A highly potent ISVD compound that can replace FVIII(a)

Abstract

The problem that the present invention aims to solve is to reduce the treatment burden in the treatment of blood coagulation disorders and to provide an improved compound that can be used as an alternative to FVIII(a) for use in the treatment of blood coagulation disorders such as hemophilia A and related diseases. [Solution] The present invention relates to an ISVD polypeptide derivative capable of binding coagulation factor IX(a) and factor X(a), which is highly potent and provides a sufficiently long half-life to enable effective subcutaneous and oral administration.

Description

[Technical Field]

[0001] The present invention relates to compounds that can bind to coagulation factor IX(a) and factor X(a), and to their use in the treatment of blood coagulation disorders, including various forms of hemophilia, including hemophilia A.

[0002] Inclusion by referencing the sequence list This application is submitted together with an electronic sequence listing. The entire contents of the sequence listing are incorporated herein by reference. [Background technology]

[0003] In patients with blood clotting disorders, such as those with hemophilia A (HA) and B (HB), various steps in the coagulation cascade become dysfunctional, for example, due to the absence or insufficient presence of functional clotting factors. Such dysfunction of parts of the coagulation cascade can lead to inadequate blood clotting and potentially life-threatening bleeding or damage to internal organs such as joints.

[0004] Hemophilia A, commonly known as factor VIII (FVIII) deficiency, is a congenital bleeding disorder affecting approximately 420,000 people worldwide, of which about 105,000 are currently diagnosed. Hemophilia A has three grades of severity defined by plasma factor VIII levels of 5–<40%, ranging from less than 1% (severe), 2–5% (moderate), and 6–30% (mild) ([Non-Patent Literature 1]), or according to the WFH's "Guidelines for the management of haemophilia" 2nd edition Haemophilia; Epub 6 JUL 2012. Bleeding can occur spontaneously or after trauma. About half of patients with hemophilia A are classified as having severe hemophilia A, experiencing severe bleeding beginning in early childhood and frequent episodes of spontaneous or excessive bleeding later in life. Bleeding typically occurs within the joints and muscles, and if not treated properly, recurrent bleeding can lead to irreversible hemophilic arthropathy ([Non-Patent Literature 2]).

[0005] Patients with hemophilia A may receive coagulation factor replacement therapy, such as exogenous FVIII. Conventional treatment consists of replacement therapy, provided as a preventive measure or as needed for bleeding episodes. Until recently, prophylactic measures for patients with severe hemophilia A included intravenous injections of plasma-derived FVIII, recombinant FVIII, or their long-acting variants, up to three times per week.

[0006] However, these patients are at risk of developing neutralizing antibodies, so-called inhibitors, against these exogenous factors, rendering previously effective therapies ineffective. Hemophilia A patients with inhibitors represent a non-limited example of a blood coagulation disorder that is partly congenital and partly acquired. Patients who develop inhibitors against FVIII cannot be treated with conventional replacement therapy. Exogenous coagulation factors may only be administered intravenously, which is considerably inconvenient and uncomfortable for the patient.

[0007] Reduced or absent FVIII activity, resulting in inadequate FXa formation and decreased thrombin production, is the underlying reason for bleeding diathesis in hemophilia A patients.

[0008] The proteolytic conversion of FX to its enzymatically active form, FXa, can be achieved by a unique FX activation complex containing FIXa and its cofactor-active form, FVIII (FVIIIa). Cofactor binding is thought to increase the enzymatic activity of FIXa by approximately five orders of magnitude and occurs through multiple mechanisms, as outlined in [Non-Patent Literature 3]. In particular, FVIIIa has been shown to stabilize the structure of FIXa, which has increased proteolytic activity against FX ([Non-Patent Literature 4], [Non-Patent Literature 5]).

[0009] In recent years, emicizumab (HEMLIBRA®) (also known as ACE910) has been approved for subcutaneous prophylaxis of hemophilia A with or without inhibitors of conventional replacement therapies against factors. Emicizumab is a humanized bispecific anti-full-length FIX(a) / anti-FX(a) monoclonal antibody developed by Chugai Pharmaceuticals / Roche Pharmaceuticals for the treatment of hemophilia A. Emicizumab is designed to mimic the function of FVIII cofactor (see Non-Patent Literature 6 and Patent Literature 1). Treatment with 30-50 μg of emicizumab per milliliter of plasma is estimated to correspond to at least 10-15 IU of equivalent factor VIII activity per deciliter of plasma (Non-Patent Literature 7). However, some patients develop inhibitors (anti-drug antibodies) against emicizumab, and treatment with this compound is ineffective.

[0010] In addition to the generation of inhibitors, as exemplified by emicizumab, other antibody properties are also important for achieving effective antibody-based therapy for patients. In particular, antibodies with a high tendency towards nonspecific binding have been shown to lead to clinic safety issues. Some reports have shown that high levels of nonspecific binding can reduce the circulating half-life of antibodies by several times, resulting in ineffective and cumbersome drug regimens for patients (see Non-Patent Literature 8 and Non-Patent Literature 9).

[0011] [Patent Document 2], [Patent Document 3] ([Patent Document 4]), [Patent Document 5], and [Patent Document 6] disclose anti-FIX(a) / anti-FX(a) bispecific full-length antibodies and their use as blood coagulation promoters for use in the treatment of hemophilia by subcutaneous administration. One such bispecific antibody is designated Mim8 (see [Non-Patent Document 10] and [Patent Document 7]).

[0012] However, within the hemophilia community, particularly among those with blood clotting disorders, there are still many crucial unmet medical needs, especially a need for reduced treatment burden and improved compounds that can replace FVIII(a) for use in the treatment of blood clotting disorders such as hemophilia A and related diseases. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] International Publication No. 2012 / 067176 [Patent Document 2] International Publication No. 2018 / 141863 [Patent Document 3] International Publication No. 2019 / 065795 [Patent Document 4] U.S. Patent No. 10759870 [Patent Document 5] International Publication No. 2020 / 025672 [Patent Document 6] International Publication No. 2021 / 152066 [Patent Document 7] International Publication No. 2020 / 025672 [Non-patent literature]

[0014] [Non-Patent Document 1] White et al.(2001)Thromb.Haemost.85:560 [Non-Patent Document 2] Manco-Johnson et al. (2007) N. Engl. J. Med. 357:535-44 [Non-Patent Document 3] Scheiflinger et al. (2008) J Thromb Haemost,6:315-322 [Non-Patent Document 4] Kolkman JA, Mertens K (2000)Biochemistry,39:7398-7405 [Non-Patent Document 5] Zogg T,Brandstetter H (2009)Biol Chem,390:391-400 [Non-Patent Document 6] Sampei et al.(2013)PLoS One,8,e57479 [Non-Patent Document 7] Shima et al.,N Engl J Med 2016;374:2044-53 [Non-Patent Document 8] Dobson et al.,Nature,volume 6,art.no.:38644 (2016) [Non-Patent Document 9] Avery et al.,MAbs 2018,Vol.10,No.2,244-255 [Non-Patent Document 10] Ostergaard H et al.Blood.2021;138:1258-68 [Overview of the project]

[0015] The present invention provides a highly potent and effective method for subcutaneous and oral administration, and offers a sufficiently long half-life to bind coagulation factor IX(a) and factor X(a), V H The present invention provides a single variable domain (ISVD) polypeptide derivative of a blood coagulation-promoting immunoglobulin, such as an H polypeptide derivative. Accordingly, in one embodiment, the present invention provides a first ISVD (ISVD1) that can bind to factor IX (SEQ ID NO: 1) or its activated form, a second ISVD (ISVD2) that can bind to factor X (SEQ ID NO: 2) or its activated form, one or more protraction moieties (ies) bound to one or more surface-exposed residues, and optionally a linker (L) that links ISVD1 and ISVD2. 1-2The present invention relates to a single variable domain (ISVD) polypeptide derivative of a blood coagulation-promoting immunoglobulin, comprising ) and, optionally, one or more extensions (E, extension(s)).

[0016] In one embodiment, the present invention comprises a first ISVD (ISVD1) capable of binding to factor IX (SEQ ID NO: 1) or its active form, a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or its active form, at least one extension portion bound to a surface-exposed residue, and optionally a linker (L) connecting ISVD1 and ISVD2. 1-2 The present invention relates to a blood coagulation-promoting ISVD polypeptide derivative comprising a first ISVD and optionally one or more extensions (E), wherein the first ISVD can bind to an epitope on factor IX (SEQ ID NO: 1) or its active form, which comprises at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering), and the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) which comprises at least one of the amino acid residues N173, P174, F175, L177, L178, and D179 (sequential numbering).

[0017] ISVD polypeptide derivatives include, for example, V H It may be an H polypeptide derivative.

[0018] Another aspect of the present invention relates to pharmaceutical compositions comprising ISVD polypeptide derivatives disclosed herein. Another aspect of the present invention relates to the use of ISVD polypeptide derivatives and compositions disclosed herein for the treatment of various forms of hemophilia by various routes of administration, including subcutaneous and oral administration, and in particular for the treatment of hemophilia A, hemophilia A in the presence of inhibitors, and acquired hemophilia A.

[0019] In a further embodiment, the present invention relates to a specific anti-FIX(a)V H H fragment or its specific anti-FX(a)V HISVD polypeptide derivatives such as H fragments or V H Individual components (intermediates) that are part of an H polypeptide derivative, ISVD or V H Regarding H fragments.

[0020] Further aspects of the present invention relate to the production of components (intermediates) of the compounds disclosed herein, including a method for modifying the isoelectric point of such ISVD polypeptide derivatives to which FIX(a) and FX(a) can be attached in order to improve the oral bioavailability of the polypeptide derivatives. [Brief explanation of the drawing]

[0021] [Figure 1a-h] Figures 1a-h show non-limiting examples of anti-FX(a) / FIX(a)ISVD polypeptide derivatives. E: extension, PM: extension portion, L1-2: linker connecting ISVD1 and ISVD2. Dashed lines indicate disulfide bonds. [Figure 2a] Figure 2a shows a non-limiting example of an anti-FX / FIX(a)VHH polypeptide derivative containing an extension based on a single C18 diacid fatty acid conjugated to the C-terminal extension by an extension partial linker (LP), ID:LP1. Dashed lines indicate disulfide bonds. [Figure 2b] Figure 2b shows a non-limiting example of an anti-FX / FIX(a)VHH polypeptide derivative containing an extension based on a single tetrazole, conjugated to the C-terminal extension by an extension partial linker (LP), ID:LP2. Dashed lines indicate disulfide bonds. [Figure 3a-f]Figures 3a - f show detailed illustrative descriptions of the compounds of compound number 20 (a), compound number 18 (b), compound number 15 (c), compound number 13 (d), compound number 14 (e), and compound number 12 (f), which are non - limiting examples of anti - FX / FIX(a) VHH polypeptide derivatives containing an extension part based on two C16 diacid fatty acids conjugated to the C - terminal extension (E) by an extension part linker (LP), ID LP1. The dashed lines indicate disulfide bonds. The extension part (E) (SEQ ID NO: 9) was used for these VHH polypeptide derivatives. The extension part contains two cysteine residues at positions 4 and 6 respectively, which serve as the binding points for the extension part (PM). a) In the sequence of VHH1.20~L1 - 2~VHH2.20~E, the polypeptide is represented by SEQ ID NO: 634 (compound number 20). b) In the sequence of VHH1.18~L1 - 2~VHH2.18~E, the polypeptide is represented by SEQ ID NO: 632 (compound number 18). c) In the sequence of VHH1.15~L1 - 2~VHH2.15~E, the polypeptide is represented by SEQ ID NO: 629 (compound number 15). d) In the sequence of VHH1.13~L1 - 2~VHH2.13~E, the polypeptide is represented by SEQ ID NO: 627 (compound number 13). e) In the sequence of VHH1.14~L1 - 2~VHH2.14~E, the polypeptide is represented by SEQ ID NO: 628 (compound number 14). f) In the sequence of VHH1.12~L1 - 2~VHH2.12~E, the polypeptide is represented by SEQ ID NO: 626 (compound number 12). [Figure 4] Figure 4 shows examples of the titration curves (activity as a result of compound concentration) of compound number 6, Mim8, and emicizumab SIA. [Figure 5a-b] Figures 5a and 5b show the sequence alignments of the anti - FIX(a) and anti - FX ISVD (VHH fragment) sequences respectively, and the CDR sequences are highlighted in bold and underlined.

[0022] Brief description of sequences SEQ ID NO: 1 represents the amino acid sequence of human coagulation factor IX. <00SEQ ID NO:2 represents the amino acid sequence of human coagulation factor X. SEQ ID NOs:3 to 13 and 690 represent the amino acid sequences of the extension part (E). SEQ ID NOs:14 to 26 and 691 represent L 1-2 and L P represent the amino acid sequences of the linker. SEQ ID NOs:27 to 614 represent V H represent the amino acid sequence of the VH fragment and its complementarity-determining region (CDR). SEQ ID NOs:615 to 691, 734, and 735 represent any L 1-2 linker and / or extension part-containing VH H represent the amino acid sequence of the polypeptide. SEQ ID NOs:692 to 733 represent the sequences of the peptide fragments disclosed in Example 4 of the present specification. SEQ ID NOs:736 to 739 represent the amino acid sequences of potential protractors. [[FORM FOR CARRYING OUT THE INVENTION]]

[0023] The present invention provides an ISVD polypeptide derivative capable of binding FIX(a) and FX(a), which is highly effective and provides a sufficiently long half-life to enable effective subcutaneous and oral administration. Therefore, the ISVD polypeptide derivatives disclosed herein are suitable for the treatment of various forms of hemophilia, such as the treatment of hemophilia A by various administration routes including subcutaneous and oral administration, the treatment of hemophilia A with inhibitors, and the treatment of acquired hemophilia A.

[0024] In particular, the present invention relates to a bispecific extended ISVD polypeptide such as a VH polypeptide derivative (or a VH polypeptide), which can bind coagulation factor FX leading to the formation of coagulation FIXa and activated coagulation factor FX (FXa) in a manner that mimics the cofactor activity of coagulation factor VIIIa (FVIIIa). The VH H polypeptide disclosed herein H HH polypeptide derivatives exhibit very high in vitro potency, which is on a larger scale than, for example, the bispecific antibody emicizumab sequence identity analog (SIA). H H polypeptide derivatives also exhibit half-life extension in animal models such as rats, dogs, and pigs, via introduced extension factors, such as fatty acid conjugation and albumin-binding peptide fusion. Non-extended V H H polypeptides exhibit very rapid clearance in dogs and pigs. Furthermore, V H H polypeptide derivatives have been engineered to enable clinically relevant bioavailability after oral administration through pI-reducing amino acid substitutions of surface-exposed residues, as well as formulations using excipients such as sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) and, for example, nicotinamide (NAM). Therefore, upon oral administration, the formulated, highly potent V H H polypeptide derivatives exhibit clinically relevant levels of bioavailability in rat and canine animal models. Conventionally, V polypeptide derivatives with molecular weights exceeding 10 kDa have been used. H Therapeutic polypeptides such as ISVD polypeptide derivatives, including H polypeptide derivatives, are not considered suitable for oral administration. However, the V polypeptides disclosed herein H H polypeptide derivatives, though not limited to these, are suitable for novel oral treatments of blood coagulation disorders such as hemophilia A, with or without the presence of inhibitors.

[0025] Therefore, the present invention provides a highly potent and effective solution that allows for subcutaneous and oral administration of coagulation factor IX(a) and factor X(a), and provides a long half-life. HThe present invention provides a single variable domain (ISVD) polypeptide derivative of a blood coagulation-promoting immunoglobulin, such as an H polypeptide derivative. Accordingly, in one embodiment, the present invention provides a first ISVD (ISVD1) that can bind to factor IX (SEQ ID NO: 1) or its active form, a second ISVD (ISVD2) that can bind to factor X (SEQ ID NO: 2) or its active form, one or more extensions bound to one or more surface-exposed residues, and optionally a linker (L) that links ISVD1 and ISVD2. 1-2 This relates to a single variable domain (ISVD) polypeptide derivative of a pro-coagulation immunoglobulin, comprising ( ) and, optionally, one or more extensions (E). Figures 1a-f show non-limiting examples of anti-FX / FIX(a)ISVD polypeptide derivatives.

[0026] In one embodiment, the present invention comprises a first ISVD (ISVD1) capable of binding to factor IX (SEQ ID NO: 1) or its active form, a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or its active form, at least one extension portion bound to a surface-exposed residue, and optionally a linker (L) connecting ISVD1 and ISVD2. 1-2 The present invention relates to a blood coagulation-promoting ISVD polypeptide derivative comprising a first ISVD and optionally one or more extensions (E), wherein the first ISVD can bind to an epitope on factor IX (SEQ ID NO: 1) or its active form, which comprises at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering), and the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) which comprises at least one of the amino acid residues N173, P174, F175, L177, and L178, and D179 (sequential numbering).

[0027] In another embodiment, the present invention relates to blood coagulation promotion V H Regarding H polypeptide derivatives, Factor IX (SEQ ID NO: 1) or the first V that can bind to its active form H H(V HH1) and A second V that can bind to factor X (sequence number 2) H H(V H H2) and, At least one extension attached to a surface-exposed residue, Optional, V H H1 and V H Linker connecting H2 (L 1-2 )and, Optionally, it includes one or more extensions (E), V H H1 is V H H-2.20 (Sequence ID 171), V H H-2.18 (Sequence ID 155), V H H-2.15 (Sequence ID 131), V H H-2.13 (Sequence ID 115), V H H-2.14 (Sequence ID 123), V H H-2.12 (Sequence ID 107), or V H Includes the sequence H-2.2 (sequence number 35), V H H2 is V H H-1.20 (Sequence ID 167), V H H-1.18 (Sequence ID 151), V H H-1.15 (Sequence ID 127), V H H-1.13 (Sequence ID 111), V H H-1.14 (Sequence ID 119), V H H-1.12 (Sequence ID 103), V H H-1.3 (Sequence ID 31), or V H Includes the sequence H-1.4 (sequence number 39).

[0028] In such an embodiment, V H H1 includes the sequence of V H H-2.20 (SEQ ID NO: 171), and V H H2 includes the sequence of V H H-1.20 (SEQ ID NO: 167).

[0029] In another such embodiment, V H H1 includes the sequence of V H H-2.20 (SEQ ID NO: 171), and V H H2 includes the sequence of V H H-1.18 (SEQ ID NO: 151).

[0030] In another such embodiment, V H H1 includes the sequence of V H H-2.20 (SEQ ID NO: 171), and V H H2 includes the sequence of V H H-1.15 (SEQ ID NO: 127).

[0031] In another such embodiment, V H H1 includes the sequence of V H H-2.20 (SEQ ID NO: 171), and V H H2 includes the sequence of V H H-1.13 (SEQ ID NO: 111).

[0032] In another such embodiment, V H H1 includes the sequence of V H H-2.20 (SEQ ID NO: 171), and V H H2 includes the sequence of V H H-1.12 (SEQ ID NO: 103).

[0033] In another such embodiment, V H H1 includes the sequence of V H H-2.20 (SEQ ID NO: 171), and V H H2 includes the sequence of V H H-1.3 (SEQ ID NO: 31).

[0034] In another such embodiment, V H H1 includes the sequence of V HContains the sequence of H-2.20 (SEQ ID NO: 171), V H H2 is V H Contains the sequence of H-1.4 (SEQ ID NO: 39).

[0035] In such an embodiment, V H H1 is V H Contains the sequence of H-2.20 (SEQ ID NO: 171), V H H2 is V H Contains the sequence of H-1.20 (SEQ ID NO: 167).

[0036] In another such embodiment, V H H1 is V H Contains the sequence of H-2.18 (SEQ ID NO: 155), V H H2 is V H Contains the sequence of H-1.18 (SEQ ID NO: 151).

[0037] In another such embodiment, V H H1 is V H Contains the sequence of H-2.20 (SEQ ID NO: 171), V H H2 is V H Contains the sequence of H-1.15 (SEQ ID NO: 127).

[0038] In another such embodiment, V H H1 is V H Contains the sequence of H-2.20 (SEQ ID NO: 171), V H H2 is V H Contains the sequence of H-1.13 (SEQ ID NO: 111).

[0039] In another such embodiment, V H H1 is V H Contains the sequence of H-2.20 (SEQ ID NO: 171), V H H2 is V<97>Contains the sequence of H-1.12 (SEQ ID NO: 103).

[0040] In another such embodiment, V H H1 is V H Contains the sequence of H-2.20 (SEQ ID NO: 171), V H H2 is VH Includes the H-1.3 sequence (sequence number 31).

[0041] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.4 sequence (sequence number 39).

[0042] In another such embodiment, V H H1 is V H Includes the sequence H-2.15 (sequence number 131), V H H2 is V H Includes the H-1.18 sequence (sequence number 151).

[0043] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.15 sequence (sequence number 127).

[0044] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.13 sequence (sequence number 111).

[0045] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.12 sequence (sequence number 103).

[0046] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.3 sequence (sequence number 31).

[0047] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.4 sequence (sequence number 39).

[0048] In another such embodiment, V H H1 is V H Includes the sequence H-2.13 (sequence number 115), V H H2 is V H Includes the H-1.18 sequence (sequence number 151).

[0049] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.15 sequence (sequence number 127).

[0050] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.13 sequence (sequence number 111).

[0051] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.12 sequence (sequence number 103).

[0052] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.3 sequence (sequence number 31).

[0053] In another such embodiment, V HH1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.4 sequence (sequence number 39).

[0054] In another such embodiment, V H H1 is V H Includes the sequence H-2.14 (sequence number 123), V H H2 is V H Includes the H-1.18 sequence (sequence number 151).

[0055] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.15 sequence (sequence number 127).

[0056] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.13 sequence (sequence number 111).

[0057] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.12 sequence (sequence number 103).

[0058] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.3 sequence (sequence number 31).

[0059] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), VH H2 is V H Includes the H-1.4 sequence (sequence number 39).

[0060] In another such embodiment, V H H1 is V H Includes the sequence H-2.12 (sequence number 107), V H H2 is V H Includes the H-1.18 sequence (sequence number 151).

[0061] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.15 sequence (sequence number 127).

[0062] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.13 sequence (sequence number 111).

[0063] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.12 sequence (sequence number 103).

[0064] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.3 sequence (sequence number 31).

[0065] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.4 sequence (sequence number 39).

[0066] In another such embodiment, V H H1 is V H Includes the sequence H-2.2 (sequence number 135), V H H2 is V H Includes the H-1.18 sequence (sequence number 151).

[0067] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.15 sequence (sequence number 127).

[0068] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.13 sequence (sequence number 111).

[0069] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.12 sequence (sequence number 103).

[0070] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.3 sequence (sequence number 31).

[0071] In another such embodiment, V H H1 is V H Includes the sequence H-2.20 (sequence number 171), V H H2 is V H Includes the H-1.4 sequence (sequence number 39).

[0072] In some embodiments, the extension includes the following structure: [ka] * indicates a binding site on the ISVD polypeptide.

[0073] ISVD polypeptide derivatives include, for example, V H It may be an H polypeptide derivative.

[0074] In another aspect, the present invention relates to pharmaceutical compositions comprising ISVD polypeptide derivatives disclosed herein. Another aspect of the present invention relates to the use of ISVD polypeptide derivatives and compositions disclosed herein for the treatment of various forms of hemophilia by various routes of administration, including but not limited to subcutaneous and oral administration, and in particular for the treatment of hemophilia A, hemophilia A in the presence of inhibitors, and acquired hemophilia A.

[0075] In a further embodiment, the present invention relates to a specific anti-FIX(a)V H H fragment or its specific anti-FX(a)V H ISVD polypeptide derivatives such as H fragments or V H Individual components (intermediates) that are part of an H polypeptide derivative, ISVD or V H Regarding H fragments.

[0076] Further aspects of the present invention relate to the production of components (intermediates) of the compounds disclosed herein, including a method for modifying the isoelectric point of such ISVD polypeptide derivatives to which FIX(a) and FX(a) can be attached in order to improve the oral bioavailability of the polypeptide derivatives.

[0077] definition To make the present invention easier to understand, certain terms are defined below.

[0078] Greek letters may also be represented by their symbols or corresponding names, for example, α = alpha, β = beta, ε = epsilon, γ = gamma, and ω = omega. Similarly, the Greek letter μ may be represented by "u", for example, μl = ul, or μM = uM.

[0079] An asterisk (*) in a chemical formula indicates a bonding point.

[0080] The terms "a" or "an" are intended to mean "one or more." When preceding an enumeration of processes or elements, the term "comprise," and its variations such as "comprises" and "comprising," are intended to mean that the addition of further processes or elements is optional and not excluded.

