Fusion protein

Abstract

The present invention relates to a fusion protein including: a mutant ADAMTS13 that contains at least a metalloprotease domain (M domain) and is missing a spacer domain (S domain); and antigen-binding molecules that bind to a von Willebrand factor (vWF). The mutant ADAMTS13 is preferably one satisfying to (N1) or (N2). (N1) In addition to a S domain, T2-T8 domains are also missing from the ADAMTS13. (N2) In addition to a S domain, T2-T8 domains and a CUB1-2 domain are also deleted from the ADAMTS13.

Description

Fusion protein

[0001] The present invention relates to a fusion protein, a nucleic acid encoding the fusion protein, a vector containing the nucleic acid, a transformed cell obtained by introducing the vector into a host cell, a method for producing the fusion protein, and a therapeutic, preventive, and / or diagnostic agent for diseases containing the fusion protein as an active ingredient.

[0002] ADAM metalloproteinase with thrombospondin type 1 motif 13 (ADAMTS13) is a metalloproteinase present in plasma, primarily responsible for cleaving von Willebrand factor (vWF) (Non-Patent Literature 1). ADAMTS13 consists of a metalloproteinase domain, a disintegrin-like domain, a thrombospondin type 1 repeat, a cysteine-rich domain, a spacer domain, seven thrombospondin type 1 repeats, and two CUB domains, starting from the N-terminus (Non-Patent Literature 2). ADAMTS13 cleaves polymerized vWF, preventing abnormal platelet aggregation. Dysfunction or deficiency of ADAMTS13 leads to blood coagulation abnormalities and causes serious thrombotic diseases.

[0003] vWF is a multimeric glycoprotein that has initial platelet adhesion and platelet aggregation effects at the site of vascular injury (Non-Patent Literature 3). vWF is released from vascular endothelial cells and promotes hemostasis by rapidly attracting platelets at the site of injury. vWF is self-associating, and particularly large multimers are called ultra-large vWF (UL-vWF). UL-vWF has extremely high adhesion and, if not properly controlled, can cause abnormal thrombus formation. Therefore, the proper degradation of UL-vWF by ADAMTS13 is extremely important for maintaining normal blood circulation (Non-Patent Literature 4).

[0004] Defense or dysfunction of ADAMTS13 causes thrombotic thrombocytopenia purpura (TTP). ADAMTS13 deficiency leads to the accumulation of UL-vWF in the blood and abnormal platelet aggregation. Thrombi form in microvessels, causing thrombocytopenia and hemolytic anemia. TTP presents with acute symptoms and has an extremely poor prognosis, with over 90% of patients dying without treatment. Furthermore, TTP can cause serious damage to major organs such as the kidneys, heart, and brain, requiring early diagnosis and rapid treatment (Non-Patent Literature 5).

[0005] TTP is classified into two types: congenital and acquired. Congenital TTP is a rare disease caused by dysfunction of the gene encoding ADAMTS13, accounting for about 5% of all cases (Non-Patent Literature 5). The standard treatment for congenital TTP is fresh frozen plasma infusion or recombinant ADAMTS13 replacement therapy (Non-Patent Literature 6).

[0006] Acquired TTP is classified into primary and secondary types. The majority of acquired TTP patients have primary TTP, and the exact cause remains unclear. Secondary causes have been reported to include infections, pregnancy, and autoimmune diseases (e.g., systemic lupus erythematosus) (Non-patent document 7).

[0007] One of the causes of acquired platelet-thinning platelet (TTP) is the presence of autoantibodies against ADAMTS13. These autoantibodies have been reported to inhibit the enzymatic activity of ADAMTS13 (hereinafter abbreviated as "ADAMTS13 enzymatic activity," "ADAMTS13 activity," or "enzymatic activity") and to have an effect of clearing ADAMTS13 from plasma (Non-Patent Literature 8). Current treatments for acquired TTP generally involve plasma exchange and the use of immunosuppressants. In recent years, in addition to these, caplacizumab, an anti-vWF nanobody dimer that competitively inhibits the adsorption of vWF to platelets, has been used.

[0008] Patients with acquired TTP possess ADAMTS13 autoantibodies, and attempts have been made to create ADAMTS13 variants that do not bind to these autoantibodies (Patent Document 1, Non-Patent Document 9). Most patients with acquired TTP possess autoantibodies against the spacer domain (hereinafter also abbreviated as the S domain) of ADAMTS13 (Non-Patent Documents 10, 11). Several variants have been reported in which amino acid mutations have been introduced into the spacer domain of ADAMTS13 to inhibit binding to autoantibodies.

[0009] vWF is cleaved not only by ADAMTS13 but also by plasmin. Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are enzymes that convert plasminogen to plasmin. There are reports confirming that fusion proteins of tPA or uPA with vWF antibodies degrade vWF and platelet aggregates (Patent Document 2).

[0010] International Publication No. 2022 / 108148, Japanese Patent Publication No. 2024-28711

[0011] Fujikawa K et al. Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood. 2001 Sep 15;98(6):1662-6. doi: 10.1182 / blood.v98.6.1662. PMID: 11535495.Zheng XL. Structure-function and regulation of ADAMTS-13 protease. J Thromb Haemost. 2013 Jun;11 Suppl 1(01):11-23. doi: 10.1111 / jth.12221. PMID: 23809107; PMCID: PMC3713533.Lenting PJet al. von Willebrand factor biosynthesis, secretion, and clearance: connecting the far ends. Blood. 2015 Mar 26;125(13):2019-28. doi: 10.1182 / blood-2014-06-528406. Epub 2015 Feb 23. PMID: 25712991.CRAWLEY, James TB, et al. Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor. Blood, The Journal of the American Society of Hematology, 2011, 118.12: 3212-3221.JOLY, Berangere S et al. Thrombotic thrombocytopenic purpura. Blood, The Journal of the American Society of Hematology, 2017, 129.21: 2836-2846.Heo YA. Apadamtase Alfa: First Approval. Drugs. 2024 Apr;84(4):467-472. doi: 10.1007 / s40265-024-02007-6. PMID: 38418772.SCULLY, M et al. Consensus on the standardization of terminology in thrombotic thrombocytopenic purpura and related thrombotic microangiopathies. Journal of thrombosis and haemostasis, 2017, 15.2: 312-322.KREMER HOVINGA et al. Nature reviews Disease primers, 2017, 3.1: 1-17.GRACA, Nuno et al. Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura. Haematologica, 2019, 105.11: 2619-2630.Klaus C et al. Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura. Blood. 2004 Jun 15;103(12):4514-9. doi: 10.1182 / blood-2003-12-4165. Epub 2004 Feb 19. PMID: 14976043.KANGRO, Kadri et al. Anti-ADAMTS13 autoantibody profiling in patients with immune-mediated thrombotic thrombocytopenic purpura. Blood advances, 2021, 5.17: 3427-3435.;

[0012] As mentioned above, TTP presents with acute symptoms and has an extremely poor prognosis if left untreated, requiring early diagnosis and rapid treatment. Current treatments for acquired TTP generally involve plasmapheresis and the use of immunosuppressants, but these methods are burdensome for patients. On the other hand, since one of the causes of acquired TTP is the presence of autoantibodies against ADAMTS13, modified ADAMTS13 with reduced reactivity to autoantibodies through point mutations has been developed. However, these modified ADAMTS13 can only avoid some autoantibodies, and tend to have reduced efficacy due to decreased ADAMTS13 enzyme activity, as well as a short half-life.

[0013] Therefore, the present disclosure aims to provide a means for rapidly and safely replenishing ADAMTS13 enzyme activity.

[0014] As a means to solve the above problems, this disclosure provides a fusion protein comprising a mutant ADAMTS13 and a vWF antigen-binding molecule. That is, this disclosure relates to the following: 1. A fusion protein comprising a mutant ADAMTS13 containing at least a metalloproteinase domain (M domain) and lacking a spacer domain (S domain), and an antigen-binding molecule that binds to von Willebrand factor (vWF). 2. The fusion protein according to 1, wherein the mutant ADAMTS13 is (N1) or (N2) below: (N1) In addition to the S domain of ADAMTS13, the T2-T8 domain is further lacking. (N2) In addition to the S domain of ADAMTS13, the T2-T8 domain and the CUB1-2 domain are further lacking. 3. The fusion protein according to 1, wherein the mutant ADAMTS13 is (D1) or (D2) below. (D1) Consists of the M domain of ADAMTS13 (D2) Consists of the M domain and a disintegrin-like domain (D domain) of ADAMTS13 4. The fusion protein according to 1, wherein the M domain in the mutant ADAMTS13 contains an amino acid sequence that has 70% or more homology to the amino acid sequence from position 80 to 286 of the amino acid sequence represented by SEQ ID NO: 194, and the fusion protein has ADAMTS13 enzyme activity. 5. The fusion protein according to 1, wherein the amino acid sequence of the M domain in the mutant ADAMTS13 contains an amino acid sequence that corresponds to the amino acid sequence from position 80 to 286 of the amino acid sequence represented by SEQ ID NO: 194, and further contains at least one of the substitutions (M1) to (M13) below. (M1) Substitution of the amino acid residue corresponding to the 159th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to R. (M2) Substitution of the amino acid residue corresponding to the 167th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to M. (M3) Substitution of the amino acid residue corresponding to the 207th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to N. (M4) Substitution of the amino acid residue corresponding to the 209th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to V.(M5) Substitution of L from the amino acid residue corresponding to the 230th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M6) Substitution of A from the amino acid residue corresponding to the 238th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M7) Substitution of K from the amino acid residue corresponding to the 243rd amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M8) Substitution of A from the amino acid residue corresponding to the 271st amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M9) Substitution of E from the amino acid residue corresponding to the 271st amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M10) Substitution of L from the amino acid residue corresponding to the 279th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M11) Substitution of L from the amino acid residue corresponding to the 282nd amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M12) Substitution of G from the amino acid residue corresponding to the 283rd amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M13) Substitution of L from the amino acid residue corresponding to the 285th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 6. The fusion protein described in 5 above, wherein the amino acid sequence of the mutant ADAMTS13 is any one of the following: (m1) including the substitutions of (M1) and (M2). (m2) including the substitutions of (M1) and (M3). (m3) including the substitutions of (M8) and (M13). (m4) including the substitutions of (M9) and (M13). (m5) including the substitutions of (M1), (M8) and (M13). (m6) including the substitutions of (M1), (M9) and (M13). (m7) including the substitutions of (M4), (M5), (M8) and (M13). (m8) including the substitutions of (M4), (M6), (M8) and (M13). (m9) including the substitutions of (M4), (M7), (M8) and (M13). (m10) Includes substitution of (M5), (M6), (M8) and (M13). (m11) Includes substitution of (M5), (M7), (M8) and (M13). (m12) Includes substitution of (M6), (M7), (M8) and (M13). (m13) Includes substitution of (M10), (M11) and (M13). (m14) Includes substitution of (M10), (M12) and (M13).(m15) Includes substitutions of (M11), (M12), and (M13). (m16) Includes substitutions of (M1), (M10), (M11), and (M13). (m17) Includes substitutions of (M1), (M10), (M12), and (M13). (m18) Includes substitutions of (M1), (M11), (M12), and (M13). 7. The fusion protein according to 1, wherein the mutant ADAMTS13 consists of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 99. 8. The fusion protein according to 1, wherein the antigen-binding molecule comprises at least the heavy chain variable region (VH) and the light chain variable region (VL) of an anti-vWF antibody. 9. The fusion protein according to 8, wherein in the antigen-binding molecule, the VH and the VL are any one of (c1) to (c37) below. (c1) The VH includes Complementarity Determinating Regions (CDRs) 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 565 to 567, and the VL includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 568 to 570. (c2) The VH includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 573 to 575, and the VL includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 576 to 578. (c3) The VH includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 581 to 583, and the VL includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 584 to 586. (c4) The VH includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 587 to 589, and the VL includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 590 to 592. (c5) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 595 to 597, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 598 to 600. (c6) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 603 to 605, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 606 to 608.(c7) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 611 to 613, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 614 to 616, respectively. (c8) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 619 to 621, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 622 to 624, respectively. (c9) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 627 to 629, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 630 to 632, respectively. (c10) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 635 to 637, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 638 to 640, respectively. (c11) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 643 to 645, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 646 to 648, respectively. (c12) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 651 to 653, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 654 to 656, respectively. (c13) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 659 to 661, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 662 to 664, respectively. (c14) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 667 to 669, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 670 to 672, respectively. (c15) The VH comprises CDR1 to CDR3 each containing amino acid sequences represented by SEQ ID NOs. 675 to 677, and the VL comprises CDR1 to CDR3 each containing amino acid sequences represented by SEQ ID NOs. 678 to 680. (c16) The VH comprises CDR1 to CDR3 each containing amino acid sequences represented by SEQ ID NOs. 683 to 685, and the VL comprises CDR1 to CDR3 each containing amino acid sequences represented by SEQ ID NOs. 686 to 688.(c17) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 691 to 693, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 694 to 696, respectively. (c18) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 699 to 701, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 702 to 704, respectively. (c19) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 707 to 709, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 710 to 712, respectively. (c20) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 715 to 717, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 718 to 720, respectively. (c21) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 723 to 725, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 726 to 728. (c22) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 731 to 733, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 734 to 736. (c23) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 795 to 797, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 798 to 800. (c24) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 803 to 805, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 806 to 808. (c25) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 811 to 813, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 814 to 816. (c26) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 819 to 821, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 822 to 824.(c27) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 827 to 829, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 830 to 832, respectively. (c28) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 835 to 837, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 838 to 840, respectively. (c29) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 843 to 845, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 846 to 848, respectively. (c30) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 851 to 853, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 854 to 856, respectively. (c31) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 859 to 861, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 862 to 864. (c32) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 867 to 869, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 870 to 872. (c33) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 875 to 877, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 878 to 880. (c34) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 883 to 885, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 886 to 888. (c35) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 891 to 893, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 894 to 896. (c36) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 899 to 901, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 902 to 904.(c37) The VH comprises CDR1 to CDR3 each containing amino acid sequences represented by SEQ ID NOs: 907 to 909, and the VL comprises CDR1 to CDR3 each containing amino acid sequences represented by SEQ ID NOs: 910 to 912. 10. The fusion protein according to 9, wherein the antigen-binding molecule is one of the following (v1) to (v37): (v1) The VH comprises an amino acid sequence represented by SEQ ID NOs: 563, and the VL comprises an amino acid sequence represented by SEQ ID NOs: 564. (v2) The VH comprises an amino acid sequence represented by SEQ ID NOs: 571, and the VL comprises an amino acid sequence represented by SEQ ID NOs: 572. (v3) The VH comprises an amino acid sequence represented by SEQ ID NOs: 579, and the VL comprises an amino acid sequence represented by SEQ ID NOs: 580. (v4) The VH comprises an amino acid sequence represented by SEQ ID NOs: 100, and the VL comprises an amino acid sequence represented by SEQ ID NOs: 101. (v5) The VH includes the amino acid sequence represented by SEQ ID NO: 593, and the VL includes the amino acid sequence represented by SEQ ID NO: 594. (v6) The VH includes the amino acid sequence represented by SEQ ID NO: 601, and the VL includes the amino acid sequence represented by SEQ ID NO: 602. (v7) The VH includes the amino acid sequence represented by SEQ ID NO: 609, and the VL includes the amino acid sequence represented by SEQ ID NO: 610. (v8) The VH includes the amino acid sequence represented by SEQ ID NO: 617, and the VL includes the amino acid sequence represented by SEQ ID NO: 618. (v9) The VH includes the amino acid sequence represented by SEQ ID NO: 625, and the VL includes the amino acid sequence represented by SEQ ID NO: 626. (v10) The VH includes the amino acid sequence represented by SEQ ID NO: 633, and the VL includes the amino acid sequence represented by SEQ ID NO: 634. (v11) The VH includes the amino acid sequence represented by SEQ ID NO: 641, and the VL includes the amino acid sequence represented by SEQ ID NO: 642. (v12) The VH includes the amino acid sequence represented by SEQ ID NO: 649, and the VL includes the amino acid sequence represented by SEQ ID NO: 650. (v13) The VH includes the amino acid sequence represented by SEQ ID NO: 657, and the VL includes the amino acid sequence represented by SEQ ID NO: 658. (v14) The VH includes the amino acid sequence represented by SEQ ID NO: 665, and the VL includes the amino acid sequence represented by SEQ ID NO: 666.(v15) The VH includes the amino acid sequence represented by SEQ ID NO: 673, and the VL includes the amino acid sequence represented by SEQ ID NO: 674. (v16) The VH includes the amino acid sequence represented by SEQ ID NO: 681, and the VL includes the amino acid sequence represented by SEQ ID NO: 682. (v17) The VH includes the amino acid sequence represented by SEQ ID NO: 689, and the VL includes the amino acid sequence represented by SEQ ID NO: 690. (v18) The VH includes the amino acid sequence represented by SEQ ID NO: 697, and the VL includes the amino acid sequence represented by SEQ ID NO: 698. (v19) The VH includes the amino acid sequence represented by SEQ ID NO: 705, and the VL includes the amino acid sequence represented by SEQ ID NO: 706. (v20) The VH includes the amino acid sequence represented by SEQ ID NO: 713, and the VL includes the amino acid sequence represented by SEQ ID NO: 714. (v21) The VH includes the amino acid sequence represented by SEQ ID NO: 721, and the VL includes the amino acid sequence represented by SEQ ID NO: 722. (v22) The VH includes the amino acid sequence represented by SEQ ID NO: 729, and the VL includes the amino acid sequence represented by SEQ ID NO: 730. (v23) The VH includes the amino acid sequence represented by SEQ ID NO: 793, and the VL includes the amino acid sequence represented by SEQ ID NO: 794. (v24) The VH includes the amino acid sequence represented by SEQ ID NO: 801, and the VL includes the amino acid sequence represented by SEQ ID NO: 802. (v25) The VH includes the amino acid sequence represented by SEQ ID NO: 809, and the VL includes the amino acid sequence represented by SEQ ID NO: 810. (v26) The VH includes the amino acid sequence represented by SEQ ID NO: 817, and the VL includes the amino acid sequence represented by SEQ ID NO: 818. (v27) The VH includes the amino acid sequence represented by SEQ ID NO: 825, and the VL includes the amino acid sequence represented by SEQ ID NO: 826. (v28) The VH includes the amino acid sequence represented by SEQ ID NO: 833, and the VL includes the amino acid sequence represented by SEQ ID NO: 834. (v29) The VH includes the amino acid sequence represented by SEQ ID NO: 841, and the VL includes the amino acid sequence represented by SEQ ID NO: 842. (v30) The VH includes the amino acid sequence represented by SEQ ID NO: 849, and the VL includes the amino acid sequence represented by SEQ ID NO: 850.(v31) The VH includes the amino acid sequence represented by SEQ ID NO: 857, and the VL includes the amino acid sequence represented by SEQ ID NO: 858. (v32) The VH includes the amino acid sequence represented by SEQ ID NO: 865, and the VL includes the amino acid sequence represented by SEQ ID NO: 866. (v33) The VH includes the amino acid sequence represented by SEQ ID NO: 873, and the VL includes the amino acid sequence represented by SEQ ID NO: 874. (v34) The VH includes the amino acid sequence represented by SEQ ID NO: 881, and the VL includes the amino acid sequence represented by SEQ ID NO: 882. (v35) The VH includes the amino acid sequence represented by SEQ ID NO: 889, and the VL includes the amino acid sequence represented by SEQ ID NO: 890. (v36) The VH includes the amino acid sequence represented by SEQ ID NO: 897, and the VL includes the amino acid sequence represented by SEQ ID NO: 898. (v37) The VH includes the amino acid sequence represented by SEQ ID NO: 905, and the VL includes the amino acid sequence represented by SEQ ID NO: 906. 11. The fusion protein according to claim 1, wherein the antigen-binding molecule is an antigen-binding molecule containing any one of (B1) to (B4) below. (B1) An antigen-binding molecule containing scFv, which includes VH and VL. (B2) An antigen-binding molecule containing Fab, which includes a heavy chain containing VH and a CH1 domain, and a light chain containing VL and a constant light chain (CL) region. (B3) An antigen-binding molecule containing the following first polypeptide and second polypeptide, wherein mutations have been introduced into the Cys residue involved in the intermolecular disulfide bond between the light chain and heavy chain in the IgG antibody in the CH1 domain and CL. First polypeptide: A polypeptide in which VH, CH1 domain, hinge domain, CH2 domain and CH3 domain are linked in this order. Second polypeptide: A polypeptide in which VL, CL, hinge domain, CH2 domain and CH3 domain are linked in this order. (B4) An antigen-binding molecule containing IgG, which includes two heavy chains containing VH, CH1 domain, hinge domain, CH2 domain and CH3 domain, and two light chains containing VL and CL. 12. The fusion protein according to claim 1, wherein the mutant ADAMTS13 consists of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 99, and the antigen-binding molecule is an antigen-binding molecule containing any one of the following (B1) to (B4).(B1) An antigen-binding molecule containing scFv, which includes VH and VL. (B2) An antigen-binding molecule containing Fab, which includes a heavy chain containing VH and a CH1 domain, and a light chain containing VL and a constant light chain (CL) region. (B3) An antigen-binding molecule containing the following first polypeptide and second polypeptide, wherein mutations have been introduced into the Cys residue involved in the intermolecular disulfide bond between the light chain and heavy chain in the IgG antibody in the CH1 domain and CL. First polypeptide: A polypeptide in which VH, CH1 domain, hinge domain, CH2 domain and CH3 domain are linked in this order. Second polypeptide: A polypeptide in which VL, CL, hinge domain, CH2 domain and CH3 domain are linked in this order. (B4) An antigen-binding molecule containing IgG, which includes two heavy chains containing VH, CH1 domain, hinge domain, CH2 domain and CH3 domain, and two light chains containing VL and CL. 13. The fusion protein according to 1, wherein the mutant ADAMTS13 is linked to the antigen-binding molecule via a linker. 14. The fusion protein according to 13, wherein the linker is 1 to 35 amino acids long. 15. The fusion protein according to 13, wherein the linker is a linker containing at least one repeating unit selected from (L1) to (L3) below: (L1) A polypeptide containing the amino acid sequence (GGGGS) represented by SEQ ID NO: 925 as a repeating unit. (L2) A polypeptide containing the amino acid sequence (PAPAP) represented by SEQ ID NO: 926 as a repeating unit. (L3) A polypeptide containing the amino acid sequence (EAAAK) represented by SEQ ID NO: 927 as a repeating unit. 16. The fusion protein according to 14, wherein the antigen-binding molecule contains VH and VL of an anti-vWF antibody, and the C-terminus of the mutant ADAMTS13 is linked to the N-terminus of the VH or VL in the antigen-binding molecule via the linker. 17. The fusion protein described in 11 above, which is any one of (p3-1) to (p3-3), (p4-1), and (p4-2) below.(p3-1) The antigen-binding molecule is an antigen-binding molecule comprising (B3), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of the mutant ADAMTS13 is linked to the N-terminus of VH in the antigen-binding molecule via a linker, and comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [VL-CL-hinge domain-CH2-CH3] (p3-2) The antigen-binding molecule is an antigen-binding molecule comprising (B3), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of the mutant ADAMTS13 is linked to the N-terminus of the VL in the antigen-binding molecule via a linker, and comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [mutant ADAMTS13-peptide linker-VL-CL-hinge domain-CH2-CH3] (p3-3) The antigen-binding molecule is an antigen-binding molecule comprising (B3), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the N-terminus of the mutant ADAMTS13 is linked to the C-terminus of the CH3 domain in the antigen-binding molecule via a linker, and comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [VL-CL-hinge domain-CH2-CH3-peptide linker-mutant ADAMTS13](p4-1) The antigen-binding molecule is an antigen-binding molecule containing (B4), and is a fusion protein formed by linking two mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of each mutant ADAMTS13 and the N-terminus of each VH in the antigen-binding molecule are linked via a linker, and the fusion protein contains the following first to fourth polypeptides, wherein the CL of the first polypeptide and the hinge domain or CH1 of the second polypeptide are linked via a disulfide bond, the hinge domain or CH1 of the third polypeptide and the CL of the fourth polypeptide are linked via a disulfide bond, and the second polypeptide and the third polypeptide are linked via a disulfide bond of the hinge domain. First and fourth polypeptides: [VL-CL] Second and third polypeptides: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] (p4-2) The antigen-binding molecule is an antigen-binding molecule containing (B4), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of mutant ADAMTS13 and the N-terminus of VH in the antigen-binding molecule are linked via a linker, and the fusion protein contains the following first to fourth polypeptides, wherein the CL of the first polypeptide and the hinge domain or CH1 of the second polypeptide are linked via a disulfide bond, the hinge domain or CH1 of the third polypeptide and the CL of the fourth polypeptide are linked via a disulfide bond, and the second polypeptide and the third polypeptide are linked via a disulfide bond of the hinge domain. First and fourth polypeptides: [VL-CL] Second polypeptide: [VH-CH1-hinge domain-CH2-CH3] Third polypeptide: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] 18. The fusion protein described in 17, which is any one of the following: (t1) The fusion protein of (p3-1), wherein the first polypeptide contains an amino acid sequence represented by any one of SEQ ID NOs: 333, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, and 545.A fusion protein wherein the second polypeptide contains the amino acid sequence represented by SEQ ID NO: 334. (t2) The fusion protein of (p4-1) wherein the first polypeptide and the fourth polypeptide are the same and contain the amino acid sequence represented by SEQ ID NO: 114, and the second polypeptide and the third polypeptide are the same and contain the amino acid sequence represented by any one of SEQ ID NOs: 113, 352, 354, 356, 358, 360, and 362. (t3) A fusion protein of the above (p4-1), wherein the first polypeptide and the fourth polypeptide are the same and include the amino acid sequence represented by SEQ ID NO: 110, and the second polypeptide and the third polypeptide are the same and include the amino acid sequence represented by any one of SEQ ID NOs: 109, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406 and 408. (t4) A fusion protein of the above (p4-1), wherein the first polypeptide and the fourth polypeptide are the same and include the amino acid sequence represented by SEQ ID NO: 126, A fusion protein in which the second polypeptide and the third polypeptide are identical and contain an amino acid sequence represented by any one of SEQ ID NOs: 125, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, and 512. 19. The fusion protein according to claim 1, wherein the antigen-binding molecule in the fusion protein specifically binds to the A2 domain of vWF. 20. The fusion protein according to claim 1, wherein the antigen-binding molecule in the fusion protein specifically binds to the polypeptide of (α1) or (α2) below. (α1) A polypeptide consisting of the amino acid sequence represented by Sequence ID No. 9141. A polypeptide comprising the amino acid sequence represented by (α2) Sequence ID No. 915. 21. The fusion protein according to 19 or 20, wherein the antigen-binding molecule has the ability to bind to both vWF with the A2 domain folded and vWF without the A2 domain folded. 22. A nucleic acid encoding the fusion protein according to any one of 1 to 21. 23. A vector containing the nucleic acid according to 22. 24. A transformed cell obtained by introducing the vector according to 23 into a host cell. 25. A method for producing the fusion protein according to any one of 1 to 21, characterized by culturing the transformed cell according to 24 in a culture medium, producing and accumulating the fusion protein according to any one of 1 to 21 in the culture obtained by the culture, and collecting the fusion protein from the culture. 26. A therapeutic agent for thrombotic diseases containing the fusion protein according to any one of 1 to 21 as an active ingredient. 27. A pharmaceutical composition containing the fusion protein described in any one of items 1 to 21 above as an active ingredient, used to suppress recurrence of thrombotic thrombocytopenic purpura in patients with thrombotic thrombocytopenic purpura. 28. A pharmaceutical composition containing the fusion protein described in any one of items 1 to 21 above as an active ingredient, used to supplement ADAMTS13 enzyme activity in patients with thrombotic thrombocytopenic purpura exhibiting recurrent episodes.

[0015] The fusion protein of this disclosure comprises a mutant ADAMTS13 containing at least an M domain and lacking a spacer domain (S domain), and an antigen-binding molecule that binds to vWF. The fusion protein of this disclosure can suppress the generation of autoantibodies against the S domain due to the S domain deficiency in the mutant ADAMTS13. The fusion protein of this disclosure includes an antigen-binding molecule that binds to vWF together with the mutant ADAMTS13, so that despite the S domain deficiency in the mutant ADAMTS13, it can access vWF via the antigen-binding molecule that binds to vWF and effectively cleave vWF. Therefore, the fusion protein of this disclosure may enable rapid, safe, and sustained replenishment of ADAMTS13 enzyme activity.

[0016] Figure 1 shows one embodiment of the structure of full-length ADAMTS13. Figure 2 shows a schematic diagram of one embodiment of IgG1 as an antigen-binding molecule. Figures 3(a) to (c) show schematic diagrams of one embodiment of an antigen-binding molecule containing scFv. Figures 4(a) and (b) show schematic diagrams of one embodiment of an antigen-binding molecule containing Fab. Figure 5 shows a schematic diagram of one embodiment of mvG1. Figure 6 shows a schematic diagram of vWF. Figure 7 shows a schematic diagram of one embodiment of the fusion protein of the present disclosure. Figures 8(a) to (d) show schematic diagrams of one embodiment of the fusion protein of the present disclosure in which the antigen-binding molecule is an antigen-binding molecule containing (B1)scFv. Figures 9(a) to (g) show schematic diagrams of one embodiment of the fusion protein of the present disclosure in which the antigen-binding molecule is an antigen-binding molecule containing (B2)Fab. Figures 10(a) to (c) show schematic diagrams of one embodiment of the fusion protein of this disclosure in which the antigen-binding molecule is an antigen-binding molecule containing (B3)mvG1. Figures 11(a) and (b) show schematic diagrams of one embodiment of the fusion protein of this disclosure in which the antigen-binding molecule is an antigen-binding molecule containing (B4)IgG1. Figure 12 shows the results of evaluating the vWF degradation activity of non-fusion compounds (anti-human vWF antibodies or various domain-deficient compounds of ADAMTS13). Figure 13 shows the results of evaluating the inhibition rate by autoantibodies for each fusion protein. Figures 14(a) to (e) show the results of evaluating the ADAMTS13 enzyme activity when the PEP01 region, PEP05 region, or PEP06 region is used as the epitope region, respectively.

