Method for treating TTP with a single variable immunoglobulin domain, and its use

Abstract

The present invention is based on the finding that administration of a polypeptide comprising at least one immunoglobulin single variable domain against vWF to human TTP patients provides a significantly reduced time to response. The present invention provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) directed against von Willebrand factor (vWF) for use in treating vWF-associated diseases in a human in need thereof. The present invention further relates to dosage unit forms, kits, and medical uses for treating TTP.

Description

Technical Field

[0001] 1. Field of the Invention The present invention is based on the finding that administration of a polypeptide comprising at least one immunoglobulin single variable domain against vWF to human TTP patients provides a significantly shorter time to response and fewer complications. The present invention provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF) for use in treating vWF-related diseases in humans who need it. The present invention further relates to dosage unit forms, kits and medical uses for treating TTP.

Background Art

[0002] 2. Background of the Invention 2.1 Role of vWF in Platelet Aggregation The multimeric plasma protein von Willebrand factor (vWF) is essential for mobilizing circulating platelets to the damaged vascular wall upon vascular injury. This mobilization is mediated through the binding of the vWF A1 domain to the platelet receptor glycoprotein GPIb-IX-V. After being expressed by endothelial cells, vWF is secreted into the circulation as ultra-large multimers or ultra-large vWF (ULvWF). These multimers are processed into smaller, normal-sized multimers through enzymatic cleavage by ADAMTS13. In these normal-sized vWF multimers, the GPIb-IX-V platelet receptor binding site in the A1 domain is hidden and does not react spontaneously with platelets. Conformational activation of the GPIb-IX-V platelet receptor binding site in the A1 domain is caused by immobilization or under shear stress conditions, leading to platelet adhesion and subsequent thrombus formation.

[0003] 2.2 Role of vWF and vWF Processing in the Pathophysiology of TTP Thrombotic thrombocytopenic purpura (「TTP」) is a rare and life - threatening disorder of the blood - clotting system thought to be associated with ULvWF multimers, leading to an increased risk of thrombus formation in small blood vessels due to excessive platelet aggregation. The condition is characterized by systemic platelet aggregation in the microcirculation, resulting in fluctuating ischaemia in many organs. If persistent, this can cause tissue infarction with severe thrombocytopenia and fragmentation of red blood cells. ULvWF multimers have the natural ability to interact spontaneously with the platelet receptor GPIb - IX - V. In healthy subjects, these ULvWF multimers are immediately processed into normal - sized vWF multimers via cleavage by the vWF protease ADAMTS13. However, ADAMTS13 activity has been found to be markedly deficient in both hereditary TTP and acquired idiopathic TTP. Many patients with TTP have autoantibodies against ADAMTS13, which cause a disruption in the processing of ULvWF multimers. As a result, the A1 domain of ULvWF is constantly active and readily interacts with the GPIb - IX - V platelet receptor. This ultimately leads to the characteristic platelet - plug formation found in the TTP patient population.

[0004] Current TTP treatment by plasma exchange (abbreviated herein as 「PE」 or 「PEX」) and transfusion provides replacement ADAMTS13 and removes antibodies against the enzyme, thereby gradually normalizing the processing of ULvWF. However, this treatment requires multiple exchanges and transfusions over several days, during which there is no direct pharmacological targeting of the active process of ULvWF - mediated platelet aggregation.

[0005] While the introduction of pulmonary embolism (PE) and blood transfusions has significantly reduced mortality from idiopathic TTP (TTP) over the past 30 years, the condition still carries significant risks of mortality and morbidity. The mortality rate from acute attacks in acute idiopathic TTP in patients treated with current therapies remains around 10%–30% (Vesely et al. Blood 2003;102: 60-68; Allford et al. Br.J.Haematol. 2003;120: 556-573; Sadler et al. Hematology. Am.Soc.Hematol.Educ.Program. 2004;407-423). In cases of secondary TTP, PE and blood transfusions have been found to be less effective, with significantly higher mortality rates. While PE is considered reasonably effective in cases where the disease is secondary to pregnancy, the mortality rate for acute attacks of TTP is approximately 25%, and rises to over 40% in cases with pre-eclampsia (Martin et al. Am.J.Obstet.Gynecol. 2008;199: 98-104). However, in cases secondary to underlying malignancies or bone marrow transplants, for example, the mortality rate remains 40% to 60% despite the use of such treatment regimens (Sadler et al. 2004, see above; Elliott et al. Mayo Clin.Proc. 2003;78:421-430; Kremer Hovinga and Meyer Curr.Opin.Hematol. 2008;15:445-450.).

[0006] Given the persistently high level of mortality from TTP, as well as the observed PE and transfusion complications, there is a clear need for the development of further therapeutic approaches to supplement or potentially reduce the need for these treatment methods. Research conducted on TTP over the past 30 years has improved our understanding of the disease's pathophysiology and enabled the potential development of novel agents targeting the underlying disease processes. However, there are currently no approved treatments for TTP, and while there are newer treatments currently under evaluation, research into these potential treatments is still in its early stages.

[0007] Immunoglobulin single variable domains (ISVDs) for vWF are described, for example, in WO2004 / 015425, WO2004 / 062551, WO2006 / 074947, WO2006 / 122825, WO2009 / 115614 and WO2011 / 067160. ISVD (e.g., ALX 0081) to vWF has been shown to actively bind to multimeric vWF, thereby blocking the interaction of multimeric vWF of any size and activity level with the GPIb-IX-V platelet receptor. The interaction between ALX 0081 and vWF is highly specific, and it does not interact with human hematopoiesis or platelets. Furthermore, its interference with the platelet GPIb-IX-V receptor is selective, through binding to the vWF A1 domain, and it does not affect vWF's ability to interact with fibrous collagen or type VI collagen. In addition, ISVD (e.g., ALX 0081) to vWF has been shown not to affect the activity of (the remaining) vWF-proteases ADAMTS13, and they do not interfere with the binding of FVIII to vWF.

[0008] In Phase I studies, ALX 0081 was shown to be safe and well-tolerated in healthy volunteers. However, healthy human volunteers are generally unpredictable regarding the efficacy of ISVD against vWF, or specifically ALX 0081, in the underlying pathophysiology of TTP patients. vWF is abnormal not only in quantity but also in quality in TTP patients. While it is accepted that ULvWF does not function normally in hemostasis in TTP patients, the underlying mechanisms are not understood. Higher vWF levels are predicted during acute episodes in TTP patients (Lotta et al. 2011 J Thromb Haemost 9:1744-51; Stufano et al. 2012 J Thromb Haemost 10:728-730). Due to the lack of suitable animal models, the efficacy of ALX 0081 in neutralizing ULvWF has not been demonstrated in vivo.

[0009] Therefore, it remains unclear whether polypeptides containing at least one ISVD for vWF, such as ALX 0081, are beneficial in TTP patients, whether polypeptides containing at least one ISVD for vWF, such as ALX 0081, have a positive effect on PE, and what treatment and dosage regimens are effective. There is a need for improved treatments for TTP patients. [Overview of the Initiative]

[0010] 3. Summary of the Invention This invention is based on the unexpected finding that administration of a polypeptide containing at least one ISVD to vWF to human TTP patients provides a 2-day reduction in time-to-response, objectified by the recovery of platelets ≥ 150,000 / μL. The increase in platelet count is a sign of pathological reduction in platelet aggregation, thereby reducing the thrombotic process initiated by the platelet-vWF complex, which is characteristic of this disease. The hazard ratio ("HR") for placebo to the polypeptide of this invention was surprisingly 2.2, at 95% CI (1.28, 3.78), p=0.013. This response was confirmed up to 48 hours after the time to response. Thus, the proof of concept of the polypeptide of this invention is demonstrated by statistical significance and a clinically meaningful reduction in time to confirmed platelet response. Furthermore, the number of exacerbations decreased from 11 in the placebo group to 3 in the treatment group. There were no deaths in the treatment group compared to 2 deaths in the placebo group.

[0011] Furthermore, this clinical study in TTP patients also demonstrates that the polypeptide of the present invention (e.g., ALX 0081) is well-tolerated, and in particular, that the potential risk of bleeding, while present, is low and manageable. The data currently available therefore demonstrate that reduction of PE and transfusions, and associated complications, can be achieved without significant adverse effects from the use of the polypeptide of the present invention itself. This represents a clear safety benefit of using the polypeptide of the present invention in the treatment of patients with TTP.

[0012] Therefore, administration of polypeptides containing at least one ISVD to vWF to human TTP patients provides an unexpected reduction in time to response, sustained and long-lasting effects, reduced exacerbations, reduced hospitalizations, reduced morbidity, reduced mortality, and reduced number of PEs.

[0013] Current treatment of TTP with PE and transfusions provides replacement ADAMTS13, which removes antibodies against the enzyme, thereby gradually normalizing ULvWF processing. However, this procedure requires multiple exchanges and transfusions over several days, during which time there is no direct pharmacological targeting of the active process of platelet aggregation mediated by ULvWF.

[0014] Now, even more unexpectedly, it has been shown that the polypeptides of the present invention do not interfere with enzymes exchanged by plasma transfusion. The polypeptides of the present invention (e.g., ALX 0081) have been shown to be usable in combination with PE and transfusion to directly inhibit the formation of persistent small thrombi and platelet consumption in the microvascular system. This allows for more rapid control of the basal thrombotic process and associated platelet consumption, which has the benefit of reducing the degree of ischemic and hemorrhagic complications. It also results in faster clinical recovery and lower morbidity, thereby shortening the duration and frequency of PE and transfusions. Indeed, analysis of LDH, troponin T or I, and creatinine, which are specific and clinically relevant organ injury biomarkers, suggests that more rapid reduction of microvascular tissue ischemia is predicted to have clinical benefits. In addition, the polypeptide of the present invention (e.g., ALX 0081) demonstrated inhibition of ULvWF-mediated platelet interactions and exhibited antithrombotic effects, raising the possibility of its longer-term use to prevent disease recurrence after patients have recovered from acute TTP attacks. A reduction in the frequency of acute TTP attacks represents significant benefits, with the potential for lower TTP-related mortality and morbidity, and further, a reduction in the lifetime need for PE and transfusions in patients.

[0015] While faster recovery from TTP and reduced exacerbations and relapses represent clear clinical benefits in terms of treatment efficacy, reduced duration and frequency of PE and blood transfusions also offer further benefits in terms of patient safety. Although PE and blood transfusions are now considered standard procedures in the management of TTP (Scully et al. Br.J.Haem. 2012;158:323-335), the techniques carry a significant risk of complications. The PE technique requires high fluid volume and flow rate, which necessitates the use of a central venous dual-lumen hemodialysis catheter. Complications from this procedure include bleeding from catheter insertion, sepsis, catheter thrombosis, pneumothorax, fluid overload, hypoxia, and hypotension (Fontana et al. Semin. Hematol. 2004; 41: 48-59; George J. Intensive Care Med. 2007; 22: 82-91; Howard et al. Transfusion 2006; 46: 154-156; Rizvi et al. Transfusion 2000; 40: 896-901; Nguyen et al. Transfusion 2009; 49: 392-394). Anaphylactic-like reactions occur in 0.25% to 0.5% of cases (Allford et al 2003, see above; George 2007, see above). In addition, infusion of plasma containing blood products can cause non-infectious TRALI. This condition is recognized as one of the most frequent causes of transfusion-related death, with an estimated incidence of 0.02% to 0.05% per plasma unit, based on an average of 17 plasma units per day, and a daily risk of 0.34% to 0.85%. Most patients with TTP require multiple PEs and transfusions. Patients with acute idiopathic TTP require daily treatment, with an average of approximately 16 treatments needed to achieve remission (Allford et al. 2003, see above). In refractory cases, the frequency of treatment may be increased to twice a day (Allford et al. 2003, see above).In patients with familial TTP, regular prophylactic plasma infusions at 2-3 week intervals are recommended (Lammle et al. J.Thromb.Haemost. 2005;3:1663-1675). Thus, anaphylaxis and TRALI represent a clear risk for patients with TTP who require such frequency and regularity of PE and transfusions. This risk is thought to be lower when solvent / detergent (S / D) treated plasma is used instead of fresh frozen plasma, however, the use of large volumes of S / D plasma may be associated with an increased risk of venous thromboembolism (Allford et al. 2003, see above; Fontana et al. 2004, see above). Overall, it is estimated that approximately 30%–40% of patients will experience adverse effects from PE and blood transfusions, and the mortality rate from this method is around 2%–3% (George et al. Semin. Hematol. 2004;41:60-67; George 2007, see above). Therefore, reducing the duration and frequency of PE and blood transfusions also provides further benefits in terms of patient safety.

[0016] After recovering from a TTP attack, many patients report long-term cognitive impairment, including troublesome problems with memory, concentration, energy levels, and fatigue. These symptoms negatively impact the patient's quality of life. Furthermore, this quality-of-life impairment can occur in all patients with TTP, regardless of etiology or severity (Lewis et al. Transfusion 2009;49:118-124). These symptoms may reflect the residual effects of tissue ischemia. Based on this, it can be reasonably proposed that faster recovery from TTP and limited thrombus formation in the microvascular system, provided by polypeptides of the present invention such as ALX 0081, may result in improved long-term prognosis for patients in terms of their quality of life.

[0017] Accordingly, the present invention provides a method for treating or mitigating vWF-related disease in a subject by administering to the subject a polypeptide comprising at least one ISVD against vWF, wherein the amount of polypeptide administered is effective in shortening the time to response, reducing exacerbations, reducing hospitalizations, reducing ischemia, reducing the number of deaths, and / or reducing the number of required PEs. The present invention provides specific dose ranges and administration schedules for the polypeptide of the present invention that produce one or more of these effects against vWF-related disease. In particular, the present invention provides a pharmacologically active agent, composition, method, and / or administration schedule that has specific advantages compared to agents, compositions, methods, and / or administration schedules currently in use and / or known in the art, including a less frequent need to perform PEs. These advantages will become apparent from the further description below.

[0018] Accordingly, the present invention provides a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF) for use in treating vWF-related diseases in humans in need, comprising administering to the human a first dose of the polypeptide of 1 to 80 mg, for example, 5 to 40 mg, preferably 10 mg. The present invention provides a polypeptide as described herein, wherein a first plasma exchange (PE) is performed within 5 minutes to 8 hours after administration of the polypeptide. The present invention provides polypeptides as described herein, wherein prior plasma exchange (PE) is performed before administration of the first dose, preferably within 36 hours of the first PE, for example, within 32 hours, 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, or 8 hours, for example, within 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or as little as 5 minutes.

[0019] The present invention provides a polypeptide as described herein, wherein a second dose of the polypeptide, 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg, of the polypeptide is administered after the first PE, preferably by subcutaneous injection, preferably within 1 to 60 minutes, more preferably within 30 minutes, of the first PE. The present invention provides a polypeptide as described herein, wherein the prior PE is carried out within 36 hours, preferably within 32, 30, 28, 26, 24, 22, 20, 18 or 16 hours, preferably within about 24 hours, of the first PE.

[0020] The present invention provides polypeptides as described herein, which are administered parenterally, preferably by subcutaneous, intraperitoneal, intravenous or intramuscular injection, and preferably by intravenous (iv) bolus push injection. The present invention provides a polypeptide as described herein, wherein PE is performed within 5 minutes to 8 hours after administration of the polypeptide, for example, within 10 minutes to 6 hours or within 15 minutes to 4 hours, for example, within 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes or as little as 5 minutes.

[0021] The present invention provides polypeptides as described herein, which are used to treat vWF-related diseases in humans requiring such polypeptides, further as follows: (i) Perform PE; and (afterwards) (ii) Administer a dose of 1 to 80 mg of the polypeptide, for example, 5 to 40 mg, 5 minutes to 4 hours after the PE in step (i); and (iii) Optionally, measure the platelet count and / or ADAMTS13 activity of the patient; Includes, Here, steps (i) and (ii) are preferably repeated once per day until the patient's platelet count is ≥150,000 / μl and / or the ADAMTS13 activity is at least 10% of the ADAMTS13 reference activity, for example, at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50%.

[0022] The present invention provides a polypeptide as described herein, further comprising administering a dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg, of the polypeptide once daily for 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, or 120 days, after the patient's platelet count first becomes ≥ 150,000 / μl. The present invention provides a polypeptide as described herein, further comprising administering a dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg, of the polypeptide once daily until the human is in remission. The present invention provides a polypeptide as described herein, comprising administering the polypeptide until the ADAMTS13 activity is at least 10% of the ADAMTS13 reference activity, for example, at least 15%, 20%, 25%, 30%, 35%, 45%, or 50%.

[0023] The present invention provides polypeptides as described herein, wherein the dose is about 1 to 80 mg, or 5 to 40 mg, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, or 80 mg, preferably about 10 mg of the polypeptide. The present invention provides a polypeptide as described herein, wherein the human being suffers from an acute episode of TTP, an exacerbation of TTP, or a relapse of TTP.

[0024] In a preferred aspect, the present invention provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF) for use in treating vWF-related diseases in humans where it is needed, which comprises: (1) Optionally, perform pre-procedure plasma exchange (PE); (2) Administer to the human a first dose of the polypeptide of 1 to 80 mg, for example, 5 to 40 mg, preferably 10 mg (if step (1) is performed, preferably within 36 hours of the completion of step (1), for example, within 32 hours, 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, for example, within 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or just 5 minutes); (3) Perform plasma exchange (PE) (optionally, within 5 minutes to 8 hours of step (2), for example, 10 minutes to 6 hours or 15 minutes to 4 hours, for example, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes or just 5 minutes); (4) Administer a further dose of the polypeptide of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg (preferably within 5 minutes to 8 hours of the end of step (3), for example 10 minutes to 6 hours or 15 minutes to 4 hours, for example 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes or just 5 minutes); (5) Repeat steps (3) and (4) once per day; optionally, until the patient's platelet count is ≥150,000 / μl and / or until the ADAMTS13 activity is at least 10% of the ADAMTS13 reference activity, e.g., at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50%. (6) Optionally, administer a dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg, of the polypeptide once daily for at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, or 120 days, after the patient's platelet count first becomes ≥ 150,000 / μl, or until the ADAMTS13 activity becomes at least 10% of the ADAMTS13 reference activity, for example at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50%.

[0025] In addition, the present invention provides two anti-human vWF ISVD polypeptides for use in preventing recurrence of vWF-related disease (symptoms) in humans by administering a dose of 1 to 80 mg, for example, 5 to 40 mg, preferably 10 mg, of the polypeptide to humans. The present invention provides a polypeptide as described herein, wherein the ISVD for vWF comprises at least one immunoglobulin monovariate domain bound to SEQ ID NO: 20.

[0026] The present invention provides polypeptides as described herein, wherein the ISVD for vWF comprises a heavy chain variable domain derived from a conventional quadruple-chain antibody, or a heavy chain variable domain derived from a heavy chain antibody or Nanobody. The present invention provides a polypeptide as described herein, wherein the Nanobody is VHH.

[0027] The present invention provides a polypeptide as described herein, wherein the ISVD for the vWF essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), where: a) CDR1 is as follows: - Amino acid sequence YNPMG; or - Amino acid sequences that differ from the amino acid sequence YNPMG in only two or one amino acid; including or essentially derived from these, Furthermore b) CDR2 is as follows: - Amino acid sequence AISRTGGSTYYPDSVEG; or -An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the amino acid sequence AISRTGGSTYYPDSVEG; or - Amino acid sequences that differ from the amino acid sequence AISRTGGSTYYPDSVEG by only 2 or 1 amino acid; including or essentially derived from these, Furthermore c) CDR3 is as follows: - Amino acid sequence AGVRAEDGRVRTLPSEYTF; or -An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the amino acid sequence AGVRAEDGRVRTLPSEYTF; or - Amino acid sequences that differ from the amino acid sequence AGVRAEDGRVRTLPSEYTF by only 2 or 1 amino acid It includes or is essentially derived from these.

[0028] The present invention provides polypeptides as described herein, wherein: a) CDR1 is YNPMG (sequence number 21); b) CDR2 is AISRTGGSTYYPDSVEG (Sequence ID 22); and c) CDR3 is AGVRAEDGRVRTLPSEYTF (Sequence ID 23). The present invention provides polypeptides as described herein, where ISVD for vWF is represented by Sequence ID No. 19 (12A02H1). The present invention provides a polypeptide, as described herein, comprising or consisting of at least two ISVDs for vWF.

[0029] The present invention provides a polypeptide as described herein, wherein each of the at least two ISVDs for a vWF essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), where: a) CDR1 is as follows: - Amino acid sequence YNPMG; or - Amino acid sequences that differ from the amino acid sequence YNPMG in only two or one amino acid; including or essentially derived from these, Furthermore b) CDR2 is as follows: - Amino acid sequence AISRTGGSTYYPDSVEG; or -An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the amino acid sequence AISRTGGSTYYPDSVEG; or - Amino acid sequences that differ from the amino acid sequence AISRTGGSTYYPDSVEG by only 2 or 1 amino acid; including or essentially derived from these, Furthermore c) CDR3 is as follows: - Amino acid sequence AGVRAEDGRVRTLPSEYTF; or -An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the amino acid sequence AGVRAEDGRVRTLPSEYTF; or - Amino acid sequences that differ from the amino acid sequence AGVRAEDGRVRTLPSEYTF by only 2 or 1 amino acid It includes or is essentially derived from these.

[0030] The present invention provides polypeptides as described herein, where each ISVD for vWF essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), where: a) CDR1 is YNPMG (sequence number 21); b) CDR2 is AISRTGGSTYYPDSVEG (Sequence ID 22); and c) CDR3 is AGVRAEDGRVRTLPSEYTF (Sequence ID 23). The present invention provides polypeptides as described herein, wherein the polypeptide comprises or is essentially comprised of SEQ ID NOs: 1 to 19, preferably SEQ ID NO: 19.

[0031] The present invention provides polypeptides as described herein, wherein the ISVD for vWF is a single-chain polypeptide comprising one or more immunoglobulin monovariable domains. The present invention provides polypeptides as described herein, wherein the ISVD with respect to vWF is monovalent or polyvalent. The present invention provides polypeptides as described herein, wherein the ISVD for vWF is monospecific or polyspecific. The present invention provides polypeptides as described herein, wherein one or more immunoglobulin monovariate domains are selected by CDR grafting, humanization, camelization, deimmunization, or phage display.