[0081] The term "approximately" is used herein to mean roughly, roughly, or around that. When the term "approximately" is used in conjunction with a numerical range, the range is modified by extending the boundaries above and below the stated number. Generally, the term "approximately" can be modified by 10 percent above or below (higher or lower) the stated value.

[0082] Where used herein, the term "skeleton" is any L 1-2 ISVD polypeptide or V including linker and extension H This refers to the amino acid sequence of the H polypeptide, but excludes the extended portion and any other extended portion that is suitable for fusion with the skeleton to avoid any suspicion of exclusion.

[0083] The term "binding affinity" is V H It is a measure of the strength of non-covalent interactions between two molecules, such as a H fragment and an antigen, for example, between ISVD. The term is used to describe monovalent interactions. HThe binding affinity between two molecules, such as H and ISVD, is determined by the equilibrium dissociation constant (K). D It can be quantified by determining K. D The dynamics of complex formation and dissociation can be determined, for example, by measuring surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). The rate constants corresponding to the association and dissociation of monovalent complexes are the association rate constant k, respectively. a (or k on ), and the dissociation rate constant k d (or k off ) is called K D is, formula K D =k d / k a Through, k a and k d It is related to this.

[0084] According to the definition above, binding affinity, which is related to different molecular interactions such as the binding affinity of different ISVDs to a given antigen, is the K of individual antibody / antigen complexes. D They can be compared by comparing their values.

[0085] The value of the dissociation constant can be directly determined by well-known methods. Standard assays for evaluating the binding ability of ligands such as ISVD toward a target are known in the art and include, for example, ELISA, Western blotting, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of ISVD can also be evaluated by standard assays known in the art, such as SPR.

[0086] V with the target H A competitive binding assay can be performed in which the binding of an ISVD such as H is compared to the binding of the target in the presence of another ligand of that target, such as another ISVD.

[0087] Unless it contradicts the context, K D This is preferably determined by surface plasmon resonance as described herein (see Example 7).

[0088] Preferably, ISVD K which can be coupled to FIX(a) D The value is 3 μM or less, for example, 15 nM or less, for example, 11.7 nM or less.

[0089] Preferably, ISVD's K can be coupled to FX(a). D The value is 3 μM or less, for example, 350 nM or less, for example, 300 nM or less.

[0090] "Cross-species reactivity" ISVD (or V H The H fragment is, for example, equivalent affinity to FIX from all indicated species (e.g., humans and cynomolgus monkeys), particularly within a range of 100 coefficients, such as within a coefficient range of 50, within a coefficient range of 20, or within a coefficient range of 10. D The coefficients X and K are defined. D Within the range, it means that the highest affinity for a particular enumerated species does not exceed X times the lowest affinity measured for binding to a different enumerated species. Those skilled in the art can use any method for measuring affinity to determine if the interspecies reactivity ISVD is K for all enumerated species under the same conditions. D To the extent applicable to the measurement, a given K as described herein D It will be understood that it is possible to verify that it binds to target antigens from all enumerated species within the coefficient range. Preferably, K D The value is measured using SPR, particularly at 25°C. Preferably, affinity is V H It is measured using ISVD for heterogeneous reactivity such as H.

[0091] Amino acids are molecules that optionally contain an amine group, a carboxylic acid group, and one or more additional groups, often called side chains.

[0092] The term "amino acid" includes standard amino acids (genetically encoded) and non-natural amino acids. Non-exclusive examples of non-natural amino acids include Aib (α-aminoisobutyric acid), deaminohistidine (also known as 3-(imidazole-4-yl)propanoic acid, abbreviated as Imp (imidazolpropionyl)), and d-isomers of standard amino acids. In this specification, all amino acid residues in polypeptides for which optical isomers are not listed should be understood to mean the l-isomer unless otherwise specified.

[0093] In this specification, the term "antibody" refers to a protein comprising or derived from an immunoglobulin sequence that can bind to an antigen or a portion thereof.

[0094] The term "antibody" includes, but is not limited to, full-length antibodies comprising at least four polypeptide chains, each consisting of two heavy chains (HC) and two light chains (LC) linked by disulfide bonds, as well as antibodies comprising at least three polypeptide chains, each consisting of two heavy chains (HC) and one light chain (LC) linked by disulfide bonds. One class of immunoglobulins is IgG. In humans, the IgG class may be divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the sequence of their heavy chain constant regions. The light chains can be divided into two types, kappa chains and lambda chains, based on differences in their sequence composition. An IgG molecule consists of two heavy chains linked by two or more disulfide bonds, and two light chains, each attached to the heavy chains by disulfide bonds. The term "antibody" is also used in V H This includes single-domain antibodies such as H fragments and V-NAR fragments.

[0095] As used herein, the term “hypervariable region” refers to an amino acid residue of an antibody involved in antigen binding. The hypervariable region includes the “complementarity-determining region,” also known as “CDR.”

[0096] The "framework" or "FR" region is a variable domain region other than the hypervariable region residues. Therefore, antibodies or ISVDs contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N to the C terminus. CDR3 of the heavy chain is typically the region that contributes most to antigen binding.

[0097] As used herein, the term “epitope” refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of surface elements of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural properties as well as specific charge properties. Structural epitopes and non-structural epitopes are distinguished in that the latter, but not the former, lose their binding in the presence of denaturants that can disrupt the structure of a protein.

[0098] For example, ISVD, V H In the context of X-ray-derived crystal structures, defined by the spatial coordinates of the complex between the H fragment and its target, the term epitope as used herein is specifically defined as an ISVD residue having a heavy atom (i.e., a non-hydrogen atom) within a distance of 4 Å from a heavy atom in FIX / FIXa or FX / FXa, unless otherwise specified or inconsistent with the context.

[0099] The epitope of a given ISVD / antigen pair can be identified by routine methods, such as those described in the examples. For example, the ISVD and antigen may be combined, and the ISVD / antigen complex may be crystallized. The crystal structure of the complex may be determined and used to identify the specific site of interaction between the ISVD and its antigen.

[0100] In one embodiment, the ISVD polypeptide derivative described herein includes a first ISVD that can bind to an epitope on FIX or its active form, comprising at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (SEQ ID NO: 1), and a second ISVD that can bind to an epitope on FX (SEQ ID NO: 2), comprising at least one of the amino acid residues N173, P174, F175, L177, and L178 (sequential numbering).

[0101] In one embodiment, the ISVD polypeptide derivative is a first ISVD capable of binding to an epitope on factor IX (SEQ ID NO: 1) or its active form, comprising at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering), and It contains a second ISVD that can bind to an epitope on factor X (SEQ ID NO: 2) that contains at least one of the amino acid residues N173, P174, F175, L177, L178 (sequential numbering), or at least one of the amino acid residues N173, P174, F175, L177, L178, and D179.

[0102] Coagulation factor IX (FIX) is a vitamin K-dependent coagulation factor that shares structural similarities with factor VII, prothrombin, factor X, and protein C. FIX circulates in plasma as a single-chain enzyme precursor (SEQ ID NO: 1). The circulating enzyme precursor consists of 415 amino acids divided into four distinct domains, including an N-terminal γ-carboxyglutamic acid-rich (Gla) domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. Activation of FIX occurs through limited proteolysis at Arg145 and Arg180, releasing an activating peptide (residues 146-180 of SEQ ID NO: 1). Therefore, activated FIX (FIXa) consists of residues 1-145 (light chain) and residues 181-415 (heavy chain) of SEQ ID NO: 1.

[0103] Therefore, the circulating FIX molecule comprises the FIX enzyme precursor and the active form of FIX, which are generally referred to herein as FIX and FIXa with respect to Sequence ID No. 1.

[0104] The activated form of factor IX is called 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)”.

[0105] FIXa is a trypsin-like serine protease that plays a crucial role in hemostasis by producing most of the factor Xa necessary to support proper thrombin formation during coagulation, as part of the tenase complex.

[0106] FIX is represented herein by Sequence ID No. 1, which corresponds to the Ala148 allele of human FIX (Ansn et al. EMBO J. 1984 3:1053-1060, McGraw et al., Proc Natl Acad Sci USA. 1985 82:2847-2851, Graham et al. Am.J.Hum.Genet. 1988 42:573-580). In this invention, FIX is intended to encompass all natural variants of FIX, such as the T148 variant (Uniprot ID P00740).

[0107] FX is a vitamin K-dependent coagulation factor that has structural similarities to factor VII, prothrombin, FIX, and protein C. FX circulates in plasma as a double-chain enzyme precursor containing residues 1-139 (light chain) and 143-448 (heavy chain) of SEQ ID NO: 2. The human FX enzyme precursor contains four distinct domains, including an N-terminal gamma-carboxyglutamic acid-rich (Gla) domain (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 via limited proteolysis at Arg194, resulting in the release of the activating peptide (residues 143-194). Therefore, activated FX (FXa) consists of residues 1-139 (light chain) of SEQ ID NO: 2 and residues 195-448 (activating heavy chain) of SEQ ID NO: 2. Thus, the circulating factor X molecule includes the FX enzyme precursor and the active form of FX, which are referred to herein as FX and FXa, respectively, with respect to SEQ ID NO: 2. In this invention, FX is intended to encompass all natural variants of FX. The terms “FX (SEQ ID NO: 2) and / or its active form (FXa)” may also be referred to as “FX / FXa” or “FX(a)”.

[0108] As used herein, the term “conservative substitution” means that an amino acid may be substituted for another amino acid having similar biochemical properties, for example, a basic amino acid may be substituted for another basic amino acid (e.g., lysine to arginine), an acidic amino acid may be substituted for another acidic amino acid (e.g., glutamate to aspartate), a neutral amino acid may be substituted for another neutral amino acid (e.g., threonine to serine), a charged amino acid may be substituted for another charged amino acid (e.g., glutamate to aspartate), a hydrophilic amino acid may be substituted for another hydrophilic amino acid (e.g., asparagine to glutamine), a hydrophobic amino acid may be substituted for another hydrophobic amino acid (e.g., alanine to valine), a polar amino acid may be substituted for another polar amino acid (e.g., serine to threonine), an aromatic amino acid may be substituted for another aromatic amino acid (e.g., phenylalanine to tryptophan), and an aliphatic amino acid may be substituted for another aliphatic amino acid (e.g., leucine to isoleucine).

[0109] As used herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient.

[0110] Examples of excipients, though not limited to them, include calcium carbonate, calcium phosphate, various types of sugars and starches, L-arginine, nicotinamide, SNACs, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0111] As used herein, the term "extension" (E) refers to a peptide or polypeptide suitable for binding to the ISVD polypeptide.

[0112] The extension may consist of several amino acids, 1 to 10 amino acids, or it may be longer, consisting of 10 to 30 amino acids, or even a very long extension, consisting of more than 30 amino acids. The extension is preferably an ISVD polypeptide, an ISVD polypeptide derivative, or V H H polypeptide, or V H It is present at the N-terminus (N-terminal extension) or C-terminus (C-terminal extension), or both, of the H polypeptide derivative. Alternatively, the extension may be one or more framework regions or linker L 1-2 For example, it binds somewhere on the ISVD polypeptide outside the CDR sequence.

[0113] The extension is preferably recombined and fused with an ISVD polypeptide. In other embodiments, the extension is conjugated with an ISVD polypeptide.

[0114] Non-limiting examples include compound number 22, which has a 6-amino acid C-terminal extension composed of GQACPC (sequence number 9); compound number 6, which has a 13-amino acid C-terminal extension composed of GGGGCSCHHHHHH (sequence number 8); and a 11-amino acid C-terminal extension composed of GGGGSHHHHHH (sequence number 7).

[0115] The purpose of the extension is to provide a bonding point for the extension portion and / or a means for purification. Therefore, the term extension does not encompass the extension factor and the extension portion.

[0116] For example, any extension factor linker (L) that is recombinantly fused to the N-terminus or C-terminus of a polypeptide. P Extension factors that include ) are not considered extensions.

[0117] 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 a fusion polypeptide DNA sequence results in 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 may be artificially constructed by standard molecular biology methods such as overlap PCR or DNA ligation. The resulting fusion polypeptide DNA sequence may be inserted into a suitable expression vector that supports heterologous fusion protein expression in a host organism such as bacteria, yeast, fungi, insect cells, or mammalian cells. The extended portion may be, for example, an ISVD polypeptide or V H It may be fused to the C-terminus or N-terminus of the H polypeptide backbone.

[0118] As used herein, the term “host cell” encompasses any type of cell line that can be manipulated to produce the ISVDs disclosed herein. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells including 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.

[0119] The term “identity” as known in the art refers to the relationship between sequences of two or more polypeptides, determined by comparing their sequences. In the art, “identity” also means the degree of sequence relevance between polypeptides, such as that determined by the number of matches between strings of two or more amino acid residues. “Identity” measures the percentage of perfect matches between the smaller of two or more sequences, with gap adjustments (if any) addressed by a specific mathematical model or computer program (i.e., “algorithm”). The identity of related polypeptides can be readily calculated by known methods. In this invention, similarity and identity were determined using Needleman from EMBOSS-6.6.0 (Needleman et al, J.Mol.Biol.1970;48:443-453), with parameters 10 and 0.5 for gap start and gap extend, respectively (gapopen=10, gapextend=0.5).

[0120] The terms "Immunoglobulin Monovariable Domain" or "ISVD" are used as general terms to refer to antigen-binding domains or fragments, such as the VH domain and VL domain, respectively.

[0121] Therefore, ISVD may be a light chain variable domain sequence (e.g., a VL sequence) or a heavy chain variable domain sequence (e.g., a VH sequence), such as a heavy chain variable domain sequence derived from a conventional quad antibody, or a light chain variable domain sequence derived from a conventional quad antibody.

[0122] Certain types of ISVDs are originally identified as types of immunoglobulins defined as antibody fragments consisting of a single monomeric variable antibody domain. H This is an H fragment. H The H fragment is a single-domain antibody that contains the antigen-binding variable region of a heavy-chain-only antibody and can be obtained from camels. H H fragments are approximately 15 kDa in size. They contain single-chain molecules that can bind their homologous antigens using a single domain. HThe antigen-binding surface of the H fragment is more convex (or protruding) than the surface of conventional antibodies, which are typically flat or concave. H The H fragment consists of four framework regions (or FRs) and three complementarity-determining regions (or CDRs), whose sequence and structure are defined as conserved. It exhibits high variability in both sequence content and structural conformation, participating in antigen binding and providing antigen specificity.

[0123] Another type of ISVD can be obtained from IgNAR in cartilaginous fish, and its single-domain antibody is indicated as "V-NAR fragment".

[0124] V H The H and V-NAR fragments do not contain a constant domain and therefore typically lack the Fc region that is part of partial / full-length and / or manipulated / natural heavy chain-only antibodies [Dooley et al. (2006) Dev. Comp. Immunol. 30:43-56, Muyldermans S. (2013) Annu Rev Biochem. 82:775-97].

[0125] Camel V H A general description of the H fragments and methods for their production and / or isolation and / or use can be found in particular in the following references, International Publication Nos. 94 / 04678 and International Publication Nos. 97 / 49805. A general description of heavy chain immunoglobulins and their variable regions derived from cartilaginous fish, and methods for their production and / or isolation and / or use can be found in particular in International Publication No. 2005 / 118629.

[0126] V H The total number of amino acid residues in the H fragment is typically in the range of 110–140. However, V H It should be noted that parts, fragments, or analogues of H are not particularly limited in terms of their length and / or size, provided that such parts, fragments, or analogues satisfy the further requirements outlined below herein and are also suitable for the purposes described herein.H The molecular weight of the H fragment is typically in the range of 12–15 kDa. H The pI of the H fragment is generally basic, as is generally the case with antibodies, meaning that the pI value is generally above 7, often between 7.5 and 8.5. H The H fragment typically contains at least one disulfide bridge, usually formed by conserved cysteine ​​pairs located in framework regions 1 and 3. Such a disulfide bridge is V H Ensure the correct folding and stability of the H fragment, for example, V H When side-chain modification and / or conjugation is performed in a manner that targets the introduced unpaired cysteine ​​in the H polypeptide, it is desirable to retain such disulfide bridges.

[0127] In this document, V H The CDR sequences of ISVDs, such as H fragments, are determined using the Kabat definition (Kontermann and Dubel, 2010, Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, the CDRs of the variable domain are defined as positions 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3). However, unless otherwise specified, the ISVD polypeptide or V H This term is used to refer to specific amino acid residue positions in polypeptide compounds described herein, including the CDR and framework (FR) regions in the sequential numbering of H polypeptides.

[0128] As used herein, the term "ISVD polypeptide" means, for example, direct domain fusion, and any suitable composition and length of linker (L) as needed. 1-2Refers to a polypeptide containing two or more ISVDs, such as a first ISVD (ISVD1) and a second ISVD (ISVD2), which are linked by a linker or have no linker at all.

[0129] 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 linked only via a peptide bond, the linker may be called a “peptide linker.” Otherwise, the linker may be called a “chemical linker.”

[0130] An example of an ISVD polypeptide is a linker (L 1-2 These are two ISVDs linked via ).

[0131] Another example of an ISVD polypeptide is a linker (L) that further includes one or more extensions. 1-2 These are two ISVDs linked via ).

[0132] Another example of an ISVD polypeptide is a linker (L 1-2 Two linked ISVDs, without the C-terminal and / or N-terminal extensions, further comprising one or more extensions, such as but not limited to these.

[0133] Linker (L 1-2 ) may consist of, for example, an amino acid sequence, and may contain no repeats or contain multiple repeats.

[0134] For example, a linker may contain 2 to 50 amino acids, 5 to 40 amino acids, or 10 to 30 amino acids.

[0135] Non-restrictive examples of linkers include the *-GGGGS-* linker, *-GQAPGQ-* linker (SEQ ID NO: 20), *-QAPGQA-* linker (SEQ ID NO: 16), *-GI-* linker, *-GV-* linker, *-GT-* linker, *-GL-* linker, or other amino acid complex linkers. Two examples of linkers are the 2- and 6-repeated GGGGS complexes, with lengths of 10 and 30 amino acids, respectively.

[0136] To avoid any doubt, L 1-2 subscript 1-2 This refers to the specific direction of the connected ISVD, i.e., L 1-2 This does not imply that ISVD1 can be concatenated to ISVD2, or that ISVD2 can be concatenated to ISVD1 (from the N-terminus to the C-terminus).

[0137] Sequence numbers 14-24 are L 1-2 This represents a non-restrictive example of a linker.

[0138] In some embodiments, the ISVD polypeptide includes an extension (E) as outlined in one of the following formulas. ISVD1-ISVD2, or ISVD1-ISVD2-E, or ISVD2-ISVD1-E, or E-ISVD1-ISVD2, or ISVD1-L 1-2 -ISVD2-E, or ISVD2-L 1-2 -ISVD1-E, or E-ISVD1-L 1-2 -ISVD2 The extension (E) may be bound to the ISVD polypeptide, for example, as outlined in one of the following formulas (N-terminus to C-terminus). ISVD1-ISVD2, or ISVD1-ISVD2-E, or ISVD2-ISVD1-E, or E-ISVD1-ISVD2, or ISVD1-L 1-2 -ISVD2-E, or ISVD2-L 1-2 -ISVD1-E, or E-ISVD1-L 1-2 -ISVD2

[0139] The ISVD polypeptide preferably includes an extended portion. In such cases, the ISVD polypeptide is called an ISVD polypeptide derivative.

[0140] In some embodiments, the first ISVD serves as a connection point for one or more extensions.

[0141] In some embodiments, the second ISVD serves as a connection point for one or more extensions.

[0142] In some embodiments, L 1-2 A linker acts as a connecting point for one or more extensions.

[0143] To avoid any ambiguity, I should add that the extended portion is ISVD1-L 1-2 - Linker (L) coupled to extension (E) to ISVD2 p ) if it includes Linker L P This is not considered part of the "extension."

[0144] In some embodiments, the ISVD polypeptide is chemically conjugated with a non-ISVD, such as a small chemical non-polypeptide molecule, carbohydrate, fatty acid, or oligopeptide or polypeptide, or a protein, such as an antibody or preferably an antibody fragment.

[0145] In one embodiment, the ISVD polypeptide is linked to the Fc domain from an IgG antibody without the use of a linker.

[0146] In another embodiment, the ISVD polypeptide is linked from an IgG antibody to an Fc domain or a fragment thereof by a linker.

[0147] In a preferred embodiment, the extension is fused to the ISVD polypeptide and is therefore not linked to the ISVD polypeptide by chemical conjugation.

[0148] In some embodiments, the extended portion is fused to the ISVD polypeptide and is therefore not linked to the ISVD polypeptide by chemical conjugation.

[0149] As used herein, the term “isoelectric point” or “pI” refers to the pH value at which the overall net charge of a protein, such as an antibody, is zero. Proteins have many charged groups, and at the isoelectric point, the sum of all these charges is zero. Above the isoelectric point, the overall net charge of a protein is negative, while below the isoelectric point, the overall net charge of a protein is positive.

[0150] pI may be either the theoretically or experimentally determined isoelectric point.

[0151] Those skilled in the art are familiar with methods for determining the isoelectric point of a protein. Most commonly, the isoelectric point of a protein is calculated based on the amino acid sequence of the protein. Numerous (online) tools are available that enable the 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 algorithm of Skoog & Wichman, 1986. The amino acid residues of the pKa are used to calculate the pI.

[0152] pl may also be determined experimentally, and charge variants can be separated using charge-based separation techniques such as isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, etc.

[0153] In one embodiment, the first and second ISVDs in the ISVD polypeptide are V-NAR fragments, and such a compound is referred to as "V-NAR polypeptide".

[0154] In another embodiment, the first and second ISVDs in the ISVD polypeptide are V H It is an H fragment, and such a compound is "V H This is indicated as "H polypeptide". In one such embodiment, the first V H H fragments can be joined to FIX / FIXa, and the second V H The H fragment can bind FX / FXa. In a preferred embodiment, V H H polypeptides have bispecificity V H It is an H polypeptide.

[0155] To avoid any ambiguity, the terms multiple singularity, triple singularity, or bisingularity are V H It is intended to reflect the number of antigens bound by ISVD, such as H fragments, and does not include molecules bound by extensions (if any), such as but not limited to albumin.

[0156] In one embodiment, V does not have an extended portion. H The molecular weight of H polypeptides is in the range of 27-29 kDa.

[0157] In a preferred embodiment, one or more extensions (i.e., V H H polypeptide derivatives), and optionally V including one or more extensions H The molecular weight of the H polypeptide is in the range of 28 to 33 kDa.

[0158] Those skilled in the art will understand that the above embodiments are similarly applicable to V-NAR polypeptides.

[0159] As used herein, the term “free cysteine” refers to 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 crosslink. Essentially, while free cysteine ​​can be used for conjugation, it is not the case that free cysteine ​​residues, including recombinantly introduced free cysteine, are often blocked by small thiols such as cysteine, homocysteine, or glutathione during recombinant expression of polypeptides in host cells. This is also observed in the recombinant production of ISVD polypeptides in which one or more free cysteine ​​have been introduced. Therefore, free cysteine ​​can be released and prepared for conjugation to desired moieties, such as extension moieties such as C18 diacitol gamma-Glu 2xOEG fatty acid moieties, etc., using reduction reactions with appropriate reducing agents such as bis(p-sulfonatophenyl)phenylphosphine dihydrate or tris(2-carboxyethyl)phosphine hydrochloride.

[0160] V disclosed herein H ISVD polypeptides, such as H polypeptides, may have multispecificity, including but not limited to bispecificity or tripspecificity.

[0161] As used herein, the term "bispecific ISVD polypeptide" or "bispecific V H The term "H polypeptide" refers to an ISVD polypeptide or V polypeptide, each capable of binding to two different antigens or two different epitopes on the same antigen. H This refers to H polypeptide.

[0162] As used herein, "triple-specific ISVD polypeptide" or "triple-specific V H The term "H polypeptide" refers to an ISVD polypeptide or V polypeptide that can bind to three different antigens, or to three different epitopes on the same antigen, or to three different epitopes present on two different antigens. H This refers to H polypeptide.

[0163] As used herein, "multispecific ISVD polypeptide" or "multispecific V H The term "H polypeptide" refers to an ISVD polypeptide or V polypeptide, each capable of binding to two or more different antigens or two or more different epitopes on the same antigen. H Refers to H polypeptide. Also known as multiplicative ISVD polypeptide or multiplicative V polypeptide. H H polypeptides are therefore bispecific ISVD polypeptides and triplicate ISVD polypeptides or V polypeptides, respectively. H Contains H polypeptide.