[0017] This disclosure relates to a fusion protein (hereinafter also referred to as "this fusion protein") comprising a mutant ADAMTS13 containing at least an M domain and lacking an S domain, and an antigen-binding molecule that binds to vWF.

[0018] Unless otherwise specified, the homology values ​​in this disclosure may be values ​​calculated using homology search programs known to those skilled in the art. Examples include values ​​calculated using default parameters in BLAST [J. Mol. Biol., 215, 403 (1990)] for nucleotide sequences, and values ​​calculated using default parameters in BLAST2 [Nucleic Acids Res., 25, 3389 (1997), Genome Res., 7, 649 (1997), http: / / www.ncbi.nlm.nih.gov / Education / BLASTinfo / information3.html] for amino acid sequences.

[0019] The default parameters are: G (Cost to open gap) is 5 for nucleotide sequences and 11 for amino acid sequences; -E (Cost to extend gap) is 2 for nucleotide sequences and 1 for amino acid sequences; -q (Penalty for nucleotide mismatch) is -3; -r (reward for nucleotide match) is 1; -e (expect value) is 10; -W (wordsize) is 11 residues for nucleotide sequences and 3 residues for amino acid sequences; -y [Dropoff(X) for blast extensions in bits] is 20 for blastn and 7 for programs other than blastn; -X (X dropoff value for The value of -Z (final X dropoff value for gapped alignment in bits) is 15 and -Z is 50 for blastn and 25 for programs other than blastn (http: / / www.ncbi.nlm.nih.gov / blast / htmL / blastcgihelp.htmL).

[0020] <Mutant ADAMTS13> In this disclosure, "ADAMTS13" or "A13" refers to a metalloproteinase of the ADAMTS (disintegrin and metalloproteinase having a thrombospondin type 1 motif) family that cleaves von Willebrand factor (vWF) between Tyr1605 and Met1606.

[0021] In this disclosure, "ADAMTS13" includes, for example, ADAMTS13 derived from mammals such as primates, humans (GenBank accession number: NP620594), monkeys, rabbits, pigs, and cattle (GenBank accession number: XP610784), rodents, mice (GenBank accession number: NP001001322), rats (GenBank accession number: XP342396), hamsters, gerbils, dogs, cats, frogs (GenBank accession number: NP001083331), and chickens (GenBank accession number: XP415435), as well as its biologically active derivatives.

[0022] As the fusion protein in this disclosure, the amino acid sequences of the M domain of ADAMTS13 from mammals such as green monkeys (Chlorocebus sabaeus), Yunnan hognose monkeys (Rhinopitecus biti), Nancy's maznight monkeys (Aotus nancymaae), dogs, and rats may be used as mutant ADAMTS13. In one embodiment, the amino acid sequences represented by SEQ ID NOs. 48 to 52 may be used as the M domain of ADAMTS13 from these animals. For example, the rat-derived M domain has 80% homology to the human ADAMTS13 M domain represented by SEQ ID NO. 99, and the dog-derived M domain has 86% homology to the human-derived M domain. When these M domains from other species are used as mutant ADAMTS13 in this fusion protein, they may further contain 1 to 20 amino acid point mutations.

[0023] ADAMTS13 is translated into a full-length amino acid protein within the cell, but when it is subsequently processed and secreted outside the cell, the signal peptide and propeptide are usually cleaved. Examples of processed polypeptides include polypeptides from which the signal peptide or a portion thereof has been removed, and / or polypeptides from which the propeptide or a portion thereof has been removed. Preferably, the processed polypeptide is one from which both the signal peptide and the propeptide have been removed.

[0024] The mutant ADAMTS13 in this fusion protein is preferably the amino acid sequence represented by SEQ ID NOs: 1 to 99, but during the processing process, it may be generated as an amino acid sequence in which a signal peptide or a part thereof and / or a propeptide or a part thereof is added to the N-terminus, and these amino acid sequences are also included in the mutant ADAMTS13 of the present invention. For example, the mutant ADAMTS13 in this fusion protein may have 1 to 5 amino acids of a propeptide added to the N-terminus. Alternatively, in another embodiment, the mutant ADAMTS13 in this fusion protein may be generated during the processing process as an amino acid sequence in which 1 to 10 amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NOs: 1 to 99 are removed, and these amino acid sequences are also included in the mutant ADAMTS13 of the present invention.

[0025] Human ADAMTS13 is not particularly limited, but includes a polypeptide (SEQ ID NO: 194) which is the full-length amino acid sequence of UniPro accession number Q76LX8-1, or a processed polypeptide thereof. In human ADAMTS13, the signal peptide is a polypeptide containing amino acids 1 to 29 of the amino acid sequence represented by SEQ ID NO: 194, and the propeptide is a polypeptide containing amino acids 30 to 74 of the amino acid sequence represented by SEQ ID NO: 194.

[0026] In this specification, the amino acid numbers in the amino acid sequence of mutant ADAMTS13 are expressed using the amino acid numbers corresponding to the amino acid sequence represented by SEQ ID NO: 194. The amino acid numbers corresponding to the amino acid sequence represented by SEQ ID NO: 194 refer to the amino acid numbers corresponding to the amino acid numbers in the amino acid sequence represented by SEQ ID NO: 194 when the amino acid sequence of mutant ADAMTS13 is aligned with the amino acid sequence represented by SEQ ID NO: 194.

[0027] Amino acid sequence alignments can be created using the well-known alignment program CrystalW (Nucleic Acids Research 22, 4673, (1994)). CrystalW is available from the European Bioinformatics Institute website. When creating alignments using CrystalW, default values ​​can be used for parameters, for example.

[0028] Many natural variants of human ADAMTS13 are known in the art, and ADAMTS13 of this disclosure may have some of these amino acid mutations. Some of these include mutations selected from R7W, V88M, H96D, R102C, R193W, T196I, H234Q, A250V, R268P, W390C, R398H, Q448E, Q456H, P457L, P475S, and C508Y.

[0029] FIG. 1 shows one embodiment of a schematic diagram of the structure of full-length ADAMTS13. As shown in FIG. 1, ADAMTS13, from the N-terminal side, consists of a metalloprotease (M) domain, a disintegrin-like (D) domain, a thrombospondin type 1 motif (T1) domain, a cysteine-rich (C) domain, a spacer (S) domain, seven consecutive T domains (T2-T8 domains), and two CUB domains (CUB1-2 domains) (Thrombosis and Hemostasis 2015; 26(4): 433-438). The mutant ADAMTS13 of the present disclosure contains at least its M domain and lacks the S domain. The black line present further on the N-terminal side of the M domain in FIG. 1 represents the signal peptide and propeptide that are cleaved by processing.

[0030] The M domain of ADAMTS13 has metalloprotease activity that requires zinc ions and is a domain having the structure and function necessary for catalyzing the cleavage of von Willebrand factor (vWF) in particular. The M domain of human ADAMTS13 preferably contains at least the amino acid sequence from positions 80 to 286 of the amino acid sequence represented by SEQ ID NO: 194.

[0031] The S domain of ADAMTS13 is a region present between the M domain and the T2 domain. It is a domain that controls the distance and interaction between the M domain and the T2 domain and plays a role in maintaining the normal function of ADAMTS13 (Blood. 102(9): 3232-7). Blood. 102(9): 3232-7 describes that the S domain of ADAMTS13 is functionally essential for the cleavage of vWF.

[0032] The fusion protein in the present disclosure contains an antigen-binding molecule that binds to vWF together with the mutant ADAMTS13. Thus, although the mutant ADAMTS13 lacks the S domain, it can access vWF via the antigen-binding molecule that binds to vWF, and the cleavage of vWF may be possible. Furthermore, since the mutant ADAMTS13 lacks the S domain, it can suppress the generation of autoantibodies against the S domain, and thus has low autoantibody reactivity.

[0033] The mutant ADAMTS13 in this disclosure lacks an S domain. For example, if ADAMTS13 is human ADAMTS13, in this embodiment, it is preferable that the mutant ADAMTS13 does not contain amino acid sequences 556 to 685 of the amino acid sequence represented by SEQ ID NO: 194 as the S domain.

[0034] The mutant ADAMTS13 in this disclosure can further reduce autoantibody reactivity by further deleting the T2-8 domain and / or the CUB1-2 domain in addition to the S domain. One embodiment of the mutant ADAMTS13 in this disclosure is, for example, (N1) or (N2) below. (N1) In addition to the S domain of ADAMTS13, the T2-T8 domain is further deleted. For example, when ADAMTS13 is human ADAMTS13, in this embodiment, it is preferable that the mutant ADAMTS13 does not contain amino acid sequences 556 to 1191 of the amino acid sequence represented by SEQ ID NO: 194 as the S domain and T2-8 domain. (N2) In addition to the S domain of ADAMTS13, the T2-8 domain and the CUB1-2 domain are deleted. For example, if ADAMTS13 is human ADAMTS13, in this embodiment, it is preferable that the mutant ADAMTS13 does not contain amino acid sequences 556 to 1427 of the amino acid sequence represented by Sequence ID No. 194 as the S domain, T2-8 domain, and CUB1-2 domain.

[0035] Examples of mutant ADAMTS13 in this disclosure include (D1) to (D4) below. From the viewpoint of reactivity with autoantibodies, (D1) and (D2) are preferred, and (D1) is more preferred. (D1) Consists of the M domain of ADAMTS13. (D2) Consists of the M domain and disintegrin-like (D) domain of ADAMTS13. (D3) Consists of the M domain, D domain and thrombospondin type 1 motif (T1) domain of ADAMTS13. (D4) Consists of the M domain, D domain, thrombospondin type 1 motif (T1) domain and cysteine-rich (C) domain of ADAMTS13.

[0036] In one aspect, the M domain of ADAMTS13 consists of an amino acid sequence (SEQ ID NO: 931) composed of amino acids at positions 75 to 286 of the amino acid sequence represented by SEQ ID NO: 194, the D domain consists of an amino acid sequence (SEQ ID NO: 932) composed of amino acids at positions 287 to 383 of the amino acid sequence represented by SEQ ID NO: 194, the T1 domain consists of an amino acid sequence (SEQ ID NO: 933) composed of amino acids at positions 384 to 439 of the amino acid sequence represented by SEQ ID NO: 194, the C domain consists of an amino acid sequence (SEQ ID NO: 934) composed of amino acids at positions 440 to 555 of the amino acid sequence represented by SEQ ID NO: 194, and the S domain consists of an amino acid sequence (SEQ ID NO: 935) composed of amino acids at positions 556 to 685 of the amino acid sequence represented by SEQ ID NO: 194, respectively. The definition of the number or range of amino acid residues constituting each domain allows for an increase or decrease of several to several tens of amino acids depending on the known literature or sequence database referred to, and fusion proteins using these variations are also included in the fusion proteins of the present disclosure as long as the effects of the present disclosure can be obtained.

[0037] In the fusion protein of the present disclosure, the mutant ADAMTS13 may have the same ADAMTS13 enzyme activity as in the case where the amino acids at positions 75 to 85 and / or 286 to 290 of the amino acid sequence represented by SEQ ID NO: 194 are deleted. Alternatively, in the fusion protein of the present disclosure, the mutant ADAMTS13 may have the same ADAMTS13 enzyme activity as in the present invention even if the amino acids at positions 70 to 74 of the amino acid sequence represented by SEQ ID NO: 194 are added to the N-terminus.

[0038] Based on these characteristics, the amino acid sequence of the M domain of the mutant ADAMTS13 may have a signal peptide and / or a part of a propeptide added to its N-terminus, and the amino acid sequences at positions 75 to 85 and / or 286 to 290 of the amino acid sequence represented by SEQ ID NO: 194 may be deleted. Furthermore, the mutant ADAMTS13 of the present invention may use the amino acid sequence of the M domain of ADAMTS13 derived from mammals other than humans or a derivative thereof.

[0039] Based on the above, if the mutant ADAMTS13 has ADAMTS13 enzyme activity as a fusion protein, then an amino acid sequence with at least 70% homology to the amino acid sequence from position 80 to 286 of the amino acid sequence represented by Sequence ID No. 194 is acceptable. The homology is preferably 70% or more, more preferably 75% or more, 80% or more, 85% or more, 90% or more, and even more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, and 97% or more.

[0040] In this specification, ADAMTS13 enzyme activity refers to the protease activity of ADAMTS13. ADAMTS13 enzyme activity can be measured by conventionally known methods such as the FRET substrate method and the ELISA method. One embodiment of a method for measuring ADAMTS13 enzyme activity is a method including the following steps (ai) to (aiii). (ai) Mix the FRET substrate solution and the fusion protein. (aiii) Immediately after mixing, measure the fluorescence at a wavelength of 450 nm using excitation light at a wavelength of 340 nm every 5 minutes for 60 minutes using a plate reader. (aiii) Calculate the substrate degradation rate from the measured fluorescence intensity and the calibration curve of the standard fluorescent dye, and convert it to the concentration of hADAMTS13 (nM).

[0041] In this disclosure, having ADAMTS13 enzyme activity means, in one embodiment, having ADAMTS13 activity of preferably 4% or more, more preferably 10% or more, and even more preferably 20% or more, compared to 100% of the ADAMTS13 activity of wild-type ADAMTS13. Here, the percentage of ADAMTS13 enzyme activity in this specification is expressed as a percentage obtained by dividing the value (nM) of the substrate degradation rate of the fusion protein converted to the concentration of wild-type hADAMTS13 by the final concentration of the fusion protein used for evaluation. For example, if the value of the substrate degradation rate converted to the hADAMTS13 concentration is 1.0 nM or more, and the fusion protein is evaluated at a final concentration of 10 nM, it can be considered to have 10% or more ADAMTS13 activity.

[0042] Another embodiment of the method for measuring ADAMTS13 enzyme activity is, for example, a method comprising the following steps (bi) to (biii): (bi) Plot the reaction time (min) and product concentration (μmol / L) at each substrate concentration, and calculate the initial velocity (V0, μmol / L・min) by the slope from 0 to 5 minutes. (biii) Using the data for each substrate concentration and initial velocity, perform curve fitting to the Michaelis-Menten equation to calculate the maximum reaction rate (Vmax, μmol / L・min) and the dissociation constant (Km, μmol / L). (biii) Calculate kcat ( / min) by dividing Vmax by the enzyme concentration (fusion protein concentration in this embodiment).

[0043] In this disclosure, having ADAMTS13 enzyme activity means, in one embodiment, preferably 0.15 ( / min) or more, more preferably 0.3 ( / min) or more, and even more preferably 1.0 ( / min) or more.

[0044] In one embodiment, the mutant ADAMTS13 is preferably composed of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 99. More preferably, the amino acid sequence is the amino acid sequence represented by SEQ ID NOs: 68, 69, 82 to 85, 87 to 98, or 99.

[0045] When ADAMTS13 is human ADAMTS13, one embodiment of the mutant ADAMTS13 preferably includes one amino acid sequence selected from (A1) to (A4) below: (A1) The amino acid sequence from position 75 to 286 of SEQ ID NO: 194 (A2) The amino acid sequence from position 75 to 386 of SEQ ID NO: 194 (A3) The amino acid sequence from position 75 to 440 of SEQ ID NO: 194 (A4) The amino acid sequence from position 75 to 556 of SEQ ID NO: 194

[0046] From the viewpoint of enhancing ADAMTS13 enzyme activity, in this embodiment, when mutant ADAMTS13 is human ADAMTS13, it is preferable that the amino acid sequence of the M domain in mutant ADAMTS13 includes an amino acid sequence corresponding to the amino acid sequence from position 80 to 286 of the amino acid sequence represented by SEQ ID NO: 194, and further includes at least one of the following substitutions (M1) to (M13). (M1) Substitution of the amino acid residue corresponding to the 159th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to R (M2) Substitution of the amino acid residue corresponding to the 167th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to M (M3) Substitution of the amino acid residue corresponding to the 207th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to N (M4) Substitution of the amino acid residue corresponding to the 209th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to V (M5) Substitution of the amino acid residue corresponding to the 230th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to L (M6) Substitution of the amino acid residue corresponding to the 238th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to A (M7) Substitution of the amino acid residue corresponding to the 243rd amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to K (M8) Substitution of the amino acid residue corresponding to the 271st amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to A (M9) Substitution of the amino acid residue corresponding to the 271st amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to E (M10) Substitution of L from the amino acid residue corresponding to the 279th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M11) Substitution of L from the amino acid residue corresponding to the 282nd amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M12) Substitution of G from the amino acid residue corresponding to the 283rd amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M13) Substitution of L from the amino acid residue corresponding to the 285th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194

[0047] The substituted amino acid residues described above may be mutually substituted amino acids. Examples of mutually substituted amino acids are shown below. Amino acids in the same group are mutually substituted. Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutanoic acid, Methionine, O-Methylserine, t-Butylglycine, t-Butylalanine, Cyclohexylalanine Group B: Aspartic acid, Glutamic acid, Isoaspartic acid, Isoglutamic acid, 2-Aminoadipic acid, 2-Aminosveric acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine, Ornithine, 2,4-Diaminobutanoic acid, 2,3-Diaminopropionic acid Group E: Proline, 3-Hydroxyproline, 4-Hydroxyproline Group F: Serine, Threonine, Homoserine Group G: Phenylalanine, Tyrosine

[0048] The amino acids to be substituted as described above may be either natural or unnatural. Examples of natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine. Examples of unnatural amino acids include various amino acids having an amino group and a carboxyl group, but derivatives of various natural amino acids are preferred. Many unnatural amino acids are available from various reagent companies (Sigma-Aldrich, TCI). Much information on non-natural amino acids can be found in the literature (Chem. Today 2003, 65.; Curr Opin Chem Biol. 2000, 6, 645.).

[0049] The amino acid sequence of mutant ADAMTS13 is preferably one of the following in one embodiment: (m1) Includes the substitutions of (M1) and (M2). In one embodiment, an amino acid sequence is provided in which the amino acid residue corresponding to the 159th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with R, and the amino acid residue corresponding to the 167th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with M. An example of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 68. (m2) Includes the substitutions of (M1) and (M3). In one embodiment, an amino acid sequence is provided in which the amino acid residue corresponding to the 159th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with R, and the amino acid residue corresponding to the 207th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with N. An example of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 69. (m3) Includes the substitutions of (M8) and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 82. (m4) Includes the substitutions of (M9) and (M13) above. One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with E, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 83. (m5) Includes the substitutions of (M1), (M8) and (M13) above. One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 159th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with R, the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L.One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 84. (m6) Includes the substitutions of (M1), (M9) and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 159th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with R, the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with E, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 85. (m7) Includes the substitutions of (M4), (M5), (M8) and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 209th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with V, the amino acid residue corresponding to the 230th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 87. (m8) Includes the substitutions of (M4), (M6), (M8) and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 209th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with V, the amino acid residue corresponding to the 238th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by Sequence ID No. 88, which includes the substitutions of (M4), (M7), (M8), and (M13) (m9).One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 209th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with V, the amino acid residue corresponding to the 243rd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with K, the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 89. (m10) Includes the substitutions of (M5), (M6), (M8) and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 230th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, the amino acid residue corresponding to the 238th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 90. (m11) Includes the substitutions of (M5), (M7), (M8) and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 230th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, the amino acid residue corresponding to the 243rd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with K, the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 91. (m12) Includes the substitutions of (M6), (M7), (M8) and (M13).One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 238th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, the amino acid residue corresponding to the 243rd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with K, the amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with A, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment is an amino acid sequence represented by SEQ ID NO: 92. (m13) Includes the substitutions of (M10), (M11) and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 279th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, the amino acid residue corresponding to the 282nd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment is an amino acid sequence represented by SEQ ID NO: 93. (m14) Includes the substitutions of (M10), (M12) and (M13). In one embodiment, an amino acid sequence is provided in which the amino acid residue corresponding to the 279th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, the amino acid residue corresponding to the 283rd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with G, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. An example of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 94. (m15) Includes the substitutions of (M11), (M12) and (M13). In one embodiment, an amino acid sequence is provided in which the amino acid residue corresponding to the 282nd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, the amino acid residue corresponding to the 283rd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with G, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. An example of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 95.(m16) Includes the substitutions of (M1), (M10), (M11), and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 159th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with R, the amino acid residue corresponding to the 279th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, the amino acid residue corresponding to the 282nd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 96. (m17) Includes the substitutions of (M1), (M10), (M12), and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 159th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with R, the amino acid residue corresponding to the 279th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, the amino acid residue corresponding to the 283rd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with G, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One embodiment of such an amino acid sequence is the amino acid sequence represented by SEQ ID NO: 97. (m18) Includes the substitutions of (M1), (M11), (M12) and (M13). One embodiment is an amino acid sequence in which the amino acid residue corresponding to the 159th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with R, the amino acid residue corresponding to the 282nd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L, the amino acid residue corresponding to the 283rd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with G, and the amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 is substituted with L. One example of such an amino acid sequence is the amino acid sequence represented by Sequence ID No. 98.

[0050] ADAMTS13 may be further modified, for example, by post-translational modifications (e.g., glycosylation of one or more amino acids selected from the 142nd, 146th, and 552nd amino acids of the amino acid sequence represented by SEQ ID NO: 194, or any other natural or genetically modified sites), or by ex vivo chemical modifications or enzymatic modifications, including but not limited to glycosylation, modification with water-soluble polymers (e.g., pegylation, sialic acid addition, HESylation, etc.), tagging, etc.

[0051] <Antigen-binding molecule> (Definition) In this specification, "antigen-binding molecule" refers to any molecule containing an antigen-binding domain, or any substructure containing any molecule having antigen-binding activity, that is included in the fusion protein of the present invention, and may further refer to molecules such as peptides or proteins having a length of about 5 amino acids or more. The peptides and proteins are not limited to those of biological origin; for example, they may be polypeptides produced from artificially designed sequences. They may be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Figure 2 is a schematic diagram showing one embodiment of an antigen-binding molecule.

[0052] In this specification, “antigen-binding domain” means a region that specifically and complementarily binds to part or all of an antigen. “Antigen-binding domain” means a domain that includes at least part of the heavy chain variable region (VH) and / or part of the light chain variable region (VL) of an antibody, insofar as it has activity to bind to part or all of an antigen, each containing four framework regions (FRs) and three adjacent complementarity-determining regions (CDRs). In this disclosure, it is preferable that the antigen-binding domain includes either the light chain variable region (VL) or the heavy chain variable region (VH).

[0053] In this disclosure, the antigen-binding domain specifically refers to, for example, an antibody fragment, an antibody variable region (hereinafter sometimes abbreviated as V), a single chain Fv (scFv), Fab, Fab', and F (ab'). ２Examples include peptides containing diabody, disulfide-stabilized Fv (dsFv), and CDR, single variable domain (VHH), ligand proteins, and receptor proteins.

[0054] Examples of antigen-binding molecules include antibodies or antibody fragments or derivatives thereof, non-antibody proteins or their fragments or derivatives thereof. Specifically, examples of antigen-binding molecules include diabody (Db), single-chain antibodies, or sc(Fab'). ２ Examples of antigen-binding molecules include those having an antigen-binding domain as defined above, which contains a heavy chain variable region (VH) and a light chain variable region (VL) in a single polypeptide chain linked by one or more linkers, but lacking an Fc region.

[0055] In this invention, antibodies are also referred to as immunoglobulins (hereinafter referred to as Ig), and human antibodies are classified into isotypes IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM according to differences in molecular structure. IgG1, IgG2, IgG3, and IgG4, which have relatively high homology in amino acid sequences, are collectively referred to as IgG.

[0056] Antibodies are composed of polypeptides called heavy chains (also known as H chains) and light chains (also known as L chains). The H chain is composed of a variable region (also known as VH) and a constant region (also known as CH) from the N-terminus, while the L chain is composed of a variable region (also known as VL) and a constant region (also known as CL) from the N-terminus. For each subclass, α, δ, ε, γ, and μ chains are known for the CH chain. For the CL chain, λ and κ chains are known.

[0057] A domain refers to a functional structural unit that constitutes each polypeptide of an antibody molecule. In this invention, Fc and Fc region refer to a partial sequence and substructure of the constant H chain region, which consists of a hinge domain, a CH2 domain, and a CH3 domain.

[0058] CH is further composed of the following domains from the N-terminus: the CH1 domain, the hinge domain, the CH2 domain, and the CH3 domain. In this invention, the CH1 domain, the hinge domain, the CH2 domain, the CH3 domain, and the Fc region can be identified by the number of amino acid residues from the N-terminus using the EU index [Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].

[0059] Specifically, CH1 is identified as the amino acid sequence corresponding to EU index 118-215, Hinge as the amino acid sequence corresponding to EU index 216-230, CH2 as the amino acid sequence corresponding to EU index 231-340, and CH3 as the amino acid sequence corresponding to EU index 341-447.

[0060] Unless otherwise specified, the amino acid numbers for the CH1 domain, hinge domain, CH2 domain, CH3 domain, Fc region, and CL domain are based on the EU index by Kabat et al. [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. The number before the amino acid residue indicates the residue before substitution, and the number after the substitution indicates the amino acid residue after substitution.

[0061] In this disclosure, antibodies include not only monoclonal antibodies obtained from hybridomas, but also recombinant antibodies produced by genetic recombination technology. Recombinant antibodies include chimeric antibodies in which the constant region of a human antibody is conjugated to the variable region of a non-human antibody, humanized antibodies (or CDR-transplanted antibodies) produced by inserting the complementarity determining region (CDR) of the H chain and L chain of the non-human antibody variable region into the framework region (FR) of the human antibody variable region, and human antibodies produced using human antibody-producing animals.

[0062] Chimeric antibodies can be produced by obtaining cDNA encoding VH and VL from hybridomas derived from non-human animal cells that produce monoclonal antibodies, inserting these cDNAs into animal cell expression vectors containing nucleic acids (e.g., DNA or RNA) encoding the CH and CL of human antibodies, respectively, to construct a human-type chimeric antibody expression vector, and then introducing it into animal cells for expression.

[0063] Humanized antibodies are antibodies in which the amino acid sequences of the CDRs (Cellular Derived Ratios) of the VH and VL (Very Longitudinal) parts of non-human animal antibodies have been transplanted into the corresponding CDRs of the VH and VL parts of human antibodies. The regions of the VH and VL parts other than the CDRs are called framework regions (hereinafter referred to as FR).

[0064] Humanized antibodies can be produced by constructing a cDNA encoding the VH amino acid sequence, which consists of the CDR amino acid sequence of the VH of a non-human animal antibody and the FR amino acid sequence of the VH of any human antibody, and a cDNA encoding the VL amino acid sequence, which consists of the CDR amino acid sequence of the VL of a non-human animal antibody and the FR amino acid sequence of the VL of any human antibody. These cDNAs are then inserted into an animal cell expression vector containing nucleic acids encoding the CH and CL of a human antibody, respectively, to construct a humanized antibody expression vector, which is then introduced into animal cells for expression.

[0065] Human antibodies originally refer to antibodies that naturally exist in the human body, but recent advances in genetic engineering, cell engineering, and developmental engineering technologies have also led to the creation of human antibody phage libraries, cloned immortalized human peripheral blood lymphocytes, and antibodies obtained from human antibody-producing transgenic animals.

[0066] Human antibodies can be obtained by immunizing mice carrying human immunoglobulin genes with the desired antigen. Furthermore, by using a phage display library in which antibody genes are amplified from human B cells, human antibodies with the desired binding activity can be selected, thereby obtaining human antibodies without immunization. Additionally, by immortalizing human B cells using EB virus, cells that produce human antibodies with the desired binding activity can be created, allowing for the acquisition of human antibodies.

[0067] Antibodies present in the human body can be obtained, for example, by immortalizing lymphocytes isolated from human peripheral blood by infecting them with EB virus or the like, then cloning them to culture lymphocytes that produce the antibodies, and finally purifying the antibodies from the culture.

[0068] A human antibody phage library is a library of phages in which antibody fragments such as Fab or scFv are expressed on the surface by inserting antibody genes prepared from human B cells into phage genes. From this library, phages expressing antibody fragments with desired antigen-binding activity can be recovered, using the binding activity to an antigen-immobilized substrate as an indicator. These antibody fragments can further be converted into human antibody molecules consisting of two complete H chains and two complete L chains using genetic engineering techniques.

[0069] Human antibody-producing transgenic animals are animals in which human antibody genes have been incorporated into the chromosomes of a host animal. Specifically, human antibody-producing transgenic animals can be created by introducing human antibody genes into mouse ES cells, transplanting these ES cells into early-stage embryos of other mice, and then allowing them to develop.

[0070] A method for producing human antibodies from human antibody-producing transgenic animals involves obtaining human antibody-producing hybridomas using the same hybridoma production methods used for non-human mammals, and then culturing them to produce and accumulate human antibodies in the culture.