[0032] The present invention provides a polypeptide as described herein, wherein the ISVD for vWF comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. The present invention provides polypeptides, as described herein, comprising two anti-human vWF immunoglobulin monovariable domains (ISVDs) and one anti-human serum albumin (HSA) ISVD. The present invention provides polypeptides as described herein, wherein the polypeptides are formulated in a pharmaceutically acceptable formulation. The present invention provides polypeptides as described herein, wherein the formulation comprises citrate or phosphate buffer having a pH in the range of 5.0 to 7.5.

[0033] The present invention provides polypeptides as described herein, wherein the formulations are suitable for parenteral administration, such as by intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection (one or more selected from these). The present invention provides polypeptides as described herein, wherein the formulations are in liquid form, lyophilized, spray-dried, or reconstituted lyophilized or frozen form. The present invention provides a kit or product comprising a container containing a polypeptide or formulation as described herein, and instructions for use.

[0034] The present invention provides a kit or product as described herein, wherein the formulation is contained in a vial or syringe for injection. The present invention provides a kit or product as described herein, wherein the formulation is present in a pre-filled syringe for injection. The present invention provides a kit or product as described herein, wherein the syringe or vial is made of glass, plastic, or a polymer material selected from cyclic olefin polymers or copolymers.

[0035] The present invention relates to the use of the polypeptide in humans in need of the following treatment of vWF-related diseases by administering a dose of 1 to 80 mg, for example, 5 to 40 mg, preferably 10 mg, of the polypeptide to humans: (a) vWF binder at a concentration of approximately 0.1 mg / mL to approximately 80 mg / mL; (b) Excipients selected from sucrose, glycine, mannitol, trehalose, or NaCl in a concentration of approximately 1% to approximately 15% (w / v); (c) Tween-80 at a concentration of approximately 0.001% to 0.5% (v / v); and (d) A buffer selected from citrate buffer at a concentration of approximately 5 mM to approximately 200 mM such that the pH of the formulation is approximately 6.0 to 7.0, and phosphate buffer at a concentration of approximately 10 mM to approximately 50 mM such that the pH of the formulation is approximately 6.5 to 7.5. A formulation is provided, wherein a first plasma exchange (PE) is performed within 5 minutes to 8 hours, for example, within 15 minutes to 4 hours, after the dose.

[0036] The present invention provides a pharmaceutical unit dosage form suitable for parenteral administration to a patient, preferably a human patient, comprising a polypeptide or formulation as described herein. The present invention provides polypeptides as described herein, wherein the vWF-related disease is selected from acute coronary syndrome (ACS), transient ischemic attack, unstable or stable angina, stroke, myocardial infarction, or thrombotic thrombocytopenic purpura (TTP).

[0037] The present invention provides a method for treating human patients who are susceptible to or diagnosed with a disease characterized by vWF-related disease, the method comprising administering an effective amount of a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF) to a human patient. The present invention provides a method for treating or preventing vWF-related diseases such as TTP, the method comprising administering to a human being a dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg, of a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF), thereby alleviating one or more symptoms associated with vWF-related diseases.

[0038] The present invention provides a procedure as described herein, wherein a first plasma exchange (PE) is performed within 5 minutes to 8 hours, for example, within 15 minutes to 4 hours, after administering a polypeptide as described herein. The present invention provides a procedure as described herein, wherein a prior plasma exchange (PE) is performed within 36 hours, preferably within 32, 30, 28, 26, 24, 22, 20, 18 or 16 hours, preferably within about 24 hours, of the first PE before administering a polypeptide as described herein.

[0039] The present invention provides a treatment as described herein, wherein, after the first PE, within 5 minutes to 8 hours, for example, within 10 minutes to 6 hours or 15 minutes to 4 hours, for example, within 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes or just 5 minutes, a second dose of a polypeptide as described herein, of 1 to 80 mg, for example, 5 to 40 mg, preferably 10 mg, is administered, for example, within 1 to 60 minutes, for example, within 30 minutes of the first PE, preferably by subcutaneous injection.

[0040] The present invention further provides treatments as described herein, including: (i) Perform PE; (then) (ii) Administer a dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg, of a polypeptide as described herein, 15 minutes to 4 hours after the PE in step (i); and, (iii) Optionally, measure the platelet count and / or ADAMTS13 activity of the patient. Here, steps (i) and (ii) are optionally repeated once per day until the patient's platelet count is ≥150,000 / μl and / or the ADAMTS13 activity is at least 10% of the ADAMTS13 reference activity, for example, at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50%.

[0041] The present invention also provides a treatment as described herein, further comprising administering a dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg, of a polypeptide as described herein, once daily for at least 5, 10, 15, 20, 25, or further 30 days, after the patient's platelet count has reached ≥ 150,000 / μl. The present invention provides a treatment as described herein, further comprising administering a polypeptide as described herein in a dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg, once daily until the person achieves remission. The present invention provides a treatment as described herein, comprising administering the polypeptide until the ADAMTS13 activity is at least 10% of the ADAMTS13 reference activity, for example, at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50%.

[0042] In one embodiment, the present invention relates to a method for reducing the risk of vWF-related disease and / or preventing an acute episode thereof in a person in need thereof, the method comprising (i) administering to the person a dose of 5 to 40 mg, preferably 10 mg, of a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF); wherein the administration of the polypeptide comprises or comprises reducing the risk of and / or preventing an acute episode of vWF-related disease. Preferably, the risk is reduced by a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 times or more, for example, 3, 4, 5, 6, 7, 8, 9 times or 10 times, or more, for example, 20, 50 or 100 times. Preferably, the risk is reduced by 10% or more, for example, 20%, 30%, 40%, 50%, 60%, or even higher, for example, up to 80% or 100%.

[0043] In one embodiment, the present invention relates to a method as described herein, wherein step (i) of administering the polypeptide of the present invention is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or more than 10 times, for example 20 times, preferably more than 30 times or more. In one embodiment, the present invention relates to a method as described herein, wherein step (i) of administering the polypeptide of the present invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or longer than 10 days, for example 20 days, preferably longer than 30 days, for example 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In one embodiment, the present invention relates to a method as described herein, wherein the dose is administered once or twice a day.

[0044] In one embodiment, the present invention further includes the following, by a method as described herein: (ii) Measure the ADAMTS13 activity of the patient; (iii) comparing the ADAMTS13 activity with the reference ADAMTS13 activity; and (iv) If the ADAMTS13 activity is lower than the reference ADAMTS13 activity of 30%, for example, 20%, 15%, or 10%, repeat step (i) of administering the polypeptide of the present invention.

[0045] In one embodiment, the present invention relates to a method as described herein, wherein the ADAMTS13 activity of the patient is measured daily, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably at least once a week. In one embodiment, the present invention relates to a method as described herein, wherein step (i) of administering the polypeptide of the present invention is repeated until the ADAMTS13 activity is at least 10%, 15%, for example 20%, or 30%, or higher than the reference ADAMTS13 activity.

[0046] In one embodiment, the present invention relates to a method as described herein, wherein step (i) is repeated until the ADAMTS13 activity is at least 10%, 15%, for example 20%, or 30% of the reference ADAMTS13 activity in at least two consecutive measurements. Preferably, the two consecutive measurements are separated by at least 24 hours, more preferably 48 hours, for example, at least 3 days, or longer, for example, 4, 5, 6, or 7 days, preferably 1 week. In one embodiment, the present invention relates to a method as described herein, wherein step (i) of administering the polypeptide of the present invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or longer than 10 days, for example 20 days, preferably longer than 30 days, or longer, after the ADAMTS13 activity has become at least 10% or 15% of the reference activity, for example 20% or 30%, in at least two consecutive measurements.

[0047] In one embodiment, the present invention further includes the following, by a method as described herein: -Measure the ADAMTS13 activity of the aforementioned patient; -Comparing the aforementioned ADAMTS13 activity with the reference ADAMTS13 activity; and - If the ADAMTS13 activity is ≥10% of the reference ADAMTS13 activity, for example, higher than 15%, or higher than 20% or 30%, then step (i) of administering the polypeptide of the present invention is repeated for a maximum of 30 days, for example, a maximum of 20 days, or for 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or just 1 day.

[0048] In one embodiment, the present invention relates to a method for reducing the risk of vWF-related disease and / or preventing an acute episode thereof in a person in need thereof, the method comprising at least the following steps: (i) Measure the ADAMTS13 activity of the patient; (ii) Comparing the ADAMTS13 activity with the reference ADAMTS13 activity; and (iii) If the ADAMTS13 activity is lower than 30%, 20%, 15%, or 10% of the reference activity, administer to the human a dose of 5 to 40 mg of a polypeptide containing at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF).

[0049] In one embodiment, the present invention relates to a method as described herein, wherein - The risk of organ damage, ischemic injury, and / or microthrombus formation is reduced by 10%, 20%, 30%, preferably at least 40%, or at least 50%, for example, up to 60%, 70%, 80%, 90%, or 100%; - The risk of organ damage, ischemic injury, and / or microthrombus formation is reduced by 1.2, 1.3, 1.4, 1.5, 1.75, 2 times or more, for example, 3, 4, 5, 6, 7, 8, 9 times or 10 times, or even higher, for example, 20, 50 or 100 times; - Organ damage, ischemic injury, and / or microthrombus formation are preferably reduced by at least 10%, 20%, 30%, 40%, or at least 50%, for example, up to 60%, 70%, 80%, 90%, or 100%; - Organ damage, ischemic injury, and / or microthrombus formation are reduced by a factor of 2 or more, for example, 3, 4, 5, 6, 7, 8, 9 or 10, or even more, for example, 20, 50 or 100; - Organ damage markers, such as LDH levels, troponin T levels, troponin I levels, and / or creatinine levels, recover to at least 40% of normal levels, or at least 50%, for example, up to 60%, 70%, 80%, 90%, or even up to 100%; - Organ damage markers, such as LDH levels, troponin T levels, troponin I levels, and / or creatinine levels, improve by at least 20% of normal levels, for example 30% or higher, for example 40%, or even as much as 50%, for example 60%, 70%, 80%, 90%, or 100%. Preferably, the organ damage markers, such as LDH levels, troponin T levels, troponin I levels, and / or creatinine levels, improve with treatment for less than 30 days, preferably less than 20 days, for example less than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or within 1 day. - The platelet count is maintained at ≥150,000 / μl. - The risk of exacerbation decreases by at least 10%, 20%, 30%, 40%, or even 50%, and can even decrease to, for example, 60%, 70%, 80%, 90%, or 100%; - The risk of exacerbation decreases by a factor of 2 or more, for example, 3, 4, 5, 6, 7, 8, 9 or 10 times, or even more, for example, 20, 50 or 100 times; - The mortality rate attributable to the vWF-related disease is reduced to 10%, 20%, 30%, preferably at least 40%, or at least 50%, and even to, for example, 60%, 70%, 80%, 90%, or 100%; - The mortality rate due to the aforementioned vWF-related disease decreases by a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 times or more, for example, 3, 4, 5, 6, 7, 8, 9 times or 10 times, or even more, for example, 20, 50 or 100 times.

[0050] In one embodiment, the present invention relates to a method as described herein, further comprising measuring the platelet count; and, if the platelet count is less than 150,000 / μl, repeating step (i) of administering the polypeptide of the present invention. In one embodiment, the present invention relates to a method as described herein, wherein the platelet count of the patient is measured daily, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably at least weekly. In one embodiment, the present invention relates to a method as described herein, wherein step (i) of administering the polypeptide of the present invention is repeated until the platelet count is at least 150,000 / μl.

[0051] In one embodiment, the present invention relates to a method as described herein, wherein step (i) of administering the polypeptide of the present invention is repeated until the platelet count is at least 150,000 / μl in at least two consecutive measurements. Preferably, the two consecutive measurements are separated by at least 24 hours, more preferably 48 hours, for example, at least 3 days, or longer, for example, 4, 5, 6, or 7 days, preferably 1 week. In one embodiment, the present invention relates to a method as described herein, wherein, after the platelet count has reached at least 150,000 / μl in at least two consecutive measurements, step (i) of administering the polypeptide of the present invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or longer than 10 days, for example 20 days, preferably longer than 30 days, or longer. Preferably, the two consecutive measurements are separated by at least 24 hours, more preferably 48 hours, for example at least 3 days, or longer, for example 4, 5, 6, or 7 days, preferably 1 week.

[0052] In one embodiment, the present invention relates to a method as described herein, further comprising measuring the platelet count of the patient; and, if the platelet count is ≥ 150,000 / μl, repeating step (i) of administering the polypeptide of the present invention for a maximum of 30 days, for example, a maximum of 20 days, or for as little as 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or as little as 1 day.

[0053] In one embodiment, the present invention relates to reducing the risk of vWF-related disease and / or preventing an acute episode thereof in a person who needs it, and the method includes at least the following steps: (i) to measure the platelet count of the patient; and (ii) If the platelet count is less than 150,000 / μl, administer to the person a dose of 5 to 40 mg of a polypeptide containing at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF); Herein, administration of the polypeptide reduces and / or prevents the risk of acute episodes of vWF-related disease.

[0054] In one embodiment, the present invention relates to a method for treating an acute episode of a vWF-related disease such as TTP in a human being who needs it, the method comprising at least the following steps; (i) Administer to the human being a first dose of 5 to 40 mg, preferably 10 mg, of a polypeptide containing at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF); (ii) The first plasma exchange (PE) is preferably performed within 5 minutes to 8 hours of step (i).

[0055] In one embodiment, the present invention relates to a method for treating an acute episode of a vWF-related disease, such as TTP, in a human being in need, as described herein, wherein step (i), i.e., before administering the polypeptide of the present invention to the human being, preferably step (ii), i.e., a pre-PE is performed within 24 hours of the first PE. In one embodiment, the present invention relates to a method for treating an acute episode of a vWF-related disease, such as TTP, in a person in need thereof, the method comprising at least the following steps: (i) performing plasma exchange (PE); (ii) administering to the person a dose of 5 to 40 mg, preferably 10 mg, of a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF). Preferably, step (i), i.e., performing PE, and step (ii), i.e., administering to the person the polypeptide of the present invention, are repeated once or twice per day for a maximum of 1, 2, 3, 4, 5, 6, or 7 days.

[0056] In one embodiment, the present invention relates to a method for treating an acute episode of a vWF-related disease, such as TTP, in a human being as described herein, wherein step (ii), i.e., administering the polypeptide of the present invention to the human being, is performed within 15 minutes to 4 hours of step (i), i.e., PE. In one embodiment, the present invention relates to a method for treating an acute episode of a vWF-related disease, such as TTP, in a human being as required thereto, the method further comprising: measuring the platelet count of the human, preferably after step (ii), i.e., administering the polypeptide of the present invention to the human; and, if the platelet count is <150,000 / μl, repeating step (i), i.e., performing PE, and step (ii), i.e., administering the polypeptide to the human.

[0057] In one embodiment, the present invention relates to a method for treating an acute episode of a vWF-related disease, such as TTP, in a human being in need, as described herein, the method further comprising: measuring the platelet count of the human being [preferably in step (ii), i.e., after administering the polypeptide of the present invention to the human being]; and repeating step (i), i.e., performing PE, and step (ii), i.e., administering the polypeptide to the human being [once / twice per day] until the platelet count is at least 150,000 / μl in at least two consecutive measurements. Preferably, the two consecutive measurements are separated by at least 24 hours, more preferably 48 hours, for example, at least 3 days, or longer, for example, 4, 5, 6, or 7 days, preferably 1 week.

[0058] In one embodiment, the present invention relates to a method for treating an acute episode of a vWF-related disease, such as TTP, in a human being as described herein, the method further comprising administering a dose of 5 to 40 mg, preferably 10 mg, of the polypeptide once daily for at least 1 to 30 days, after the human's platelet count first becomes ≥ 150,000 / μl. In one embodiment, the present invention relates to a method for treating an acute episode of a vWF-related disease, such as TTP, in a human being as described herein, the method further comprising measuring the ADAMTS13 activity of the human, preferably in step (ii), i.e., after administering the polypeptide to the human.

[0059] In one embodiment, the present invention relates to a method for treating an acute episode of a vWF-related disease, such as TTP, in a human being as needed, as described herein, wherein step (i), i.e., performing PE, and step (ii), i.e., administering the polypeptide of the present invention to the human being, are repeated until the ADAMTS13 activity is [for the first time] higher than 15% of the reference ADAMTS13 activity, or even 20% or 30%.

[0060] In one embodiment, the present invention relates to a method for reducing and / or preventing the risk of ischemic injury, organ damage and / or microthrombus formation [which may be caused by vWF-related disease] in a person who needs it, the method comprising at least the following steps: (i) administer to the human being a dose of 5 to 40 mg / day, preferably 10 mg / day, of a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF); wherein the administration of the polypeptide reduces and / or prevents the risk of ischemic injury, organ injury and / or microthrombus formation by 10%, 20%, 30%, preferably at least 40%, or at least 50%, or even up to, for example, 60%, 70%, 80%, 90%, or 100%. Preferably, administration of the polypeptide reduces and / or prevents the risk of ischemic injury, organ injury and / or microthrombus formation at a multiplier of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 times or higher, for example, 3, 4, 5, 6, 7, 8, 9 times or 10 times, or higher, for example, 20, 50 or 100 times.

[0061] In one embodiment, the present invention relates to a method for repeating step (i) of administering the polypeptide for at least 1, 2, 3, 4, 5, 6, or 7 days, or longer, for example, 1 week, 2 weeks, 3 weeks, or longer, for example, 1 month or 2 months. In one embodiment, the present invention further comprises measuring the ADAMTS13 activity of the patient, preferably once every week. In one embodiment, the present invention relates to a method for repeating step (i) of administering the polypeptide when the ADAMTS13 activity is [for the first time] ≥10%, for example 15%, or ≥20%, of the reference ADAMTS13 activity, for at least 1, 2, 3, 4, 5, 6, 7 days, or longer, for example 1 week, 2 weeks, 3 weeks, or longer, for example 1 month or 2 months.

[0062] In one embodiment, the present invention relates to a method for treating symptoms of vWF-related diseases such as TTP in a person suffering from the disease, the method comprising administering an effective amount to treat the symptoms of vWF-related diseases in a person suffering from the disease. In one embodiment, the present invention relates to a method for inhibiting the onset or progression of vWF-related diseases such as TTP in humans, wherein the inhibition is achieved by conjugating a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) to von Willebrand factor (vWF), which comprises administering an effective inhibitory dose of the polypeptide to a human at prescribed intervals, wherein each administration of the polypeptide delivers to the human at a dose of 0.1 mg to 25 mg per kg of human body weight, thereby inhibiting the onset or progression of the disease in the human.

[0063] In one embodiment, the present invention relates to a method for reducing the likelihood of a human being to suffer ischemic organ injury due to vWF-related disease, the method comprising administering to a human being a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF) in a prescribed dose, wherein each administration of the antibody delivers to the human being 0.1 mg to 25 mg per kg of human body weight, thereby reducing the likelihood of the human being to suffer ischemic organ injury. [Brief explanation of the drawing]

[0064] 4. Drawing Description [Figure 1] Procedure flowchart. [Figure 2] The curve showing the time to normalization of the first LDH (ITT group = subjects with abnormally high levels at baseline). [Figure 3] Curves showing the time to normalization of troponin T or I in subjects with abnormally high baseline levels in an intent-to-treat population. [Figure 4] Curve showing the time to normalization of creatinine levels in subjects with abnormally high baseline levels within the treatment-intended population.

[0065] [Figure 5] vWF levels in TTP patients: Percentage decrease from baseline in free vWF levels as a function of daily dose levels at the end of period 2, including patients treated with placebo, as predicted by the model. Median, 25th and 75th percentiles are shown. [Figure 6] PK / PD model of caplacizumab and free total vWF and complexed vWF: (A) Model-predicted caplacizumab concentration profile after daily sc administration during periods 1 and 2 (with and without daily PE concomitant administration). (B) Model-predicted free vWF levels during daily 10 mg caplacizumab sc administration and after the treatment period. (C) Approximate complexed caplacizumab-vWF levels during daily 10 mg caplacizumab sc administration and after the treatment period. (D) Model-predicted total vWF levels during daily 10 mg caplacizumab sc administration and after the treatment period. Median profiles, 5th and 95th percentiles are shown. [Modes for carrying out the invention]

[0066] 5. Detailed explanation Unless otherwise indicated, all methods, steps, techniques and operations not specifically described can and have been performed in a manner known in itself, as will be apparent to those skilled in the art. For example, references are made to standard handbooks and the general background art mentioned herein, and to the further references cited therein, as well as to the following reviews: Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6):640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12; Gonzales et al., Tumour Biol., 2005, 26(1), 31-43. These describe techniques for protein engineering, such as affinity maturation, as well as other techniques for improving the specificity and other desired properties of proteins, such as immunoglobulins.

[0067] It should be noted that, as used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Therefore, for example, a reference to “a reagent” includes one or more different reagents, and a reference to “the method” includes references to equivalent steps and methods known to those skilled in the art that may modify or substitute for the method described herein. Unless otherwise indicated, the term “at least” preceding a set of elements should be understood to refer to all elements within that set. Those skilled in the art can understand or confirm, by conventional experimentation alone, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the present invention.

[0068] Wherever the terms “and / or” are used herein, they include the meanings of “and,” “or,” and “all or any other combination of the elements that are joined by the terms.” When used herein, the terms “about” or “approximately” mean within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range. Throughout this specification and the subsequent claims, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” shall be understood to include the integer or step or group of integers or steps described, but not to exclude any other integer or group of integers or steps. Where used herein, the term “comprising” may be replaced by the term “containing” or “including” or, at times, the term “having.”

[0069] The potential therapeutic capacity of the polypeptides of the present invention, such as ALX 0081, in the setting of TTP was demonstrated by in vitro experiments in flow chamber experiments using plasma from TTP patients. In these experiments, endothelial cells were stimulated to produce strings of ULvWF on their surface (see Example 7.2). It was shown that the polypeptides of the present invention, such as ALX 0081, could inhibit platelet-vWF interactions and, in particular, ULvWF-mediated platelet interactions in vitro, and that they did not affect the function of ADAMTS13. In particular, it was shown that the polypeptides of the present invention, such as ALX 0081, can interact with vWF in the active stage (i.e., functional for interaction with GPIb-IX-V as a normal-sized multimer and as a hypermacro-multimer) and with vWF in its inactive stage (a normal-sized multimer before the steric change of the A1 domain). The study provided proof of concept that the polypeptides of the present invention, such as ALX 0081, can be used to treat TTP patients. The study also demonstrates that the polypeptides of the present invention, such as ALX 0081, do not interfere with ADAMTS13 activity.