[0164] Those skilled in the art will understand that the above also applies to polypeptide derivatives (i.e., including extended portions).

[0165] As used herein, the terms “oral bioavailability” or “peroral bioavailability” refer to the amount of the drug administered in the systemic circulation after oral administration (estimated as the area under plasma concentration of the drug-time curve) relative to the amount of the drug administered in the systemic circulation after intravenous administration.

[0166] Therefore, as used herein, the term “paratope” refers to an area or region on the ISVD to which the antigen specifically binds, i.e., to which the antigen has physical contact.

[0167] For example, ISVD, V H In the context of X-ray-derived crystal structures, defined by the spatial coordinates of the complex between the H fragment and its target, the term "paratope" as used herein is specifically defined as an ISVD residue characterized by having a heavy atom (i.e., a non-hydrogen atom) within a distance of 4 Å from a heavy atom in FIX / FIXa or FX / FXa, unless otherwise specified or inconsistent with the context.

[0168] The paratopes (and epitopes) of a given ISVD / antigen pair can be identified by routine methods, such as those described in the examples. For example, the ISVD and antigen may be combined, and the ISVD / antigen complex may be crystallized. The crystal structure of the complex may be determined and used to identify the specific site of interaction between the ISVD and its antigen.

[0169] The term "pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and acceptable for human medicinal use, and is useful for preparing a pharmaceutical composition. Such excipients may be, for example, solid, liquid, or semi-solid.

[0170] 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 removed from the patient’s serum or plasma by normal biological processes.

[0171] As used herein, the term “selectively combine” means V H It should be understood that a binding region on an ISVD, such as an H fragment, binds to one component (e.g., activated FIX) in preference to or in support of another component. Therefore, "selective binding" does not necessarily require exclusive or undetectable binding to other components. For example, anti-FIX(a)V H Anti-FIX(a)ISVDs, such as H fragments, can selectively bind to activated FIX compared to inactivated FIX.

[0172] The term "blood coagulation-promoting antibody" refers to an antibody that enhances blood coagulation, for example, by accelerating the blood coagulation process and / or by increasing the enzymatic activity of one or more coagulation factors.

[0173] As used herein, the term “pro-coagulation 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 by increasing the enzymatic activity of one or more coagulation factors. Therefore, the term “pro-coagulation activity” encompasses (but is not limited to) one or more of the activities listed below.

[0174] Enhancement of factor IXa-mediated factor X activation, measured by an amidolytic (chromogenic or fluorescent) assay based on FIXa-mediated FX activation. The assay measures FXa through cleavage of an FXa-specific peptide substrate. The substrate is produced, yielding a color that can be photometrically measured by absorbance.

[0175] A reduction in coagulation time, measured by coagulation assays such as activated partial thromboplastin time (APTT), measures the activity of intrinsic and common coagulation pathways. Plasma is pre-incubated with APTT reagents containing contact activators, e.g., ellagic acid or kaolin, and phospholipids. Calcium chloride is added to promote fibrin thrombus formation. Possible readings are coagulation time or coagulation waveform.

[0176] Enhancement of thrombinogenesis, measured by thrombinogenesis assays such as calibrated automated thrombography (CAT). A thrombogram describes the concentration of thrombin in coagulated plasma and is therefore a functional test of the hemostatic system. The assay is based on the measurement of fluorescence produced by the cleavage of the fluorescent substrate ZGR AMC by thrombin over time. See also the method used in Example 8 of this specification.

[0177] Enhancement of the global viscoelastic properties of thrombus formation, measured, for example, in whole blood under shear stress, by viscoelastic hemostasis methods such as ROTEM (rotational thromboelastometry). Inside the instrument, a ball bearing pin rotates within a fixed cup. Fibrin chains in the sample form between the cup wall and the pin during coagulation, and the strength of these chains affects the movement of the pin, which is then detected.

[0178] A reduction in whole blood closure time (WBCT), as measured by a platelet function analyzer, is based on von Willebrand factor (VWF)-mediated platelet adhesion to collagen after platelet activation. High shear stress is generated, which leads to platelet adhesion and aggregation. The time from the start to the end of blood flow is measured.

[0179] The ISVD polypeptides disclosed herein preferably comprise one, two, three, or four “extension portions” (PMs). More preferably, one or two extension portions.

[0180] As used herein, the term “extension portion” means having half-life extension properties and having an “extension factor” (P) and an optional “linker” (L). P This refers to the portion containing ).Therefore, the term “extension” refers to the extension of the half-life, and thus the extension factor or extension portion serves the purpose of extending the half-life of the ISVD polypeptide disclosed herein.

[0181] The extension portion (PM) consists of the extension factor "P" and an optional linker (L P ) includes.

[0182] Each extension preferably binds to a surface-exposed lysine or cysteine ​​residue within the polypeptide backbone of the compound. The binding site is generally called R1 (and R1 ≠ R2 ≠ R3, etc., in the case of binding to more than one extension R2, R3, etc.).

[0183] Those skilled in the art will be able to identify other surface-exposed residues suitable for binding.

[0184] The extension can consist of a single extension factor.

[0185] The extension section has one linker (L P ) and may include one extension factor (P).

[0186] The extension may include one linker and two or more extension factors.

[0187] Linker (L P If ) exists, the extension part is L P It binds to the ISVD polypeptide backbone via the linker (L P If ) is absent, P binds to the polypeptide backbone.

[0188] In one embodiment, there is a first extension portion and a second extension portion, and the first extension portion includes the following structure: [ka] The second extension includes the following structure: [ka] "*" (R1) represents a binding site on the ISVD polypeptide to the first extension portion. "**" (R2) represents the binding site on the ISVD polypeptide to the second extension portion. "*" is the Cys at position 257 of sequence number 629. "**" is the Cys at position 259 of sequence number 629.

[0189] The ISVD polypeptides disclosed herein may contain a single cysteine ​​residue or a lysine residue to which a single extension is conjugated.

[0190] The ISVD polypeptides disclosed herein may comprise two lysine residues and two extensions.

[0191] The ISVD polypeptides disclosed herein may comprise two lysine residues and two identical extensions.

[0192] The ISVD polypeptides disclosed herein may comprise two lysine residues and two non-identical extensions.

[0193] The ISVD polypeptides disclosed herein may comprise three lysine residues and three extensions.

[0194] The ISVD polypeptides disclosed herein may comprise three lysine residues and three identical extensions.

[0195] The ISVD polypeptides disclosed herein may comprise three lysine residues and three non-identical extensions (for example, the first and second extensions may be identical, while the third extension may be different from the first and second extensions).

[0196] The ISVD polypeptides disclosed herein may comprise two cysteine ​​residues and two extensions.

[0197] The ISVD polypeptides disclosed herein may comprise two cysteine ​​residues and two identical extensions.

[0198] The ISVD polypeptides disclosed herein may comprise two cysteine ​​residues and two non-identical extensions.

[0199] The ISVD polypeptides disclosed herein may comprise three cysteine ​​residues and three extensions.

[0200] The ISVD polypeptides disclosed herein may comprise three cysteine ​​residues and three identical extensions.

[0201] The ISVD polypeptides disclosed herein may comprise three cysteine ​​residues and three non-identical extensions (for example, the first and second extensions may be identical, while the third extension may be different from the first and second extensions).

[0202] If the ISVD polypeptide contains two or three extensions, the extensions are preferably similar, more preferably substantially identical, or most preferably identical.

[0203] With respect to chemical parts such as extensions disclosed herein, similarity and / or identity can be determined using any suitable computer program and / or algorithm known in the art.

[0204] Compounds containing an extended portion can be called "derivatives." For example, an "ISVD polypeptide derivative" is an ISVD polypeptide containing an extended portion, "V H "H polypeptide derivative" is a V including the extended portion. H H polypeptides and "V-NAR polypeptide derivatives" are understood to be V-NAR polypeptides that include the extended portion.

[0205] The extended portion may be capable of non-covalent binding to albumin, thereby promoting the circulation of ISVD polypeptide derivatives in the bloodstream and extending their half-life. Therefore, in one embodiment, the extended portion is an albumin-binding portion.

[0206] The extension factor (P) may contain an acyl group. The acyl group may be branched or unbranched. The acyl group may be saturated or unsaturated. The extension factor may contain a fatty acyl group. The acyl group may be branched or unbranched. The acyl group may be saturated or unsaturated.

[0207] The extension factor may include a distal carboxylic acid group.

[0208] The extension factor may contain a fatty acid group.

[0209] The extension factor may include fatty acid groups and amide groups.

[0210] The extension factors may include distal carboxylic acid groups and amide groups.

[0211] The extension factor may include alkyl groups.

[0212] The extension factor may include an aryl group.

[0213] The extension factor may contain a tetrazole group.

[0214] The extension factor may contain a sulfonic acid group.

[0215] The extension factor may contain a phenoxy group.

[0216] The extension factor may contain a benzoic acid group.

[0217] The extension factor may contain 8 to 30 carbon atoms. The extension factor 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.

[0218] The extension factor may contain 6 to 30 consecutive -CH2- groups. The extension factor may contain a carbon chain containing 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.

[0219] The extension factor may contain 12 to 26 carbon atoms. The "extension factor" may contain 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms.

[0220] The extension factor may contain 10 to 26 consecutive -CH2- groups. The extension factor may contain a carbon chain containing 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive -CH2- groups.

[0221] The extension factor can contain 16 to 22 carbon atoms.

[0222] The extension factor may contain 14 to 20 consecutive -CH2- groups. The extension factor may contain a carbon chain containing 14, 15, 16, 17, 18, 19, or 20 consecutive -CH2- groups.

[0223] The extension factor may contain 16 to 22 consecutive carbon atoms and 14 to 20 consecutive -CH2- groups.

[0224] The extension factor may contain 16 consecutive carbon atoms and 14 consecutive -CH2- groups.

[0225] The extension factor may contain 18 consecutive carbon atoms and 16 consecutive -CH2- groups.

[0226] The extension factor may contain 20 consecutive carbon atoms and 18 consecutive -CH2- groups.

[0227] The extension factor may contain 22 consecutive carbon atoms and 20 consecutive -CH2- groups.

[0228] In some embodiments, the extension factor is as follows: Chemical formula a:HOOC-(CH2) n It contains a group defined by -CO-* (where n is an integer in the range of 8 to 30), which is a C(n+2) diacid, or Chemical formula a1: [ka]

[0229] The extension factor may include an oligopeptide. In one embodiment, the extension factor oligopeptide consists of 10 to 40 amino acids, for example 10 to 30 amino acids, for example 15 to 25 amino acids, and preferably 20 amino acids. The extension factor oligopeptide sequence composition may be, for example, QRLMEDICLPRWGCLWEDDF (SEQ ID NO: 736), exemplified as the fusion sequence of SEQ ID NOs. 3 and 4 [see also International Publication No. 01 / 45746A2]. Compound number 71 includes a 50-amino acid N-terminal extension portion consisting of, for example, QRLMEDICLPRWGCLWEDDFGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4), where residues 1 to 20 are the extension factor (P) and residues 21 to 50 are the linker (L P )

[0230] The extension factor is amino acid linker L P It may be bound to ISVD or ISVD polypeptide via this method.

[0231] In one embodiment, L P This allows for the attachment of an extension factor (P) to the side chain of a lysine or cysteine ​​residue in the ISVD polypeptide backbone.

[0232] The ISVD polypeptide derivative may contain two extensions, each containing 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The ISVD polypeptide may contain two extensions, each extension factor (P) containing 12, 13, 14, 15, 16, 17, or 18 consecutive -CH2- groups.

[0233] ISVD polypeptide derivatives may contain two C14 diacides, two C16 diacides, or two C18 diacides.

[0234] The ISVD polypeptide derivative may contain three extensions, each containing an extension factor comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The ISVD polypeptide may contain three extensions, each containing 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive -CH2- groups.

[0235] To avoid any ambiguity, it should be added that, for example, even if albumin can be bound, the extension is not considered a binder in terms of defining whether the ISVD polypeptide disclosed herein is bispecific, triplicate, or multispecific.

[0236] In some embodiments, the ISVD polypeptides disclosed herein may include an extension factor selected from any one of those shown in Table 1.

[0237] In Table 2, L P R1 represents an optional linker that connects the indicated extension factor (P) to the ISVD polypeptide backbone. R1 represents the binding site in the ISVD polypeptide. [Table 1-1] [Table 1-2]

[0238] In one embodiment, the extension factor is directly conjugated onto the ISVD polypeptide, i.e., linker L P Do not use (i.e., by covalent bonding).

[0239] In other embodiments, the extension factor is linker L P It is directly conjugated onto the ISVD polypeptide using [this method]. A non-restrictive example is provided below.

[0240] L PThe linker, if present, may include Ado, Aeep, or Aeeep, sulfonamide, Trx, ε-Lys, Ahx, Glu, γGlu, Gly, Ser, Ala, and / or Thr.

[0241] L P Linker, Chemical formula 1:*-NH-(CH2)2-(O-(CH2)) k -O-(CH2) n -CO-* or [ka] It may include at least a portion that can be represented by the chemical formula (wherein k is an integer in the range of 1 to 5, and n is an integer in the range of 1 to 5).

[0242] When k=1 and n=1, the linker element may be designated as Ado, or 8-amino-3,6-dioxaoctanoyl, and may be represented by the following chemical formula: Chemical formula 3:*-NH-(CH2)2-O-(CH2)2-O-CH2-CO-* or [ka]

[0243] For k=1 and n=2, the linker element may be designated Aeep and may be represented by the following chemical formula. Chemical formula 5:*-NH-(CH2)2-O-(CH2)2-O-(CH2)2-CO-* or [ka]

[0244] For k=2 and n=2, the linker element may be designated Aeeep and may be represented by the following chemical formula. Chemical formula 7:*-NH-(CH2) 2- O-(CH2)2O-(CH2)2-O-(CH2)2-CO-* or [ka]

[0245] Any linker (L P ) may contain an 8-amino-3,6-dioxa-octanoic acid (OEG) group having the following chemical formula. [ka]

[0246] Optional linker (L P ) may contain a sulfonamide-C4 moiety. The sulfonamide-C4 group is a sulfonamide group bonded to a 4-butanoyl group and has the following chemical formula. Chemical formula 9:*-NH-S(O)2-CH2-CH2-CH2-CO-* or [ka]

[0247] Optional linker L P It may contain Trx. Trx is also known as tranexamic acid, trans-4-(aminomethyl)cyclohexanecarboxylic acid, and has the following chemical formula: Chemical formula 11:*-NH-CH2-(C6H 10 )-CO-* or [ka]

[0248] Linker L P It may contain epsilon-lysine (ε-Lys).

[0249] Linker L P It may contain lysine (Lys).

[0250] Linker L PThis may include Ahx. Ahx, also known as aminocaproic acid or 6-aminohexanoic acid, is defined as follows: Chemical 13:*-NH-(CH2)5-CO-* or [ka]

[0251] Linker L P teeth, [ka] The formula may include Glu diradicals such as (wherein the formula may contain p times, and p is an integer in the range of 1 to 3).

[0252] Chemical formula 15 is also sometimes referred to as gamma-Glu, or simply γGlu, as it is the gamma-carboxyl group of the amino acid glutamic acid used herein for linking with the epsilon-amino group of another lysine. As described above, other linker elements may be, for example, another Glu residue or an Ado molecule. The amino group of Glu then forms an amide bond with the carboxyl group of the extended portion, or, if present, the carboxyl group of, for example, an Ado molecule, or, if present, the gamma-carboxyl group of another Glu.

[0253] Alternatively, the ISVD polypeptide derivatives disclosed herein may be linked with a linker (L) selected from any one of those shown in Table 2 below. P ) may include. R1 represents a residue in the ISVD polypeptide to which the extension portion is bound, and P represents the extension factor. [Table 2]

[0254] Based on the disclosure herein, a person skilled in the art may, at their discretion, determine after some limited routine experiments the optimal L for use with the specific ISVD polypeptide derivatives disclosed herein.P and L 1-2 The linker can be determined. For example, the linker is preferably V in the ISVD polypeptide. H Each ISVD, such as an H fragment, is designed to bind to its target. Furthermore, based on the disclosure herein, those skilled in the art may, at their discretion, determine the optimal linker for use with a particular ISVD polypeptide derivative disclosed herein after several limited routine experiments.

[0255] The amino acid residue in the ISVD or ISVD polypeptide backbone where binding occurs is denoted as R1 in this specification. If more than one extension is bound, further binding sites may be denoted as R2, R3, and so on.

[0256] The extended portion may be bound to a cysteine ​​or lysine residue of the first ISVD portion of the ISVD polypeptide backbone.

[0257] The extended portion may be bound to a cysteine ​​or lysine residue of the second ISVD portion of the ISVD polypeptide backbone.

[0258] The extended portion is an optional linker (L) of the ISVD polypeptide backbone. 1-2 The ) portion may be bound to a cysteine ​​residue or a lysine residue.

[0259] The extended portion can be covalently bonded to a lysine residue within the ISVD polypeptide backbone. The extended portion can also be bonded via an amide bond formed between the carboxylic acid group in the extended portion and the epsilon-amino group of the lysine residue.

[0260] The extended portion can be covalently bonded to a cysteine ​​residue within the ISVD polypeptide backbone. The extended portion can also be bonded via a thioether bond formed between the extended portion and the sulfur atom of the cysteine ​​residue in the polypeptide.

[0261] Accordingly, in some embodiments, the compounds disclosed herein may comprise one, two, or three lysine or cysteine ​​residues and one, two, or three extensions, each extension being bound to a side chain of a single lysine or cysteine ​​residue.

[0262] When the binding occurs via a cysteine ​​residue, the cysteine ​​is preferably free cysteine.

[0263] In some embodiments, free cysteine ​​may be introduced by recombinant DNA technology and function as a conjugation site for binding one or more C16 diacitors, C17 diacitors, or C18 diacitors gamma-Glu 2xOEG fatty acid moieties.

[0264] In a preferred embodiment, free cysteine ​​may optionally be introduced by recombinant DNA technology and function as a conjugation site for binding one, two, or three C18 diacitol gamma-Glu 2xOEG fatty acid moieties. (IUPAC name S{beta-AA#}-[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 bond).

[0265] In another embodiment, free cysteine ​​may be optionally introduced by recombinant DNA technology and function as a conjugation site for binding one, two, or three C17 diacitic acid gamma-Glu 2xOEG fatty acid moieties. (IUPAC name S{beta-AA#}-[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 bond).

[0266] In the most preferred embodiment, free cysteine ​​may be introduced by recombinant DNA technology and function as a conjugation site for binding one, two, or three C16 diacitic acid gamma-Glu 2xOEG fatty acid moieties. (IUPAC name S{beta-AA#}-[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 bond).

[0267] As used herein, the term “standard chromatography” encompasses standard chromatography methods such as protein A, cation exchange, anion exchange, hydrophobic interactions, and hydroxyapatite chromatography.

[0268] As used herein, the term “surface-exposed amino acid residue” refers to an amino acid residue whose side chain may come into contact with a solvent molecule (generally, most likely to be water molecules). However, the side chain does not necessarily have to be in complete contact with the solvent molecule; even if only a portion of the side chain is in contact with the solvent molecule, the amino acid residue is defined as an “amino acid located on the surface.” Amino acid residues located on the surface of a polypeptide may also include amino acid residues located near the ISVD surface, thereby having the influence of cross-charges from other amino acid residues whose side chains are in partial contact with solvent molecules. Those skilled in the art can prepare homology models or machine learning-based three-dimensional molecular models of polypeptides or antibodies, for example, by performing homology modeling or machine learning using commercially available or publicly available software. Alternatively, methods such as X-ray crystallography can be used to generate three-dimensional molecular models. Amino acid residues that may be exposed on the surface can be determined, for example, using computer programs such as MOE (Chemical Computing Group) or Bioluminate (Schrodinger) with coordinates from the three-dimensional molecular model of the antibody. The surface exposure sites may be determined using algorithms known in the art (see, for example, Lee and Richards (1971) J. Mol. Biol. 55:379-400, Connolly, J. Appl. Cryst. (1983) 16:548-558). Surface exposure sites can be determined using software suitable for protein modeling and analysis of three-dimensional structural information obtained from antibodies. Software available for such purposes includes, for example, MOE (Chemical Computing Group) or Bioluminate (Schrodinger). Surface accessible to the solvent (Å) 2The area is calculated using a water probe with a probe radius of 1.4 Å. Furthermore, a method for determining the surface exposure region and area using software for personal computers has been described by Pacios (Pacios, Comput. Chem. 18(4):377-386 (1994), J. Mol. Model. 1:46-53 (1995)). Based on the information described above, appropriate amino acid residues located on the surface of the antibody in contact with the solvent can be selected.

[0269] Pharmaceutical composition V disclosed herein H H polypeptide derivatives may be prepared in pharmaceutical compositions. In some embodiments, such compositions include at least one pharmaceutically acceptable excipient.

[0270] As used herein, the term “excipient” broadly refers to any component other than the active therapeutic ingredient (API). Excipients may serve a variety of purposes, for example, as carriers, vehicles, fillers, binders, lubricants, disintegrants, flow regulators, crystallization inhibitors, solubilizers, stabilizers, colorants, flavorings, surfactants, emulsifiers, delivery agents, hydrotropes, or combinations thereof, and / or to improve the administration and / or absorption of the active pharmaceutical ingredient.

[0271] The amount of each excipient used may vary within the limits of what is practiced in the art. Techniques and excipients that may be used to formulate oral dosage forms are described in Handbook of Pharmaceutical Excipients, 8th edition, Sheskey et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2017), and Remington: the Science and Practice of Pharmacy, 22nd edition, Remington and Allen, Eds., Pharmaceutical Press (2013).

[0272] In a preferred embodiment, V H The composition containing the H polypeptide further comprises a delivery agent and a hydrotrope.

[0273] A preferred delivery agent is a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid (NAC).

[0274] In some embodiments, the delivery agent is a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid, as described in International Publication No. 2007 / 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-(salicyroylamino)octanoate.

[0275] The pharmaceutical composition preferably contains one or more hydrotropes. Like surfactants, hydrotropes contain both hydrophilic and hydrophobic portions, can form micelles and self-aggregate, but solubilize solutes without micelle solubilization. In one embodiment, the hydrotrope can increase the solubility of SNACs. In one embodiment, the hydrotrope is nicotinamide (NAM).

[0276] In one embodiment, the composition is a solid composition. The composition may be in a form suitable for oral administration, such as a tablet, pouch, or capsule. In such one embodiment, the composition is formulated as a tablet. The solid compositions provided herein allow for enhanced dissolution, thereby enabling rapid uptake of the active pharmaceutical ingredient.

[0277] Dosage and Administration Compounds disclosed herein, for example, V H H polypeptide derivatives may be administered parenterally in a suitable pharmaceutical composition, for example, intravenously, intramuscularly, or subcutaneously. The compounds may be administered via non-injectable routes, preferably orally (PO). The compounds may be administered prophylactically. The compounds may be administered therapeutically (as required).

[0278] Subcutaneous administration The dose of the compound delivered by subcutaneous administration may be approximately 0.01 mg to 1 mg of the compound per day, preferably approximately 0.05 mg to 5 mg per day, and more preferably approximately 0.1 mg to 10 mg per day, depending on the severity of the condition, administered every other day, every 3 days, every 4 days, every 5 days, every 6 days, or once a week. Furthermore, the preferred dose may be adjusted for a particular compound based on its properties, including its in vivo half-life or mean residence time and its biological activity.

[0279] In one embodiment, the present invention relates to an injection device having the contents of the composition.

[0280] Oral (PO) administration The orally delivered dose of the compound may range from approximately 1 mg to approximately 300 mg, delivered daily, every other day, or every three days, depending on the severity of the condition. Furthermore, the preferred dose may be adjusted for a particular compound based on its properties, including its in vivo half-life or mean residence time and its biological activity.

[0281] Compositions containing the compounds disclosed herein may be administered for prophylactic treatment and / or, in some embodiments, for therapeutic treatment. In therapeutic use, the composition is administered to a subject suffering 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 is defined as the “therapeutic effective dose.” As will be understood by those skilled in the art, the effective dose for this purpose depends on the severity of the disease or injury, as well as the subject’s weight and overall condition.