[0071] In this disclosure, “antibody fragment” refers to a molecule other than the antibody that has antigen-binding properties and contains a portion of the antibody. Antibody fragments in this disclosure are not limited to, but include, for example, Fab, Fab', F(ab'). ２ Examples include a peptide containing scFv, diabody, dsFv, VHH, and multiple CDRs, preferably a peptide containing six CDRs of an antibody.

[0072] In this specification, when a polypeptide chain is represented as [A-B-C] (where A, B, and C are regions constituting an antigen-binding molecule or fusion protein), it indicates that A, B, and C are linked in this order, starting from the N-terminus of a single-chain polypeptide. In this specification, the heavy chain variable region and the light chain variable region are also simply abbreviated as "VH" and "VL," respectively.

[0073] In this specification, “heavy chain variable region” or “VH” is not limited to heavy chain variable regions or VH present in IgG antibodies. Variable regions in variations of other antibody formats (e.g., VHH, scFv, scFab, VNAR, etc., of single-chain antibodies) are also included in “heavy chain variable region” or “VH” as used herein.

[0074] In this specification, “light chain variable region” or “VL” is not limited to light chain variable regions or VL present in IgG antibodies. Variable regions in other antibody format variations are also included in “light chain variable region” or “VL” as defined herein.

[0075] "Fab" consists of one light chain and a CH1 domain and variable region derived from one heavy chain. A portion of the hinge domain may be attached to the CH1. The cysteine ​​residue of the CL of the light chain forms a disulfide bond with the cysteine ​​residue contained in the CH1 domain or a portion of the hinge domain. For example, Fab derived from IgG1 forms a disulfide bond between the CL and the hinge domain. More specifically, Fab derived from IgG1 forms a disulfide bond between the cysteine ​​residue at position 214 of the CL and the cysteine ​​residue at position 220 of the hinge domain. Similarly, Fab derived from IgG4 forms a disulfide bond between the CL and CH1. More specifically, Fab derived from IgG4 forms a disulfide bond between the cysteine ​​residue at position 114 of the CL and the cysteine ​​residue at position 131 of the CH1. The heavy chain of a wild-type Fab molecule cannot form disulfide bonds with another heavy-chain molecule. Fab as used herein also includes Fab variants in which amino acid residues in a wild-type Fab molecule are modified by substitution, addition, or deletion, depending on the purpose. In certain embodiments, the mutant amino acid residues in a Fab variant (e.g., cysteine ​​or lysine residues after substitution, addition, or insertion) can form disulfide bonds with another heavy-chain molecule or a portion thereof (e.g., a Fab molecule). In another embodiment, Fab as used herein includes Fab molecules in which the heavy and light chains do not form disulfide bonds and are non-covalently associated primarily by electrostatic interactions.

[0076] F(ab') ２ This is an antibody fragment with antigen-binding activity, having a molecular weight of approximately 100,000, which is slightly larger than the fragment obtained by treating IgG with the proteolytic enzyme pepsin (cleaved at the amino acid residue at position 234 of the H chain) in which Fab is linked via an S-S bond in the hinge domain.

[0077] Fab' is the above F(ab') ２ This is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 50,000, obtained by cleaving the S-S bond in the hinge domain.

[0078] scFv is an antibody fragment having antigen-binding activity, which is a VH-P-VL or VL-P-VH polypeptide formed by linking one VH molecule and one VL molecule using a suitable peptide linker (P), such as a linker peptide consisting of any number of linkers (G4S) composed of four Gly residues and one Ser residue.

[0079] A Diabody is an antibody fragment in which two scFvs with the same or different antigen-binding specificities form a dimer, and which has bivalent antigen-binding activity against the same antigen or bispecific antigen-binding activity against different antigens.

[0080] dsFv refers to a polypeptide in which one amino acid residue in VH and VL is replaced with a cysteine ​​residue, and these polypeptides are linked together via S-S bonds between the cysteine ​​residues.

[0081] VHH (Variable domain of Heavy chain of Heavy chain antibody) is the antigen-binding domain in heavy chain antibodies derived from camelid animals.

[0082] A peptide containing a CDR is composed of at least one region of a VH or VL CDR. Multiple CDRs in a peptide can be linked together directly or via a suitable peptide linker.

[0083] In this specification, the term "hinge domain" refers to the polypeptide portion that links the CH1 and CH2 domains in the wild-type antibody heavy chain, and means the region from approximately position 216 to 230 according to the EU index. Figure 2 shows a schematic diagram of an IgG1 antibody. In the natural IgG1 antibody, it is known that the cysteine ​​residue at EU numbering position 220 in the hinge domain forms a disulfide bond with the cysteine ​​residue at position 214 in the antibody light chain. Furthermore, it is known that disulfide bonds are formed between the cysteine ​​residues at position 226 and at position 229 in the hinge domain of the two antibody heavy chains. In this specification, the hinge domain includes not only the wild type but also modified versions in which amino acid residues are substituted, added, or deleted from the wild type. In this disclosure, "disulfide bond of hinge domain" means a disulfide bond consisting of cysteine ​​residues of each molecule when molecules having hinge domains are joined by their hinge domains. For example, when using the hinge domain of IgG1, it means two disulfide bonds consisting of the cysteine ​​residues at position 226 and the cysteine ​​residues at position 229.

[0084] In the present invention, the "Fc region" refers to a region in an antibody molecule that includes a hinge or a part thereof, as well as a fragment consisting of CH2 and CH3 domains. The IgG class Fc region is not limited to, but for example, the region from cysteine ​​226 to the C-terminus, or from proline 230 to the C-terminus.

[0085] (Antigen-binding molecule that binds to vWF) The antigen-binding molecule that binds to vWF in this disclosure (hereinafter also simply referred to as "the antigen-binding molecule") preferably comprises at least VH and VL of an anti-vWF antibody. In one embodiment, the antigen-binding molecule preferably comprises any one of the following (c1) to (c37): (c1) The VH comprises CDR1 to 3 containing amino acid sequences represented by SEQ ID NOs. 565 to 567, and the VL comprises CDR1 to 3 containing amino acid sequences represented by SEQ ID NOs. 568 to 570, respectively. (c2) The VH comprises CDR1 to 3 containing amino acid sequences represented by SEQ ID NOs. 573 to 575, respectively, and the VL comprises CDR1 to 3 containing amino acid sequences represented by SEQ ID NOs. 576 to 578, respectively. (c3) The VH comprises CDR1 to 3 containing amino acid sequences represented by SEQ ID NOs. 581 to 583, respectively, and the VL comprises CDR1 to 3 containing amino acid sequences represented by SEQ ID NOs. 584 to 586, respectively. (c4) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 587 to 589, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 590 to 592, respectively. (c5) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 595 to 597, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 598 to 600, respectively. (c6) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 603 to 605, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 606 to 608, respectively. (c7) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 611 to 613, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 614 to 616, respectively. (c8) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 619 to 621, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 622 to 624. (c9) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 627 to 629, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 630 to 632.(c10) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 635 to 637, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 638 to 640, respectively. (c11) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 643 to 645, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 646 to 648, respectively. (c12) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 651 to 653, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 654 to 656, respectively. (c13) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 659 to 661, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 662 to 664, respectively. (c14) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 667 to 669, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 670 to 672, respectively. (c15) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 675 to 677, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 678 to 680, respectively. (c16) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 683 to 685, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 686 to 688, respectively. (c17) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 691 to 693, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 694 to 696, respectively. (c18) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 699 to 701, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 702 to 704. (c19) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 707 to 709, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 710 to 712.(c20) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 715 to 717, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 718 to 720, respectively. (c21) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 723 to 725, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 726 to 728, respectively. (c22) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 731 to 733, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 734 to 736, respectively. (c23) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 795 to 797, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 798 to 800, respectively. (c24) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 803 to 805, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 806 to 808, respectively. (c25) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 811 to 813, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 814 to 816, respectively. (c26) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 819 to 821, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 822 to 824, respectively. (c27) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 827 to 829, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 830 to 832, respectively. (c28) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 835 to 837, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 838 to 840. (c29) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 843 to 845, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 846 to 848.(c30) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 851 to 853, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 854 to 856, respectively. (c31) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 859 to 861, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 862 to 864, respectively. (c32) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 867 to 869, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 870 to 872, respectively. (c33) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 875 to 877, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 878 to 880, respectively. (c34) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 883 to 885, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 886 to 888, respectively. (c35) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 891 to 893, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 894 to 896, respectively. (c36) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 899 to 901, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 902 to 904, respectively. (c37) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 907 to 909, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 910 to 912, respectively.

[0086] In one embodiment, the antigen-binding molecule is preferably such that VH and VL are any one of the following (v1) to (v37): (v1) VH includes the amino acid sequence represented by SEQ ID NO: 563, and VL includes the amino acid sequence represented by SEQ ID NO: 564. (v2) VH includes the amino acid sequence represented by SEQ ID NO: 571, and VL includes the amino acid sequence represented by SEQ ID NO: 572. (v3) VH includes the amino acid sequence represented by SEQ ID NO: 579, and VL includes the amino acid sequence represented by SEQ ID NO: 580. (v4) VH includes the amino acid sequence represented by SEQ ID NO: 100, and VL includes the amino acid sequence represented by SEQ ID NO: 101. (v5) VH includes the amino acid sequence represented by SEQ ID NO: 593, and VL includes the amino acid sequence represented by SEQ ID NO: 594. (v6) VH includes the amino acid sequence represented by SEQ ID NO: 601, and VL includes the amino acid sequence represented by SEQ ID NO: 602. (v7) The VH includes the amino acid sequence represented by SEQ ID NO: 609, and the VL includes the amino acid sequence represented by SEQ ID NO: 610. (v8) The VH includes the amino acid sequence represented by SEQ ID NO: 617, and the VL includes the amino acid sequence represented by SEQ ID NO: 618. (v9) The VH includes the amino acid sequence represented by SEQ ID NO: 625, and the VL includes the amino acid sequence represented by SEQ ID NO: 626. (v10) The VH includes the amino acid sequence represented by SEQ ID NO: 633, and the VL includes the amino acid sequence represented by SEQ ID NO: 634. (v11) The VH includes the amino acid sequence represented by SEQ ID NO: 641, and the VL includes the amino acid sequence represented by SEQ ID NO: 642. (v12) The VH includes the amino acid sequence represented by SEQ ID NO: 649, and the VL includes the amino acid sequence represented by SEQ ID NO: 650. (v13) The VH includes the amino acid sequence represented by SEQ ID NO: 657, and the VL includes the amino acid sequence represented by SEQ ID NO: 658. (v14) The VH includes the amino acid sequence represented by SEQ ID NO: 665, and the VL includes the amino acid sequence represented by SEQ ID NO: 666. (v15) The VH includes the amino acid sequence represented by SEQ ID NO: 673, and the VL includes the amino acid sequence represented by SEQ ID NO: 674.(v16) The VH includes the amino acid sequence represented by SEQ ID NO: 681, and the VL includes the amino acid sequence represented by SEQ ID NO: 682. (v17) The VH includes the amino acid sequence represented by SEQ ID NO: 689, and the VL includes the amino acid sequence represented by SEQ ID NO: 690. (v18) The VH includes the amino acid sequence represented by SEQ ID NO: 697, and the VL includes the amino acid sequence represented by SEQ ID NO: 698. (v19) The VH includes the amino acid sequence represented by SEQ ID NO: 705, and the VL includes the amino acid sequence represented by SEQ ID NO: 706. (v20) The VH includes the amino acid sequence represented by SEQ ID NO: 713, and the VL includes the amino acid sequence represented by SEQ ID NO: 714. (v21) The VH includes the amino acid sequence represented by SEQ ID NO: 721, and the VL includes the amino acid sequence represented by SEQ ID NO: 722. (v22) The VH includes the amino acid sequence represented by SEQ ID NO: 729, and the VL includes the amino acid sequence represented by SEQ ID NO: 730. (v23) The VH includes the amino acid sequence represented by SEQ ID NO: 793, and the VL includes the amino acid sequence represented by SEQ ID NO: 794. (v24) The VH includes the amino acid sequence represented by SEQ ID NO: 801, and the VL includes the amino acid sequence represented by SEQ ID NO: 802. (v25) The VH includes the amino acid sequence represented by SEQ ID NO: 810, and the VL includes the amino acid sequence represented by SEQ ID NO: 811. (v26) The VH includes the amino acid sequence represented by SEQ ID NO: 818, and the VL includes the amino acid sequence represented by SEQ ID NO: 819. (v27) The VH includes the amino acid sequence represented by SEQ ID NO: 825, and the VL includes the amino acid sequence represented by SEQ ID NO: 826. (v28) The VH includes the amino acid sequence represented by SEQ ID NO: 833, and the VL includes the amino acid sequence represented by SEQ ID NO: 834. (v29) The VH includes the amino acid sequence represented by SEQ ID NO: 841, and the VL includes the amino acid sequence represented by SEQ ID NO: 842. (v30) The VH includes the amino acid sequence represented by SEQ ID NO: 849, and the VL includes the amino acid sequence represented by SEQ ID NO: 850. (v31) The VH includes the amino acid sequence represented by SEQ ID NO: 857, and the VL includes the amino acid sequence represented by SEQ ID NO: 858.(v32) The VH includes the amino acid sequence represented by SEQ ID NO: 865, and the VL includes the amino acid sequence represented by SEQ ID NO: 866. (v33) The VH includes the amino acid sequence represented by SEQ ID NO: 873, and the VL includes the amino acid sequence represented by SEQ ID NO: 874. (v34) The VH includes the amino acid sequence represented by SEQ ID NO: 881, and the VL includes the amino acid sequence represented by SEQ ID NO: 882. (v35) The VH includes the amino acid sequence represented by SEQ ID NO: 889, and the VL includes the amino acid sequence represented by SEQ ID NO: 890. (v36) The VH includes the amino acid sequence represented by SEQ ID NO: 897, and the VL includes the amino acid sequence represented by SEQ ID NO: 898. (v37) The VH includes the amino acid sequence represented by SEQ ID NO: 905, and the VL includes the amino acid sequence represented by SEQ ID NO: 906.

[0087] In one embodiment, examples of the antigen-binding molecules include the following: (B1) an antigen-binding molecule containing scFv (B2) an antigen-binding molecule containing Fab (B3) an antigen-binding molecule containing a first polypeptide containing VH and CH1 domains, hinge domains, and CH2 and CH3 domains, and a second polypeptide containing VL and CL domains, hinge domains, and CH2 and CH3 domains (B4) IgG These will be explained below. In the following classification of antigen-binding molecules, the numbering of polypeptide chains such as the first polypeptide and second polypeptide is defined for each format of the antigen-binding molecule. The numbering of polypeptide chains in each format of the antigen-binding molecule is shown in Figures 3 to 5 or Figures 7 to 11.

[0088] (B1) Antigen-binding molecule containing scFv In this disclosure, the antigen-binding molecule containing scFv includes one VH and one VL of an anti-vWF antibody. One embodiment of the antigen-binding molecule containing scFv is shown in Figures 3(a) to 3(c). (B-1-1) Antigen-binding molecule containing one scFv One embodiment is an antigen-binding molecule in which the VL and VH are linked by a peptide linker. [Figure 3(a)] (B-1-2) An antigen-binding molecule in which two scFv and one Fc region are linked is an antigen-binding molecule (hereinafter also referred to as "Fv-Fc") comprising the following first and second polypeptides, wherein in each polypeptide chain, the scFv, in which VL and VH are linked by a peptide linker, is linked to the constant region of an antibody consisting of a CH2 domain and a CH3 domain via a hinge domain, and the first polypeptide and the second polypeptide are associated by a disulfide bond of the hinge domain. [Figure 3(b)] First and second polypeptides: [VH-peptide linker-VL-hinge domain-CH2-CH3] (B-1-3) An antigen-binding molecule in which one scFv and one Fc region are linked. One embodiment includes the following first and second polypeptides, wherein the first polypeptide consists of an scFv in which VH and VL are linked by a peptide linker, and the constant region of an antibody including a CH2 domain and a CH3 domain is linked via a hinge domain, and the second polypeptide consists of a hinge domain, a CH2 domain and a CH3 domain, and the first and second polypeptides are associated by a disulfide bond of the hinge domain. [Figure 3(c)] First polypeptide: [VH-peptide linker-VL-hinge domain-CH2-CH3] Second polypeptide: [hinge domain-CH2-CH3]

[0089] (B2) Fab In this disclosure, Fab comprises a heavy chain [VH-CH1] containing the VH and CH1 domains of an anti-vWF antibody, and a light chain [VL-CL] containing the VL and CL domains of an anti-vWF antibody. One embodiment of the antigen-binding molecule containing Fab is shown in Figures 4(a) and 4(b). The following are examples of embodiments of the antigen-binding molecule containing Fab. (B-2-1) One embodiment of an antigen-binding molecule containing one Fab is an antigen-binding molecule containing the following first polypeptide and second polypeptide. [Figure 4(a)] First polypeptide: [VH-CH1] Second polypeptide: [VL-CL] (B-2-2) Antigen-binding molecule comprising one Fab and one Fc region. One embodiment includes the following first to third polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain, and the second polypeptide and the third polypeptide are linked via a disulfide bond between CL and CH1 or the hinge domain. [Figure 4(b)] First polypeptide: [Hinge domain-CH2-CH3] Second polypeptide: [VH-CH1-Hinge domain-CH2-CH3] Third polypeptide: [VL-CL] One embodiment includes the first polypeptide comprising a domain other than VH-CH1 of the IgG heavy chain, the second polypeptide comprising the IgG heavy chain, and the third polypeptide comprising the IgG light chain. For example, in Figure 4(b), if the subclass of IgG is IgG1, the CL of the third polypeptide can be bound to the hinge domain of the second polypeptide via a disulfide bond. If the subclass of IgG is IgG4, the CL of the third polypeptide can be bound to the CH1 of the second polypeptide via a disulfide bond. In one embodiment, for example, in Figure 4(b), if the subclass of IgG is IgG1, the cysteine ​​residue at position 214 of the CL of the third polypeptide and the cysteine ​​residue at position 220 of the hinge domain of the second polypeptide can form a disulfide bond.

[0090] (B3) An antigen-binding molecule (hereinafter also referred to as "mvG1") comprising the following first polypeptide and second polypeptide, wherein the first polypeptide and the second polypeptide are linked by a disulfide bond in the hinge domain. mvG1 can be exemplified by the embodiment shown in Figure 5. First polypeptide: [VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [VL-CL-hinge domain-CH2-CH3] Preferably as mvG1, mutations are introduced in the CH1 or hinge domain in the first polypeptide and in the CL in the second polypeptide to the Cys residue involved in the intermolecular disulfide bond between the light chain and heavy chain in the IgG antibody. In this specification, a more preferred embodiment as mvG1 is mvG1-2 described in International Publication No. 2014 / 054804.

[0091] (Cys residues involved in intermolecular disulfide bonding between L-H chains in mvG1) In this disclosure, mvG1 is characterized by the absence or substitution of Cys residues involved in intermolecular disulfide bonding between L-H chains in IgG antibodies, thereby suppressing the amount of macromers, halomers, and macromer / halomer formation that occur during the production of heterodimer proteins, and enabling efficient and stable production. In this disclosure, due to the absence or substitution of the Cys residues, disulfide bonds are formed between the first polypeptide and the second polypeptide only at the hinge domain.

[0092] The Cys residues involved in the L-H chain disulfide bond typically refer to the Cys residues required for the CH1 or hinge domain of the IgG antibody and CL to form an intermolecular disulfide bond. In one embodiment, it is preferable that a mutation is introduced in the Cys residues involved in the disulfide bond between CL and CH1 or the hinge domain in the CH1 or hinge domain of the first polypeptide of mvG1 and CL in the second polypeptide, and it is more preferable that the Cys residues are deleted or substituted. By deleting or substituting the Cys residues, the disulfide bond between CL and CH1 or the hinge domain is inhibited, and the generation of unwanted polypolymers can be reduced.

[0093] Cys residues involved in the L-H chain disulfide bond in the first polypeptide of mvG1 according to this disclosure include the Cys residue at position 220 of the heavy chain hinge domain when CH1 or the hinge domain is derived from human IgG1, and the Cys residue at position 131 of CH1 when it is derived from an IgG4 antibody. Cys residues involved in the L-H chain disulfide bond in the second polypeptide of mvG1 include, for example, the Cys residue at EU index 214 of the human κ chain and λ chain (CL). In addition to these Cys residue mutations, deletions or substitutions of amino acid residues may also be included. In one embodiment, in addition to substitution or deletion of Cys residues in CL, Cys residues in CH1 or the hinge domain may also be substituted or deleted. As an example of such a combination of Cys residue substitution or deletion, when mvG1 utilizes each domain of the IgG1 antibody, the amino acid C at EU index 220 in the hinge domain of the first polypeptide of mvG1 is substituted with S, the amino acid residue C at EU index 214 in CL of the second polypeptide is substituted with S, and the amino acid residue EPKSC at EU index 216-220 in the hinge domain of the second polypeptide is deleted.

[0094] (Mutations to reduce or eliminate binding activity to CH conjugates) In mvG1 in this disclosure, from the viewpoint of improving separation and purification efficiency, the second polypeptide may be a polypeptide in which binding activity to CH conjugates is reduced or eliminated.

[0095] CH conjugates refer to any protein, chemical substance, resin, etc., that specifically binds to the constant region (CH) of an antibody heavy chain. Examples include Fc-binding proteins and antibodies that bind to CH. Specific examples of Fc-binding proteins include, for example, Protein A from Staphylococcus aureus, Protein G from hemolytic Streptococcus, Fc receptors, their subclasses (FcγRI, IIA, IIB, IIIIA, IIIIB), and binding fragments of the aforementioned proteins.

[0096] In the fusion protein of the present invention, when the antigen-binding molecule is mvG1, a second polypeptide with reduced or absent binding activity to the CH conjugate means that the binding activity is substantially reduced or absent due to a decrease in affinity to the CH conjugate. A second polypeptide with reduced or absent binding activity is a second polypeptide that has reduced or absent binding activity to the CH conjugate compared to the original second polypeptide without the addition, deletion, or substitution of amino acid residues, achieved by adding, deleting, or substituting amino acid residues to a second polypeptide that has binding activity to the CH conjugate. The binding activity to the CH conjugate can be confirmed using measurement systems such as binding ELISA and surface plasma resonance (SPR) described below.

[0097] The first polypeptide containing CH in mvG1 in this disclosure specifically binds to the CH conjugate. By reducing or eliminating the binding activity of the second polypeptide in mvG1 to the CH conjugate, only the heterodimer protein consisting of the first and second polypeptides binds to the CH conjugate, allowing for more specific isolation and purification of mvG1.

[0098] With respect to mvG1 in this disclosure, in order to make the second polypeptide a polypeptide in which the binding activity to the CH conjugate is reduced or absent, for example, it is preferable to introduce a CL-Fc in which at least one amino acid residue selected from EU index positions 253, 310, 433, 435, and 436 is missing or substituted in the second polypeptide, preferably a substitution of the amino acid residue at EU index position 435, or substitution of the amino acid residues at EU index positions 435 and 436, more preferably a substitution of His at position 435 to Arg and a substitution of Tyr at position 436 to Phe, or a substitution of His at position 435 to Arg.

[0099] In one aspect of this disclosure, when mvG1 utilizes each format of the IgG1 antibody, mvG1 may be introduced with the following mutations: Introducing the (γ1) or (γ2) mutation can improve purification efficiency. Introducing the (γ3) mutation can easily suppress effector function. (γ1) Mutations in the first and second polypeptides as follows (γ1-1) to (γ1-2) (Nature Biotechnology volume 16, 1998, pp. 677-681) (γ1-1) Mutations in which the amino acid residue at EU index position 354 is replaced with cysteine, and mutations in which the amino acid residue at EU index position 366 is replaced with tryptophan (hereinafter also abbreviated as "Knob"). (γ1-2) Mutations in which the amino acid residue at EU index position 349 is replaced with cysteine, mutations in which the amino acid residue at EU index position 366 is replaced with serine, mutations in which the amino acid residue at EU index position 368 is replaced with alanine, and mutations in which the amino acid residue at EU index position 407 is replaced with valine (hereinafter also abbreviated as "Hole"). (γ2) Mutations of (γ2-1) and (γ2-2) in the first and second polypeptides (International Publication No. 2010 / 151792) The following (γ2-1) and (γ2-2) mutations are preferably heteromutations, and in one embodiment, it is preferable that the (γ2-1) and / or (γ2-2) mutations are introduced into the second polypeptide. (γ2-1) A mutation in which the amino acid residue at EU index position 435 is replaced with arginine. (γ2-2) A mutation in which the amino acid residue at EU index position 436 is replaced with phenylalanine. (γ3) The following mutations in the first and second polypeptides (γ3-1) to (γ3-3) (hereinafter also abbreviated as "LALAGA"): (γ3-1) A mutation in which the amino acid residue at EU index position 234 is replaced with alanine. (γ3-2) A mutation in which the amino acid residue at EU index position 235 is replaced with alanine. (γ3-3) A mutation in which the amino acid residue at EU index position 237 is replaced with alanine.

[0100] (B4) Antigen-binding molecule containing IgG The antigen-binding molecule containing IgG includes the following first to fourth polypeptides, wherein the first polypeptide and the second polypeptide, and the third polypeptide and the fourth polypeptide are linked by a disulfide bond between CL and CH1 or the hinge domain, respectively, and the second and third polypeptides are linked by a disulfide bond of the hinge domain. For example, the embodiment shown in Figure 2 can be cited. First and fourth polypeptides: polypeptides containing [VL-CL] Second and third polypeptides: polypeptides containing [VH-CH1-hinged domain-CH2-CH3] In one embodiment, the first and fourth polypeptides are polypeptides containing the light chain of IgG, and the second and third polypeptides are polypeptides containing the heavy chain of IgG.

[0101] The antigen-binding molecules in (B1) to (B4) above may be any subclass selected from antigen-binding molecules derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the antigen-binding molecule in (B3) above is preferably an antigen-binding molecule derived from IgG1, and the antigen-binding molecule in (B4) above is preferably an antigen-binding molecule derived from IgG1 or IgG4. For example, when the subclass of IgG is IgG1, two disulfide bonds are formed between cysteine ​​residues at position 226 and cysteine ​​residues at position 229 of the hinge domain.

[0102] In the cases where CH1, CL, CH2, and CH3 derived from IgG1 are used in (B1) to (B4) above, they may include any amino acid mutation selected from (γ1) to (γ3) above.

[0103] When CH1, CL, CH2, and CH3 derived from IgG4 are used in (B1) to (B4) above, the following amino acid mutations (γ4) may be included. (γ4) Mutations in the first and second polypeptides described below (γ4-1) to (γ4-3) (hereinafter also abbreviated as "IgG4PEK") (γ4-1) S228P in the IgG4 constant region (γ4-2) L235E in the IgG4 constant region (γ4-3) R409K in the IgG4 constant region

[0104] (Binding mode between antigen-binding molecule and vWF) vWF (von Willebrand factor) is a plasma protein involved in blood coagulation, playing an important role in platelet adhesion to the subendothelial tissue of blood vessels exposed during bleeding, as well as in the protection and transport of blood coagulation factor VIII. Abnormalities in vWF cause von Willebrand disease (VWD), a hemorrhagic disorder. The vWF in this disclosure has the amino acid sequence represented by UniPro access number: P04275.

[0105] Figure 6 shows a schematic diagram of vWF. As shown in Figure 6, vWF has multiple domains from the N-terminus to the C-terminus. vWF needs to form a specific three-dimensional structure in order to fold properly in the cell and exert its function. The A2 domain plays an important role in the regulation of vWF. The amino acid sequence from position 1495 to 1670 of vWF shown in Figure 6 is the amino acid sequence of the A2 domain (SEQ ID NO: 916).

[0106] In this disclosure, "vWF with folded A2 domain" refers to vWF that has a native three-dimensional structure under physiological conditions. During the synthesis and secretion of vWF by endothelial cells and megakaryocytes, appropriate folding occurs via the endoplasmic reticulum and Golgi apparatus, and the formation of disulfide bonds, glycosylation, dimerization, and multimerization are controlled. In particular, the functional multimer of vWF is formed by disulfide bonds between D3 domains, and the higher-order structure is maintained.

[0107] When the A2 domain of vWF is folded, its higher-order structure unfolds in the bloodstream, resulting in an extended state. This allows it to bind to glycoprotein Ib (GPIb) receptors on the platelet membrane and collagen in the blood vessel wall. Furthermore, ADAMTS13 in plasma plays a role in preventing abnormal platelet aggregation by binding to and cleaving the A2 domain of unfolded vWF due to the high shear stress of blood flow.

[0108] In this disclosure, "vWF in which the A2 domain is not folded" refers to a vWF in which the A2 domain is unfolded due to shear stress from blood flow.

[0109] In this disclosure, the mode of binding between the antigen-binding molecule and vWF is not particularly limited as long as the effects of the present invention are achieved. However, in one embodiment, it is preferable that the antigen-binding molecule specifically binds to (α1) or (α2) below. (α1) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 914 (hereinafter also referred to as "PEP05" in this specification) (α2) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 915 (hereinafter also referred to as "PEP06" in this specification) The amino acid sequence represented by SEQ ID NO: 914 in (α1) and the amino acid sequence represented by SEQ ID NO: 915 in (α2) are regions located in the A2 domain of vWF. The regions consisting of these amino acid sequences are regions close to the cleavage site by ADAMTS13 in vWF, and their positional relationship is also shown in Figure 6. The mutant ADAMTS13 contained in the fusion protein of the present invention exhibits extremely low ADAMTS13 enzyme activity on its own. However, by linking it with an antigen-binding molecule, the antigen-binding site binds to the region of vWF, and the mutant ADAMTS13 can exhibit high ADAMTS13 enzyme activity by being close to the cleavage site of vWF. Therefore, the antigen-binding molecule that specifically binds to (α1) or (α2) is positioned as one form of an antigen-binding molecule that binds to the A2 domain of vWF.

