ADAMTS13 CUB Domain Variant and Uses

Abstract

The present invention provides an ADAMTS13 protein variant comprising one or more N-linked glycosylation sites in a portion of a CUB domain, for example, wherein the CUB domain comprises amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 (defined by SEQ ID NO. 43). The present invention also provides a composition comprising the ADAMTS13 protein variant, a method for preparing the same, and a use thereof.

Description

Technology Field

[0001] The present invention relates to the field of treatment. More specifically, the present invention relates to the field of treatment for disorders involving von Willebrand Factor (VWF). The present invention relates to a modified protease involved in maintaining hemostasis, in particular a modified protease in which the binding of autoantibodies is significantly reduced while maintaining activity, and to the use thereof in the treatment of diseases. Background Technology

[0002] Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare but life-threatening autoimmune disease caused by the production of autoantibodies against ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13). ADAMTS13 is a metalloproteinase that proteolytically degrades the Tyr1605-Met1606 binding within the A2 domain of von Willebrand factor (VWF). VWF is a multiplexed protein that mediates the adhesion of platelets to damaged blood vessels. The multiplexed size of VWF is directly proportional to its biological activity, and larger multiplexes are highly active in promoting platelet adhesion to damaged vessel walls. In healthy individuals, the multiplexed size of VWF is regulated by ADAMTS13, a VWF-degrading protease. In iTTP patients, the processing of VWF multiplexes is impaired due to the presence of pathogenic autoantibodies against ADAMTS13 that occur in these patients. The persistence of high molecular weight VWF multiplexes in iTTP patients with autoantibodies against ADAMTS13 is associated with excessive intravascular thrombus formation, manifesting as life-threatening microvascular thrombosis.

[0003] Currently, the treatment of iTTP consists of plasma exchange (PEX) and immunosuppression using high-dose glucocorticoids. Although plasma exchange supplies exogenous ADAMTS13, its effect is short-lived due to the persistent presence of circulating pathogenic antibodies targeting ADAMTS13, including exogenous ADAMTS13. In addition to plasma exchange, rituximab, a monoclonal antibody for B-cell depletion anti-CD20 therapy, is used in the treatment of iTTP and for the prevention of recurrence in iTTP patients. Recently, caplacizumab, a humanized anti-VWF nanoantibody that blocks platelet binding to VWF, was shown to accelerate the normalization of platelet counts by 1.55 times (Scully et al., 2019). Bleeding has been reported as a side effect of caplacizumab treatment (Mazepa et al., 2019). Mazepa et al. reported that bleeding was the primary adverse event of caplacizumab treatment, occurring in 65% of the caplacizumab group (compared to 48% in the placebo group) during the HERCULES clinical trial. Mucocutaneous bleeding, including nosebleeds and gingival bleeding, was the most common event, and most bleeding was mild to moderate and resolved without intervention. Three subjects who experienced severe bleeding during caplacizumab administration received VWF concentrate (severe nosebleed), tranexamic acid (gingival bleeding), and red blood cell transfusions (upper gastrointestinal bleeding), respectively. Overall, most caplacizumab-related bleeding resolves without intervention (although drug retention may be necessary); however, local vasoconstrictors and antifibrinolytics are effective in some cases, and VWF concentrate is retained in patients with severe, refractory bleeding (e.g., Dutt et al, 2021).

[0004] Despite advancements in the treatment of iTTP patients, current therapeutic regimens are still not the best. There is a clear need for a treatment that rapidly restores ADAMTS13 activity to help accelerate the normalization of platelet counts in iTTP patients.

[0005] ADAMTS13 variants accommodating resistance to autoantibodies have been reported in the literature (Jian et al., 2012). The spacer domain of ADAMTS13 provides a key site for the binding of pathogenic autoantibodies. Conservative mutations in five residues within the spacer domain produced a so-called Gain-of-Function (GoF) variant claimed to be resistant to the binding of pathogenic autoantibodies occurring in iTTP patients (Jian et al., 2012). This GoF variant of ADAMTS13 is also described in US Patent US 9,546,360. However, subsequent experiments revealed that this GoF variant of ADAMTS13 remains a target of patient-derived autoantibodies and is not resistant to their inhibitory effects (Graca et al., 2019). WO 2021 / 242092 describes an ADAMTS13 variant containing an N-linked glycan that reduces autoantibody binding to the spacer domain. However, pathogenic autoantibodies binding to other domains of ADAMTS13 have also been reported, for example, found in 20–40% of iTTP patients (see, e.g., Thomas et al. 2015 and Pos et al. 2011). Prior art literature

[0006] Austin SK, Starke RD, Lawrie AS, Cohen H, Machin SJ, Mackie IJ. The VWF / ADAMTS13 axis in the antiphospholipid syndrome: ADAMTS13 antibodies and ADAMTS13 dysfunction. Br J Haematol. 2008 May;141(4):536-44. doi: 10.1111 / j.1365-2141.2008.07074.x Bitsadze V, Bouvier S, Khizroeva J, Cochery-Nouvellon , Mercier , Perez-Martin A, Makatsariya A, Gris JC. Early ADAMTS13 testing associates with pre-eclampsia occurrence in antiphospholipid syndrome. Thromb Res. 2021 Apr 27;203:101-109. doi: 10.1016 / j.thromres.2021.04.021Burbelo PD1, O'Hanlon TP. New autoantibody detection technologies yield novel insights into autoimmune disease. Curr Opin Rheumatol. 2014 Nov;26(6):717-23Cabin, D. E., Shimazu, K., Murphy, D., Cole, N. B., Gottschalk, W., McIlwain, K. L., Orrison, B., Chen, A., Ellis, C. E., Paylor, R., Lu, B., & Nussbaum, R. L. (2002). Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 22(20), 8797-8807. https: / / doi.org / 22 / 20 / 8797 [pii]Deforche, L., Roose, E., Vandenbulcke, A., Vandeputte, N., Feys, H. B., Springer, T. A., Mi, L. Z., Muia, J., Sadler, J. E., Soejima, K., Rottensteiner, H., Deckmyn, H., De Meyer, S. F., & Vanhoorelbeke, K. (2015). Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13. Journal of Thrombosis and Haemostasis, 13(11), 2063-2075. https: / / doi.org / 10.1111 / jth.13149Dong, J. F. (2005). Cleavage of ultra-large von Willebrand factor by ADAMTS-13 under flow conditions. Journal of Thrombosis and Haemostasis, 3(8), 1710-1716. https: / / doi.org / https: / / doi.org / 10.1111 / j.1538-7836.2005.01360.xDutt et al, Real-world experience with caplacizumab in the management of acute TTP. Blood 2021;137(13):1731-1740. 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Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura. 2019; Haematology. 2019 Nov 21. more: haematol. doi:10.3324 / haematol.2019.226068Jian, C., Xiao, J., Gong, L., Skipwith, CG, Jin, SY, Kwan, HC, & Zheng, XL (2012). Gain-of-function ADAMTS13 variants that are resistant to autoantibodies against ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura. Blood, 119(16), 3836-3843. https: / / doi.org / 10.1182 / blood-2011-12-399501Joshua, M., Jian, Z., Garima, G., L., HS, D., FK, B., FH, Louis, D., Karen, V., A., WL, Robyn, R., Harish, TN, E., HJ, & Evan, SJ (2014). Allosteric activation of ADAMTS13 by von Willebrand factor. Proceedings of the National Academy of Sciences, 111(52), 18584-18589. https: / / doi.org / 10.1073 / pnas Kangro, K., Roose, E., Joly, B. S., Sinkovits, G., Falter, T., von Auer, C., Rossmann, H., Reti, M., Voorberg, J., Prohszka, Z., Lmmle, B., Coppo, P., Veyradier, A., De Meyer, S. F., Mnnik, A., & Vanhoorelbeke, K. (2021). Anti-ADAMTS13 autoantibody profiling in patients with immune-mediated thrombotic thrombocytopenic purpura. Blood Advances, 5(17), 3427-3435. https: / / doi.org / 10.1182 / bloodadvances.2020004172Kelwick R, Desanlis I, Wheeler GN, Edwards DR. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family. Genome Biol. 2015 May 30;16:113Kokame K, Nobe Y, Kokubo Y, Okayama A, Miyata T. FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br J Haematol. 2005 Apr;129(1):93-100Kim, H. J., Xu, Y., Petri, A., Vanhoorelbeke, K., Crawley, J. T. B., & Emsley, J. (2021). Crystal structure of ADAMTS13 CUB domains reveals their role in global latency. Science Advances, 7(16), 1-12. https: / / doi.org / 10.1126 / sciadv.abg4403Klaus, C., Plaimauer, B., Studt, J.-D., Dorner, F., Lammle, B., Mannucci, P. M., & Scheiflinger, F. (2004). Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura. Blood, 103(12), 4514-4519. https: / / doi.org / 10.1182 / blood-2003-12-4165Long Zheng, X., Wu, H. M., Shang, D., Falls, E., Skipwith, C. G., Cataland, S. R., Bennett, C. L., & Kwaan, H. C. (2010). Multiple domains of ADAMTS13 are targeted by autoantibodies against ADAMTS13 in patients with acquired idiopathic thrombotic thrombocytopenic purpura. Haematologica, 95(9), 1555-1562. https: / / doi.org / 10.3324 / haematol.2009.019299De Maeyer B, De Meyer SF, Feys HB, Pareyn I, Vandeputte N, Deckmyn H, Vanhoorelbeke K. The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet-decorated VWF strings in vivo. J Thromb Haemost. 2010 Oct;8(10):2305-12Mazepa MA, Masias C, Chaturvedi S. How targeted therapy disrupts the treatment paradigm for acquired TTP: the risks, benefits, and unknowns. Blood.2019 Aug 1;134(5):415-420Muia, J., Gao, W., Haberichter, S. L., Dolatshahi, L., Zhu, J., Westfield, L. A., Covill, S. C., Friedman, K. D., & Sadler, J. E. (2013). An optimized fluorogenic ADAMTS13 assay with increased sensitivity for the investigation of patients with thrombotic thrombocytopenic purpura. Journal of Thrombosis and Haemostasis, 11(8), 1511-1518. https: / / doi.org / https: / / doi.org / 10.1111 / jth.12319Ostertag, E. M., Kacir, S., Thiboutot, M., Gulendran, G., Zheng, X. L., Cines, D. B., & Siegel, D. L. (2016). ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 1. Structural and functional characterization in vitro. Transfusion, 56(7), 1763-1774. https: / / doi.org / https: / / doi.org / 10.1111 / trf.13584Pos, W., Crawley, J. T. B., Fijnheer, R., Voorberg, J., Lane, D. A., & Luken, B. M. (2010). An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF.Blood, 115(8), 1640-1649. https: / / doi.org / 10.1182 / blood-2009-06-229203Pos W, Crawley JTB, Fijnheer R, et al. An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF. Blood. 2010;115(8):1640-1649Pos W, Sorvillo N, Fijnheer R, et al. Residues Arg568 and Phe592 contribute to an antigenic surface for anti-Adamts13 antibodies in the spacer domain. Haematologica. 2011;96(11):1670-1677. Postmus, T., Graa, N. A. G., Ferreira de Santana, J., Ercig, B., Langerhorst, P., Luken, B., Joly, B. S., Vanhoorelbeke, K., Veyradier, A., Coppo, P., & Voorberg, J. (2023). Impact of N-glycan mediated shielding of ADAMTS-13 on the binding of pathogenic antibodies in immune thrombotic thrombocytopenic purpura. Journal of Thrombosis and Haemostasis. https: / / doi.org / https: / / doi.org / 10.1016 / j.jtha.2023.08.017Roose et al. Open ADAMTS13, induced by antibodies, is a biomarker for subclinical immune-mediated thrombotic thrombocytopenic purpura Blood (2020) 16;136(3):353-361Ramsi M, Al Ali AS. Thrombocytopenia-associated multiple-organ failure (TAMOF): recognition and management. BMJ Case Rep. 2018 Aug 27;2018:bcr2018225594. doi: 10.1136 / bcr-2018-225594Schelpe, A. S., Petri, A., Roose, E., Pareyn, I., Deckmyn, H., De Meyer, S. F., Crawley, J. T. B., & Vanhoorelbeke, K. (2020). Antibodies that conformationally activate ADAMTS13 allosterically enhance metalloprotease domain function.Blood Advances, 4(6), 1072-1080. https: / / doi.org / 10.1182 / bloodadvances.2019001375. Scully M, Cataland SR, Peyvandi F, Coppo P, Knbl P, Kremer Hovinga JA, Metjian A, de la Rubia J, Pavenski K, Callewaert F, Biswas D, De Winter H, Zeldin RK; HERCULES Investigators. Caplacizumab Treatment for Acquired Thrombotic Thrombocytopenic Purpura. N Engle J Med. 2019 Jan 24;380(4):335-346Thomas, MR, de Groot, R., Scully, MA, & Crawley, JTB (2015). Pathogenicity of Anti-ADAMTS13 Autoantibodies in Acquired Thrombotic Thrombocytopenic Purpura. EBioMedicine, 2(8), 942-952. https: / / doi.org / 10.1016 / j.ebiom.2015.06.007Sins JWR, Schimmel M, Luken BM, Nur E, Zeerleder SS, van Tuijn CFJ, Brandjes DPM, Kopatz WF, Urbanus RT, Meijers JCM, Biemond BJ, Fijn Dvannat factor of K. reactivity in sickle cell disease during vaso-occlusive crisis and steady state. J Thromb Haemost. 2017 Jul;15(7):1392-1402. doi: 10.1111 / jth.13728.South, K., Luken, BM, Crawley, JTB, Phillips, R., Thomas, M., Collins, RF, Deforche, L., Vanhoorelbeke, K., & Lane, DA (2014).Conformational activation of ADAMTS13. Proceedings of the National Academy of Sciences of the United States of America, 111(52), 18578-18583. https: / / doi.org / 10.1073 / pnas.1411979112Turecek PL, Peck RC, Rangarajan S, Reilly-Stitt C, Laffan MA, Kazmi R, James I, Dushianthan A, Schrenk G, Gritsch H, Ewenstein BM, Mellgard B, Erdlenbruch W, Jain N, Binder NB, Mumford AD. Recombinant ADAMTS13 reduces abnormally up-regulated von Willebrand factor in plasma from patients with severe COVID-19. Thromb Res. 2021 May;201:100-112. doi: 10.1016 / j.thromres.2021.02.012Verbij et al. Identification of glycans on plasma-derived ADAMTS13 Blood. 2016 Nov 24;128(21):e51-e58. doi: 10.1182 / blood-2016-06-720912.Xiao J, Jin SY, Xue J, Sorvillo N, Voorberg J, Zheng XL. Essential domains of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 metalloprotease required for modulation of arterial thrombosis. Arterioscler Thromb Vasc Biol. 2011 Oct;31(10):2261-9. 해결하려는 과제

[0007] The present invention provides an improved ADAMTS13 protein variant having reduced binding by autoantibodies compared to wild-type ADAMTS13. Accordingly, the provided ADAMTS13 variant effectively evades autoantibody binding while maintaining ADAMTS13 activity. means of solving the problem

[0008] The present invention provides an ADAMTS13 protein variant comprising one or more N-linked glycosylation sites in a CUB domain portion comprising amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0009] In some embodiments, the ADAMTS13 protein variant is a full-length ADAMTS13 protein variant comprising amino acids 75 to 1427 of the ADAMTS13 amino acid sequence shown in FIG. 1 (defined by SEQ ID NO. 43), or comprising one or more N-linked glycosylation sites in part of the CUB domain and optionally additional mutations described herein.

[0010] In some embodiments, the ADAMTS13 protein variant comprises one or more additional N-linked glycosylation sites compared to the wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites compared to the wild-type ADAMTS13 are shifted in a spacer domain comprising amino acid residues S556 to A685 of ADAMTS13 as shown in FIG. 1 or defined by SEQ ID NO. 43.

[0011] In some embodiments, the present invention provides an ADAMTS13 protein variant comprising one or more N-linked glycosylation sites in a CUB domain portion comprising 1320-1322 amino acid residues of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, and further comprising one or more additional mutations that reduce binding by autoantibodies toward the spacer domain.

[0012] In some embodiments, the present invention provides an ADAMTS13 protein variant comprising one or more N-linked glycosylation sites in a CUB domain portion comprising 1320-1322 amino acid residues of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, wherein one or more N-linked glycosylation sites are added and / or one or more existing N-linked glycosylation sites are shifted compared to wild-type ADAMTS13 in a spacer domain comprising amino acid residues S556 to A685 of ADAMTS13 shown in FIG. 1 or defined by SEQ ID NO. 43.

[0013] In some embodiments, the present invention provides a nucleic acid structure comprising a nucleic acid sequence encoding an ADAMTS13 protein variant according to the present invention.

[0014] In some embodiments, the present invention provides a pharmaceutical composition comprising an ADAMTS13 protein variant or nucleic acid structure according to the present invention and one or more pharmaceutically acceptable carriers, adjuvants, excipients and / or diluents.

