGATKNYEY (SEQ ID NO: 134) (Kabat definition); and CDR1: RLAMG (SEQ ID NO:128), CDR2: AISRRGGSTNYADSVKG (SEQ ID NO: 129), and CDR3: DDSVGDGYLDY (SEQ ID NO: 130) (Kabat definition); or V selected from the following group H H fragment: V H H-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), V H H-1.12(SEQ ID NO:103), V H H-1.3(SEQ ID NO:31), V H H-1.4(SEQ ID NO:39), 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), V H H-2.14(SEQ ID NO:123), V H H-2.12(SEQ ID NO:107), V H H-2.2 (SEQ ID NO: 35), and V H H-1.20(SEQ ID NO:167); It is used as an intermediate for preparing V that can bind to FIX (SEQ ID NO: 1) or its activated form (FIXa) and can bind to FX (SEQ ID NO: 2). H H polypeptide or V H H polypeptide derivatives.

24. A method for improving the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative, wherein the ISVD polypeptide or ISVD polypeptide derivative comprises a first ISVD (ISVD1) capable of binding to Factor IX (SEQ ID NO: 1) or an activated form thereof, a second ISVD (ISVD2) capable of binding to Factor X (SEQ ID NO: 2) or an activated form thereof, Extension part, Optionally, a linker capable of connecting ISVD1 and ISVD2 ("L 1-2 "),as well as Optionally, one or more extensions ("E") The method comprises the following steps a. Modification of the amino acid residues encoding the ISVD polypeptide or ISVD polypeptide derivative Acid, so that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is reduced, b. culturing the host cell 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, d. purifying said ISVD polypeptide or ISVD polypeptide derivative from said host cell culture using standard chromatography methods, and e. Attaching a prolongation moiety to the ISVD polypeptide, unless such a moiety is already present.

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