[0110] The specific binding affinity of the antigen-binding molecule to the polypeptides (α1) to (α2) can be confirmed, for example, by the following test: Test: The binding affinity between the immobilized polypeptides (α1) to (α2) and the antigen-binding molecule is measured by ELISA using a protein containing the amino acid sequence of the vWF-A2 domain as a substrate, and the absorbance value at a wavelength of 450 nm is measured.

[0111] In the fusion protein of this embodiment, the antigen-binding molecule is thought to bind to the A2 domain of vWF, thereby physically bringing the ADAMTS13 enzyme domain closer to the cleavage site in vWF (near the Tyr1605-Met1606 binding site). As a result, even enzyme domains that have insufficient binding activity to vWF on their own, such as the M domain, have their distance from the enzyme domain to the substrate reduced, promoting efficient cleavage. In other words, by targeting the A2 domain of the antigen-binding molecule, it can guide ADAMTS13 to its original cleavage site, thereby promoting the degradation of vWF higher-order multimers. Furthermore, it is expected that vWF cleavage at sites other than the original cleavage site and non-specific cleavage to other proteins will be suppressed.

[0112] In one embodiment, the antigen-binding molecule in this disclosure, when the polypeptide in the test consists of the (α1) or (α2) amino acid sequence, preferably has an absorbance value measured by the test that is at least twice, more preferably five times, and even more preferably ten times, the background signal or noise. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d. ed. 1989).

[0113] In one embodiment, the antigen-binding molecule in this disclosure preferably specifically binds to the (α1) or (α2) amino acid sequence and further has the ability to bind to both vWF with folded A2 domain and vWF without folded A2 domain.

[0114] The ability to bind to both vWF with folded A2 domain and vWF without folded A2 domain can be confirmed by the following tests including the procedures (x1) to (x3). Test: (x1) First, in order to investigate the three-dimensional structure of vWF in blood in the absence of blood flow and in the presence of shear stress, polypeptides consisting of the amino acid sequence represented by SEQ ID NO: 194 (Open-type substrate) and polypeptides consisting of the amino acid sequence represented by SEQ ID NO: 195 (Close-type substrate) are prepared as constructs that mimic the Open and Close structures, respectively (Blood. 2011; 117(17):4623-31). Each construct is added to a 96-well ELISA plate at 50 μL / well and allowed to stand overnight at 4°C to solidify. (x2) Next, in order to measure the vWF binding activity of each antigen-binding molecule, 0.01 to 10 μg / mL of each antigen-binding molecule is added to the plate, and washing, blocking, dilution, and addition of secondary antibody are performed. (x3) After color development, the absorbance at 450 nm and 570 nm is measured using a plate reader, and the absorbance at 570 nm is subtracted from the absorbance at 450 nm to analyze the absorbance as the binding strength.

[0115] In one embodiment, the antigen-binding molecule in this disclosure preferably exhibits a binding strength of 0.2 to 5.0 for both open-type and closed-type substrates when the antigen-binding molecule is at a concentration of 0.1 μg / mL, more preferably 0.5 to 5.0, and even more preferably 0.6 to 5.0. When the binding strength of the antigen-binding molecule is 0.2 or higher at a concentration of 0.1 μg / mL, it can exhibit binding ability to both vWF with folded A2 domains and vWF without folded A2 domains.

[0116] <Fusion Protein> This fusion protein includes mutant ADAMTS13 and an antigen-binding molecule that binds to vWF. In one embodiment, a preferred combination of mutant ADAMTS13 and antigen-binding molecule in the fusion protein of this disclosure is one in which the mutant ADAMTS13 contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 99, and the antigen-binding molecule contains any one of (B1) to (B4).

[0117] In one embodiment, it is preferable that the antigen-binding molecule comprises VH and VL of the anti-vWF antibody, and the C-terminus of mutant ADAMTS13 is directly or via a linker to the N-terminus of the VH or VL in the antigen-binding molecule. In a preferred embodiment, it is preferable that the C-terminus of mutant ADAMTS13 is directly or via a linker to the N-terminus of the VH in the antigen-binding molecule.

[0118] (Linker) Figure 7 shows a schematic diagram of one embodiment of the fusion protein. As shown in Figure 7, in one embodiment, the mutant ADAMTS13 is preferably linked to the antigen-binding molecule via a linker. The linker is preferably 1 to 40 amino acids long, more preferably 5 to 30 amino acids long, and even more preferably 5 to 20 amino acids long. In another embodiment, the linker may be 1 to 5 amino acids long.

[0119] In one aspect of this disclosure, the linker preferably comprises at least one repeating unit selected from (L1) to (L3) below. The type and length of the linker repeating unit can be optimized considering the distance between the cleavage site in the vWF and the binding site of the antigen-binding molecule to the vWF. (L1) A polypeptide comprising the amino acid sequence (GGGGS) represented by SEQ ID NO: 925 as a repeating unit. (L2) A polypeptide comprising the amino acid sequence (PAPAP) represented by SEQ ID NO: 926 as a repeating unit. (L3) A polypeptide comprising the amino acid sequence (EAAAK) represented by SEQ ID NO: 927 as a repeating unit.

[0120] The repeating units (L1) to (L3) described above are preferably repeated 1 to 10 times, more preferably 1 to 6 times, and even more preferably 1 to 4 times. Different repeating units may also be combined. In this specification, the (repeating units) used in the linker are... ｘ In this expression, x is an integer greater than or equal to 1. In this case, it indicates that the repeating unit within the parentheses ( ) is repeated x times. For example, (GGGGS) ３ In this case, the linker contains an amino acid in which GGGGS is repeated three times.

[0121] (Combination of mutant ADAMTS13 and antigen-binding molecule) There are no particular restrictions on the linkage between mutant ADAMTS13 and the antigen-binding molecule, but in one embodiment, it is preferable that the antigen-binding molecule contains VH and VL of an anti-vWF antibody, and that the C-terminus of mutant ADAMTS13 is linked directly or via a linker to the N-terminus of VH in the antigen-binding molecule.

[0122] As one embodiment of the fusion protein of this disclosure, the following combinations of mutant ADAMTS13 and an antigen-binding molecule are listed below. (p1) The following are examples of embodiments in which the antigen-binding molecule is an antigen-binding molecule containing (B1) scFv. (p1-1) A fusion protein comprising one mutant ADAMTS13 and one scFv, wherein the N-terminus of mutant ADAMTS13 is directly or via a linker linked to the C-terminus of VL in scFv. [Figure 8(a)] (p1-2) A fusion protein comprising one mutant ADAMTS13 and one scFv, wherein the N-terminus of mutant ADAMTS13 is directly or via a linker linked to the C-terminus of VH in scFv. [Figure 8(b)] (p1-3) A fusion protein comprising one mutant ADAMTS13 and one scFv, wherein the C-terminus of the mutant ADAMTS13 is directly or via a linker linked to the N-terminus of the VH in the scFv. [Figure 8(c)] (p1-4) A fusion protein comprising two mutant ADAMTS13, two scFv, and an Fc region, wherein two molecules, each with the C-terminus of the mutant ADAMTS13 directly or via a linker linked to the N-terminus of the VH in the scFv, are linked to the Fc region via a hinge domain. In one embodiment, a fusion protein comprising the following first and second polypeptides, wherein the first and second polypeptides are linked via a disulfide bond in the hinge domain. [Figure 8(d)] First and second polypeptides: [mutant ADAMTS13 - peptide linker - scFv - hinge domain - CH2 - CH3]

[0123] (p2) The following are examples of an antigen-binding molecule in which the antigen-binding molecule is (B2)Fab. One example is the following: (p2-1) A fusion protein comprising one mutant ADAMTS13 and one Fab, wherein the C-terminus of the mutant ADAMTS13 is directly or via a linker to the N-terminus of VH in Fab. [Figure 9(a)] One example is a fusion protein in which the following first polypeptide and second polypeptide are linked by a disulfide bond between the CH1 domain or hinge domain and CL. Another example is a fusion protein in which the following first polypeptide and second polypeptide are non-covalently associated. First polypeptide: [mutant ADAMTS13-peptide linker-VH-CH1] Second polypeptide: [VL-CL] (p2-2) A fusion protein comprising one mutant ADAMTS13 and one Fab, wherein the C-terminus of mutant ADAMTS13 is directly or via a linker to the N-terminus of VL in Fab. [Figure 9(b)] In one embodiment, a fusion protein is provided in which the first polypeptide and the second polypeptide described below are linked by a disulfide bond between the CH1 domain or hinge domain and CL. In another embodiment, a fusion protein is provided in which the first polypeptide and the second polypeptide described below are associated non-covalently. First polypeptide: [VH-CH1] Second polypeptide: [mutant ADAMTS13-peptide linker-VL-CL] (p2-3) A fusion protein comprising one mutant ADAMTS13 and one Fab, wherein the C-terminus of CL in Fab is directly or via a linker to the N-terminus of mutant ADAMTS13. [Figure 9(c)] In one embodiment, a fusion protein is provided in which the first polypeptide and the second polypeptide described below are linked by a disulfide bond between the CH1 domain and CL. In another embodiment, a fusion protein is provided in which the first polypeptide and the second polypeptide described below are associated non-covalently.First polypeptide: [VH-CH1] Second polypeptide: [VL-CL-peptide linker-mutant ADAMTS13] (p2-4) A fusion protein in which two Fabs, two mutant ADAMTS13s and an Fc region are linked, wherein molecules linked directly or via a linker between Fab and mutant ADAMTS13 are linked to the Fc region via a hinge domain. [Figure 9(d)] In one embodiment, a fusion protein is provided which comprises the following first to fourth polypeptides, wherein the first polypeptide and the second polypeptide, and the third polypeptide and the fourth polypeptide are linked via disulfide bonds between CL and CH1 or the hinge domain, and the second polypeptide and the third polypeptide are linked by disulfide bonds of the hinge domain. First polypeptide and fourth polypeptide: [VL-CL] Second polypeptide and third polypeptide: [VH-CH1-peptide linker-mutant ADAMTS13-hinge domain-CH2-CH3] (p2-5) A fusion protein in which one mutant ADAMTS13, one Fab and an Fc region are linked, wherein the C-terminus of mutant ADAMTS13 is linked directly or via a linker to the N-terminus of VH in Fab, and the CH1 domain in Fab is linked to the Fc region via a hinge domain. [Figure 9(e)] In one embodiment, a fusion protein is provided which comprises the following first to third polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain, and the CH1 domain or hinge domain in the second polypeptide and CL in the third polypeptide are linked via a disulfide bond. Polypeptide 1: [Hinge domain - CH2 - CH3] Polypeptide 2: [Mutant ADAMTS13 - Peptide linker - VH - CH1 - Hinge domain - CH2 - CH3] Polypeptide 3: [VL - CL] (p2-6) A fusion protein in which one mutant ADAMTS13, one Fab and Fc region are linked, the C-terminus of mutant ADAMTS13 is linked directly or via a linker to the N-terminus of VL in Fab, and the C-terminus of the CH1 domain in Fab is linked to the N-terminus of the Fc region via a hinge domain.[Figure 9(f)] As one embodiment, a fusion protein is provided which comprises the following first to third polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain, and the CH1 domain or hinge domain of the second polypeptide and CL of the third polypeptide are linked via a disulfide bond. First polypeptide: [Hinge domain - CH2 - CH3] Second polypeptide: [VH - CH1 - Hinge domain - CH2 - CH3] Third polypeptide: [Mutant ADAMTS13 - Peptide linker - VL - CL] (p2-7) A fusion protein in which one mutant ADAMTS13, one Fab and an Fc region are linked, wherein the C-terminus of mutant ADAMTS13 is linked to the N-terminus of the Fc region via a hinge domain or linker and a hinge domain, and the CH1 domain of Fab is linked to the N-terminus of the Fc region via a hinge domain. [Figure 9(g)] One embodiment is a fusion protein comprising the following first to third polypeptides, wherein the CL of the first polypeptide and the CH1 domain or hinge domain of the second polypeptide are linked via a disulfide bond, and the second polypeptide and the third polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [VL-CL] Second polypeptide: [VH-CH1-hinged domain-CH2-CH3] Third polypeptide: [mutant ADAMTS13-peptide linker-hinged domain-CH2-CH3].

[0124] (p3) The following are examples of an antigen-binding molecule in which the antigen-binding molecule is (B3) an antigen-binding molecule containing mvG1. One example is the following: (p3-1) A fusion protein in which one mutant ADAMTS13 and one mvG1 are linked, wherein the C-terminus of mutant ADAMTS13 is linked directly to the N-terminus of VH in mvG1 or via a linker. [Figure 10(a)] One example is a fusion protein comprising the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [VL-CL-hinge domain-CH2-CH3] (p3-2) A fusion protein in which one mutant ADAMTS13 and one mvG1 are linked, wherein the C-terminus of mutant ADAMTS13 is linked directly or via a linker to the N-terminus of VL in mvG1. [Figure 10(b)] In one embodiment, a fusion protein comprising the first and second polypeptides described below is provided, in which the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [mutant ADAMTS13-peptide linker-VL-CL-hinge domain-CH2-CH3] (p3-3) A fusion protein in which one mutant ADAMTS13 and one mvG1 are linked, wherein the N-terminus of mutant ADAMTS13 is linked directly or via a linker to the C-terminus of the CH3 domain in mvG1. [Figure 10(c)] In one embodiment, a fusion protein is provided which comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [VL-CL-hinge domain-CH2-CH3-peptide linker-mutant ADAMTS13]

[0125] In preferred embodiments of (p3-1) to (p3-3) above, mutations are introduced in the CH1 or hinge domain of the first polypeptide and in the CL of the second polypeptide, in which the Cys residue involved in the intermolecular disulfide bond between the light and heavy chains of the IgG antibody is modified. In this specification, a more preferred embodiment of mvG1 is mvG1-2 as described in International Publication No. 2014 / 054804.

[0126] In embodiments of the fusion protein in this disclosure (p3-1) to (p3-3), the Cys residue involved in the disulfide bond between the CH1 or hinge domain in the first polypeptide and CL in the second polypeptide refers to the Cys residue required for the CH1 or hinge domain of the IgG antibody and CL to form an intermolecular disulfide bond. In one embodiment, it is preferable that a mutation is introduced in the Cys residue involved in the disulfide bond between CL and CH1 or hinge domain in the CH1 or hinge domain of the first polypeptide and CL in the second polypeptide, and it is more preferable that the Cys residue is deleted or substituted. The deletion or substitution of the Cys residue inhibits the formation of an intermolecular disulfide bond between CL and CH1 or hinge domain. Examples of Cys residues involved in disulfide bonding between CH1 or the hinge domain in the first polypeptide (p3-1) to (p3-3) and CL in the second polypeptide include the Cys residue at EU index 220 of the heavy chain hinge domain of human IgG1. Examples of Cys residues involved in disulfide bonding between CH1 or the hinge domain in the CL in the second polypeptide (p3-1) to (p3-3) include the Cys residue at EU index 214 of the human κ and λ chains (CL). In addition to these Cys residue mutations, deletions or substitutions of amino acid residues may also be included. In one embodiment, in addition to substitution or deletion of Cys residues in CL, Cys residues in CH1 or the hinge domain may also be substituted or deleted. As an example of such a combination of Cys residue substitution or deletion, when mvG1 in (p3-1) to (p3-3) utilizes each domain of the IgG1 antibody, the amino acid C at EU index 220 in the hinge domain of the first polypeptide (p3-1) to (p3-3) is substituted with S, the amino acid residue C at EU index 214 in CL of the second polypeptide is substituted with S, and the amino acid residue EPKSC at EU index 216 to 220 in the hinge domain of the second polypeptide is deleted.

[0127] In one embodiment of (p3-1) to (p3-3), when each domain derived from IgG1 is used, at least one of the following mutations may be introduced. (ε1) Mutations in the first and second polypeptides as follows (ε1-1) to (ε1-2) (Nature Biotechnology volume 16, 1998, p. 677-681) (ε1-1) Mutations in which the amino acid residue at EU index position 354 is replaced with cysteine, and mutations in which the amino acid residue at EU index position 366 is replaced with tryptophan (hereinafter also abbreviated as "Knob"). (ε1-2) Mutations in which the amino acid residue at EU index position 349 is replaced with cysteine, mutations in which the amino acid residue at EU index position 366 is replaced with serine, mutations in which the amino acid residue at EU index position 368 is replaced with alanine, and mutations in which the amino acid residue at EU index position 407 is replaced with valine (hereinafter also abbreviated as "Hole"). (ε2) Mutations of (γ2-1) and (γ2-2) below in the first and second polypeptides (International Publication No. 2010 / 151792) The following mutations of (ε2-1) and (ε2-2) are preferably heteromutations, and in one embodiment, it is preferable that the mutations of (ε2-1) and / or (ε2-2) are introduced into the second polypeptide. (ε2-1) A mutation in which the amino acid residue at EU index position 435 is replaced with arginine. (ε2-2) A mutation in which the amino acid residue at EU index position 436 is replaced with phenylalanine. (ε3) Mutations of (ε3-1) to (ε3-3) below in the first and second polypeptides (hereinafter also abbreviated as "LALAGA"): (ε3-1) A mutation in which the amino acid residue at EU index position 234 is replaced with alanine. (ε3-2) A mutation in which the amino acid residue at EU index position 235 is replaced with alanine. (ε3-3) Mutation in which the amino acid residue at EU index position 237 is replaced with alanine.

[0128] (p4) The following are examples of embodiments in which the antigen-binding molecule is an antigen-binding molecule containing (B4) IgG. Here, for example, in IgG1, CL is bound to the hinge domain via a disulfide bond, and in IgG4, CL is bound to CH1 via a disulfide bond. Figures 11(a) and (b) of this specification illustrate embodiments of the IgG4 type, but the embodiments are not limited to these examples and include other subclasses and modifications. (p4-1) A fusion protein in which two mutant ADAMTS13 and one IgG are linked, wherein the C-terminus of each mutant ADAMTS13 and the N-terminus of each VH in IgG are linked directly or via a linker. [Figure 11(a)] As one embodiment, a fusion protein is provided which comprises the following first to fourth polypeptides, wherein the CL of the first polypeptide and the hinge domain or CH1 of the second polypeptide are linked via a disulfide bond, the hinge domain or CH1 of the third polypeptide and the CL of the fourth polypeptide are linked via a disulfide bond, and the second polypeptide and the third polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide and fourth polypeptide: [VL-CL] Second polypeptide and third polypeptide: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] (p4-2) A fusion protein in which one mutant ADAMTS13 and one IgG1 are linked, wherein the C-terminus of mutant ADAMTS13 and the N-terminus of VH in IgG1 are linked directly or via a linker. [Figure 11(b)] One embodiment is a fusion protein comprising the following first to fourth polypeptides, wherein the CL of the first polypeptide and the hinge domain or CH1 of the second polypeptide are linked via a disulfide bond, the hinge domain or CH1 of the third polypeptide and the CL of the fourth polypeptide are linked via a disulfide bond, and the second polypeptide and the third polypeptide are linked via a disulfide bond of the hinge domain.Polypeptide 1 and 4: [VL-CL] Polypeptide 2: [VH-CH1-hinged domain-CH2-CH3] Polypeptide 3: [Variant ADAMTS13-peptide linker-VH-CH1-hinged domain-CH2-CH3].

[0129] The antigen-binding molecule in (p1) to (p4) above may be derived from any subclass selected from antigen-binding molecules derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, IgG in (p3) above is preferably IgG1, and the antigen-binding molecule in (p4) above is preferably an antigen-binding molecule derived from IgG1 or IgG4. For example, when the subclass of IgG is IgG1, two disulfide bonds are formed between the cysteine ​​residues at position 226 and cysteine ​​residues at position 229 of the hinge domain.

[0130] In the cases where CH1, CL, CH2, and CH3 derived from IgG1 are used in (p1) to (p4) above, they may include any amino acid mutation selected from (ε1) to (ε3) above.

[0131] When CH1, CL, CH2, and CH3 derived from IgG4 are used in (p1) to (p4) above, the following amino acid mutations (ε4) may be included. (ε4) Mutations in the first and second polypeptides described below (ε4-1) to (ε4-3) (hereinafter also abbreviated as "IgG4PEK") (ε4-1) S228P in the IgG4 constant region (ε4-2) L235E in the IgG4 constant region (ε4-3) R409K in the IgG4 constant region

[0132] One of the following embodiments of the fusion protein is preferred: (t1) The fusion protein of (p3-1) wherein the first polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 333, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, and 545, and the second polypeptide comprises an amino acid sequence represented by SEQ ID NOs: 334. (t2) The fusion protein of (p4-1) wherein the first polypeptide and the fourth polypeptide are the same and comprise an amino acid sequence represented by SEQ ID NOs: 114, and the second polypeptide and the third polypeptide are the same and comprise an amino acid sequence represented by any one of SEQ ID NOs: 113, 352, 354, 356, 358, 360, and 362. (t3) A fusion protein of the above (p4-1), wherein the first polypeptide and the fourth polypeptide are the same and include the amino acid sequence represented by SEQ ID NO: 110, and the second polypeptide and the third polypeptide are the same and include the amino acid sequence represented by any one of SEQ ID NOs: 109, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406 and 408. (t4) A fusion protein of the above (p4-1), wherein the first polypeptide and the fourth polypeptide are the same and include the amino acid sequence represented by SEQ ID NO: 126, A fusion protein in which the second polypeptide and the third polypeptide are the same and contain an amino acid sequence represented by any one of SEQ ID NOs: 125, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, and 512.

[0133] (vWF activity in the presence of autoantibodies) In one embodiment, the fusion protein of this disclosure has a vWF degradation activity measured by the following tests, preferably 40% or more, and more preferably 50% or more, 60% or more, 70% or more, and 80% or more, respectively, when the degradation activity in the absence of the anti-spacer domain antibody is taken as 100%. Here, the anti-spacer domain antibody refers to an antibody that specifically binds to the S domain in ADAMTS13. In one embodiment, an equal mixture of a protein containing the amino acid sequence represented by SEQ ID NO: 559, a protein containing the sequence represented by SEQ ID NO: 560, and a protein containing the sequence represented by SEQ ID NO: 561 is used as the anti-spacer domain antibody cocktail. In another embodiment, an equal mixture of a protein containing the amino acid sequence represented by SEQ ID NO: 559 and a protein containing the sequence represented by SEQ ID NO: 560 is used as the anti-spacer domain antibody cocktail.

[0134] <Method for producing a fusion protein> One aspect of the present disclosure is a method for producing the fusion protein of the present disclosure as described above, which includes the following: (s1) producing an expression vector containing nucleic acids encoding at least one polypeptide constituting the fusion protein; (s2) introducing the expression vector produced in (s1) into a host cell, culturing the host cell so that the polypeptide is expressed, and obtaining the polypeptide from the culture supernatant; (s3) producing the fusion protein from the polypeptide obtained in (s2). (s1) to (s3) will be described below.

[0135] <<(s1) Prepare an expression vector containing nucleic acids encoding at least one polypeptide that constitutes this fusion protein>> The polypeptides constituting this fusion protein are described in Molecular Cloning, Second Edition, Current Protocols in Molecular Biology, Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, 1988, Monoclonal Antibodies: principles and practice, Third Edition, Acad. Using methods described in Press, 1993, Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996, etc., it can be obtained, for example, by expression in host cells as follows.

[0136] The proteins that constitute this fusion protein are the polypeptides that make up this fusion protein. If there are multiple polypeptides that make up this fusion protein, each polypeptide may be present in the same expression vector or in separate expression vectors.

[0137] A vector containing nucleic acids encoding polypeptides can, in one embodiment, be constructed by cloning a gene encoding a polypeptide of a heterodimer protein molecule contained in a heterodimer protein composition into an expression vector for animal cells. Examples of nucleic acids include DNA and RNA.

[0138] Furthermore, it is possible to synthesize all DNA using synthetic DNA, and synthesis by polymerase chain reaction (PCR) is also possible (Molecular Cloning, 2nd Edition). In addition, by combining several of these methods, it is possible to create genes that encode polypeptides.

[0139] When using animal cells as a host, any expression vector that can function in animal cells can be used, for example, pcDNAI, pCDM8 (Funakoshi Corporation), pAGE107 [Japanese Patent Publication No. 3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Patent Publication No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pcDNA3.1 (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Examples include Biochemistry, 101, 1307 (1987), pAGE210, pME18SFL3, pKANTEX93 (International Publication No. 97 / 10354), N5KG1val (U.S. Patent No. 6,001,358), and Tol2 transposon vectors (International Publication No. 2010 / 143698).

[0140] Any promoter capable of functioning in animal cells can be used, such as the promoter of the cytomegalovirus (CMV) immediate early (IE) gene, the SV40 early promoter, retrovirus promoters, metallothionein promoters, heat shock promoters, SRα promoters, or the promoter or enhancer of Moloney's mouse leukemia virus. The enhancer of the human CMV IE gene may also be used in conjunction with the promoter.

[0141] <<(s2) The expression vector prepared in (s1) is introduced into host cells, the host cells are cultured to express the polypeptide, and the polypeptide is obtained from the culture supernatant.>> By introducing the expression vector prepared in (s1) into suitable animal cells, a transformed cell line that transiently or stably produces the polypeptide constituting this fusion protein can be obtained. Any host cell line capable of expressing a heterodimer protein can be used as the host cell into which the expression vector is introduced, but for example, COS-7 cells [American Type Culture Collection (ATCC) number: CRL1651] are used (Methods in Nuclear Acids Res., CRC press, 283, 1991).

[0142] To introduce expression vectors into COS-7 cells, methods such as the DEAE-dextran method (Methods in Nucleic Acids Res., CRC press, 1991) or the lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413, 1987) are used. After introducing the expression vector, the expression level and antigen-binding activity of the polypeptide constituting the fusion protein in the culture supernatant can be measured using enzyme immunoassay [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)].

[0143] By introducing the expression vector obtained in (s1) into a suitable host cell, a transformed cell line that stably expresses the heterodimer protein can be obtained. Any method for introducing DNA into host cells can be used to introduce expression vectors into host cells. Examples include electroporation (Cytotechnology, 3, 133, 1990), calcium phosphate method (Japanese Patent Publication No. 2-227075), lipofection method (Proc. Natl. Acad. Sci. U.S.A., 84, 7413, 1987), injection method [Manipulating the Mouse Embryo: A Laboratory Manual], particle gun method (Japanese Patent No. 2606856, Japanese Patent No. 2517813), DEAE-dextran method [Biomanual Series 4 - Gene Introduction and Expression / Analysis Methods (Yodosha), edited by Takashi Yokota and Kenichi Arai (1994)], and viral vector method (Manipulating the Mouse Embryo, 2nd edition).

[0144] Any host cell capable of expressing the polypeptides constituting this fusion protein can be used as the host cell into which the expression vector is introduced. Examples of such host cells include human leukemia cells (Namalwa cells), monkey COS cells, Chinese hamster CHO cells, HBT5637 (Japanese Patent Publication No. 63-299), rat myeloma cells, mouse myeloma cells, Syrian hamster kidney-derived cells, embryonic stem cells, and fertilized egg cells.

[0145] Specifically, for example, PER. C6, CHO-K1 (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat # 11619), Lec13 cells, rat myeloma cells YB2 / 3HL. P2. G11.16Ag. Cells that can be used include 20 (ATCC number: CRL1662, also known as YB2 / 0), mouse myeloma cells NS0, mouse myeloma cells SP2 / 0-Ag14 (ATCC number: CRL1581), mouse P3X63-Ag8.653 cells (ATCC number: CRL1580), CHO cells (CHO / DG44) lacking the dihydrofolate reductase gene (Dihydrofolate Reductase, hereafter referred to as dhfr) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], Syrian hamster cells BHK, HBT563 cells, sub-cell lines of the above-mentioned cell lines, cells adapted from the above-mentioned cell lines under serum-free culture conditions, and cells adapted under non-adherent culture conditions.

[0146] After introducing the expression vector, transformed cells that stably express heterodimer proteins are selected by culturing them in animal cell culture medium containing drugs such as G418 sulfate (hereinafter referred to as G418), cycloheximide (hereinafter abbreviated as CHX), and methotrexate (hereinafter abbreviated as MTX) (Japanese Patent Publication No. 2-257891).

[0147] For animal cell culture, you can use RPMI1640 medium (Invitrogen), GIT medium (Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium, EX-CELL302, EX-CELL325 medium (JRH Corporation), IMDM medium (Invitrogen), Hybridoma-SFM medium (Invitrogen), or any of these media to which various additives such as fetal bovine serum (hereinafter abbreviated as FBS) have been added.

[0148] The resulting transformed strain is cultured in a culture medium to express and accumulate the polypeptide constituting the fusion protein in the culture supernatant. The expression level and antigen-binding activity of the polypeptide in the culture supernatant can be measured by ELISA or other methods. Furthermore, the expression level of the polypeptide produced by the transformed strain can be increased using a dhfr gene amplification system (Japanese Patent Publication No. 2-257891).

[0149] The above describes a method for expressing the polypeptide using animal cells as a host. However, heterodimer protein compositions can also be produced in yeast, insect cells, plant cells, or individual animals or individual plants by the same method as in animal cells, based on known techniques.