[0282] Embodiment 1. A single variable domain (ISVD) polypeptide derivative of a blood coagulation-promoting immunoglobulin, A first ISVD (ISVD1) that can bind to factor IX (SEQ ID NO: 1) or its active form, A second ISVD (ISVD2) that can bind to factor X (SEQ ID NO: 2) or its active form, At least one extension section, Optionally, a linker (L) connects ISVD1 and ISVD2. 1-2 )and, A blood coagulation-promoting ISVD polypeptide derivative comprising, optionally, one or more extensions (E). 2. An ISVD polypeptide derivative according to Embodiment 1, having the following formula. ISVD1-ISVD2, or ISVD1-ISVD2-E, or ISVD2-ISVD1-E, or E-ISVD1-ISVD2, or ISVD1-L1-2-ISVD2-E, or ISVD2-L1-2-ISVD1-E, or E-ISVD1-L1-2-ISVD2 3. An ISVD polypeptide derivative according to Embodiment 1, having the following formula (from N-terminus to C-terminus). ISVD1-ISVD2, or ISVD2-ISVD1, or ISVD1-ISVD2-E, or ISVD2-ISVD1-E, or E-ISVD1-ISVD2, or E-ISVD2-ISVD1, or ISVD1-L1-2-ISVD2, or ISVD2-L1-2-ISVD1, or ISVD1-L1-2-ISVD2-E, or ISVD2-L1-2-ISVD1-E, or E-ISVD1-L1-2-ISVD2, or E-ISVD2-L1-2-ISVD1 4. An ISVD polypeptide derivative according to any one of embodiments 1 to 3, wherein at least one extension portion is bound to one or more surface-exposed amino acid residues. 5. Having the following formula (from N-terminus to C-terminus), ISVD2-L 1-2 -ISVD1-E An ISVD polypeptide derivative according to any one of Embodiments 1 to 4, wherein one or more extension portions are bound to one or more surface-exposed residues. 6. Having the following formula (from N-terminus to C-terminus), ISVD2-L 1-2 -ISVD1-E An ISVD polypeptide derivative according to any one of Embodiments 1 to 5, wherein two extension portions are bound to one or more surface-exposed residues on E. 7. Having the following formula (from N-terminus to C-terminus), ISVD2-L 1-2 -ISVD1-E An ISVD polypeptide derivative according to any one of Embodiments 1 to 6, further comprising a first extension and a second extension attached to a first surface-exposed residue and a second surface-exposed residue in E, respectively. 8. L 1-2 The ISVD polypeptide derivative according to any one of Embodiments 1 to 7, wherein the ratio of hydrophobic amino acids to hydrophilic amino acids is "40-60%" to "60-40%". 9. An ISVD polypeptide derivative according to any one of Embodiments 1 to 8, wherein the first ISVD can bind to an epitope on factor IX (SEQ ID NO: 1) or its active form, comprising at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering). 10. An ISVD polypeptide derivative according to any one of Embodiments 1 to 9, wherein the first ISVD can bind to an epitope on factor IX (SEQ ID NO: 1) or its active form, comprising at least 4, 5, 6, 7, or 8 amino acid residues from E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering). 11. An ISVD polypeptide derivative according to any one of Embodiments 1 to 10, wherein the first ISVD can bind to an epitope on factor IX (SEQ ID NO: 1) or its active form, comprising amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering). 12. An ISVD polypeptide derivative according to any one of Embodiments 1 to 11, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising at least one of the amino acid residues N173, P174, F175, L177, L178, and D179 (sequential numbering). 13. An ISVD polypeptide derivative according to any one of Embodiments 1 to 12, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising amino acid residues N173, P174, F175, L177, and L178 (sequential numbering). 14. An ISVD polypeptide derivative according to any one of Embodiments 1 to 13, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising at least one of the amino acid residues N173, P174, F175, L177, L178, and D179 (sequential numbering). 15. An ISVD polypeptide derivative according to any one of Embodiments 1 to 14, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising at least 3, 4, 5, or 6 amino acid residues N173, P174, F175, L177, L178, and D179 (sequential numbering). 16. An ISVD polypeptide derivative according to any one of Embodiments 1 to 15, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising amino acid residues N173, P174, F175, L177, L178, and D179 (sequential numbering). 17. The first ISVD can bind to an epitope on factor IX (SEQ ID NO: 1) or its active form, which contains at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering). An ISVD polypeptide derivative according to any one of Embodiments 1 to 16, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising at least one of the amino acid residues N173, P174, F175, L177, and L178 (sequential numbering). 18. The first ISVD can bind to an epitope on factor IX (SEQ ID NO: 1) or its active form, which includes amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering). An ISVD polypeptide derivative according to any one of Embodiments 1 to 17, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising amino acid residues N173, P174, F175, L177, L178, and D179 (sequential numbering). 19. The first ISVD contains a paratope comprising amino acid residues F29, N30, Y32, T54, D99, R100, S101, F102, L103, F104, Q106, A107, and N113 (SEQ ID NO: 35), The second ISVD is an amino acid residue a) D32, A33, M34, G35, Y37, L47, V48, A49, G50, I51, M52, N57, T58, N59, Y60, T61, K97, V99, R101, and P102 (Sequence ID 27), or b) A33, M34, G35, W47, V48, A49, A50, I51, S52, S57, T58, N59, Y60, A61, A97, A98, D99, G105, L107, and Y109 (Sequence ID 734) An ISVD polypeptide derivative according to any one of Embodiments 1 to 9, comprising a paratope containing (continuous numbering). 20. The first ISVD, 1) CDR1: Optionally, IYTMS (SEQ ID NO: 172) containing one or two substitutions. CDR2: Optionally, GLRWTDSSTEYADSVKG (SEQ ID NO: 173) containing one, two, or three amino acid substitutions. CDR3: An ISVD polypeptide derivative according to any one of Embodiments 1 to 9, comprising DRSFLFAQALGATKNYEY (SEQ ID NO: 174) (Kabat definition), which optionally contains one, two, or three amino acid substitutions. 21. The second ISVD, (A) CDR1: Optionally, RYAMG (SEQ ID NO: 168) containing one or two substitutions. CDR2: Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 169) containing one, two, or three amino acid substitutions. CDR3: Optionally, DDSVGDGYLDY (SEQ ID NO: 170) containing one, two, or three amino acid substitutions, or (B) CDR1: Optionally, RLAMG (SEQ ID NO: 128) containing one or two amino acid substitutions. CDR2:Optionally, AISRRGGSTNYADSVKG (Sequence ID 129) containing one, two, or three substitutions. CDR3: Optionally, containing DDSVGDGYLDY (SEQ ID NO: 130) (Kabat definition) with one, two, or three amino acid substitutions. An ISVD polypeptide derivative according to any one of Embodiments 1 to 9. 22. The first ISVD, 1) CDR1: Optionally, IYTMS (SEQ ID NO: 172) containing one or two amino acid substitutions. CDR2: Optionally, GLRWTDSSTEYADSVKG (SEQ ID NO: 173) containing one, two, or three amino acid substitutions. CDR3: Optionally, contains DRSFLFAQALGATKNYEY (SEQ ID NO: 174) with one, two, or three amino acid substitutions, or The second ISVD said, (A) CDR1: Optionally, RYAMG (sequence number 168) containing one or two substitutions. CDR2:Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 169) containing one, two, or three substitutions. CDR3: Optionally, DDSVGDGYLDY (SEQ ID NO: 170) containing one, two, or three amino acid substitutions, or (B) CDR1: Optionally, RLAMG (sequence number 128) containing one or two substitutions. CDR2:Optionally, AISRRGGSTNYADSVKG (Sequence ID 129) containing one, two, or three substitutions. CDR3: Optionally, containing DDSVGDGYLDY (SEQ ID NO: 130) (Kabat definition) with one, two, or three amino acid substitutions. An ISVD polypeptide derivative according to any one of Embodiments 1 to 9. 23. An ISVD polypeptide derivative according to any one of Embodiments 20 to 22, wherein the substitution is a conservative substitution. 24. An ISVD polypeptide derivative according to any one of Embodiments 1 to 23, 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 identified by SEQ ID NOs: 107, 115, 123, 131, 155, or 171, respectively. 25. An ISVD polypeptide derivative according to any one of Embodiments 1 to 24, 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 identified by SEQ ID NOs: 103, 111, 119, 127, 151, or 167. 26. An ISVD polypeptide derivative according to any one of Embodiments 1 to 25, 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 identified by SEQ ID NOs. 107, 115, 123, 131, 155, or 171, and the sequence of the second ISVD is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the sequence identified by SEQ ID NOs. 103, 111, 119, 127, 151, or 167. 27. An ISVD polypeptide derivative according to any one of Embodiments 1 to 26, wherein the first ISVD comprises the following sequence. V H H-2.20 (Sequence ID 171), V H H-2.18 (Sequence ID 155), V H H-2.15 (Sequence ID 131), V H H-2.13 (Sequence ID 115), V H H-2.14 (Sequence ID 123), or V H H-2.12 (Sequence ID 107) 28. An ISVD polypeptide derivative according to any one of Embodiments 1 to 27, wherein the second ISVD comprises the following sequence. V H H-1.20 (Sequence ID 167), V H H-1.18 (Sequence ID 151), V H H-1.15 (Sequence ID 127), V H H-1.13 (Sequence ID 111), V H H-1.14 (Sequence ID 119), or V H H-1.12 (Sequence ID 103) 29. The first ISVD contains the following sequence: V H H-2.20 (Sequence ID 171), V H H-2.18 (Sequence ID 155), V H H-2.15 (Sequence ID 131), V H H-2.13 (Sequence ID 115), V H H-2.14 (Sequence ID 123), or V H H-2.12 (Sequence ID 107) The ISVD polypeptide derivative according to any one of Embodiments 1 to 28, wherein the second ISVD comprises the following sequence. V H H-1.20 (Sequence ID 167), V H H-1.18 (Sequence ID 151), VH H-1.15 (Sequence ID 127), V H H-1.13 (Sequence ID 111), V H H-1.14 (Sequence ID 119), or V H H-1.12 (Sequence ID 103) 30. The first ISVD is V H Includes the sequence H-2.20 (sequence number 171), The second ISVD is V H An ISVD polypeptide derivative according to Embodiment 29, comprising the sequence H-1.20 (SEQ ID NO: 167). 31. The first ISVD is V H Includes the sequence H-2.18 (sequence number 155), The second ISVD is V H An ISVD polypeptide derivative according to Embodiment 29, comprising the sequence H-1.18 (SEQ ID NO: 151). 32. The first ISVD is V H Includes the sequence H-2.15 (sequence number 131), The second ISVD is V H An ISVD polypeptide derivative according to Embodiment 29, comprising the sequence H-1.15 (SEQ ID NO: 127). 33. The first ISVD is V H Includes the sequence H-2.13 (sequence number 115), The second ISVD is V H An ISVD polypeptide derivative according to Embodiment 29, comprising the sequence H-1.13 (SEQ ID NO: 111). 34. The first ISVD, V H Includes the sequence H-2.14 (sequence number 123), The second ISVD is V H An ISVD polypeptide derivative according to Embodiment 29, comprising the sequence H-1.14 (SEQ ID NO: 119). 35. The first ISVD, V H Includes the sequence H-2.12 (sequence number 107), The second ISVD is V HAn ISVD polypeptide derivative according to Embodiment 29, comprising the sequence H-1.12 (SEQ ID NO: 103). 36. An ISVD polypeptide derivative according to any one of Embodiments 1 to 35, wherein the extended portion comprises an extension factor selected from the group consisting of the following. a) C16 dioxide, C17 dioxide, C18 dioxide, C19 dioxide, C20 dioxide, C21 dioxide, or C22 dioxide, b) Tetrazole, c) Albumin binder peptides such as RLIEDICLPRGWCLWEDD (SEQ ID NO: 737), d) FcRn binder peptides such as QRFCTGHFGGLYPCNG (SEQ ID NO: 738), and e) Fc binder peptides such as FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDDN (SEQ ID NO: 739) 37. An ISVD polypeptide derivative according to any one of Embodiments 1 to 36, wherein the extension portion comprises an extension factor selected from the group consisting of C16 diacid, C17 diacid, C18 diacid, C19 diacid, and C20 diacid. 38. The ISVD polypeptide derivative according to Embodiment 34, wherein the extension factor is a C16 diacid extension factor. 39. The extension part is ISVD1, L 1-2 Linker L to which the extension factor is attached to ISVD2 or E P An ISVD polypeptide derivative according to any one of Embodiments 1 to 38, comprising: 40. L P However, the ISVD polypeptide derivative according to Embodiment 39 is selected from the group consisting of the following: [Table 3] 41. An ISVD polypeptide derivative according to any one of Embodiments 1 to 40, wherein the extension is bonded to position 13 of ISVD2 and / or one or more of positions 12, 14, 15, 42, 44, 63, and / or 85 (sequential numbering) of ISVD1. 42. An ISVD polypeptide derivative according to any one of Embodiments 1 to 40, wherein the ISVD polypeptide derivative includes a C-terminal extension, and the extension is bonded to the C-terminal extension. 43. The ISVD polypeptide derivative according to any one of Embodiments 1 to 42, wherein the ISVD polypeptide derivative comprises first and second extension portions and a C-terminal extension portion, the first and second extension portions being bonded to the C-terminal extension portion. 44. The ISVD polypeptide derivative according to Embodiment 43, wherein the C-terminal extension is bonded to ISVD1. 45. The ISVD polypeptide derivative according to Embodiment 43, wherein the C-terminal extension is bonded to ISVD2. 46. ​​An ISVD polypeptide derivative according to any one of Embodiments 1 to 45, wherein the extended portion is identical. 47. An ISVD polypeptide derivative according to any one of Embodiments 1 to 46, wherein one or more extensions are bound to a surface-exposed residue. 48. An ISVD polypeptide derivative according to any one of Embodiments 1 to 47, wherein one or more extensions are bound to a surface-exposed residue, and the surface-exposed residue is not a residue in the CDR region. 49. An ISVD polypeptide derivative according to any one of Embodiments 1 to 48, wherein one or more extensions are linked by one or more cysteine ​​or lysine residues. 50. The ISVD polypeptide derivative comprises SEQ ID NO: 629, The first extension includes the following structure: [ka] The second extension includes the following structure: [ka] "*" (R1) represents the binding site on the ISVD polypeptide to the first extension portion. "**" (R2) represents the binding site on the ISVD polypeptide to the second extension portion. "*" is the Cys at position 257 of sequence number 629. The ISVD polypeptide derivative according to Embodiment 43, wherein "**" is the Cys at position 259 of Sequence ID No. 629. 51. An ISVD polypeptide derivative according to any one of Embodiments 1 to 50, wherein the isoelectric point of the polypeptide is 6.5 or less when determined using isoelectric focusing electrophoresis. 52. The ISVD polypeptide derivative according to any one of Embodiments 1 to 51, wherein the polypeptide derivative is a multispecific polypeptide derivative such as a bispecific or tripspecific polypeptide derivative. 53. The ISVD polypeptide derivative according to any one of Embodiments 1 to 52, wherein the polypeptide derivative is a bispecific polypeptide derivative. 54. The polypeptide derivative is in the range of 12 to 50 kDa, for example 12 to 48 kDa, for example 12 to 43 kDa, for example 12 to 37 kDa, for example 12 to 35 kDa, for example 12 to 32 kDa, for example 12 to 27 kDa, for example 12 to 22 kDa, for example 12 to 18 kDa, for example 14 to 50 kDa, for example 14 to 48 kDa, for example 14 to 43 kDa, For example, 14-37kDa, for example, 14-35kDa, 14-32kDa, for example, 14-27kDa, for example, 14-22kDa, for example, 14-18kDa, for example, 22-50kDa, 22-48kDa, for example, 22-43kDa, for example, 22-37kDa, for example, 22-35kDa, for example, 22-32kDa, for example, 22-31kDa, for example, 22-30 kDa, e.g., 22-29kDa, e.g., 22-28kDa, e.g., 22-27kDa, e.g., 24-50kDa, 24-48kDa, e.g., 24-43kDa, e.g., 24-37kDa, e.g., 24-35kDa, e.g., 24-32kDa, e.g., 24-31kDa, 24-30kDa, 24-29kDa, 24-28kDa, e.g., 24-27kDa, e.g., example An ISVD polypeptide derivative according to any one of Embodiments 1 to 53, having a molecular weight of, for example, 36-48 kDa, for example, 36-43 kDa, for example, 36-37 kDa, for example, 28-36 kDa, for example, 29-33 kDa, for example, 29-30, for example, about 29, for example, about 30, for example, 30, for example, 30-32 kDa, or for example, 31 kDa. 55. An ISVD polypeptide derivative according to any one of Embodiments 1 to 54, which is not a membrane-targeted ISVD polypeptide derivative. 56. An ISVD polypeptide derivative according to any one of Embodiments 1 to 55, wherein the extended portion is unable to bind to plasma membrane components such as aminophospholipids, such as phosphatidylserine and / or phosphatidylethanolamine. 57. The extended portion contains GPlb-1X, collagen chaperone HSP47, ephrin B1, thiol isomerase protein ERP5, hematopoietic precursor kinase 1-interacting protein (HIP-55), glycoprotein V1, platelet glycoprotein 1b, platelet-derived growth factor receptor, platelet endothelial aggregation receptor I, CD31, CD36, MARKS, multimelin, integrin alpha-1b / beta-3, trigger receptor (TREM)-like transcript-1 (TLT-1) expressed in bone marrow cells, integrin-binding kinase (ILK), dixin, collagen, P-selectin, factor XIII, and P-selectin glycoprotein ligand-1. ISVD polypeptide derivatives according to any one of Embodiments 1 to 56, which are unable to bind platelet surface proteins such as integrin alpha-6 beta-1, 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. 58. An ISVD polypeptide derivative according to any one of Embodiments 1 to 57, wherein the extended portion is unable to bind membrane-related polypeptides such as glycoproteins, GPIIb / IIIa, β2GP1, TLT-1, selectins, coagulation factors or coagulation factor complexes, and / or selectins. 59. An ISVD polypeptide derivative according to any one of Embodiments 1 to 58, wherein the mean residence time (terminal phase half-life) in the blood is extended by at least 12 hours, at least 24 hours, at least 48 hours, and at least 3, 4, 5, or 7 days compared to an ISVD polypeptide without the extended portion. 60. The first and / or second ISVD, H An ISVD polypeptide derivative according to any one of Embodiments 1 to 59, which is an H fragment. 61. An ISVD polypeptide derivative according to any one of Embodiments 1 to 59, wherein the first and / or second ISVD is a V-NAR fragment. 62. The ISVD polypeptide derivative is V H An ISVD polypeptide derivative according to any one of Embodiments 1 to 60, which is an H polypeptide derivative. 63. Blood coagulation promotion V H H polypeptide derivative, Factor IX (SEQ ID NO: 1) or the first V that can bind to its active form H H and, A second V that can bind to factor X (sequence number 2) H H and, The first V H H and the second V H Linker connecting H (L 1-2 )and, C-terminal extension (E), Including one or two extensions, The following formula (from N-terminus to C-terminus) "The second V H H"-L 1-2 - "The first V H H"-E I) The first V H H is CDR1:IYTMS (Sequence ID 172), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 173) CDR3:DRSFLFAQALGATKNYEY (SEQ ID NO: 174) The second V H H is CDR1:RYAMG (Sequence ID 168) CDR2:AISRRGGSTNYADSVKG (Sequence ID 169) CDR3:DDSVGDGYLDY (Sequence ID 170) contains, or II) The first V H H is CDR1:IYTMS (Sequence ID 132), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 133) CDR3:DRSFLFAQALGATKNYEY (SEQ ID NO: 134) The second V H H is CDR1:RLAMG (Sequence ID 128), CDR2:AISRRGGSTNYADSVKG (Sequence ID 129) CDR3:DDSVGDGYLDY (Sequence ID 130) contains, or (Kabat definition) including blood coagulation promoter V H H polypeptide derivative. 64. I) The first V H H, V H Includes the sequence H-2.20 (sequence number 171), The second V H H, V H Includes the sequence H-1.20 (sequence number 167), II) The first V H H, V H Includes the sequence H-2.18 (sequence number 155), The second V H H, V H Includes the sequence H-1.18 (sequence number 151), (Spro) The first V H H, V H Includes the sequence H-2.15 (sequence number 131), The second V H H, V H Includes the sequence H-1.15 (sequence number 127), IV) The first V H H, V H Includes the sequence H-2.13 (sequence number 115), The second V H H, V H Includes the sequence H-1.13 (sequence number 111), V) The first V HH, V H Includes the sequence H-2.14 (sequence number 123), The second V H H, V H Includes the sequence H-1.14 (sequence number 119), VI) The first V H H, V H Includes the sequence H-2.12 (sequence number 107), The second V H H, V H The blood coagulation promoter V according to Embodiment 63, which includes the sequence H-1.12 (SEQ ID NO: 103) H H polypeptide derivative. 65. The relevant V H H polypeptide derivatives, bispecificity V H A blood coagulation promoter V according to any one of embodiments 1 to 60 and 62 to 64, which is an H polypeptide derivative. H H polypeptide derivative. 66. Linker L 1-2 However, blood coagulation promoter V according to any one of embodiments 63 to 65, which contains the amino acid residue GQAPGQ (SEQ ID NO: 20) H H polypeptide derivative. 67. Blood coagulation promoter V according to any one of embodiments 63 to 65, wherein the extension (E) contains the amino acid residue GQACPC (SEQ ID NO: 9) H H polypeptide derivative. 68. Blood coagulation promoting V according to any one of embodiments 63 to 67, wherein two extensions are bonded to E at positions 4 and 6 (SEQ ID NO: 9), respectively. H H polypeptide derivative. 69. Blood coagulation promoter V according to any one of embodiments 63 to 68, wherein the two extension portions are identical. H H polypeptide derivative. 70. Blood coagulation promoting V according to any one of embodiments 63 to 69, wherein the two extension portions include the following structure. H H polypeptide derivative. [ka] 71. Including the first and second extensions, The first extension portion includes the following structure: [ka] The second extension includes the following structure: [ka] "*" (R1) is V relative to the first extension. H This represents a binding site on an H polypeptide derivative. "**" (R2) is V relative to the second extension. H This represents a binding site on an H polypeptide derivative. "*" is the Cys at position 257 of sequence number 629. Blood coagulation promoter V according to any one of embodiments 63 to 70, wherein "**" is the Cys at position 259 of sequence number 629. H H polypeptide derivative. 72. ISVD polypeptide derivative or V described in any one of Embodiments 1 to 71 H A pharmaceutical composition containing an H polypeptide derivative. 73. ISVD polypeptide derivative or V described in any one of Embodiments 1 to 71 H A pharmaceutical composition for oral administration containing an H polypeptide derivative. 74. ISVD polypeptide derivatives are V H The pharmaceutical composition according to embodiment 72 or 73, which is an H polypeptide derivative. 75. The pharmaceutical composition according to Embodiment 72 or 73, wherein the ISVD polypeptide derivative is a V-NAR polypeptide derivative. 76. The pharmaceutical composition according to any one of Embodiments 72 to 75, wherein the composition comprises a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid. 77. The 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). 78. The pharmaceutical composition according to embodiment 76 or 77, further comprising nicotinamide (NAM). 79. The composition is V H A pharmaceutical composition according to any one of Embodiments 74 to 78, comprising an H polypeptide derivative, sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC), nicotinamide (NAM), and magnesium stearate. 80. The composition contains approximately 7% w / w V A pharmaceutical composition according to any one of Embodiments 74 to 78, comprising an HH polypeptide derivative, about 55% w / w sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC), about 36% w / w nicotinamide (NAM), and about 0.5% w / w magnesium stearate. 81. The pharmaceutical composition according to any one of embodiments 73 to 80, wherein the composition is a liquid composition. 82. The pharmaceutical composition according to any one of embodiments 73 to 80, wherein the composition is a solid composition. 83. The pharmaceutical composition according to Embodiment 82, wherein the composition is a tablet, lozenge, or capsule. 84. A method for performing treatment in a patient requiring treatment, comprising an ISVD polypeptide derivative according to any one of Embodiments 1 to 83, V H A method comprising administering an H polypeptide derivative or a pharmaceutical composition. 85. The treatment method according to Embodiment 84, wherein the patient is a patient suffering from hemophilia A or acquired hemophilia A with or without inhibitors. 86. The treatment method according to embodiment 84 or 85, wherein the treatment is a preventive measure. 87. ISVD polypeptide derivative, V H A therapeutic method according to any one of embodiments 84 to 86, wherein an H polypeptide derivative or pharmaceutical composition is administered orally. 88. ISVD polypeptide derivative, V HA therapeutic method according to any one of embodiments 84 to 86, wherein an H polypeptide derivative or pharmaceutical composition is administered by subcutaneous injection. 89. ISVD polypeptide derivatives according to any one of Embodiments 1 to 83, for use in pharmaceuticals, V H H polypeptide derivatives, or pharmaceutical compositions. 90. ISVD polypeptide derivatives according to any one of Embodiments 1 to 83, for use in the treatment of hemophilia such as hemophilia A with or without inhibitors, or acquired hemophilia A. H H polypeptide derivatives or compositions. 91. ISVD polypeptide derivatives according to any one of Embodiments 1 to 83, for use in the treatment of hemophilia A, with or without inhibitors. H H polypeptide derivatives or compositions. 92. ISVD polypeptide derivative for use according to any one of embodiments 89-92, where the treatment is a preventive measure, V H H polypeptide derivatives or compositions. 93. The derivative or composition is an ISVD polypeptide derivative for use according to any one of Embodiments 89 to 92, administered orally. H H polypeptide derivatives or compositions. 94. ISVD polypeptide derivative for use according to any one of Embodiments 89 to 92, the derivative or composition is administered by subcutaneous injection. H H polypeptide derivatives or compositions. 95. A method for producing a single variable domain (ISVD) polypeptide derivative of a blood coagulation-promoting immunoglobulin, A first ISVD (ISVD1) that can bind to factor IX (SEQ ID NO: 1) or its active form, A second ISVD (ISVD2) that can bind to factor X (SEQ ID NO: 2) or its active form, One or more extensions, Optionally, a linker (L) connects ISVD1 and ISVD2.1-2 )and, Optionally, it includes one or more extensions (E), a. A step of modifying a nucleic acid encoding an amino acid residue of an ISVD polypeptide or ISVD polypeptide derivative such that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is reduced. b. A step of culturing host cells to express nucleic acids encoding ISVD polypeptides or ISVD polypeptide derivatives, c. A step of collecting ISVD polypeptides or ISVD polypeptide derivatives from host cells, d. A step of purifying ISVD polypeptides or ISVD polypeptide derivatives from host cell cultures using standard chromatography, e. A method comprising the step of binding an extension portion to an ISVD polypeptide, unless such portion already exists. 96. A method for producing a single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative, comprising: a first ISVD (ISVD1) capable of binding to factor IX (SEQ ID NO: 1) or its active form; a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or its active form; an extension; and optionally a linker (L) capable of linking ISVD1 and ISVD2. 1-2 ) and optionally one or more extensions (E), a. A step of modifying a nucleic acid encoding an amino acid residue of an ISVD polypeptide or ISVD polypeptide derivative such that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is 6.5 or less, b. A step of culturing host cells to express nucleic acids encoding ISVD polypeptides or ISVD polypeptide derivatives, c. A step of collecting ISVD polypeptides or ISVD polypeptide derivatives from host cells, d. A step of purifying ISVD polypeptides or ISVD polypeptide derivatives from host cell cultures using standard chromatography, e. A method comprising the step of binding an extension portion to an ISVD polypeptide, unless such portion already exists. 97. A method for increasing the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative, comprising: a first ISVD (ISVD1) capable of binding to factor IX (SEQ ID NO: 1) or its active form; a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or its active form; an extension; and optionally a linker (L) capable of linking ISVD1 and ISVD2. 1-2 ) and optionally one or more extensions (E), a. A step of modifying a nucleic acid encoding an amino acid residue of an ISVD polypeptide or ISVD polypeptide derivative such that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is reduced. b. A step of culturing host cells to express nucleic acids encoding ISVD polypeptides or ISVD polypeptide derivatives, c. A step of collecting ISVD polypeptides or ISVD polypeptide derivatives from host cells, d. A step of purifying ISVD polypeptides or ISVD polypeptide derivatives from host cell cultures using standard chromatography, e. A method comprising the step of binding an extension portion to an ISVD polypeptide, unless such portion already exists. 98. A method for increasing the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative, comprising: a first ISVD (ISVD1) capable of binding to factor IX (SEQ ID NO: 1) or its active form; a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or its active form; an extension; and optionally a linker (L) capable of linking ISVD1 and ISVD2. 1-2 ) and optionally one or more extensions (E), a. A step of modifying a nucleic acid encoding an amino acid residue of an ISVD polypeptide or ISVD polypeptide derivative such that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is 6.5 or less, b. A step of culturing host cells to express nucleic acids encoding ISVD polypeptides or ISVD polypeptide derivatives, c. A step of collecting ISVD polypeptides or ISVD polypeptide derivatives from host cells, d. A step of purifying ISVD polypeptides or ISVD polypeptide derivatives from host cell cultures using standard chromatography, e. A method comprising the step of binding an extension portion to an ISVD polypeptide, unless such portion already exists. 99. The method according to any one of embodiments 95 to 98, wherein the isoelectric point is reduced by at least 0.5 pH units, such as 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4 pH units. 100. The method according to any one of Embodiments 95 to 99, wherein step e) does not lead to an increase in the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative. 101. The method according to any one of Embodiments 95 to 100, wherein the isoelectric point is determined using isoelectric focusing electrophoresis. 102. The method according to any one of Embodiments 95 to 101, wherein the ISVD polypeptide or ISVD polypeptide derivative is encoded by a single nucleic acid. 103. The method according to any one of embodiments 95 to 101, wherein an ISVD polypeptide or ISVD polypeptide derivative is a conjugate of a first and a second ISVD, and the first and a second ISVD are encoded by separate nucleic acids. 104. ISVD polypeptide or ISVD polypeptide derivative is V HH polypeptide or V H The method according to any one of embodiments 95 to 103, wherein the H polypeptide derivative is an H polypeptide derivative. 105. An ISVD polypeptide or ISVD polypeptide derivative produced using the method described in any one of Embodiments 95 to 103. 106. ISVD polypeptides or ISVD polypeptide derivatives that can be obtained using the method described in any one of Embodiments 95 to 103. 107. V produced using the method described in any one of embodiments 95 to 104. H H polypeptide or V H H polypeptide derivative. 108. V that can bind to FIX (SEQ ID NO: 1) or its active form (FIXa) H H fragment, a. The CDR1 sequence is identified by Sequence ID No. 172, which optionally contains one or two amino acid substitutions. The CDR2 sequence is identified by Sequence ID No. 173, which optionally contains one, two, or three amino acid substitutions. The CDR3 sequence is identified by Sequence ID No. 174, which optionally contains one, two, or three amino acid substitutions, or b. The CDR1 sequence is identified by Sequence ID No. 156, which optionally contains one or two amino acid substitutions. The CDR2 sequence is identified by Sequence ID No. 157, which optionally contains one, two, or three amino acid substitutions. The CDR3 sequence is identified by Sequence ID No. 158, which optionally contains one, two, or three amino acid substitutions, or The c.CDR1 sequence is identified by Sequence ID No. 132, which optionally contains one or two amino acid substitutions. The CDR2 sequence is identified by Sequence ID No. 133, which optionally contains one, two, or three amino acid substitutions. The CDR3 sequence is identified by Sequence ID No. 134, which optionally contains one, two, or three amino acid substitutions, or d. The CDR1 sequence is identified by Sequence ID No. 116, which optionally contains one or two amino acid substitutions. The CDR2 sequence is identified by Sequence ID No. 117, which optionally contains one, two, or three amino acid substitutions. The CDR3 sequence is identified by Sequence ID No. 118, which optionally contains one, two, or three amino acid substitutions, or The e.CDR1 sequence is identified by Sequence ID No. 124, which optionally contains one or two amino acid substitutions. The CDR2 sequence is identified by Sequence ID No. 125, which optionally contains one, two, or three amino acid substitutions. The CDR3 sequence is identified by Sequence ID No. 126, which optionally contains one, two, or three amino acid substitutions, or The f.CDR1 sequence is identified by Sequence ID No. 108, which optionally contains one or two amino acid substitutions. The CDR2 sequence is identified by Sequence ID No. 109, which optionally contains one, two, or three amino acid substitutions. The CDR3 sequence is identified by sequence number 110 (Kabat definition), which optionally contains one, two, or three amino acid substitutions. H H fragment. 109. V can be coupled to FX (sequence number 2). H H fragment, a. The CDR1 sequence is identified by Sequence ID No. 152, which optionally contains one or two amino acid substitutions. The CDR2 sequence is identified by Sequence ID No. 153, which optionally contains one, two, or three amino acid substitutions. The CDR3 sequence is identified by Sequence ID No. 154, which optionally contains one, two, or three amino acid substitutions, or b. The CDR1 sequence is identified by Sequence ID No. 128, which optionally contains one or two amino acid substitutions. The CDR2 sequence is identified by Sequence ID No. 129, which optionally contains one, two, or three amino acid substitutions. The CDR3 sequence is identified by Sequence ID No. 130, which optionally contains one, two, or three amino acid substitutions. H H fragment. 110. The V described in Embodiment 108 or 109, wherein the substitution is a conservative substitution. H H fragment. 111. The relevant V H The H fragment can bind to FIX (SEQ ID NO: 1) or its active form (FIXa), and can also bind to FX (SEQ ID NO: 2) or its active form (FXa), V H H polypeptide or V H V described in any one of embodiments 108 to 110 is an intermediate for use in the production of H polypeptide derivatives. H H fragment. 112. The relevant V H The V described in any one of embodiments 108 to 110 is an intermediate for use in the production of an ISVD polypeptide derivative. H H polypeptide. 113. ISVD or V as described in Embodiment 109 or 110, in which the activity is improved in that it makes FX more susceptible to proteolysis by FIXa compared to any of the anti-FX ISVD compounds described in International Publication No. 2019 / 096874. H H. 114. A single variable domain (ISVD) polypeptide derivative of a blood coagulation-promoting immunoglobulin, A first ISVD (ISVD1) that can bind to factor IX (SEQ ID NO: 1) or its active form, A second ISVD (ISVD2) that can bind to factor X (SEQ ID NO: 2) or its active form, At least one extension section, Optionally, a linker (L) connects ISVD1 and ISVD2. 1-2 )and, Optionally, it includes one or more extensions (E), A blood coagulation-promoting ISVD polypeptide derivative, wherein the ISVD polypeptide is selected from the group consisting of SEQ ID NOs. 615-691, 734, or 735. 115. V containing one of the sequences described in sequence numbers 27-614 H H fragment. 116. V containing any sequence number 615-691 or 734-735 H H polypeptide derivative. 117. A single variable domain (ISVD) polypeptide derivative of a blood coagulation-promoting immunoglobulin, A first ISVD (ISVD1) that can bind to factor IX (SEQ ID NO: 1) or its active form, A second ISVD (ISVD2) that can bind to factor X (SEQ ID NO: 2) or its active form, At least one extension section, Optionally, a linker (L) connects ISVD1 and ISVD2. 1-2 )and, A blood coagulation-promoting ISVD polypeptide derivative comprising, optionally, one or more extensions (E). 118. Having the following formula (from N-terminus to C-terminus), ISVD2-L 1-2 -ISVD1-E An ISVD polypeptide derivative according to Embodiment 117, wherein one or more extension portions are bound to one or more surface-exposed residues. 119. Having the following formula (from N-terminus to C-terminus), ISVD2-L 1-2 -ISVD1-E In the formula, the two extended portions are bound to one or more surface-exposed residues on E, An ISVD polypeptide derivative according to Embodiment 118 or 119, wherein the molecular weight of the derivative is in the range of 20 to 35 kDa. 120. The first ISVD can bind to an epitope on factor IX (SEQ ID NO: 1) or its active form, which includes amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering). An ISVD polypeptide derivative according to any one of embodiments 117 to 119, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) or its active form, comprising amino acid residues N173, P174, F175, L177, and L178 (sequential numbering). 121. The first ISVD, 1) CDR1: Optionally, IYTMS (sequence number 172) containing one or two substitutions. CDR2: Optionally, GLRWTDSSTEYADSVKG (SEQ ID NO: 173) containing one, two, or three substitutions. CDR3:Optionally, DRSFLFAQALGATKNYEY (SEQ ID NO: 174) containing one, two, or three substitutions, or 2) CDR1: Optionally, IYTMS (sequence number 156) containing one or two substitutions. CDR2: Optionally, GLRWTDSSTEYADSVKG (SEQ ID NO: 157) containing one, two, or three substitutions. CDR3:Optionally, DRSFLFAQALGATKNYEY (SEQ ID NO: 158) containing one, two, or three substitutions, or 3) CDR1: Optionally, IYTMS (sequence number 132) containing one or two substitutions. CDR2: Optionally, GLRWTDSSTEYADSVKG (SEQ ID NO: 133) containing one, two, or three substitutions. CDR3:Optionally, DRSFLFAQALGATKNYEY (SEQ ID NO: 134) containing one, two, or three substitutions, or 4) CDR1: Optionally, IYTMS (sequence number 116) containing one or two substitutions. CDR2: Optionally, GLRWTDSSTEYADSVKG (SEQ ID NO: 117) containing one, two, or three substitutions. CDR3:Optionally, DRSFLFAQALGATKNYEY (SEQ ID NO: 118) containing one, two, or three substitutions, or 5) CDR1: Optionally, IYTMS (sequence number 124) containing one or two substitutions. CDR2: Optionally, GLRWTDSSTEYADSVKG (SEQ ID NO: 125) containing one, two, or three substitutions. CDR3:Optionally, DRSFLFAQALGATKNYEY (SEQ ID NO: 126) containing one, two, or three substitutions, or 6) CDR1: Optionally, IYTMS (sequence number 108) containing one or two substitutions. CDR2: Optionally, GLRWTDSSTEYADSVKG (SEQ ID NO: 109) containing one, two, or three substitutions. CDR3: optionally includes DRSFLFAQALGATKNYEY (sequence number 110) with one, two, or three substitutions. The second ISVD said, (A) CDR1: Optionally, RYAMG (sequence number 168) containing one or two substitutions. CDR2:Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 169) containing one, two, or three substitutions. CDR3:Optionally, DDSVGDGYLDY (SEQ ID NO: 170) containing one, two, or three substitutions, or (B) CDR1: Optionally, RYAMG (sequence number 152) containing one or two substitutions. CDR2:Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 153) containing one, two, or three substitutions. CDR3:Optionally, DDSVGDGYLDY (sequence number 154) containing one, two, or three substitutions, or (C) CDR1: Optionally, RLAMG (sequence number 128) containing one or two substitutions. CDR2:Optionally, AISRRGGSTNYADSVKG (Sequence ID 129) containing one, two, or three substitutions. CDR3:Optionally, DDSVGDGYLDY (SEQ ID NO: 130) containing one, two, or three substitutions, or (D) CDR1: Optionally, RLAMG (sequence number 112) containing one or two substitutions. CDR2:Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 113) containing one, two, or three substitutions. CDR3:Optionally, DDSVGDGYLDY (sequence number 114) containing one, two, or three substitutions, or (E) CDR1: Optionally, RLAMG (sequence number 120) containing one or two substitutions. CDR2:Optionally, AISRRGGSTNYADSVKG (Sequence ID 121) containing one, two, or three substitutions. CDR3:Optionally, DDSVGDGYLDY (sequence number 122) containing one, two, or three substitutions, or (F) CDR1: Optionally, RLAMG (sequence number 104) containing one or two substitutions. CDR2:Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 105) containing one, two, or three substitutions. CDR3: An ISVD polypeptide derivative according to any one of embodiments 117 to 119, comprising DDSVGDGYLDY (SEQ ID NO: 106) (Kabat definition), which optionally includes one, two, or three substitutions. 122. The ISVD polypeptide derivative according to Embodiment 121, wherein the substitution is a conservative substitution. 123. The first ISVD contains the following sequence: V H H-2.20 (Sequence ID 171), V H H-2.18 (Sequence ID 155), V H H-2.15 (Sequence ID 131), V H H-2.13 (Sequence ID 115), V H H-2.14 (Sequence ID 123), or V H H-2.12 (Sequence ID 107) The ISVD polypeptide derivative according to any one of embodiments 117 to 122, wherein the second ISVD comprises the following sequence. V H H-1.20 (Sequence ID 167), V H H-1.18 (Sequence ID 151), V H H-1.15 (Sequence ID 127), V H H-1.13 (Sequence ID 111), V H H-1.14 (Sequence ID 119), or V H H-1.12 (Sequence ID 103) 124. Blood coagulation promotion V H H polypeptide derivative, Factor IX (SEQ ID NO: 1) or the first V that can bind to its active form H H and, A second V that can bind to factor X (SEQ ID NO: 2) or its active form. H H and, The first V H H and the second V H Linker connecting H (L 1-2 )and, C-terminal extension (E), Including one or two extensions, The following formula (from N-terminus to C-terminus) "The second V H H"-L 1-2 - "The first V H H"-E I) The first V H H is CDR1:IYTMS (Sequence ID 172), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 173) CDR3:DRSFLFAQALGATKNYEY (SEQ ID NO: 174) The second V H H is CDR1:RYAMG (Sequence ID 168) CDR2:AISRRGGSTNYADSVKG (Sequence ID 169) CDR3:DDSVGDGYLDY (Sequence ID 170) contains, or II) The first V H H is CDR1:IYTMS (Sequence ID 156), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 157) CDR3:DRSFLFAQALGATKNYEY (SEQ ID NO: 158) The second V H H is CDR1:RYAMG (Sequence ID 152), CDR2:AISRRGGSTNYADSVKG (Sequence ID 153) CDR3:DDSVGDGYLDY (Sequence ID 154) contains, or (Spro) The first V H H is CDR1:IYTMS (Sequence ID 132), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 133) CDR3:DRSFLFAQALGATKNYEY (SEQ ID NO: 134) The second V H H is CDR1:RLAMG (Sequence ID 128), CDR2:AISRRGGSTNYADSVKG (Sequence ID 129) CDR3:DDSVGDGYLDY (Sequence ID 130) contains, or IV) The first V H H is CDR1:IYTMS (Sequence ID 116), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 117) CDR3:DRSFLFAQALGATKNYEY (SEQ ID NO: 118) The second V H H is CDR1:RLAMG (Sequence ID 112), CDR2:AISRRGGSTNYADSVKG (Sequence ID 113) CDR3:DDSVGDGYLDY (Sequence ID 114) contains, or V) The first V H H is CDR1:IYTMS (Sequence ID 124), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 125) CDR3:DRSFLFAQALGATKNYEY (SEQ ID NO: 126) The second V H H is CDR1:RLAMG (Sequence ID 120), CDR2:AISRRGGSTNYADSVKG (Sequence ID 121) CDR3:DDSVGDGYLDY (Sequence ID 122) contains, or VI) The first V H H is CDR1:IYTMS (Sequence ID 108), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 109) CDR3:DRSFLFAQALGATKNYEY (SEQ ID NO: 110) The second V H H is CDR1:RLAMG (Sequence ID 104), CDR2:AISRRGGSTNYADSVKG (Sequence ID 105) CDR3:DDSVGDGYLDY (SEQ ID NO: 106) (Kabat definition) contains pro-coagulation V H H polypeptide derivative. 125. ISVD polypeptide derivative or V described in any one of Embodiments 117 to 124 H A pharmaceutical composition containing an H polypeptide derivative. 126. The pharmaceutical composition according to Embodiment 125, wherein the composition comprises a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid. 127. The 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). 128. A pharmaceutical composition according to any one of embodiments 125 to 127, further comprising nicotinamide (NAM). 129. The pharmaceutical composition according to any one of embodiments 125 to 128, wherein the composition is a solid composition. 130. ISVD polypeptide derivatives according to any one of Embodiments 117 to 129, for use in the treatment of hemophilia such as hemophilia A with or without inhibitors, or acquired hemophilia A. H H polypeptide derivatives or compositions. 131. The polypeptide derivative or composition is an ISVD polypeptide derivative for use according to any one of Embodiments 1 to 130, administered orally. H H polypeptide derivatives or compositions. 132. A method for increasing the oral bioavailability of a procoagulant immunoglobulin single variable domain (ISVD) polypeptide or an ISVD polypeptide derivative, comprising: a first ISVD (ISVD1) capable of binding to factor IX (SEQ ID NO: 1) or its active form; a second ISVD (ISVD2) capable of binding to factor X (SEQ ID NO: 2) or its active form; an extension; and optionally a linker (L) capable of linking ISVD1 and ISVD2. 1-2) and optionally one or more extensions (E), a. A step of modifying a nucleic acid encoding an amino acid residue of an ISVD polypeptide or an ISVD polypeptide derivative so that its isoelectric point is reduced, b. A step of culturing host cells to express nucleic acids encoding ISVD polypeptides or ISVD polypeptide derivatives, c. A step of collecting ISVD polypeptides or ISVD polypeptide derivatives from host cells, d. A step of purifying ISVD polypeptides or ISVD polypeptide derivatives from host cell cultures using standard chromatography, e. A method comprising the step of binding an extension portion to an ISVD polypeptide, unless such portion already exists.