[0070] The present invention is at least in part based on the finding that administration of a polypeptide containing at least one ISVD to vWF (also referred herein as "the polypeptide of the present invention") to human TTP patients provides an unexpected two-day reduction in time to response. The time to response is objectified by the time required for platelet count to recover to ≥150,000 / μL. In addition, the present invention provides unexpectedly sustained long-term effects, reduced exacerbations, reduced hospitalizations, reduced morbidity, reduced number of required PEs, reduced ischemia, reduced organ damage, and reduced mortality.

[0071] Accordingly, the present invention relates to the use of the polypeptide of the present invention to treat or alleviate vWF-related disease in patients by an unexpectedly large reduction in the time to response, as demonstrated by the promotion of platelet recovery. The present invention also provides a lower frequency of platelet recovery while still maintaining platelet recovery for an unexpectedly long period in human patients. Therefore, a method is provided for shortening the time to response in human patients by administering the polypeptide of the present invention to a patient, wherein the amount of polypeptide administered is effective in altering one or more disease markers of TTP, such as platelet count, thrombocytopenia, neurocognitive function, ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin repeats 13) levels and anti-ADAMTS13 antibody titer, ADAMTS13 activity level, cardiac markers (troponin T or troponin I), BNP (brain natriuretic peptide) or N-terminal pro-brain natriuretic peptide (NT proBNP), and brain injury markers (e.g., NSE (neuron-specific enolase) and Sβ100 (S100 beta)), preferably an increase in platelet count.

[0072] In addition, the polypeptides of the present invention were tested for safety when administered to human TTP patients using safety markers, such as RICO, vWF, and FVIII chromogens. While there is a possibility of an increased risk of bleeding, this was fully manageable. Markers can be measured using standard methods known and used by those skilled in the art, such as various immunological assays (enzyme-linked immunosorbent assays (ELISA; also known as enzyme immunoassays (EIAs)), radioimmunoassays, or immunoenzymatic assays). Chemical, colorimetric, and enzyme-based assays can also be used where appropriate.

[0073] Accordingly, the present invention provides a polypeptide comprising at least one ISVD for vWF for use in treating vWF-related diseases in humans in need by administering a dose of 5 to 40 mg of the polypeptide to a human, wherein a first plasma exchange (PE) is performed 15 minutes to 4 hours after the dose. The polypeptide of the present invention was administered as an adjunct treatment at a specific time point relative to the PE method to treat or prevent vWF-related diseases, such as TTP (e.g., to alleviate or reduce one or more associated symptoms).

[0074] The term “treatment” refers to administering a treatment in an effective amount, form, and / or mode to improve a disease-related condition, symptom, or parameter, or to prevent disease progression, to a statistically significant extent or to a degree detectable by a person skilled in the art. In therapeutic use, the treatment may improve, cure, maintain, or shorten the duration of the disease or condition in the subject. In therapeutic use, the subject may have partial or complete signs of symptoms, in typical cases. The treatment improves the disease or condition in the subject to a degree detectable by a physician, or prevents the worsening of the disease or condition. For example, the clinical features and signs of an acute episode of TTP (Scully et al. 2012, above) improve, as shown in Table 1 or provided in the TTP treatment guidelines. For example, the treatment may result in normalization of platelet count, a decrease in the titer of ADAMTS13 autoantibody, and / or an increase in ADAMTS13 activity. These are all publicly known in the art and / or are further described herein (see below). The effective quantity, form, or mode may vary depending on the subject and can be adapted to the subject.

[0075] As used herein, the term “prevention” means alleviating the symptoms of the disorder in question. In particular, the term encompasses the full range of therapeutic positive effects of administering the polypeptides of the present invention to the subject, including reduction, alleviation, and relief from vWF-related disorders, e.g., TTP, and their symptoms. The term “prevention” includes preventing or delaying the onset of a disease, preventing or delaying the onset of symptoms, and / or reducing the severity of symptoms that would develop or are expected to develop. These further include relieving existing symptoms, preventing further symptoms, and relieving or preventing the underlying causes of symptoms.

[0076] As used herein, the terms “subject” and “patient” are interchangeable. As used herein, “subject” and “patient” refer to mammals, including animals such as non-primates (e.g., cattle, pigs, horses, donkeys, goats, camels, cats, dogs, guinea pigs, rats, mice, sheep) and primates (e.g., monkeys such as crab-eating macaques, gorillas, chimpanzees, and humans). “Patient” preferably refers to humans. The patient may include elderly, adults, adolescents and children of all ages, such as children in the range of 2 to under 12 years, adolescents in the range of 12 to under 18 years, adults in the range of 18 to under 65 years, and elderly people 65 years and older.

[0077] Non-limited examples of vWF-related diseases that can be treated include, but are not limited to, acute coronary syndrome (ACS), transient ischemic attack, unstable or stable angina, stroke, myocardial infarction, thrombotic thrombocytopenic purpura (TTP), and Upshaw-Schulman syndrome, preferably TTP. For example, PE's approach to managing vWF-related diseases such as TTP is described in "the Guidelines on the diagnosis and management of TTP and other thrombotic microangiopathies" (Scully et al. 2012, above), which is expressly incorporated herein by reference. Complete remission is defined as a normal platelet count, i.e., ≥150,000 / μl, and optionally, the absence of exacerbations (see Scully et al. 2012 above).

[0078] As used herein, “time to response” is the time between a first treatment of a patient having an acute episode of TTP and a platelet count of ≥150,000 / μl, where the first treatment is the administration of PE or the polypeptide of the present invention, or both, which may be the earliest. The term "plasma exchange" ("PE") refers to a therapeutic technique used to treat a variety of diseases, including TTP, through bulk removal of plasma, specifically the removal of a large volume of plasma, typically 1 to 1.5 plasma volumes, and replacement with a replacement fluid (Winters 2012 Hematology ASH Education Book 1:7-12). Through bulk removal and replacement of plasma, PE removes pathogenic substances such as autoantibodies against ADAMTS13 and ULvWF, but also removes some platelets. Plasma is used as a replacement fluid to replace ADAMTS13 when treating thrombotic thrombocytopenic purpura (McLeod Best Pract Res Clin Haematol. 2006;19:157-167). Bulk removal and replacement of plasma also has implications for clinical testing and complicates patient examination.

[0079] Since PE involves bulk removal of plasma, anything circulating in the plasma is removed. Therefore, this method is non-selective and removes both normal and pathological plasma components, as well as any medications administered prior to PE to treat TTP. Those skilled in the art are well aware of how to determine the number of platelets. Platelet counts can be performed by any method known in the art, for example, manually using a hematologist or by an automated analyzer, such as electronic counting. Counts can also be estimated during blood smear testing. Microscopy involves observing blood on a hematologist slide using a phase-contrast microscope. Electronic counting of platelets is the most common method. Two types of electronic counting exist: voltage pulse and electro-optical counting systems. For example, platelet counts can be obtained using an ADVIA hematology analyzer, and the obtained counts can be verified by estimating the count in a Wright-stained blood smear. ADVIA measures platelets by flow cytometry based on the principle of light scattering. For example, platelets are identified by their size (<30 FL, small-angle light scattering) and refractive index (n=1.35~n=1.40 or high-angle light scattering).

[0080] In a diverse range of patients, the polypeptide of the present invention, e.g., ALX 0081, containing at least one ISVD against vWF, was administered after an acute episode of TTP, following the patient's administration of pre-epilation ("prior pre-epilation"; pre-epilation preceding a first dose of the polypeptide of the present invention). In the group of subjects who received prior pre-epilation (also referred to as "one pre-randomization PEX"), the median time to response was unexpectedly reduced by 2 days, from 4.31 days for the placebo group to 2.44 days for the treatment group: a 43% reduction (Table 5; pre-randomization PEX = present).

[0081] Accordingly, the present invention relates to administering a pre-epileptic pulmonary embolism (PE) to a patient in need, for example, a patient having an acute episode of TTP, and then administering a subsequent PE within 24 hours of the pre-epileptic PE, with the polypeptide of the present invention ("first dose") administered approximately 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or just 5 minutes before initiating the subsequent PE ("first PE"), for example, 6 hours to 15 minutes before initiating the subsequent PE ("first PE"). In the present invention, the term "first dose" means the first administration of the polypeptide of the present invention to a patient in need, for example, after an acute episode of TTP or all acute episodes.

[0082] In one embodiment, post-epilation (PE) is performed within 5 minutes to 8 hours, for example, 10 minutes to 6 hours or 15 minutes to 4 hours, for example, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or just 5 minutes, after administration of the polypeptide of the present invention, preferably a first dose, to the patient. In the present invention, the term "first PE" means the first PE after (or in some cases concurrently with) the administration of a first dose of the polypeptide of the present invention to a patient. The polypeptides of the present invention may be administered or used for administration in liquid form (e.g., injectable and injectable solutions). Such compositions may be administered parenterally (e.g., by subcutaneous, intraperitoneal, or intramuscular injection) or by inhalation. The phrases “parenteral administration” and “administered parenterally,” as used herein, mean modes of administration other than enteral and topical administration, usually by injection, including subcutaneous (sc) or intramuscular administration, as well as intravenous (iv), intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subepidermal, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Preferably, second or further doses of the polypeptides of the present invention as described herein are administered subcutaneously.

[0083] Preferably, the administration of a first dose of the polypeptide of the present invention after an acute episode of TTP is an intravenous bolus injection, which delivers, for example, the polypeptide through an intravenous line, and is administered all at once over a period of 1 or 2 minutes. More preferably, the administration of a first dose of the polypeptide of the present invention after an acute episode of TTP is an intravenous push injection, which delivers, for example, the polypeptide through an intravenous line, and is administered all at once over a period of about 30 seconds or less.

[0084] Surprisingly, the polypeptide of the present invention, administered prior to PE (at which time there is no direct pharmacological targeting of the active process of platelet aggregation mediated by ULvWF, and the PE is expected to remove the polypeptide), was found to still reduce the median time to response by an unexpectedly large 2-day reduction, from 4.92 days in the placebo group to 3.00 days in the caplacizumab group: a 39% reduction (Table 5: PEX=none before randomization).

[0085] The inventors of the present invention have demonstrated that the polypeptide of the present invention is safe to use, as demonstrated in previous studies in healthy volunteers and in this study in TTP patients (see Example 7.5.3). Considering that TTP, particularly acute attacks of TTP, can be difficult to diagnose, and that any time lost before initiating treatment can be unfortunate, the inventors have concluded that this finding has the benefit of allowing treatment with the polypeptide of the present invention to be initiated in a timely manner, even before the patient enters the hospital, for example, in an ambulance. Preferably, the polypeptide of the present invention, e.g., ALX 081, is administered by intravenous push injection, because this can be easily done outside the hospital and therefore does not waste valuable time.

[0086] Therefore, the present invention relates to administering the polypeptide of the present invention to patients who require it, for example, patients having an acute episode (acute attack) of TTP, at approximately 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or just 5 minutes before initiating PE, for example, 6 hours to 15 minutes before initiating PE ("first dose"). In one embodiment, postnatal administration (PE) is performed after administration of a first dose of the polypeptide of the present invention following an acute episode of TTP ("first PE"). This first PE is followed by administration of a second or further dose of the polypeptide of the present invention ("second dose" or "further dose"), regardless of whether a prior PE has been performed beforehand. Preferably, the second or further dose is administered within 120, 90, or 60 minutes after the first PE, for example, within 1 to 60 minutes, for example, within 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or just 1 minute. In some cases, it may be advantageous to administer the second or further dose together with or concurrently with a replacement fluid, for example, the plasma of the PE.

[0087] In further embodiments, the first, second, or further dose of the polypeptide of the present invention is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40, 50, 60, 70 or 80 mg, preferably 5 to 40 mg, and more preferably 10 mg, which can be administered to a patient in need, preferably per day. For administration to young patients, such as children or adolescents, the dose can be adjusted relative to the patient's body weight. In certain embodiments, the dose is approximately 0.01, 0.025, 0.05, 0.075, 0.1, 0.12, 0.14, 0.15, 0.16, 1.08, 0.2, 0.22, 0.24, or 0.25 mg / kg, preferably 0.143 mg / kg, which corresponds to a dose of 10 mg in a 70 kg adult. In one embodiment, the present invention relates to the administration of about 5 to 40 mg, preferably 10 mg, of the polypeptide of the present invention, such as ALX 0081, within 1 to 60 minutes after a PE procedure, such as a first PE, a second PE, or further PE procedures.

[0088] In one embodiment, the polypeptide of the present invention, for example ALX 0081, is administered once or twice daily to TTP patients who require it, preferably patients with a platelet count of less than 100,000 per μl of plasma, and / or patients with ADAMTS13 activity of ≤10%, for example ≤5%: In a further embodiment, TTP patients who require it are as follows: (i)PE; and, (ii) 60 minutes to 1 minute after step (i) the PE, a dose of 5 to 40 mg, preferably 10 mg, of the polypeptide, The patient is treated in this manner, and steps (i) and (ii) are repeated once or twice per day until the patient's platelet count reaches at least 50,000 per μl of plasma, for example, 75,000, 100,000, 125,000, or 150,000 per μl of plasma.

[0089] In some cases, it may be beneficial to repeat steps (i) and (ii) for at least two days after complete remission (a platelet count of ≥150,000 per μl of plasma). In one embodiment, 5 to 40 mg of the polypeptide of the present invention is administered daily or twice daily for at least 5, 10, 15, 20, 25, 30, 60, 90, or 120 days, after the patient's platelet count reaches ≥150,000 per μl of plasma, or after the last PE (especially if the patient's ADAMTS13 activity is ≤10%, e.g., ≤5%).

[0090] When evaluating the data by stratification (one PEX before randomization: with & without), the overall hazard ratio for the entire population aggregated to 2.197, with a p-value of 0.013. This hazard ratio means that, at any given time, the proportion of subjects receiving the polypeptide of the present invention who achieve the confirmed primary endpoint of platelet recovery is more than twice that of subjects receiving placebo. In addition, this platelet recovery is achieved two days earlier in the treatment group. Therefore, administration of a polypeptide containing at least one ISVD to vWF (such as ALX 0081) to human TTP patients after an acute episode of TTP results in an unexpected reduction in time to response, regardless of the order of the polypeptide administration and the PE administration, whether performed before or after, for example, the administration of a first dose of the polypeptide of the present invention.

[0091] Even more surprisingly, we found that the number of exacerbations decreased from 11 in the placebo group to 3 in the caplacizumab group. Therefore, there were three times more exacerbations in the placebo group (i.e., TTP patients receiving PE and placebo instead of the polypeptide of the present invention) compared with the treatment group (i.e., TTP patients receiving PE and the polypeptide of the present invention; also indicated as the caplacizumab group). The term “exacerbation,” as used herein, refers to a recurrent thrombocytopenia following a confirmed platelet response that requires the restart of daily PE treatment at least one day but no more than 30 days after the last PE. This indicates that polypeptides of the present invention, such as ALX 0081, can, on their own, cause treatment and / or alleviation of TTP (symptoms).

[0092] Accordingly, the present invention relates to a polypeptide (such as ALX 0081) containing at least one ISVD for vWF, for use in treating vWF-related diseases such as TTP in humans who require it, by administering to humans a dose of 1 to 80 mg or 5 to 40 mg of the polypeptide, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, or 80 mg, preferably 10 mg. Based on this remarkable finding, the inventors designed a further optimized treatment protocol based on the idea that, essentially, the observed platelet response time distribution in the CAP group was shorter, not sloping to the right (time to longer response), and not biased compared to the placebo group. In a further optimized treatment protocol, all subjects are treated for a fixed PE treatment period, which is set to 3 to 5 days, for example, 3, 4, or 5 days, preferably 3 days. In this case, the PE treatment period may be independent of platelet recovery (≧150,000 / μl). In a further optimized treatment protocol, patient burden and cost are reduced.

[0093] Accordingly, the present invention relates to a polypeptide (such as ALX 0081) comprising at least one ISVD for vWF for use in treating vWF-related diseases in humans in need thereof, wherein the use comprises (i) performing PE; and (ii) administering a dose of 5 to 40 mg, e.g., 10 mg, of the polypeptide of the present invention 15 minutes to 4 hours after the PE of step (i), wherein steps (i) and (ii) are repeated once per day for 3 to 5 days, e.g., 3, 4, or 5 days, preferably 3 days; and thereafter, after the patient's platelet count is first ≥ 150,000 / μl, a dose of 5 to 40 mg, e.g., 10 mg, of the polypeptide once per day for at least 10 days, e.g., at least 20 or at least 30 days, and / or for at least 10 days, e.g., at least 20 or at least 30 days.

[0094] In this study, TTP patients were followed up for one year for remission. The term "remission," as used herein, refers to the absence of a confirmed platelet response and exacerbation. The term "confirmed platelet response," as used herein, refers to the time to treatment for response, which is defined as a recovery of platelets ≥150,000 / μL, and this response must be confirmed 48 hours after the first report of a platelet recovery exceeding 150,000 / μL by a new measurement of platelets ≥150,000 / μL, and preferably by LDH ≤ 2 x ULN. As shown herein (Example 7.5.5; Table 8), overall, 29 patients in the treatment group achieved remission compared to 18 patients in the placebo group. Therefore, the treatment group represents 1.6 times more patients with complete remission than the placebo group.

[0095] As stated above, platelet counting is a primary tool for assessing remission. Measuring ADAMTS13 activity in patients with a history of classical TTP is important because low levels have been shown to be a predictor of relapse. However, it is currently unclear (and the data are inconsistent) whether the titer of inhibitory antibodies against ADAMTS13 is important, i.e., whether individuals with high titers of anti-ADAMTS13 antibodies are more likely to relapse than those with low titers. Those skilled in the art will understand that current tests for ADAMTS13 are performed under static conditions and do not always accurately reflect the physiological changes that occur in vivo (http: / / practical-haemostasis.com / Miscellaneous / Miscellaneous%20Tests / adamts13_assays.html). The inventors have now unexpectedly observed that remission is more pronounced in a subgroup of subjects with low (i.e., less than 10%, e.g., less than 5%) baseline ADAMTS13 activity when initiating treatment, e.g., a first dose of the polypeptide of the present invention, such as ALX 0081 (see Example 7.5.8).

[0096] Accordingly, the present invention relates to a polypeptide comprising at least one ISVD to vWF for use in treating vWF-related diseases in humans in need, by administering a first dose of 1 to 40 mg, preferably 10 mg, of the polypeptide to the human until the human platelet count is ≥150,000 / μl. In a preferred aspect, the human has less than 10%, for example less than 5%, ADAMTS13 activity when administered the polypeptide. In this study, TTP patients were followed up for up to one year for relapse. The term "relapse," as used herein, refers to a new event of TTP occurring more than 30 days after the last daily PE, for example, 0-2 days after a TTP patient achieved complete remission. In this study, the "more than 30 days" coincides with the last administration of the polypeptide of the present invention. No further administration of the polypeptide of the present invention was performed, but the number of relapses in the caplacizumab group was comparable to that in the placebo group (see Table 7 in Example 7.5.4).

[0097] The inventors observed that in all treatment groups, relapse was more pronounced in patients with <10%, e.g., <5%, baseline ADAMTS13 activity, although ADAMTS13 activity was only available in a subset of patients. The inventors hypothesized that this could indicate that patients with <10%, e.g., <5%, baseline ADAMTS13 activity are more likely to relapse (or worsen) upon discontinuation of the polypeptide of the present invention, such as ALX 0081.

[0098] In particular, the data support the use of ADAMTS13 activity as a predictive marker for TTP recurrence and its potential for treatment decisions. ADAMTS13 activity can predict recurrences occurring shortly after discontinuation of caplacizumab treatment. These recurrences are thought to be recurrences of symptomatic TTP episodes (unresolved disease activity based on persistently low ADAMTS13 activity). A 30-day treatment period with caplacizumab (post-PE) showed a significant impact on the number of exacerbations. Therefore, extending the duration of caplacizumab treatment in patients at risk of recurrence (i.e., those with underlying disease activity based on ADAMTS13 activity) would maintain the protective effect of caplacizumab until the underlying disease is adequately treated and resolved. Conversely, prophylactic treatment with caplacizumab would reduce the risk of new acute episodes of TTP.

[0099] Therefore, treatment with the polypeptide of the present invention, such as ALX 0081, should be continued for a longer period compared to patients with high activity. The polypeptide of the present invention should be administered to TTP patients until ADAMTS13 activity is at least 10% of normal activity or reference activity, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, in order to reduce the risk of relapse and / or prevent the opportunity for relapse.

[0100] Accordingly, the present invention relates to a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF) for use in reducing the risk of vWF-related disease (e.g., TTP) and / or preventing an acute episode thereof in a person requiring it, comprising step (i): administering to the person a dose of 5 to 40 mg, preferably 10 mg, of the polypeptide. Preferably, the risk is reduced by at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 times or higher, for example 3, 4, 5, 6, 7, 8, 9 times or 10 times, or higher, for example 20, 50 or 100 times. Preferably, the risk is reduced by 10% or higher, for example 20%, 30%, 40%, 50%, 60% or higher, for example up to 80% or 100%.

[0101] Accordingly, the present invention relates to a polypeptide as described herein, wherein step (i) of administering the polypeptide to the human is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or more than 10 times, for example 20 times, preferably more than 30 times or more. The method according to claim 1, wherein step (i) of administering the polypeptide to the human is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, or longer than 10 days, for example 20 days, preferably longer than 30 days, for example 2 months, 3 months, 4 months, 5 months, 6 months or longer.

[0102] Accordingly, the present invention relates to polypeptides as described herein, wherein the dose is administered once or twice daily. Therefore, the present invention further relates to polypeptides such as those described herein, including: (ii) Measure the ADAMTS13 activity of the patient; (iii) comparing the ADAMTS13 activity with the reference ADAMTS13 activity; and (iv) If the ADAMTS13 activity is less than 30% of the reference ADAMTS13 activity, for example 20%, 15%, 10%, or 5%, repeat step (i) of administering the polypeptide to the human.