[0150] When yeast is used as the host cell, microorganisms belonging to genera such as Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, and Schwanniomyces can be used, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, and Schwanniomyces alluvius.

[0151] Any method for introducing recombinant vectors into yeast that involves introducing DNA can be used. Examples include electroporation (Methods. Enzymol., 194, 182, 1990), spheroplast (Proc. Natl. Acad. Sci. U.S.A., 84, 1929, 1978), and lithium acetate (J. Bacteriology, 153, 163, 1983, Proc. Natl. Acad. Sci. U.S.A., 75, 1929, 1978).

[0152] When insect cells are used as a host, the polypeptide can be expressed by methods described, for example, in Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York, 1992, Bio / Technology, 6, 47, 1988, etc.

[0153] <<Production of the fusion protein from the polypeptide obtained in (s3) and (s2)>> If the host cell has the ability to express the polypeptide that constitutes the fusion protein, the fusion protein can be produced by introducing the gene encoding the polypeptide into the host cell, culturing the cell, and purifying the target polypeptide from the culture.

[0154] Furthermore, by redifferentiating the cells of genetically modified animals or plants, genetically modified animal individuals (transgenic non-human animals) or plant individuals (transgenic plants) can be created, and these individuals can be used to produce this fusion protein.

[0155] If the transformant is an animal or plant, the heterodimer protein composition can be produced by rearing or cultivating it according to conventional methods to generate and accumulate the fusion protein, and then collecting the fusion protein from the animal or plant.

[0156] One method for producing this fusion protein using animal organisms is to introduce genes into animals according to known methods [American Journal of Clinical Nutrition, 63, 639S, 1996; American Journal of Clinical Nutrition, 63, 627S, 1996; Bio / Technology, 9, 830, 1991] and then produce the target fusion protein in those animals.

[0157] In the case of individual animals, for example, the fusion protein can be produced by raising transgenic non-human animals into which nucleic acids (e.g., DNA or RNA) encoding the polypeptide constituting the fusion protein have been introduced, allowing the fusion protein to be generated and accumulated in the animals, and then collecting the heterodimer protein composition from the animals.

[0158] Examples of production and accumulation sites in the animal include the animal's milk (Japanese Patent Publication No. 63-309192) or eggs. Any promoter that can be expressed in animals can be used in this case. For example, α-casein promoter, β-casein promoter, β-lactoglobulin promoter, and whey acid protein promoter, which are mammary gland cell-specific promoters, are preferably used.

[0159] One method for producing the fusion protein using individual plants is to cultivate transgenic plants into which nucleic acids encoding heterodimer protein molecules have been introduced, in accordance with known methods [Tissue Culture, 20 (1994); Tissue Culture, 21 (1995); Trends in Biotechnology, 15, 45 (1997)], to generate and accumulate the fusion protein in the plants, and then to produce the fusion protein by collecting the heterodimer protein composition from the plants.

[0160] The fusion protein of the present invention can be purified as follows. The polypeptide or fusion protein produced by a transformant into which the gene encoding the polypeptide constituting the fusion protein has been introduced can be purified in the following manner: For example, if the polypeptide or fusion protein is expressed as a soluble protein in cells, after the culture is complete, the cells are collected by centrifugation, suspended in an aqueous buffer, and then the cells are disrupted using an ultrasonic disruptor, French press, Manton Gaurine homogenizer, Dynomil, etc., to obtain a cell-free extract.

[0161] From the supernatant obtained by centrifugation of the cell-free extract, a purified sample of this fusion protein can be obtained using conventional enzyme isolation and purification methods, namely solvent extraction, salting out with ammonium sulfate, desalting, precipitation with organic solvents, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 [manufactured by Mitsubishi Chemical Corporation], cation exchange chromatography using resins such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis methods such as isoelectric focusing, either alone or in combination.

[0162] In this disclosure, affinity chromatography using CH conjugates is used as the affinity chromatography method (Monochromatic Antibodies—Principles and Practice, Third Edition, Academic Press (1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

[0163] Furthermore, if the fusion protein is expressed in an insoluble form within cells, the cells are similarly harvested, disrupted, and centrifuged to recover the insoluble fusion protein as a precipitate fraction. The recovered fusion protein is then solubilized with a denaturing agent. By diluting or dialyzing the solubilized solution, the fusion protein is returned to its normal three-dimensional structure, and a purified sample of the fusion protein can then be obtained by the same isolation and purification method as described above.

[0164] If this fusion protein is secreted extracellularly, it can be recovered in the culture supernatant. That is, the culture supernatant can be obtained by processing the culture using the same centrifugation method as described above, and a purified sample of this fusion protein can be obtained from the culture supernatant using the same isolation and purification method as described above.

[0165] Specifically, the purification method for this fusion protein involves, for example, loading the culture supernatant obtained from a transformed cell line onto a Protein A column or Protein G column, and then washing the column with phosphate buffer saline (PBS). Subsequently, the heterodimer protein is eluted from the column with a low pH (pH 2.0 to 6.0) citrate buffer, and the eluate is neutralized with an alkaline Tris buffer. The neutralized eluate is then dialyzed with a sufficient amount of PBS to obtain the purified heterodimer protein.

[0166] <Therapeutic and / or prophylactic and / or diagnostic agents> One embodiment of the present disclosure is a therapeutic and / or prophylactic and / or diagnostic agent for thrombotic diseases, comprising the fusion protein described above as an active ingredient. Examples of thrombotic diseases, but not limited to, include congenital or acquired thrombotic thrombocytopenic purpura (hereinafter also referred to as TTP), arterial thrombosis, acute myocardial infarction (AMI), stroke, sepsis, and disseminated intravascular coagulation (DIC), and the fusion protein can be used for the treatment and / or prevention and / or diagnosis of any of these diseases. In a preferred embodiment, the fusion protein is used for the treatment of congenital or acquired TTP.

[0167] This fusion protein has the potential to contribute to supplementing or maintaining ADAMTS13 activity in thrombotic thrombocytopenic purpura (TTP), in which decreased ADAMTS13 activity is involved. In acquired TTP, it is known that residual or re-elevated autoantibodies against ADAMTS13 lead to decreased ADAMTS13 activity, resulting in insufficient cleavage of ultra-high molecular weight von Willebrand factor (UL-vWF), which in turn leads to the recurrence of microthrombus formation and thrombocytopenia. Because this fusion protein (i) has structural properties that make it less reactive with autoantibodies, and (ii) can maintain relatively stable ADAMTS13 enzyme activity, it is suggested that it may contribute to supplementing ADAMTS13 activity and suppressing the accumulation of UL-vWF even in the presence of autoantibodies. Therefore, this fusion protein may be useful in patients with acquired TTP, particularly for conditions related to the suppression of TTP recurrence. Furthermore, this fusion protein may also influence disease progression in TTP patients experiencing recurrent episodes by supplementing ADAMTS13 activity.

[0168] In another embodiment, since this fusion protein can be used as a therapeutic agent for TTP by cleaving vWF to remove platelet thrombi, it can be used to treat or prevent symptoms such as thrombocytopenia, thrombosis, and coagulation disorders through a similar mechanism.

[0169] As described above, the following configurations are disclosed in this specification: 1. A fusion protein comprising a mutant ADAMTS13 containing at least a metalloproteinase domain (M domain) and lacking a spacer domain (S domain), and an antigen-binding molecule that binds to von Willebrand factor (vWF). 2. The fusion protein according to 1, wherein the mutant ADAMTS13 is (N1) or (N2) below: (N1) In addition to the S domain of ADAMTS13, the T2-T8 domain is further lacking. (N2) In addition to the S domain of ADAMTS13, the T2-T8 domain and the CUB1-2 domain are further lacking. 3. The fusion protein according to 1 or 2, wherein the mutant ADAMTS13 is (D1) or (D2) below. 1. A fusion protein according to any one of 1 to 3, wherein the M domain in the mutant ADAMTS13 comprises an amino acid sequence having 70% or more homology to the amino acid sequence from position 80 to 286 of the amino acid sequence represented by SEQ ID NO: 194, and the fusion protein has ADAMTS13 enzyme activity. 2. A fusion protein according to any one of 1 to 4, wherein the amino acid sequence of the M domain in the mutant ADAMTS13 comprises an amino acid sequence corresponding to the amino acid sequence from position 80 to 286 of the amino acid sequence represented by SEQ ID NO: 194, and further comprises at least one of the substitutions (M1) to (M13) below. (M1) Substitution of the amino acid residue corresponding to the 159th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to R. (M2) Substitution of the amino acid residue corresponding to the 167th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to M. (M3) Substitution of the amino acid residue corresponding to the 207th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to N. (M4) Substitution of the amino acid residue corresponding to the 209th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to V. (M5) Substitution of the amino acid residue corresponding to the 230th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to L.(M6) Substitution of an amino acid residue corresponding to the 238th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 to A (M7) Substitution of an amino acid residue corresponding to the 243rd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 to K (M8) Substitution of an amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 to A (M9) Substitution of an amino acid residue corresponding to the 271st amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 to E (M10) Substitution of an amino acid residue corresponding to the 279th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 to L (M11) Substitution of an amino acid residue corresponding to the 282nd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 to L (M12) Substitution of an amino acid residue corresponding to the 283rd amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 to G (M13) Substitution of an amino acid residue corresponding to the 285th amino acid residue of the amino acid sequence represented by SEQ ID NO: 194 6. The fusion protein described in 5 above, wherein the amino acid sequence of the mutant ADAMTS13 is any one of the following. (m1) Includes substitution of (M1) and (M2). (m2) Includes substitution of (M1) and (M3). (m3) Includes substitution of (M8) and (M13). (m4) Includes substitution of (M9) and (M13). (m5) Includes substitution of (M1), (M8) and (M13). (m6) Includes substitution of (M1), (M9) and (M13). (m7) Includes substitution of (M4), (M5), (M8) and (M13). (m8) Includes substitution of (M4), (M6), (M8) and (M13). (m9) Includes substitution of (M4), (M7), (M8) and (M13). (m10) Includes substitution of (M5), (M6), (M8) and (M13). (m11) Includes substitution of (M5), (M7), (M8) and (M13). (m12) Includes substitution of (M6), (M7), (M8) and (M13). (m13) Includes substitution of (M10), (M11) and (M13). (m14) Includes substitution of (M10), (M12) and (M13). (m15) Includes substitution of (M11), (M12) and (M13). (m16) Includes substitution of (M1), (M10), (M11) and (M13).(m17) Includes substitutions of (M1), (M10), (M12), and (M13). (m18) Includes substitutions of (M1), (M11), (M12), and (M13). 7. The fusion protein according to any one of 1 to 6, wherein the mutant ADAMTS13 consists of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 99. 8. The fusion protein according to any one of 1 to 7, wherein the antigen-binding molecule comprises at least the heavy chain variable region (VH) and the light chain variable region (VL) of an anti-vWF antibody. 9. The fusion protein according to 8, wherein in the antigen-binding molecule, the VH and the VL are any one of (c1) to (c37) below. (c1) The VH includes Complementarity Determinating Regions (CDRs) 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 565 to 567, and the VL includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 568 to 570. (c2) The VH includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 573 to 575, and the VL includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 576 to 578. (c3) The VH includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 581 to 583, and the VL includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 584 to 586. (c4) The VH includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 587 to 589, and the VL includes CDRs 1 to 3, each containing the amino acid sequence represented by SEQ ID NOs. 590 to 592. (c5) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 595 to 597, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 598 to 600. (c6) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 603 to 605, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 606 to 608. (c7) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 611 to 613, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 614 to 616.(c8) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 619 to 621, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 622 to 624, respectively. (c9) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 627 to 629, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 630 to 632, respectively. (c10) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 635 to 637, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 638 to 640, respectively. (c11) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 643 to 645, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 646 to 648, respectively. (c12) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 651 to 653, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 654 to 656, respectively. (c13) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 659 to 661, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 662 to 664, respectively. (c14) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 667 to 669, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 670 to 672, respectively. (c15) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 675 to 677, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 678 to 680, respectively. (c16) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 683 to 685, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 686 to 688. (c17) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 691 to 693, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 694 to 696.(c18) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 699 to 701, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 702 to 704, respectively. (c19) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 707 to 709, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 710 to 712, respectively. (c20) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 715 to 717, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 718 to 720, respectively. (c21) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 723 to 725, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 726 to 728, respectively. (c22) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 731 to 733, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 734 to 736, respectively. (c23) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 795 to 797, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 798 to 800, respectively. (c24) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 803 to 805, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 806 to 808, respectively. (c25) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 811 to 813, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 814 to 816, respectively. (c26) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 819 to 821, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 822 to 824. (c27) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 827 to 829, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 830 to 832.(c28) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 835 to 837, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 838 to 840, respectively. (c29) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 843 to 845, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 846 to 848, respectively. (c30) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 851 to 853, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 854 to 856, respectively. (c31) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 859 to 861, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 862 to 864, respectively. (c32) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 867 to 869, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 870 to 872, respectively. (c33) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 875 to 877, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 878 to 880, respectively. (c34) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 883 to 885, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 886 to 888, respectively. (c35) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 891 to 893, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 894 to 896, respectively. (c36) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs: 899 to 901, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs: 902 to 904. (c37) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs: 907 to 909, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs: 910 to 912.10. The fusion protein according to 9, wherein the antigen-binding molecule is such that the VH and VL are any one of the following (v1) to (v37): (v1) The VH comprises an amino acid sequence represented by SEQ ID NO: 563, and the VL comprises an amino acid sequence represented by SEQ ID NO: 564. (v2) The VH comprises an amino acid sequence represented by SEQ ID NO: 571, and the VL comprises an amino acid sequence represented by SEQ ID NO: 572. (v3) The VH comprises an amino acid sequence represented by SEQ ID NO: 579, and the VL comprises an amino acid sequence represented by SEQ ID NO: 580. (v4) The VH comprises an amino acid sequence represented by SEQ ID NO: 100, and the VL comprises an amino acid sequence represented by SEQ ID NO: 101. (v5) The VH comprises an amino acid sequence represented by SEQ ID NO: 593, and the VL comprises an amino acid sequence represented by SEQ ID NO: 594. (v6) The VH comprises an amino acid sequence represented by SEQ ID NO: 601, and the VL comprises an amino acid sequence represented by SEQ ID NO: 602. (v7) The VH includes the amino acid sequence represented by SEQ ID NO: 609, and the VL includes the amino acid sequence represented by SEQ ID NO: 610. (v8) The VH includes the amino acid sequence represented by SEQ ID NO: 617, and the VL includes the amino acid sequence represented by SEQ ID NO: 618. (v9) The VH includes the amino acid sequence represented by SEQ ID NO: 625, and the VL includes the amino acid sequence represented by SEQ ID NO: 626. (v10) The VH includes the amino acid sequence represented by SEQ ID NO: 633, and the VL includes the amino acid sequence represented by SEQ ID NO: 634. (v11) The VH includes the amino acid sequence represented by SEQ ID NO: 641, and the VL includes the amino acid sequence represented by SEQ ID NO: 642. (v12) The VH includes the amino acid sequence represented by SEQ ID NO: 649, and the VL includes the amino acid sequence represented by SEQ ID NO: 650. (v13) The VH includes the amino acid sequence represented by SEQ ID NO: 657, and the VL includes the amino acid sequence represented by SEQ ID NO: 658. (v14) The VH includes the amino acid sequence represented by SEQ ID NO: 665, and the VL includes the amino acid sequence represented by SEQ ID NO: 666. (v15) The VH includes the amino acid sequence represented by SEQ ID NO: 673, and the VL includes the amino acid sequence represented by SEQ ID NO: 674.(v16) The VH includes the amino acid sequence represented by SEQ ID NO: 681, and the VL includes the amino acid sequence represented by SEQ ID NO: 682. (v17) The VH includes the amino acid sequence represented by SEQ ID NO: 689, and the VL includes the amino acid sequence represented by SEQ ID NO: 690. (v18) The VH includes the amino acid sequence represented by SEQ ID NO: 697, and the VL includes the amino acid sequence represented by SEQ ID NO: 698. (v19) The VH includes the amino acid sequence represented by SEQ ID NO: 705, and the VL includes the amino acid sequence represented by SEQ ID NO: 706. (v20) The VH includes the amino acid sequence represented by SEQ ID NO: 713, and the VL includes the amino acid sequence represented by SEQ ID NO: 714. (v21) The VH includes the amino acid sequence represented by SEQ ID NO: 721, and the VL includes the amino acid sequence represented by SEQ ID NO: 722. (v22) The VH includes the amino acid sequence represented by SEQ ID NO: 729, and the VL includes the amino acid sequence represented by SEQ ID NO: 730. (v23) The VH includes the amino acid sequence represented by SEQ ID NO: 793, and the VL includes the amino acid sequence represented by SEQ ID NO: 794. (v24) The VH includes the amino acid sequence represented by SEQ ID NO: 801, and the VL includes the amino acid sequence represented by SEQ ID NO: 802. (v25) The VH includes the amino acid sequence represented by SEQ ID NO: 809, and the VL includes the amino acid sequence represented by SEQ ID NO: 810. (v26) The VH includes the amino acid sequence represented by SEQ ID NO: 817, and the VL includes the amino acid sequence represented by SEQ ID NO: 818. (v27) The VH includes the amino acid sequence represented by SEQ ID NO: 825, and the VL includes the amino acid sequence represented by SEQ ID NO: 826. (v28) The VH includes the amino acid sequence represented by SEQ ID NO: 833, and the VL includes the amino acid sequence represented by SEQ ID NO: 834. (v29) The VH includes the amino acid sequence represented by SEQ ID NO: 841, and the VL includes the amino acid sequence represented by SEQ ID NO: 842. (v30) The VH includes the amino acid sequence represented by SEQ ID NO: 849, and the VL includes the amino acid sequence represented by SEQ ID NO: 850. (v31) The VH includes the amino acid sequence represented by SEQ ID NO: 857, and the VL includes the amino acid sequence represented by SEQ ID NO: 858.(v32) The VH includes the amino acid sequence represented by SEQ ID NO: 865, and the VL includes the amino acid sequence represented by SEQ ID NO: 866. (v33) The VH includes the amino acid sequence represented by SEQ ID NO: 873, and the VL includes the amino acid sequence represented by SEQ ID NO: 874. (v34) The VH includes the amino acid sequence represented by SEQ ID NO: 881, and the VL includes the amino acid sequence represented by SEQ ID NO: 882. (v35) The VH includes the amino acid sequence represented by SEQ ID NO: 889, and the VL includes the amino acid sequence represented by SEQ ID NO: 890. (v36) The VH includes the amino acid sequence represented by SEQ ID NO: 897, and the VL includes the amino acid sequence represented by SEQ ID NO: 898. (v37) The VH includes the amino acid sequence represented by SEQ ID NO: 905, and the VL includes the amino acid sequence represented by SEQ ID NO: 906. 11. The fusion protein according to any one of 1 to 10, wherein the antigen-binding molecule is an antigen-binding molecule containing any one of (B1) to (B4) below. (B1) An antigen-binding molecule containing scFv, which includes VH and VL. (B2) An antigen-binding molecule containing Fab, which includes a heavy chain containing VH and a CH1 domain, and a light chain containing VL and a constant light chain (CL) region. (B3) An antigen-binding molecule containing the following first polypeptide and second polypeptide, wherein mutations have been introduced into the Cys residue involved in the intermolecular disulfide bond between the light chain and heavy chain in the IgG antibody in the CH1 domain and CL. First polypeptide: A polypeptide in which VH, CH1 domain, hinge domain, CH2 domain and CH3 domain are linked in this order. Second polypeptide: A polypeptide in which VL, CL, hinge domain, CH2 domain and CH3 domain are linked in this order. (B4) An antigen-binding molecule containing IgG, which includes two heavy chains containing VH, CH1 domain, hinge domain, CH2 domain and CH3 domain, and two light chains containing VL and CL. 12. The fusion protein according to any one of 1 to 11, wherein the mutant ADAMTS13 consists of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 99, and the antigen-binding molecule is an antigen-binding molecule containing any one of (B1) to (B4) below. (B1) An antigen-binding molecule containing scFv including VH and VL.(B2) An antigen-binding molecule comprising Fab, comprising a heavy chain comprising VH and CH1 domains, and a light chain comprising VL and a constant light chain (CL) region. (B3) An antigen-binding molecule comprising the following first polypeptide and second polypeptide, wherein mutations have been introduced into the Cys residues involved in the intermolecular disulfide bond between the light chain and heavy chain in the IgG antibody in the CH1 domain and CL. First polypeptide: A polypeptide in which VH, CH1 domain, hinge domain, CH2 domain and CH3 domain are linked in this order. Second polypeptide: A polypeptide in which VL, CL, hinge domain, CH2 domain and CH3 domain are linked in this order. (B4) An antigen-binding molecule comprising IgG, comprising two heavy chains comprising VH, CH1 domain, hinge domain, CH2 domain and CH3 domains, and two light chains comprising VL and CL. 13. A fusion protein according to any one of 1 to 12, wherein the mutant ADAMTS13 is linked to the antigen-binding molecule via a linker. 14. The fusion protein according to 13, wherein the linker has a length of 1 to 35 amino acids. 15. The fusion protein according to 13 or 14, wherein the linker is a linker comprising at least one repeating unit selected from (L1) to (L3) below: (L1) A polypeptide comprising the amino acid sequence (GGGGS) represented by SEQ ID NO: 925 as a repeating unit. (L2) A polypeptide comprising the amino acid sequence (PAPAP) represented by SEQ ID NO: 926 as a repeating unit. (L3) A polypeptide comprising the amino acid sequence (EAAAK) represented by SEQ ID NO: 927 as a repeating unit. 16. The fusion protein according to 14 or 15, wherein the antigen-binding molecule comprises VH and VL of an anti-vWF antibody, and the C-terminus of the mutant ADAMTS13 is linked to the N-terminus of the VH or VL in the antigen-binding molecule via the linker. 17. A fusion protein according to any one of 11 to 16, which is any one of (p3-1) to (p3-3), (p4-1), and (p4-2) below.(p3-1) The antigen-binding molecule is an antigen-binding molecule comprising (B3), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of the mutant ADAMTS13 is linked to the N-terminus of VH in the antigen-binding molecule via a linker, and comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [VL-CL-hinge domain-CH2-CH3] (p3-2) The antigen-binding molecule is an antigen-binding molecule comprising (B3), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of the mutant ADAMTS13 is linked to the N-terminus of the VL in the antigen-binding molecule via a linker, and comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [mutant ADAMTS13-peptide linker-VL-CL-hinge domain-CH2-CH3] (p3-3) The antigen-binding molecule is an antigen-binding molecule comprising (B3), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the N-terminus of the mutant ADAMTS13 is linked to the C-terminus of the CH3 domain in the antigen-binding molecule via a linker, and comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [VL-CL-hinge domain-CH2-CH3-peptide linker-mutant ADAMTS13](p4-1) The antigen-binding molecule is an antigen-binding molecule containing (B4), and is a fusion protein formed by linking two mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of each mutant ADAMTS13 and the N-terminus of each VH in the antigen-binding molecule are linked via a linker, and the fusion protein contains the following first to fourth polypeptides, wherein the CL of the first polypeptide and the hinge domain or CH1 of the second polypeptide are linked via a disulfide bond, the hinge domain or CH1 of the third polypeptide and the CL of the fourth polypeptide are linked via a disulfide bond, and the second polypeptide and the third polypeptide are linked via a disulfide bond of the hinge domain. First and fourth polypeptides: [VL-CL] Second and third polypeptides: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] (p4-2) The antigen-binding molecule is an antigen-binding molecule containing (B4), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of mutant ADAMTS13 and the N-terminus of VH in the antigen-binding molecule are linked via a linker, and the fusion protein contains the following first to fourth polypeptides, wherein the CL of the first polypeptide and the hinge domain or CH1 of the second polypeptide are linked via a disulfide bond, the hinge domain or CH1 of the third polypeptide and the CL of the fourth polypeptide are linked via a disulfide bond, and the second polypeptide and the third polypeptide are linked via a disulfide bond of the hinge domain. First and fourth polypeptides: [VL-CL] Second polypeptide: [VH-CH1-hinge domain-CH2-CH3] Third polypeptide: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] 18. The fusion protein described in 17, which is any one of the following: (t1) The fusion protein of (p3-1), wherein the first polypeptide contains an amino acid sequence represented by any one of SEQ ID NOs: 333, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, and 545.A fusion protein wherein the second polypeptide contains the amino acid sequence represented by SEQ ID NO: 334. (t2) The fusion protein of (p4-1) wherein the first polypeptide and the fourth polypeptide are the same and contain the amino acid sequence represented by SEQ ID NO: 114, and the second polypeptide and the third polypeptide are the same and contain the amino acid sequence represented by any one of SEQ ID NOs: 113, 352, 354, 356, 358, 360, and 362. (t3) A fusion protein of the above (p4-1), wherein the first polypeptide and the fourth polypeptide are the same and include the amino acid sequence represented by SEQ ID NO: 110, and the second polypeptide and the third polypeptide are the same and include the amino acid sequence represented by any one of SEQ ID NOs: 109, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406 and 408. (t4) A fusion protein of the above (p4-1), wherein the first polypeptide and the fourth polypeptide are the same and include the amino acid sequence represented by SEQ ID NO: 126, A fusion protein in which the second polypeptide and the third polypeptide are identical and contain an amino acid sequence represented by any one of SEQ ID NOs: 125, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, and 512. 19. The fusion protein according to any one of 1 to 18, wherein the antigen-binding molecule in the fusion protein specifically binds to the A2 domain of vWF. 20. The fusion protein according to any one of 1 to 19, wherein the antigen-binding molecule in the fusion protein specifically binds to the polypeptide of (α1) or (α2) below. (α1) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9141. A polypeptide comprising the amino acid sequence represented by (α2) Sequence ID No. 915. 21. The fusion protein according to 19 or 20, wherein the antigen-binding molecule has the ability to bind to both vWF with the A2 domain folded and vWF without the A2 domain folded. 22. A nucleic acid encoding the fusion protein according to any one of 1 to 21. 23. A vector containing the nucleic acid according to 22. 24. A transformed cell obtained by introducing the vector according to 23 into a host cell. 25. A method for producing the fusion protein according to any one of 1 to 21, characterized by culturing the transformed cell according to 24 in a culture medium, producing and accumulating the fusion protein according to any one of 1 to 21 in the culture obtained by the culture, and collecting the fusion protein from the culture. 26. A therapeutic agent for thrombotic diseases containing the fusion protein according to any one of 1 to 21 as an active ingredient. 27. A pharmaceutical composition containing the fusion protein described in any one of items 1 to 21 above as an active ingredient, used to suppress recurrence of thrombotic thrombocytopenic purpura in patients with thrombotic thrombocytopenic purpura. 28. A pharmaceutical composition containing the fusion protein described in any one of items 1 to 21 above as an active ingredient, used to supplement ADAMTS13 enzyme activity in patients with thrombotic thrombocytopenic purpura exhibiting recurrent episodes.

[0170] Example 1 Production of anti-human vWF monoclonal antibodies (1) Preparation of human vWF A2 domain protein antigen and peptide antigen For immunization of animals, a human vWF A2 domain protein (GST fusion) antigen (SEQ ID NO: 928) or a partial sequence of the human vWF A2 domain (GST fusion) antigen (SEQ ID NO: 929), in which a GST tag was fused to the A2 domain of human vWF (SEQ ID NO: 916) or a partial region consisting of 73 residues in the A2 domain (SEQ ID NO: 195), was transiently expressed in Expi293F cells as host cells and purified by affinity. For screening, a human vWF A2 domain protein antigen (His fusion) (SEQ ID NO: 930), in which a His tag and a FRET probe were fused to the A2 domain of human vWF, was transiently expressed in Expi293F cells as host cells and purified by affinity. Furthermore, PEP01 (SEQ ID NO: 913), PEP05 (SEQ ID NO: 914), and PEP06 (SEQ ID NO: 915) were synthesized from partial sequences of the A2 domain of human vWF. For immunization of animals, KLH conjugates or BSA conjugates were created by conjugating keyhole limpet hemocyanin (KLH) or BSA via the Cys at the terminals of PEP01, PEP05, and PEP06, respectively.

[0171] (2) Immunization of animals and preparation of antibody-producing cells Human antibody-producing mice [Ishida & Lomber, IBC's 11th Antibody Engineering, Abstract 2000; Ishida, I. et al., Cloning & Stem Cells 4, 91-102 (2002), and Ishida, I. (2002) Experimental Medicine 20, 6, 846-851] were administered as immunogens at doses of 20 μg / mice or 50 μg / mice for a total of 4 to 6 doses. For the initial immunization only, an alum gel (0.25 mg / animal or 2 mg / animal) and an inactivated Bordetella pertussis suspension (Nacalai Tesque) (1 x 10) should be administered as adjuvants. ９Individuals / animals) were added. Immunization was performed 4 times at intervals of 1 to 2 weeks from the first immunization, and dissection was performed 3 or 4 days after the final immunization to surgically remove lymph nodes or spleen. Lymphocyte cells were prepared from the removed lymph nodes or spleen and used for the production of hybridomas or screening for anti-human vWF antibodies by the B cell cloning method.