[0015] In some embodiments, the present invention provides a method for reducing autoantibody binding, particularly binding by an anti-CUB domain antibody, to ADAMTS13 or a variant thereof, comprising introducing one or more, preferably one or two N-linked glycosylation sites, to the CUB domain portion comprising amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43 to reduce autoantibody binding to ADAMTS13 or a variant.

[0016] In some embodiments, the present invention provides a method for reducing plasma clearance of ADAMTS13 or a variant thereof, the method comprising introducing one or more N-linked glycosylation sites to the CUB domain portion comprising amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, thereby reducing plasma clearance of ADAMTS13 or a variant.

[0017] In some embodiments, ADAMTS13 is an ADAMTS13 protein variant in which binding to autoantibodies specific to the spacer domain is reduced compared to wild-type ADAMTS13.

[0018] In some embodiments, the present invention provides an ADAMTS13 protein variant or nucleic acid structure according to the present invention for use in treatment.

[0019] In some embodiments, the present invention provides an ADAMTS13 protein variant or nucleic acid structure according to the present invention for use as an antithrombotic agent.

[0020] In some embodiments, the present invention provides an ADAMTS13 protein variant or nucleic acid construct according to the present invention for use in the treatment of disorders characterized by abnormal von Willebrand factor (VWF) activity and / or VWF treatment.

[0021] In some embodiments, the present invention provides a method for treating a disorder characterized by abnormal VWF activity and / or VWF treatment, the method comprising administering a therapeutically effective amount of an ADAMTS13 protein variant according to the present invention to a subject in need thereof.

[0022] In some embodiments, the present invention provides a method for treating a disorder characterized by abnormal VWF activity and / or VWF treatment in a patient who responds insufficiently to treatment that reduces ADAMTS13 autoantibody binding to a spacer domain, the method comprising administering a therapeutically effective amount of an ADAMTS13 protein variant according to the present invention to the patient. Effects of the invention

[0023] The present invention provides a novel ADAMTS13 protein variant that is less susceptible to binding by autoantibodies than wild-type ADAMTS13. The ADAMTS13 variant according to the present invention reduces autoantibody binding to the CUB domain, thereby allowing functional ADAMTS13 to remain in the patient for a longer period and reducing plasma clearance of ADAMTS13. Furthermore, an ADAMTS13 variant that reduces autoantibody binding to both the spacer domain and the CUB domain has the potential to improve the overall clinical efficacy of treatment for impaired ADAMTS13 activity by up to 100% in iTTP patients when combined with a single ADAMTS13 protein variant. Brief explanation of the drawing

[0024] Fig. 1: Amino acid sequence of ADAMTS13; UniProt accession number Q76LX8. Corresponds to sequence number 43. Fig. 2: N-linked glycans identified in ADAMTS13 (modified from Verbij et al. 2016). Square: GlcNAc; Circle: Mannose; Inner cross circle: Galactose; Triangle: Fuchsus; Diamond: Sialic acid. Fig. 3: Overview of the CUB-NGLY variants. A) Table of mutated amino acids for each individual CUB N-glycan variant. B) Visualization of the positions of each individual CUB N-glycan mutation in the CUB domain crystal structure of ADAMTS13 (PDB: 7B01). Fig. 4: N-glycan analysis of the CUB-NGLY variant. List of deglycosylated peptides identified after PNGaseF treatment and digestion with chymotrypsin or thermorisin. Values ​​represent signal intensity identified using PEAKS. -10LgP provides the confidence score of the identified peptides. Start and end indicate the positions of the identified peptides within the ADAMTS13 sequence. Figure 5: Activity of CUB-NGLY variants in FRETS-VWF73 activity assay. All mutants were able to efficiently process the FRETS-VWF73 substrate. 1251N+1368N showed a twofold increase in activity compared to wild-type ADAMTS13 (WT Ctrl). Fig. 6: Activity of CUB-NGLY variants against VWF multiples. All CUB-NGLY variants were able to digest high molecular weight VWF multiples with efficiency equivalent to wild-type ADAMTS13 (WT Ctrl). Fig. 7: Characterization of patient-derived monoclonal anti-CUB antibodies. A) Epitope mapping of the produced antibodies. All antibodies bind strongly to ADAMTS13. None of the antibodies bind to the MDTCS variant, indicating that all antibodies target the C-terminal domain of ADAMTS13. B) Activity of wild-type ADAMTS13 in the presence of anti-CUB antibodies and anti-spacer antibodies (1-416). None of the anti-CUB antibodies inhibited ADAMTS13 activity. 1-440 can significantly increase ADAMTS13 activity. The spacer antibody (1-416) can completely inhibit ADAMTS13, indicating that the concentration used is sufficient for inhibition. Fig. 8: Heatmap showing the relative reactivity (binding) of human monoclonal patient-derived antibodies to CUB-NGLY based on two alternative calculation methods. A) Data are based on optical density (OD) binding data. B) Binding data were calculated using a calibration curve based on human monoclonal anti-ADAMTS13 antibody II-1. A) and B) Binding to wild-type ADAMTS13 (WT) is set to 100% of binding. Spacer antibody (1-416) is used as a loading control. 1251N or a combination of 1255N and 1368N can prevent the binding of 6 out of 7 anti-CUB antibodies. Fig. 9: Reactivity of patient samples to CUB-NGLY variants of ADAMTS13. A) and D) Heatmaps showing the relative reactivity of 9 patient samples to the CUB-NGLY combination. Binding to wild-type ADAMTS13 (WT) was set to 100% of the binding. Two alternative calculation methods were used. Since NGLY3 and FL-5ALA (5ALA) prevent the binding of anti-spacer antibodies, the effects of CUB-NGLY can be studied independently. Adding 1251N+1368N or 1255N+1368N to NGLY3 or 5ALA further reduces antibody binding in 8 out of 9 patient samples. B) and E) Adding both CUB-NGLY combinations significantly reduces antibody binding. C) and F) The effect of adding CUB-NGLY to NGLY3 and 5ALA is equivalent, indicating that the CUB domain modification has an effect independent of the spacer mutant. Fig. 10: Activity of CUB-NGLY variants in the FRETS-VWF73 activity assay at pH 6.0 and pH 7.5. All mutants were able to efficiently process the FRETS-VWF73 substrate at both pH. At pH 7.5, the activity of 1251N, 1255N, 1320N, 1321N, and 1322N was reduced compared to the activity at pH 6.0, suggesting that they are a closed form similar to wild-type ADAMTS13 (WT). On the other hand, 1295N, 1368N, 1251N+1368N, and 1255N+1368N showed the same activity at both pH, ​​suggesting that they are an open, more active form at pH 7.5 compared to wild-type ADAMTS13. Fig. 11: Activity of CUB-NGLY variants in VWF multiplet cleavage under flow conditions (primary human umbilical vein endothelial cells, HUVEC). A) 1251N+1368N and 1255N+1368N variants exhibited activity similar to wild-type ADAMTS13. B) Representative images of the negative control, wild-type ADAMTS13, and variants at time points 0, 2, and 9 min. Fig. 12: Western blot of ADAMTS13 variants using HRP-conjugated anti-V5 antibody after expression by ExpiCHO cells. A) and B) ADAMTS13 CUB-NGLY variants are expressed at levels similar to wild-type ADAMTS13. Specific details for implementing the invention

[0025] The present invention provides a novel ADAMTS13 protein variant that is less susceptible to binding by autoantibodies than wild-type ADAMTS13. Essentially, all iTTP patients have autoantibodies against the spacer domain, but modifications to the spacer domain alone may not be sufficient to remove autoantibody binding to ADAMTS13. This is believed to be due to antibodies against other domains of ADAMTS13 that occur in 20–40% of iTTP patients (e.g., see Thomas et al. (2015) and Pos et al. 2011). One of these is the CUB domain (an acronym for complement C1r / C1s proteases, sea Urchin, and Bone morphogenic protein), which can be subdivided into the CUB1 domain and the CUB2 domain. Antibodies against the CUB domain do not directly inhibit ADAMTS13, but lower the functional level of ADAMTS13 by increasing the rate of clearance of ADAMTS13 from plasma. In addition, these antibodies can convert the form of ADAMTS13 into an open form, which increases the accessibility of the spacer domain to the immune-dominant epitope (Roose et al. 2020).

[0026] In its resting state, ADAMTS13 circulates in the blood in a closed form. Upon binding to its substrate, von Willebrand factor, ADAMTS13 is converted to an open form (South et al., 2014). The conformational state of ADAMTS13 is pH-dependent; it exists in an open form at pH 6.0 and in a closed form at pH 7.5 (Joshua et al., 2014; Muia et al., 2013). It has been previously shown that ADAMTS13 activity in the closed form (pH 7.5) (measured by the conversion of the synthetic peptide substrate FRETS-VWF73) is lower than that in the open form (pH 6.0) (Joshua et al., 2014; Muia et al., 2013).

[0027] The inventors introduced an N-glycan to residues around the spacer-CUB interface of ADAMTS13 to reduce autoantibody binding. Preventing autoantibody binding to the CUB domain allows functional ADAMTS13 to remain in patients for a longer period. Residues for N-glycan addition were selected based on two criteria. First, the N-glycan was preferably introduced around the modeled spacer-CUB interface to minimize the potential for conformational changes within this part of the molecule. Second, the amino acid side chain had to be outwardly oriented, as mutating an inwardly oriented side chain could potentially affect protein stability and / or N-glycan attachment efficiency. Based on these selection criteria, several sites for N-glycosylation site insertion were selected. As demonstrated in the examples of this specification, several ADAMTS13 protein variants having one of the selected N-glycosylation sites were unsuccessful (e.g., due to absence of expression or inability to reduce autoantibody binding), but several ADAMTS13 protein variants were designed to reduce autoantibody binding to the CUB domain without negatively affecting ADAMTS13 activity.

[0028] Furthermore, it is expected that 20–40% of iTTP patients will not fully respond to treatment using ADAMTS13 protein variants with reduced binding by autoantibodies targeting the spacer domain, even though patients with such antibodies account for more than 95%. In the embodiments of this specification, it was confirmed that these ADAMTS13 protein variants remain vulnerable to autoantibodies against the CUB domain, which can lead to rapid clearance and consequently reduced in vivo activity. The strategy of reducing autoantibody binding to the CUB domain of the present invention has been demonstrated to increase the activity of ADAMTS13 protein variants with reduced autoantibody binding to the spacer domain. By reducing anti-CUB domain autoantibody binding, the present invention allows for the possibility of increasing the overall clinical efficacy of treatment for impaired ADAMTS13 activity by up to 100% in iTTP patients, preferably when a strategy of reducing autoantibody binding to both the spacer and CUB domains is combined in a single ADAMTS13 protein variant.

[0029] In some embodiments, the present invention provides an ADAMTS13 protein variant comprising one or more N-linked glycosylation sites in a CUB domain portion comprising amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43. In some embodiments, one or more N-linked glycosylation sites are 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1272, 1273, 1274, 1275, 1276, 1295, 1296, 1297, 1326, 1327, 1328, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1392, 1393 and / or as defined in SEQ ID NO. 43. It is present at the 1394 amino acid residue.

[0030] As used herein, "comprising" and its conjugations are used in a non-restrictive sense, meaning that items following the word are included but not specifically mentioned are excluded. Additionally, the verb "to be composed of" may be replaced with "essentially composed of," which implies that the compounds or auxiliary compounds defined herein may include additional components other than those specifically identified, and that said additional components do not alter the inherent properties of the invention.

[0031] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. When the words “approximately” or “about” are used with a number (e.g., approximately 10, about 10), they preferably include ±5%, preferably ±1%, of the value (e.g., 10). When the term “at least” is used with a number or percentage (e.g., 90%), it includes the specified number and any higher value (90% or more). The use of an alternative (e.g., “or”) means one, both, or any combination thereof of the alternatives.

[0032] The term "protein" in this specification refers to a compound comprising amino acids linked by peptide bonds. Proteins encoded by a gene are not limited to the amino acid sequence encoded by the gene and may include post-translational modifications.

[0033] The term "subject" in this specification refers to a recipient of the ADAMTS13 protein variant or coding nucleic acid according to the present invention and includes humans and animals. The subject is preferably a mammal, more preferably a human.

[0034] The term "therapeutic effective dose" refers to an amount of ADAMTS13 protein variant sufficient to alleviate one or more of the symptoms of the disease or condition being treated to some extent. This may be a reduction or alleviation of symptoms, a reduction or alleviation of the cause of the disease or condition, or any other desired therapeutic effect.

[0035] In this specification, the term “treatment” refers to inhibiting a disorder, that is, stopping or reducing its development or at least one clinical symptom, and / or alleviating the symptoms of a disease or pathological condition.

[0036] As used herein, the terms "ADAMTS13" and "ADAMTS13 protein" refer to the protein encoded by the ADAMTS13 gene. ADAMTS13 is a member of the ADAM (a disintegrin and metalloproteinase) family of metalloproteinase genes, a family composed of membrane-fixed proteases with different functions. Members of the ADAMTS family are characterized by having one or more thrombospondin 1-like (TSP1) domains at the C-terminus and lacking the EGF repeats, transmembrane domains, and cytoplasmic tails present in ADAM metalloproteinases. ADAMTS13 is the only ADAMTS member to possess two C-terminal CUB domains (complement C1r / C1s proteases, sea urchin, and bone morphogenic protein) and possesses VWF cleavage protease activity (Kelwick et al, 2005). "Wild-type ADAMTS13" and "wild-type ADAMTS13 protein" refer to naturally occurring human ADAMTS13, and FIG. 1 provides the amino acid sequence of full-length wild-type ADAMTS13 defined by SEQ ID NO. 43.

[0037] The "CUB (Complement component Clr / Cls, Uegf, and Bone morphogenic protein 1) domain" of ADAMTS13 refers to a portion containing residues C1192 to T1427, which is the C-terminal portion of the ADAMTS13 amino acid sequence shown in FIG. 1 or defined by SEQ ID NO. 43. The CUB domain may be subdivided into a CUB1 domain and a CUB2 domain. In some embodiments, the CUB1 domain refers to a portion of ADAMTS13 containing amino acid residues 1192-1298 of SEQ ID NO. 43, and the CUB2 domain refers to a portion of ADAMTS13 containing amino acid residues 1299-1427 of SEQ ID NO. 43.

[0038] The term "ADAMTS13 protein variant" refers to a variant of ADAMTS13 that differs in amino acid sequence from wild-type ADAMTS13 in that it has at least one N-glycosylation site not present in wild-type ADAMTS13. In some embodiments, the ADAMTS13 protein variant according to the present invention comprises at least amino acids 75 to 1427 of the ADAMTS13 amino acid sequence shown in FIG. 1 (or defined by SEQ ID NO. 43) and comprises one or more N-linked glycosylation sites in a portion of the CUB domain. In some embodiments, the ADAMTS13 protein variant comprises additional mutations, preferably as described herein.

[0039] Residues 1-74 of SEQ ID NO. 43 encode a signal peptide and a precursor peptide. In some embodiments, the ADAMTS13 protein variant according to the present invention is a full-length ADAMTS13 protein variant. In some embodiments, the full-length ADAMTS13 protein variant comprises a whole amino acid sequence comprising residues 1 to 1427 of ADAMTS13 as shown in FIG. 1 or defined by SEQ ID NO. 43, comprises one or more N-linked glycosylation sites in part of the CUB domain, optionally comprises additional mutations, preferably as described herein. An ADAMTS13 protein variant comprising one or more N-linked glycosylation sites as defined herein is also referred to herein as a "CUB-NGLY protein variant" or "CUB-NGLY ADAMTS13 protein variant".

[0040] In the amino acid sequences or protein variants shown in this specification, amino acids are represented by a single letter or a three-letter symbol. These symbols are well known to ordinary skilled technicians and have the following meanings: A(Ala) is alanine, C(Cys) is cysteine, D(Asp) is aspartic acid, E(Glu) is glutamic acid, F(Phe) is phenylalanine, G(Gly) is glycine, H(His) is histidine, I(Ile) is isoleucine, K(Lys) is lysine, L(Leu) is leucine, M(Met) is methionine, N(Asn) is asparagine, P(Pro) is proline, Q(Gln) is glutamine, R(Arg) is arginine, S(Ser) is serine, T(Thr) is threonine, V(Val) is valine, W(Trp) is tryptophan, and Y(Tyr) is tyrosine.

[0041] All positions of amino acid residues shown in this specification refer to the amino acid residue numbering in the wild-type ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0042] Mutations, particularly the substitution of one amino acid with another, are indicated herein in the standard manner of the art, namely by indicating the amino acid present in the wild-type ADAMTS13 sequence, the position of the amino acid within the sequence, and the amino acid introduced at that position. For example, “R568K” indicates that arginine at position 568 is substituted with lysine. As another example, 1251N indicates that asparagine is introduced at position 1251. As yet another example, “568REY570 to 568NET570” indicates that the arginine-glutamic acid-tyrosine sequence at positions 568-570 is substituted with the asparagine-glutamic acid-threonine sequence.