[0103] Accordingly, the present invention relates to polypeptides as described herein, wherein the ADAMTS13 activity of the patient is measured daily, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably at least once a week. Accordingly, the present invention relates to polypeptides as described herein, wherein step (i) is repeated until the ADAMTS13 activity is at least 5%, 10%, 15%, for example 20%, or 30%, or even higher, of the reference ADAMTS13 activity.

[0104] Accordingly, the present invention relates to a polypeptide as described herein, wherein step (i) is repeated, in which the ADAMTS13 activity is at least 5%, 10%, 15%, for example 20%, or 30% of the reference ADAMTS13 activity in at least two consecutive measurements. Preferably, the two consecutive measurements are separated by at least 24 hours, more preferably 48 hours, for example, at least 3 days, or longer, for example, 4, 5, 6, or 7 days, preferably 1 week. Accordingly, the present invention relates to a polypeptide as described herein, wherein step (i) is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or longer than 10 days, for example 20 days, preferably longer than 30 days, after the ADAMTS13 activity has become at least 5%, at least 10%, at least 15%, for example 20%, or at least 30% of the reference activity in at least two consecutive measurements.

[0105] Accordingly, the present invention relates to a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF) for use in reducing the risk of vWF-related diseases such as TTP and / or preventing acute episodes thereof in humans who need it, wherein such use comprises step (i): administering to the human a dose of 5 to 40 mg, preferably 10 mg, of the polypeptide, and further comprising: -Measure the ADAMTS13 activity of the aforementioned patient; -Comparing the aforementioned ADAMTS13 activity with the reference ADAMTS13 activity; and - If the ADAMTS13 activity is ≥5%, for example ≥10%, or ≥15%, or higher than 20%, or 30%, of the reference ADAMTS13 activity, then step (i) is repeated for a maximum of 30 days, for example ≥20 days, or for 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or just 1 day.

[0106] Accordingly, the present invention relates to a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF) for use in reducing the risk of vWF-related diseases such as TTP and / or preventing acute episodes thereof in humans who require such use, the use comprising at least the following steps: (i) Measure the ADAMTS13 activity of the patient; (ii) Comparing the ADAMTS13 activity with the reference ADAMTS13 activity; and (iii) If the ADAMTS13 activity is lower than 30%, 20%, 15%, 10%, or 5% of the reference activity, administer to the human a dose of 5 to 40 mg, preferably 10 mg, of the polypeptide containing at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF).

[0107] As used herein, reducing risk or incidence includes reducing the likelihood or incidence of signs, symptoms or consequences of vWF-related diseases such as TTP in a subject compared to an appropriate, for example, untreated, control population, or in the same subject before treatment according to the present invention. Signs, symptoms, or consequences of vWF-related diseases such as TTP, as used herein, include organ injury, ischemic injury, microthrombosis, exacerbation, death, and relapse, and one or more disease markers of vWF-related diseases such as TTP include platelet count, thrombocytopenia, neurocognitive function, ADAMTS13 levels and anti-ADAMTS13 antibody titer, ADAMTS13 activity level, cardiac markers (troponin T or troponin I), BNP (brain natriuretic peptide) or N-terminal pro-brain natriuretic peptide (NT proBNP), creatinine, and brain injury markers (e.g., NSE (neuron-specific enolase) and Sβ100 (S100 beta)), preferably organ injury markers, e.g., LDH levels, troponin T and / or troponin I levels, and / or creatinine levels.

[0108] A reduction in risk or incidence may include delaying or preventing the onset of signs, symptoms, or consequences of vWF-related disorders such as TTP. A reduction in risk or incidence may also occur if the severity of signs, symptoms, or consequences of vWF-related disorders such as TTP decreases to a clinically irrelevant level; that is, signs, symptoms, or consequences of vWF-related disorders such as TTP may be present, but not at a level that threatens the subject's life, activities, and / or well-being. In some circumstances, the incidence of vWF-related disorders such as TTP decreases to the extent that the subject does not present any signs of vWF-related disorders such as TTP during and / or thereafter.

[0109] It will be understood that it is not possible to obtain actual evidence that the risk has decreased for any given individual. This is because, when a treatment is provided, it is not possible to say whether or not signs, symptoms, or consequences of vWF-related diseases such as TTP would have occurred, or occurred sooner, in the absence of such treatment. Therefore, the concepts of risk, increased risk, or decreased risk refer only to statistical values. Furthermore, a reduction in the risk of signs, symptoms, or consequences of vWF-related diseases such as TTP may be reflected in a reduction in the severity of signs, symptoms, or consequences of vWF-related diseases such as TTP, as well as in the absence or delay of observation of signs, symptoms, or consequences of vWF-related diseases such as TTP.

[0110] It will be understood that the polypeptides of the present invention reduce and / or prevent the risk of acute episodes of vWF-related diseases such as TTP. Therefore, the signs, symptoms, or consequences of vWF-related diseases such as TTP will also be reduced. Given the pathophysiology of acquired TTP, in which strings of ULvWF consume platelets in the formation of microthrombi, recovery of platelet counts is an indirect measure of prevention of further microthrombus formation. The morbidity and acute mortality associated with acquired TTP are consequences of these microthrombi.

[0111] In fact, this reasoning is supported by the normalization of organ damage markers. In particular, the results show that organ damage markers, such as troponin I and T, LDH, and creatinine, return to normal levels faster in subjects treated with the polypeptide of the present invention, e.g., ALX 0081, than in subjects treated with placebo (see Example 7.5.7). Therefore, the results suggest that a faster rate of normalization of these organ damage markers is associated with better clinical outcomes, namely a reduced risk of organ damage caused by organ ischemia induced by microthrombi, and that these damages are less frequent.

[0112] Therefore, the present invention relates to methods as described herein, - The risk of organ damage, ischemic injury, and / or microthrombus formation is reduced by 10%, 20%, 30%, preferably at least 40%, or at least 50%, and even up to, for example, 60%, 70%, 80%, 90%, or 100% (e.g., absence of organ damage, ischemic injury, and / or microthrombus formation due to vWF-related disease); - The risk of organ damage, ischemic injury, and / or microthrombus formation is reduced by a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 times or more, for example, 3, 4, 5, 6, 7, 8, 9 times or 10 times, or even more, for example, 20, 50 or 100 times; - Organ damage, ischemic injury, and / or microthrombus formation are preferably reduced by at least 10%, 20%, 30%, 40%, or even 50%, for example, 60%, 70%, 80%, 90%, or even 100%; - Organ damage, ischemic injury and / or microthrombus formation are reduced by a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 times or more, e.g., 3, 4, 5, 6, 7, 8, 9 times or 10 times, or more, e.g., 20, 50 or 100 times; - Organ damage markers, such as LDH levels, troponin T levels, troponin I levels, and / or creatinine levels, may return to at least 40% of normal levels, or even at least 50%, or even 60%, 70%, 80%, 90%, or 100%; - Organ damage markers, such as LDH levels, troponin T levels, troponin I levels, and / or creatinine levels, improve to at least 20% of normal levels, for example 30% or higher, for example 40%, or even 50% or more, for example 60%, 70%, 80%, 90%, or 100%. Preferably, the organ damage markers, such as LDH levels, troponin T levels, troponin I levels, and / or creatinine levels, improve within 30 days of treatment, preferably within 20 days of treatment, for example, within 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or within 1 day. - The platelet count is maintained at ≥150,000 / μl. - The time to normalization of platelet count (≧150,000 / μl) is shortened by at least 10%, 20%, 30%, 35%, 39%, preferably at least 40%, or even 50%, for example, 60%, 70%, or 80%. - The risk of exacerbation decreases by at least 10%, 20%, 30%, 40%, or even 50%, and can even decrease to, for example, 60%, 70%, 80%, 90%, or 100%; - The risk of exacerbation decreases by a factor of 2 or more, for example, 3, 4, 5, 6, 7, 8, 9 or 10 times, or even more, for example, 20, 50 or 100 times; - The mortality rate attributable to the vWF-related disease can be reduced to 10%, 20%, 30%, preferably at least 40%, or at least 50%, or even to, for example, 60%, 70%, 80%, 90%, or 100%; -The mortality rate attributable to the vWF-related disease decreases by a factor of 2 or more, for example, 3, 4, 5, 6, 7, 8, 9 or 10, or even more, for example, 20, 50 or 100; and / or - Remission increases at multipliers of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 times or higher, for example, 3, 4, 5, 6, 7, 8, 9 times or 10 times, or even higher, for example, 20, 50 or 100 times.

[0113] The term "reference activity," as used herein, refers to the average ADAMTS13 activity of five healthy subjects in the assay performed, and is set to 100%. For example, in the FRETS-vWF73 assay, the slope of the regression curve is calculated for each calibration sample in the calibration curve prepared using a pool of normal human plasma, and is used to construct the calibration curve (trend line: y=ax+b; x=ADAMTS13(%) and y=delta RFU / delta time). The ADAMTS13 activity (%) of the sample is then calculated as (yb) × 1 / a. In practice, generally, patients who relapsed had lower ADAMTS13 activity than patients who did not relapse.

[0114] Accordingly, the present invention relates to a polypeptide for reducing and / or preventing the risk of ischemic injury, organ damage and / or microthrombus formation that may be caused by vWF-related diseases such as TTP in humans who need it, comprising at least the following steps: (i) administering to the human a dose of 5 to 40 mg / day, preferably 10 mg / day, of a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF); wherein the administration of the polypeptide reduces and / or prevents the risk of ischemic injury, organ damage and / or microthrombus formation by at least 10%, 20%, 30%, preferably at least 40%, or at least 50%, or even up to 60%, 70%, 80%, 90%, or 100%. Preferably, administration of the polypeptide reduces and / or prevents the risk of ischemic injury, organ damage and / or microthrombus formation by 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 times or more, for example, 3, 4, 5, 6, 7, 8, 9 times or 10 times, or more, for example, 20, 50 or 100 times.

[0115] Accordingly, the present invention relates to a polypeptide for reducing and / or preventing the risk of ischemic injury, organ injury and / or microthrombus formation as described herein, wherein step (i) of administering the polypeptide is repeated for at least 1, 2, 3, 4, 5, 6, 7 days, or longer, for example, 1 week, 2 weeks, 3 weeks, or longer, for example, 1 month or 2 months. Accordingly, the present invention relates to a polypeptide for reducing and / or preventing the risk of ischemic injury, organ injury and / or microthrombus formation as described herein, further comprising measuring the ADAMTS13 activity of the patient, preferably once every week.

[0116] Accordingly, the present invention relates to a polypeptide for reducing and / or preventing the risk of ischemic injury, organ injury and / or microthrombus formation as described herein, wherein, when the ADAMTS13 activity becomes ≥5%, e.g., ≥10%, or ≥15% of the reference ADAMTS13 activity for the first time, the step (i) of administering the polypeptide is repeated for at least 1, 2, 3, 4, 5, 6, or 7 days, or longer, e.g., 1 week, 2 weeks, 3 weeks, or longer, e.g., 1 month or 2 months. Accordingly, the present invention relates to a polypeptide for treating symptoms of vWF-related diseases such as TTP in a person suffering from the said disease, which comprises administering the polypeptide to a subject in an amount effective for treating symptoms of vWF-related diseases in a person suffering from the said disease.

[0117] Accordingly, the present invention relates to a polypeptide for inhibiting the onset or progression of vWF-related diseases such as TTP in humans (the inhibition being brought about by the binding of a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) to von Willebrand factor (vWF) to vWF), comprising administering to humans an effective inhibitory dose of the polypeptide at prescribed intervals, wherein each administration of the antibody delivers to the human 0.1 mg to 25 mg per kg of human body weight in order to thereby inhibit the onset or progression of the disease in the human.

[0118] Accordingly, the present invention relates to a polypeptide for reducing the likelihood of a human being to suffer ischemic organ injury due to vWF-related disease, comprising administering to a human being a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF) in a prescribed dose, wherein each administration of the antibody delivers to the human being 0.1 mg to 25 mg per kg of human body weight in order to thereby reduce the likelihood of the human being to suffer ischemic organ injury. Modeling based on these results indicates that long-term administration of the polypeptide of the present invention is effective in preventing acute episodes. This favorable profile leads to a reduction in health threats. Therefore, it can be concluded that the polypeptide of the present invention prevents recurrence.

[0119] Accordingly, the present invention relates to administering the polypeptide of the present invention at doses ranging from 1 to 80 mg, for example, 5 to 40 mg, every 1, 2, 3, 4, 5, 6, or 7 days, or every 2, 4, 6, or 8 weeks, preferably in order to prevent acute episodes of TTP. Particularly effective doses are 10 to 20 mg. In certain embodiments, the dose comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, or 80 mg, preferably 10 mg, of a polypeptide (such as ALX 0081) containing at least one ISVD relative to vWF.

[0120] In one embodiment, the present invention relates to a method for preventing recurrence in TTP patients, the method comprising: (1) Measuring ADAMTS13 activity from TTP patients by assay, such as a direct or indirect assay; (2) Comparing the activity in step (1) with a reference value (normal value); and (3) If the ADAMTS13 activity in step (1) is less than 15% of the reference value, for example less than 10% and less than 5%, administer the polypeptide of the present invention, for example ALX 0081, thereby preventing relapse.

[0121] The results in preparation suggest that administration of the first dose of the polypeptide of the present invention prior to the first PE already results in an increase in platelet count. Accordingly, the present invention relates to administering a dose of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, or 80 mg, preferably 10 mg, of the polypeptide of the present invention, a polypeptide comprising at least one ISVD relative to vWF (such as ALX 0081), to patients who need it, for example, patients experiencing an acute episode of TTP.

[0122] The polypeptide of the present invention, comprising at least one ISVD for vWF, e.g., ALX 0081, can be administered to a subject (e.g., a human subject) alone or in combination with a second agent, e.g., a second therapeutically or pharmacologically active agent, to treat or prevent vWF-related diseases, e.g., TTP (e.g., to alleviate or induce remission of one or more associated symptoms). Non-limiting examples of agents that can be co-prescribed with the polypeptide of the present invention (e.g., ALX 0081) containing at least one ISVD against vWF include, for example, adjuvant immunosuppressive treatments (e.g., corticosteroids such as (methyl)prednisolone or (methyl)-prednisone; or rituximab), antiplatelet agents (e.g., aspirin), supportive therapy with red blood cell transfusion or folic acid supplementation, treatment with vincristine or cyclosporine, anti-autologous ADAMTS13 antibodies, or ADAMTS13. Such combination therapies can advantageously utilize lower doses than those of the therapeutic agents administered, thereby avoiding the potential toxicity or complications associated with various monotherapies.

[0123] In one aspect, the present invention relates to a combination therapy of the polypeptide of the present invention together with rituximab for immunosuppressive treatment, particularly for efficiently preventing relapse in patients with TTP. Preferably, the combination therapy is provided until ADAMTS13 activity is at least ≥5%, for example ≥10%, ≥15%, ≥20%, 25%, 30%, 35%, 40%, 45%, or normalized, for example ≥50% of normal activity.

[0124] TTP is still diagnosed based on clinical history, patient and blood film. The ADAMTS13 assay helps confirm the diagnosis and monitor the course of the disease and the possible need for further intervention. Acute episodes of TTP can be diagnosed according to Table 1 and the guidelines, for example, Scully et al. (2012, above). Table 1 Clinical features and signs in acute episodes of TTP. [Table 1]

[0125] The efficacy or administration regimen of any particular polypeptide of the present invention can be determined by methods available to those skilled in the art. Briefly, during clinical trials, patients may be observed by healthcare professionals and their disease state assessed using any combination of criteria. Improvement in the patient's disease state is determined at multiple time points based on these criteria, and the efficacy of the treatment is evaluated by plotting combinations of these decisions for the patient population.

[0126] In exemplary embodiments, the assessment of effectiveness may be measured by any or all of the following criteria: • Treatment time to response defined by recovery of platelets ≥ 150,000 / μL. This response must be confirmed 48 hours after the first report of platelet recovery exceeding 150,000 / μL by a new measurement of platelets ≥ 150,000 / μL, and preferably by LDH ≤ 2 x ULN. • Number of subjects with complete remission • The number of subjects experiencing TTP exacerbations and the time to the first TTP exacerbation. An exacerbation is defined as a recurrent thrombocytopenia after a response that occurs at least one day after the last daily PE but within 30 days, requiring the restart of daily PE treatment. • The number of targets experiencing a recurrence of TTP over a maximum of one year (defined as a new TTP event occurring more than 30 days after the last daily PE), and the time until the first TTP recurrence. Daily PE data, including serious adverse events (SAEs) associated with daily PE procedures. • Neurocognitive function as measured by the neurocognitive test battery during complete remission and during a one-year follow-up. Prior to this test, the Glasgow Coma Score is administered to measure the subject's state of consciousness. • Improvement of organ dysfunction and improvement of signs and symptoms associated with TTP. • Total mortality during the daily PE treatment period and the subsequent study drug treatment period (including tapering). Determination of biomarkers for TTP, including, but not limited to, ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin repeats 13) levels and titers of anti-ADAMTS13 antibodies.

[0127] Those skilled in the art are adept at determining the effect. For example, ADAMTS13 activity can be evaluated by detecting uncleaved ultralarge multimers using electrophoresis of vWF multimers (Moake et al. (1982) The New England journal of medicine 307, 1432-1435; Furlan, et al. (1997) Blood 89, 3097-3103 7, 8). ADAMTS13 activity can also be tested using FRETS-vWF73, a vWF fragment chemically modified to emit fluorescence when cleaved by ADAMTS13. In the assay, FRETS-vWF73 is added to a patient's plasma sample, and ADAMTS13 activity is determined by measuring the change in fluorescence over time. If an inhibitor is present, it often neutralizes the IgG antibody against ADAMTS13, which can be measured by ELISA (Kokame et al. (2005) British journal of haematology 129, 93-100). Alternatively, or in addition, ADAMTS13 activity can be determined as described, for example, in Vesely et al. (2003, above), Fontana et al. (2004, above) or Remuzzi et al. (Blood 2002;100:778-7852002). For example, an indirect ADAMTS13 activity assay involves the detection of cleavage of either the full-length VWF molecule or a VWF fragment containing the ADAMTS13 cleavage site in the A2 domain of VWF. (1) Collagen binding assay. Normal plasma or purified VWF is incubated with the test plasma sample in the presence of BaCl2 and 1.5 M urea to denature the VWF. VWF is cleaved by ADAMTS13, and the remaining VWF is measured by its binding to type III collagen. The bound VWF is quantified using an ELISA assay with a conjugated anti-VWF antibody. (2) Ristocetin-induced agglutination. This is similar to the collagen binding assay described above, but the remaining VWF is measured by ristocetin-induced platelet aggregation using a platelet agglutinometer. (3) Functional ELISA assay.In this assay, recombinant VWF fragments are immobilized on an ELISA plate using an antibody against a tag on the VWF. The VWF fragments encode the A2 domain and ADAMTS13 cleavage site at Tyr1605–Met1606 and are tagged with S-transferase [GST]-histidine [GST-VWF73-His]. Plasma is added to the immobilized GST-VWF73-His fragment, and cleavage of the immobilized fragment occurs at the ADAMTS13 cleavage site. The remaining cleaved VWF fragments are measured using a second monoclonal antibody that recognizes the cleaved VWF fragments but not the complete fragments. ADAMTS13 activity is therefore inversely proportional to the concentration of the remaining substrate. This method forms the basis for the TECHNOZYM® ADAMTS13 activity ELISA.

[0128] Those skilled in the art are familiar with determining autoantibodies against ADAMTS13, for example, anti-ADAMTS13 autoantibodies can be determined by ELISA, such as TECHNOZYM® ADAMTS13 INH ELISA (Technoclone). Those skilled in the art are familiar with determining ristocetin cofactor activity in human samples, for example, ristocetin cofactor can be determined using Bio / Data corp.'s vW Select on a platelet aggregation analyzer PAP-8E (Bio / Data corp.). Those skilled in the art are familiar with determining Coamatic Factor VIII (Chromogenix) in human samples using, for example, a STA-R evolution analyzer (Diagnostica Stago). Those skilled in the art are familiar with determining von Willebrand factor antigen in human samples using, for example, immunoturbidometric assays (e.g., using the STA Lia test vWF:Ag).

[0129] Those skilled in the art are familiar with determining LDH levels. Many methods are based on enzymatic analysis using lactate dehydrogenase in a spectrophotometer. A useful review is provided by Medbo et al. (2000) "Examination of four different instruments for measuring blood lactate concentration," Scand J Clin Lab Invest 60:367-380. Various companies offer assays such as Abnova (catalog no. KA1653) for measuring the LDH-mediated catalysis of the interconversion of lactate and pyruvate, namely, a non-radioactive colorimetric LDH assay for the reduced form of tetrazolium salt MTT, which exhibits maximum absorbance at 565 nm, based on the reduction of MTT in an enzymatic reaction coupled with NADH. The resulting purple absorbance is directly proportional to the enzyme activity. Similarly, in Sigma Aldrich's kit (MAK066-1KT), LDH reduces NAD to NADH, which is specifically detected by a colorimetric (450 nm) assay. Normal values ​​are provided in Table 1.1 below.

[0130] Those skilled in the art are familiar with determining troponin I and T. Generally, troponin T and I are measured by immunoassay methods, which are available in many different immunoassay platforms, such as DPC Immulite, Abbott AxSYM, Bayer ACS:Centaur, Ortho Vitros, Roche Elecsys, and third-generation. A helpful review is provided by Wu et al. (1999) National Academy of Clinical Biochemistry Standards of Laboratory Practice: recommendations for the use of cardiac markers in coronary artery diseases. Clin Chem. Jul 1999;45(7):1104-21. Normal values ​​are provided in Table 1.1 below.