[0283] In preferred embodiments, the ISVD polypeptide derivative is anti-FIX(a) V in the N-terminus to C-terminus direction. H Includes anti-FX ISVD connected to H ISVD.

[0284] In one preferred embodiment, the ISVD polypeptide derivative is anti-FIX(a)V in the N-terminus to C-terminus direction. H Anti-FXV connected to H fragment H This includes fragment H; see, for example, Figures 2a, 2b, and 3a-f.

[0285] In a preferred embodiment, ISVD or V can be coupled to FIX(a). H The H fragment selectively binds to activated FIX (FIXa).

[0286] In a preferred embodiment, ISVD or V can be coupled to FX. H The H fragment selectively binds to the FX enzyme precursor, i.e., inactive FX.

[0287] In one embodiment, the ISVD polypeptide (derivative) or V of the present invention HThe H polypeptide (derivative) can stimulate the enzymatic activity of FIXa toward FX. In one such embodiment, the ISVD polypeptide (derivative) or V H The stimulating properties of H polypeptides (derivatives) are that they can bind FIX(a) to ISVD or V H It originates from H.

[0288] Figures 5a and 5b show preferred anti-FIX(a) and anti-FX ISVD(V) respectively. H The sequence alignment of the H fragment sequence is shown, and the CDR sequence is highlighted in bold and underlined.

[0289] The present invention is subject to the ISVD or V disclosed herein. H The H fragment substitution variants are included, and they may contain 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 the individual sequences disclosed herein. In some embodiments, such substitutions and / or deletions and / or insertions are in one or more of the CDR sequences disclosed herein. In some embodiments, one or more of the CDR sequences are subjected to amino acid substitutions. In some embodiments, each individual CDR sequence may contain 0, 1, 2, or 3 amino acid substitutions. For example, V by SEQ ID NO: 559 H In the H fragment, CDR1 may not contain substitutions, CDR2 may contain three substitutions, and CDR3 may contain one substitution. The "substituted" variant preferably contains one or more amino acid substitutions having the same number of amino acids. In some embodiments, the substitutions are conservative substitutions.

[0290] In one embodiment, V H ISVD polypeptide derivatives, such as H polypeptide derivatives, include at least one extension that cannot bind to plasma membrane components such as aminophospholipids, such as phosphatidylserine and / or phosphatidylethanolamine.

[0291] In some embodiments, the extension includes the following structure: [ka] "*" is V H This represents the binding site to the H polypeptide.

[0292] In one embodiment, V H ISVD polypeptide derivatives such as H polypeptide derivatives include GPlb-1X, collagen chaperone HSP47, ephrin B1, thiol isomerase protein ERP5, 55 hematopoietic progenitor kinase 1-interacting protein (HIP-55), glycoprotein V1, platelet glycoprotein 1b, platelet-derived growth factor receptor, platelet endothelial aggregation receptor I, CD31, CD36, MARKS, multimelin, integrin alpha Ib / beta 3, trigger receptor (TREM)-like transcript-1 (TLT-1) expressed in bone marrow cells, integrin-binding kinase (ILK), dixin, collagen, P-selectin, and XII Includes an extended portion that cannot bind platelet surface proteins such as Factor I, P-selectin glycoprotein ligand-1, integrin alpha-6 beta-1, thrombospondin, von Willebrand factor, G6B, CD42b, syntaxin-binding protein 2, phosphatidylethanolamine, fibrinogen / fibrin, filamin, stomatin, sphingolipids, CD31, CD36, CD40, CD41, CD42c, CD42, CD49b, CD61, CD62P, CD63, CD69, CD107a, CD107b, CD109, CD154, PECAM-1, and / or ERPS.

[0293] In one embodiment, V H ISVD polypeptide derivatives, such as H polypeptide derivatives, include an extended portion that cannot bind glycoproteins, GPIIb / IIIa, β2GP1, TLT-1, selectins, coagulation factors or coagulation factor complexes, and / or membrane-related polypeptides such as selectins.