[0043] Mutations introduced into an ADAMTS13 protein variant according to the present invention, comprising mutations having an addition and / or shift of one or more N-linked glycosylation sites and mutations such as any additional mutation described in the present invention compared with wild-type ADAMTS13, preferably produce an ADAMTS13 protein variant having an amino acid sequence that is at least 90% identical to the corresponding amino acid sequence of wild-type ADAMTS13.

[0044] In some embodiments, the ADAMTS13 protein variant of the present invention has at least 90% sequence homology with wild-type ADAMTS13. In some embodiments, the full-length ADAMTS13 protein variant of the present invention has at least 90% sequence homology with full-length wild-type ADAMTS13. In some embodiments, the ADAMTS13 protein variant according to the present invention comprises at least amino acids 75 to 1427 of the ADAMTS13 amino acid sequence shown in FIG. 1 or defined by SEQ ID NO. 43, has the indicated mutation, and preferably has at least 90% sequence identity with amino acids 75 to 1427 of wild-type ADAMTS13. The sequence homology is preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, and more preferably at least 98%.

[0045] The term "% sequence homology" is defined herein as the percentage of amino acids within an amino acid sequence that are identical to the amino acids of a reference amino acid sequence or an amino acid sequence of interest, after the sequences have been aligned to achieve maximum sequence homology. Methods and computer programs for alignment are well known in the art. A person skilled in the art understands that consecutive amino acid residues of one amino acid sequence are compared with consecutive amino acid residues of another amino acid sequence.

[0046] "N-linked glycosylation" typically refers to the attachment of an oligosaccharide moiety to the nitrogen atom, N4, of an asparagine residue. The terms "N-linked glycosylation site" and "N-glycosylation site" are used interchangeably to refer to sites capable of N-linked glycosylation in ADAMTS13 protein variants. These sites possess the amino acid sequence NXT or NXS, where X is any amino acid excluding P. N-linked glycosylation is a post-translational modification that can regulate protein folding, cell adhesion, and / or function. N-linked glycans can consist of various combinations of mannose, N-acetylglucosamine (GlcNAc), galactose, fucose, and sialic acid residues. In ADAMTS13, several N-linked glycans were identified attached to asparagine residues 142, 146, 552, 579, 614, 667, 707, 828, 1235, and 1354 (Verbij et al. 2016) (see Fig. 2), and several other types of glycosylation, including O-glycosylation and S- and C-mannosylation, were also identified. Fig. 2 also shows the most common structures of N-glycans identified in ADAMTS13 and other structures. In the ADAMTS13 protein variant according to the present invention, one or more N-linked glycosylation sites present in addition to the N-glycosylation sites present in wild-type ADAMTS13 are indicated by reference to the residues of the ADAMTS13 sequence shown in Fig. 1 or defined by SEQ ID NO. 43. In particular, residues corresponding to asparagine of the NXT or NXS sequences are indicated. For example, the N-linked glycosylation site at the 1251 amino acid residue means that asparagine is present at residue 1251, X at residue 1252, and T or S at residue 1253.

[0047] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises one or more N-linked glycosylation sites that are not present in wild-type ADAMTS13. This means that an N-glycosylation site (NXT or NXS, where X is any amino acid excluding P) is present at an amino acid residue that is not present in wild-type ADAMTS13. This is achieved by introducing one or more N-linked glycosylation sites into the ADAMTS13 sequence. Preferably, the ADAMTS13 protein variant according to the present invention comprises 1 to 5, more preferably 1 to 3, more preferably 1 or 2 N-linked glycosylation sites in a CUB domain portion comprising amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43. That is, the above 1 to 5, between 1 and 3, and 1 or 2 N-linked glycosylation sites are 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1272, 1273, 1274, 1275, 1276, 1295, 1296, 1297, 1326, 1327, 1328, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, as shown in FIG. 1 or SEQ ID NO. 43. It exists in 1370, 1392, 1393 and / or 1394.

[0048] In some embodiments, the introduction of an N-linked glycosylation site means that an N-linked glycosylation site not present in wild-type ADAMTS13 is introduced into the ADAMTS13 protein variant of the present invention, and in some embodiments, a naturally occurring N-linked glycosylation site present in wild-type ADAMTS13 is introduced without removal. In some embodiments, the introduction of an N-linked glycosylation site may be achieved by introducing one or more mutations into the amino acid sequence compared to wild-type ADAMTS13, thereby introducing the N-linked glycosylation site. Such mutations may consist of one or more amino acid residue substitutions by other amino acid residues, one or more amino acid residue insertions or deletions, or a combination thereof, thereby introducing an N-linked glycosylation site not present in wild-type ADAMTS13 into the ADAMTS13 sequence. In some embodiments, the N-linked glycosylation site is introduced by substituting one or more amino acid residues with other amino acid residues. In some embodiments, any amino acid may be substituted with an asparagine (N) residue to introduce asparagine (N) as the first residue of the N-linked glycosylation site NXS or NXT. In some embodiments, proline may be substituted with any other amino acid to remove potential proline as the second residue. Both the NXT and NXS sites may be introduced into the ADAMTS13 protein variant according to the present invention. In some embodiments, any amino acid other than serine (S) and threonine (T) may be substituted with serine (S) or threonine (T) to introduce serine (S) or threonine (T) as the third residue of the N-linked glycosylation site NXS or NXT as defined herein, or a combination thereof. Both the NXS and NXT sites may be introduced into the ADAMTS13 protein variant according to the present invention or used according to the present invention. Without being bound by a specific theory, NXT appears to result in more efficient N-glycan addition compared to NXS.In some embodiments, one or more N-linked glycosylation sites have an NXT sequence. In some embodiments, one or more N-linked glycosylation sites defined herein have an NXT sequence. A person skilled in the art can design a suitable mutation in the ADAMTS13 sequence to introduce one or more N-linked glycosylation sites into the residues defined herein.

[0049] In some embodiments, the ADAMTS13 protein variant according to the present invention has reduced binding by autoantibodies compared to wild-type ADAMTS13. In some embodiments, the ADAMTS13 protein variant according to the present invention is characterized by reduced binding by autoantibodies. In some embodiments, the ADAMTS13 protein variant is characterized by reduced binding by autoantibodies, indicating reduced binding of autoantibodies specific to the ADAMTS13 protein compared to the wild-type ADAMTS13 protein when evaluated by ELISA analysis under the conditions described in Examples 5 and 6. That is, as used herein, "reduced binding by autoantibodies" means that the binding of autoantibodies specific to ADAMTS13 is reduced for the ADAMTS13 protein variant of the present invention or for the ADAMTS13 protein variant used according to the present invention compared to wild-type ADAMTS13. In particular, autoantibodies are autoantibodies present in the serum of patients suffering from immune-mediated thrombotic thrombocytopenic purpura (iTTP). Preferably, the autoantibody is an anti-CUB antibody, that is, an antibody that binds to the CUB1 domain and / or CUB2 domain of ADAMTS13.

[0050] "Reduction" in this specification preferably means that binding is reduced by at least 10%, preferably at least 15%, more preferably at least 20%, more preferably at least 25%, more preferably at least 50%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%. Accordingly, "reduction in binding by autoantibodies compared to wild-type ADAMTS13" preferably means that binding by autoantibodies, as evidenced by the reactivity of the ADAMTS13 protein variant according to the present invention with iTTP patient serum as described in detail below, is reduced by at least 10%, more preferably at least 15%, 20%, 25%, 50%, 75%, 80%, 85%, 90%, or 95% compared to binding of the wild-type ADAMTS13 protein.

[0051] The reduction in binding by autoantibodies in the serum or plasma of iTTP patients can be determined, for example, by measuring the reactivity of the iTTP patient's serum or serums with the ADAMTS13 protein variant in the analysis described in the examples of this specification. Briefly, the binding of autoantibodies to ADAMTS13 can be detected by immobilizing ADAMTS13 directly on the surface, or indirectly via a monoclonal antibody or a polyclonal antibody (or a V5-tag, a His-tag, or another tag). Subsequently, the immobilized ADAMTS13 is incubated with a patient-derived biological fluid, preferably plasma or serum, to allow anti-ADAMTS13 immunoglobulin to bind to the immobilized ADAMTS13. The bound patient-derived immunoglobulin can then be detected using a conjugated or labeled antibody that specifically recognizes human immunoglobulin. An example of such an analysis is presented in Example 7. Alternatively, a monoclonal patient-derived anti-CUB antibody can be prepared to test its binding to ADAMTS13 or variants according to the present invention by ELISA. Examples are presented in Examples 5 and 6.

[0052] Other methods for detecting antigen-specific antibodies in biological fluids are extensively described in the literature and can be applied to the detection of antibodies against ADAMTS13 (e.g., Burbelo PD and O'Hanlon TP, 2014). Since autoantibodies in iTTP patient serum are heterogeneous, it is preferable to determine the binding of autoantibodies to the ADAMTS13 protein variant of the present invention from multiple iTTP patient serums, e.g., serum or plasma samples from at least five different iTTP patients.

[0053] In some embodiments, the ADAMTS13 protein variant according to the present invention has proteolytic activity against von Willebrand factor (VWF) corresponding to at least 10% of the proteolytic activity against VWF of the wild-type ADAMTS13 protein. In some embodiments, the ADAMTS13 protein variant according to the present invention has proteolytic activity against von Willebrand factor (VWF) corresponding to at least 10% of the proteolytic activity against VWF of the wild-type ADAMTS13 protein in the absence of autoantibodies. In some embodiments, the ADAMTS13 protein variant according to the present invention has proteolytic activity against von Willebrand factor (VWF) corresponding to at least 10% of the proteolytic activity against VWF of the wild-type ADAMTS13 protein in the presence of autoantibodies. In some embodiments, the ADAMTS13 protein variant has a proteolytic activity against VWF corresponding to at least 20%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50% of the proteolytic activity against VWF of the wild-type ADAMTS13 protein.

[0054] As used herein, "proteolytic activity against VWF" refers to the ability of ADAMTS13 or ADAMTS13 protein variants to cleave VWF. As used herein, "x% of the proteolytic activity against VWF of wild-type ADAMTS13 protein" means x% of the proteolytic activity against VWF compared to recombinant wild-type ADAMTS13 under the same conditions. That is, the proteolytic activity against VWF of the ADAMTS13 protein variant according to or used according to the present invention is compared with the proteolytic activity against VWF of the recombinant wild-type ADAMTS13 protein under the same conditions, including the use of the same analytical method, the same duration, and the same protein concentration. A person skilled in the art can evaluate the proteolytic activity of wild-type ADAMTS13 and the ADAMTS13 protein variant according to or used according to the present invention so as to be compared under the same conditions. Proteolytic activity against VWF can be determined, for example, using FRETS-VWF73 (described in Kokame et al. 2005) and VWF multiplex analysis (described in Graca et al. 2019) in the examples of this specification. Proteolytic activity against VWF can be measured, for example, using a commercially available FRETS-VWF73 substrate (AnaSpec, Fremont, Ca, USA) according to the FRETS-VWF73 substrate analysis protocol described in the examples of this specification.

[0055] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites in a CUB domain portion comprising amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43. That is, the one or more N-linked glycosylation sites shown in FIG. 1 or defined in SEQ ID NO. 43 are 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1272, 1273, 1274, 1275, 1276, 1295, 1296, 1297, 1326, 1327, 1328, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1392, 1393 and / or It is present at amino acid residue 1394. In some embodiments, one or more N-glycosylation sites (NXT or NXS, where X is any amino acid excluding P) are present at amino acid residues within the CUB domain where no N-glycosylation site is present in the CUB domain of wild-type ADAMTS13.

[0056] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites in a CUB domain portion comprising amino acid residues 1248-1257, 1274-1276, 1295-1297, 1326-1328, 1364-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0057] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites in a CUB domain portion comprising amino acid residues 1248-1255, 1274-1275, 1295-1296, 1326-1327, 1364-1367 and / or 1392-1393 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0058] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites at amino acid residues 1248, 1249, 1250, 1251, 1255, 1274, 1295, 1326, 1364, 1365, 1366, 1368, and / or 1392 within the CUB domain of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0059] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites at amino acid residues 1251, 1255, 1295 and / or 1368 within the CUB domain of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0060] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites in the CUB domain of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, at amino acid residues 1248, 1249, 1250, 1251, 1255, 1295 and / or 1368.

[0061] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites at amino acid residues 1251, 1255, 1274, 1295 and / or 1368 within the CUB domain of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0062] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites in the CUB domain of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, at amino acid residues 1251, 1255, 1295, 1364, 1365, 1366 and / or 1368.

[0063] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites in the CUB domain of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, at amino acid residues 1251, 1255, 1295, 1364, 1365, 1366, 1368 and / or 1392.

[0064] As demonstrated in the examples, in some embodiments, the ADAMTS13 protein variant of the present invention comprises one or more N-linked glycosylation sites within the CUB1 domain and / or one or more N-linked glycosylation sites within the CUB2 domain. These ADAMTS13 protein variants were shown to significantly reduce binding by anti-CUB domain autoantibodies (e.g., see Fig. 8). Accordingly, in some embodiments, the ADAMTS13 protein variant comprises one or more N-linked glycosylation sites in the CUB1 domain portion comprising 1246-1258, 1272-1276 and / or 1295-1297 amino acid residues of ADAMTS13 shown in FIG. 1 (defined by SEQ ID NO. 43) and one or more N-linked glycosylation sites in the CUB2 domain portion comprising 1326-1328, 1361-1370 and / or 1392-1394 amino acid residues of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43. That is, one or more N-linked glycosylation sites are present on amino acid residues 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1272, 1273, 1274, 1275, 1276, 1295, 1296, and 1297 shown in FIG. 1 (or SEQ ID NO. 43), and 1326, 1327, 1328, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, and 1369 shown in FIG. 1 or SEQ ID NO. 43 One or more N-linked glycosylation sites are present on amino acid residues 1370, 1392, 1393 and / or 1394.

[0065] In some embodiments, the ADAMTS13 protein variant comprises one or two N-linked glycosylation sites in a portion of the CUB1 domain of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43 comprising 1246-1258, 1272-1276 and / or 1295-1297 and one or two N-linked glycosylation sites in a portion of the CUB2 domain comprising amino acid residues 1326-1328, 1361-1370 and / or 1392-1394. In some embodiments, the ADAMTS13 protein variant comprises one N-linked glycosylation site in the portion of the CUB1 domain of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43 containing amino acid residues 1246-1258, 1272-1276 and / or 1295-1297 and one N-linked glycosylation site in the portion of the CUB2 domain containing amino acid residues 1326-1328, 1361-1370 and / or 1392-1394.

[0066] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites in a CUB domain portion comprising amino acid residues 1248-1257, 1274-1276 and / or 1295-1297 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, and one or more N-linked glycosylation sites in a CUB domain portion comprising amino acid residues 1326-1328, 1364-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0067] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in a CUB domain portion comprising amino acid residues 1248-1255, 1274-1275 and / or 1295-1296 of the ADAMTS13 sequence shown in FIG. 1 (defined by SEQ ID NO. 43) and one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in a CUB domain portion comprising amino acid residues 1326-1327, 1364-1367 and / or 1392-1393 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0068] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in a CUB domain portion comprising 1248, 1249, 1250, 1251, 1255, 1274 and / or 1295 amino acid residues as shown in FIG. 1 (defined by SEQ ID NO. 43) and one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in a CUB domain portion comprising 1326, 1364, 1365, 1366, 1368 and / or 1392 amino acid residues as shown in FIG. 1 or defined by SEQ ID NO. 43.

[0069] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in the CUB domain portion comprising 1251, 1255 and / or 1295 amino acid residues shown in FIG. 1 (defined by SEQ ID NO. 43) and an N-linked glycosylation site in the 1368 amino acid residue shown in FIG. 1 or defined by SEQ ID NO. 43.

[0070] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in the CUB domain portion comprising 1248, 1249, 1250, 1251, 1255, and 1295 amino acid residues shown in FIG. 1 (defined by SEQ ID NO. 43), and an N-linked glycosylation site in the 1368 amino acid residue shown in FIG. 1 or defined by SEQ ID NO. 43.

[0071] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in the CUB domain portion comprising 1251, 1255, 1274 and / or 1295 amino acid residues shown in FIG. 1 (defined by SEQ ID NO. 43) and an N-linked glycosylation site in the 1368 amino acid residue shown in FIG. 1 or defined by SEQ ID NO. 43.

[0072] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in a CUB domain portion comprising 1251, 1255 and / or 1295 amino acid residues as shown in FIG. 1 (defined by SEQ ID NO. 43) and one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in a CUB domain portion comprising 1364, 1365, 1366 and / or 1368 amino acid residues as shown in FIG. 1 or defined by SEQ ID NO. 43.