[0131] Those skilled in the art are familiar with determining creatinine levels. A useful review is provided in Peake and Whiting, "Measurement of Serum Creatinine—Current Status and Future Goals," Clin Biochem Rev. 2006 Nov;27(4): 173-184. For example, creatinine levels can be determined using the Abcam creatinine assay kit (ab65340) or the BioVision creatinine assay kit. In the assay, creatinine is converted to creatine by creatininase, and creatine is converted to sarcosine, which is specifically oxidized to produce a product that reacts with a probe that emits red (λmax=570nm) and fluorescence (Ex / Em=538 / 587nm). Normal values ​​are provided in Table 1.1 below. Since the amount of creatinine in the blood increases with muscle mass, men usually have higher creatinine levels than women. Table 1.1: Normal values [Table 1-1] It will be understood that the normal values ​​provided in Table 1.1 may vary by laboratory, between males and females, and by age. However, those skilled in the art will likely consider that, depending on the assay used, the normal values ​​provided by the manufacturer can generally be used as a reference or as a normal value assessed by a physician in a particular setting.

[0132] The polypeptide of the present invention typically comprises at least one ISVD for vWF. The immunoglobulin monovariate domain of the present invention binds to vWF and / or has affinity for vWF. With respect to the present invention, "vWF" includes, but is not limited to, vWF from cynomolgus monkeys, baboons, pigs, guinea pigs, mice, and / or humans, and most preferably human vWF, i.e., SEQ ID NO: 20 or GenBank entry: NP_000543.

[0133] Preferably, the ISVD for vWF consists of four framework domains (FR1 to FR4, respectively) and three complementarity determination domains (CDR1 to CDR3, respectively), where: a) CDR1 is as follows: - Amino acid sequence YNPMG; or - Amino acid sequences that differ from the amino acid sequence YNPMG in only two or one amino acid; including or essentially derived from these, Furthermore b) CDR2 is as follows: - Amino acid sequence AISRTGGSTYYPDSVEG; or -An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the amino acid sequence AISRTGGSTYYPDSVEG; or - Amino acid sequences that differ from the amino acid sequence AISRTGGSTYYPDSVEG by only 2 or 1 amino acid; including or essentially derived from these Furthermore c) CDR3 is as follows: - Amino acid sequence AGVRAEDGRVRTLPSEYTF; or -An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the amino acid sequence AGVRAEDGRVRTLPSEYTF; or - Amino acid sequences that differ from the amino acid sequence AGVRAEDGRVRTLPSEYTF by only 2 or 1 amino acid It includes or is essentially derived from these.

[0134] More preferably, the ISVD for vWF consists of four framework domains (FR1 to FR4, respectively) and three complementarity determination domains (CDR1 to CDR3, respectively), where: a) CDR1 is YNPMG (sequence number 21); b) CDR2 is AISRTGGSTYYPDSVEG (Sequence ID 22); and c) CDR3 is AGVRAEDGRVRTLPSEYTF (Sequence ID 23). More preferably, the ISVD for vWF is represented by sequence number 19 (12A02H1).

[0135] Preferably, the polypeptide of the present invention comprises or consists of at least two ISVDs for vWF. More preferably, the polypeptide of the present invention comprises or consists of two ISVDs (ALX 0081; INN caplacizumab) targeting vWF as defined by SEQ ID NOs: 1 to 18, and most preferably SEQ ID NO: 1. ALX 0081 is a bivalent nanobody consisting of two identical monovalent components that target vWF. Polypeptides containing at least one ISVD for vWF, such as Sequence IDs 1-19, can be used in the treatment of vWF-related diseases, particularly thrombotic thrombocytopenic purpura (TTP). The terms "polypeptide" and "amino acid sequence" are interchangeable in this specification.

[0136] Therefore, for example, polypeptides suitable for use in the present invention may include compounds in Table A-1, such as SEQ ID NOs: 1-19 or 20-22, or compounds having 80% or more, more preferably 85% or more, and most preferably 90%, 95%, 96%, 97%, 98%, or 99% or more of amino acid sequence identity with respect to the compounds in Table A-1 (see the section on the definition of "sequence identity").

[0137] Preferably, the ISVD for vWF for use in the polypeptide of the present invention is a 12A02H1-like compound. For the purposes of describing the present invention, a 12A02H1-like compound is a compound comprising 12A02H1 (i.e., SEQ ID NO: 19), or a compound having 80% or more, more preferably 85% or more, and most preferably 90%, 95%, 96%, 97%, 98%, or 99% or more amino acid sequence identity with respect to 12A02H1 (SEQ ID NO: 19). A particularly preferred polypeptide comprising two ISVDs for vWF is ALX 0081 (SEQ ID NO: 1).

[0138] Immunoglobulin monovariable domains, such as camelid VHH domains, camelid VH domains, or humanized VHH domains, represent a rapidly growing class of therapeutic agents. For example, immunoglobulin monovariable domains for vWF are described in WO2004 / 015425, WO2004 / 062551, WO2006 / 074947, WO2006 / 122825, WO2009 / 115614, and WO2011 / 067160. A more preferred immunoglobulin monovariable domain for use in polypeptides of the present invention includes the improved Nanobody described in WO06 / 122825.

[0139] Unless otherwise indicated, the term “immunoglobulin sequence” is used herein as a general term to include a complete-sized antibody, its individual chains, and all its parts, domains, or fragments (including, but not limited to, antigen-binding domains or fragments, such as the VHH domain or VH / VL domain, respectively), whether used to refer to a heavy-chain antibody or a conventional quadruple-chain antibody. In addition, the term “sequence,” as used herein (for example, in terms such as “immunoglobulin sequence,” “antibody sequence,” “variable domain sequence,” “VHH sequence,” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding it, unless the context requires a more specific interpretation.

[0140] The term “Immunoglobulin Single Variable Domain” ("ISVD") is used interchangeably with “single variable domain” and defines a molecule formed by the presence of an antigen-binding site on a single immunoglobulin domain. This distinguishes an immunoglobulin single variable domain from “conventional” immunoglobulins or fragments in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, complementarity-determining regions (CDRs) in both the VH and VL contribute to the antigen-binding site; that is, a total of six CDRs are involved in the formation of the antigen-binding site. In contrast, the binding site of a single immunoglobulin variable domain is formed by a single VH or VL domain. Therefore, the antigen-binding site of a single immunoglobulin variable domain is formed by just three CDRs.

[0141] The term “immunoglobulin single variable domain” therefore does not include conventional immunoglobulins or fragments thereof that require the interaction of at least two variable domains for the formation of an antigen-binding site. This is also true in embodiments of the present invention that “comprise / contain” an immunoglobulin single variable domain. With respect to the present invention, such embodiments exclude conventional immunoglobulins or fragments thereof. Therefore, a polypeptide or composition that “comprises / contains” an immunoglobulin single variable domain may, for example, relate to a construct that includes one or more immunoglobulin single variable domains. Alternatively, there may be further components other than immunoglobulin single variable domains, such as various types of adjuvants, protein tags, colorants, dyes, etc. However, these terms do not include conventional immunoglobulin fragments whose antigen-binding site is formed by a single variable domain.

[0142] Generally, a single variable domain is an amino acid sequence essentially consisting of four framework regions (FR1-FR4, respectively) and three complementarity-determining regions (CDR1-CDR3, respectively). Such single variable domains and fragments are most preferably folded or contain immunoglobulins capable of forming immunoglobulin folds under optimal conditions. Therefore, a single variable domain may contain a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or VHH sequence) or a suitable fragment thereof, insofar as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit essentially consisting of a single variable domain, thereby eliminating the need for a single antigen-binding domain to interact with another variable domain to form a functional antigen-binding unit; this is the case, for example, with the variable domains present in conventional antibodies and scFv fragments, which require interaction with another variable domain to form a functional antigen-binding domain (e.g., through VH / VL interaction)).

[0143] In one embodiment of the present invention, the immunoglobulin monovariate domain is a light chain variable domain sequence (e.g., a VL sequence) or a heavy chain variable domain sequence (e.g., a VH sequence); more specifically, the immunoglobulin monovariate domain may be a heavy chain variable domain sequence derived from a conventional quadruple-chain antibody, or a heavy chain variable domain sequence derived from a heavy chain antibody (e.g., a VHH).

[0144] For a general description of heavy chain antibodies and their variable domains, references are made, in particular, to the prior art cited herein, to the prior art mentioned on page 59 of WO 08 / 020079, and to the list of references mentioned on pages 41-43 of international application WO 06 / 040153, which are incorporated herein by reference. As described in these references, Nanobodies (especially VHH sequences and partially humanized Nanobodies) can be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. Further descriptions of nanobodies, including humanization and / or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusions," polyvalent constructs (including some non-limiting examples of linker sequences), and various modifications and preparations thereof to extend the half-life of nanobodies, can be found, for example, in WO 08 / 101985 and WO 08 / 142164.

[0145] For example, a single variable domain or an immunoglobulin single variable domain (or an amino acid sequence suitable for use as an immunoglobulin single variable domain) may be a (single) domain antibody (or an amino acid sequence suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), or a Nanobody (as defined herein, but including a VHH sequence, but not limited to one); another single variable domain, or any suitable fragment of any one of them. For a general description of (single) domain antibodies, references are made to the prior art cited herein and to EP 0 368 684. The term "dAb" is referenced, for example, to Ward et al., 1989 (Nature 341 (6242): 544-6), to Holt et al., 2003 (Trends Biotechnol. 21(11): 484-490), and to other published patent applications of Domantis Ltd., such as WO 04 / 068820, WO 06 / 030220, WO 06 / 003388, and other such publications. It should also be noted that, with respect to the present invention, single variable domains can be derived from certain sharks, although this is less preferable as they are not of mammalian origin (e.g., the so-called "IgNAR domain," see, for example, WO 05 / 18629).

[0146] In particular, the immunoglobulin monovariate domain may be Nanobody® (as defined herein) or a preferred fragment thereof [Note: Nanobody®, Nanobodies®, and Nanoclone® are registered trademarks of Ablynx NV]. For a general description of Nanobody, refer to the following further descriptions and to the prior art cited herein (e.g., as described in WO 08 / 020079 (page 16)).

[0147] The amino acid sequences and structures of immunoglobulin sequences, particularly those of immunoglobulin monovariate domains, can be considered (but not limited to) four framework regions or "FRs," which are referred to in this art and herein as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4," respectively; these framework regions are separated by three complementarity-determining regions or "CDRs," which are referred to in this art as "complementarity-determining region 1" or "CDR1"; "complementarity-determining region 2" or "CDR2"; and "complementarity-determining region 3" or "CDR3," respectively.

[0148] The total number of amino acid residues in an immunoglobulin monovariable domain is in the range of 110 to 120, preferably 112 to 115, and most preferably 113. However, it should be noted that the portion, fragment, analog, or derivative of an immunoglobulin monovariable domain is not particularly limited in terms of its length and / or size, as long as such portion, fragment, analog, or derivative meets the further requirements outlined herein and is also preferably suitable for the purposes described herein. Therefore, in the sense of the present invention, the terms “immunoglobulin monovariate domain” or “monovariate domain” include peptides derived from non-human sources, preferably from camelids, preferably from camel heavy chain antibodies. These may be humanized, as previously described, for example, in WO 08 / 101985 and WO 08 / 142164. Furthermore, the terms include polypeptides derived from non-camelid sources, such as mouse or human, that are “camelized,” as previously described, for example, in WO 08 / 101985 and WO 08 / 142164.

[0149] The term “immunoglobulin monovariable domain” encompasses immunoglobulin sequences of various origins, including those from mouse, rat, rabbit, donkey, human, and camelid. It also includes fully human, humanized, or chimeric immunoglobulin sequences. For example, it includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelid immunoglobulin monovariable domains, such as camelidized dAb as described by Ward et al. (see, e.g., WO 94 / 04678 and Davies and Riechmann, 1994, Febs Lett. 339: 285, and 1996, Protein Engineering 9: 531). All ISVDs (or vWF binders) for the aforementioned VWFs are well known from the scientific literature, including their manufacture (see, in particular, WO2006 / 122825 and WO2004 / 062551). For example, ALX 0081 can be prepared as described, for example, in WO2006 / 122825 or WO2009 / 115614.

[0150] The immunoglobulin monovariable domains provided by the present invention are preferably in isolated or essentially isolated forms, or form a portion of a protein or polypeptide of the present invention, which may comprise or be essentially derived from one or more immunoglobulin monovariable domains, and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, one or more immunoglobulin monovariable domains can be used as binding units in such protein or polypeptide, which may optionally comprise one or more further amino acid sequences that can serve as binding units (i.e., for one or more other targets other than cell-associated antigens) to provide monovalent, polyvalent, or polyspecific polypeptides of the present invention, each as described herein. Such protein or polypeptide may also be in isolated or essentially isolated forms. Thus, according to the present invention, an immunoglobulin monovariable domain comprises a construct comprising two or more antigen-binding units in the form of a single domain, as outlined above. For example, two (or more) immunoglobulin monovariable domains having the same or different antigen specificities can be joined to form constructs that are, for example, bivalent, trivalent, or polyvalent. By combining two or more immunoglobulin monovariable domains with different specificities, constructs such as bispecific, trispecific, etc., can be formed. For example, the polypeptide according to the present invention may include two immunoglobulin monovariable domains directed toward target A and one immunoglobulin monovariable domain directed toward target B, which makes it bivalent for A and monovalent for B. Those skilled in the art will readily recall such constructs and their modifications, all of which are encompassed by the present invention. In certain embodiments, the present invention relates to a bi-paratopic construct comprising at least two immunoglobulin monovariable domains directed toward different epitopes in the same target antigen.

[0151] These molecules are also referred to as “polypeptides of the present invention,” which is synonymous with “immunoglobulin sequences” or “immunoglobulin monovariate domains” of the present invention. In addition, the term “sequence,” as used herein (for example, in terms such as “immunoglobulin sequence,” “antibody sequence,” “variable domain sequence,” “VHH sequence,” or “protein sequence”), should generally be understood to include the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding it, unless the context requires a more specific interpretation. In one non-limiting aspect of the present invention, the immunoglobulin sequence, Nanobody®, or polypeptide of the present invention is glycosylated. In another non-limiting aspect of the present invention, the immunoglobulin sequence, Nanobody®, or polypeptide of the present invention is not glycosylated.

[0152] As described above, the present invention typically relates to polypeptides comprising at least one, for example, two or three or more ISVDs to vWF, i.e., ISVDs that bind to and / or have affinity for antigens as defined herein, such as von Willebrand factor (vWF) and preferably human vWF (SEQ ID NO: 20). With respect to the present invention, "binding to and / or having affinity for" a particular antigen has the ordinary meaning in the art, as understood, for example, with respect to antibodies and their corresponding antigens. In certain embodiments of the present invention, the term “to bind to and / or have affinity for these” means that the immunoglobulin sequence specifically interacts with the antigen and is used interchangeably with an immunoglobulin sequence “against” the antigen.

[0153] The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular immunoglobulin sequence, antigen-binding molecule or antigen-binding protein (such as a Nanobody® or a polypeptide of the invention) can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity. Affinity is represented by the equilibrium dissociation constant (KD) for the dissociation of the antigen and the antigen-binding protein, and is a measure of the strength of the binding between the antigenic determinant and the antigen-binding site on the antigen-binding protein: the lower the value of KD, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule (alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD). As will be apparent to those skilled in the art (e.g., based on further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is a measure of the strength of the binding between an antigen-binding molecule (such as a Nanobody® or a polypeptide of the invention) and an antigen associated therewith. Avidity is related to both the affinity between the antigenic determinant and its antigen-binding site on the antigen-binding molecule, and the number of related binding sites present on the antigen-binding molecule.

[0154] Typically, the immunoglobulin sequences (such as amino acid sequences, Nanobodies® and / or polypeptides of the invention) of the present invention have dissociation constants (KD) for their antigens of less than 10 -5 ~10 -12 mol / litre, and preferably less than 10 -7 ~10 -12 mol / litre, and more preferably less than 10 -8 ~10 -12 mol / litre (i.e., 10 5 ~10 12 litres / mol or more, and preferably 10 7 ~10 12 litres / mol or more, and more preferably 10 8 ~10 12It binds to and / or to cell-associated antigens as defined herein, at an association constant (KA) of liters / moles, and / or 10 2 M -1 s -1 ~about 10 7 M -1 s -1 Preferably 10 3 M -1 s -1 ~10 7 M -1 s -1 , more 10 4 M -1 s -1 ~10 7 M -1 s -1 For example, 10 5 M -1 s -1 ~10 7 M -1 s -1 It binds at a rate of 1 s; and / or to cell-associated antigens as defined herein, 1 s -1 (t1 / 2=0.69s)~10 -6 s -1 (Provides a nearly irreversible complex having t1 / 2 of multiple days), preferably 10 -2 s -1 ~10 -6 s -1 , more 10 -3 s -1 ~10 -6 s -1 For example, 10 -4 s -1 ~10 -6 s -1 The coupling is performed at the koff speed.

[0155] 10 -4 Any KD value higher than M (or 10 4 M -1 Lower KA values ​​(any lower value) are generally considered to indicate nonspecific binding. Preferably, the monovalent immunoglobulin sequence of the present invention will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example less than 500 pM. The specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any preferred manner known by itself, including, for example, scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIAs), enzyme immunoassays (EIAs), and sandwich competitive assays, and various variants thereof known by itself in the art; as well as other techniques referred to herein.

[0156] It will be apparent to those skilled in the art that the dissociation constant (KD) may be the actual dissociation constant or an apparent dissociation constant. Methods for determining the dissociation constant are obvious to those skilled in the art and include, for example, the techniques referred to herein. In this regard, also 10 -4 moles / liter or 10 -3 Higher than moles / liter (for example, 10 -2 It will be apparent that measuring the dissociation constant (in moles / liter) may not always be possible. Optionally, and this too will be apparent to those skilled in the art, the (actual or apparent) dissociation constant can be calculated based on the (actual or apparent) association constant (KA) using the relationship [KD = 1 / KA].

[0157] Affinity represents the strength or stability of molecular interactions. Affinity is generally expressed in terms of the KD or dissociation constant, which has units of moles / liter (or M). Affinity can also be expressed as the association constant, KA, which is equal to 1 / KD, (moles / liter) -1 (or M -1) has units of ). In this specification, the stability of the interaction between two molecules (e.g., an amino acid sequence, an immunoglobulin sequence, Nanobody®, or a polypeptide of the present invention and its intended target) will be expressed primarily in terms of the KD value of their interaction; it will be apparent to those skilled in the art that, considering the relationship KA = 1 / KD, specifying the strength of the molecular interaction by its KD value can also be used to calculate the corresponding KA value. The KD value also characterizes the strength of the molecular interaction in a thermodynamic sense, for it relates to the free energy (DG) of the bond by the well-known relationship DG = RT.ln(KD) (equivalently DG = RT.ln(KA)), where R is equal to the gas constant, T is equal to the absolute temperature, and ln represents the natural logarithm.

[0158] The KD for biological interactions considered meaningful (e.g., specific), such as the binding of the immunoglobulin sequence of the present invention to cell-associated antigens as defined herein, is typically 10. -10 M(0.1nM)~10 -5 The M (10,000 nM) range is used. The stronger the interaction, the lower the KD (Knockdown Distance). KD can also be expressed as the ratio of the dissociation rate constant of the complex, represented as koff, to the association rate of the complex, represented as kon (hence KD = koff / kon and KA = kon / koff). The off-rate koff is expressed in units s -1 It has (where s is the SI unit symbol for second). The velocity kon is in units M -1 s -1 It has. Regarding the immunoglobulin sequence of the present invention, the on rate is 10 2 M -1 s -1 ~about 10 7 M -1 s -1 It can change and approaches the association rate constant, which is limited to diffusion for bimolecular interactions. The off rate is related to the half-life of a given molecular interaction by the relationship t1 / 2 = ln(2) / koff. The off rate of the immunoglobulin sequence of the present invention is 10-6 s -1 (A nearly irreversible complex with t1 / 2 of multiple days) ~1s -1 It can change in (t1 / 2 = 0.69 s).

[0159] The affinity of molecular interactions between two molecules can be measured through various techniques known to the public, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, e.g., Ober et al., Intern. Immunology, 13, 1551-1559, 2001), where one molecule is immobilized on a biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions to obtain a kon, koff measurement, thereby obtaining a KD (or KA) value. This can be done, for example, using a well-known Biacore instrument.

[0160] It will also be apparent to those skilled in the art that if the measurement process affects the intrinsic binding affinity of the molecules being measured by some means, for example, artifacts related to the coating of one molecule onto the biosensor, the measured KD can be made to correspond to the apparent KD. Furthermore, if one molecule contains more than one recognition site for the other molecule, the apparent KD can be measured. In such situations, the measured affinity may be affected by the avidity of the interaction between the two molecules.

[0161] Another approach that can be used to assess affinity is the two-step ELISA (enzyme-linked immunosorbent assay) method by Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This method establishes solution-phase binding equilibrium measurements and avoids possible artifacts related to the adsorption of any of the molecules onto a support such as plastic. However, the accurate measurement of KD is extremely labor-intensive, and as a result, an apparent KD value is often determined to evaluate the binding strength of two molecules. It should be noted that as long as all measurements are carried out in a consistent manner (e.g., keeping the assay conditions unchanged), the measurement of the apparent KD can be used as an approximation of the true KD, and thus, in this specification, KD and apparent KD should be treated with equal importance or appropriateness.

[0162] Finally, it should be noted that in many situations, an experienced scientist may determine that it is convenient to determine the binding affinity relative to several reference molecules. For example, to evaluate the binding strength between molecules A and B, a reference molecule C, which is preferably labeled with a fluorophore or chromophore group or other chemical moiety such as biotin (fluorophore for fluorescence detection, chromophore for absorbance detection, biotin for streptavidin-mediated ELISA detection) for easy detection in ELISA or FACS (fluorescence-activated cell sorting) or other formats and is known to bind to B, may be used. Typically, the reference molecule C is maintained at a fixed concentration, and for a given concentration or amount of B, the concentration of A is varied. As a result, an IC50 value is obtained that coincides with the concentration of A at which the signal measured for C in the absence of A is halved. Assuming that the KD of the reference molecule, KDref, and the total concentration of the reference molecule, cref, are known, the apparent KD for the interaction A - B can be obtained from the following equation: KD = IC50 / (1 + cref / KDref). Note that when cref << KDref, KD ≈ IC50. Assuming that the measurement of IC50 for the binding agents being compared is carried out in a consistent manner (e.g., continuing to fix cref), the strength or stability of the molecular interaction can be evaluated by IC50, and throughout this text, this measurement is judged to be equivalent to KD or apparent KD.