[0172] (3) Production of hybridomas The 8-azaguanine-resistant mouse myeloma cell line P3-U1 [P3X63Ag8U.1, ATCC: CRL-1597, European Journal of Immunology, 6, 511 (1976)] and the mouse lymphocyte cells obtained in (2) were mixed so that the cell number ratio was 1:2, and cell fusion was performed using an electrofusion apparatus ECM2001 (manufactured by BTX). After fusion, the cells were suspended in a medium (hereinafter referred to as HAT medium) prepared by adding HAT supplement (manufactured by Thermo Fisher Scientific) and gentamicin (50 μg / mL) to cloning medium CM-B (manufactured by Sekisui Medical Co., Ltd.), seeded in a 96-well plate, and cultured at 37°C and 5% CO ２ under the conditions for 8 to 10 days. The medium was changed using HAT medium 1 to 2 days before screening, and screening of the hybridomas described below was performed using the culture supernatant 1 to 2 days after the medium change.

[0173] (4) Screening of anti-human vWF antibody-producing hybridomas Screening of anti-human vWF antibody-producing hybridomas was performed by the ELISA method. The BSA conjugate of human vWF A2 domain protein antigen (His fusion) or human vWF peptide antigen prepared in (1) was prepared to 5 μg / mL with D-PBS(-) (Nacalai Tesque), and dispensed into 96-well or 384-well ELISA plates (MAXISORP NUNC-IMMNO PLATE, Thermo Fisher Scientific), and allowed to stand overnight at 4°C to adsorb. The plates were washed with PBS, 1% BSA-PBS (Nacalai Tesque) was dispensed, and the plates were allowed to stand at room temperature for 1 hour to block. Next, after removing the blocking solution, the hybridoma supernatant was added and the plates were allowed to stand at room temperature for 1 hour. After washing the plate with 0.05% Tween-PBS (PBST), HRP-labeled Goat Anti-Human IgG (Fc) antibody (IBL) diluted with 1% BSA-PBS was added and allowed to stand at room temperature for 1 hour. After that, the plate was washed with PBST, and color development was performed using ABTS (Wako) substrate solution, followed by stopping the color development with 1% SDS solution, or color development was performed using TMB substrate solution (Thermo Fisher Scientific), followed by stopping the color development with 0.5 mol / L sulfuric acid. When using ABTS, the absorbance at a sample wavelength of 415 nm and a reference wavelength of 490 nm was measured using a plate reader (Enspire, PerkinElmer, or SPARK 10M, TECAN). When using TMB, the absorbance at a sample wavelength of 450 nm and a reference wavelength of 570 nm was measured using a plate reader.

[0174] (5) Isolation of hybridomas and analysis of antibody gene sequences Hybridomas selected from the positive wells by hybridoma screening were seeded into 96-well plates dispensed with culture medium using a cell sorter (SONY SH800) and monoclonalized. 37°C, 5% CO2 ２Under these conditions, the cells in the wells were cultured until the number of cells suitable for screening was reached. Using the obtained monoclonal hybridoma culture supernatant, screening was performed again using the method described in (4) to establish anti-human vWF monoclonal antibody-producing hybridomas.

[0175] Total RNA was extracted from the obtained hybridomas, and cDNA was synthesized using the 5'RACE method. Heavy chain antibody gene fragments and light chain antibody gene fragments were amplified by performing a PCR reaction using a forward primer that anneals to the 5' end of the synthesized cDNA and a reverse primer that anneals to the human IgG heavy chain constant region or light chain constant region. The amplified heavy chain antibody gene fragments and light chain antibody gene fragments were sequenced using subcloning with the Zero Blunt TOPO PCR Cloning Kit for Sequencing (Thermo Fisher Scientific) or by using the next-generation sequencer ION S5™ system (Thermo Fisher Scientific).

[0176] Based on DNA sequencing analysis results, the nucleotide sequences encoding the full-length VH or VL were identified, and the amino acid sequences of the antibodies against human vWF expressed by each hybridoma were determined. The correspondence between each clone name and immunogen is shown in Table 1-1. As mentioned above, the PEP01, PEP05, PEP06, A2 domain, and subdomain of the A2 domain (VWF73) of human vWF are represented by the amino acid sequences 913, 914, 915, 916, and 195, respectively. The positional relationship of the PEP01, PEP05, and PEP06 regions in human vWF is shown in Figure 6.

[0177]

[0178] (6) Screening of anti-human vWF antibodies by B cell cloning method B cells were isolated from the mouse lymphocytes obtained in (2) using the EasySep Mouse Pan-B Cell Isolation Kit (STEMCELL). The isolated B cells were stained with a fluorescently labeled human vWF A2 domain (SEQ ID NO: 141) and rabbit Fc fusion protein antigen, as well as anti-mouse CD19 antibody (BioLegend) and anti-mouse CD138 antibody (BioLegend), and sorted using a cell sorter (SONY SH800). Antigen-bound B cells were sorted at a rate of 1 cell / well in a 96-well PCR plate dispensed with cell lysis buffer, and cDNA was synthesized from the cell suspension using the 5'RACE method. Heavy chain antibody gene fragments and light chain antibody gene fragments were amplified by performing a PCR reaction combining a forward primer that anneals to the 5' end of cDNA and a reverse primer that anneals to the constant region of the human IgG heavy chain or light chain. Subsequently, antibody expression cassettes were prepared using a procedure similar to that described in J Virol Methods. (2009) 158(1-2): 171-179. Using these cassettes, gene transfer was performed using Expi293F cells as the host, and the binding affinity to human vWF was evaluated using the ELISA method described in (4) with the obtained transient expression cell culture supernatant.

[0179] For wells that showed positive binding activity using the method described above, sequence analysis was performed using the next-generation sequencer ION S5™ system (Thermo Fisher Scientific). From the DNA sequencing results, the base sequence encoding the full-length VH or VL was identified, and the amino acid sequence of the antibody against human vWF expressed by each B cell was determined. The correspondence between each clone name and immunogen is shown in Table 1-2.

[0180]

[0181] Example 2: Preparation of fusion proteins and confirmation of pharmacokinetic activity (1) vWF degradation activity of non-fusion proteins (various domain knockouts of ADAMTS13) Human IgG1 antibody against vWF (clone name: Vf1005, amino acid sequences of heavy chain variable region and light chain variable region: SEQ ID NOs. 100 and 101), or various domain knockout proteins of ADAMTS13 were prepared by adding His tags to the C-terminus of the M domain (SEQ ID NOs. 931), MD domain (SEQ ID NOs. 936), MDT domain (SEQ ID NOs. 937), MDTC domain (SEQ ID NOs. 938), or MDTCS domain (SEQ ID NOs. 939) of ADAMTS13, and their vWF degradation activity was confirmed. The amino acid sequences of the M domain, MD domain, MDT domain, MDTC domain, and MDTCS domain proteins fused with the His tag are shown in SEQ ID NOs: 102, 103, 104, 105, and 106, respectively.

[0182] ADAMTS13 is composed of the Peptidase M12B (M), Disintegrin (D), TSP type-1 (T), Cysteine-rich (C), and Spacer (S) domains from the N-terminus, and the above-mentioned domain-deficient organisms are created by deleting the domains in order from the C-terminus.

[0183] Furthermore, human IgG1 antibodies against vWF were obtained for use in subsequent mouse efficacy studies (Example 2 (5), Table 2-2). The pCI-OtCAG_hG1 vector, containing a signal sequence and a human γ-chain constant region sequence, was used as the heavy chain expression vector. The pCI-OtCMV_hK vector, containing a signal sequence and a human K-chain constant region sequence, was used as the light chain expression vector. These vectors were constructed by introducing restriction enzyme sites necessary for expressing human antibody genes, using the Promega pCl vector as a common main skeleton. For each vector, DNA was synthesized by total synthesis and subcloned as inserts containing the necessary protein-coding nucleotide sequences. Expression vectors were constructed by transformation into E. coli DH5α Competent Cells (Takara Bio, 9057). The inserted gene region was amplified by colony PCR or plasmid extraction by miniprep, and then subjected to sequencing analysis. Colonies with the correct sequence were selected, and plasmids of the quantity and quality required for antibody expression were prepared using the CompactPrep Plasmid Maxi Kit (QIAGEN, #12863). These plasmids were then dissolved in the TE buffer provided with the kit and stored at -30°C.

[0184] The obtained expression vector and the ExpiFectamine™ 293 Transfection Kit (Gibco, # A14525) were used to transiently express the protein in Expi293F cells, followed by affinity purification. The transfection procedure followed the product protocol. Cells were kept at 37°C, 120 rpm, and 5% or 8% CO2. ２ Cells were cultured under controlled conditions, and the culture was terminated on the fourth day after vector introduction. The cell culture medium was centrifuged, and the culture supernatant was collected through a 0.22 μm filter. Purified antibodies or various domain-deficient forms of ADAMTS13 were obtained from the culture supernatant by affinity purification using MabSelect SuReTM (Cytiva, #17543802) or completeTM His-Tag Purification Resin (Roche, #5893801001).

[0185] Purification was performed according to the standard procedure described by the manufacturer. For anti-human vWF antibodies, a elution buffer of 20 mmol / L citrate, 50 mmol / L NaCl, pH 3.4 was used. For various domain-deficient forms of ADAMTS13, the elution buffer included with the His Buffer Kit (cytiva, #11003400) was used. After elution, the antibodies were rapidly neutralized with 1 mol / L phosphate-NaOH, pH 7.0. Subsequently, the buffer was replaced with TBS (BioRad, #1706435) using a NAP-25 column (cytiva, #17085201). For ADAMTS13 domain-deficient forms, the buffer was replaced with the storage buffer included with the His Buffer Kit (cytiva, #11003400). The absorbance at 280 nm was measured using NanoDrop, and the concentration and preparation of protein solutions were determined. The molecular extinction coefficient was calculated from the amino acid composition and molecular weight of the protein.

[0186] For the evaluation of vWF degradation activity, FRETS-vWF73 (Peptide Institute, #3224-s) was used. For each protein of various domain-deficient ADAMTS13 proteins, 3.5 μmol / L FRETS-vWF73, 50 nmol / L FRETS buffer (5 mmol / L Bis-Tris-HCl, 150 mmol / L NaCl, 25 mmol / L CaCl) were used. ２ The reaction was carried out at room temperature for 24 hours in a pH of 7.0 solution. The reaction solution was measured using an absorbance microplate reader ARVO X3 (Perkin Elmer) at an excitation wavelength of 340 nm and an fluorescence wavelength of 450 nm.

[0187] The measurement results are shown in Figure 12. This experiment revealed that the vWF degradation activity of ADAMTS13 decreased each time a domain was removed from the C-terminal side. It was also found that the M domain alone does not exhibit vWF degradation activity.

[0188] (2) Production of fusion protein (IgG1 type) The heavy chain N-terminus of the IgG1 type anti-human vWF antibody is (GGGGS) ４ The fusion proteins shown in Table 2-1 were created by fusing the M domain sequence of human ADAMTS13 via an amino acid linker consisting of R.

[0189]

[0190] DNA encoding each protein was synthesized and subcloned into an expression vector with the pCI system vector described in Example 2(1) as the parent skeleton, and the expression vector was constructed using the method described in Example 2(1).

[0191] The fusion protein was transiently expressed by introducing each expression plasmid into Expi293F cells in a 1:1 ratio. Plasmid introduction was performed using ExpiFectamine293 (Thermofischer Scientific, #A14525) according to the instructions. Cells were cultured for 3 days using Expi293 Expression Medium (Thermofischer Scientific, #A14351) as the culture medium. Subsequently, the culture supernatant was collected and affinity purified using MabSelect SuRe (GE Healthcare, #17-5438-01) by the following method. The culture supernatant was passed through a resin-packed column, washed with D-PBS(-), and then eluted with elution buffer (100 mM citrate, pH 3.5). Immediately after elution, elution was performed on a NAP column (GE Healthcare, #17-0852-01) using acetate buffer (50 mM sodium acetate, 250 mM D-sorbitol, 50 mM L-arginine, 7.15 μg / mL ZnSO4). ４ Direct substitution was performed (at pH 5.0).

[0192] Anti-human vWF antibodies were prepared by the method described in Example 2(1). For the control substance, hADAMTS13, Recombinant Human ADAMTS13 (Full Length) (R&D systems, #6156-AD) (hereinafter also referred to as recombinant human full-length ADAMTS13 or hADAMTS13) was used.

[0193] These samples were concentrated as needed using Amicon Ultra-4, PLTK Ultracel-PL membrane, 30 kDa (Merck, #UFC803096). If purity was low, gel filtration purification was performed using a chromatography system (AKTA pure 25, Cytiva) with a Superdex 200 Increase 10 / 300 GL column (Merck, #28-9909-44). The mobile phase for gel filtration purification was sugar-free acetate buffer (50 mM sodium acetate, 50 mM L-arginine, 7.15 μg / mL ZnSO4). ４ (pH 5.0) was used.

[0194] (3) Comparison of inhibition rates by autoantibodies The vWF degradation activity in the presence of autoantibodies against ADAMTS13 was compared for each of the prepared fusion proteins (A-1, A-4), recombinant human full-length ADAMTS13 (hADAMTS13), and the His-tagged fusion of the MDTCS domain (SEQ ID NO: 939) of ADAMTS13 described in Example 2(1). The autoantibodies used were those cloned from patients with acquired TTP. Since many patients with acquired TTP have autoantibodies that recognize the Spacer domain, and it has been reported that binding inhibits ADAMTS13 enzyme activity, in this study, an equal mixture of three antibodies that recognize the Spacer domain [scFv_1_4_16_Fc (SEQ ID NO: 559), scFv_1_4_20_Fc (SEQ ID NO: 560), and scFv_3_3_01_Fc (SEQ ID NO: 561)] was used as the autoantibody. (PNAS. 2015; 112(31): 9620-9625)

[0195] Patient-derived autoantibodies were subcloned into expression vectors using the pCI-Ot2.01 vector as the parent skeleton, with the DNA containing the base sequence encoding each protein being converted to the expression vector as described in Example 2(1). The obtained expression vectors were transiently expressed in Expi293F cells using the ExpiFectamine 293 Transfection Kit, and affinity purification was performed. The transfection procedure followed the product protocol. Cells were incubated at 37°C, 120 rpm, and under 5% or 8% CO2. ２ The cells were cultured under specified conditions, and the culture was terminated on the fourth day after vector introduction. The cell culture medium was centrifuged, and the culture supernatant was collected through a 0.22 μm filter.

[0196] Purified antibodies were obtained from the culture supernatant by affinity purification using MabSelect SuRe. Purification was performed according to the standard procedure described by the manufacturer. The elution buffer used was 20 mmol / L citrate, 50 mmol / L NaCl, pH 3.4. After elution, the antibodies were rapidly neutralized with 1 mol / L phosphate-NaOH, pH 7.0. Subsequently, the buffer was replaced with PBS(-) using a NAP25 column.

[0197] To measure the ADAMTS13 enzyme activity using the full-length vWF-A2 domain as a substrate in ELISA, a substrate (vWF-A2-FRET) was prepared by fusing an RFP variant to the N-terminus and a GFP variant to the C-terminus of the vWF-A2 domain sequence (sequence 141), and adding a His tag to the N-terminus. DNA containing the base sequence encoding the amino acid sequence of vWF-A2-FRET was synthesized, and an expression vector was constructed using the pCI-OtCMV vector as the backbone according to the method described in Example 2(1). The obtained expression vector was transiently expressed in Exp293F cells using the ExpIFectamine 293 Transfection Kit, and affinity purification was performed. The transfection procedure followed the product protocol. Cells were heated at 37°C, 120 rpm, and 5% or 8% CO2. ２Cells were cultured under specified conditions, and the culture was terminated on the fourth day after vector introduction. The cell culture medium was centrifuged, and the culture supernatant was collected through a 0.22 μm filter. vWF-A2-FRET was obtained from the culture supernatant by affinity purification using complete His-Tag Purification Resin. Purification was performed according to the standard method described by the manufacturer. The elution buffer used was the elution buffer included with the His Buffer Kit. Subsequently, the buffer was replaced with TBS (pH 7.4) (Nippon Gene, #317-90371) using a NAP-25 column.

[0198] RFP Antibody Pre-adsorbed Rabbit Polyclonal (ROCKLAND, #600-401-379), which can bind to the above RFP variant, was diluted to 5 μg / mL using PBS(-), and 50 μL / well was added to a maxisorb 96 well plate and solidified overnight at 4°C. The plate was washed three times with PBST, 100 μL / well of 2% FBS, 1% BSA PBS(-) was added, and the mixture was blocked at room temperature for 2 hours, after which the plate was washed three times with PBST. vWF-A2-FRET was diluted to 2 μg / mL with blocking buffer, added at 100 μL / well, and reacted at room temperature for 2 hours. The plate was washed three times with PBST, and the fusion protein, or hADAMTS13, or MDTCS, diluted with FRET Buffer to a final concentration of 25 nmol / L, was mixed in equal volumes with the autoantibody or FRET Buffer, each diluted to a final concentration of 2 μg / mL, and reacted at room temperature for 20 minutes. 80 μL / well of each reaction was added and reacted at room temperature for 3 hours. The plates were washed three times with PBST, and Human vWF cleavage site recognition antibody Human vWF-A2 (ADAMTS13-cleaved) Antibody (R&D SYSTEMS, Cat#MAB27642), which was labeled with Peroxidase Labeling Kit-NH2 (Dojin Institute of Science, #LK11) for HRP, was added at a rate of 100 μL / well after being diluted to 0.25 μg / mL with blocking buffer and allowed to bind at room temperature for 1 hour. The plates were washed three times with PBST, and TMB (Dako #S1599) was added at a rate of 100 μL / well to induce color development. The color reaction was then stopped by adding 100 μL / well of 0.5 M sulfuric acid. The absorbance at 450 nm / 570 nm was measured using a plate reader. The results are shown in Figure 13.

[0199] As shown in Figure 13, recombinant human full-length ADAMTS13 (ADAMTS13 in Figure 13) and the MDTCS variant (ADAMTS13_MDTCS in Figure 13) were significantly inhibited by autoantibodies. On the other hand, the fusion proteins (A-1 and A-4 in Figure 13) did not contain an S domain in their components, and therefore showed less inhibition by autoantibodies.

[0200] (4) Effect of differences in antibody epitope regions on ADAMTS13 enzyme activity The effect of differences in the epitope region in the vWF-A2 domain to which the anti-human vWF antibody binds on ADAMTS13 enzyme activity in the fusion protein prepared in Example 2 (2) was measured using the evaluation system described in Example 2 (3) with the full-length vWF-A2 domain as the substrate. Anti-RFP antibody was immobilized on a blocking plate, and 50 μL / well of vWF-A2-FRET, diluted to 2 μg / mL with blocking buffer, was added and reacted at room temperature for 2 hours. The plate was washed three times with PBST, and 50 μL / well of each fusion protein with different binding epitope regions, diluted with FRET Buffer to vary the concentration, was added and reacted at room temperature for 4 hours. The plate was washed three times with PBST, and 100 μL / well of an HRP-labeled vWF cleavage site recognition antibody, diluted to 0.25 μg / mL with blocking buffer, was added and conjugated at room temperature for 1 hour. The plate was washed three times with PBST, 100 μL / well of TMB was added to induce color development, and then 100 μL / well of 0.5 M sulfuric acid was added to stop the color reaction. The absorbance at 450 nm / 570 nm was measured using a plate reader. The results are shown in Figures 14(a) to (e).

[0201] As shown in Figures 14(a) to (e), when the amino acid sequence of a monoclonal antibody obtained by immunization with the PEP01 region (amino acid sequence represented by SEQ ID NO: 913) located at the N-terminus of the vWF-A2 domain was used, all of the resulting fusion proteins showed very weak ADAMTS13 enzyme activity. However, fusion proteins containing the amino acid sequences of monoclonal antibodies obtained by immunization with the PEP05 region (amino acid sequence represented by SEQ ID NO: 914) or the PEP06 region (amino acid sequence represented by SEQ ID NO: 915) located at the C-terminus of the vWF-A2 domain all showed ADAMTS13 enzyme activity. Furthermore, the M domain (M domain in the figure), which was not fused with a vWF-binding molecule, showed almost no ADAMTS13 enzyme activity. From this, it is considered important to appropriately select the epitope of the anti-human vWF antibody in order to confer ADAMTS13-like protease activity to the fusion protein.

[0202] (5) Using a fusion protein (IgG1-type fusion protein) A-2, whose mouse antigen-binding molecule is IgG1, we evaluated its effect in suppressing platelet count decline (pharmacological efficacy) in a mouse model of acquired TTP-like disease induced by activity inhibition using an anti-ADAMTS13 antibody derived from a patient, as well as in a normal mouse C57BL / 6J (Jackson Laboratory Japan). In addition, an anti-vWF antibody without an M domain fusion was also evaluated as a control group.

[0203] After acclimatization for more than one week, the mice were divided into groups of four based on their weight on the grouping day, ensuring that the average weight of each group was approximately the same. The two aforementioned anti-ADAMTS13 antibodies [scFv_1_4_16_Fc (SEQ ID NO: 559) and scFv_1_4_20_Fc (SEQ ID NO: 560)], which are autoantibodies that inhibit endogenous ADAMTS13 activity, were administered to wild-type mice via tail vein at a dose of 5 mg / kg each, or D-PBS(-) (Nacalai Tesque, #14249-24) at a dose of 5 mL / kg. Approximately 24 hours after administration of the anti-ADAMTS13 antibody, administer 1 mg / kg or 10 mg / kg of each fusion protein, or 10 mg / kg of anti-vWF antibody (Vf1005), or acetate buffer (50 mM sodium acetate, 250 mM D-sorbitol, 50 mM L-arginine, 7.15 μg / mL ZnSO4). ４ 5 mL / kg of (pH 5.0) was administered intravenously in the tail vein. Then, within 5 minutes, 600 U / kg of hvWF or glycine buffer (1.13 mg / mL glycine, 20 mg / mL D-mannitol, 10 mg / mL trehalose hydrate, 0.1 mg / mL polysorbate 80, pH 7.35) was administered intravenously in the tail vein.

[0204] Approximately 24 hours after administration of the test substance, mice were anesthetized with isoflurane (Muromachi Instruments, Small Animal Anesthesizer MKA110, flow rate 2.5 L / min, concentration 2.5%), and laparotomy was performed. 630 μL of peripheral blood was collected from the posterior vena cava using a syringe pre-added with 70 μL of sodium citrate buffer pH 5.5 (concentration 3.2%). The blood was transferred to a 1.5 mL tube (Eppendorf, Safe-Lock Tube), mixed by inversion, and 170 μL was dispensed into a blood collection tube (BD MicroTina micro blood collection tube / EDTA 2K) for platelet count measurement. The measurement was performed using a multi-parameter automated hematology analyzer XN-2000 (Sysmex). The mouse platelet count was calculated by correcting for dilution from the obtained platelet count.

[0205] Table 2-2 summarizes each treatment group and its results. The groups included a buffer-only group (no ADAMTS13 antibody or hvWF administration), a hvWF-only group (no anti-ADAMTS13 antibody administration), and a group receiving either buffer, A-2 (two doses), or anti-vWF antibody at the onset of the disease.

[0206]

[0207] As shown in Table 2-2, a decrease in platelet count was observed in the disease-induced group, but the decrease in platelet count was suppressed in the A-2 administration group. In the group administered anti-vWF antibodies, which are known to be effective in treating TTP, at the time of disease induction, the platelet count decreased similarly to the disease-induced group. This suggests that what is important in suppressing the decrease in platelet count is not the binding of the test substance to the A2 domain of vWF, but rather the ADAMTS13-like enzyme activity that can cleave vWF in mouse blood. Based on these results, the fusion protein of the present invention is expected to have a therapeutic effect in acquired TTP patients in whom ADAMTS13 activity is deficient due to autoantibodies.

[0208] Example 3: Production and performance evaluation of fusion proteins with different antibody clones (1) Production Based on the results of Example 2 (4), it was decided to use the amino acid sequence of a monoclonal antibody obtained by immunization using the peptide sequence of the PEP05 or PEP06 region of the vWF-A2 domain. In this example, fusion proteins containing various antibodies with PEP05 or PEP06 as epitopes were produced by the following method, and their ADAMTS13 enzyme activity was compared. A list of the fusion proteins produced and whose activity was compared in this example is shown in Table 3-1.

[0209]

[0210] The enzyme fusion proteins described above were prepared by the following method. DNA containing the base sequence encoding each protein was subcloned into the pCI-based expression vector described in Example 2(1), and the expression vector was constructed using the method described in Example 2(1).

[0211] Fusion proteins were transiently expressed by introducing each expression plasmid into Expi293F cells in a 1:1 ratio. Plasmid introduction was performed using ExpiFectamine293 (Thermofischer Scientific, #A14525) according to the instructions. Cells were cultured for 3 days in Expi293 Expression Medium (Thermofischer Scientific, #A14351) supplemented with 0.1 mol / L zinc sulfate solution to a final zinc concentration of 7.15 μg / mL. Subsequently, the culture supernatant was collected and affinity purified using MabSelect SuRe (GE Healthcare, #17-5438-01) by the following method. The culture supernatant was passed through a resin-packed column, washed with D-PBS(-), and then eluted with elution buffer (100 mM citrate, pH 3.5). Immediately after elution, elution was performed on a NAP column (GE Healthcare, #17-0852-01) using acetate buffer (50 mM sodium acetate, 250 mM D-sorbitol, 50 mM L-arginine, 7.15 μg / mL ZnSO4). ４ Direct substitution was performed (at pH 5.0). In selecting antibody clones, the expression titer of each antibody clone was checked to select those with higher expression titers, and the desired IgG1-type fusion protein was confirmed by SEC-HPLC.

[0212] Anti-human vWF antibodies (with amino acid sequences of the heavy chain variable region and light chain variable region of SEQ ID NOs: 100 and 101, respectively) were prepared by the method described in Example 1. For the control substance hADAMTS13, Recombinant Human ADAMTS13 (Full Length) (R&D systems, #6156-AD) was used.

[0213] Human vWF-A2-WT (open) (amino acid sequence: SEQ ID NO: 192) and human vWF-A2-CC (closed) (amino acid sequence: SEQ ID NO: 193) (Blood. 2011; 117 (17): 4623-31), for evaluation of vWF binding activity, were prepared by the following method. First, DNA containing the base sequences encoding each protein was subcloned into a pCI expression vector, and the expression vector was constructed using the method described in Example 2 (1). The obtained expression vector was transiently expressed in Expi293F cells using the ExpiFectamine 293 Transfection Kit, and affinity expression purification was performed. The transfection procedure followed the product protocol. Cells were heated at 37°C, 120 rpm, and 5% or 8% CO2. ２ The cells were cultured under specified conditions, and the culture was terminated on the fourth day after vector introduction. The cell culture medium was centrifuged, and the culture supernatant was collected through a 0.22 μm filter.

[0214] Human vWF-A2-WT (open) and human vWF-A2-CC (closed) were obtained from the culture supernatant by affinity purification using complete His-Tag Purification Resin. Purification was performed according to the standard method described by the manufacturer. The elution buffer used was the elution buffer included with the His Buffer Kit. Subsequently, the buffer was directly replaced with TBS (pH 7.4) (Nippon Gene, #317-90371) using a NAP25 column.

[0215] Anti-human ADAMTS13 antibodies derived from acquired TTP patients [scFv_1_4_16_Fc (SEQ ID NO: 559), scFv_1_4_20_Fc (SEQ ID NO: 560)] were prepared by the method described in Example 2(3).

[0216] These samples were concentrated as needed using Amicon Ultra-4, PLTK Ultracel-PL membrane, 30 kDa (Merck, #UFC803096). If purity was low, gel filtration purification was performed using a chromatography system (AKTA pure 25, Cytiva) with a Superdex 200 Increase 10 / 300 GL column (Merck, #28-9909-44). The mobile phase for gel filtration purification was a sugar-free acetate buffer (50 mM sodium acetate, 50 mM L-arginine, 7.15 μg / mL ZnSO4). ４ (pH 5.0) was used.

[0217] SEC data was obtained for the prepared samples using ultra-high performance liquid chromatography (Nexera X2, Shimadzu Corporation) (ACQUITY UPLC Protein BEH SEC Column 200 Å 1.7 μm 4.6 mm × 150 mm 1 / pkg (Waters, #186005225)). Endotoxin analysis was performed on samples used in animal studies using nexgene-PTS (PTS-150k) (Charles River) and PTS cartridge FDA (0.5-0.005 EU / mL) (Charles River, #PTS20005F). Proteins used in animal studies were selected based on SEC data showing monomer purity of 85% or higher and endotoxin levels of less than 5.0 EU / kg upon administration.

[0218] (2) Measurement of specific activity of fusion proteins with different antibody clones Specific activity measurements were performed on 34 clones of the fusion proteins listed in Table 3-1 above. The test substance and substrate, FRETS-vWF73 (Peptide Institute, #3224-s), were respectively tested in FRET buffer [5 mM Bis-Tris, 150 mM NaCl, 25 mM CaCl]. ２The solutions were diluted with 0.005 v / v% Tween®-20, pH 7.2, and 50 μL of each solution was mixed. The reaction was carried out at room temperature, and the fluorescence of the cleavage products produced by the degradation of FRETS-vWF73 was measured over time. FRETS-vWF73 was dissolved to 100 μM in 25 v / v% DMSO distilled water, and then a 2-fold dilution series from 32 μM to 0.25 μM was prepared in FRET buffer. The various fusion proteins, which were the test substances, were prepared to 200 nM, and rhADAMTS13 to 60 nM. Since these solutions were mixed in a 1:1 ratio, the final concentrations of each were halved.