[0073] In some embodiments, the ADAMTS13 protein variant comprises one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in a CUB domain portion comprising 1251, 1255 and / or 1295 amino acid residues as shown in FIG. 1 (defined by SEQ ID NO. 43) and one or more, e.g., 1 or 2, e.g., 1 N-linked glycosylation site in a CUB domain portion comprising 1364, 1365, 1366, 1368 and / or 1392 amino acid residues as shown in FIG. 1 or defined by SEQ ID NO. 43.

[0074] In some embodiments, the ADAMTS13 protein variant of the present invention comprises an N-linked glycosylation site at 1251 amino acid residues and an N-linked glycosylation site at 1368 amino acid residues. As demonstrated in the examples herein, these ADAMTS13 protein variants exhibit significantly reduced binding by anti-CUB domain autoantibodies and increased proteolytic activity comparable to wild-type ADAMTS13.

[0075] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises an N-linked glycosylation site at 1255 amino acid residues and an N-linked glycosylation site at 1368 amino acid residues. As demonstrated in the examples herein, such ADAMTS13 protein variants have significantly reduced binding by anti-CUB domain autoantibodies.

[0076] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises one or more N-linked glycosylation sites selected from Table 1 or Table 2, e.g., 1 to 5, e.g., 1 or 2.

[0077] As illustrated in the examples of this specification, when a CUB-NGLY variant is combined with an ADAMTS13 protein variant that reduces or prevents the binding of anti-spacer target autoantibodies, particularly in patients with diverse autoantibody populations, the ADAMTS13 protein variant of the present invention has been shown to significantly reduce binding to antibodies in the serum of iTTP patients. The introduction of one or more N-glycosylation sites in the CUB domain according to the present invention can be advantageously combined with other mutations in ADAMTS13, particularly other mutations aimed at reducing binding by autoantibodies. Accordingly, in some embodiments, the ADAMTS13 protein variant of the present invention further comprises one or more additional mutations that reduce autoantibody binding to the ADAMTS13 protein variant compared to autoantibody binding to wild-type ADAMTS13, particularly compared to autoantibody binding to ADAMTS13 domains other than the CUB domain. In some embodiments, the ADAMTS13 protein variant comprises amino acids 75 to 1427 of the ADAMTS13 amino acid sequence shown in FIG. 1 (or defined by SEQ ID NO. 43), or is the full-length ADAMTS13 protein variant defined by SEQ ID NO. 43, comprises one or more N-linked glycosylation sites in part of the CUB domain, and optionally comprises additional mutations, preferably mutations as described herein.

[0078] Accordingly, the introduction of one or more N-glycosylation sites in the CUB domain according to the present invention can be advantageously combined with other mutations aimed at reducing binding by autoantibodies that bind to ADAMTS13 domains other than the CUB domain.

[0079] In some embodiments, the ADAMTS13 protein variant of the present invention further comprises one or more additional mutations that reduce autoantibody binding to the ADAMTS13 protein variant compared to autoantibody binding to wild-type ADAMTS13.

[0080] In some embodiments, the introduction of one or more N-glycosylation sites into the CUB domain may be combined with a mutation of ADAMTS13 that reduces binding of autoantibodies to the spacer domain. The spacer domain is 130 amino acids long and is known to mediate several important interactions required for the protease activity of ADAMTS13. It has been shown that truncated variants of ADAMTS13 containing amino acids including the spacer domain, namely amino acids 1-685, also exhibit proteolytic activity (e.g., Xiao et al. 2011 and De Maeyer et al. 2010). The spacer domain contains surface-exposed residues that form a major epitope recognized by anti-ADAMTS13 autoantibodies, which is also referred to as the exocyt-3 domain. This domain contains amino acid residues R568, F592, R660, Y661, and Y665. Alanine mutations in R660, Y661, and Y665 impair ADAMTS13 recognition by VWF (Pos et al. 2010; Pos et al. 2011). However, conservative amino acid substitutions of exocy-3 residues were shown to result in an acquired variant of ADAMTS13 (Jian et al. 2012). Initially, this variant was thought to be resistant to autoantibodies, but subsequent experiments revealed that it remains a target of patient-derived autoantibodies (Graca et al. 2019).

[0081] Accordingly, in some embodiments, the present invention provides an ADAMTS13 protein variant comprising one or more additional mutations that reduce binding by autoantibodies toward the spacer domain. Additionally, an ADAMTS13 protein variant is provided comprising one or more N-linked glycosylation sites in the CUB domain portion comprising amino acid residues 1320–1322 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, and further comprising one or more additional mutations that reduce binding by autoantibodies toward the spacer domain. The reduction in binding by antibodies is compared with ADAMTS13 that does not contain the one or more mutations. The spacer domain comprises amino acid residues S556 to A685 of ADAMTS13 shown in FIG. 1 or defined by SEQ ID NO. 43. One or more mutations in the spacer domain may be mutations that reduce binding by autoantibodies. In some embodiments, the ADAMTS13 protein variant comprises amino acids 75 to 1427 of the ADAMTS13 amino acid sequence shown in FIG. 1 (defined by SEQ ID NO. 43), or the full-length ADAMTS13 protein variant defined by SEQ ID NO. 43, comprising one or more N-linked glycosylation sites in part of the CUB domain, optionally comprising additional mutations, preferably mutations as described herein.

[0082] In some embodiments, in the ADAMTS13 protein variants of the present invention, one or more N-linked glycosylation sites are additionally added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites compared to wild-type ADAMTS13 are shifted in the spacer domain comprising amino acid residues S556 to A685 of ADAMTS13 as shown in FIG. 1 or defined by SEQ ID NO. 43. The addition or shifting of such N-linked glycosylation sites is described in WO 2021 / 242092, which is incorporated herein by reference.

[0083] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises 1 to 5 N-linked glycosylation sites added to or moved to the spacer domain defined herein, e.g., 1 to 3, e.g., 1 or 2, more preferably 1.

[0084] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises 1 to 5 N-linked glycosylation sites, e.g., 1 to 3, e.g., 1 or 2, in the CUB domain portion defined herein, and 1 to 5 N-linked glycosylation sites, more preferably 1 to 3, more preferably 1 or 2, added or moved to the spacer domain defined herein.

[0085] In some embodiments, in an ADAMTS13 protein variant according to or used according to the present invention, one or more N-linked glycosylation sites are added or moved by introducing an N-glycosylation site (NXT or NXS, where X is any amino acid excluding P) into the spacer domain compared to wild-type ADAMTS13 or by moving an N-glycosylation site within the spacer domain to a different location.

[0086] Adding or shifting an N-linked glycosylation site in the spacer domain means that the N-glycosylation site (NXT or NXS, where X is any amino acid excluding P) is present at an amino acid residue that is not present in the spacer domain of wild-type ADAMTS13.

[0087] As used herein, "addition" of an N-linked glycosylation site refers to the introduction of an N-linked glycosylation site that is not present in wild-type ADAMTS13. That is, in ADAMTS13 protein variants with one or more N-linked glycosylations added, the total number of N-linked glycosylation sites is greater than the number of N-linked glycosylation sites in wild-type ADAMTS13.

[0088] As used herein, "shift" of an N-linked glycosylation site means that an N-linked glycosylation site located at a specific amino acid residue of wild-type ADAMTS13 is shifted to another amino acid residue of the ADAMTS13 amino acid sequence. That is, the total number of N-linked glycosylation sites in the ADAMTS13 protein variant with the shifted N-linked glycosylation is equal to the number of N-linked glycosylation sites of wild-type ADAMTS13. In some embodiments, the N-linked glycosylation site is shifted by 1 to 10 amino acid residues. The shift may occur downstream or upstream relative to the location of the N-linked glycosylation site in the wild-type ADAMTS13 sequence. In some embodiments, the N-linked glycosylation site is shifted by 1 to 7, e.g., 1 to 5, e.g., 1 to 4, e.g., 1 to 3, e.g., 1, 2, or 3 amino acid residues, and can be either upstream or downstream relative to the position of the N-linked glycosylation site in the wild-type ADAMTS13 sequence. In some embodiments, the N-linked glycosylation site is shifted by 1 or 2, e.g., 1 amino acid residue.

[0089] In some embodiments, one or more N-linked glycosylation sites added and / or moved in the spacer domain are added and / or moved to a portion of the spacer domain comprising amino acid residues R568 to R670 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43.

[0090] In some embodiments, one or more N-linked glycosylation sites added or moved from the spacer domain defined herein are added to and / or moved to amino acid residues selected from the group consisting of R568, L591, V604, V605, A606, G607, K608, M609, R636, L637, P638, R639, Y665, L668, and combinations thereof. In some embodiments, the ADAMTS13 protein variant according to or used according to the present invention comprises an N-glycan in an amino acid residue mutation selected from the group consisting of R568N, L591N, V604N, V605N, A606N, G607N, K608N, M609N, R636N, L637N, P638N, R639N, Y665N, L668N, and combinations thereof. In some embodiments, additional mutations may be introduced to introduce an N-linked glycosylation site to these amino acids, that is, to introduce the N-linked glycosylation sequence NXT or NXS described above. That is, by introducing a mutation selected from this group, one or more N-linked glycosylation sites are added compared to wild-type ADAMTS13.

[0091] In some embodiments, one or more N-linked glycosylation sites added or moved from the spacer domain defined herein are added to and / or moved to amino acid positions selected from the group consisting of positions 568, 591, 608, 609, 636, 637, 665, 668 and combinations thereof shown in FIG. 1 (defined by SEQ ID NO. 43), more preferably to positions 591, 608, 609, 636, 665, 668 and combinations thereof shown in FIG. 1, more preferably to positions 608, 609, 665 and combinations thereof shown in FIG. 1 (defined by SEQ ID NO. 43).

[0092] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises a mutation selected from the group consisting of 568REY570 to 568NET570 (NGLY1), 591LFT593 to 591NFT593 (NGLY2), 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), 636RLPR639 to 636NLSR639 (NGLY5), 636RLPR639 to 636RNAS639 (NGLY6), 665YGNL668 to 665NVTL668 (NGLY7), 667NLTRP671 to 667LNVTA671 (NGLY8), and combinations thereof. That is, by introducing a mutation selected from this group, one or more N-linked glycosylation sites are added and / or shifted compared to wild-type ADAMTS13. Designations in parentheses, such as "NGLY1", "NGLY2", etc., refer to the variants shown in Tables 3 and 4. The designations "N-glyx" and "NGLYx" are used interchangeably herein as "N-gly1" and "NGLY1" or "N-gly2" and "NGLY2", etc.

[0093] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises a mutation selected from the group consisting of 591LFT593 to 591NFT593 (NGLY2), 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), 665YGNL668 to 665NVTL668 (NGLY7), and 667NLTRP671 to 667LNVTA671 (NGLY8), and more preferably, a mutation selected from the group consisting of 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), and 665YGNL668 to 665NVTL668 (NGLY7). It includes, most preferably, 608KMSI611 to 608NMSI611 (including NGLY3_ mutations. That is, by introducing a mutation selected from this group, one or more N-linked glycosylation sites are added and / or moved compared to wild-type ADAMTS13.

[0094] In some embodiments, an ADAMTS13 protein variant comprising an N-linked glycosylation site at 1251, 1255, 1368 amino acid residues or any combination thereof, preferably comprising an N-linked glycosylation site at 1251, 1255, 1368, 1251+1255, 1251+1368, 1255+1368, or 1251+1255+1368, further comprises one or more N-linked glycosylation sites added to or moved to a spacer domain as defined herein.

[0095] In some embodiments, an ADAMTS13 protein variant according to or used according to the present invention comprises an N-linked glycosylation site at amino acid residues 1251, 1255, 1368, or any combination thereof of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, preferably at amino acid residues 1251, 1255, 1368, 1251+1255, 1251+1368, 1255+1368, or 1251+1255+1368, further comprising one or more N-linked glycosylation sites added to or moved to a spacer domain defined herein.

[0096] In some embodiments, the ADAMTS13 protein variant comprises a full-length ADAMTS13 protein variant, i.e., an ADAMTS13 protein variant having amino acids 1-1427 as shown in FIG. 1 or defined by SEQ ID NO. 43, and comprises one or more mutations provided herein. In some embodiments, the ADAMTS protein variant comprises a portion of the full-length ADAMTS13 protein variant and comprises one or more mutations provided herein.

[0097] In addition to one or more N-linked glycosylation sites provided herein, the ADAMTS13 protein variant according to the present invention may include additional mutations in one or more amino acid residues. In some embodiments, said mutations do not introduce glycosylation sites into the protein variant. In some embodiments, said mutations or mutations are mutations or mutations in the amino acid sequence of the protein variant compared to the amino acid sequence of the wild-type ADAMTS13 protein. For example, one or more mutations that further reduce binding by autoantibodies to the ADAMTS13 protein variant, one or more mutations that increase proteolytic activity against VWF, and / or one or more mutations that increase the stability of the ADAMTS13 protein variant may be introduced.

[0098] In some embodiments, the mutation may be the substitution of one amino acid with another, the insertion of one or more amino acids, or the deletion of one or more amino acids. In some embodiments, one or more mutations are the substitution of one or more amino acids with other amino acids. In some embodiments, the mutation may be introduced throughout the sequence of the protein variant. In some embodiments, one or more mutations, preferably substitutions, are introduced into sites within various domains of ADAMTS13 that are targeted by autoantibodies. In some embodiments, the mutation, e.g., substitution, is introduced into one or more amino acid residues within a spacer domain comprising residues S556 to A685 as shown in FIG. 1 or defined by SEQ ID NO. 43.

[0099] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises a mutation, preferably a substitution, in one or more amino acid residues selected from the group consisting of R568, L591, F592, R636, L637, R660, Y661, Y665, and L668. In some embodiments, one or more amino acids mutated in the gain-of-function mutant, namely R568, F592, R660, Y661, and Y665, e.g., R660, Y661, and Y665, or R568, F592, R660, and Y661, or R568, F592, R660, Y661, and Y665, are mutated in the ADAMTS13 protein variant according to the present specification.

[0100] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises one or more mutations selected from the group consisting of R568K, R568A, R568N, L591A, F592Y, F592A, F592N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A, Y661N, Y665F, Y665A, Y665N, and L668A. As shown in WO 2021 / 242092 and Jian et al. 2012, Pos et al. 2010, and Graca et al. 2019, these mutants maintain or increase the proteolytic activity of ADAMTS13.

[0101] In some embodiments, the ADAMTS13 protein variant according to the present invention includes R568A and Y665A mutations.

[0102] In some embodiments, the ADAMTS13 protein variant according to the present invention includes L591A, R636A, L637A, and L668A mutations.

[0103] In some embodiments, the ADAMTS13 protein variant according to the present invention includes the R568A and Y665A mutations or the L591A, R636A, L637A and L668A mutations.

[0104] In some embodiments, ADAMTS13 protein variants according to the present invention include R660K, R660A, or R660N; Y661F, Y661A, or Y661N; and Y665F, Y665A, or Y665N mutations.

[0105] In some embodiments, ADAMTS13 protein variants according to the present invention include R568K, R568A, or R568N; F592Y, F592A, or F592N; R660K, R660A, or R660N; and Y661F, Y661A, or Y661N mutations.

[0106] In some embodiments, ADAMTS13 protein variants according to the present invention include R568K, R568A, or R568N; F592Y, F592A, or F592N; R660K, R660A, or R660N; Y661F, Y661A, or Y661N; and Y665F, Y665A, or Y665N mutations.

[0107] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises an N-linked glycosylation site at 1251, 1255 and / or 1368 amino acid residues, preferably comprising an N-linked glycosylation site at 1251, 1255, 1368, 1251 and 1255, 1251 and 1368, 1255 and 1368 amino acid residues of the ADAMTS13 sequence as shown in FIG. 1 or defined by SEQ ID NO. 43, and further comprises one or more of the additional mutations.

[0108] In some embodiments, the ADAMTS13 protein variant is a full-length ADAMTS13 protein variant, i.e., having amino acids 1-1427 as shown in FIG. 1 or defined by SEQ ID NO. 43, and includes the mutation provided herein.

[0109] In some embodiments, the ADAMTS13 protein variant according to the present invention comprises one or more N-linked glycosylation sites selected from Table 1 and / or Table 2, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites, and one or more N-linked glycosylation sites selected from Table 3. In some embodiments, the ADAMTS13 protein variant comprises one or more N-linked glycosylation sites selected from Table 1, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites. In some embodiments, the N-linked glycosylation sites selected from Table 1 are 1251N, 1255N and / or 1368N, more preferably 1251N, 1255N, 1251N+1255N, 1368N, 1251N+1368N and / or 1255+1368N.