[0163] The present invention relates to immunoglobulin monovariable domains that can be obtained by methods described or disclosed in WO2004 / 015425, WO2004 / 062551, WO2006 / 074947, WO2006 / 122825, WO2009 / 115614 or WO2011 / 067160, all under the name of the present applicant. The present invention also encompasses optimized variants of these amino acid sequences. Generally, an “optimized variant” of an amino acid sequence according to the present invention is a variant containing one or more beneficial substitutions, e.g., substitutions that increase i) the degree of “humanization,” ii) chemical stability, and / or iii) the level of expression; on the other hand, the potency (e.g., measured by a potency assay as described in the experimental part of WO2006 / 122825) is still comparable to wild-type 12A02 (as defined in WO2006 / 122825) (i.e., a variation of no more than 10%) or comparable to variant 12A02H1 (SEQ ID NO: 19) (also defined in WO2006 / 122825). Preferably, compared to the wild-type sequence of 12A02, the amino acid sequence of the present invention contains at least one such substitution, preferably at least two such substitutions, preferably at least three humanization substitutions, and preferably at least ten such humanization substitutions.

[0164] In certain aspects, the amino acid sequence of the present invention includes a total of 1 to 15, preferably 2 to 14, e.g., 9 to 13, e.g., 10, 11, or 12 amino acid substitutions compared to the wild-type sequence 12A02. As mentioned, these differences preferably include at least 1, and preferably at least 2, e.g., 3, 4, or 5, or 10 humanization substitutions, and optionally include one or more further substitutions (e.g., any one of the further substitutions (a) to (c) mentioned herein, or any preferred combination of any two or three or more). Again, based on the disclosure herein, and optionally after a limited degree of trial and error, a person skilled in the art can select one or more such preferred humanizations and / or further substitutions (or preferred combinations thereof).

[0165] The present invention encompasses polypeptide sequences that are highly similar to any of the specific examples provided herein or any of the specific examples defined in the above references. Highly similar means at least 90%, e.g., 95, 97, 98, or 99% amino acid identity. Highly similar polypeptide sequences will have the same function as the sequences from which they are derived; that is, they will bind to vWF, bind more specifically to vWF, and inhibit the interaction between vWF and platelets.

[0166] In certain embodiments, the present invention relates to any one of sequence numbers 1 to 19, particularly sequences highly similar to sequence number 1. However, for each variant, the stability of the sequence in a formulation as defined herein must be evaluated so that the present invention refers to a variant or highly similar sequence that is particularly stable in a formulation as defined herein. Methods for producing the polypeptide sequences of the present invention are widely known and include, for example, recombinant expression or synthesis. Those skilled in the art are familiar with suitable expression techniques, such as suitable recombinant vectors and host cells, such as bacterium or yeast host cells. Those skilled in the art are also familiar with suitable purification techniques and protocols.

[0167] The present invention also provides a polypeptide formulation comprising at least one immunoglobulin monovariable domain relative to vWF, such as ALX 0081, which is stable and preferably suitable for pharmaceutical applications including the preparation of pharmaceuticals (also referred to as "the pharmaceutical formulation of the present invention" or "the formulation of the present invention"). In a particular embodiment, the formulation comprises one or more polypeptides selected from SEQ ID NOs: 1 to 19, preferably SEQ ID NO: 1.

[0168] The term "pharmaceutical formulation" refers to a preparation in a form that enables the biological activity of the active ingredient (polypeptide of the present invention) to be effective, and which does not contain further ingredients that are unacceptable or toxic to the subject to which the formulation will be administered. Such formulation is sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that can be reasonably administered to the target mammal in order to provide an effective dose of the active ingredient used. The term “excipient,” as used herein, refers to an inert substance commonly used as a diluent, vehicle, preservative, cryoprotectant, surfactant, binder, carrier, or stabilizer for a compound, which imparts beneficial physiological properties to the formulation. Those skilled in the art are familiar with excipients suitable for pharmaceutically acceptable purposes that may have specific functions in a formulation, such as cryoprotection, stabilization, or preservation.

[0169] A “sterile” formulation is sterile or essentially free of all live microorganisms and their spores. This is easily achieved by filtration through a sterile filtration membrane. A “sterile” formulation is one in which the proteins contained therein essentially retain their physical and / or chemical stability and / or biological activity during storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity during storage.

[0170] The storage period is generally selected based on the expiration date of the intended formulation. A variety of analytical techniques for measuring protein stability are available in the art and are outlined, for example, in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature over a selected period of time. In some embodiments, the formulation is stable at about 40°C for at least about 1, 2, 3, 4, 5, 6, 7, or 8 weeks or longer. Furthermore, the formulation is preferably stable after the following freezing (e.g., down to -20°C or -70°C) and thawing of the formulation, for example, after 1, 2, 3, 4, or 5 cycles of freeze-thaw. Stability can be evaluated quantitatively and / or qualitatively in a variety of different methods known to those skilled in the art. Stability studies have shown that ALX 0081 is stable at -20°C for at least 3 years.

[0171] The formulation contains an aqueous carrier. The aqueous carrier is, in particular, a buffer. As used herein, “buffer” refers to a buffered solution that resists changes in pH due to the action of its acid-base conjugated components. The formulations of the present invention include a buffer selected from at least one of citrate or phosphate buffer, preferably citrate buffer. As previously determined, these buffers enhance the stability of the vWF binder.

[0172] The formulation according to the present invention contains citrate buffer at a concentration in the range of 5 to 200 mM, preferably 7.5 to 80 mM, more preferably 10 to 50 mM, for example 10, 15, 20, 25 or 30 mM, and most preferably 20 mM, where each value is understood to optionally include a range of ±5 mM. Alternatively, the formulation according to the present invention contains phosphate buffer at a concentration in the range of 5 to 200 mM, preferably 5 to 80 mM, more preferably 7.5 to 60 mM, more preferably 10 to 40 mM, for example 10, 15, 20, 25 or 30 mM, and most preferably 10 mM, where each value is understood to optionally include a range of ±5 mM. It will be understood that lower concentrations of buffer will have an effect on the final osmotic pressure and on any further solutes that may need to be added.

[0173] The pH of the formulations of the present invention is in the range of 5.0 to 7.5, where it is understood that each value encompasses a range of ±2 mM. The most advantageous pH will depend on the buffer contained in the formulation. Accordingly, the present invention relates in particular to formulations containing phosphate buffer, which preferably have a pH in the range of 6.5 to 7.5, preferably 6.9, 7.0, 7.1, for example 7.1. Formulations containing citrate buffer have been shown to be remarkably suitable for storage and use. Accordingly, the present invention relates to formulations containing citrate buffer, which have a pH of 6.0 to 7.0, more preferably 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 or 6.9, for example 6.5, where it is understood that each value optionally encompasses a range of ±2 mM.

[0174] The formulation of the present invention contains the polypeptide of the present invention, in particular a polypeptide comprising at least one immunoglobulin monovariable domain or a polypeptide comprising at least one immunoglobulin monovariable domain relative to vWF, e.g., ALX 0081, at concentrations suitable for clinical purposes, including concentrations used in stock solutions for dilution before use in patients. In addition to improved stability, the formulation of the present invention enables polypeptides comprising at least one ISVD relative to vWF at high concentrations, e.g., ALX 0081. Typical concentrations of the activator in the formulation of the present invention, such as a polypeptide containing at least one ISVD relative to vWF, such as ALX 0081, include non-limiting examples of concentrations in the range of 0.1 to 150 mg / mL, e.g., 1 to 100 mg / mL, 5 to 80 mg / mL, or 10 to 40 mg / mL, preferably 10 mg / mL, where each value is understood to optionally encompass a range of ±20% (for example, a value of 10 optionally encompasses a range of 8 to 12 mg / mL). In further embodiments of the present invention, formulations according to any aspect of the present invention may further include detergents or surfactants.

[0175] In this specification, “surfactant” refers to a surfactant, preferably a nonionic surfactant. Examples of surfactants in this specification include: polysorbate; poloxamer (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamide Examples include propyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl taurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, NJ); polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). In one embodiment, the surfactant as used herein is polysorbate 80. Preferred detergents or surfactants for use according to the present invention include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, such as polysorbate-20, -40, -60, -65, -80, or -85. Common trade names for polysorbates include Alkest, Canarcel, and Tween. Those skilled in the art are familiar with further non-limiting examples of detergents, such as those listed in WO2010 / 077422. In a preferred embodiment, the detergent is a nonionic detergent. More specifically, the detergent is polysorbate-80, which will hereafter be referred to herein as Tween-80. Those skilled in the art can readily determine a suitable concentration of the detergent for the formulations of the present invention.Typically, the concentration is kept as low as possible while maintaining the beneficial effects of the detergent (e.g., stabilizing effect under shear stress conditions (e.g., stirring) that reduces aggregation of the formulated polypeptide of the present invention). In exemplary, non-limiting embodiments, the concentration of the detergent is in the range of 0.001 to 0.5%, for example, 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%, preferably 0.01 to 0.05%, more preferably 0.01 to 0.02%, for example, a concentration of 0.01% (v / v).

[0176] The formulation of the present invention may further contain excipients such as preservatives. "Preservative" means a compound that may optionally be included in a formulation to substantially reduce bacterial activity, thereby facilitating the preparation of a formulation for multiple uses, for example. Examples of possible preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, chlorohexanol, 3-pentanol, and m-cresol. In one embodiment, the preservative as used herein is benzyl alcohol.

[0177] The formulation of the present invention may further contain a stabilizer such as a polyol. "Polyol" refers to a substance having a plurality of hydroxyl groups, including sugars (including reducing sugars and non-reducing sugars), sugar alcohols, and sugar acids. Polyols may optionally be included in the formulation, for example, to improve stability. In certain embodiments, the polyols herein have a molecular weight of less than about 600 kD (e.g., in the range of about 120 to about 400 kD). "Reducing sugar" refers to a substance that contains a hemiacetal group and can reduce metal ions in proteins or react covalently with lysine and other amino groups, and "non-reducing sugar" refers to a substance that does not have these properties of reducing sugars. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Examples of non-reducing sugars include sucrose, trehalose, sorbose, melibiose, and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol, and glycerol are examples of sugar alcohols. Regarding sugar acids, these include L-gluconic acid and its metal salts. If desired, the formulation is stable to freeze-thaw, and it is preferred that the polyol does not crystallize to destabilize the antibody in the formulation at the freezing temperature (e.g., -20°C). In certain embodiments, non-reducing sugars such as sucrose and trehalose are examples of polyols, and sucrose is preferred despite the solution stability of trehalose.

[0178] The therapeutic compounds of the present invention used according to the present invention are prepared in the form of a lyophilized formulation or an aqueous solution by mixing a polypeptide having a desired degree of purity for storage with any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A.

[1980] ). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosages and concentrations used. Thus, the formulations according to the present invention may also optionally contain one or more excipients.

[0179] Commonly used stabilizers and preservatives are well known to those skilled in the art (see, for example, WO2010 / 077422). Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphoric acid, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, hydrophilic polymers such as polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylic acid, wax, gelatin, polyethylene polyoxypropylene block copolymers, polyethylene glycol and lanolin. Antioxidants containing ascorbic acid and methionine; preservatives; polypeptides of low molecular weight (less than about 10 residues); proteins; and amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine. In a preferred embodiment, the excipient may be one or more selected from the list consisting of NaCl, trehalose, sucrose, mannitol or glycine.

[0180] The active ingredient may also be encapsulated in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, for example, in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxyethylmethylcellulose or gelatin microcapsules and poly-(methylmethacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, edited by Osol, A. (1980). The polypeptides of the present invention can be formulated in any pharmaceutically acceptable formulation. The formulation may be liquid or dry. The formulation can be prepared by mixing, drying, freeze-drying, vacuum-drying, or any known method for formulating pharmaceutical compositions.

[0181] A preferred formulation of the present invention comprises a polypeptide (such as ALX 0081) containing at least one ISVD for vWF in a phosphate buffer solution (pH 7.1). More preferably, the formulation of the present invention comprises a polypeptide (such as ALX 0081) containing at least one ISVD for vWF in a phosphate buffer solution (pH 7.1), glycine (0.2 M), and polysorbate 80 (0.02% v / v). The polypeptide of the present invention can further be formulated as described in PCT / EP14 / 060107.

[0182] Preferred formulations include: (a) A polypeptide containing at least one ISVD relative to vWF at a concentration of approximately 0.1 mg / mL to approximately 80 mg / mL (e.g., ALX 0081); (b) Excipients selected from sucrose, glycine, mannitol, trehalose, or NaCl in a concentration of approximately 1% to approximately 15% (w / v); (c) Tween-80 at a concentration of approximately 0.001% to 0.5% (v / v); and (d) A citrate buffer with a concentration of approximately 5 mM to 200 mM, such that the pH of the formulation is approximately 6.0 to 7.0.

[0183] A further preferred formulation of the present invention comprises a polypeptide containing at least one ISVD relative to vWF (such as ALX 0081), preferably at a concentration of 10 mg / ml, citrate buffer at a concentration of 20 mM (pH 6.5), and further comprising 7% sucrose (w / v) and Tween-80 at a concentration of 0.01% (v / v). In some embodiments, the formulation is stored as a liquid. In other embodiments, the formulation is prepared as a liquid and then dried before storage, for example, by freeze-drying or spray-drying. The dried formulation may be used as a dry compound, for example, as an aerosol or powder, or it may be reconstituted to its original concentration or another concentration using water, a buffer or other suitable liquid.

[0184] The present invention also relates to a vial filled with a lyophilized product containing 12.5 mg of caplacizumab and excipients for a solution for injection. Excipients (per 1 mL of reconstituted solution): 0.21 mg of citrate, 5.58 mg of trisodium citrate dihydrate, 70 mg of sucrose, and 0.11 mg of polysorbate-80 (pH 6.5 + / - 0.5) per vial. After reconstitution with 1 mL of water for injection (WFI), the strength is 12.5 mg / mL of caplacizumab (for an apparent dose of 10 mg administered).

[0185] The present invention also includes products that can be obtained by further processing of liquid formulations, such as freeze-dried, lyophilized, or spray-dried products. Through reconstitution, these solid products can become liquid formulations as (but not limited to) described herein. Thus, in its broadest sense, the term “formulation” encompasses both liquid and solid formulations. However, solid formulations are understood as derivable from liquid formulations (e.g., by freeze-drying, lyophilization, or spray-drying) and therefore have a variety of characteristics defined by the characteristics specified for liquid formulations herein. The present invention does not exclude reconstitutions that result in compositions derived from the original composition, for example, before lyophilization or spray-drying. Thus, depending on the amount of water or diluent added to the lyophilized product compared to the volume of the originally lyophilized liquid, a lyophilized formulation may be reconstituted to produce a formulation with a different concentration than the original (i.e., before lyophilization) concentration. Suitable formulations can be identified by assaying one or more parameters of antibody integrity.

[0186] In a preferred embodiment, the formulation according to the present invention is isotonic with respect to human blood. The isotonic solution has the same osmotic pressure as plasma and can therefore be intravenously injected into the subject without altering the osmotic pressure of the subject's plasma. It can be expressed in terms of gravimetric osmolality, which may be a theoretical gravimetric osmolality or preferably an experimentally determined gravimetric osmolality. Typically, the gravimetric osmolality will be in the range of 290 ± 60 mOsm / kg, preferably 290 ± 20 mOsm / kg.

[0187] The formulations of the present invention may also contain compounds particularly useful for protecting the polypeptides of the present invention during freeze-drying. Such compounds are known as cryoprotective agents and are well known to those skilled in the art. Specific examples, but not limited to, include sugars such as sucrose, sorbitol, or trehalose; amino acids such as glutamic acid, particularly monosodium glutamate, or histidine; betaine, magnesium sulfate, sugar alcohols, propylene glycol, polyethylene glycol, and combinations thereof. By understanding the present invention, the required amount of such compounds to be added can be easily determined by those skilled in the art, taking into account the stability of the formulation in liquid form and when freeze-dried. Formulations particularly suitable for freeze-drying may further contain bulking agents. Suitable agents are widely known to those skilled in the art. Formulations containing sucrose have been shown to be particularly suitable not only for maintaining the physical stability of the vWF binder during storage and freeze-drying, but also as cryoprotective agents.

[0188] As outlined, any of the above formulations may be further processed, for example, by freeze-drying, spray-drying, or freezing, for example, bulk freezing. The resulting processed product will have characteristics derived from the liquid starting formulation, as defined above. If necessary, further agents, such as cryoprotectants, may be included for further processing. The formulations of the present invention have the effect of maintaining the chemical and physical integrity of the polypeptide of the present invention, particularly ALX 0081, after lyophilization. That is, even after long-term storage at temperatures of -70°C to +40°C, for example over the period defined above, the purity / impurity profile of the product does not change essentially. For example, long-term storage after lyophilization did not have a significant effect on the RP-HPLC, SE-HPLC, or cIEF profile.

[0189] The polypeptides of the present invention can be produced by any commonly used method. Typical examples include recombinant expression in a suitable host system, such as bacteria or yeast. The polypeptides of the present invention undergo a suitable purification regimen before being formulated according to the present invention. Generally, the polypeptides of the present invention are produced by living host cells genetically engineered to produce the polypeptides. Methods for genetically engineering cells to purify proteins are well known in the art. See, for example, Ausubel et al. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods involve introducing nucleic acids that encode the polypeptide and enable its expression into living host cells. These host cells may be bacterial, fungal, or animal cells grown in culture. Examples of bacterial host cells, but not limited to, Escherichia coli cells. Examples of suitable E. coli strains include HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that cannot cleave foreign DNA. Examples of usable fungal host cells, but not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Aspergillus cells. Some examples of animal cell lines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and WI38. Novel animal cell lines may be established using methods well known to those skilled in the art (e.g., by transformation, viral infection, and / or selection). Optionally, polypeptides may be secreted into the culture medium by the host cell.

[0190] In some embodiments, polypeptides can be produced in bacterial cells, such as E. coli cells. For example, if the polypeptide is encoded by a sequence in a phage display vector, and the sequence includes a controllable stop codon between the display entity and the bacteriophage protein (or its fragment), the vector nucleic acid can be transferred into bacterial cells that cannot suppress the stop codon. In this case, the polypeptide does not fuse with the gene III protein and is secreted into the periplasm and / or culture medium. Polypeptides can also be produced in eukaryotic cells. In one embodiment, polypeptides are expressed in yeast cells such as Pichia (see, e.g., Powers et al. J Immunol Methods 251:123-35 (2001)), Hansenula, or Saccharomyces.

[0191] In one embodiment, polypeptides are produced in mammalian cells. Typical mammalian host cells for expressing clonal antibodies or their antigen-binding fragments include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980), used with a DHFR-selectable marker such as those described in Kaufman and Sharp, Mol. Biol. 159:601-621 (1982)), lymphocyte cell lines such as NS0 myeloma cells and SP2 cells, COS cells, and cells from transgenic animals, such as transgenic mammals. For example, mammary epithelial cells are used.

[0192] In addition to the nucleic acid sequence encoding the polypeptide, the recombinant expression vector may carry further sequences, such as sequences that control the replication of the vector in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216; 4,634,665; and 5,179,017). Typically, for example, selectable marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced.

[0193] Using standard molecular biology techniques, recombinant expression vectors can be prepared, host cells transformed, transformants selected, host cells cultured, and antibody molecules recovered from the culture medium. For example, the polypeptide of the present invention can be isolated by affinity chromatography. In one embodiment, the polypeptide of the present invention is purified as described in WO 10 / 056550. In an exemplary embodiment, the polypeptide is purified from one or more impurities by: contacting a mixture of the polypeptide and impurities with a Protein A-based support and / or an ion-exchange support under conditions that allow the polypeptide to bind to or adsorb to the support; removing one or more impurities by washing the bound support under conditions that allow the polypeptide to remain bound to the support; and selectively eluting the polypeptide from the support by eluting the adsorbed polypeptide molecules with an elution buffer.

[0194] The polypeptides of the present invention can also be produced by transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed comprising a milk-specific promoter and a nucleic acid encoding an antibody molecule and a signal sequence for secretion. The milk produced by such a female transgenic mammal contains the single domain of interest as secreted therein. The antibody molecule may be purified from the milk or used directly for some applications. The present invention encompasses methods for producing formulations as defined herein.

[0195] For example, when eluting the polypeptide of the present invention from a column using the buffer according to the present invention, the purification and formulation steps may be performed simultaneously. Alternatively, the formulation of the present invention can be prepared by exchanging the buffer by any suitable means, such as dialysis or ultrafiltration, which are widely used in the art. In some embodiments, the method of manufacturing a formulation of the invention may also relate to the reconstitution of a lyophilized or spray-dried formulation by the addition of, for example, water or a suitable buffer, which may optionally contain further excipients.

[0196] The method for preparing a formulation according to the invention may include further steps, such as filling it into a vial suitable for clinical use, such as a sealed container, and / or formulating it into a unit dosage form. The method may also include further steps such as spray drying, lyophilization, or freezing, such as bulk freezing. The invention also encompasses containers, unit dosage forms, or other products obtainable by any of the methods described herein. The formulations of the invention can be used to store a polypeptide of the invention as defined herein, such as a polypeptide comprising at least one ISVD against a polypeptide of the invention, such as vWF, for example ALX 0081. Thus, the invention encompasses a method of storing a polypeptide of the invention as used herein, characterized by the use of a formulation as defined herein. More specifically, the invention encompasses a method for stabilizing a polypeptide of the invention for storage, the method including, for example, the preparation of a formulation as described herein. Storage may optionally be at a temperature of -70°C to +40°C, such as -70°C, -20°C, +5°C, +25°C or +40°C, preferably at a temperature of -70°C to +25°C, more preferably at a temperature of -20°C to +5°C, for 1 to 36 months, such as 1, 1.5, 3, 6, 9, 12, 18, 24, 30 or 36 months, for example at least 12 months. Thus, storage may include freezing, freeze-drying (lyophilization) and / or spray drying. The storage method may further include an assessment of the physical and chemical integrity of a vWF binder as defined herein.