[0219] To calculate the absolute amount of product from the fluorescence intensity, calibration curves were created using small molecules FRETS-25-STD1 (Peptide Institute, #3720-v) and FRETS-25-STD2 (Peptide Institute, #3720-v), which have a fluorescent group and a quenching group bound to FRETS-vWF73, respectively. Stock solutions of FRETS-25-STD1 and FRETS-25-STD2 were prepared at 1 mM in DMSO, then equal volumes were mixed, and a dilution series with a 2x common ratio was prepared in FRET buffer so that the concentrations ranged from 32 μM to 0.25 μM.

[0220] Mixing was performed using a plate shaker (TITRAMAX1000, Heidolph) by stirring at 1350 rpm for 30 seconds. Immediately after mixing, fluorescence at 450 nm was measured every 5 minutes for 60 minutes using a plate reader (Ensight, PerkinElmer) with excitation light at 340 nm. For the obtained data, the reaction time (minutes) was plotted on the x-axis and the product concentration (μmol / L) on the y-axis, and the slope from 0 to 5 minutes for each substrate concentration was calculated as the initial velocity (V0, μmol / L・min). Using the data for each substrate concentration and initial velocity, the Michaelis-Menten equation was fitted in JMP (JMP Statistical Discovery) to calculate the maximum reaction rate (Vmax, μmol / L・min) and the dissociation constant (Km, μmol / L). Furthermore, kcat was calculated by dividing Vmax by the concentration of the test substance.

[0221] The results of the measurements of 34 clones are shown in Table 3-2.

[0222]

[0223] As shown in Table 3-2, among the 34 molecules, A-2, whose antibody clone is Vf1005, showed the highest specific activity. The specific activity ranged from 0.10 to 2.40 ( / min), and ADAMTS13 enzyme activity was confirmed in all molecules. On the other hand, the average specific activity of the fusion protein group containing antibody clones that bind to PEP05 (molecules A7-10, B-22, 23, 25, 27-29) and the fusion protein group containing antibody clones that bind to PEP06 (molecules A-1-6, B-1-12, 20, 21) was calculated, and the result was that the PEP05 region had an average specific activity of 0.62 ( / min), while the PEP06 region had an average specific activity of 1.26 ( / min). Compared to the PEP05 region, the average specific activity of the fusion protein containing antibody clones that bind to the PEP06 region tended to be higher. As shown in Figure 6, in the A2 domain of vWF, the PEP05 region is closer to the cleavage site of ADAMTS13 than the PEP06 region. The results in Table 3-2 suggest that the positional relationship between the binding site and the enzyme cleavage site when the fusion protein binds to vWF may be important for ADAMTS13 enzyme activity.

[0224] (3) The binding affinity of vWF with the A2 domain in a closed structure (folded state) and vWF with the A2 domain in an open structure (unfolded state) affects platelet count (pharmacokinetics) <ELISA evaluation system> When there is no shear stress due to blood flow, vWF in the blood takes on a closed structure in which the A2 domain forms a three-dimensional structure, and when there is shear stress, it takes on an open structure in which the three-dimensional structure of the A2 domain is resolved. ADAMTS13 is said to bind to vWF in an open structure via the D domain, C domain and S domain, and cleave vWF with the M domain (Blood. 2011;117 (17):4623-31). To investigate the binding state of the Open and Close structures of the fusion protein disclosed herein to vWF, the vWF binding activity of each anti-vWF antibody listed in Tables 1-1 and 1-2 was evaluated using the ELISA system. Two constructs were used: human vWF-A2-WT (Open-type substrate) (amino acid sequence: SEQ ID NO: 192), prepared in Example 3(1), which mimics the Open and Close structures, and human vWF-A2-CC (Close-type substrate) (amino acid sequence: SEQ ID NO: 193), which forms a three-dimensional structure by disulfide bonds between the N-terminus and C-terminus.

[0225] Human vWF-A2-WT (Open-type substrate) and human vWF-A2-CC (Close-type substrate) were each diluted to 160 μM with PBS, and then added at a rate of 50 μL / well to a 96-well ELISA plate (F96 MAXISORP NUNC-IMMUNO PLATE, Thermo, #442404) and immobilized overnight at 4°C. After washing three times with 350 μL / well of PBS-T (Nacalai Tesque, #15675-04), 100 μL / well of 2 v / v% FBS / 1 v / v% BSA / PBS (2% blocking solution) was added, and the plates were blocked at 37°C for 1 hour.

[0226] After washing three times with PBS-T 350 μL / well, each anti-vWF antibody prepared in Example 1, or the culture supernatant of a hybridoma, was diluted with 2% blocking solution to final concentrations of 0.001, 0.01, 0.1, and 1.0 μg / mL. 50 μL / well of each antibody or hybridoma supernatant was added and allowed to stand at room temperature for 2 hours. After washing five times with PBS-T 350 μL / well, 50 μL / well of a secondary antibody (Anti-Human IgG (Fc) Goat IgG Fab' HRP, IBL, #17507) diluted 800-fold with 2% blocking solution was added and allowed to stand at room temperature for 2 hours.

[0227] After washing five times with PBS-T 350 μL / well, 50 μL / well of TMB colorant (Dako, #S1599) was added, and the mixture was allowed to stand at room temperature for approximately 3 minutes until sufficient color development was visible. After stopping the color development by adding 50 μL / well of 0.5 M sulfuric acid (Wako, #192-04755), the absorbance at 450 nm and 570 nm was measured using a plate reader (Ensight, PerkinElmer). The absorbance at 570 nm was subtracted from the absorbance at 450 nm, and the resulting absorbance was analyzed as the binding strength. The results are shown in Table 3-3.

[0228]

[0229] As shown in Table 3-3, the binding of each anti-vWF antibody used in this fusion protein to vWF-A2 in its open form was confirmed to be effective, as a binding strength of at least 0.2 was measured at a concentration of 0.1 μg / mL for all vWF antibody clones. On the other hand, for antibody clones A-3 and A-4, which are capable of binding to the PEP06 region located at the C-terminus of the vWF-A2 domain, a binding strength of less than 0.2 was observed for the closed form of vWF-A2 at a concentration of 0.1 μg / mL, indicating low binding activity. It is known that a portion of the C-terminal side of the PEP06 region forms an α-helix in the closed state (Blood. 2011;117 (17):4623-31), and it was speculated that the decrease in closed vWF-A2 binding activity in some antibody clones that recognize this portion may have been the cause. On the other hand, since the PEP05 region does not form a secondary structure even in the closed state, there was no difference between the open and closed states, and it was hypothesized that antibody clones capable of binding to the PEP05 region maintained their binding activity even in the closed state.

[0230] <Mouse Efficacy> We investigated how significantly differences there were in platelet count (efficacy) or residual ADAMTS13 enzyme activity (kinetics) after a certain period of time, depending on the characteristics of the antibody clone, such as the mode of binding to vWF. As test substances, we selected fusion proteins using three clones (molecular numbers A-2, 4, and 10): Vf1005, whose epitope is contained in the PEP06 region and can bind to Close vWF-A2; Vf1010, which has weak binding activity to Close vWF-A2; and Vf1040, whose epitope is contained in the PEP05 region and can bind to Close vWF-A2. The evaluation was carried out using the WT mouse TTP model described in Example 2(4), which exhibits an acquired TTP-like pathology and is induced by administration of anti-ADAMTS13 antibody and hvWF. Platelet count (pharmacological efficacy) and ADAMTS13 enzyme activity in plasma were evaluated 24 hours after administration. The results are shown in Tables 3-4A and 3-4B and 3-5A and 3-5B.

[0231] Similar to Example 2(4), two anti-ADAMTS13 antibodies (scFv_1_4_16_Fc, scFv_1_4_20_Fc) were mixed in equal amounts to a total of 30 mg / kg as autoantibodies that inhibit endogenous ADAMTS13 activity, and D-PBS(-) was administered to wild-type mice via tail vein at 5 mL / kg. Approximately 24 hours after administration of the anti-ADAMTS13 antibodies, each fusion protein was administered via tail vein at 1 mg / kg or 5 mg / kg, or acetate buffer at 5 mL / kg.

[0232] Subsequently, within 5 minutes, hvWF (500 U / kg) or glycine buffer was administered intravenously via the tail vein. Approximately 24 hours after administration of the test substance, the mice were laparotomed under isoflurane anesthesia, and 630 μL of peripheral blood was collected from the posterior vena cava using a syringe pre-added with 70 μL of sodium citrate buffer pH 5.5 (3.2% concentration). After transferring to a 1.5 mL tube and mixing by inversion, 170 μL was dispensed into an EDTA blood collection tube and used for platelet count measurement. The measurement was performed using a multi-parameter automated hematology analyzer XN-2000, and the mouse platelet count was calculated by correcting for dilution from the obtained platelet count.

[0233] The remaining blood was centrifuged (800g, 4°C, 10 minutes), and the ADAMTS13 activity was measured using the collected plasma. FRET buffer [5 mM Bis-Tris, 150 mM NaCl, 25 mM CaCl] ２ Plasma obtained at [0.005 v / v% Tween®-20, pH 7.2] was prepared to 15 v / v% to form a plasma sample, and hADAMTS13, prepared to 0.1-300 nmol / L in FRET buffer as a standard substance, was reacted with a FRET substrate (FRETS-vWF73), and the product concentration was calculated from the fluorescence value using the method described in Example 3(2). Furthermore, the ADAMTS13 enzyme activity of each fusion protein was calculated as activity equivalent to the hADAMTS13 concentration (nM) using the hADAMTS13 calibration curve. The calculated value (nM) represents the vWF degradation rate of a hADAMTS13 solution of the same concentration. The ADAMTS13 activity in mouse plasma from disease-induced and buffer-administered mice was used as the endogenous ADAMTS13 activity at the time of disease induction.

[0234] The summaries and results for each group are shown in Tables 3-4A and 3-5A. Three groups were prepared: a group receiving only the buffer (Buffer group) without administering either the ADAMTS13 antibody or hvWF; a group receiving only hvWF (hvWF group) without administering the anti-ADAMTS13 antibody; and a group receiving either the buffer or the test substance (two doses each of A-2, A-4, and A-10) when the disease state was induced. In addition, a separate study was conducted using the same procedure as above, administering 5 mg / kg of each test substance (A-2, A-3, and A-10), and the results are shown in Tables 3-4B and 3-5B.

[0235]

[0236]

[0237]

[0238]

[0239] As shown in Tables 3-4A and 3-4B, in the group administered with hvWF in addition to anti-ADAMTS13 antibody (disease induction group), the platelet count decreased to less than 10% compared to the buffer group, confirming the induction of acquired TTP-like disease. In addition, as shown in Tables 3-5A and 3-5B, a decrease in endogenous mouse ADAMTS13 activity was confirmed even 24 hours after disease induction. In the groups administered with fusion proteins (A-2 and A-10) containing Vf1005 and Vf1040, which can also bind to Close vWF-A2, at the time of disease induction, the decrease in platelet count was suppressed. On the other hand, in the groups administered with fusion proteins (A-3 and A-4) using Vf1009 and Vf1010, which have weak binding activity to Close vWF-A2, at the time of disease induction, the effect of suppressing platelet decrease was extremely small compared to the two clones mentioned above.

[0240] As shown in Tables 3-5A and 3-5B, serum enzyme activity (sauna ADAMTS13 enzyme activity), including endogenous mouse ADAMTS13 activity, was measured 24 hours after administration of the fusion protein using the FRET substrate (FRETS-vWF73). The results showed that A-3 and A-4 had higher serum enzyme activity compared to A-2. Although molecules that maintain enzyme activity in the blood 24 hours after administration existed for A-3 and A-4, which have weak binding activity to the A2 domain in the closed state, they had a low inhibitory effect on platelet count reduction. From this, it was considered that for the fusion protein to cleave full-length vWF in the circulating blood, it is important that it can bind to the A2 domain from the closed state before the structure of vWF opens due to shear stress.

[0241] Example 4 Preparation and performance evaluation of fusion proteins with different antibody formats (1) The above-mentioned fusion proteins, which are the test substances, were prepared by the method described in Example 3 (1). The schematic diagrams of the antibody formats of each fusion protein and the numbering of the peptide chains in each format are shown in Tables 4-1A and 4-1B. In the tables, the format name "mvG1" in C-21, C-18, C-28, and C-31 represents the antibody format disclosed as mvG1-2 in International Publication No. 2014 / 054804, and hereafter, "mvG1" in this specification refers to mvG1-2. Tables 4-1A and 4-1B show amino acid mutations that were further added based on mvG1-2.

[0242]

[0243]

[0244] (2) As an initial study of the ADAMTS13 enzyme activity constant region format of fusion proteins with different antibody formats, samples with different constant regions were prepared using the antibody clone Vf1005. The following formats were evaluated: a format with the M domain on only one side (C-1), a format with Fab on one side and the M domain on the other (C-2), a format with CH1 removed from IgG (C-3), a format with the M domain between Fab and the hinge (C-4), and an IgG1 type (A-2).

[0245] Based on the ELISA test system for detecting the vWF-A2 domain cleavage sequence described in Example 2(4), the ADAMTS13 enzyme activity was evaluated under conditions that would cause saturation if sufficient ADAMTS13 enzyme activity was present. To measure the ADAMTS13 enzyme activity using the full-length vWF-A2 domain as a substrate, an anti-RFP antibody was diluted to 10 μg / mL using PBS(-), added to a 96-well plate at 50 μL / well, and immobilized overnight at 4°C. The plate was washed three times with PBST, 200 μL / well of 2% FBS, 1% BSA PBS(-) was added, and the plate was blocked at 37°C for 2 hours, then washed three times with PBST. The vWF-A2-FRET described in Example 2(3) was diluted to 2 μg / mL with blocking buffer and added at 50 μL / well, and reacted at room temperature for 2 hours. The plate was washed three times with PBST, and 100 μL / well of each of the fusion proteins in different formats, diluted to 0.5 mg / mL with FRET Buffer, were added to each plate at 2 wells, and reacted overnight at room temperature.

[0246] The plate was washed three times with PBST, and 100 μL / well of HRP-labeled vWF cleavage site recognition antibody, diluted to 0.25 μg / mL with blocking buffer, was added and conjugated at room temperature for 1 hour. The plate was washed three times with PBST, 100 μL / well of TMB was added to induce color development, and then 100 μL / well of 0.5 M sulfuric acid was added to stop the color reaction. Absorbance measurements were taken at 450 nm / 570 nm using a plate reader, and the average value of 2 wells was taken. The results are shown in Table 4-2.

[0247]

[0248] As shown in Table 4-2, ADAMTS13 enzyme activity was observed in all evaluated formats. However, ADAMTS13 enzyme activity was partial for C-2 and C-4.

[0249] For monovalent formats with only one M domain in the molecule, further investigations were conducted using the antibody clone Vf1005. Formats in which the M domain was fused only to Fab (C-5 to C-7), mvG1 format (C-8 to C-10), and format in which the M domain was fused only to scFv (C-11 to C-13) were investigated.

[0250] Furthermore, we investigated the fusion site of the M domain, considering whether it was on the heavy chain side (C-5, C-8, C-12, C-13), the light chain side (C-6, 7, 9, 10, 11), the N-terminal side (C-5, 6, 8, 9, 13), or the C-terminal side (C-7, 10, 11, 12).

[0251] The results of measuring the specific activity using the method described in Example 3(2) are shown in Table 4-3.

[0252]

[0253] As shown in Table 4-3, the fusion site of the M domain tended to be higher when it was at the N-terminus of the heavy chain, but ADAMTS13 enzyme activity was observed in all of these formats.

[0254] For heterogeneous monovalent formats, Knobs-into-holes mutations (KIH; Y349C, T366S, L368A, Y407V) were introduced to suppress light-light-light and heavy-heavy-heavy chain expression, and further studies were conducted using the antibody clone Vf1040. Formats in which the light chain side is divided into an Fc region and a Fab region (C-14, 15), a format with an M domain on only one side (C-16), a format with a Fab region on one side and an M domain on the other (C-17), and the mvG1 format (C-18) were investigated.

[0255] For these fusion proteins, the ADAMTS13 enzyme activity was measured using FRETS-vWF73 (Peptide Institute, #3224-s) as a substrate, similar to the specific activity measurement. FRETS-vWF73 was dissolved to 100 μM in 25 v / v% DMSO distilled water, and an 8 μM dilution was prepared in FRET buffer. Each of the test fusion proteins was prepared to 20 nM. These sample solutions were mixed with the substrate solution in a 1:1 ratio, so the final concentrations were halved. To calculate the absolute amount of product from the fluorescence intensity, a calibration curve was created using the small molecules FRETS-25-STD1 (Peptide Institute, #3720-v) and FRETS-25-STD2 (Peptide Institute, #3720-v), which possess the fluorescent group and quenching group bound to FRETS-vWF73, respectively.

[0256] Stock solutions of FRETS-25-STD1 and FRETS-25-STD2 were prepared in DMSO at a concentration of 1 mM. Equal volumes were then mixed, and a 2x common ratio dilution series was prepared in FRET buffer to range from 32 μM to 0.25 μM. In addition, as a comparison of the enzyme activity of ADAMTS13, a dilution series of 0.1, 0.3, 1, 3, 10, 30, 100, and 300 nM of hADAMTS13 was prepared in FRET buffer. Mixing was performed using a plate shaker (TITRAMAX1000, Heidolph) by stirring at 1350 rpm for 30 seconds. Immediately after mixing, fluorescence at 450 nm was measured every 5 minutes for 60 minutes using a plate reader (Ensight, PerkinElmer) with excitation light at 340 nm. The results were calculated by converting the substrate degradation rate to the concentration (nM) of hADAMTS13. The results are shown in Table 4-4.

[0257]

[0258] As shown in Table 4-4, ADAMTS13 enzyme activity was observed in all of these formats.

[0259] To reduce unnecessary effector activity in the mechanism of action of fusion proteins, we compared and examined two formats: one with LALAGA mutations (L234A, L235A, G237A) added to the Fc region of human IgG1 (hereinafter referred to as LALAGA type; C-19, 22, 24, 26, 29), and another with mutations (hereinafter referred to as IgG4PEK type; C-20, 23, 25, 27, 30) introduced to the Fc region of human IgG4 (S228P for half-antibody production suppression, L235E for reduced FcgRs binding, and R409K for improved thermal stability), against the mvG1 format (C-18, 21, 28, 31) containing LALAGA and KIH mutations. The antibody clones used were Vf1005, Vf1038, Vf1040, Vf0138, and Vf0179.

[0260] The ADAMTS13 enzyme activity of each fusion protein was measured in terms of its concentration (nM) adjusted to a final concentration of 15 nM using the method described above. The results are shown in Table 4-5.

[0261]

[0262] As shown in Table 4-5, when fusion proteins with mutations that reduce effector activity were compared with IgG1 type (A-2, 8 and 10, and B-28 and 29), the LALAGA type and IgG4PEK type were comparable to the IgG1 type, and there were no significant changes.

[0263] Example 5 Preparation and performance evaluation of fusion proteins with different linkers (1) As test materials, the fusion proteins shown in Tables 5-1A to 5-1C were prepared by the method described in Example 3 (1).

[0264]

[0265]

[0266]

[0267] (2) The ADAMTS13 enzyme activity linker sequence of fusion proteins with different linkers is a sequence that connects the C-terminus of the M domain and the N-terminus of the antibody variable region, and is related to the binding site with vWF and the position with respect to the M domain. When a fusion protein is formed, antibody clones that show vWF cleavage activity include clones that can bind to the PEP05 region located near the C-terminus of the vWF A2 domain cleavage site, and antibody clones that can bind to the PEP06 region sequence located further towards the C-terminus. Therefore, in order to examine the length of the linker sequence for each bindable region, Vf1040 and Vf0138 were selected as antibody clones that can bind to the PEP05 region, and Vf1005 and Vf1010 were selected as antibody clones that can bind to the PEP06 region. In parallel with the format study described in Example 4, the change in ADAMTS13 enzyme activity was evaluated by changing the length of the IgG1 type linker sequence.

[0268] In addition, since the molecular motion of the M domain is thought to affect the ADAMTS13 enzyme activity, we also investigated fusion proteins with a relatively rigid PAPAP repeat sequence (PA linker) (D-3, 4, 5, 12-14, 23-24, 37, 38) and fusion proteins with an EAAAK repeat sequence (EK linker) that forms an α-helix within a single unit (D-6-8, 15, 16, 25-29, 39, 40), in addition to the currently used flexible GGGGS repeat sequence (GS linker) fusion proteins.

[0269] The results of measuring the specific activity using the method described in Example 3 (2) are shown in Table 5-2.

[0270]

[0271] As shown in Table 5-2, ADAMTS13 enzyme activity was observed in all of these antibody clone and linker combinations. Furthermore, the specific activity of linker sequences modified using Vf1005 (A-2, D-1 to 8) and Vf1010 (A-4, D-11 to 16), which can bind to the PEP06 region, tended to improve when using shorter linkers with a peak at 1 unit, and rigid PA and EK linkers.

[0272] On the other hand, when the specific activity of linker sequence modified products using Vf1040, which can bind to the PEP05 region (A-10, D-17 to 29), was evaluated, the trend of improved specific activity with the use of rigid linker sequences (PA linker, EK linker) was the same, but longer linkers with a peak of 4 units showed higher specific activity. Similarly, the ADAMTS13 enzyme activity of linker sequence modified products using Vf0138, which can bind to the PEP05 region (B-28, D-39 to 42) showed a similar trend.

[0273] Next, to confirm the effects of changing the format, Vf1040 was used as the antibody clone for the LALAGA type (D-30 to 32) and IgG4PEK type (D-33 to 35) examined in Example 4, and Vf1040 (D-36 to 38) and Vf0138 (D-43 to 47) were used as the antibody clone for the mvG1 type format, and the fusion proteins with modified linker sequences were evaluated.

[0274] The ADAMTS13 enzyme activity was measured in terms of the concentration (nM) of the fusion protein hADAMTS13 adjusted to a final concentration of 15 nM using the method described in Example 4(2). The results are shown in Table 5-3.

[0275]

[0276] As shown in Table 5-3, ADAMTS13 enzyme activity was observed in all combinations, even when the format was changed in addition to the linker. Furthermore, as in the study with IgG1 type, an improvement in activity was observed by changing to a rigid linker sequence.

[0277] Example 6 Preparation and performance evaluation of a fusion protein with a modified M domain (1) The above fusion protein, which is the test substance, was prepared by the method described in Example 3 (1). In Tables 6-1A to 6-1E, CHLSB is amino acids 73-284 of ADAMTS13 (UniProt access number A0A0D9RRZ0) derived from Chlorocebus sabaeus, RHIBE is amino acids 86-297 of ADAMTS13 (UniProt access number A0AAJ7DF33) derived from Rhinopitecus biti, AOTNA is amino acids 79-290 of ADAMTS13 (UniProt access number A0A2K5E765) derived from Aotus nancymaae, and DOG is Canis lupus RAT represents the amino acid sequence from 225 to 437 of ADAMTS13 (UniPro access number A0A8P0T9G5) derived from familiaris, and RAT represents the amino acid sequence from 77 to 291 of ADAMTS13 (UniPro access number D4A0T9) derived from Rattas norvegicus.

[0278]

[0279]

[0280]

[0281]

[0282]

[0283] (2) Modified M-domain fusion protein ADAMTS13 enzyme activity We investigated the improvement of ADAMTS13 enzyme activity by modifying the amino acid sequence of the M-domain derived from ADAMTS13 used in the fusion protein. Based on the fusion proteins used in Example 2 (A-2, A-4, A-10, D-37; antibody clones Vf1005, Vf1010 or Vf1040. IgG1 type or mvG1 type. GS linker 4 units or PA linker 4 units), we investigated the modification of the M-domain.

[0284] The ADAMTS13 enzyme activity of the fusion protein was measured in nM equivalent to the final concentration of 10 nM (Tables 6-2, 6-3) or 15 nM (Tables 6-4, 6-5, 6-6, 6-7) using the method described in Example 4(2). The results are shown in Tables 6-2 to 6-7.

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291] As shown in Tables 6-2 to 6-7, ADAMTS13 enzyme activity was observed in all modified compounds, regardless of antibody clone, linker sequence, or format. Furthermore, it was confirmed that the fusion protein possessed ADAMTS13 enzyme activity even when the M-domain sequence was derived from a non-human mammal, such as E-48 to E-52. The amino acid sequences of the M-domains of E-48 to E-52 (SEQ ID NOs. 48 to 52) have approximately 80-99% homology to the amino acid sequence of the M-domain of human-derived ADAMTS13, such as A-4 (SEQ ID NOs. 99). These test results also indicate that the M-domain may contain amino acid mutations. Additionally, activity was demonstrated even with 80% homology to the human M-domain sequence.

[0292] Example 7 In vivo test results (1) Evaluation of drug efficacy in a congenital TTP-like mouse model using KO mice Platelet count was evaluated using a congenital TTP-like disease model associated with decreased ADAMTS13 enzyme activity in ADAMTS13 gene-deficient mice (ADAMTS13 KO mice) using an IgG1 format fusion protein (A-2). For the evaluation, an animal model of congenital TTP-like disease using ADAMTS13 KO C57BL / 6CrSlc[Homo] (Banno et al., Blood 107(8), 3161-3166 (2006)) was used. After acclimatization for more than one week, the mice were divided into groups of three based on their body weight on the group division day, so that the average body weight of each group was approximately the same. Each fusion protein was administered intravenously at a dose of 1 mg / kg or 5 mg / kg, or at a dose of 5 mL / kg of acetate buffer (50 mM sodium acetate, 250 mM D-sorbitol, 50 mM L-arginine, 7.15 μg / mL ZnSO4, pH 5.0) via tail vein.

[0293] Subsequently, within 5 minutes, hvWF (400 U / kg) or glycine buffer (1.13 mg / mL glycine, 20 mg / mL D-mannitol, 10 mg / mL trehalose hydrate, 0.1 mg / mL polysorbate 80, pH 7.35) was administered intravenously via tail vein. Approximately 24 hours after administration of the test substance, the mice were anesthetized with isoflurane (Muromachi Instruments, Small Animal Anesthesizer MKA110, flow rate 2.5 L / min, concentration 2.5%), and 630 μL of peripheral blood was collected from the posterior vena cava using a syringe pre-added with 70 μL of sodium citrate buffer pH 5.5 (concentration 3.2%). The solution was transferred to a 1.5 mL tube (Eppendorf, Safe-Lock Tube), mixed by inversion, and then 170 μL was dispensed into a blood collection tube (BD MicroTina micro-blood collection tube / EDTA 2K) and used for platelet count measurement.

[0294] The measurement involved calculating the mouse platelet count by converting the platelet count obtained using a multi-parameter automated blood cell analyzer XN-2000 (Sysmex) to the value before dilution. A summary of each group and its results are shown in Table 7-1. Three groups were prepared: a group administered only buffer without hvWF (Buffer group), a group administered hvWF and buffer (Pathogenesis-inducing group), and a group administered A-2 (3 doses) or hADAMTS13 (2 doses) before hvWF administration. The results are shown in Table 7-1.

[0295]

[0296] As shown in Table 7-1, a decrease in platelet count was observed in the disease induction group, but in the group administered A-2 at the time of disease induction, the decrease in platelet count was suppressed, similar to the hADAMTS13 administration group. From these results, the fusion protein is expected to have a therapeutic effect on congenital TTP patients who are genetically deficient in ADAMTS13 activity.

[0297] In Example 5, improvement in ADAMTS13 enzyme activity was observed by modifying the linker sequence. To confirm whether this contributes to the suppression of platelet count decline, an evaluation was performed using an ADAMTS13 KO mouse model resembling congenital TTP. Fusion proteins with modified linker sequences (A-10 or D-17, 23, or 28) were administered at a dose of 1 mg / kg, and platelet count (pharmacological efficacy) and plasma ADAMTS13 enzyme activity were evaluated 24 hours later. The results are shown in Table 7-2.

[0298]

[0299] As shown in Table 7-2, there was a certain correlation between the intensity of ADAMTS13 enzyme activity in the in vitro test in Example 5(2) and the in vivo test results in this mouse model. Specifically, in the in vitro activity test of Example 5(2), when comparing fusion proteins using Vf1040 as the antibody clone, D-17, which used one unit of a shorter linker, showed weaker ADAMTS13 enzyme activity compared to A-2, while D-23 and D-28, which had their sequences modified to PAPAP or EAAAK, tended to show stronger ADAMTS13 enzyme activity. A similar trend was observed in this mouse model test in terms of the inhibitory effect on platelet count reduction and the residual ADAMTS13 enzyme activity in the blood. From these results, it was confirmed that the strength of ADAMTS13 enzyme activity determined by the linker sequence, as confirmed in in vitro evaluation, is reflected in the pharmacological efficacy of the fusion protein.

[0300] In Example 6, the fusion protein in which improved ADAMTS13 enzyme activity was observed due to modification of the M domain was evaluated for its effect in suppressing platelet count decline using an acquired TTP-like mouse model. First, the L271A, D165C_D168C, and R278A modified forms (E-31, 32, 34) of the IgG type basic format were compared with the wild type (A-10). Each was administered at a dose of 1 mg / kg, and the platelet count (pharmacological effect) and the ADAMTS13 enzyme activity in the plasma at that time were evaluated. The results are shown in Table 7-3.

[0301]

[0302] As shown in Table 7-3, there was no decrease in platelet count from the wild type (A-10), and the 24-hour duration of blood enzyme activity showed more than three times improvement from the wild type (A-10) in L271A and R278A (E-31, 34), which also showed improved ADAMTS13 enzyme activity in vitro in Example 6(2). These results confirm that the changes in ADAMTS13 enzyme activity due to M-domain modification, as confirmed in in vitro evaluation, are also reflected in vivo.