[0110] In some embodiments, the ADAMTS13 protein variant comprises one or more N-linked glycosylation sites selected from Table 1 and / or Table 2, preferably 1 to 5, more preferably 1 or 2, and one or more N-linked glycosylation sites selected from Table 3. In some embodiments, the ADAMTS13 protein variant comprises one or more N-linked glycosylation sites selected from Table 1, preferably 1 to 5, more preferably 1 or 2, and one or more N-linked glycosylation sites selected from Table 3. In some embodiments, the N-linked glycosylation sites selected from Table 1 are 1251N, 1255N and / or 1368N, more preferably 1251N, 1255N, 1251N+1255N, 1368N, 1251N+1368N and / or 1255+1368N. In some embodiments, the N-linked glycosylation site selected in Table 3 is NGLY3, NGLY7 and / or NGLY8, and more preferably NGLY3 or NGLY3 and NGLY7.

[0111] In some embodiments, the ADAMTS13 protein variant comprises one or more selected from Table 1 and / or Table 2, e.g., 1 to 5, e.g., 1 or 2 N-linked glycosylation sites, optionally one or more N-linked glycosylation sites selected from Table 3, and the R568A / F592A / R660A / Y661A / Y665A mutation. In some embodiments, the ADAMTS13 protein variant comprises one or more selected from Table 1, preferably 1 to 5, more preferably 1 or 2 N-linked glycosylation sites, optionally one or more N-linked glycosylation sites selected from Table 3, and the R568A / F592A / R660A / Y661A / Y665A mutation. In some embodiments, the N-linked glycosylation site selected from Table 1 is 1251N, 1255N and / or 1368N, more preferably 1251N, 1255N, 1251N+1255N, 1368N, 1251N+1368N and / or 1255+1368N. In some embodiments, the N-linked glycosylation site selected from Table 3 is NGLY3, NGLY7 and / or NGLY8, more preferably NGLY3 or NGLY3 and NGLY7.

[0112] In some embodiments, the ADAMTS13 protein variants of the present invention are selected from Table 4.

[0113] In some embodiments, the ADAMTS13 protein variant comprises amino acids 75 to 1427 of the ADAMTS13 amino acid sequence shown in FIG. 1 (defined by SEQ ID NO. 43), or the full-length ADAMTS13 protein variant defined by SEQ ID NO. 43, comprising one or more N-linked glycosylation sites in part of the CUB domain, optionally comprising additional mutations, preferably mutations as described herein.

[0114] Exemplary N-linked glycosylation site of the ADAMTS13 CUB domain mutant Original hierarchy Mutated sequence 1251N 1250-W R K M C-1254 1250-W N K T C-1254 1255N 1254-C R K L L-1258 1254-C N K T L-1258 1295N 1294-T F Y R E-1298 1294-T N Y T E-1298 1368N 1367-R T T A F-1371 1367-R N T T F-1371

[0115] Exemplary N-linked glycosylation site of the ADAMTS13 CUB domain mutant Original hierarchy Mutated sequence 1248N 1247-R L T W R-1251 1247-R N T T R-1251 1249N 1248-L T W R K-1252 1248-L N W T R-1253 1250N 1249-T W R K M-1253 1249-T N R T M-1253 1274N 1276-Q R C G R-1280 1276-Q N C T R-1280 1326N 1325-C R L F I-1329 1325-C N L T I-1329 1364N 1363-T H S L R-1367 1363-T N S T R-1367 1365N 1364-H S L R T-1368 1364-H N L T T-1368 1366N 1365-S L R T T-1369 1365-S N R T T-1369 1392N 1391-S E G F L-1395 1391-S N G T L-1395

[0116] Exemplary N-linked glycosylation site of the ADAMTS13 spacer domain mutant Original hierarchy Mutated sequence NGLY1 567-A REY V-571 567-A NET V-571 NGLY2 590-P LFT H-594 590-P NFT H-594 NGLY3 607-G KMS I-611 607-G NMS I-611 NGLY4 608-K MSI S-612 608-K NST S-612 NGLY5 635-D RLP R-639 635-D NLS R-639 NGLY6 636-R LPR L-640 636-R NAS L-640 NGLY7 (glycan shift from N667 to Y665) 664-EYG NLT -669 664-E NVT LT-669 NGLY8 (glycan transfer from N667 to L668) 667- NLT RP-671 667-L NVT A-671 NGLY3 + NGLY7 607-G KMS I-611 / 664-EYG NLT -669 607-G NMS I-611 / 664-E NVT LT-669 NGLY3+ NGLY8 607-G KMS I-611 / 667- NLT RP-671 607-G NMS I-611 / 667-L NVT A-671

[0117] Exemplary ADAMTS13 N-linked glycosylated variant of the present invention mutant Original hierarchy Mutated sequence 1251N 1250-W R K M C-1254 1250-W N K T C-1254 1255N 1254-C R K L L-1258 1254-C N K T L-1258 1295N 1294-T F Y R E-1298 1294-T N Y T E-1298 1368N 1367-R T T A F-1371 1367-R N T T F-1371 1251N+1368N 1250-W R K M C-1254 / 1367-R T T A F-1371 1367-R N T T F-1371 / 1367-R N T T F-1371 1255N+1368N 1254-C R K L L-1258 / 1367-R T T A F-1371 1254-C N K T L-1258 / 1367-R N T T F-1371 5ALA+1251N R568 / F592 / R660 / Y661 / Y665 / 1250-W R K M C-1254 A568 / A592 / A660 / A661 / A665 / 1250-W N K T C-1254 5ALA+1368N R568 / F592 / R660 / Y661 / Y665 / 1367-R T T A F-1371 A568 / A592 / A660 / A661 / A665 / 1367-R N T T F-1371 5ALA+1251N+1368N R568 / F592 / R660 / Y661 / Y665 / 1250-W R K M C-1254 / 1367-R T T A F-1371 A568 / A592 / A660 / A661 / A665 / 1250-W N K T C-1254 / 1367-R N T T F-1371 5ALA+1255N+1368N R568 / F592 / R660 / Y661 / Y665 / 1254-C R K L L-1258 / 1367-R T T A F-1371 A568 / A592 / A660 / A661 / A665 / 1254-C N K T L-1258 / 1367-R N T T F-1371 NGLY3+1251N+1368N 607-G KMS I-611 / 1250-W R K M C-1254 / 1367-R T T A F-1371 607-G NMS I-611 / 1250-W N K T C-1254 / 1367-R N T T F-1371 NGLY3+1255N+1368N 607-G KMS I-611 / 1254-C R K L L-1258 / 1367-R T T A F-1371 607-G NMS I-611 / 1254-C N K T L-1258 / 1367-R N T T F-1371 NGLY3+NGLY7+ 1251N+1368N 607-G KMS I-611 / 664-EYG NLT -669 / 1250-W R K M C-1254 / 1367-R T T A F-1371 607-G NMS I-611 / 664-E NVT LT-669 / 1250-W N K T C-1254 / 1367-R N T T F-1371 NGLY3+NGLY7+ 1255N+1368N 607-G KMS I-611 / 664-EYG NLT -669 / 1254-C R K L L-1258 / 1367-R T T A F-1371 607-G NMS I-611 / 664-E NVT LT-669 / 1254-C N K T L-1258 / 1367-R N T T F-1371

[0118] In some embodiments, the ADAMTS13 protein variant of the present invention does not include an N-linked glycosylation site that is not present in wild-type ADAMTS13, in addition to the N-linked glycosylation site defined herein. In some embodiments, the ADAMTS13 protein variant of the present invention further includes one or two of the N-linked glycosylation sites defined herein.

[0119] The present invention also provides an ADAMTS13 protein variant comprising an N-linked glycan at one or more N-linked glycosylation sites defined herein. In some embodiments, the ADAMTS13 protein variant comprises amino acids 75 to 1427 of the ADAMTS13 amino acid sequence shown in FIG. 1 (or defined by SEQ ID NO. 43), or is a full-length ADAMTS13 protein variant defined by SEQ ID NO. 43, comprising one or more N-linked glycosylation sites in part of the CUB domain, and optionally comprises additional mutations, preferably mutations as described herein.

[0120] As used herein, the term “N-linked glycan” refers to a carbohydrate portion connected to a protein or protein variant via a nitrogen linkage at an N-glycosylation site. As described in detail above, various N-linked glycans exist, and an N-linked glycan may be any glycan that can be attached to an N-linked glycosylation site as defined herein. A person skilled in the art is well aware of the glycans that can be attached to an N-linked glycosylation site. FIG. 2 shows suitable general N-linked glycan structures and other structures. In some embodiments, the N-linked glycan is an N-glycan selected from the N-linked glycans shown in FIG. 2. Attachment of an N-linked glycan to the ADAMTS13 protein variant described herein may be achieved by methods known in the art, such as recombinantly expressing the protein variant in a suitable host cell capable of producing a glycoprotein containing an N-linked glycan. Suitable host cells include eukaryotic host cells, particularly mammalian cells, such as CHO cells, NS0 cells, SP2 / 0 cells, PERC.6 cells, or HEK293 cells. Alternatively, in vitro glycosylation pattern modification is also possible.

[0121] The present invention also provides a nucleic acid encoding an ADAMTS13 protein variant according to the present invention. Additionally, a nucleic acid structure comprising a nucleic acid sequence encoding an ADAMTS13 protein variant according to the present invention is provided. The nucleic acid sequence and structure according to the present invention are useful for therapeutic applications as well as for the production of ADAMTS13 protein variants according to the present invention. In some embodiments, the nucleic acid encodes amino acids 75 to 1427 of the ADAMTS13 amino acid sequence shown in FIG. 1 (defined by SEQ ID NO. 43), or encodes the full-length ADAMTS13 protein variant defined by SEQ ID NO. 43, comprises one or more N-linked glycosylation sites in part of the CUB domain, and optionally comprises additional mutations, preferably mutations described herein.

[0122] As used herein, the term “nucleic acid” refers to DNA and RNA, including mRNA or cDNA, and synthetic variants thereof. Nucleic acids may be recombinant or synthetic nucleic acids.

[0123] The nucleic acid construct according to the present invention is preferably present in a vector, such as an expression vector or a plasmid. The expression vector may be a viral vector, a nonviral vector, or a plasmid. Non-limiting examples of suitable expression vectors include retroviruses, adenoviruses, adeno-associated, herpes simplex, and lentivirus vectors, nonviral vectors and plasmids, and recombinant vectors. Nonviral expression vectors and plasmids include nucleic acids packaged in synthetic or engineered compositions such as nude DNA, liposomes, polymers, nanoparticles, and molecular conjugates. Methods for preparing such nonviral expression vectors are well known in the art. The expression vector preferably comprises a potent promoter / enhancer, such as a CMV or SV40 promoter, an optimal translation initiation sequence, such as a ribosome binding site and an initiation codon, and / or a transcription termination sequence (including a poly(A) signal when the protein is expressed in eukaryotic cells). A person skilled in the art will understand that the expression vector to be used is determined by the host cell used for the expression of the ADAMTS13 protein variant according to the present invention, preferably a protein variant containing an N-linked glycan. The expression vector is suitable for the expression of the nucleic acid molecules of the present invention in preferably eukaryotic host cells, more preferably mammalian host cells, and more preferably CHO cells, NS0 cells, SP2 / 0 cells, PERC.6 cells and / or HEK293 cells.

[0124] Alternatively, the nucleic acid sequence according to the present invention may be provided to a subject by gene editing technology including CRISPR / Cas, zinc-finger nucleases, and transcriptional activator-like effector nucleases-TALENs to insert a receptor transposable gene at a specific genomic location in or without the presence of an exogenous promoter. Preferred genomic locations include the AAVS1 locus and the PD-1 locus, as are known to those skilled in the art.

[0125] In addition, the present specification provides a pharmaceutical composition comprising an ADAMTS13 protein variant according to the present invention and one or more pharmaceutically acceptable carriers, adjuvants, excipients, and / or diluents. “Pharmaceutically acceptable” means that the adjuvant, carrier, diluent, or excipient is compatible with other components of the formulation and is not harmful, e.g., toxic to the recipient. In general, any pharmaceutically suitable additive that does not interfere with the function of the active compound may be used. The pharmaceutical composition according to the present invention is preferably suitable for human use.

[0126] In some embodiments, the pharmaceutical composition according to the present invention is suitable for parenteral administration or is adapted for such administration. In some embodiments, administration is intravenous, intra-arterial, subcutaneous, and / or intramuscular administration.

[0127] In some embodiments, administration may be performed by injection or infusion. In some embodiments, the composition is a solution containing the ADAMTS13 protein variant of the present invention in a sterile aqueous solution. In some embodiments, the aqueous solution is an isotonic aqueous buffer, an oil solution, a dispersion, an emulsion, and / or a suspension.

[0128] In some embodiments, the pharmaceutical composition comprises one or more of a buffering system, a preservative, an isotonic agent, a chelating agent, a stabilizer, a surfactant, and a solubilizer, and combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. For reference, Remington: The Science and Practice of Pharmacy, 19th edition, 1995 may be used. The pharmaceutical composition may include a local anesthetic for relieving pain at the injection or infusion site.

[0129] In some embodiments, the pharmaceutical composition according to the present invention is a liquid pharmaceutical composition. In some embodiments, the pharmaceutical composition is an aqueous composition. Such compositions are typically solutions or suspensions, but also include colloids, dispersions, emulsions, and multiphase materials. The term “aqueous composition” is defined as a composition containing at least 50% w / w water. Likewise, the term “aqueous solution” is defined as a solution containing at least 50% w / w water, and the term “aqueous suspension” is defined as a suspension containing at least 50% w / w water. In some preferred embodiments, the composition is a sterile aqueous solution, such as an isotonic aqueous buffer.

[0130] In some embodiments, the pharmaceutical composition according to the present invention is a solid composition, such as a lyophilized composition. Such solids, particularly lyophilized compositions, can be reconstituted into a liquid composition before administration to a subject. In particular, a solvent and / or diluent may be added before administration to the subject.

[0131] An appropriate administration regimen, i.e., dosage and administration interval, can be determined by a person skilled in the art based on the condition to be treated and the desired effect, e.g., short-term effect or long-term treatment. The precise dosage and regimen of these compounds and their compositions are determined based on the biological activity of the ADAMTS13 protein variant, the age, body weight, and sex of the subject, the needs of the individual subject being administered, the severity or need of the disease, and the judgment of a medical professional. Examples of suitable dosages are doses of the ADAMTS13 protein variant of the present invention ranging from 0.1 mg to 15 g, e.g., 1 to 10 g. In some embodiments, the pharmaceutical composition comprises an aqueous solution of the ADAMTS13 protein variant according to the present invention and a buffer in which the ADAMTS13 protein variant is present at a concentration of 1 mg / mL or higher, and said composition has a pH of about 5.0 to about 8.0. In a further embodiment, the pharmaceutical composition comprises an aqueous solution of an ADAMTS13 protein variant according to the present invention, and comprises a buffer in which the ADAMTS13 protein variant is present at a concentration of 1 mg / mL to 150 mg / mL, and the composition has a pH of about 5.0 to about 8.0.

[0132] In some embodiments, a pharmaceutical kit or component kit is provided comprising one or more containers filled with one or more pharmaceutical compositions. Various written materials, such as instructions for use or notices in a format prescribed by government agencies regulating the manufacture, use, or sale of pharmaceuticals, may be attached to these container(s), and such notices reflect the agency’s authorization for manufacture, use, or sale for human or veterinary administration. Preferably, the pharmaceutical kit or component kit includes instructions for use.

[0133] In some embodiments, the ADAMTS13 protein variant according to the present invention may be administered to a subject by various routes. For example, the ADAMTS13 protein variant may be administered by a suitable route including parenteral routes such as intra-articular, intramuscular, intravenous, intraventricular, intra-arterial, intrathecal, subcutaneous, or intraperitoneal administration, or non-parenteral routes such as topical (e.g., cream, ointment, eye drops) or nasal (e.g., solution, suspension). Parenteral administration may include, for example, intra-arterial, intramuscular, intravenous, intraventricular, intra-arterial, intrathecal, subcutaneous, or intraperitoneal administration. Intravenous and subcutaneous administration may be most advantageous. In some embodiments, the ADAMTS13 protein variant may be administered to a subject in a hospital by infusion or injection by a medical professional.

[0134] In some embodiments, the ADAMTS13 protein variant according to the present invention may be prepared by methods generally known in the art. For example, a person skilled in the art will understand a method for generating a DNA sequence encoding an amino acid sequence of the protein variant according to the present invention and a method for preparing and isolating a nucleic acid molecule having said DNA sequence using generally known recombinant DNA technology.

[0135] The introduction, addition, or shifting of an N-linked glycosylation site can be achieved by introducing one or more mutations into the amino acid sequence such that the N-linked glycosylation site shifts relative to wild-type ADAMTS13. Such mutations may involve substituting one or more amino acid residues with other amino acid residues, inserting one or more amino acid residues, deleting one or more amino acid residues, or a combination thereof, in such a way that the position of the N-linked glycosylation site is introduced, added, or shifted relative to the position of the N-linked glycosylation site in the wild-type ADAMTS13 sequence. In a preferred embodiment, the N-linked glycosylation site is introduced, added, or shifted by substituting one or more amino acid residues with other amino acid residues. In particular, any amino acid may be substituted with an asparagine residue to introduce asparagine as the first residue of the N-linked glycosylation site NXS or NXT defined herein, proline may be substituted with any other amino acid to remove potential proline as the second residue of the N-linked glycosylation site NXS or NXT defined herein, and any amino acid other than serine and threonine may be substituted with serine or threonine to introduce serine or threonine as the third residue of the N-linked glycosylation site NXS or NXT defined herein, or a combination thereof. Alternatively or additionally, asparagine may be substituted with any other amino acid to remove the N-linked glycosylation site present in wild-type ADAMTS13.