[0197] The present invention also relates to a method for analyzing formulations comprising at least one of the vWF binders as defined herein. The formulations can be analyzed for any indication of vWF binder chemical or physical instability as defined herein. For example, the formulations can be evaluated for the presence of degradation products, such as low molecular weight derivatives such as proteolytic fragments; and / or compound derivatives, such as pyroglutamic acid variants; and / or high molecular weight derivatives such as aggregates and clumps. The formulations can also be evaluated for total protein content and / or titer. Each of the various assay methods referred to herein can be used in the analytical method of the present invention.

[0198] Accordingly, the present invention also relates to a method for monitoring and / or evaluating the quality and / or stability of a formulation during, for example, one or more of the following: manufacturing, storage, and use. The present invention also relates to a method for controlling the quality of a formulation to evaluate whether the formulation conforms to product specifications, for example, as further described herein. In any of these aspects, the present invention includes one or more selected from comparison with one or more reference samples, batch or batch variation, and monitoring in progress of the manufacturing process.

[0199] The present invention relates, without limitation, to any product relating to the formulation of the present invention, for example, by including the formulation of the present invention, or by being necessary for the manufacture or formulation of the formulation of the present invention. For example, the present invention relates to a sealed container containing a product, such as one or more formulations according to the present invention. The present invention also relates to a pharmacokinetic dosage form, for example, a dosage form suitable for parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous) to a patient, preferably a human patient, comprising one or more formulations in any embodiment described herein. The dosage unit may be in the form of, for example, a pre-filled syringe, ampoule, cartridge, or vial.

[0200] Also provided is a kit or product, including the formulation of the present invention and instructions for use, for example, by a healthcare professional. The kit or product includes a vial or syringe containing the formulation as described herein. Preferably, the vial or syringe is made of glass, plastic, or a polymer material selected from cyclic olefin polymers or copolymers. Syringes, ampoules, cartridges, or vials can be manufactured from any suitable material, e.g., glass or plastic, and may include rubber materials, e.g., rubber stoppers for vials, and rubber stoppers and rubber seals for syringes and cartridges. The present invention also relates to a kit comprising one or more formulations according to the present invention. The kit may further include instructions for use and / or a clinical package insert. In any embodiment of the product as defined herein, the present invention also includes packaging materials, instructions for use, and / or a clinical package insert as required by regulatory requirements, for example.

[0201] For the purpose of comparing two or more amino acid sequences, the percentage of “sequence identity” (also referred to herein as “amino acid identity”) between a first amino acid sequence and a second amino acid sequence can be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], where each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (position), i.e., an “amino acid difference” as defined herein.

[0202] Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as those mentioned above, for determining the degree of sequence identity between nucleotide sequences, also using standard settings. Typically, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences using the calculation method outlined above in this specification, the amino acid sequence having the largest number of amino acid residues is designated as the "first" amino acid sequence, and the other amino acid sequence as the "second" amino acid sequence.

[0203] Furthermore, in determining the degree of sequence identity between two amino acid sequences, those skilled in the art can consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which one amino acid residue is replaced by another amino acid residue of a similar chemical structure that has little or no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art from, for example, WO 04 / 037999, GB-A-3 357 768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and (preferred) types and / or combinations of such substitutions can be selected based on relevant teachings from WO 04 / 037999 and WO 98 / 49185, as well as from further references cited herein. Such conservative substitutions are preferably those in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.Any amino acid substitutions applied to the polypeptides described herein may also be based on an analysis of the frequency of amino acid changes between homologous proteins of different species, developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978; or on an analysis of structure formation potentials, developed by Chou and Fasman, Biochemistry 13:211, 1974 and Adv. Enzymol., 47:45-149, 1978; or on Eisenberg et al., Proc. Natl. Acad. Sci. USA 81:140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157:105-132, 1981 and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353. The analysis may also be based on the development of hydrophobic patterns in proteins in 1986, all of which are incorporated herein by reference in their entirety. Information on the primary, secondary, and tertiary structures of Nanobody® is given in the description herein and in the general background art cited above. For this purpose, the crystal structure of the VHH domain from llama is given, for example, in Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further information on some of the amino acid residues that form the VH / VL interface in conventional VH domains, and on possible camelid substitutions at these positions, can be found in the prior art cited above.

[0204] The present invention also relates to methods for treating or preventing vWF-related disorders, such as acute coronary syndrome (ACS), transient ischemic attack, unstable or stable angina, stroke, myocardial infarction, or thrombotic thrombocytopenic purpura (TTP); the methods include administering to a subject a pharmaceutical composition comprising the formulation of the present invention, thereby alleviating one or more symptoms associated with the vWF-related disorder. In particular, the vWF-related disorder is TTP.

[0205] In addition, the present invention relates to a method for treating human patients who are susceptible to or have been diagnosed with a disease characterized by vWF-related disease, the method comprising administering to a human patient an effective amount of a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF). The present invention provides a method for treating or preventing vWF-related diseases such as TTP, the method comprising administering to a human being a polypeptide comprising at least one immunoglobulin monovariate domain (ISVD) against von Willebrand factor (vWF) in a dose of 5 to 40 mg, thereby alleviating one or more symptoms associated with the vWF-related disease.

[0206] The present invention provides a procedure as described herein, wherein a first plasma exchange (PE) is performed within 5 minutes to 8 hours after administration of a polypeptide as described herein. The present invention provides a procedure as described herein, wherein a prior plasma exchange (PE) is performed within 36 hours, preferably within 32, 30, 28, 26, 24, 22, 20, 18 or 16 hours, preferably within about 24 hours, of the first PE before administering the polypeptide as described herein. The present invention provides a treatment as described herein, wherein a second dose of 1 to 40 mg, preferably 10 mg, of a polypeptide as described herein is administered within 5 minutes to 8 hours, for example, 10 minutes to 6 hours or 15 minutes to 4 hours, for example, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or just 5 minutes after the first PE, for example, wherein the second dose of the polypeptide is administered within 1 to 60 minutes, for example, 30 minutes, preferably by subcutaneous injection.

[0207] The present invention (i) Perform PE; (then) (ii) Administer a dose of 5 to 40 mg of a polypeptide as described herein 15 minutes to 4 hours after the PE in step (i); and (iii) Optionally, measure the platelet count and / or ADAMTS13 activity of the patient. The treatment provided herein includes, further including, Steps (i) and (ii) are repeated once per day until the patient's platelet count is ≥150,000 / μl and / or the ADAMTS13 activity is at least 10% of the ADAMTS13 reference activity, for example, at least 15%, 20%, 25%, 30%, 35%, 45%, or 50%.

[0208] The present invention also provides a treatment as described herein, further comprising administering a dose of 5 to 40 mg of a polypeptide as described herein once daily for at least 5, 10, 15, 20, 25 or further 30 days, after the patient's platelet count has reached ≥ 150,000 / μl. The present invention provides a treatment as described herein, further comprising administering a dose of 5 to 40 mg of a polypeptide as described herein once daily until the person achieves remission. The present invention provides a treatment as described herein, comprising administering the polypeptide until the ADAMTS13 activity is at least 10% of the ADAMTS13 reference activity, for example, at least 15%, 20%, 25%, 30%, 35%, 45%, or 50%.

[0209] In another embodiment of the present invention, a product is provided comprising materials useful for treating the diseases described above. The product comprises a container, a label, and an accompanying leaflet. Suitable containers include, for example, bottles, vials, and syringes. The container may be made of a variety of materials, such as glass or plastic. The container may hold a composition effective in treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous injection needle). At least one activator in the composition is a polypeptide of the present invention, such as ALX 0081. A label on or associated with the container indicates that the composition is used to treat a selected condition. The product may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline or citrate-buffered saline, as described herein. It may further comprise other materials desirable from the user or commercial standpoint, including other buffers, diluents, filters, needles, and syringes.

[0210] The present invention provides a kit or product comprising a container containing a polypeptide or formulation as described herein, and instructions for use. The present invention provides a kit or product as described herein, wherein the formulation is contained in a vial or syringe for injection. The present invention provides a kit or product as described herein, wherein the formulation is present in a pre-filled syringe for injection. The present invention provides a kit or product as described herein, wherein the syringe or vial is made of glass, plastic, or a polymer material selected from cyclic olefin polymers or copolymers.

[0211] The embodiments described and discussed herein are intended solely to teach those skilled in the art the best methods known to the inventors for manufacturing and using the invention. It will be understood, in consideration of the above teachings, that modifications and variations of the above embodiments of the invention are possible without departing from the invention. Therefore, it will be understood that within the scope of the claims and their equivalents, the invention may be carried out in ways different from those specifically described. The present invention will now be further described by the following non-limiting preferred aspects, examples, and drawings. The entire contents of all references cited throughout this application (scientific literature, published patents, published patent applications, and concurrently pending patent applications) are expressly incorporated herein, in particular with respect to the teachings referred to above.

[0212] 6. Abbreviations AE harmful events ACS acute coronary syndrome ADAMTS13 Disintegrin-like and meta having thrombospongin repeats Loprotease 13 (a disintegrin-like and metalloprotease with thrombospondin repeats 13) ALX 0081 Caplacizumab AMI (Acute Myocardial Infarction) BNP (Brain Natriuretic Peptide) Body Mass Index (BMI) BU (Bethesda Units) CDR Complementarity Determination Region cIEF capillary isoelectric focusing electrophoresis dAb single-domain antibody ELISA enzyme-linked immunosorbent assay HR Hazard Ratio ISVD Immunoglobulin Monovariable Domain ITT (Intention to Treat) iv intravenous FR Framework Domain KA meeting number KD dissociation constant LDH lactate dehydrogenase NSE (Neuronal-Specific Enolase) NT proBNP (N-terminal pro-brain natriuretic peptide) PE or PEX plasma exchange Per PP protocol RICO ristocetin cofactor activity RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) SAE (Serious Adverse Event) sc subcutaneous scFv Single-chain variable fragment S / D Solvent / Detergent SE-HPLC (Self-Efficiency Sieving High-Performance Liquid Chromatography) SPR Surface Plasmon Resonance TnI (Troponin I) TnT (Troponin T) TRALI (Transfusion-Related Acute Lung Injury) TTP (Thrombotic Thrombocytopenic Purpura) TTR (Time to Response) ULN Upper Limit of Normal Values ULvWF Super huge vWF VH Heavy Chain Variable Domain Heavy chain variable domain sequence derived from VHH heavy chain antibody VL (Light Chain Variable Domain) vWF von Willebrand factor

[0213] 7. Example 7.1 Applicable Regulations All human samples used in the example section were obtained from either commercially available sources or human volunteers (after obtaining all necessary consent and approval) and were used in accordance with applicable legal and regulatory requirements, including those relating to medical secrecy and patient privacy. Clinical trials were conducted in accordance with applicable laws and regulations (including the Declaration of Helsinki and the principles of protecting medical secrets and patient privacy) after obtaining all necessary approvals (including approval by relevant ethics committees) and informed consent (including informed consent from those included). The purpose, content, and results of this clinical study are treated as confidential and have not been made available to third parties. All personnel participating in the study were bound by confidentiality agreements. All unused drugs were returned to the applicant or destroyed.

[0214] 7.2 Effects of ALX 0081 on platelet adhesion to endothelial cell-derived ULvWF and on ADAMTS13 activity Goal: The goal of this study was to evaluate whether the polypeptides of the present invention, such as ALX 0081, can inhibit platelet adhesion to ULvWF. This could then serve as a proof of concept for the use of the polypeptides of the present invention, e.g., ALX-081, for the treatment of TTP patients in acute episodes. The study also determines the effect of the polypeptides of the present invention, such as ALX 0081, on the activity of ADAMTS13. Methods: Following Sixma (Sixma et al. 1998 Thromb Res 92: S43-S46), a flow chamber test was used to investigate whether the polypeptides of the present invention, such as ALX 0081, could inhibit the interaction between platelets and ULvWF. Briefly, endothelial cells were cultured and stimulated on coverslips to induce ULvWF secretion. Platelets were supplemented with plasma from TTP patients and perfused over the stimulated cells. Platelet strings adhering to ULvWF were visualized using a real-time video microscope. The experiment was repeated while increasing the concentration of the polypeptides of the present invention, such as ALX 0081, that were present. Two types of experiments were used to determine the effect of the polypeptides of the present invention, such as ALX 0081, on ULvWF cleavage by ADAMTS13. In the first experiment, platelet string cleavage by ADAMTS13 was evaluated in the absence of excess polypeptides of the present invention, such as ALX 0081. In the second experiment, the inhibitory effect of polypeptides of the present invention, such as ALX 0081, on ADAMTS13 was evaluated using a recombinant fragment consisting of the A1-A2-A3 domains of vWF.

[0215] Results: ALX 0081 inhibited platelet string formation in ULvWF at all tested concentrations and had no effect on platelet string detachment by ADAMTS13. The polypeptides of the present invention, including ALX 0081, also had no effect on the cleavage of recombinant A1-A2-A3 domain fragments of vWF by ADAMTS13. Furthermore, the polypeptides of the present invention, including ALX 0081, were unable to detach platelets from already formed strings in this experiment. Conclusion: This study provides proof of concept that the polypeptides of the present invention, such as ALX 0081, can be used to treat TTP patients. This study also demonstrates that the polypeptides of the present invention, such as ALX 0081, do not interfere with ADAMTS13 activity.

[0216] 7.3 Eligibility Criteria Patients must meet all of the following criteria to be eligible for approval of the study: Calculation criteria 1. Ages 18 and over 2. Men or women who wish to accept acceptable contraceptive regimens 3. Patients with a clinical diagnosis of TTP 4. The patient requires a PE (a single PE session is permitted before randomization to the study). 5. Subjects accessible for follow-up observation. 6. An informed consent that has been obtained, signed, and dated.

[0217] Exclusion criteria 1. Platelet count of 1,100,000 / μL or higher 2. Severe, active infection indicated by sepsis (with or without positive blood cultures, and requiring vasopressors) 3. Clinical evidence of intestinal infection by E. coli O157 or related organisms 4. Antiphospholipid syndrome 5. Diagnosis of DIC 6. Pregnancy or breastfeeding 7. Thrombotic microangiopathy associated with hematopoietic stem cell or bone marrow transplantation 8. Known congenital TTP 9. Active bleeding or high risk of bleeding 10. Uncontrolled arterial hypertension 11. Known anticoagulant treatments that cannot be safely discontinued (including, but not limited to, vitamin K antagonists, heparin, or LMWH), and chronic treatments with nonacetylsalicylic acid nonsteroidal anti-inflammatory molecules. 12. Other serious or life-threatening clinical conditions that would be an impediment to participation in the clinical trial. Subjects with malignant lesions resulting in an average life expectancy of less than 13.3 months. 14. Subjects with known or suspected myelocarcinomatosis 15. Subjects who cannot comply with the requirements and procedures of the research protocol. 16. Known hypersensitivity to the active substance or excipients of the research drug. 17. Severe hepatic impairment corresponding to Grade 3 severity as defined by the CTCAE scale. Regarding key liver parameters, this is defined as follows: • Bilirubin > 3 × ULN (It is necessary to distinguish isolated increases in bilirubin due to hemolysis, which is not an exclusion parameter but is associated with the disease.) • ALT / AST>5×ULN • AP>5×ULN Gamma-glutamyl transpeptidase (GGT) > 5 × ULN 18. Severe chronic renal impairment defined by GFR < 30 mL / min

[0218] 7.4 Research Design This study was designed as a Phase II, multicenter, single-blind, parallel-design, randomized, placebo-controlled study (Titan trial). The study population consisted of symptomatic patients with an acute episode of acquired TTP requiring treatment with PE. After confirmation of eligibility to participate in the study (see Example 7.3), patients were randomized in a 1:1 ratio to receive either ALX 0081 or placebo as adjunctive treatment for PE (Figure 1). Patients were randomized before the initiation of PE treatment. However, in exceptional cases (due to the need or ability to initiate PE within a timeframe that does not allow all necessary screenings and / or baseline study procedures to be performed), patients were randomized after a single prior PE session ("prior PE") but before the initiation of the next PE session ("first PE"). This subsequent PE session as a whole was initiated within 24 hours of the end of the prior PE session and was considered the first PE in the study ("first PE").

[0219] Patients were followed at different phases throughout this study: • Post-admission screening and baseline measurement • Treatment phase ○ A single IV bolus study agent administered via push injection ○ Daily supplemental sc treatment phase for PE ○ Daily post-PE treatment phase (including tapering of PE and post-PE study drugs for 30 days after the last PE, if applicable) • Observation period

[0220] Patients received the best medical care and treatment deemed appropriate by researchers at their respective locations, in accordance with guidelines for the management of TTP. The maximum total duration of individual study participation was up to 15 months: a treatment phase of up to 90 days, and a follow-up period of up to 1 year after remission or 90 days of treatment, whichever came first. Generally, patients were hospitalized for at least one day after their last daily PE. The study drug was administered as an adjunct treatment at specific time points relative to the PE procedure. The study drug consisted of 10 mg of caplacizumab ("treatment group") or placebo ("placebo group") once or twice daily.

[0221] 7.4.1 First drug administration Patients received a first IV bolus of 10 mg of ALX 0081 or placebo via push injection 15 minutes to 6 hours before the start of the first PE. Following this first PE, a 10 mg sc dose of the study drug was administered within 30 minutes of the completion of the PE procedure. complete PE treatment period During this period (including PE administered for tapering and exacerbation), the study drug was administered daily via sc injection.

[0222] If a PE (penetration) was planned once per day, 10 mg of the study drug was administered within 30 minutes of the completion of the PE procedure. When two PE (prenatal administration) procedures were planned per day, 10 mg of the study drug was administered within 30 minutes of the completion of each PE procedure. The maximum total daily dose of the study drug was therefore 20 mg. When fewer than one PE per day were planned (i.e., during tapering regimens), 10 mg of the study drug was administered daily. On days when PE was performed, the study drug was administered within 30 minutes of the completion of the PE procedure; on days when PE was not performed, the study drug was administered 24 hours (±1 hour) after the previous dose. Daily administration of 10 mg of the study drug at sc was continued for 30 days after the last PE (including tapering).

[0223] 7.4.2 Primary Endpoint The primary endpoint of this Phase II study was time to response (TTR) based on the following criteria: platelet recovery ≥ 150,000 / μL. To qualify as meeting the endpoint, the response had to be confirmed by a new platelet measurement ≥ 150,000 / μL and lactate dehydrogenase (LDH) ≤ 2 × upper limit of normal (ULN), i.e., a "confirmed platelet response," 48 hours after the first report of platelet recovery equal to or higher than 150,000 / μL. Platelet count is an important research marker for treatment decision-making in patients with TTP. This is based on the fact that ULvWF-mediated platelet aggregation is a common pathophysiological mechanism behind TTP and leads to severe thrombocytopenia and microangiogenic hemolytic anemia, which are key features in the diagnosis of TTP (Scully et al., above).

[0224] 7.4.3 Determination of ADAMTS13 activity The activity of ADAMTS13 and its functional inhibitors was measured by fluorescence assay using the FRETS-VWF73 substrate (Kokame et al. 2005. Br J Haematol 129(1):93-100; Kremer Hovinga et al. 2006 J Thromb Haemost 4(5):1146-8). Briefly, the FRETS-VWF73 assay was performed essentially as described (Kokame et al. 2005, above), with the following modifications: Pefabloc SC (Boehringer, Mannheim, Germany) was added to assay buffer (5 mmol L-1 Bis-Tris, 25 mmol L-1 CaCl2, 0.005% Tween-20, pH 6.0) at a final concentration of 1 mmol L-1. Assay calibration was performed using a pool of normal human plasma (NHP; Swiss Red Cross Blood Services, Bern, Switzerland) diluted 1:25 in assay buffer (100%). Further calibration samples were obtained by serial pre-dilutions of NHP in heat-inactivated NHP (incubated at 56°C for 30 minutes, followed by centrifugation at 15,000 × g for 15 minutes) at 3:4 (75%), 1:2 (50%), 1:4 (25%), 1:10 (10%), 1:20 (5%), 1:50 (2%), and 1:100 (1%) to correct for plasma matrix effects in the lower activity range of the standard curve. All of these standard samples, as well as heat-inactivated NHP (0% ADAMTS13 activity) and all test samples, were then diluted 1:25 in assay buffer. Next, 25 μL of each diluted standard or patient sample was incubated at 37°C in a 384-well white plate (NUNC, Roskilde, Denmark). After 10 minutes, 4 μmol of L-1 FRETS-VWF73 peptide substrate, dissolved in 25 μL of assay buffer, was added to each well, and fluorescence emission was recorded at 37°C using a fluorescence microplate reader (GENios, Tecan, Zurich, Switzerland) equipped with a 340 nm excitation filter (bandwidth 35 nm) and a 450 nm emission filter (bandwidth 25 nm). Fluorescence emission was measured over time (42 cycles every 5 minutes). The reaction rate was calculated by linear regression analysis (Passing-Bablok) of fluorescence emission over time from 5 minutes (cycle 2) to 60 minutes (cycle 13).For each calibration sample, the slope of the regression curve was calculated and used to create the calibration curve (trend line): y = ax + b, where x = ADAMTS13 (%) and y = delta RFU / delta time). Next, the ADAMTS13 activity (%) of the sample was calculated as (yb) × 1 / a.

[0225] The activity of functional inhibitors of ADAMTS13 was determined by the same fluorescence-generating FRETS-VWF73 method, by determining the residual ADAMTS13 activity in normal human plasma after 1:1 (v:v) incubation at 37°C for 2 hours, using patient plasma that had been thermally inactivated (56°C for 30 minutes). For each analysis batch, a calibration curve was prepared using (100%) normal human plasma pool (NHP; Swiss Red Cross Blood Services, Bern, Switzerland) diluted 1:25 in assay buffer. Further calibration samples were obtained by serial pre-dilutions of NHP in thermoactivated NHP at 1:2 (50%), 1:4 (25%), 1:10 (10%), 1:20 (5%), 1:50 (2%), and 1:100 (1%). All calibration points were applied once. Acceptance criteria: (1) the slope of the final regression of the standard curve must be >6.0; and (2) the R² of the regression in the final plot must be >0.98 (or R>0.9899). Otherwise, the assay is rejected.