[0303] (2) The efficacy of the fusion protein and the persistence of blood enzyme activity: Because the ADAMTS13 enzyme activity and efficacy change depending on the region containing the acquired TTP-like mouse model epitope and the binding activity with Open / Close vWF-A2, the efficacy of fusion proteins with modified antibody clones (A-1 to 2 or 5 to 10 or B-2, 4, 8, 15, 17, 20, 21, 23, 25 or 27 to 29) was evaluated by platelet count when the anti-ADAMTS13 antibody dose to the acquired TTP-like mouse model described in Example 3 (3) was changed to 30 mg / kg and the fusion protein was administered at 5 mg / kg, and compared with the buffer administration group. In addition, blood ADAMTS13 activity was measured 24 hours after administration using plasma collected from the remaining blood.

[0304] FRET Buffer [5 mM Bis-Tris, 150 mM NaCl, 25 mM CaCl] ２ Using plasma obtained at [0.005 v / v% Tween®-20, pH 7.2], plasma samples were prepared to 15 v / v%, and hADAMTS13 prepared to 0.1-300 nmol / L in FRET buffer as a standard substance. Using the same method as described in Example 4(2), blood enzyme activity was calculated as activity equivalent to the hADAMTS13 concentration (nM). Furthermore, the ADAMTS13 activity in the blood of mice in the disease-free group (buffer administration group) was considered as the endogenous ADAMTS13 activity of mice, and the difference was taken from the measured values ​​to determine the blood enzyme activity derived from the fusion protein. The results are shown in Tables 7-4 to 7-6.

[0305]

[0306]

[0307]

[0308] As shown in Tables 7-4 to 7-6, suppression of platelet count reduction was confirmed in all fusion proteins with modified antibody clones (A-1, A-2 and 5-10, and B-2, 4, 8, 15, 17, 20, 21, 23, 25, and 27-29). These antibody clones were able to bind to Close vWF-A2, confirming that the strength of binding affinity to Close vWF-A2 in Example 3(3) affects the drug efficacy.

[0309] The ADAMTS13 enzyme activity remaining in the blood 24 hours after administration differed among antibody clones. Of the fusion proteins evaluated (A-1, A-2, 5-10, B-2, 4, 8, 15, 17, 20, 21, 23, 25, 27-29), clones A-10, B-28, and B-29, which showed high plasma enzyme activity after 24 hours, were antibody clones with the PEP05 epitope.

[0310] Next, for the eight clones that showed high efficacy in the aforementioned tests (A-2, 7, 8, and 10, and B-4 and 27-29), the platelet count 24 hours after administration of the fusion protein at a lower dose was evaluated in the aforementioned acquired TTP-like mouse model at a dose of 1 mg / kg. The results are shown in Table 7-7.

[0311]

[0312] As shown in Table 7-7, by reducing the dosage to 1 mg / kg, the efficacy of A-8 and B-27, which had almost completely suppressed platelet reduction at a 5 mg / kg dose, became partial, and antibody clones with more potent efficacy were identified.

[0313] To confirm the effect of the modified format in Example 4 on drug efficacy, an evaluation was performed using the acquired TTP-like mouse model used in Example 3(3). Platelet count (drug efficacy) 24 hours after administration of 1 mg / kg of fusion protein (A-10, C-18~22, 24~27, 29) and the ADAMTS13 enzyme activity in plasma at that time were evaluated to compare these with the initial elimination. The results are shown in Table 7-8.

[0314]

[0315] As shown in Table 7-8, in the groups administered with LALAGA-type and IgG4PEK-type fusion proteins (C-19, 20, 22, 24-27, and 29), the decrease in platelet count was suppressed by more than 90% with a 1 mg / kg dose for all antibody clones. On the other hand, the effect of suppressing the decrease in platelet count with mvG1-type fusion proteins (C-18 and 21) was partial, and in particular, with C-21 using the Vf1005 antibody clone, the platelet count was approximately 65% ​​of the normal level. Meanwhile, blood enzyme activity after 24 hours was higher with mvG1-type proteins (C-18 and 21).

[0316] To confirm the maximum efficacy of the mvG1 type with a monovalent M domain, the maximum efficacy of the fusion proteins (D-30, 36, and 43) was confirmed using the aforementioned acquired TTP-like mouse model, with doses ranging from 0.3, 1.0, and 3.0 mg / kg. The results are shown in Table 7-9.

[0317]

[0318] As shown in Table 7-9, even in mvG1 type, increasing the dosage to 1-3 mg / kg completely suppressed the decrease in platelet count.

[0319] In addition, to confirm that platelet count reduction can be similarly suppressed even with mvG1 type and enzyme modification, the efficacy of the fusion protein (E-87, 96) was confirmed using the aforementioned acquired TTP-like mouse model, with dosages of 0.1, 0.3, and 1.0 mg / kg. The results are shown in Table 7-10.

[0320]

[0321] As shown in Table 7-10, even with the mvG1 type and enzyme modification, a dose of 1.0 mg / kg completely suppressed the decrease in platelet count.

[0322] As shown in Table 7-10, improvement in the persistence of enzyme activity in the blood is expected due to M-domain modification. Therefore, the persistence of enzyme activity in the blood was evaluated in more detail for the various modified compounds evaluated in Example 6. Using the same ADAMTS13 KO mice as in Example 7(1), the residual ADAMTS13 enzyme activity of the fusion protein was measured under conditions where there was no endogenous ADAMTS13 activity in the mouse. Each fusion protein (A-10, D-37, E-87, 91, 93, 96, 97) was administered to ADAMTS13 KO mice at 3 mg / kg, and the ADAMTS13 enzyme activity in the plasma 72 hours and 168 hours after administration was calculated as activity equivalent to the hADAMTS13 concentration (nM) using the same method as described in Example 4(2). The results are shown in Table 7-11.

[0323]

[0324] As shown in Table 7-11, the fusion proteins with amino acid modifications (E-87, 91, 93, 96, and 97) showed improved sustained enzyme activity in the blood compared to the unmodified wild-type fusion proteins (A-10, D-37).

[0325] Example 8 Preparation of a fusion compound with an additional Dis domain (1) Preparation Since it has been reported that ADAMTS13 undergoes allosteric changes by the binding of the D domain adjacent to the M domain to the A2 domain of vWF (Nat Commun. 2019 Aug 22;10(1):3781.), a fusion protein was designed in which the full amino acid sequence of the D domain (SEQ ID NO: 932), or a part of the sequence, was added to the C-terminal side of the M domain. The fusion protein is shown in Table 8-1. The fusion protein, which is the test substance, was prepared by the method described in Example 3(1).

[0326] In Table 8-1, the enzyme domain in F-1 is an amino acid sequence consisting of an M domain and a D domain, and is also referred to as the MD domain in this specification (SEQ ID NO: 936). The enzyme domain in F-2 consists of an M domain (SEQ ID NO: 931) and a part of the D domain, and the part of the D domain consists of amino acids 287 to 310 in the amino acid sequence represented by SEQ ID NO: 194 in human ADAMTS13 (UniProt access number Q76LX8-1). The enzyme domain in F-3 consists of an M domain (SEQ ID NO: 931) and a part of the D domain, and the part of the D domain consists of amino acids 287 to 315 in human ADAMTS13 (UniProt access number Q76LX8-1), with Cys at position 311 being replaced with Ala. The enzyme domain in F-4 consists of the M domain (SEQ ID NO: 931) and a portion of the D domain, the portion of which consists of amino acids 287 to 373 in human ADAMTS13 (UniProt access number Q76LX8-1).

[0327]

[0328] (2) Enzyme activity The ADAMTS13 enzyme activity of each fusion protein was measured by converting it to the concentration (nM) of hADAMTS13 adjusted to a final concentration of 10 nM using the method described in Example 4 (2). The results are shown in Tables 8-2 and 8-3.

[0329]

[0330]

[0331] As shown in Tables 8-2 and 8-3, fusion proteins F-1 to F-4, each containing the full-length or partial D-domain, exhibited ADAMTS13 enzyme activity equivalent to or greater than that of enzyme fusion A-2, which contained only the M-domain. This result indicates that ADAMTS13 enzyme activity can be obtained even when using an enzyme domain in which the D-domain is added to the M-domain in the fusion protein lacking the Spacer domain of the present invention. In particular, an increase in ADAMTS13 enzyme activity was confirmed in fusion proteins fused with the full-length D-domain (F-1) and a fragment close to the full-length (F-4).

[0332] Since many patients with acquired TTP possess autoantibodies against the Spacer domain of ADAMTS13, which is known to be the cause of decreased ADAMTS13 enzyme activity in ADAMTS13 replacement therapy, it is suggested that the fusion protein of this disclosure, which lacks the Spacer domain, maintains ADAMTS13 enzyme activity while being unaffected by inhibition by autoantibodies.

[0333] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications are possible without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2025-024453, filed on 18 February 2025, which is incorporated herein by reference in its entirety.

[0334]

Claims

1. A fusion protein comprising a mutant ADAMTS13 containing at least a metalloproteinase domain (M domain) and lacking a spacer domain (S domain), and an antigen-binding molecule that binds to von Willebrand factor (vWF).

2. The fusion protein according to claim 1, wherein the mutant ADAMTS13 is (N1) or (N2) as follows: (N1) In addition to the S domain of ADAMTS13, the T2-T8 domain is further missing. (N2) In addition to the S domain of ADAMTS13, the T2-T8 domain and the CUB1-2 domain are further missing.

3. The fusion protein according to claim 1, wherein the mutant ADAMTS13 is (D1) or (D2) below: (D1) consisting of the M domain of ADAMTS13; (D2) consisting of the M domain and the disintegrin-like domain (D domain) of ADAMTS13.

4. The fusion protein according to claim 1, wherein the M domain in the mutant ADAMTS13 contains an amino acid sequence having 70% or more homology to the amino acid sequence from position 80 to 286 of the amino acid sequence represented by Sequence ID No. 194, and the fusion protein has ADAMTS13 enzyme activity.

5. The fusion protein according to claim 1, wherein the amino acid sequence of the M domain in the mutant ADAMTS13 includes an amino acid sequence corresponding to the amino acid sequence from position 80 to 286 of the amino acid sequence represented by SEQ ID NO: 194, and further includes at least one of the substitutions (M1) to (M13) below. (M1) Substitution of the amino acid residue corresponding to the 159th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to R (M2) Substitution of the amino acid residue corresponding to the 167th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to M (M3) Substitution of the amino acid residue corresponding to the 207th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to N (M4) Substitution of the amino acid residue corresponding to the 209th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to V (M5) Substitution of the amino acid residue corresponding to the 230th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to L (M6) Substitution of the amino acid residue corresponding to the 238th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to A (M7) Substitution of the amino acid residue corresponding to the 243rd amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to K (M8) Substitution of the amino acid residue corresponding to the 271st amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to A (M9) Substitution of the amino acid residue corresponding to the 271st amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 to E (M10) Substitution of L from the amino acid residue corresponding to the 279th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M11) Substitution of L from the amino acid residue corresponding to the 282nd amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M12) Substitution of G from the amino acid residue corresponding to the 283rd amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 (M13) Substitution of L from the amino acid residue corresponding to the 285th amino acid residue in the amino acid sequence represented by SEQ ID NO: 194 6. The fusion protein according to claim 5, wherein the amino acid sequence of the mutant ADAMTS13 is any one of the following: (m1) including the substitutions of (M1) and (M2); (m2) including the substitutions of (M1) and (M3); (m3) including the substitutions of (M8) and (M13); (m4) including the substitutions of (M9) and (M13); (m5) including the substitutions of (M1), (M8) and (M13); (m6) including the substitutions of (M1), (M9) and (M13); (m7) including the substitutions of (M4), (M5), (M8) and (M13); (m8) including the substitutions of (M4), (M6), (M8) and (M13); (m9) including the substitutions of (M4), (M7), (M8) and (M13). (m10) Includes substitution of (M5), (M6), (M8) and (M13). (m11) Includes substitution of (M5), (M7), (M8) and (M13). (m12) Includes substitution of (M6), (M7), (M8) and (M13). (m13) Includes substitution of (M10), (M11) and (M13). (m14) Includes substitution of (M10), (M12) and (M13). (m15) Includes substitution of (M11), (M12) and (M13). (m16) Includes substitution of (M1), (M10), (M11) and (M13). (m17) Includes substitution of (M1), (M10), (M12) and (M13). (m18) Includes substitution of (M1), (M11), (M12) and (M13) above.

7. The fusion protein according to claim 1, wherein the mutant ADAMTS13 consists of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 99.

8. The fusion protein according to claim 1, wherein the antigen-binding molecule comprises at least the heavy chain variable region (VH) and the light chain variable region (VL) of an anti-vWF antibody.

9. The fusion protein according to claim 8, wherein the antigen-binding molecule is such that the VH and VL are any one of the following (c1) to (c37): (c1) The VH comprises Complementarity Determinating Regions (CDRs) 1 to 3 each containing an amino acid sequence represented by SEQ ID NOs. 565 to 567, and the VL comprises CDRs 1 to 3 each containing an amino acid sequence represented by SEQ ID NOs. 568 to 570. (c2) The VH comprises CDRs 1 to 3 each containing an amino acid sequence represented by SEQ ID NOs. 573 to 575, and the VL comprises CDRs 1 to 3 each containing an amino acid sequence represented by SEQ ID NOs. 576 to 578. (c3) The VH comprises CDRs 1 to 3 each containing an amino acid sequence represented by SEQ ID NOs. 581 to 583, and the VL comprises CDRs 1 to 3 each containing an amino acid sequence represented by SEQ ID NOs. 584 to 586. (c4) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 587 to 589, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 590 to 592, respectively. (c5) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 595 to 597, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 598 to 600, respectively. (c6) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 603 to 605, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 606 to 608, respectively. (c7) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 611 to 613, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 614 to 616, respectively. (c8) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 619 to 621, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 622 to 624. (c9) The VH comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 627 to 629, and the VL comprises CDR1 to CDR3 each containing the amino acid sequence represented by SEQ ID NOs. 630 to 632.(c10) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 635 to 637, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 638 to 640, respectively. (c11) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 643 to 645, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 646 to 648, respectively. (c12) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 651 to 653, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 654 to 656, respectively. (c13) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 659 to 661, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 662 to 664, respectively. (c14) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 667 to 669, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 670 to 672, respectively. (c15) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 675 to 677, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 678 to 680, respectively. (c16) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 683 to 685, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 686 to 688, respectively. (c17) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 691 to 693, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 694 to 696, respectively. (c18) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 699 to 701, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 702 to 704. (c19) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 707 to 709, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 710 to 712.(c20) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 715 to 717, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 718 to 720, respectively. (c21) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 723 to 725, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 726 to 728, respectively. (c22) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 731 to 733, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 734 to 736, respectively. (c23) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 795 to 797, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 798 to 800, respectively. (c24) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 803 to 805, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 806 to 808, respectively. (c25) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 811 to 813, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 814 to 816, respectively. (c26) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 819 to 821, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 822 to 824, respectively. (c27) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 827 to 829, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 830 to 832, respectively. (c28) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 835 to 837, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 838 to 840. (c29) The VH comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 843 to 845, and the VL comprises CDR1 to CDR3 each containing the amino acid sequences represented by SEQ ID NOs. 846 to 848.(c30) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 851 to 853, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 854 to 856, respectively. (c31) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 859 to 861, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 862 to 864, respectively. (c32) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 867 to 869, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 870 to 872, respectively. (c33) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 875 to 877, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 878 to 880, respectively. (c34) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 883 to 885, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 886 to 888, respectively. (c35) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 891 to 893, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 894 to 896, respectively. (c36) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 899 to 901, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 902 to 904, respectively. (c37) The VH comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 907 to 909, respectively, and the VL comprises CDR1 to CDR3 containing amino acid sequences represented by SEQ ID NOs. 910 to 912, respectively.

10. The fusion protein according to claim 9, wherein the antigen-binding molecule is such that the VH and VL are any one of the following (v1) to (v37): (v1) The VH comprises an amino acid sequence represented by SEQ ID NO: 563, and the VL comprises an amino acid sequence represented by SEQ ID NO:

564. (v2) The VH comprises an amino acid sequence represented by SEQ ID NO: 571, and the VL comprises an amino acid sequence represented by SEQ ID NO:

572. (v3) The VH comprises an amino acid sequence represented by SEQ ID NO: 579, and the VL comprises an amino acid sequence represented by SEQ ID NO:

580. (v4) The VH comprises an amino acid sequence represented by SEQ ID NO: 100, and the VL comprises an amino acid sequence represented by SEQ ID NO:

101. (v5) The VH comprises an amino acid sequence represented by SEQ ID NO: 593, and the VL comprises an amino acid sequence represented by SEQ ID NO:

594. (v6) The VH comprises an amino acid sequence represented by SEQ ID NO: 601, and the VL comprises an amino acid sequence represented by SEQ ID NO:

602. (v7) The VH includes the amino acid sequence represented by SEQ ID NO: 609, and the VL includes the amino acid sequence represented by SEQ ID NO:

610. (v8) The VH includes the amino acid sequence represented by SEQ ID NO: 617, and the VL includes the amino acid sequence represented by SEQ ID NO:

618. (v9) The VH includes the amino acid sequence represented by SEQ ID NO: 625, and the VL includes the amino acid sequence represented by SEQ ID NO:

626. (v10) The VH includes the amino acid sequence represented by SEQ ID NO: 633, and the VL includes the amino acid sequence represented by SEQ ID NO:

634. (v11) The VH includes the amino acid sequence represented by SEQ ID NO: 641, and the VL includes the amino acid sequence represented by SEQ ID NO:

642. (v12) The VH includes the amino acid sequence represented by SEQ ID NO: 649, and the VL includes the amino acid sequence represented by SEQ ID NO:

650. (v13) The VH includes the amino acid sequence represented by SEQ ID NO: 657, and the VL includes the amino acid sequence represented by SEQ ID NO:

658. (v14) The VH includes the amino acid sequence represented by SEQ ID NO: 665, and the VL includes the amino acid sequence represented by SEQ ID NO:

666. (v15) The VH includes the amino acid sequence represented by SEQ ID NO: 673, and the VL includes the amino acid sequence represented by SEQ ID NO: 674.(v16) The VH includes the amino acid sequence represented by SEQ ID NO: 681, and the VL includes the amino acid sequence represented by SEQ ID NO:

682. (v17) The VH includes the amino acid sequence represented by SEQ ID NO: 689, and the VL includes the amino acid sequence represented by SEQ ID NO:

690. (v18) The VH includes the amino acid sequence represented by SEQ ID NO: 697, and the VL includes the amino acid sequence represented by SEQ ID NO:

698. (v19) The VH includes the amino acid sequence represented by SEQ ID NO: 705, and the VL includes the amino acid sequence represented by SEQ ID NO:

706. (v20) The VH includes the amino acid sequence represented by SEQ ID NO: 713, and the VL includes the amino acid sequence represented by SEQ ID NO:

714. (v21) The VH includes the amino acid sequence represented by SEQ ID NO: 721, and the VL includes the amino acid sequence represented by SEQ ID NO:

722. (v22) The VH includes the amino acid sequence represented by SEQ ID NO: 729, and the VL includes the amino acid sequence represented by SEQ ID NO:

730. (v23) The VH includes the amino acid sequence represented by SEQ ID NO: 793, and the VL includes the amino acid sequence represented by SEQ ID NO:

794. (v24) The VH includes the amino acid sequence represented by SEQ ID NO: 801, and the VL includes the amino acid sequence represented by SEQ ID NO:

802. (v25) The VH includes the amino acid sequence represented by SEQ ID NO: 809, and the VL includes the amino acid sequence represented by SEQ ID NO:

810. (v26) The VH includes the amino acid sequence represented by SEQ ID NO: 817, and the VL includes the amino acid sequence represented by SEQ ID NO:

818. (v27) The VH includes the amino acid sequence represented by SEQ ID NO: 825, and the VL includes the amino acid sequence represented by SEQ ID NO:

826. (v28) The VH includes the amino acid sequence represented by SEQ ID NO: 833, and the VL includes the amino acid sequence represented by SEQ ID NO:

834. (v29) The VH includes the amino acid sequence represented by SEQ ID NO: 841, and the VL includes the amino acid sequence represented by SEQ ID NO:

842. (v30) The VH includes the amino acid sequence represented by SEQ ID NO: 849, and the VL includes the amino acid sequence represented by SEQ ID NO:

850. (v31) The VH includes the amino acid sequence represented by SEQ ID NO: 857, and the VL includes the amino acid sequence represented by SEQ ID NO: 858.(v32) The VH includes the amino acid sequence represented by SEQ ID NO: 865, and the VL includes the amino acid sequence represented by SEQ ID NO:

866. (v33) The VH includes the amino acid sequence represented by SEQ ID NO: 873, and the VL includes the amino acid sequence represented by SEQ ID NO:

874. (v34) The VH includes the amino acid sequence represented by SEQ ID NO: 881, and the VL includes the amino acid sequence represented by SEQ ID NO:

882. (v35) The VH includes the amino acid sequence represented by SEQ ID NO: 889, and the VL includes the amino acid sequence represented by SEQ ID NO:

890. (v36) The VH includes the amino acid sequence represented by SEQ ID NO: 897, and the VL includes the amino acid sequence represented by SEQ ID NO:

898. (v37) The VH includes the amino acid sequence represented by SEQ ID NO: 905, and the VL includes the amino acid sequence represented by SEQ ID NO:

906.

11. The fusion protein according to claim 1, wherein the antigen-binding molecule is an antigen-binding molecule comprising any one of (B1) to (B4) below. (B1) An antigen-binding molecule containing scFv, which includes VH and VL. (B2) An antigen-binding molecule containing Fab, which includes a heavy chain containing VH and a CH1 domain, and a light chain containing VL and a constant light chain (CL) region. (B3) An antigen-binding molecule containing the following first polypeptide and second polypeptide, wherein mutations have been introduced into the Cys residue involved in the intermolecular disulfide bond between the light chain and heavy chain in the IgG antibody in the CH1 domain and CL. First polypeptide: A polypeptide in which VH, CH1 domain, hinge domain, CH2 domain and CH3 domain are linked in this order. Second polypeptide: A polypeptide in which VL, CL, hinge domain, CH2 domain and CH3 domain are linked in this order. (B4) An antigen-binding molecule containing IgG, which includes two heavy chains containing VH, CH1 domain, hinge domain, CH2 domain and CH3 domain, and two light chains containing VL and CL.

12. The fusion protein according to claim 1, wherein the mutant ADAMTS13 consists of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 99, and the antigen-binding molecule is an antigen-binding molecule containing any one of the following (B1) to (B4). (B1) An antigen-binding molecule containing scFv, which includes VH and VL. (B2) An antigen-binding molecule containing Fab, which includes a heavy chain containing VH and a CH1 domain, and a light chain containing VL and a constant light chain (CL) region. (B3) An antigen-binding molecule containing the following first polypeptide and second polypeptide, wherein mutations have been introduced into the Cys residue involved in the intermolecular disulfide bond between the light chain and heavy chain in the IgG antibody in the CH1 domain and CL. First polypeptide: A polypeptide in which VH, CH1 domain, hinge domain, CH2 domain and CH3 domain are linked in this order. Second polypeptide: A polypeptide in which VL, CL, hinge domain, CH2 domain and CH3 domain are linked in this order. (B4) An antigen-binding molecule containing IgG, which includes two heavy chains containing VH, CH1 domain, hinge domain, CH2 domain and CH3 domain, and two light chains containing VL and CL.

13. The fusion protein according to claim 1, wherein the mutant ADAMTS13 is linked to the antigen-binding molecule via a linker.

14. The fusion protein according to claim 13, wherein the linker has a length of 1 to 35 amino acids.

15. The fusion protein according to claim 13, wherein the linker is a linker comprising at least one repeating unit selected from (L1) to (L3) below: (L1) A polypeptide comprising the amino acid sequence (GGGGS) represented by SEQ ID NO: 925 as a repeating unit. (L2) A polypeptide comprising the amino acid sequence (PAPAP) represented by SEQ ID NO: 926 as a repeating unit. (L3) A polypeptide comprising the amino acid sequence (EAAAK) represented by SEQ ID NO: 927 as a repeating unit.

16. The fusion protein according to claim 14, wherein the antigen-binding molecule comprises VH and VL of an anti-vWF antibody, and the C-terminus of the mutant ADAMTS13 is linked to the N-terminus of the VH or VL in the antigen-binding molecule via the linker.

17. The fusion protein according to claim 11, wherein the fusion protein is any one of (p3-1) to (p3-3), (p4-1), and (p4-2) below. (p3-1) The antigen-binding molecule is an antigen-binding molecule comprising (B3), and a fusion protein is formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of the mutant ADAMTS13 is linked to the N-terminus of VH in the antigen-binding molecule via a linker, and comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [VL-CL-hinge domain-CH2-CH3] (p3-2) The antigen-binding molecule is an antigen-binding molecule comprising (B3), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of the mutant ADAMTS13 is linked to the N-terminus of the VL in the antigen-binding molecule via a linker, and comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [mutant ADAMTS13-peptide linker-VL-CL-hinge domain-CH2-CH3] (p3-3) The antigen-binding molecule is an antigen-binding molecule comprising (B3), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the N-terminus of the mutant ADAMTS13 is linked to the C-terminus of the CH3 domain in the antigen-binding molecule via a linker, and comprises the following first and second polypeptides, wherein the first polypeptide and the second polypeptide are linked via a disulfide bond of the hinge domain. First polypeptide: [VH-CH1-hinge domain-CH2-CH3] Second polypeptide: [VL-CL-hinge domain-CH2-CH3-peptide linker-mutant ADAMTS13](p4-1) The antigen-binding molecule is an antigen-binding molecule containing (B4), and is a fusion protein formed by linking two mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of each mutant ADAMTS13 and the N-terminus of each VH in the antigen-binding molecule are linked via a linker, and the fusion protein contains the following first to fourth polypeptides, wherein the CL of the first polypeptide and the hinge domain or CH1 of the second polypeptide are linked via a disulfide bond, the hinge domain or CH1 of the third polypeptide and the CL of the fourth polypeptide are linked via a disulfide bond, and the second polypeptide and the third polypeptide are linked via a disulfide bond of the hinge domain. First and fourth polypeptides: [VL-CL] Second and third polypeptides: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] (p4-2) The antigen-binding molecule is an antigen-binding molecule containing (B4), and is a fusion protein formed by linking one mutant ADAMTS13 and one of the antigen-binding molecules, wherein the C-terminus of mutant ADAMTS13 and the N-terminus of VH in the antigen-binding molecule are linked via a linker, and the fusion protein contains the following first to fourth polypeptides, wherein the CL of the first polypeptide and the hinge domain or CH1 of the second polypeptide are linked via a disulfide bond, the hinge domain or CH1 of the third polypeptide and the CL of the fourth polypeptide are linked via a disulfide bond, and the second polypeptide and the third polypeptide are linked via a disulfide bond of the hinge domain. First and fourth polypeptides: [VL-CL] Second polypeptide: [VH-CH1-hinge domain-CH2-CH3] Third polypeptide: [mutant ADAMTS13-peptide linker-VH-CH1-hinge domain-CH2-CH3] 18. The fusion protein according to claim 17, which is any one of the following: (t1) The fusion protein of (p3-1), wherein the first polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 333, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, and 545, and the second polypeptide comprises an amino acid sequence represented by SEQ ID NOs:

334. (t2) The fusion protein of (p4-1), wherein the first polypeptide and the fourth polypeptide are the same and comprise an amino acid sequence represented by SEQ ID NOs: 114, and the second polypeptide and the third polypeptide are the same and comprise an amino acid sequence represented by any one of SEQ ID NOs: 113, 352, 354, 356, 358, 360, and 362. (t3) A fusion protein of the above (p4-1), wherein the first polypeptide and the fourth polypeptide are the same and include the amino acid sequence represented by SEQ ID NO: 110, and the second polypeptide and the third polypeptide are the same and include the amino acid sequence represented by any one of SEQ ID NOs: 109, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406 and 408. (t4) A fusion protein of the above (p4-1), wherein the first polypeptide and the fourth polypeptide are the same and include the amino acid sequence represented by SEQ ID NO: 126, A fusion protein in which the second polypeptide and the third polypeptide are the same and contain an amino acid sequence represented by any one of SEQ ID NOs: 125, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, and 512.

19. The fusion protein according to claim 1, wherein the antigen-binding molecule in the fusion protein specifically binds to the polypeptide of (α1) or (α2) below: (α1) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 914 (α2) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 915 20. The fusion protein according to claim 19, wherein the antigen-binding molecule specifically binds to the (α1) or (α2) polypeptide, and further has the ability to bind to both vWF in which the A2 domain is folded and vWF in which the A2 domain is not folded.

21. A nucleic acid encoding a fusion protein according to any one of claims 1 to 20.

22. A vector containing the nucleic acid described in claim 21.

23. A transformed cell obtained by introducing the vector according to claim 22 into a host cell.

24. A method for producing a fusion protein according to any one of claims 1 to 20, characterized by culturing the transformed strain according to claim 23 in a culture medium, producing and accumulating the fusion protein according to any one of claims 1 to 20 in the culture obtained by said culture, and collecting said fusion protein from said culture.

25. A therapeutic agent for thrombotic diseases, comprising a fusion protein as described in any one of claims 1 to 20 as an active ingredient.

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