[0136] The sequence of the nucleic acid molecule may be codon-optimized for expression in a suitable host cell. The nucleic acid molecule may be inserted into the expression vector described herein using recombinant DNA technology known to a person skilled in the art. In the context of the present invention, the expression vector induces the expression of the protein variant according to the present invention in the suitable host cell described herein. Alternatively, the nucleic acid molecule may be inserted into the genome of the host cell using suitable gene editing technology. Such insertion is preferably made at a locus or region that ensures the expression of the nucleic acid molecule of the present invention in the host cell.

[0137] As used herein, the term “host cell” refers to any cell capable of expressing a heterologous protein, polypeptide, or peptide. In a preferred embodiment, the host cell may attach an N-linked glycan to the protein, peptide, or polypeptide. In a further preferred embodiment, the host cell is selected from the group consisting of eukaryotic host cells, more preferably mammalian cells, more preferably CHO cells, NS0 cells, SP2 / 0 cells, PERC.6 cells, and HEK293 cells. Suitable transfection techniques are known in the art (e.g., Green & Sambrook., 2012. “Molecular Cloning: A Laboratory Manual”, 4 th Edition, CSHL Press; Cold Spring Harbor Protocols, www.cshprotocols.cshlp.org).

[0138] In some embodiments, the ADAMTS13 protein variant according to the present invention is less susceptible to binding of anti-CUB domain autoantibodies compared to wild-type ADAMTS13 and known ADAMTS13 protein variants. In some embodiments, therefore, the present invention provides a method for reducing autoantibody binding to ADAMTS13 or a variant thereof, particularly binding by anti-CUB domain antibodies, which comprises introducing one or more, preferably one or two, N-linked glycosylation sites to the CUB domain portion comprising amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, to reduce autoantibody binding to ADAMTS13 or a variant.

[0139] As described in detail in this specification, an anti-CUB domain autoantibody binding to ADAMTS13 is believed to enhance the plasma clearance of ADAMTS13. In some embodiments, therefore, the present invention provides a method for reducing the plasma clearance of ADAMTS13 or a variant thereof, particularly antibody-mediated plasma clearance, which comprises introducing one or more, preferably one or two, N-linked glycosylation sites into a portion of the CUB domain comprising amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of ADAMTS13 as shown in FIG. 1 or defined by SEQ ID NO. 43, thereby reducing the plasma clearance of ADAMTS13 or a variant thereof, particularly reducing the plasma clearance of ADAMTS13 or a variant thereof. That is, the one or more N-linked glycosylation sites are 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1272, 1273, 1274, 1275, 1276, 1295, 1296, 1297, 1326, 1327, 1328, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1392, 1393 and / or as shown in FIG. 1 or SEQ ID NO. 43. It is present at amino acid residue 1394. As used herein, the term “plasma removal” refers to the rate at which ADAMTS13 or a variant thereof is removed from the blood after administration to a subject.

[0140] In some embodiments, the ADAMTS13 protein variant into which one or more N-linked glycosylation sites are introduced is wild-type ADAMTS13. In some embodiments, the ADAMTS13 into which one or more N-linked glycosylation sites are introduced is an ADAMTS13 protein variant. In some embodiments, the ADAMTS protein variant is an ADAMTS13 protein variant having reduced binding to autoantibodies specific to the spacer domain compared to the wild-type ADAMTS13, and comprises, for example, the addition of one or more N-linked glycosylation sites compared to the wild-type ADAMTS13 as defined herein and / or a shift of one or more existing N-linked glycosylation sites compared to the wild-type ADAMTS13 in the spacer domain comprising amino acid residues S556 to A685 of ADAMTS13 shown in FIG. 1 (defined by SEQ ID NO. 43) and / or one or more mutations in one or more residues in the spacer domain comprising residues 556 to 685 of ADAMTS13 shown in FIG. 1 (defined by SEQ ID NO. 43). In some embodiments, the N-linked glycosylation site is added to and / or moved to amino acid residues 568, 591, 608, 609, 636, 637, 665 and / or 668, preferably to amino acid residues 608, 609 and / or 665, and / or comprises a mutation in an amino acid residue selected from the group consisting of R568, L591, F592, R636, L637, L668, L591, F592, R636, L637, R660, Y661, Y665.

[0141] Accordingly, in some aspects, the ADAMTS13 protein variant resulting in reduced autoantibody binding and / or reduced (autoantibody-mediated) plasma clearance compared to wild-type ADAMTS13 is an ADAMTS13 protein variant according to or used according to the present invention, wherein one or more N-linked glycosylation sites are added and / or one or more existing N-linked glycosylation sites are shifted compared to wild-type ADAMTS13 in a spacer domain comprising amino acid residues S556 to A685 of ADAMTS13 shown in FIG. 1 (defined by SEQ ID NO. 43), and / or additional mutations are added to one or more amino acid residues within a spacer domain comprising residues 556 to 685 of ADAMTS13 shown in FIG. 1 or SEQ ID NO. 43 defined herein.

[0142] In some embodiments, the ADAMTS13 protein variant according to the present invention has VWF proteolytic activity and is therefore particularly useful for the treatment of disorders characterized by abnormal VWF activity and / or VWF treatment and / or thrombotic disease.

[0143] The present invention provides an ADAMTS13 protein variant according to the present invention for use in treatment. The ADAMTS13 protein variant or a nucleic acid structure encoding such ADAMTS13 protein variant is also provided for use as an antithrombotic agent. As used herein, the term “antithrombotic agent” refers to a compound that prevents the formation of a blood clot, reduces or delays the formation of a blood clot and / or neutralizes an existing blood clot, reduces its effects, or breaks down the clot.

[0144] The present invention also provides an ADAMTS13 protein variant according to the present invention or a nucleic acid construct encoding such ADAMTS13 protein variant for use in the treatment of disorders characterized by abnormal von Willebrand factor (VWF) activity and / or VWF treatment. Furthermore, a method for treating disorders characterized by abnormal VWF activity and / or VWF treatment is provided, comprising administering an ADAMTS13 protein variant according to the present invention or a nucleic acid construct encoding such ADAMTS13 protein variant to a subject in need thereof.

[0145] In some embodiments, the ADAMTS13 protein variant of the present invention has reduced binding by anti-CUB domain autoantibodies compared to wild-type ADAMTS13. In some embodiments, this reduced autoantibody binding results in reduced anti-CUB autoantibody-mediated ADAMTS13 clearance. In some embodiments, the subject treated according to the present invention is characterized by having autoantibodies against the ADAMTS13 CUB domain. In some embodiments, the subject treated according to the present invention is characterized by additionally having autoantibodies against the ADAMTS13 spacer domain. In some embodiments, the subject treated according to the present invention is characterized by having autoantibodies against both the spacer domain and the CUB domain. In some embodiments, the subject suffers from iTTP.

[0146] As described in the embodiments of this specification, not all patients with autoantibodies against the spacer domain, particularly iTTP patients, are expected to respond sufficiently to treatment with ADAMTS13 protein variants that reduce autoantibody binding to this domain. For example, they may not respond sufficiently to treatment with ADAMTS13 protein variants that include an N-linked glycosylation site at the 608 amino acid residue of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, particularly those containing a mutation from 607-GKMSI-611 to 607-GNMSI-611 (also referred to herein as the NGLY3 variant). By identifying ADAMTS13 protein variants that reduce autoantibody binding to the CUB domain and variants that reduce autoantibody binding to both the spacer domain and the CUB domain, the inventors have made it possible to treat these patients as well.

[0147] Accordingly, an ADAMTS13 protein variant or nucleic acid construct according to the present invention is provided for use in the treatment of a disorder characterized by abnormal von Willebrand factor (VWF) activity and / or VWF treatment in patients who respond insufficiently to treatment that reduces ADAMTS13 autoantibody binding to the spacer domain. Additionally, a method for treating a disorder characterized by abnormal von Willebrand factor (VWF) activity and / or VWF treatment in patients who respond insufficiently to treatment that reduces ADAMTS13 autoantibody binding to the spacer domain is provided, the method comprising administering a therapeutically effective amount of the ADAMTS13 protein variant according to the present invention to the patient. In some embodiments, the treatment for reducing ADAMTS13 autoantibody binding to the spacer domain is treatment with an ADAMTS13 protein variant comprising an N-linked glycosylation site at amino acid residue 608 of the ADAMTS13 sequence shown in FIG. 1 or defined by SEQ ID NO. 43, particularly a mutation from 607-GKMSI-611 to 607-GNMSI-611 (also referred to herein as the NGLY3 variant). In some embodiments, the patient is a TTP patient, such as an iTTP patient. In this specification, "insufficient response" generally means insufficient efficacy of such treatment. In particular, this means that the response to treatment is insufficient to achieve meaningful clinical results, as evidenced, for example, by insufficient reduction of autoantibody binding and / or insufficient proteolytic activity against VWF. Generally, proteolytic activity against VWF of less than 30% of wild-type ADAMTS13 activity is considered insufficient, especially in the absence of autoantibodies.As used herein, “proteolytic activity for insufficient VWF” refers to proteolytic activity for VWF that is less than 30%, preferably less than 25%, more preferably less than 20%, more preferably less than 15%, and more preferably less than 10% of wild-type ADAMTS13 activity, particularly in the absence of autoantibodies. Such proteolytic activity may be determined as described above in an in vitro assay using, for example, FRETS-VWF73 or VWF multiplex assay. In some embodiments, an insufficient response to treatment that reduces ADAMTS13 autoantibody binding to the spacer domain may be identified by evaluating the treatment outcome after the patient has received treatment that reduces ADAMTS13 autoantibody binding to the spacer domain. In some embodiments, an insufficient response to treatment that reduces ADAMTS13 autoantibody binding to the spacer domain may be pre-evaluated before treatment based on the patient’s autoantibody profile. For example, it is possible to pre-evaluate whether the subject possesses autoantibodies against ADAMTS13 and, if so, which domain of ADAMTS13 these autoantibodies target. Based on this, it is possible to assess whether the patient is likely to respond insufficiently to treatment that reduces ADAMTS13 autoantibody binding to the spacer domain.

[0148] Whether a subject has autoantibodies against ADAMTS13 and, if so, which domain of ADAMTS13 these autoantibodies target can be determined using samples of the subject, such as plasma and / or serum samples.

[0149] This can be determined, for example, by using an assay including an ELISA assay that determines whether the eftope to which the autoantibody binds is present in the sample. For instance, an assay may be used in which ADAMTS13 is immobilized on a plate, for instance, such as a Maxisorb plate, and incubated with a patient plasma sample. After washing, the bound autoantibody can be detected using a labeled anti-human IgG antibody. Suitable methods are described in Thomas et al. 2015, which is incorporated herein by reference. ADAMTS13 and its variants can also be immobilized by coating a plate, such as a Maxisorb plate, with a monoclonal antibody targeting the protease domain of ADAMTS13. After incubation with a patient sample, the bound anti-ADAMTS13 antibody can be detected using a labeled anti-human Ig antibody. Suitable methods are described in Thomas et al. 2015 and Postmus et al. 2023.

[0150] As used herein, the term “von Willebrand factor” or “VWF” refers to a plasma glycoprotein that mediates platelet adhesion and aggregation. VWF is a long multiplex with a molecular weight of up to 20,000 kDa or more, synthesized by endothelial cells and megakaryocytes. Most of the circulating VWF is synthesized by endothelial cells. Most of the secreted VWF consists of a thrombolytic extra-large VWF (ULVWF) multiplex. As described above in this specification, the thrombolytic activity of VWF is regulated by restrictive cleavage by ADAMTS13 during normal hemostasis. As used herein, “abnormal VWF activity” means that VWF activity, particularly the thrombolytic activity of VWF, exhibits a deviation from VWF activity in healthy subjects, preferably an increase compared to VWF activity in healthy subjects. Such deviation or increase is a level that results in particularly adverse health effects, namely disease or impairment. "Abnormal VWF treatment" means that the treatment of VWF, particularly VWF multiples, specifically cleavage, exhibits a deviation compared to VWF treatment in healthy subjects, and is reduced, in particular compared to VWF treatment in healthy subjects. As understood by a person skilled in the art, ADAMTS13 protein variants can be used to correct ADAMTS13 deficiency in subjects. Therefore, in principle, any disorder of abnormal VWF activity or treatment can be treated with ADAMTS13 protein variants according to the present invention. The term "ADAMTS13 deficiency" in this specification refers to a condition in which ADAMTS13 fails to perform its role in hemostasis (controlling VWF multiple size through cleavage) as in healthy subjects. This may be caused by low ADAMTS13 protein levels, an excess of substrate VWF, or the presence of autoantibodies against ADAMTS13. Preferably, it is ADAMTS13 deficiency caused by the presence of autoantibodies in subjects.

[0151] In some embodiments, an ADAMTS13 protein variant according to the present invention or a nucleic acid construct encoding the same is provided for use in the treatment of thrombotic diseases, including both acquired and congenital thrombotic diseases. Additionally, a method for treating thrombotic diseases, including both acquired and congenital thrombotic diseases, is provided, comprising administering an ADAMTS13 protein variant according to the present invention or a nucleic acid construct encoding such an ADAMTS13 protein variant to a subject in need thereof. The ADAMTS13 protein variant reduces the activity of VWF, e.g., the thrombopromoting activity of VWF, by cleaving VWF.

[0152] In some embodiments, the disorder is thrombotic microangiopathy. In some embodiments, the disorder is selected from the group consisting of thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), ischemic stroke, systemic thrombosis, COVID19, antiphospholipid syndrome, preeclampsia / HELLP syndrome, sepsis, and sickle cell disease.

[0153] ADAMTS13 is known to have systemic antithrombotic effects, and as described in Chauhan et al. (J Exp Med. 2006 Mar 20;203(3):767-76), spontaneous thrombus formation was observed in Adamts13- / - mice, and it was revealed that ADAMTS13 has potent natural antithrombotic activity and that recombinant ADAMTS13 can be used as an antithrombotic agent. Therefore, the ADAMTS13 protein variant according to the present invention can be advantageously used for the treatment of systemic thrombosis.

[0154] Thrombotic microangiopathy includes thrombotic thrombocytopenic purpura (TTP). Thrombotic thrombocytopenic purpura (TTP) includes both immune-mediated TTP (iTTP) and congenital TTP (cTTP). In a preferred embodiment, thrombotic microangiopathy is TTP. In a more preferred embodiment, the disorder is iTTP. ADAMTS13 levels are significantly reduced in both iTTP and congenital TTP. Recombinant wild-type ADAMTS13 has been approved for the treatment of both cTTP and iTTP (Scully et al, N Engl J Med. 2019 Jan 24;380(4):335-). Therefore, the ADAMTS13 protein variant according to the present invention can be used for the treatment of both iTTP and congenital TTP. Autoantibodies present in iTTP patients limit the effectiveness of treatment with wild-type ADAMTS13, which is present in human plasma or produced as wild-type recombinant ADAMTS13 protein in eukaryotic expression systems. Autoantibody-resistant ADAMTS13 protein variants can immediately restore functional ADAMTS13 levels, thereby alleviating severe thrombotic complications and other thrombotic disorders observed in iTTP patients.

[0155] HUS is characterized by hemolytic anemia, thrombocytopenia, systemic thrombotic microangiopathy (TMA), and renal failure. Partial ADAMTS13 deficiency may be found in patients with HUS. Therefore, the ADAMTS13 protein variant according to the present invention can be advantageously used in the treatment of HUS, particularly HUS associated with partial ADAMTS13 deficiency.

[0156] Thrombosis is the primary underlying mechanism of acute ischemic stroke (AIS). Several studies have confirmed that ADAMTS13 levels are significantly reduced in patients with ischemic stroke, with the lowest levels found in patients with acute stroke. As described in the review by Chen et al. (Front Neurol. 2019 Jul 17;10:772), available evidence suggests that ADAMTS13 is closely associated with the onset, development, and prognosis of ischemic stroke and protects the brain from ischemia-reperfusion injury. The VWF:ADAMTS13 ratio is strongly correlated with stroke risk. The activity and levels of ADAMTS13 have good predictive value for the onset and prognosis of ischemic stroke. Furthermore, in animal studies regarding ADAMTS13 in the treatment of AIS, injecting recombinant ADAMTS13 into wild-type mice 7 days after stroke onset resulted in increased neovascularization and vascular repair, and significantly improved the 14-day prognosis. It is concluded that ADAMTS13 will serve as a new therapeutic agent for ischemic stroke. Therefore, the ADAMTS13 protein variant according to the present invention can be advantageously used for the treatment of ischemic stroke.