[0294] In one embodiment, V HISVD polypeptide derivatives, such as H polypeptide derivatives, do not interfere with the effects of recombinant FVIII or other FVIII antibodies administered to patients with hemophilia A when used at clinically relevant doses in the treatment of hemophilia A.

[0295] While certain features of the present invention are illustrated and described herein, many modifications, substitutions, alterations, and equivalents will be conceivable to those skilled in the art. It should therefore be understood that the appended claims are intended to encompass all such modifications and alterations that fall within the true spirit of the invention. [Examples]

[0296] List of Abbreviations [Table 4]

[0297] Example 1: General method and V H Preparation of H fragments and other recombinant proteins General molecular biology For general molecular biology techniques, see Molecular Cloning: A Laboratory Manual (4 th See Edition, 2014, Sambrook, Fritsch and Maniatis eds., CSHL Press, Cold Spring Harbor, NY, USA.

[0298] Immunization and Library Following approval from the Ethical Committee of the Ablynx Camelid Facility (LA1400575), two llamas and two alpacas were immunized with human FIX and FX (Haemotologic Technologies, VT USA), respectively.

[0299] Cloning of antibody fragment repertoire consisting solely of heavy chains and preparation of phage immunotherapy libraries were carried out as follows.

[0300] Blood samples were collected after the final immunogen injection. 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 the PBMCs and used as a starting material for RT-PCR, essentially as described in International Publication No. 2005 / 044858. H The H fragment coding DNA segment was amplified. In short, V H H fragment coding DNA fragments were cloned into the phagemide vector pAX212 and fused with His6 and FLAG3 tags. H This enabled the generation of phage particles displaying the H fragment. Subsequently, the phages were prepared and stored according to a standard protocol.

[0301] Synthesis Library The synthetic library was obtained from immunization and cloned synthetic V H This was generated by cloning the H gene fragment into the phagemide vector pAX190. It has the same characteristics as the aforementioned pAX212, but differs in several cloning sites.

[0302] V to combine with FX or FIX H Library screening of H fragments V H H fragment phage display selection was performed on the generated immunoassay and synthetic libraries. The libraries underwent 1 to 4 consecutive enrichment rounds to different concentrations of immobilized human FIX and FX (Haemotologic Technologies, VT USA) and cynomolgus monkey FIX and FX (Novo Nordisk in-house produced) to enrich clonal screening for binders to FIX and FX.

[0303] V selective for FIXa and FXa H To specifically enrich the H fragments, in certain experiments, excess soluble FIX and FXa were used due to competition during library incubation with immobilized FIXa and FXa.

[0304] V is selective for FIX and FX compared to other structurally related coagulation factors. H To specifically enrich the H fragments, in certain experiments, excess soluble FX and FIX were used, respectively, due to competition during library incubation with immobilized FIX and FX.

[0305] Approximately 4500 distinct clones from the selected output were screened for conjugation to human and cynomolgus monkey FX, FXa, FIX, and FIXa via ELISA (V H (Using periplasmic extracts from E. coli cells expressing the H fragment). Approximately 1500 clones were identified that showed specific binding to human FX and FIXa / FIX, the majority of which showed cross-binding to cynomolgus monkey FX and FIX. Some clones showed selective binding to FXa for FX and to FIXa for FIX. Sequence analysis of ELISA-positive clones showed that V binds to FIXa / FIX or FX / FXa. H Approximately 700 unique sequences of the H fragment were identified. To further optimize the compound, we added extension factors, linkers, and combined the extensions with aggressive V H Mutagenesis of H CDR and FR is performed with a focus on enhancing hemostatic efficacy through mutation screening using thrombin generation assays, target-mediated drug elimination is avoided through screening of compounds with altered affinity, immunogenicity is reduced through screening of risk sites using MHC-related peptide proteomics assays, and in vivo oral bioavailability is enhanced via pI that reduces substitution of surface-exposed residues.

[0306] V to combine with FX or FIX HH fragment expression construct generation V from phage display selected output H Sequence analysis of the H fragment was performed according to a generally known procedure (Pardon et al. (2014) Nat Protoc 9:674).

[0307] V obtained by PCR using specific combinations of forward FR1 and reverse FR4 primers, each carrying a unique restriction site. H The H fragment-containing DNA fragment is digested with an appropriate restriction enzyme and expressed in a His6- and / or FLAG3-tagged format for E. coli or P. pastoris expression. H The ligation mixture was then ligated into matching cloning cassettes containing H polypeptide expression vectors. The ligation mixture was then transformed into electrocompetent Escherichia coli TG1 (60502, Lucigen, Middleton, WI) or TOP10 (C404052, ThermoFisher Scientific, Waltham, MA), which were then grown under appropriate antibiotic selective pressure (kanamycin or zeosin). Resistant clones were validated by Sanger sequencing of plasmid DNA (LGC Genomics, Berlin, Germany).

[0308] V that connects to FX or FIX in E.coli H General expression of H fragments V H The H fragment is preceded by the coding sequences of E. coli TG1, kanamycin resistance gene, E. coli source, and OmpA signal peptide from a plasmid expression vector containing the lac promoter. H It was expressed at the H fragment cloning site. H In frame with the H polypeptide coding sequence, the vector encodes the C-terminal FLAG3 and His6 tags. The signal peptide is expressed in the V H The H fragment is guided to the periplasmic compartment of the bacterial host.

[0309] Target V H E. coli TG-1 cells containing the H fragment construct were grown in a baffled shaking flask containing "5052" auto-incubation medium (0.5% glycerol, 0.05% glucose, 0.2% lactose + 3 mM MgSO4) for 2 hours at 37°C, followed by 29 hours at 30°C. Cell pellets from the E. coli expression culture, frozen overnight, were then dissolved in PBS (1 / 12.5 of the original culture volume) and incubated at 4°C for 1 hour with gentle agitation. Finally, the cells were pelleted again, and the supernatant containing secreted proteins into the periplasmic space was stored.

[0310] V in P. pastoris H General expression of H fragments Target V H P. pastoris cells containing the H fragment construct were grown in BGCM medium for 2 days (30°C, 200 rpm). On the 3rd day, the medium was switched to BMCM, and the construct was further grown (30°C, 200 rpm), and induced with 0.5% v / v methanol after 8 hours. The following day, the construct was induced with 0.5% v / v methanol in the morning, at noon, and in the evening. On the 5th day, the cells were centrifuged, and the supernatant (secreted V) was analyzed. H We collected (containing H fragments).

[0311] In HEK cells, V binds to FX or FIX and other recombinant proteins. H General expression of H and antibodies Expression plasmids for transient expression in HEK293 cells were purchased from either Twist Biosciences or Thermo Fisher 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 Thermo Fisher Scientific was based on the pcDNA34-Topo vector (Thermo Fisher Scientific). His6-tagged or untagged V H H polypeptide compound, V H HEK293 suspension cells were transfected for transient expression of H polypeptide compounds. Equivalent expression constructs carrying sequences encoding anti-GLA FIX antibody and anti-GLA FX antibody (Novo Nordisk in-house), emicizumab (Hoffmann-La Roche Ltd, Switzerland) sequence identity analog (SIA), and Mim8 (Novo Nordisk, Denmark) were also expressed in HEK293 using the method described below.

[0312] Transient transfection of HEK293 suspension cells (Expi293 expression system, Thermo Fisher Scientific, catalog no. A14635) was performed essentially according to the manufacturer's instructions. HEK293 cells were typically subcultured every 3-4 days in Expi293 expression medium (Gibco, catalog no. A14351-01) supplemented with 1% P / S (GIBCO, catalog no. 15140-122). HEK293 cells were transfected with Expifectamine at a cell density of 2.5-3 million / mL. Transfection was performed by diluting a total of 1 mg of plasmid DNA in 50 mL of Optimem (GIBCO, catalog no. 51985-026, dilution A) per liter of HEK293 cells, and by diluting 2.7 mL of Expifectamine in 50 mL of Optimem (dilution B). The transfection was performed. For co-transfection (i.e., antibody), plasmids were used in a 1:1 ratio. Dilutions A and B were mixed and incubated at room temperature for 10-20 minutes. After this, the transfection mixture was 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 ExpiFectamine293 transfection accelerator 1 and 50 ml of ExpiFectamine293 transfection accelerator 2. Cell culture supernatant was typically collected 4-5 days after transfection by centrifugation and then filtered.

[0313] V that binds to FX and FIX(a) in CHO cells H General expression of H fragments and antibodies Anti-FIX and anti-FX V HH compounds and antibodies were produced in Chinese hamster ovary (CHO) cells using glutamine synthase (GS) selection. CHO cells were transfected with GS expression plasmids using electroporation and then subjected to selection using glutamine depletion along with MSX supplement in CD-CHO medium (Thermo Fisher Scientific, catalog no. 10743029). A stable CHO cell pool was typically obtained after 3 weeks of culture, and the pool was subsequently subjected to single-cell cloning into 384-well plates. CHO clones resulting from the 384-well plates were typically grown into 96-well plates and screened for productivity. Using predefined proprietary cell culture media (Novo Nordisk A / S), selected productive clones were scaled up for culture in bioreactors from 1-L to 15-L scales. Cell viability remained high during culture and then gradually decreased until cell culture harvesting. Here, the cell supernatant was removed by centrifugation and / or depth filtration using MD0HC23CL3 and MX0HC01FS1 filters (Millipore), depending on the culture scale, and then chromatography-based protein purification was performed.

[0314] V to combine with FX or FIX H General purification and characterization of H fragments His6 tagged or untagged V H The H compound was purified using either MabSelectSure Protein-A resin (Cytiva) or immobilized metal affinity chromatography (IMAC) with either imidazole (in the latter case) or acid-eluting Ni-Excel (Cytiva) resin (in the former case), followed by a desalting step (PD column using Sephadex G25 resin, Cytiva), and, if necessary, gel filtration chromatography in PBS or HBS (Superdex200 column, Cytiva). Non-His6 tagged V HThe H compounds, as well as antibodies targeting the GLA domains of FIX and FX (developed in-house by Novo Nordisk), respectively, and emicizumab SIA (Hoffmann-La Roche Ltd, Switzerland) were purified using acid-eluting Protein-A resin MabSelectSure (Cytiva) or multimodal resin (Cytiva). This was followed by a desalting process (e.g., using a PD column containing Sephadex G25 resin, Cytiva), and, if necessary, gel filtration chromatography in PBS or HBS (Superdex200 column, Cytiva). Protein integrity was analyzed using size exclusion high-performance liquid chromatography (SE-HPLC) on an Agilent LC 1100 / 1200 system with a BIOSEP (Column for Separation of Biomolecules)-SEC-53000 300×7.8 mm column (Phenomenex, catalog no. OOH-2146-K0) and a running buffer consisting of 200 mM sodium phosphate pH 6.9, 300 mM NaCl, and 10% isopropanol. Purified V H The molecular weight of the H polypeptide batch was analyzed using electrospray ionization and time-of-flight mass spectrometry (ESI-TOF-MS) on a 6280 Agilent system (Agilent Technologies) with a MassPREP Desalt (Waters) column running at 0.4 ml / min in buffer A consisting of MQ-H2O / 0.1% formic acid and buffer B consisting of acetonitrile / 0.1% formic acid for stepwise elution. To determine the final protein concentration, a NanoDrop® spectrophotometer (Thermo Scientific) was used with theoretically calculated extinction coefficients.

[0315] Example 2: V H Conjugation of the extended portion to the H polypeptide cysteine ​​residue V H To extend the H polypeptide, V HThe H polypeptide compounds were manipulated to include cysteine ​​(Cys) substituents in various skeletal positions, for example, in N-terminal or C-terminal extensions having one or two introduced Cys residues used for conjugation with one or more fatty acid extensions, as further described below.

[0316] V H An intermediate reagent in the form of a modified extended portion was used to bind to the H polypeptide compound.

[0317] The intermediate reagent, including the extended portion, was prepared as described in International Publication No. 2016 / 102562, and a non-limiting example of such an intermediate reagent is shown in Table 3 below. [Table 5-1] [Table 5-2]

[0318] Conjugation, purification, and analysis V has one or more introduced cysteine ​​residues for conjugation. H To an aqueous solution containing H polypeptide, 5 equivalents of BSPP (bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt) or 1.1 equivalents of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) were added per capped cysteine. After stirring for 1-2 hours, the pH was adjusted to 8.5 with an aqueous NaOH solution, and each V H For each free cysteine ​​in the H polypeptide, for example, 5 equivalents of intermediate reagent C1 in 0.1 M NaHCO3 (aqueous solution) were added. The mixture was gently stirred in the dark for 1.5 to 16 hours. The reaction mixture was diluted with water before purification by anion exchange (AIEX) using an Akta system. V containing side chain conjugation HThe H polypeptide was purified using AIEX chromatography. Therefore, an AIEX resin Source 30Q packed into a suitable column was used with a sodium chloride gradient program set up in an Akta Avant chromatography system. The buffer systems used were an equilibrium buffer consisting of 20 mM Tris (pH 8.5) and an elution buffer consisting of 20 mM Tris and 1 M NaCl (pH 8.5). The reaction mixture was adjusted to pH 8.5 and diluted to a conductivity of less than 4 mS / cm using MilliQ-H2O or equilibrium buffer. The sample was applied to the column, and the column was washed after applying 5–10 column volumes of equilibrium buffer. Separation chromatography was then performed using a shallow gradient of 30–50 column volumes. The gradient used was for the purified V polypeptide. H Depending on the pI of the H polypeptide derivative, it ranged from 0% to a maximum of 50%. Generally, unconjugated parent V H H polypeptides elute early in the gradient and contain a single conjugation V H H polypeptide is eluted during the gradient, V H V includes multiple conjugations, meaning more than one side-chain conjugate per H polypeptide molecule. H H polypeptides eluted later in the gradient. Pooling of fractions on the main peak was performed in a certain manner, resulting in V containing single or double conjugate preparations with high purity of 90–99%. HH polypeptide was obtained. Purity analysis was performed using reversed-phase ultrahigh performance liquid chromatography (RP-UPLC) based on a HALO DiPhenyl column 1000 Å, 2.7 μm, 150 × 2.1 mm (Scantec Nordic USDPF001316) set up in a Waters Acquity UPLC system with UV and FLD detectors, and running buffers consisting of A) 0.1% v / v TFA in water and B) 0.09% v / v TFA in acetonitrile. The column temperature was set to 60°C. The gradient program was as follows: 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 H polypeptide was eluted between 4.6 and 4.8 minutes. V containing a single conjugation H The H polypeptide derivative eluted between 5.1 and 5.6 minutes, with a main peak observed at approximately 5.3 minutes. The V with double conjugation... H H polypeptide derivatives are off-site multiple conjugated V H For H polypeptides, elution occurred from 5.7-5.8 onwards. V containing conjugation. H The integrity of the H polypeptide was analyzed based on an SE-HPLC method using an Agilent LC 1100 / 1200 system and a BIOSEP-SEC-3000 300×7.8 mm column (Phenomenex, catalog no. OOH-2146-K0) and running buffer consisting of 200 mM sodium phosphate (pH 6.9), 300 mM NaCl, and 10% isopropanol. HThe molecular weight of the H polypeptide was analyzed using ESI-TOF-MS on a 6280 Agilent system (Agilent Technologies) with 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 stepwise elution. Peptide mapping for sequence validation 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. Online LC-MS analysis of the digests was performed using a CSH C-18 column, 1.7 μm, 150 × 2.1 mm, and an acetonitrile / formic acid gradient. The column temperature was 60°C. Buffer A: 0.1% FA water and Buffer B: 0.1% FA acetonitrile. The gradient program was as follows: 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, at a flow rate of 120 μl / min. Data were analyzed using the Genedata Refiner peptide mapping workflow. Complete primary sequence coverage was obtained. V using conjugation H To measure the protein concentration of batch preparations of H polypeptide derivatives, a NanoDrop® spectrophotometer (Thermo Scientific) was used with theoretically calculated extinction coefficients.

[0319] Figures 1, 2, and 3 show ISVD- and V H Non-limiting examples of H polypeptide derivatives are shown, along with increasing levels of detail.

[0320] Example 3: Sequence listing and anti-FX / anti-FIX(a)V H Hpolymer [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0321] Example 4: Anti-FX V based on ELISA experiment H H polypeptide epitopes Anti-FX V, indicated as "Compound number 1" H An ELISA assay was set up to determine whether the H polypeptide binding epitope is located inside the human FX (SEQ ID NO: 2) activating peptide (AP). Forty-one unique 12-mer peptide fragments spanning 52 residues of the activating peptide with a one-amino acid interval were immobilized in microtiter plate wells, and then the V was tested. H The H polypeptide was incubated with the antibody, and the bound ligand was detected by adding a secondary HRP-labeled antibody.

[0322] The peptide was C-terminated to biotin and immobilized in individual wells of a microtiter plate pre-coated with 1 μg / mL streptavidin using 50 μL of 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 subsequently, 50 μL of 2 μg / mL FLAG-tagged anti-FX V to be tested was added. H H polypeptide or IgG antibody was added. After 1 hour, unbound V H The H polypeptide was washed using a washing buffer.

[0323] First, an HRP-labeled secondary antibody (FLAG-tagged V) H H polypeptides were bound to 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 to detect the bound anti-FX activating peptide ligand.

[0324] Next, by identifying a common sequence coated by a set of peptides bound by each FX binder (Table 6), the minimum epitope was estimated from a set of peptides that produced a signal above baseline. The first residue of the epitope was defined as 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.

[0325] Therefore, the following table shows the initial anti-FX V compounds tested, corresponding to compound number 1. H For H polypeptides, the ELISA signal from the entire FX AP sequence is shown. The ELISA signal is normalized to the background signal.

[0326] The gray area marks the positive binding signal used to determine the minimal epitope identified as sequence NPFDLLDF (sequence number 692).

[0327] The identified epitope sequence of the FX-activating peptide relative to compound number 1 was used in a high-resolution structure to determine the epitope-paratope interaction experiment, as outlined in Example 6. [Table 8]

[0328] Example 5: Anti-FIX V H Crystallization and paratope / epitope mapping of H fragment (compound number 2). The purpose of this study is to investigate compound number 2 and designated V H The objective was to determine the paratope and epitope residues of the H fragment.

[0329] crystallization Human EGR-CMK inhibitor factor IXa Gla domain-less (wild type) (purchased from Cambridge ProteinWorks, lot number hGDFIXAWTEGR_11) mixed with V in a 1:1 molar ratio H Crystals of the H polypeptide (compound number 2) were grown at 18°C ​​using the sitting-drop vapor diffusion technique. A 100 nl solution of the 8.4 mg / ml complex protein 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), and 30% (w / v) PEG 4000 (as a precipitant), and incubated on 60 μl of precipitant. Crystals appeared within two weeks.

[0330] Diffraction data collection The crystals were cryoprotected by adding 1 μl of a precipitant containing 20% ​​ethylene glycol to the crystallization drop before flash cooling with liquid nitrogen. Diffraction data were collected at 100 K using a Dectris Pilatus 2M pixel detector at the Swiss Light Source beamline X06DA (1.0000 Å wavelength). Automated indexing, merging, and scaling of the data were performed using a program from the XDS package (diffraction data statistics are summarized in Table 7).

[0331] Determination and refinement of the structure The asymmetric unit contains two compound number 2:EGR-CMK inhibitor factor IXa Gla domain-less complexes, as determined by Matthews coefficient analysis. The structure is a predetermined V as an exploratory model. H The structure of the H:FIXa complex was determined by molecular substitution using Phaser, implemented within the Phenix program suite. The correct amino acid sequence was modeled using COOT, and the structure was then refined using the Phenix refinement process and manual reconstruction process in COOT. Refinement statistics are shown in Table 7. [Table 9]

[0332] Epitope and paratope determination V H The epitope of compound number 2, defined as a FIX(a) residue characterized by having a heavy atom (i.e., a non-hydrogen atom) within 4.0 Å of a heavy atom in the H polypeptide, is the following residue from the protease domain of FIX(a) by the FIX sequence: Includes E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering) (Sequence ID 1).

[0333] The paratope of compound number 2, defined as a residue characterized by having a heavy atom (i.e., a non-hydrogen atom) within 4.0 Å of a heavy atom in FIX(a), is the following residue from compound number 2: Includes F29, N30, Y32, T54, D99, R100, S101, F102, L103, F104, Q106, A107, and N113 (sequence number 35) (sequential numbering).

[0334] Example 6: Crystallization and Paratope / Epitope Mapping Anti-FX V H H fragment (compound number 1) The purpose of this study is to identify compound number 1 and V H The objective was to determine the paratope and epitope residues of the H fragment.

[0335] crystallization V was mixed with the synthetic peptide N-term-NPFDLLD-C-term in a molar ratio of 1:14, corresponding to the activation peptide sequences aa31-37 of human FX (purchased from Schafer-N ApS) identified in Example 4. H Crystals of the H polypeptide (compound number 1) were grown at 18°C ​​using the sitting-drop vapor diffusion technique. A 150 nl solution of the 4.0 mg / ml complex protein in 20 mM Tris-HCl (pH 7.4) and 50 mM NaCl was mixed with 50 nl of 0.2 M NaCl, 2 M ammonium sulfate, and 0.1 M sodium cacodylate (pH 6.5) (as a precipitant), and incubated with 60 μl of the precipitant. Crystals appeared within one week.

[0336] Diffraction data collection The crystals were cryoprotected by adding 1 μl of a precipitant containing 20% ​​ethylene glycol to the crystallization drop before flash cooling with liquid nitrogen. Diffraction data were collected at 100 K using a Dectris Pilatus 2M pixel detector at the Swiss Light Source beamline X06DA (1.0000 Å wavelength). Automated indexing, merging, and scaling of the data were performed using a program from the XDS package (diffraction data statistics are summarized in Table 8).

[0337] Determination and refinement of the structure The asymmetric unit contains one compound number 1:peptide complex. The structure is designated as a search model in the Protein Databank ID 4B41 (chain A). H The H polypeptide structure was determined by molecular substitution using Phaser, implemented within the Phenix program suite. The correct amino acid sequence of compound number 1 was introduced, and the differences in electron density of the synthetic peptide were identified; all models were manually constructed using COOT. The structure was then refined in COOT using Phenix refinement and manual reconstruction steps. Refinement statistics are shown in Table 8. [Table 10]

[0338] Epitope and paratope determination V H The epitope of the synthetic peptide, defined as an FX-activating peptide residue characterized by having a heavy atom within 4.0 Å of a heavy atom in the H polypeptide (compound number 1), includes the following residues from synthetic peptide N173, P174, F175, L177, and L178, according to the corresponding FX-activating peptide sequence (sequence number 2) based on sequential numbering.

[0339] V is defined as a residue of compound number 1 characterized by having a heavy atom (i.e., a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the synthetic peptide. HThe paratope of the H polypeptide (compound number 1) is derived from the following residues of compound number 1: Includes D32, A33, M34, G35, Y37, L47, V48, A49, G50, I51, M52, N57, T58, N59, Y60, T61, K97, V99, R101, and P102 (sequence number 27) (sequential numbering).

[0340] V complexed with synthetic peptides H We constructed a homology model for the H polypeptide (compound number 3) because compound number 3 is a parent of the parent polypeptide for rational sequence-based and structure-based optimization. H This is because it was used as an H polypeptide sequence.

[0341] The crystal structure of compound number 1, complexed with the synthetic peptide from the above experiment, was used as the starting model for the homology model of compound number 3, complexed with the synthetic peptide. The amino acid sequences of compound number 1 and compound number 3 were aligned, and different amino acid residues between the two sequences were mutated using COOT to create the starting model for compound number 3. This model, including compound number 3, was pre-treated, optimized, and performed through suppressed minimization in MAESTRO from SCHRODINGER.

[0342] This model showed the same epitope and paratope residues as described above because the sequences were identical at these positions.

[0343] Example 7: Anti-FX / Anti-FIX(a)V H SPR analysis of H polypeptides The purpose of this study is to identify selected anti-FX / anti-FIX(a)V cells containing human plasma-derived FX and Benefix® using surface plasmon resonance (SPR) analysis. H The objective was to estimate the binding constant of H polypeptide compounds.

[0344] Purified anti-FX / FIX(a)V HThe binding of H polypeptide compounds to human plasma-derived FX (Haematologic Technologies Inc., USA) was probed using SPR.