[0157] Sepsis is a disease in which coagulopathy is observed, and thrombotic microangiopathy may be a component thereof. Thrombotic microangiopathy in sepsis is associated with low ADAMTS13 levels. Ramsi and Al Ali (2018) described a case of sepsis-associated thrombotic thrombocytopenic multiple organ failure (TAMOF), in which ADAMTS13 activity was restored through plasma exchange, thereby halting the pathological process and organ failure. Therefore, the ADAMTS13 protein variant according to the present invention can be advantageously used for the treatment of sepsis, particularly thrombotic microangiopathy in septic patients.

[0158] A low ADAMTS13 / VWF ratio was found in sickle cell disease, and ADAMTS13 activity was lower in patients with acute chest syndrome. This suggests a quantitative decrease in ADAMTS13 levels and implies that administration of recombinant ADAMTS13 may have beneficial effects (Sins et al. 2017). This indicates that the ADAMTS13 protein variant according to the present invention can be advantageously used for the treatment of sickle cell disease.

[0159] Thrombosis affecting pulmonary and systemic blood vessels is common during severe coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Turecek et al. (2021) showed that a significant increase in plasma VWF levels is accompanied by a partial decrease in ADAMTS13, a VWF-regulating protease. Incubation of plasma samples from severe COVID-19 patients with recombinant ADAMTS13 (rADAMT13) significantly reduced abnormally high VWF activity, decreased overall multiplex size, and depleted UHMW VWF multiplexes in a time- and dose-dependent manner, suggesting that rADAMTS13 may play a therapeutic role in restoring hemostatic balance in COVID-19 patients. This indicates that the ADAMTS13 protein variant according to the present invention may be advantageously used in the treatment of COVID-19 and / or SARS-CoV-2 infection.

[0160] Antiphospholipid syndrome and preeclampsia (PEcl) are associated with reduced ADAMTS13 levels and high ADAMTS13 antibodies, and exhibit low ADAMTS13 activity and an activity-to-antigen ratio (Bitsadze et al. 2021). Additionally, thrombocytopenia and microangiogenic hemolytic anemia (TMA) are observed in HELLP syndrome. Furthermore, Austin et al. (2008) showed that ADAMTS13 autoantibodies and ADAMTS13 dysfunction can occur in antiphospholipid syndrome. This indicates that the ADAMTS13 protein variant according to the present invention can be advantageously used in the treatment of antiphospholipid syndrome and preeclampsia / HELLP syndrome, particularly in cases associated with ADAMTS13 dysfunction.

[0161] The features of this specification may be described as identical or distinct aspects or parts of embodiments for clarity and concise description. A person skilled in the art will understand that the scope of the invention may include embodiments having all or part of the features described in this specification.

[0162] The present invention will be described in more detail in the following non-limiting examples.

[0163] Examples

[0164] Example 1: Design of N-glycan variants of ADAMTS13

[0165] Patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) develop autoantibodies against ADAMTS13. In the majority of patients, these antibodies target the immune-dominant region of the ADAMTS13 spacer domain (Cabin et al., 2002; Ercig et al., 2018; Jian et al., 2012; Pos et al., 2010; Wouter Pos et al., 2011). The introduction of N-glycans into the spacer domain has been shown to reduce antibody binding in these patients (Ercig et al., 2021; Postmus et al., 2023). Nevertheless, autoantibody binding was not completely eliminated. This is because 20–40% of iTTP patients also develop antibodies against other domains of ADAMTS13, particularly the C-terminal CUB1 and CUB2 domains (Kangro et al., 2021; Klaus et al., 2004; Long Zheng et al., 2010; Pos et al., 2010; Thomas et al., 2015). These antibodies do not directly inhibit ADAMTS13 but reduce its functional level by increasing the rate of clearance of ADAMTS13 from plasma (Thomas et al., 2015). Additionally, these antibodies can convert the ADAMTS13 into an open form, which increases accessibility to the immune-dominant epitopes of the spacer domain (Roose et al., 2020).

[0166] In its resting state, ADAMTS13 circulates in the blood in a closed form. Upon binding to its substrate, von Willebrand factor, ADAMTS13 is converted to an open form (South et al., 2014). The conformational state of ADAMTS13 is pH-dependent; it exists in an open form at pH 6.0 and in a closed form at pH 7.5 (Joshua et al., 2014; Muia et al., 2013). It has been previously shown that ADAMTS13 activity in the closed form (pH 7.5) (measured by the conversion of the synthetic peptide substrate FRETS-VWF73) is lower than that in the open form (pH 6.0) (Joshua et al., 2014; Muia et al., 2013).

[0167] The present invention provides an ADAMTS13 protein variant in which an N-glycan is introduced to residues around the spacer-CUB interface of ADAMTS13 to reduce autoantibody binding while minimizing interference with spacer-CUB interactions. If the N-glycan interferes with this interaction, the spacer domain will still be more accessible to anti-spacer antibodies. Based on the structure and model of the spacer and CUB domains of ADAMTS13 and their interactions, several N-glycans were introduced into the CUB domain of ADAMTS13 (Figure 3A) (Kim et al., 2021). The selection of the N-glycan introduction site relied on two main criteria. First, the N-glycan was introduced around the modeled spacer-CUB interface to minimize the potential for morphological changes within this part of the protease. Second, because mutating inwardly oriented side chains can potentially affect protein stability and / or N-glycan attachment efficiency, the amino acid side chains in the crystal structure had to be outwardly oriented. Based on these criteria, multiple sites for N-glycan insertion were selected. An N-glycan attachment common sequence (NXT) was introduced at these selected sites. All mutations and their locations within the CUB1 or CUB2 domains are shown in Figures 3A and 3B. Additionally, combination mutants were designed in which two N-glycans were introduced into the CUB domain at amino acid positions 1251 / 1368 and 1255 / 1368.

[0168] Example 2: Expression of CUB N-glycan variant

[0169] All N-glycan variants of ADAMTS13 were expressed in ExpiCHO cells. Full-length wild-type ADAMTS13, full-length ADAMTS13 protein variants containing five alanine substitutions (R568A / F592A / R660A / Y661A / Y665A) (used herein synonymously with “ADAMTS13-5ALA” or “5ALA” or “FL-5ALA”), and full-length ADAMTS13 protein variants containing an N-glycan insertion at K608 (NGLY3; 608KMSI611 to 608NMSI611) were used as controls. As additional controls, two truncated variants were included containing wild-type amino acids 1-685 without the C-terminal domain containing the CUB1 and CUB2 domains, one containing the five alanine mutations (referred to herein as "MDTCS-5ALA") and the other not (referred to herein as "MDTCS"). All ADAMTS13 constructs were generated without a progenitor peptide; the ADAMTS13 signaling peptide was replaced with an immunoglobulin kappa signaling peptide to increase ADAMTS13 production. All constructs were generated in pcDNA3.1+ expression vectors containing a C-terminal V5 tag for detection and a Twin-Strep-tag for purification. The CUB-NGLY variant was constructed by designing and ordering a synthetic DNA fragment encoding residues R1094 through T1427 containing amino acid substitutions for the CUB-NGLY of interest from Azenta. The synthetic fragment was flanked by MreI and XhoI restriction sites and cloned into full-length wild-type ADAMTS13, ADAMTS13-5ALA, and NGLY3 constructs. The resulting plasmids were sequenced in-house to identify desired mutations and subsequently expressed in ExpiCHO cells as previously described (Postmus et al., 2023).The supernatant was collected 5 days after transfection and centrifuged at 4000g for 30 minutes to collect the supernatant. 10 mM benzamidine was added, aliquoted, and stored at -30°C. ADAMTS13 levels were determined by ELISA as previously described (Postmus et al., 2023). ADAMTS13 protein levels ranged from 1.81 to 25 μg / mL. Western blots of the ADAMTS13 variant are shown in Figures 12A-B.

[0170] Antigen levels of ADAMTS13 and variants variant Antigen level (μg / mL) Wild type ADAMTS13 16.92 Wild type ADAMTS13-5ALA (5ALA) 17.00 1251N 11.09 1255N 13.01 1265N 1.81 1295N 7.11 1320N 14.54 1321N 12.63 1322N 14.34 1368N 9.23 1382N 4.75 1251N+1368N 7.11 1255N+1368N 6.52 5ALA+1251N+1368N 4.45 5ALA+1255N+1368N 7.38 NGLY3+1251N+1368N 12.9 NGLY3+1255N+1368N 18.18

[0171] Example 3: Confirmation of N-glycan attachment using mass spectrometry

[0172] Mass spectrometry was used to determine whether the introduction of the NXT common sequence resulted in N-glycan attachment. The following CUB-NGLY variants were analyzed: 1251N, 1255N, 1295N, 1320N, 1321N, 1322N, 1368N, 1251N+1368N, and 1255N+1368N. Each of these was purified by immunoprecipitation using Strep-Tactin affinity chromatography and then treated with PNGaseF or not treated (control). After PNGaseF treatment, the CUB-NGLY variants were digested with chymotrypsin or thermorisin, depending on the enzyme that produces a peptide compatible with mass spectrometry. Treatment of the protein with PNGaseF results in the removal of N-glycans and the deamidation of N-glycan-attached asparagines, which leads to a mass shift of +0.98 Da detectable by mass spectrometry. To exclude false positives, the same deamidation events were analyzed in samples not treated with PNGaseF. For each single CUB-NGLY and double CUB-NGLY mutant, a unique deamidation event was observed at the position of interest, indicating that all N-glycans were successfully introduced (Fig. 4). In samples not treated with PNGaseF, no deamidation event was detected at the corresponding position.

[0173] Example 4: Activity of an exemplary CUB-NGLY variant

[0174] The activity of an exemplary CUB-NGLY variant (an ADAMTS13 protein variant containing one or more mutations causing an NXT site in the CUB domain) was evaluated using the FRETS-VWF73 substrate. It is a minimal peptide of 73 amino acids from the A2 domain of VWF containing the Y1605-M1606 cleavage linkage cleaved by ADAMTS13, the binding of VWF, and subsequent amino acids critical for ADAMTS13 activation. The activity of the CUB-NGLY variant was performed as previously described (Postmus et al., 2023). The activity of the diluted ADAMTS13 supernatant was performed with 1.05 nM ADAMTS13 protein variant and 4 μM FRETS-VWF73 substrate in a final volume of 200 μL of activity buffer (20 mM HEPES, 20 mM Bis-Tris, 20 mM Tris-HCl, 25 mM CaCl2, 0.005% Tween20) at pH 6.0. Activity was measured by detecting 460 nm emission every 30 seconds for 45 minutes. The initial activity slope of each mutant was measured. The activity of the CUB-NGLY mutant was calculated using the calibration curve of wild-type ADAMTS13 (concentration range of 0.13125–2.10 nM). Subsequently, the activity of the mutant was expressed as a percentage of wild-type ADAMTS13 at 1.05 nM. The results are shown in Figure 5. The activity of all CUB-NGLY variants except 1251N-1368N was equivalent to that of wild-type ADAMTS13. 1251N-1368N showed a twofold increase in activity.

[0175] The activity of the exemplary CUB-NGLY variant was also measured using a full-length VWF multiple as a substrate. This activity analysis was performed as previously described (Postmus et al., 2023). 3.8 nM ADAMTS13 was diluted in 10 mM HEPES, 10 mM Bis-Tris, 10 mM Tris-HCl, 12.5 mM CaCl2, and 0.0025% Tween20 (pH 7.5). In parallel, 80 nM VWF multiple was diluted in the same buffer and supplemented with 3 M urea. Both samples were incubated at 37°C for 30 minutes. Subsequently, ADAMTS13 was mixed with the VWF multiple at a 1:1 v / v ratio to obtain a final concentration of 1.9 nM ADAMTS13, 40 nM VWF, and 1.5 M urea. After incubation at 37°C for 10 minutes, samples were collected, and the reaction was stopped by adding 4x sample buffer (9.6 M urea, 4% SDS, 0.035 M Tris-base, 25 mM EDTA, 7.5 μM bromophenol blue). The quenched samples were subsequently incubated at 56°C for 10 minutes and loaded onto a 1.8% high-gelation temperature agarose gel. Uncleaved VWF multiples were added as a control. All CUB-NGLY variants cleaved high molecular weight VWF multiples to a level similar to that of wild-type ADAMTS13 (Fig. 6).

[0176] Example 5: Expression and Characterization of Anti-CUB Antibodies Derived from iTTP Patients

[0177] To investigate the antibody binding evasion ability of the newly introduced N-glycan, seven iTTP patient-derived anti-CUB1 / 2 IgG1 antibodies and one anti-spacer IgG1 antibody (1-416) were cloned based on published sequences (Ostertag et al., 2016; US 2019 / 0031771 A1). Synthetic gene fragments for the IgG1 heavy and light chains were ordered in Azenta and cloned into pcDNA3.1+ using HindIII and NotI. Variable domain gene fragments for each antibody were designed for both the heavy and light chains and ordered based on published sequences. The heavy chain fragments were flanked by HindIII and Esp3I restriction sites. The light chain fragments were flanked by HindIII and BsiWI restriction sites. Variable domains were cloned into the full-length heavy or light chains using the aforementioned restriction sites. For each human monoclonal antibody, a plasmid containing the heavy chain and a plasmid containing the light chain were constructed. These plasmids were co-transfected in a 1:1 ratio in an Expi293 expression system. The supernatant was collected by centrifugation at 4000g for 30 minutes 5 days after transfection, supplemented with 10 mM benzamidine, and stored at -80°C until use. Antibody concentrations were determined using a commercially available IgG ELISA (E80-104, Bethyl). Concentrations ranged from 30.1 to 137.4 μg / mL.

[0178] Antigen levels of produced monoclonal iTTP patient-derived antibodies antibodies Antigen level (μg / mL) 1-403 33.2 1-407 66.1 1-410 37.2 1-416 67.7 1-440 81.2 1-441 137.4 Z1-201 30.1 Z1-303 91.9

[0179] Once the antibodies were produced, ELISA was used to confirm that they did not target the spacer domain. Plates were coated with 1 μg / mL of 3H9 antibody and incubated overnight at 4°C. The next day, the plates were blocked with 2% BSA in PBS. Subsequently, 1.05 nmol of ADAMTS13 or MDTCS variants (truncated ADAMTS13 variants lacking the C-terminal domain containing the CUB1 and CUB2 domains) were added to each well in 1% BSA-PBS to a final volume of 175 μL. Then, 0.033 nmol of the corresponding antibody was added to a final volume of 100 μL. Detection was performed using an anti-IgG1-HRP antibody diluted 1:10000. This showed that all produced anti-CUB antibodies bound to full-length ADAMTS13 but not to the MDTCS variant (Fig. 7A). This indicates that they are indeed targeting the C-terminal domains of ADAMTS13. After the binding of the monoclonal antibodies to the C-terminal domains was confirmed, the inhibitory potential of these antibodies was also tested using the aforementioned FRETS-VWF73 activity assay. The same assay as described above was performed, except that each antibody was added at a final concentration of 9 nM. As a control, an anti-spacer antibody (1-416) was also included to determine if the usage concentration of 9 nM was sufficient to inhibit ADAMTS13 activity. Indeed, the anti-spacer antibody 1-416 completely inhibited ADAMTS13 activity, and none of the anti-CUB antibodies significantly reduced ADAMTS13 activity (Fig. 7B). Additionally, antibody 1-440 significantly increased activity, while the other antibodies showed no significant increase in ADAMTS13 activity upon addition.The 1-440 antibody significantly increased activity, which is consistent with current literature on anti-CUB antibodies that promote the conversion of ADAMTS13 from a closed form to an open form (Deforche et al., 2015; Joshua et al., 2014; Schelpe et al., 2020). This can ultimately lead to increased activity against the FRETS-VWF73 substrate as well as the exposure of normally hidden coded spacer epitopes for the binding of anti-spacer antibodies.

[0180] Example 6: The CUB-NGLY variant reduces the binding of monoclonal patient-derived antibodies.

[0181] After the characteristics of the anti-CUB antibody were tested and confirmed to match the patient's anti-CUB antibody, the CUB-NGLY variant was screened for binding to the generated human anti-CUB domain antibody panel. This was done using the same ELISA used in Example 5. Seven anti-CUB antibodies (1-403, 1-407, 1-410, 1-440, 1-441, z1-201, z1-303) and one anti-spacer antibody (1-416) were used. Wild-type ADAMTS13 was included as a 100% binding control, and MDTCS variants (truncated ADAMTS13 variants lacking the C-terminal domain containing the CUB1 and CUB2 domains) were included as negative controls for the anti-CUB antibody and positive controls for the anti-spacer antibody. Additionally, antibody 1-416 was used to verify the equal loading of ADAMTS13 protein in this ELISA-based setup. Reactivity was represented by a heatmap with binding to wild-type ADAMTS13 set to 100%, as shown in Figures 8A and 8B. The binding data in Figures 8A and 8B were calculated using two different computational methods to cross-validate conclusions derived from the data. First, the background signal obtained from wells not containing ADAMTS13 was subtracted from the antibody binding OD value. The binding data in Figure 8A is expressed as a binding percentage based on OD values. First, the background signal obtained from wells not containing ADAMTS13 was subtracted from the antibody binding OD value. Subsequently, the background subtraction OD value of the antibody binding to the CUB-NGLY or MDTCS variant was divided by the background subtraction OD value of the antibody binding to wild-type ADAMTS13. This ratio was then expressed as a binding percentage. In Fig. 8B, an internal standard was used to calibrate the intrinsic sigmoid reaction curve of the ELISA. Here, the dilution curve of human monoclonal anti-ADAMTS13 antibody II-1 (ranging from 0.11 ng / mL to 250 ng / mL) was included in all binding experiments.The calibration curve was fitted using GraphPad Prism 9.1 with a sigmoid, 4PL standard curve where X is the concentration. All data were interpolated to this binding curve. The II-1 binding equivalent of the antibody binding to the CUB-NGLY or MDTCS variant was divided by the II-1 binding equivalent of the same antibody to wild-type ADAMTS13. This ratio was subsequently expressed as a percentage of binding to wild-type ADAMTS13. Data points exceeding the calibration curve were excluded, and data points below the standard curve were set to 0% binding. Reactivity was then represented by a heatmap with binding to wild-type ADAMTS13 set to 100%.

[0182] Overall, no major differences were observed in the binding of human monoclonal antibodies to the ADAMTS13 protein variant in Figures 8A and 8B. 1251N and 1255N were able to strongly reduce the binding of 1-407, 1-410, and z1-303. They also showed a moderate effect on the binding of 1-440 and z1-201. 1251N showed a negligible effect on the binding of 1-403. 1320N, 1321N, and 1322N had no effect on antibody binding. Finally, 1295N and 1368N were able to reduce the binding of 1-440 and z1-201, with the effect of 1368N being much stronger than that of 1295N. Additionally, 1368N was able to completely reduce the binding of the 1-441 antibody. The 1251N+1368N and 1255N+1368N combination mutants exhibited significantly reduced binding to 1-407, 1-410, 1-440, 1-441, z1-201, and z1-303. The combination of 1251N and 1368N resulted in an additional reduction of the 1-403 antibody (more pronounced when using the second calculation method in Fig. 8B), even though 1368N alone had no effect, suggesting that these two N-glycans act synergistically.

[0183] Example 7: The CUB-NGLY variant of ADAMTS13 reduces autoantibody binding in iTTP plasma

[0184] Experiments using human monoclonal antibodies showed that the CUB-NGLY variant of ADAMTS13 could reduce the binding of anti-CUB antibodies. However, all human monoclonal antibodies produced were derived from a single patient. Therefore, it was important to screen the CUB-NGLY variant against a panel of plasma samples from iTTP patients. This would help determine whether the designed CUB-NGLY variant could potentially be used to reduce autoantibody binding in iTTP patients. To this end, the binding of the newly generated CUB-NGLY variant to nine patient samples was evaluated using the same ELISA described in Example 5. Previously, it was shown that the selected nine patient samples contained antibodies targeting the C-terminal TSP2-CUB2 domain of ADAMTS13 (Postmus et al., 2023). This time, instead of adding monoclonal antibodies, the patient samples were diluted and added to the ELISA plate. The optimal dilution factor was previously established and set at a level where patient samples exhibited an optical density of 1.7 at 450 nm upon binding to wild-type ADAMTS13. The dilution factors ranged from 25x to 200x (Postmus et al., 2023). Detection was performed using a mixture of anti-IgG1-HRP, anti-IgG2-HRP, anti-IgG3-HRP, and anti-IgG4-HRP antibodies. To calibrate the intrinsic sigmoid reaction curve of the ELISA, the dilution curve of the human monoclonal anti-ADAMTS13 antibody (II-1) was included in all binding experiments. Calibration curves were fitted using GraphPad Prism 9.1 with a sigmoid, 4PL standard curve where X is log(concentration). All data were extrapolated to this binding curve and expressed as the percentage of wild-type ADAMTS13 binding. Reactivity was subsequently represented by a heatmap with binding to wild-type ADAMTS13 set to 100% (Fig. 9A).In addition, an alternative calculation method was used in which raw OD values ​​were interpolated to a II-1 calibration curve (ranging from 0.11 ng / mL to 250 ng / mL) fitted with GraphPad Prism 9.1 using a sigmoid, 4PL standard curve where X is the concentration (Fig. 9D).

[0185] Compared to wild-type ADAMTS13, all patients showed average binding levels of 83% and 64% for NGLY3 and FL-5ALA (5ALA), respectively (Fig. 9A / B). Similarly, an alternative calculation showed binding levels of 81% and 60% (Fig. 9D / E). These two ADAMTS13 protein variants prevent the binding of anti-spacer target antibodies, as previously shown. This indicates that these patients still possess a population of antibodies targeting different domains of ADAMTS13. Adding CUB-NGLY combinations, such as 1251N+1368N or 1255N+1368N, to NGLY3 or FL-5ALA (5ALA) resulted in an additional reduction in antibody binding in 8 out of 9 patient samples, with the mean binding decreasing from 83% to 51% for NGLY3+CUB-NGLY and from 64% to 31% for FL-5ALA+CUB-NGLY (Fig. 9A). Under alternative calculations, NGLY3+CUB-NGLY and 5ALA+CUB-NGLY decreased from 81% to 47% and from 60% to 28%, respectively (Fig. 9D). Only LRB-18 patients did not show a reduction in binding with the addition of CUB-NGLY combinations to NGLY3 or FL-5ALA (5ALA). The difference in autoantibody binding after the addition of CUB-NGLY was significant and independent in both calculation methods described in this example (Figs. 9B and 9E), and was remarkably similar for both NGLY3 and FL-5ALA (5ALA) (32% and 33%, respectively; Fig. 9C). The alternative calculation method showed 34% and 32%, respectively (Fig. 9F). Therefore, the results obtained using the two alternative calculation methods described in this example indicate that the results obtained for CUB-NGLY are very similar. This indicates that the effect of CUB-NGLY is independent of the modification of the spacer domain and shows that a distinct antibody epitope is being targeted.This epitope is also being recognized by pathogenic antibodies present in the majority of patient samples with C-terminal antibodies that were analyzed.

[0186] Example 8: Production of additional exemplary CUB N-glycan variants

[0187] Currently, the CUB-NGLY combination mutants 1251N+1368N and 1255N+1368N can reduce the binding of monoclonal antibodies as well as antibodies present in patient samples. An additional panel of CUB-NGLY variants (Table 7) was designed to explore whether a single CUB-NGLY variant could also prevent the binding of pathogenic CUB domain antibodies occurring in iTTP patients. The criteria for selecting amino acids to mutate were the same as those mentioned in Example 1, but this time residues within the spacer-CUB interface were also selected. The new panel of single CUB-NGLY variants could potentially be used to prevent the binding of autoantibodies to the CUB domain occurring in iTTP patients. Synthetic gene fragments flanked by MreI and XhoI were ordered from Azenta, and plasmid construction was performed as described in Example 2, similar to other CUB-NGLY variants.

[0188] List of additional exemplary CUB-NGLY variants mutant Original hierarchy Mutated sequence 1248N R L T W R R N T T R 1249N L T W R K L N W T R 1250N T W R K M T N R T M 1274N Q R C G R Q N C T R 1326N C R L F I C N L T I 1364N T H S L R T N S T R 1365N H S L R T H N L T T 1366N S L R T T S N R T T 1392N S E G F L S N G T L

[0189] Example 9: Form of the CUB-NGLY variant

[0190] To evaluate the morphology of the CUB-NGLY variant, activity against VWF-73 substrates was assessed at pH 6.0 and pH 7.5. Prior to the FRETS-VWF73 activity analysis, Twin-Strep-tagged ADAMTS13 was purified from expression medium using MagStrepXT beads (2-5090-010, IBA Lifesciences) according to the manufacturer's protocol. Expression medium containing a total of 600 μL of ADAMTS13 or CUB-NGLY variants was diluted in 600 μL of binding buffer (100 mM Tris, 150 mM NaCl, pH 8.0) and added to 7.5 μL of MagStrepXT beads. The ADAMTS13 protein variants and beads were incubated in a thermomixer at 37°C and 1200 rpm for 30 minutes. Subsequently, the beads were washed with binding buffer and ADAMTS13 was eluted with elution buffer (100 mM Tris, 150 mM NaCl, 50 mM biotin, pH 8.0). After purification, the ADAMTS13 concentration was determined by ELISA as previously described (Postmus et al., 2023).

[0191] For the analysis of FRETS-VWF73 activity, purified ADAMTS13 protein variants were diluted to 0.5 nM in 100 μL of FRETS activity buffer (20 mM HEPES, 20 mM Tris-HCl, 20 mM Bis-Tris, 25 mM CaCl2, 0.005% Tween20, pH 6.0 or pH 7.5) and incubated at room temperature for 10 minutes. Subsequently, 100 μL of 2 μM FRETS-VWF in FRETS activity buffer (pH 6.0 or pH 7.5) was added to ADAMTS13. The reaction was tracked for 15 minutes at 30-second intervals at 350 nm excitation and 460 nm emission wavelengths using a TECAN Infinite 200. Initial rates were determined by linear regression using GraphPad Prism 9.1.

[0192] The pH dependence of wild-type ADAMTS13 activity was consistent with previous findings, with activity at pH 7.5 decreasing compared to activity at pH 6.0 (Fig. 10). Similar results were observed in 1251N, 1255N, 1320N, 1321N, and 1322N (Fig. 10). On the other hand, 1295N, 1368N, 1251N+1368N, and 1255N+1368N showed the same activity at both pH values, suggesting that these CUB-NGLY variants are more active forms that are open at pH 7.5 compared to wild-type ADAMTS13.

[0193] Example 10: Activation of CUB-NGLY variant under flow conditions

[0194] The activity of the CUB-NGLY variant was evaluated under flow conditions. The following experimental system was used: highly dense primary human umbilical vein endothelial cells (HUVECs) were stimulated with histamine under flow conditions to release so-called VWF strings, which efficiently capture platelets to form "bead-shaped string" structures that have been shown to occur in vascular injury. ADAMTS13 was shown to efficiently process these VWF strings (DONG, 2005; Ercig et al., 2021).

[0195] In this experimental system, we evaluated whether the CUB-NGLY variant could process the VWF variant under flow conditions. To this end, 100,000 HUVEC cells per channel were seeded on collagen-coated (50 μg / mL) Ibidi μ-slides. After seeding, the medium was replaced daily. After 4 days of clustering, a flow test was performed. The flow rate was set to 2 mL / min to induce a shear stress of 2.5 Dynes / cm². After washing the cells with M199 + 0.2% BSA for 5 minutes, the HUVECs were stimulated with 100 μM histamine for 10 minutes. After histamine stimulation, freshly isolated platelets (160 × 10⁶ 9( / L) was added for 5 minutes. After focusing on the platelet-bound VWF string, 0.1 μg / mL of ADAMTS13 or the CUB-NGLY variant was flowed over the newly formed VWF string for 9 minutes. Images were taken every 10 seconds using a Zeiss Axio Observer Z1 microscope. All experiments involving ADAMTS13 or its variants were performed in duplicate. Platelets were counted in each frame using a FIJI, and the platelet count was expressed as a percentage of the number of platelets present in the first frame.

[0196] All tested ADAMTS13 protein variants were able to effectively cleave VWF multiples under flow conditions compared to the negative control. Additionally, 1251N+1368N and 1255N+1368N exhibited activity similar to wild-type ADAMTS13, demonstrating that the CUB-NGLY variants did not impair ADAMTS13's ability to cleave VWF multiples under flow conditions (Fig. 11A). Representative images of the negative control (no ADAMTS13 added), wild-type ADAMTS13, and the 1251N+1368N and 1255N+1368N variants at time points 0, 2, and 9 minutes can be seen in Fig. 11B.

Claims

Claim 1 An ADAMTS13 protein variant comprising one or more N-linked glycosylation sites in the CUB domain portion containing amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394 of the ADAMTS13 sequence defined by SEQ ID NO.

43. Claim 2 The ADAMTS13 protein variant of claim 1, comprising one or more N-linked glycosylation sites at amino acid residues 1251, 1255, 1295 and / or 1368 of the ADAMTS13 sequence defined by SEQ ID NO.

43. Claim 3 An ADAMTS13 protein variant according to claim 1 or 2, comprising: (i) one or more N-linked glycosylation sites in a CUB1 domain portion comprising 1246-1258, 1272-1276 and / or 1295-1297 amino acid residues of ADAMTS13 defined by SEQ ID NO. 43; and / or (ii) one or more N-linked glycosylation sites in a CUB2 domain portion comprising 1326-1328, 1361-1370 and / or 1392-1394 amino acid residues of the ADAMTS13 sequence defined by SEQ ID NO.

43. Claim 4 An ADAMTS13 protein variant according to any one of claims 1 to 3, comprising: (i) an N-linked glycosylation site at the 1251 amino acid residue and the 1368 amino acid residue of the ADAMTS13 sequence defined by SEQ ID NO. 43; or (ii) an N-linked glycosylation site at the 1255 amino acid residue and the 1368 amino acid residue of the ADAMTS13 sequence defined by SEQ ID NO.

43. Claim 5 In any one of claims 1 to 4, the ADAMTS13 protein variant further comprises additional mutations in one or more amino acid residues, wherein, preferably, the mutation in one or more amino acid residues is an ADAMTS13 protein variant in a spacer domain comprising residues 556 to 685 of wild-type ADAMTS13 defined by SEQ ID NO.

43. Claim 6 In claim 5, an ADAMTS13 protein variant further comprising one or more additional mutations that reduce binding by autoantibodies toward the spacer domain, preferably having one or more additional N-linked glycosylation sites added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites in the spacer domain relocated compared to wild-type ADAMTS13. Claim 7 In claim 6, the ADAMTS13 protein variant in which one or more N-linked glycosylation sites are added and / or moved to include N-linked glycosylation sites at amino acid residues 568, 591, 608, 609, 636, 637, 665 and / or 668 of the wild-type ADAMTS13 defined by SEQ ID NO. 43, preferably at amino acid residues 608, 609 and / or 665, preferably at amino acid residue 608. Claim 8 In any one of claims 1 to 7, further comprising one or more mutations in one or more amino acid residues selected from the group consisting of R568, L591, F592, K608, R636, L637, R660, Y661, Y665, and L668, preferably comprising one or more mutations selected from the group consisting of R568K, R568A, R568N, L591A, F592Y, F592A, F592N, K608N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A, Y661N, Y665F, Y665A, Y665N, and L668A, more preferably comprising R568A and Y665A mutations or L591A, R636A, L637A, and ADAMTS13 protein variants containing the L668A mutation. Claim 9 ADAMTS13 protein variant according to any one of claims 1 to 8, comprising N-glycans at one or more N-linked glycosylation sites. Claim 10 A nucleic acid structure comprising a nucleic acid sequence encoding an ADAMTS13 protein variant according to any one of claims 1 to 9. Claim 11 A method for reducing plasma clearance of an ADAMTS13 protein variant, such as one having reduced binding to an autoantibody specific to the spacer domain compared to a wild-type ADAMTS13 protein, wherein the method comprises introducing one or more N-linked glycosylation sites into the CUB domain portion of the ADAMTS13 sequence defined by SEQ ID NO. 43, the CUB domain portion comprising amino acid residues 1246-1258, 1272-1276, 1295-1297, 1326-1328, 1361-1370 and / or 1392-1394, to reduce plasma clearance of the ADAMTS13 variant. Claim 12 ADAMTS13 protein variant according to any one of claims 1 to 9 or nucleic acid structure according to claim 10, for use as a pharmaceutical product, preferably as an antithrombotic agent. Claim 13 ADAMTS13 protein variant according to any one of claims 1 to 9 or nucleic acid construct according to claim 10, for use in the prevention and / or treatment of a disorder characterized by abnormal von Willebrand factor (VWF) activity and / or VWF treatment. Claim 14 ADAMTS13 protein variant according to any one of claims 1 to 9 or nucleic acid construct according to claim 10, intended for use in the treatment of a disorder characterized by abnormal VWF activity and / or VWF treatment in a patient who responds insufficiently to treatment that reduces ADAMTS13 autoantibody binding to a spacer domain, preferably said disorder selected from the group consisting of thrombotic thrombocytopenic purpura (TTP) including immune-mediated TTP (iTTP), hemolytic uremic syndrome (HUS), ischemic stroke, systemic thrombosis, COVID-19, antiphospholipid syndrome, preeclampsia / HELLP syndrome, sepsis, and sickle cell disease. Claim 15 An ADAMTS13 protein variant or nucleic acid construct for use according to any one of claims 12 to 14, characterized in that the subject to be treated has an autoantibody against the ADAMTS13 CUB domain.

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