[0345] In short, the recombinantly prepared anti-GLA-FX was immobilized on a CM4 sensor tip or 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 FX were measured according to Table 9. H The H polypeptide compound was injected at a flow rate of 50 μL / min for 200 seconds to allow binding to FX, followed by a 10-minute injection of 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) to allow dissociation from FX. The running buffer was also used to contain anti-FX V H It was also used for diluting H polypeptide compounds. Chip regeneration was achieved 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 a 1:1 model with BiaEvaluation 4.1 supplied by the manufacturer (Biacore AB, Uppsala, or Bruker Analyser).

[0346] In all cases, the combined sensorogram is based on K based on motion analysis. D The decision excluded rapid on and rapid off behaviors coupled to the motor profile. Therefore, the reported K D The values ​​were determined based on steady-state analysis. The analysis yielded the coupling constants reported in Table 9 below.

[0347] Purified anti-FX / FIX(a)V HThe binding of H polypeptide compounds to Benefix® (Pfizer Inc, USA) was probed by SPR. Briefly, recombinant anti-GLA-FIX, prepared as described above, was immobilized on a CM4 sensor tip or 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 H The H polypeptide compound was injected at a flow rate of 30 μL / min for 4 minutes to allow binding to FIX (Benefix®), followed by a 5-minute injection of running buffer (10 mM HEPES, 150 mM NaCl, 5 mM CaCl2, 0.05% (v / v) Surfactant P20, 1 mg / mL bovine serum albumin, Ph7.4) to allow dissociation from FIX. The running buffer also contained anti-FIX V H It was also used for diluting the H polypeptide compound. Chip regeneration was achieved 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 a 1:1 model with BiaEvaluation 4.1 supplied by the manufacturer (Biacore AB, Uppsala).

[0348] In all cases, the combined sensorogram is based on K based on motion analysis. D The decision excluded rapid on and rapid off behaviors coupled to the motor profile. Therefore, the reported K D The value is determined based on steady-state analysis.

[0349] The steady-state binding analysis of a series of compounds is reported in Table 9. The binding affinity of FIX was generally low nM K for all compounds. D On the other hand, a certain range of compounds have high nM K for FX bonding. DThis indicates that target-mediated drug placement is not expected to be present during FX, while FIX interactions are expected, but limited to the required steady-state plasma concentrations of the listed compounds. Therefore, the required plasma concentrations expected to obtain significant hemostatic coverage for the listed compounds are expected to be low nM concentrations, given the high potency of the listed compounds. [Table 11]

[0350] Example 8: Anti-FX / anti-FIX(A)V in the Thrombin Generation Test (TGT) assay H Activity of H polypeptide Anti-FX / Anti-FIX(a)V H The procoagulant activity of H polypeptide derivatives was determined based on their ability to promote thrombinogenesis in the presence of a procoagulant synthetic phospholipid membrane, according to the principle described by Hemker et al. (Pathophysiol Haemost Thromb, 2002;32:249-253). For comparison, emicizumab sequence-identical analogs (SIAs) were included. Each V H H polypeptide derivatives were tested using a TGT assay with normal human platelet-deficient plasma (NHP) supplemented with a neutralizing anti-FVIII polyclonal antibody (hereinafter referred to as HA-PPP).

[0351] TGT in NHP (from healthy volunteers) supplemented with sheep anti-human FVIII polyclonal antibody (pAb, Haematologic Technologies Inc., VT, USA) was performed using standard calibrated automated thrombography with a 96-well plate fluorometer (Fluoroscan Ascent FL, Thermolabsystems, Helsinki, Finland). The reaction mixture contains 0.1 μg / ml of anti-FVIII pAb, 4 μl of a test compound dilution (diluted with 20 mM HEPES, 140 mM NaCl, pH 7.4, and 2% BSA), 10 μl of either 1 pM tissue factor (TF, pppLow, Thrombinoscope BV, Maastricht, The Netherlands) or 1–8.3 U / ml human factor XIa (Enzyme Research Laboratories, IN, USA), and 36 μl of NHP pre-incubated with 10 μl of FluCa substrate (Thrombinoscope BV, Maastricht, The Netherlands). The TGT assay was calibrated using a thrombin calibrator (Thrombinoscope BV, Maastricht, The Netherlands). 10 μl of thrombin calibrator was mixed with 36 μl of NHP pre-incubated with 0.1 μg / ml anti-FVIII pAb, 4 μl of buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA). Generally, TGTs were performed with eight concentrations of the test compound (0.1, 0.3, 1, 3, 10, 30, 100, and 300 nM, final plasma concentrations or similar) or with only the supplemental buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) (representing the control). Normal control levels in the TGT were measured using only the NHP supplemental buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA). TGT was administered for a total of 60 minutes, and the peak thrombin height (nM) of the TGT parameters was analyzed using Thrombinoscope software (Thrombinoscope BV). Selected experiment V HFor representative titration curves of the H polypeptide derivative, compound number 6, and the comparators emicizumab SIA and Mim8, please refer to Figure 4. The difference in activity levels was evaluated using the EC25 titration of the comparator, e.g., against emicizumab SIA. 50 The difference, or given V relative to the maximum thrombin peak height for emicizumab SIA H This was performed as the difference in maximum thrombin peak height for H polypeptide compounds. The latter approach was used for more V H It was used to screen H polypeptide derivatives.

[0352] V H To boost the activity of H polypeptide derivatives, the optimal combination of CDR mutations was identified to produce the maximum activity effect. Using crystal structure models such as those described in Examples 5 and 6, key paratope residues were identified, as well as surface-exposed residues suitable for substitution with cysteine, which were then pI-decreasing substitutions to enhance bioavailability. Different linkers and different conjugation types were also investigated. The following table shows a series of Vs dealing with each of these parameters. H The activity profiles of the H polypeptide derivatives are outlined and then described individually.

[0353] Table 10 shows optimized V for activity-boosting mutations. H Efficacy EC obtained from H polypeptide derivatives 50 The values ​​are shown. The computational algorithms for the main components and the random forest algorithm were used for the mutation combinations to boost activity. V H The H polypeptide (compound number 24) is an initial derivative that does not have boost mutations, and this V H Compared to H polypeptide derivatives and emicizumab SIA, it exhibits up to 40 times and up to 76 times higher potency, respectively, making it the best developed V H It was obtained among H polypeptide derivatives. [Table 12]

[0354] Table 11 shows substitution with cysteine ​​(linker L P After conjugation with the C18 diacid fatty acid extension P3 linked via 1 (with and without), V H V that retains the activity of H polypeptide H Screening was performed to determine the optimal conjugate site on the H polypeptide (outside CDR1-3).

[0355] A total of 95 surface exposure sites were tested using Cys substitution. Sequential numbering was used with compound numbers 23 / 24 as the base (V that does not contain any boost mutations in the CDR). H Based on the H polypeptide (represented by the residue substitution), optimal conjugation sites were screened. 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, G 83C, R84C, F85C, T86C, S88C, D90C, N91C, S92C, K93C, T95C, Y97C, Q99C, M100C, N101C , S102C, L103C, R104C, P105C, E106C, D107C, T108C, G156C, V157C, V158C, Q159C, P16 0C, G161C, G162C, S163C, L164C, R165C, S167C, A169C, S171C, R184C, Q185C, A186C, P 187C, G188C, K189C, E190C, R191C, Y206C, A207C, D208C, S209C, V210C, K211C, G212C R213C, F214C, T215C, S217C, D219C, N220C, S221C, K222C, T224C, Y226C, Q228C, M229C, N230C, S231C, L232C, R233C, P234C, E235C, D236C, T237C, and Cys introduced in the C-terminal extension.

[0356] Of these, the following 41 V H H polypeptides can be expressed using the HEK293 expression system and successfully conjugate with reagent C3 containing the C18 diacid extension: Q30C, P31C, Q56C, A57C, K60C, E61C, A78C, D79C, K82C, N91C, S92C, Q99C, R104C, G156C, V158C, Q159C, P160C, G 161C, G162C, S163C, A169C, S171C, Q185C, A186C, P187C, G188C, K189C, E190C, A207C, D208C, S209C, V210C, T215C, S217C, D219C, N220C, S221C, K222C, S231C, R233C, Cys introduced in the C-terminal extension. These 41 V with and without conjugation. H The activity of the H polypeptide was tested using a TGT assay. V with and without conjugation was tested. H V of H polypeptide (derivative) relative to the maximum thrombin peak height of emicizumab SIA H The relative activity levels, expressed as the maximum thrombin peak height of the H polypeptide derivatives, are shown in Table 11. Therefore, based on the activity retained before and after conjugation, the following nine sites were identified as optimal for introducing free Cys for conjugation with the extension, for example, using C3 as a reagent: Cys introduced into the C-terminal extension (compound numbers 23 / 24), Q30C (compound numbers 25 / 26), V158C (compound numbers 27 / 28), P160C (compound numbers 29 / 30), G161C (compound numbers 31 / 32), G188C (compound numbers 33 / 34), E190C (compound numbers 35 / 36), S209C (compound numbers 37 / 38), and S231C (compound numbers 39 / 40). [Table 13-1] [Table 13-2] [Table 13-3]

[0357] Two anti-FIX and anti-FX V H Different linker L fragments are recombinantly fused between H fragments. 1-2 The composition was optimized through testing (see Table 12). H V as a function of the maximum thrombin peak height of H polypeptide emicizumab SIA H Table 12 shows the relative activity level, expressed as the maximum thrombin peak height of the H polypeptide. From these data, the linker-containing V of the hydrophobic composition is found. H H polypeptides generally showed the highest relative activity, but V polypeptides with linkers in acidic or hydrophilic compositions H The H polypeptide showed the lowest activity. V with a linker in a mixed composition consisting of QAPGQA, GQAPGQ, or similar. H The H polypeptide exhibited moderate relative activity. Since the optimal linker composition preferably contains both hydrophilic and hydrophobic amino acids, the chemical stability of the QAPGQA linker was further investigated using MS analysis, which showed high stability, for example, in isotonic buffer and neutral pH at 37°C for 2 weeks under accelerated stress conditions.

[0358] Therefore, a ratio of hydrophobic amino acids to hydrophilic amino acids of "40-60%" to "60-40%" is preferred. H All H polypeptides contained the same set of activity to boost CDR mutations, but contained different sets of pI reduction mutations outside the CDR. [Table 14]

[0359] Example 9: Anti-FX / anti-FIX(A)V with SNAC and NAM in dosage forms suitable for oral administration H Formulation of H polypeptide derivatives To prepare a liquid formulation for oral studies using N-(8-[2-hydroxybenzoyl]amino)caprylate sodium (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 2 M NaOH. Thus, purified and conjugated 2-5 mg / ml V H The batch of H polypeptide derivatives was a liquid formulation with a final concentration of 200 mg / ml SNAC and 1 M NAM.

[0360] To prepare for oral studies using tablets containing SNAC and NAM, the following procedure was followed: Purified V with or without fatty acid elongation factor conjugation. H A batch of H polypeptide (derivative) was buffered with MilliQ-H2O, and the pH was adjusted to 8.0 using either 0.1M NaOH or 0.1M formic acid. H The concentration of H polypeptide (derivative) was 0.5-4 mg / ml. H Spray drying of H polypeptide (derivative) was performed using a Mini Spray Dryer B290 (BUCHI) under the following conditions: pump setting 5-6, supply flow rate: 2 ml / min, inlet temperature: 80-85°C, outlet temperature: 45-50°C, nozzle: 1.5 mm, and suction: 100%.

[0361] To prepare the tablets, the powders were mixed to obtain the desired composition. The following tablet formulations were prepared. [Table 15] [Table 16] [Table 17]

[0362] The mixed powder was weighed and punched into homogeneous tablets using a Kilian Style One (Romaco) punch with the appropriate settings, simulating a rotary press.

[0363] Example 10: Anti-FX / anti-FIX(a)V with different PI mutations H Oral and intravenous pharmacokinetic studies of H polypeptide derivatives in rats The purpose of this study is to investigate the effects of anti-FX / FIX(a)V on pharmacokinetic parameters such as oral bioavailability. H The objective was to investigate the effect of pI alteration mutations in H polypeptide derivatives.

[0364] 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 according to Example 9 and administered orally by force-feeding to a parallel group of Sprague Dolly rats that had been acclimatized in-house at least one week prior to the study and maintained in group cages with free access to standard food and water. Rats for oral administration were fasted in grid-bottom cages with free access to water for a total of 18 hours, starting around 2 p.m. the day before administration. On the day of administration, all rats were acclimatized in the treatment room for 30 minutes.

[0365] Blood samples were collected immediately before administration and at several different time points after administration. Animals administered orally (PO) were kept fasting in grid-bottom cages for up to 4 hours post-administration. H Plasma concentrations of H polypeptide derivatives were measured using a gamma-glu-based immunoassay, where anti-V against the extended portion of the test molecule was detected. HH antibody (Novo Nordisk, Denmark) and anti-gamma-Glu (Novo Nordisk, Denmark) were used. Here, a 96-well MaxiSorp plate (Nunc, 439454) was coated with 2 μg / ml anti-gamma-Glu antibody and washed and blocked with PBS, 0.05% tween20, 1% BSA (pH 7.4). After the washing step, 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 at a minimum dilution of 100-fold were incubated on the plate. Here, V H The H polypeptide derivative was captured via the gamma-Glu extension. After an additional washing step, in-house biotinylated V H H-specific antibody (0.5 nM) was added to the plate to create a sandwich ELISA. After the final washing step, horseradish peroxidase (HRP)-streptavidin was added to the plate as a detection reagent. H The amount of biotin-labeled antibody conjugated to the H polypeptide derivative was detected after the addition of a chromogenic substrate (e.g., TMB(3,3',5,5'-tetramethylbenzidine)). Optical density was measured using a spectrometer (e.g., SpectraMax® M2 spectrometer (Molecular Devices)). The response was proportional to the peroxidase concentration, which again corresponds to V H The pharmacokinetic parameters were proportional to the concentration of the H polypeptide derivative. Based on these exposure data, non-compartmental pharmacokinetic parameters were calculated using either Phoenix WinNonlin or the open-source statistical analysis software R (package "NonCompart").

[0366] The following pI-reducing substitutions introduced at the outer surface exposed residues of CDRs, T28D, K43Q, K65Q, N84D, R148Q, N159D, K172Q, K194Q, and N213D (based on sequential numbering using compound number 4 as a template), were tested in different combinations (V H H polypeptide derivatives (leading to compound numbers 59-64 and compound number 5), non-pI adjusted VH We compared H polypeptide derivatives (compound number 4).

[0367] The results are shown in Tables 13 and 14 below. H This paper describes the effects of H derivatives on reducing pI on oral bioavailability.

[0368] Reducing the pI from approximately 8.36 to 5.85 resulted in a 5-10 fold increase in bioavailability (see Table 13).

[0369] No effect of pI reduction on half-life was observed (see Table 14). [Table 18] [Table 19]

[0370] Example 11: Intravenous pharmacokinetic study in rats: Anti-FX / anti-FIX(a)V with different prolongation factors H H polypeptide (derivative) The purpose of this study is V H The objective was to investigate the effects of different prolongation factors on pharmacokinetic parameters such as half-life on H-polypeptide derivatives.

[0371] Anti-FX / FIX(a)V prepared according to Example 9 H Liquid formulations of H polypeptide derivatives were administered intravenously (IV) to parallel groups of Sprague Dolly rats that had been acclimatized in-house at least one week prior to the study and maintained in group cages with free access to standard food and water. Rats for oral administration were fasted in grid-bottom cages with free access to water for a total of 18 hours, starting around 2 p.m. the day before administration. On the day of administration, all rats were acclimatized in the treatment room for 30 minutes. Blood samples were collected immediately before administration and at several different time points thereafter. HPlasma concentrations of H polypeptides (derivatives) are measured using His-tagged or gamma-glucan-based immunoassays, where V H Anti-V against H polypeptide derivatives H The H antibody (Novo Nordisk) was used in combination with either an anti-His tag antibody (R&D Systems, MAB050) against the His tag fused to the test molecule, or anti-gamma-Glu (Novo Nordisk, Denmark) against the extended portion of the test molecule. The two immunoassay setups exhibited similar sensitivity, with the latter being V H This 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 His tag or anti-gamma-Glu antibody and washed and blocked with PBS, 0.05% tween20, 1% BSA (pH 7.4). After the washing step, 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-fold were incubated on the plates, where V H H polypeptide derivatives were captured via their His tag or gamma-Glu extension. After an additional washing step, in-house biotinylated V H H-specific antibody (0.5 nM) was added to the plate to create a sandwich ELISA. After the final washing step, horseradish peroxidase (HRP)-streptavidin was added to the plate as a detection reagent. H The amount of biotin-labeled antibody conjugated to the H polypeptide derivative was detected after the addition of a chromogenic substrate (e.g., TMB(3,3',5,5'-tetramethylbenzidine)). Optical density readings were measured using a spectrometer (e.g., SpectraMax® M2 spectrometer (Molecular Devices)). The response was proportional to the peroxidase concentration, which again corresponds to V HThe pharmacokinetic parameters were proportional to the concentration of the H polypeptide derivative. Based on these exposure data, non-compartmental pharmacokinetic parameters were calculated using either Phoenix WinNonlin or the open-source statistical analysis software R (package "NonCompart").

[0372] V with different extension factor types H The results for H polypeptide derivatives are shown in Table 15 below. The results demonstrate the effect of using different fatty acids and albumin-binding peptides as extension factors on the mean residence time (MRTHL) of the terminal phase half-life (MRTHL).

[0373] V having C18, C20, and tetrazole fatty acid side chain conjugations H Regarding H polypeptide derivatives (compound numbers 66, 67, and 68: 3-4 times increase), as well as albumin binder peptides and V H A 30-residue GlySer L molecule C-terminally fused with an H polypeptide. P V has albumin binder peptide extension factor fusion with optimal effect using a linker. H For H polypeptide derivatives (compound numbers 70, 71, 72, and 73: 1.9 to 3.6 times increase), an increased half-life was observed compared to the non-extended polypeptide. V with short C12 fatty acids H The H polypeptide derivative (compound number 65) showed the shortest half-life. This is because V H To obtain optimal extension of the H polypeptide derivative, we have shown that fatty acids with longer half-lives than C12 are preferable for the cycling of extended half-lives (e.g., C16, C18, or C20). [Table 20]

[0374] Example 12: Oral and intravenous pharmacokinetic studies in dogs: Anti-FX / anti-FIX(a)V formulated with SNAC and NAM H H polypeptide (derivative) The purpose of this study was to investigate the effect of fatty acid extension on half-life and whether a decrease in pI leads to an increase in oral bioavailability.

[0375] Anti-FX / FIX(a)V H Appropriate formulations of H polypeptides (derivatives) were administered intravenously or prepared as tablets and orally to groups of beagle dogs. Dogs were given the single dose in the morning after an overnight fast, and fasting was maintained for 3–5 hours after the single dose. (Subset of oral study) § In this study, dogs were given a subcutaneous dose of approximately 3 nmol / kg of glucagon 10 minutes prior to oral administration. Blood samples were collected immediately before administration and at several different time points after administration. H Plasma concentrations of H polypeptide are measured using immunoassays based on His tags or gamma-glu linkers, where V H Antiviral V against H polypeptide H The H antibody (Novo Nordisk, Denmark) was used in combination with either an anti-His tag antibody (R&D Systems, MAB050) against the His tag fused to the test molecule, or an anti-gamma-Glu antibody (Novo Nordisk, Denmark) against the extension portion of the test molecule. The two immunoassay setups had similar sensitivity, with the latter being V H This 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 the antibody and washed and blocked with PBS, 0.05% tween20, 1% BSA (pH 7.4). After the washing 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 at a minimum dilution of 100-fold were incubated on the plates, where V H H polypeptide compounds were captured via their His tag or gamma-Glu linker motif. After an additional washing step, in-house biotinylated V HH-specific antibody (0.5 nM) was added to the plate to create a sandwich ELISA. After the final washing step, horseradish peroxidase (HRP)-streptavidin was added to the plate as a detection reagent. H The amount of biotin-labeled antibody bound to the H polypeptide compound was detected after the addition of a chromogenic substrate (e.g., TMB(3,3',5,5'-tetramethylbenzidine)). Optical density readings were measured using a spectrometer (e.g., SpectraMax® M2 spectrometer (Molecular Devices)). The response was proportional to the peroxidase concentration, which again corresponds to V H The pharmacokinetic parameters were proportional to the concentration of the H polypeptide compound. Based on these exposure data, non-compartmental pharmacokinetic parameters were calculated using either Phoenix WinNonlin or the open-source statistical analysis software R (package "NonCompart").

[0376] § This involves subcutaneous administration of approximately 3 nmol / kg of glucagon to dogs 10 minutes before oral administration.

[0377] The result was three C18 diacitate conjugates V H Tables 16 and 17 below show the H polypeptide derivatives. a) Effect of fatty acid extension on half-life, and b) Confirm that a decrease in pI leads to an increase in oral bioavailability.

[0378] Therefore, V H When H polypeptide derivatives were conjugated with the indicated fatty acids compared to the unextended compounds, the observed half-life in dogs increased by 370–710 times. The compound (compound number 6) with two C16 diacid fatty acid conjugations in the C-terminal linker showed the highest increase in half-life, approximately 7.6 days.

[0379] In dogs, the SNAC:NAM formulation increased oral bioavailability by 2 to 8 times compared to the SNAC formulation alone. Therefore, adding NAM to the oral formulation had a significant effect in making it possible to raise oral bioavailability to a clinically relevant level, i.e., approximately 0.1% or higher.

[0380] Overall, considering potency, mean residence time, and oral bioavailability, the V disclosed herein H H polypeptide derivatives are expected to be useful when administered orally in the treatment of hemophilia A, whether or not inhibitors are present, and in the treatment of acquired hemophilia A. [Table 21] [Table 22]

[0381] Example 13: Intravenous pharmacokinetic study in pigs: C18 conjugate anti-FX / anti-FIX(a)V H H polypeptide (derivative) The purpose of this study is to determine whether or not V has a C18 diacid extension factor group. H The objective was to investigate the pharmacokinetic effects of H polypeptides (derivatives).

[0382] Anti-FX / FIX(a)V H Appropriate formulations of H polypeptide (derivatives) were administered intravenously (IV) and subcutaneously (SC) to miniature pigs (Sus scrofa domesticus), respectively. Blood samples were collected immediately before administration and at several different time points after administration. H Plasma concentrations of H polypeptides (derivatives) are measured using immunoassays based on His tags or gamma-Glu, where V H Anti-V against H polypeptides (derivatives) HThe H antibody (Novo Nordisk) was used in combination with either an anti-His tag antibody (R&D Systems, MAB050) against the His tag fused to the test molecule, or anti-gamma-Glu (Novo Nordisk, Denmark) against the extended portion of the test molecule. The two immunoassay setups exhibited similar sensitivity, with the latter being V H This 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 His tag or anti-gamma-Glu antibody and washed and blocked with PBS, 0.05% tween20, 1% BSA (pH 7.4). After the washing step, 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, where V H H polypeptide derivatives were captured via their His tag or gamma-Glu extension. After an additional washing step, in-house biotinylated V H H-specific antibody (0.5 nM) was added to the plate to create a sandwich ELISA. After the final washing step, horseradish peroxidase (HRP)-streptavidin was added to the plate as a detection reagent. H The amount of biotin-labeled antibody conjugated to the H polypeptide derivative was detected after the addition of a chromogenic substrate (e.g., TMB(3,3',5,5'-tetramethylbenzidine)). Optical density readings were measured using a spectrometer (e.g., SpectraMax® M2 spectrometer (Molecular Devices)). The response was proportional to the peroxidase concentration, which again corresponds to V H The effect of fatty acid extender conjugations on half-life extension was confirmed in V with and without fatty acid extenders, as observed in Examples 11, 12, and 13. HThe results for H polypeptides are shown in Table 18 below.

[0383] Therefore, V H H polypeptide is non-extended V H Compared to the H compound, the half-life observed in pigs increased by approximately 15 to 20 times when conjugated with fatty acid C18 diacid extension factor. [Table 23]

[0384] Example 14: Pharmacodynamic effects of optimized anti-FX / anti-FIX(a) polypeptide derivatives in a mouse tail vein transection (TVT) bleeding model. In vivo efficacy was tested in a moderate bleeding HA mouse model. Previous studies in the TVT model have shown that FVIII administration reduces bleeding to the same level observed in wild-type mice. To overcome the lack of mouse cross-reactivity of the compound in the testes, HA mice were supplemented with human FIX and FX prior to the bleeding experiment. At the end of the bleeding period, total blood loss was determined by spectrophotometric hemoglobin measurement. H Plasma levels of H polypeptide derivatives and FIX were, respectively, anti-V H Quantification was performed by a luminescent oxygen channeling assay using H (Novo Nordisk, Denmark), anti-gamma-gamma-glucan linker antibody (Novo Nordisk, Denmark), and anti-FIX antibody (LS-B7226, LSBio, and FIX-2F24, in-house clone). FX levels were quantified by a commercially available FX ELISA (KSP134, Nordic Biosite). The V levels shown are... H By fitting the data from dose-response studies using H polypeptide derivatives to a three-parameter inverse logarithmic (dose) response equation with a shared plateau value (>0), the EC 50 The values ​​were determined. The sum of squares was weighted by applying automatic outlier removal with a 1% ROUT coefficient and using GraphPad Prism software (version 9.0.1) (blood loss).

[0385] The dose-response studies were conducted as shown in Table 19, comparing the tested V group to the vehicle group and the wild-type non-hemophilia group, respectively. H H polypeptide derivatives showed a significant reduction in blood loss, reaching the same level of blood loss observed in wild-type mice. 50 The estimated effective dose range is shown in Table 20, and all three compounds tested showed high efficacy, with a 50% effect observed at low n-moles per 1 kg dose. [Table 24] [Table 25]

[0386] Example 15: Optimized anti-FX / anti-FIX(a)V after oral administration H Determination of exvivoactivity of H polypeptide derivatives Following pharmacokinetic (PK) studies in wild-type dogs from Example 12 to characterize PO administration of compound number 6, chromogenic activity was determined at two separate time points, 30 and 90 minutes post-administration. The measured FVIII-mimicking chromogenic activity was compared to plasma exposure to compound number 6, and the activity in serum after PO administration was compared to that of compound number 6. H The proportion of H polypeptide derivatives was determined.

[0387] The chromogenic activity of compound number 6 was determined using a commercially available FVIII chromogenic activity assay (FVIII:C, Hyphen Biomed, France), and canine serum samples were analyzed. For the calibration curve, compound number 6 was spiked into 10% canine serum, and the serum samples were analyzed at a 10-fold dilution using the same assay. The final activity results were corrected 10-fold for the 10-fold dilution. Plasma exposure levels of compound number 6 were determined using an anti-VIII assay. HQuantification was performed by luminescent oxygen channeling (LOCI) assay using H (Novo Nordisk, Denmark) and anti-gamma-Glu-linker antibody (Novo Nordisk, Denmark). The data are summarized in Table 21. FVIII:C chromogenic activity was measurable in serum at 30 and 90 minutes post-administration, and the quantification of compound number 6 based on chromogenic activity is similar to the estimate based on plasma exposure measurements of compound number 6 using the LOCI assay. Any discrepancies in the quantification of compound number 6 are expected to be due to differences in assay sensitivity. The data were obtained by administering PO-VIII:C. H We demonstrate that the H polypeptide derivative is fully active upon oral administration in dogs, suggesting that the concept of creating an orally available FVIII mimetic for treating patients with hemophilia A is feasible. [Table 26]

[0388] Example 16: Crystallization and paratope / epitope mapping of anti-FX VHH fragment (compound number 76). The purpose of this study is to investigate compound number 76 and the designated V H The objective was to determine the paratope and epitope residues of the H fragment (VHH-1.15).

[0389] crystallization V was produced using CHO mixed in a 1:4 molar ratio with the synthetic peptide N-terminus-NPFDLLD-C-terminus, corresponding to the activation peptide sequences aa31-37 of human FX (purchased from Apigenex) identified in Example 4. HCrystals of the H fragment (compound number 76) were grown using the sitting drop vapor diffusion technique. 250 nl of a 5.75 mg / ml complex protein solution in 20 mM Tris-HCl (pH 7.4) and 50 mM NaCl was mixed with 250 nl of 4% (v / v) Tacsimate (1.8305 M malonic acid, 0.25 M tribasic ammonium 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 dibasic ammonium tartrate) (pH 5.0) and 12% (w / v) polyethylene glycol 3350 (as a precipitant), and incubated with 80 μl of precipitant. After incubation at 18°C ​​for 5 days, the crystallization plate was transferred to 5°C. Crystals appeared within 3 months.

[0390] Diffraction data collection The crystals were cryoprotected in a precipitant containing 20% ​​ethylene glycol before flash cooling in liquid nitrogen. Diffraction data were collected at 100K on the Swiss Light Source beamline X10SA (1.0000 Å wavelength) using a Dectris Eiger2 16M pixel detector. Automated indexing, merging, and scaling of the data were performed using a program from the XDS package (diffraction data statistics are summarized in Table 22).

[0391] Determination and refinement of the structure The asymmetric unit contains two compound number 76:FX AP 31-37 complexes. The structure is related to V as a search model. H The previously determined crystal structure of H was determined by molecular substitution using Phaser implemented within the Phenix program suite (see Example 6). The correct amino acid sequence of compound number 76 was introduced, and FX AP 31–37 were all manually constructed models using differential electron density maps with COOT. During 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. [Table 27]

[0392] Epitope and paratope determination V H The epitope of the synthetic peptide, defined as an FX-activated peptide residue characterized by having a heavy atom within 4.0 Å of a heavy atom in the H polypeptide (compound number 76), includes the following residues from the synthetic peptide N173, P174, F175, L177, L178, and D179, according to the corresponding FX-activated peptide sequence (SEQ ID NO: 2) based on sequential numbering.

[0393] V is defined as a residue of compound number 76, characterized by having a heavy atom (i.e., a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the synthetic peptide. H The paratope of the H fragment (compound number 76) is the following residues from compound number 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) (Including consecutive numbering.)

[0394] Example 17: Crystallization and paratope / epitope mapping of anti-FX VHH fragment (compound number 77). The purpose of this study is to investigate compound number 77 and the designated V H H fragment (V H The objective was to determine the paratope and epitope residues of H1.13).

[0395] crystallization V produced from CHO mixed in a 1:4 molar ratio with the synthetic peptide N-terminus-NPFDLLD-C-terminus, corresponding to the activation peptide sequences aa31-37 of human FX (synthesized by Apigenex). HCrystals of the H fragment (compound number 77) were grown using the sitting drop vapor diffusion technique. 250 nl of a 6.1 mg / ml complex protein solution in 20 mM Tris-HCl (pH 7.4) and 50 mM NaCl was mixed with 250 nl of 3% (v / v) Tacsimate (1.8305 M malonic acid, 0.25 M tribasic ammonium 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 dibasic ammonium tartrate) (pH 4.0) and 11% (w / v) PEG 3350 (as a precipitant), and incubated with 80 μl of precipitant. Crystals appeared after incubation at 5°C for 4 days.

[0396] Diffraction data collection The crystals were cryoprotected in a precipitant containing 20% ​​ethylene glycol before flash cooling in liquid nitrogen. Diffraction data were collected at 100K using a Dectris Eiger2 16M pixel detector at the Swiss Light Source beamline X10SA (1.0000 Å wavelength). Automated indexing, merging, and scaling of the data were performed using a program from the XDS package (diffraction data statistics are summarized in Table 23).

[0397] Determination and refinement of the structure The asymmetric unit contains two compound number 77:FX AP 31-37 complexes. The structure was determined by molecular substitution using Phaser implemented within the Phenix program suite, with the fully asymmetric unit from the crystal structure of compound number 76:FX AP 31-37 of Example 16 as a search model. The correct amino acid sequence of compound number 77 was introduced, and rounds of refinement in Phenix and manual reconstruction in COOT were applied. Refinement statistics are shown in Table 23. [Table 28]

[0398] Epitope and paratope determination VH The epitope of the synthetic peptide, defined as an FX-activating peptide residue characterized by having a heavy atom within 4.0 Å of a heavy atom in the H fragment (compound number 77), includes the following residues from the synthetic peptide N173, P174, F175, L177, L178, and D179, according to the corresponding FX-activating peptide sequence (SEQ ID NO: 2) based on sequential numbering.

[0399] V is defined as a residue of compound number 77, characterized by having a heavy atom (i.e., a non-hydrogen atom) within a distance of 4.0 Å from a heavy atom in the synthetic peptide. H The paratope of the H fragment (compound number 77) is the following residues from compound number 77: A33, M34, G35, W47, V48, A49, A50, I51, S52, S57, T58, N59, Y60, A61, A97, A98, D99, G105, L107, Y109 (Sequence ID 735) (Including consecutive numbering.)

[0400] Example 18: Bispecific V H Steady-state FXA generation kinetics of H polypeptide derivatives The purpose of this study is to investigate the anti-FIXa / FX bispecificity of V based on its ability to promote FX activation by FIXa in the presence of a blood coagulation-promoting phospholipid membrane. H The objective was to determine the blood coagulation-promoting activity of H polypeptide derivatives. The compounds tested are listed in Table 24, with emicizumab SIA included for comparison.

[0401] The steady-state FXa generation activity of each compound is expressed as the Michaelis-Menten kinetics (Michaelis constant (K)) at a given compound concentration. M ) and primary velocity constant (k cat / K MThe parameters are reported as follows. The compounds, whose final assay concentrations are shown in Table 24, were pre-incubated for 5 minutes in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w / v) PEG8000 (pH 7.3) + 1 mg / ml BSA) 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). Activation was then initiated by adding a 2-fold serial dilution of human plasma-derived FX (Haematologic Technologies Inc, USA) starting at 500 nM. After activation at room temperature for 7 minutes, the reactant (50 μl) was quenched by adding 25 μl of quench buffer (50 mM HEPES, 100 mM NaCl, 60 mM EDTA, 0.1% PEG8000 (pH 7.3) + 1 mg / ml BSA). 25 μl of 2 mM S-2765 chromogenic substrate (Chromogenix, Sweden) was added, and the amount of FXa produced was determined by measuring the chromogenic substrate conversion by measuring the absorbance at 405 nm (ΔOD / min) using a microplate reader. Similarly, FX activation by free FIXa was determined at a FIXa concentration of 10 nM and reaction times of 5–10 minutes. To convert the absorption measured at 405 nm to the corresponding FXa concentration, 25 μl of quench buffer and 25 μl of 2 mM S-2765 chromogenic substrate were added to a 0-5 nM (50 μl) FXa standard curve, and the absorption at 405 nm was recorded as described above. Linear regression of ΔOD / min as a function of FXa concentration generates a slope that can be used to convert the measured ΔOD / min to the FXa generation rate. FXa generation rate = (ΔOD / min) / (slope) FXa標準曲線 *Reaction time) The Michaelis-Menten steady-state dynamics parameters are determined by fitting the FXa generation rate as a function of FX (substrate) concentration to the following equation: FXa generation rate=(k cat *[FIXa]t *[FX] t ) / (K M +[FX] t ) In the formula, k cat Enzyme efficiency (minutes) -1 ) and [FIXa] t This is the total FIXa (enzyme) concentration (nM), [FX] t , the total FX (substrate) concentration (nM), K M This is the Michaelis constant (nM). Table 24 lists the steady-state kinetic constants and tested concentrations determined for each compound tested. [Table 29]

[0402] conclusion Tested anti-FIXa / FX bispecificity V H When all H polypeptide derivatives are replenished to a final concentration of 5 nM, K M This reduces the free FIXa concentration from 281.5 nM to 3.9-9.0 nM, improving the catalytic efficiency to 0.00012 min⁻¹. -1 nM -1 0.14~0.33 minutes from -1 nM -1 To further increase it. In comparison, 300nM emicizumab-SIA is K M Reduce it to 53.5 nM.

[0403] Reduced K M The value is V H This reflects the improved assembly (spatial arrangement) of FIX(a) / FX, which results in a significant increase in the potency of H polypeptide derivatives. All compounds are tested at the expected pharmacologically relevant concentrations.

Claims

1. A single variable domain (ISVD) polypeptide derivative of a blood coagulation-promoting immunoglobulin, A first ISVD (ISCVD1) that can bind to factor IX (SEQ ID NO: 1) or its active form, A second ISVD (ISCVD2) that can bind to factor X (SEQ ID NO: 2) or its active form, At least one extension section, Optionally, a linker (L) connects ISVD1 and ISVD2. １－２ )and, Optionally, it includes one or more extensions (E), The first ISVD can bind to an epitope on the IX factor (SEQ ID NO: 1) or its active form, which includes at least one of the amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering), and A blood coagulation-promoting ISVD polypeptide derivative wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising at least one of the amino acid residues N173, P174, F175, L177, and L178 (sequential numbering).

2. The first ISVD can bind to an epitope on factor IX (SEQ ID NO: 1) or its active form, which includes amino acid residues E224, T225, G226, V250, I251, R252, I253, P255, H257, and N260 (sequential numbering). The ISVD polypeptide derivative according to claim 1, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising amino acid residues N173, P174, F175, L177, and L178 (sequential numbering).

3. The ISVD polypeptide derivative according to claim 1, wherein the second ISVD can bind to an epitope on factor X (SEQ ID NO: 2) comprising amino acid residues N173, P174, F175, L177, L178, and D179 (sequential numbering).

4. The first ISVD comprises a paratope containing amino acid residues F29, N30, Y32, T54, D99, R100, S101, F102, L103, F104, Q106, A107, and N113 (SEQ ID NO: 35), and The second ISVD is an amino acid residue a) D32, A33, M34, G35, Y37, L47, V48, A49, G50, I51, M52, N57, T58, N59, Y60, T61, K97, V99, R101, and P102 (Sequence ID 27), or b) A33, M34, G35, W47, V48, A49, A50, I51, S52, S57, T58, N59, Y60, A61, A97, A98, D99, G105, L107, and Y109 (Sequence ID 734) The ISVD polypeptide derivative according to claim 1, comprising a paratope containing (continuous numbering).

5. The ISVD polypeptide derivative according to claim 1, wherein at least one of the extended portions is bound to a surface-exposed amino acid residue.

6. The ISVD polypeptide derivative according to claim 1, wherein the surface-exposed residue is not a residue in the CDR region.

7. The following formula (from N-terminus to C-terminus) IXSTD2-L １－２ -ISVD1-E In the formula, the two extended portions are bound to one or more surface-exposed amino acid residues on E, The ISVD polypeptide derivative according to claim 1, wherein the molecular weight of the ISVD polypeptide derivative is in the range of 20 to 35 kDa.

8. The first ISVD mentioned above is １） CDR1: Optionally, IYTMS containing one or two amino acid substitutions (SEQ ID NO: 172), CDR2: Optionally, GLRWTDSSTEYADSVKG (SEQ ID NO: 173) containing one, two, or three amino acid substitutions. CDR3: optionally includes DRSFLFAQALGATKNYEY (SEQ ID NO: 174) (Kabat definition) containing one, two, or three amino acid substitutions, and The second ISVD mentioned above is (A) CDR1: Optionally, RYAMG (SEQ ID NO: 152) containing one or two amino acid substitutions. CDR2: Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 153) containing one, two, or three amino acid substitutions. CDR3: Optionally, DDSVGDGYLDY (SEQ ID NO: 154) (Kabat definition) containing one, two, or three amino acid substitutions, or (B) CDR1: Optionally, RLAMG (SEQ ID NO: 128) containing one or two amino acid substitutions. CDR2: Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 129) containing one, two, or three amino acid substitutions. CDR3: Optionally, DDSVGDGYLDY (SEQ ID NO: 130) (Kabat definition) containing one, two, or three amino acid substitutions, or (C) CDR1: Optionally, RYAMG (SEQ ID NO: 32) containing one or two amino acid substitutions. CDR2: Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 33) containing one, two, or three amino acid substitutions. CDR3: Optionally, DYSSGDGYLDY (SEQ ID NO: 34) (Kabat definition) containing one, two, or three amino acid substitutions, or (D) CDR1: Optionally, RYAMG (SEQ ID NO: 40) containing one or two amino acid substitutions. CDR2: Optionally, AISRRGGSTNYADSVKG (SEQ ID NO: 41) containing one, two, or three amino acid substitutions. CDR3: The ISVD polypeptide derivative according to claim 1, comprising DDSSGDGYLDY (SEQ ID NO: 42) (Kabat definition), which optionally contains one, two, or three amino acid substitutions.

9. The ISVD polypeptide derivative according to claim 8, wherein the substitution is a conservative substitution.

10. A single variable domain (ISVD) polypeptide derivative of a blood coagulation-promoting immunoglobulin, A first ISVD (ISCVD1) that can bind to factor IX (SEQ ID NO: 1) or its active form, A second ISVD (ISCVD2) that can bind to factor X (SEQ ID NO: 2) or its active form, At least one extension portion bound to a surface-exposed amino acid residue, Optionally, a linker (L) connects ISVD1 and ISVD2. １－２ )and, Optionally, it includes one or more extensions (E), The first ISVD mentioned above is V Ｈ H-2.20 (Sequence ID 171), V Ｈ H-2.18 (Sequence ID 155), V Ｈ H-2.15 (Sequence ID 131), V Ｈ H-2.13 (SEQ ID NO: 115), V Ｈ H-2.14 (Sequence ID 123), V Ｈ H-2.12 (SEQ ID NO: 107), or V Ｈ It contains the sequence H-2.2 (sequence number 35), and, The second ISVD mentioned above is V Ｈ H-1.20 (Sequence ID 167), V Ｈ H-1.18 (Sequence ID 151), V Ｈ H-1.15 (Sequence ID 127), V Ｈ H-1.13 (Sequence ID 111), V Ｈ H-1.14 (Sequence ID 119), V Ｈ H-1.12 (Sequence ID 103), V Ｈ H-1.3 (SEQ ID NO: 31), or V Ｈ A blood coagulation-promoting ISVD polypeptide derivative containing the sequence H-1.4 (SEQ ID NO: 39).

11. The first ISVD is V Ｈ The sequence H-2.15 (sequence number 131) is included, and the second ISVD is V Ｈ The ISVD polypeptide derivative according to claim 1, comprising the sequence H-1.15 (SEQ ID NO: 127).

12. The following formula (from N-terminus to C-terminus) IXSTD2-L １－２ -ISVD1-E In the formula, the two extended portions are bound to one or more surface-exposed amino acid residues on E, The ISVD polypeptide derivative according to claim 10 or 11, wherein the molecular weight of the ISVD polypeptide derivative is in the range of 20 to 35 kDa.

13. The ISVD polypeptide derivative comprises SEQ ID NO: 629, The first extension portion includes the following structure: 【Chemistry 1】 Furthermore, the second extension portion includes the following structure: 【Chemistry 2】 In the formula, "*" represents the Cys at position 257 of sequence number 629. The ISVD polypeptide derivative according to claim 10 or 11, wherein "**" in the formula is the Cys at position 259 of Sequence ID No.

629.

14. The ISVD polypeptide derivative is V Ｈ An ISVD polypeptide derivative according to claim 1 or 10, which is an H polypeptide derivative.

15. Blood coagulation promotion V Ｈ H polypeptide derivatives, Factor IX (SEQ ID NO: 1) or a first V that can bind to its active form Ｈ H and, A second V that can bind to factor X (SEQ ID NO: 2) or its active form. Ｈ H and, The first V Ｈ H and the second V Ｈ Linker (L) connecting H １－２ )and, A C-terminal extension (E) having sequence number 9 is included, The following formula (from N-terminus to C-terminus) "The second V Ｈ H"-L １－２ - "The first V Ｈ H"-E Further including first and second extensions joined to E, １） The first V Ｈ H is, CDR1: IYTMS (Sequence ID 172), CDR2: GLRWTDSSTEYADSVKG (Sequence ID 173), Includes CDR3:DRSFLFAQALGATKNYEY (Sequence ID 174) (Kabat definition), and The second V Ｈ H is, CDR1: RYAMG (SEQ ID NO: 152), CDR2: AISRRGGSTNYADSVKG (Sequence ID 153), CDR3: Contains DDSVGDGYLDY (Sequence ID 154) (Kabat definition), or ２） The first V Ｈ H is, CDR1: IYTMS (Sequence ID 132), CDR2: GLRWTDSSTEYADSVKG (Sequence ID 133), Includes CDR3:DRSFLFAQALGATKNYEY (Sequence ID 134) (Kabat definition), and The second V Ｈ H is, CDR1: RLAMG (Sequence ID 128), CDR2: AISRRGGSTNYADSVKG (Sequence ID 129), CDR3: Includes DDSVGDGYLDY (Sequence ID 130) (Kabat definition), The first extension portion includes the following structure: 【Transformation 3】 Furthermore, the second extension portion includes the following structure: 【Chemistry 4】 In the formula, "*" represents Cys(C) at position 257 of sequence number 629. In the formula, "**" is Cys(C) at position 259 of sequence number 629, which is a blood coagulation promoter V Ｈ H polypeptide derivative.

16. The aforementioned V Ｈ The V according to claim 15, wherein the H polypeptide derivative includes SEQ ID NO:

629. Ｈ H polypeptide derivative.

17. The ISVD polypeptide derivative or V Ｈ H polypeptide derivative is a bispecific ISVD polypeptide derivative or V Ｈ The H polypeptide derivative is the ISVD polypeptide derivative according to any one of claims 1, 10, and 15 or V Ｈ H polypeptide derivative.

18. ISVD polypeptide derivative or V according to any one of claims 1, 10, and 15 Ｈ A pharmaceutical composition comprising an H polypeptide derivative and one or more pharmaceutically acceptable excipients.

19. The pharmaceutical composition according to claim 18, wherein the composition comprises a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid.

20. The salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC), and The pharmaceutical composition according to claim 19, wherein the composition further comprises nicotinamide (NAM).

21. The pharmaceutical composition according to claim 18, for use in the treatment of hemophilia A in which inhibitors are present or absent, or in the treatment of acquired hemophilia A.

22. The pharmaceutical composition according to claim 21, wherein the composition is administered orally.

23. V can bind to FIX (SEQ ID NO: 1) or its active form (FIXa), and can also bind to FX (SEQ ID NO: 2). Ｈ H polypeptide or V Ｈ An intermediate used in the production of H polypeptide derivatives, The aforementioned intermediate is as follows: CDR1: IYTMS (Sequence ID 172), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 173), and CDR3: DRSFLFAQALGATKNYEY (Sequence ID 174) (Kabat definition); CDR1: RYAMG (SEQ ID NO: 152), CDR2:AISRRGGSTNYADSVKG (Sequence ID 153), and CDR3:DDSVGDGYLDY (Sequence ID 154) (Kabat definition); CDR1: IYTMS (Sequence ID 132), CDR2:GLRWTDSSTEYADSVKG (Sequence ID 133), and CDR3:DRSFLFAQALGATKNYEY (Sequence ID 134) (Kabat definition); and CDR1: RLAMG (Sequence ID 128), CDR2:AISRRGGSTNYADSVKG (Sequence ID 129), and V containing a CDR selected from the group consisting of CDR3:DDSVGDGYLDY (Sequence ID 130) (Kabat definition) Ｈ H fragment; or below: V Ｈ H-1.18 (Sequence ID 151), V Ｈ H-1.15 (Sequence ID 127), V Ｈ H-1.13 (Sequence ID 111), V Ｈ H-1.14 (Sequence ID 119), V Ｈ H-1.12 (Sequence ID 103), V Ｈ H-1.3 (Sequence ID 31), V Ｈ H-1.4 (Sequence ID 39), V Ｈ H-2.20 (Sequence ID 171), V Ｈ H-2.18 (Sequence ID 155), V Ｈ H-2.15 (Sequence ID 131), V Ｈ H-2.13 (SEQ ID NO: 115), V Ｈ H-2.14 (Sequence ID 123), V Ｈ H-2.12 (Sequence ID 107), V Ｈ H-2.2 (SEQ ID NO: 35), and V Ｈ V selected from the group consisting of H-1.20 (Sequence ID 167) Ｈ H fragment It is an intermediate.

24. A first ISVD (ISCVD1) that can bind to factor IX (SEQ ID NO: 1) or its active form, A second ISVD (ISCVD2) that can bind to factor X (SEQ ID NO: 2) or its active form, The extension and A linker (L) can optionally connect ISVD1 and ISVD2. １－２ )and, A method for increasing the oral bioavailability of a blood coagulation-promoting immunoglobulin single variable domain (ISVD) polypeptide or ISVD polypeptide derivative comprising, optionally, one or more extensions (E), a. A step of modifying a nucleic acid encoding an amino acid residue of the ISVD polypeptide or ISVD polypeptide derivative such that the isoelectric point of the ISVD polypeptide or ISVD polypeptide derivative is reduced, b. A step of culturing host cells to express the nucleic acid encoding the ISVD polypeptide or ISVD polypeptide derivative, c. A step of collecting the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture, d. A step of purifying the ISVD polypeptide or ISVD polypeptide derivative from the host cell culture using standard chromatography, e. A method comprising the step of binding an extension portion to the ISVD polypeptide if an extension portion does not yet exist.

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