[0226] 7.5 Outcomes in TTP patients 7.5.1 Analysis of the placement and grouping of subjects. The Phase II study included a sample size of 75 patients, who were randomized as shown in Table 2. The primary analysis population was the intent-to-treat (ITT) population, which consisted of all randomized subjects assigned to randomized treatments. In addition, per-protocol populations (PP) were used for efficacy analysis. The PP population was a subset of the ITT population, consisting of all randomized subjects assigned to randomized treatments, with all major protocol deviations and violations excluded. [Table 2]

[0227] In conclusion, a uniform distribution was observed in both treatment groups, namely patients receiving caplacizumab (treatment group) and patients receiving placebo (placebo group). Furthermore, the treatment groups were well-balanced in terms of age, ethnicity / race, and BMI. Baseline characteristics of various parameters were evaluated in patients in the treatment and placebo groups. Table 3 shows baseline platelet counts and LDH levels. Elevated LDH levels are an indication of increased hemolysis and / or tissue ischemia. [Table 3]

[0228] A slightly lower mean baseline platelet count was observed in the caplacizumab group. In both groups, the mean platelet count was very low at baseline, which is consistent with the setting of severe disease, but also indicates that all present subjects were considered for inclusion and that there was no bias towards subjects with less severe thrombocytopenia. LDH is comparable for both treatment groups.

[0229] Table 4 shows baseline vWF:Ag and ADAMTS13 activity. [Table 4] Both treatment groups showed a good balance in terms of vWF:AG and ADAMTS13 activity. More than half of the subjects had idiopathic TTP, as indicated by ADAMTS13 activity of <5%.

[0230] 7.5.2 Research Results: Primary Endpoint Time to response to blood markers was monitored in a survival setting. The primary endpoint, time to response to blood markers, included platelet recovery ≥ 150,000 / μL. Platelet levels represent a reliable surrogate marker for the disease activity of TTP. The time from zero to event was set to 30 days. The results are shown in Table 5.

[0231] The data were evaluated by stratification (one pre-randomization PEX: with & without), and aggregated to a total hazard ratio of 2.197 for the complete ITT population, with a p-value of 0.013 for the total ITT population. The hazard ratio means that, at any given time point, subjects receiving placezumab were twice as likely to achieve the primary endpoint of confirmed platelet recovery compared to subjects receiving placebo. Reduced time to confirmed platelet response (primary endpoint): - In the group that did not undergo pre-randomization PEX, the median duration was reduced from 4.92 days in the placebo group to 3.00 days in the caplacizumab group: a 39% reduction (pre-randomization PEX = none). - In the group that received one pre-randomization PEX, the median time was reduced from 4.31 days in the placebo group to 2.44 days in the caplacizumab group: a 43% reduction (with pre-randomization PEX).

[0232] The median time of 4.31 days and 4.92 days for the two strata of the placebo group (with and without a single pre-randomization PEX) was lower than predicted from medical literature and researcher data (6 days; see Bandarenko et al. Journal of Clinical Apheresis 1998; 13: 133-141). This indicates that caplacizumab treatment was superior even with improved standards of medical treatment compared to historical data. The 95% confidence interval (CI) for the time to confirmed platelet response was 2–3 times narrower in the caplacizumab group compared to the placebo group. This indicates that the time to disease resolution was less variable in the caplacizumab-treated group than in the placebo group. [Table 5]

[0233] 7.5.3 Research findings: Exacerbation Within the ITT population, the proportion of subjects experiencing exacerbations was determined. An exacerbation is defined as recurrent thrombocytopenia following a confirmed platelet response, occurring at least one day after the last PE, but requiring the restart of daily PE treatment within 30 days. The results are shown in Table 6. [Table 6] There were three times more exacerbations in the placebo group than in the caplacizumab group. This confirms the finding that polypeptides of the present invention, such as ALX 0081, may, on their own, cause treatment and / or alleviation of TTP (symptoms).

[0234] 7.5.4 Research findings: Recurrence The number of subjects experiencing a relapse of TTP was evaluated. A TTP relapse is defined as a new TTP event occurring more than 30 days after the last daily PE. Table 7 shows the proportion of subjects in the ITT population who experienced exacerbations and / or relapses within one month of the end of treatment. [Table 7]

[0235] The inventors observed more relapses in the caplacizumab group than in the placebo (PLC) group, which equalizes the total number of relapses. Relapses in the caplacizumab group often occur within a few days of discontinuing caplacizumab treatment. This suggests the possibility of a "prolonged" exacerbation rather than a relapse. Therefore, caplacizumab (CAP) treatment may have been too short in some patients. In fact, early relapses in a larger number of CAP patients demonstrate a protective effect and justify longer CAP treatment in some patients. This indicates that caplacizumab treatment should be continued for a longer period. Although ADAMTS13 activity is available in only a subset of patients, it is interesting to note that in both treatment groups, relapse was more pronounced in patients with <5% baseline ADAMTS13 activity. This may indicate that patients with <5% baseline ADAMTS13 activity are more prone to relapse (or exacerbation), and that caplacizumab treatment should be continued for a longer period compared to patients with higher activity (see Example 7.5.8).

[0236] 7.5.5 Research Results: Complete Remission The number of subjects with complete remission in the ITT population was evaluated. Complete remission is defined as the absence of confirmed platelet response and exacerbations. The results are shown in Table 8. [Table 8]

[0237] The caplacizumab group represents a 1.6-fold increase in the number of patients with complete remission compared to the placebo group. The higher rate of complete remission is associated with a faster and narrower time to confirmed platelet response, which supports greater predictability of patient response to PE in the CAP group and is reflected in the duration of PE. The duration of PE was 6.6 ± 3.4 days (mean ± standard deviation) for CAP compared to 8.1 ± 6.5 days for PLC. The total plasma volume administered, including tapering, was 22.5 ± 15.9 liters for CAP compared to 28.4 ± 21.3 liters for PLC. Although baseline ADAMTS13 activity data is incomplete for the caplacizumab group, the effect is more pronounced in subgroups of patients with low baseline ADAMTS13 activity (see Example 7.5.8).

[0238] 7.5.6 Research Results: Safety Assessment The SAE assessment, which evaluated the occurrence of adverse events (AEs) and serious adverse events (SAEs) focusing on bleeding-related and immune-related SAEs, included bleeding from catheter insertion, sepsis, catheter thrombosis, pneumothorax, fluid overload, hypoxia, hypotension, anaphylactic-like reactions, and TRALI. The number of serious adverse events was similar between both treatment groups: 51% in the placebo group compared to 57% in the caplacizumab group. The number of adverse events was also similar between the treatment groups: 100% in the placebo group compared to 97% in the caplacizumab group. The number of subjects with bleeding-related AEs was slightly higher in the caplacizumab group (54%) compared to the placebo group (38%).

[0239] The most significant risk associated with currently used nonspecific antithrombotic agents is predisposition to bleeding or an apparent increase in bleeding. Besides any unpredictable effects, bleeding also represents the most relevant safety concern with caplacizumab. In this regard, caplacizumab was investigated in a preclinical surgical bleeding model. In this study, surgical blood loss in animals treated with caplacizumab was comparable to blood loss in animals treated with Heparin®, and 2-fold and 4-fold lower, respectively, than in animals treated with Plavix® and ReoPro®. This is a positive indicator that caplacizumab may be safer than Plavix® and ReoPro® in terms of bleeding risk, although this was assessed in healthy individuals. As shown above, TTP patients differ from healthy individuals in many respects with respect to vWF. The study procedure was discontinued due to adverse events in four patients treated with caplacizumab and two patients treated with placebo. However, the increased bleeding tendency was well manageable. TTP is potentially life-threatening. In the clinical trial, two deaths (5.4%) were reported, both in the placebo group. It should be noted that the number of deaths in the placebo group is lower than those reported in the literature (10-30%). From this, it can be inferred that the prognosis of TTP improved (in this study) due to more effective medical standards.

[0240] 7.5.7 Research findings: Further optimized treatment protocol Further optimized treatment protocols were designed by the inventors based on the idea that the observed platelet response time distribution in the CAP group was shorter, not skewed to the right (longer response time), and not biased compared to the placebo group. In the further optimized treatment protocol, all subjects are treated essentially as appropriately described prior to 7.4, “Study Design,” but with the following key modifications: The PE treatment period is set to 3–5 days, e.g., 3, 4, or 5 days, preferably 3 days. The PE treatment period is independent of platelet recovery (≥150,000 / μl). Daily sc research drug administration continues for at least 10 days after the last PE, e.g., 20 or 30 days, but preferably for at least 10 days after platelet recovery reaches ≥150,000 / μl, e.g., 20 or 30 days. In evaluating the further optimized treatment protocol, the primary endpoint would be the number of exacerbations as defined above. Further optimized treatment protocols reduced patient burden and costs.

[0241] 7.5.8 ADAMTS13 activity We planned to further evaluate ADAMTS13 activity as a marker for underlying disease activity and as a marker to guide the optimal duration of caplacizumab treatment to maintain treatment benefits. In this case, if ADAMTS13 activity is <10%, underlying disease activity is assumed. If ADAMTS13 activity is ≥10%, underlying disease activity is assumed not to exist. Exacerbations and relapses were associated with available ADAMTS13 activity data in patients. A relapse was considered a symptomatic episode relapse if it was preceded by consistently low ADAMTS13 (<10%) during treatment. A relapse was considered a new TTP episode if it was preceded by ≥10% of ≥10% during treatment. Patients excluded from the analysis were (i) patients with non-ADAMTS13-mediated disease (≥10% at baseline) (n=3) and (ii) patients with insufficient data (n=12).

[0242] (i) Patients who do not have an exacerbation or relapse: - Caplacizumab: 13 / 16 (81%) had ADAMST13 activity levels of ≥10% near the time of discontinuation of treatment. - Placebo: 14 / 16 (88%) had ADAMST13 activity levels of ≥10% near the end of treatment. Therefore, in the majority of patients without exacerbations or relapses, ADAMTS13 activity recovered to levels exceeding 10%, suggesting resolution of symptomatic TTP episodes. (ii) Patients with exacerbations - Caplacizumab: 2 / 3 (67%) had <10% ADAMTS13 activity near its exacerbation. - Placebo: 7 / 8 (88%) had <10% ADAMTS13 activity near exacerbation. Therefore, in the majority of patients experiencing TTP exacerbations, ADAMTS13 activity is less than 10%, suggesting that the disease activity causing the exacerbations remains unresolved.

[0243] (iii) Patients with recurrent symptomatic TTP episodes - Caplacizumab: 7 / 7 (100%) had ADAMTS13 activity levels <10% near discontinuation of treatment; relapse occurred within 10 days after discontinuation of treatment. -Placebo: N / A- It was all a "new" relapse. Therefore, all subjects in the caplacizumab group who experienced a relapse immediately after the end of treatment had persistently low ADAMTS13 activity (<10%), which is an indicator of ongoing disease.

[0244] (iv) Patients with a new relapsed episode - Caplacizumab: 4 / 4 (100%) had ADAMTS13 activity levels of ≥10% near the time of discontinuation of treatment; relapse occurred within ≥30 days after discontinuation of treatment (range: 30-167 days). All four patients also had ADAMTS13 activity <10% at the time of relapse. - Placebo: 2 / 3 (67%) had ADAMST13 activity levels of ≥10% near the discontinuation of treatment; relapse occurred within ≥30 days after discontinuation of treatment (range: 30-167 days). No data was available near the time of relapse. Therefore, in the majority of patients with a new relapsed episode, ADAMTS13 activity recovered to levels above 10% near the discontinuation of treatment, suggesting resolution of the symptomatic TTP episode; however, the value remained low (<10%) at the time of relapse, indicating a new TTP episode.

[0245] (v) Conclusion The data support the use of ADAMTS13 activity as a predictive marker for TTP recurrence and its potential for treatment decisions. ADAMTS13 activity can predict relapses that occur immediately after discontinuing caplacizumab treatment. These relapses are thought to be relapses of symptomatic TTP episodes (resulting in unresolved disease activity based on persistently low ADAMTS13 activity).

[0246] The 30-day treatment period with caplacizumab (post-PE) had a significant impact on the number of exacerbations. Extending the duration of caplacizumab treatment in patients at risk of recurrence (i.e., those with underlying disease activity based on ADAMTS13 activity) will likely maintain the protective effect of caplacizumab until the underlying disease is adequately treated and resolved. Conversely, prophylactic treatment with caplacizumab may reduce the risk of acute episodes of TTP.

[0247] 7.5.9 Research findings: Organ damage markers Characteristic microvascular occlusion in TTP patients can lead to organ ischemia throughout the body, including the brain and heart, and (to a lesser extent) the kidneys. Acute myocardial infarction (AMI) has been reported as an early complication of troponin thrombus (TTP) based on both clinical diagnosis (Patschan et al. 2006 Nephrology, dialysis, transplantation 21: p.1549-54) and autopsy findings (Hosler et al. Archives of pathology & laboratory medicine, 2003. 127(7): p.834-9). Patients clinically suspected of having TTP based on elevated serum lactate dehydrogenase (LDH) and troponin I (TnI) levels were shown to be at high risk of developing AMI. Elevated troponin T levels were associated with mortality and acute morbidity. Subjects who died had higher troponin T levels, while no deaths were reported in the group with normal troponin T levels. Histological examination confirmed extensive myocardial microvascular thrombosis in the deceased subjects (Hughes et al. 2009 J.Thromb.Haemost. 7: p.529-536). These results suggest that by more rapidly reducing further microvascular myocardial ischemia in symptomatic TTP episodes, as measured, for example by troponin, there may be clinical benefits, such as a reduction in the risk of organ damage, including the brain, heart, and kidneys. Other retrospective reports have found similar results for acute and long-term cardiac complications in TTP patients, including myocardial infarction (Wahla et al. 2008 Eur. J. Haem. 81: p.311-6), as well as other cardiac events such as infarction, arrhythmias, cardiogenic shock, and sudden cardiac death (Hawkins et al. 2008 Transfusion 48: p.382-92).

[0248] Retrospective studies have shown that high levels of LDH in symptomatic cases are associated with a worse long-term prognosis (mortality), which reflects severe multi-organ complications (Benhamou, et al. 2012 Haematologica 97: p.1181-6). Therefore, rapid normalization of LDH as a marker for hemolysis and ischemic organ injury may also be beneficial for long-term clinical outcomes and reduce the risk of organ injury.

[0249] Because ischemic injury can lead to acute complications and a worse long-term prognosis, we analyzed specific and clinically relevant organ injury biomarkers: LDH, troponin T or I, and creatinine. We considered cardiac troponin (I or T) to be a specific marker for myocardial injury, creatinine a biomarker for renal function, and LDH a marker for hemolysis, and even a marker for organ injury, primarily in this disease. In TTP patients, elevated baseline levels or slower normalization of some of these biomarkers have been associated with poorer clinical outcomes (e.g., death, refractory disease). The data suggest that a more rapid reduction in microvascular tissue ischemia, as measured by organ injury biomarkers, could lead to clinical benefits, such as a reduced risk of organ injury. However, it should be noted that the results of organ injury markers are confounded to some extent by the diluting effect of daily plasmapheresis (in both the patient and placebo groups).

[0250] (i) Lactate dehydrogenase LDH is an important marker of nonspecific tissue ischemia. The ratio was calculated based on the number of subjects in the ITT population. The number and proportion of subjects in the ITT population with LDH values ​​≤ 2 × ULN were summarized by the planned treatments for the first 5 days of the study. A summary of the results is provided in Table 9. The mean LDH / ULN ratio for each study day is provided in Table 10. On day 1, 11 subjects (30.6%) in the ALX 0081 treatment group and 9 subjects (23.1%) in the placebo group had LDH values ​​≤ 2 × ULN. The proportion of subjects in the ALX 0081 treatment group who maintained higher levels was compared to the placebo group on day 2 (28 subjects [77.8%] and 20 subjects [51.3%], respectively) and on day 3 (33 subjects [91.7%] and 29 subjects [74.4%], respectively). On days 4 and 5, the proportion of subjects with LDH values ​​≤ 2 × ULN was similar in both groups.

[0251] The mean LDH / ULN ratio was comparable between the ALX 0081 and placebo groups on day 1 (3.93 and 3.98, respectively), and was higher in the placebo group at all other time points included. Table 9: Number and percentage of subjects with lactate dehydrogenase levels ≤2 × upper limit of normal for each study day (treatment target population) [Table 9]

[0252] Table 10: Average lactate dehydrogenase / upper limit of normal ratio per study day (treatment target group) [Table 10] Kaplan-Meier analysis was performed to compare the time to normalization of LDH levels according to the planned treatment. The curves for time to LDH normalization are shown in Figure 2. Kaplan-Meier analysis suggests that subjects treated with ALX 0081 show a more rapid recovery to normal LDH levels compared to subjects treated with placebo.

[0253] (ii) troponin Kaplan-Meier analyses were performed to compare the time to normalization of troponin T or troponin I levels following planned treatments. As mentioned above, both TnT and TnI are appropriate biomarkers for cardiac cell damage. Curves of the time to normalization of troponin T or I are provided in Figure 3. Kaplan-Meier analysis suggests that subjects treated with ALX 0081 show a more rapid recovery to normal troponin T or I levels compared to subjects treated with placebo.

[0254] (iii) Creatinine Furthermore, in this case, a Kaplan-Meier analysis was performed to compare the time to normalization of creatinine levels according to the planned treatment. The curves of time to creatinine normalization are shown in Figure 4. Regarding creatinine levels, Kaplan-Meier analysis suggests that subjects treated with ALX 0081 showed a more rapid recovery to normal levels compared to those treated with placebo.

[0255] (iv) Discussion Considering the pathophysiology of acquired TTP, in which ULvWF strings consume platelets in microthrombus formation, we determined that recovery of platelet counts is an indirect measure of preventing further microthrombus formation. The morbidity and acute mortality associated with acquired TTP are consequences of these microthrombi. In fact, this assessment is supported by organ damage markers. Specifically, the results show that organ damage markers troponin I and T, LDH, and creatinine recover to normal levels more rapidly in subjects treated with ALX 0081 compared to those treated with placebo. Therefore, the results suggest that a faster rate of normalization of these organ damage markers is associated with better clinical outcomes, namely a reduced risk of organ damage caused by organ ischemia induced by microthrombi, and that these damages are less frequent.

[0256] 7.5.10 Rationale for dose selection The pharmacological effect of caplacizumab is 2x. Caplacizumab affects the functional groups of vWF, preventing vWF from binding to platelets. Caplacizumab also affects the disposal of vWF in the body, resulting in a transient decrease in total vWF:Ag levels during treatment. Using a PK / PD model, we evaluated the predicted exposure and corresponding effects on total vWF, free vWF, and complexed vWF levels for different dose scenarios in a hypothetical TTP patient population through simulations. A hypothetical population of TTP patients (n=500) with population-based features from the TITAN study was created by sampling the truncated weight distribution (mean 81.9 kg ± 22.6 SD, range 47.5–150 kg), baseline vWF from the TITAN trial TTP population estimated by the model, and the sex distribution (M:F = 40:60).

[0257] The simulated scenario included daily PE procedures for the first 8 days, followed by administration of caplacizumab in combination with sc at doses of 2.5, 5, 10, and 20 mg daily, one hour after the discontinuation of each PE (Period 1). Subsequently, it was assumed that caplacizumab was administered at the same dose level / regmen for a further 30 days in the absence of any other PE procedures (Period 2). The percentage change in free vWF from baseline increases with dose, but is less dose-proportional. Large inter-individual variability, primarily related to the large variability of target expression (vWF) at baseline, is predicted at all dose levels. Two lower doses (2.5 and 5 mg once daily) result in suboptimal target inhibition, while caplacizumab at higher daily doses than that tested in the TITAN study (20 mg) provides no substantial benefit to the simulated TTP population. Figure 5 shows the percentage decrease in free vWF:Ag levels from baseline at the end of period 2, as predicted by the model, as a function of dose level, including patients treated with placebo.

[0258] Figure 6 illustrates the plasma profiles of the free, complexed, and total vWF levels of the drug, simulated for a once-daily dose of 10 mg. Following the evaluation of changes in free vWF levels as a marker for targeted neutralization, we assessed changes in total vWF levels as a marker for bleeding risk. We used a threshold of 0.4 IU / mL (or 16 nM) associated with the highest vWF levels observed in type 1 von Willebrand disease. At these levels of vWF, sufficient levels of FVIII are still considered to remain available. Panel D indicates that total vWF levels for the proposed 10 mg once daily dose would remain higher than this threshold considered for the risk of bleeding events. Based on the results of previous in vitro studies and the levels of biomarkers for efficacy (free vWF) and safety (total vWF) predicted by the model, the dose regimen used herein (5–40 mg, preferably 10 mg per day) is considered appropriate to achieve the desired suppression of (UL)vWF platelet binding capacity in TTP patients, while bleeding events are considered to be minimal and at least controllable.

[0259] 7.6 Conclusion There is considerable variability in target expression (vWF) at the start of the study. However, the dose used results in optimal target inhibition and substantially benefits the entire TTP population being treated. Therefore, ALX 0081 represents a novel approach to the treatment of TTP and offers significant benefits in terms of efficacy, safety, and quality of life for patients with TTP. The concept of caplacizumab was demonstrated by a statistically significant and clinically meaningful reduction in time to confirmed platelet response. The median time to confirmed platelet response was 3 days for caplacizumab compared to 4.9 days for placebo. HR (placebo vs. caplacizumab) = 2.2, 95% CI (1.28, 3.78), p = 0.013.

[0260] The number of exacerbations decreased from 11 in the placebo group to 3 in the caplacizumab group. This highlights the protective effect of caplacizumab treatment. Compared to two deaths in the placebo group, there were no deaths in the caplacizumab group. AEs and SAEs were consistent with serious, potentially life-threatening conditions. Bleeding events (66 events) in the caplacizumab group were nearly twice as numerous as in the placebo group (35 events), but there were only 5 SAEs in 2 subjects in the caplacizumab group and 2 SAEs in 2 subjects in the placebo group. Overall, the benefit-risk assessment for patients with acquired TTP is very positive.

[0261] Polypeptides of the present invention, such as ALX 0081, when used in combination with PE and blood transfusion, enable more rapid control of acute TTP attacks through their inhibition of ULvWF-mediated platelet aggregation and the resulting antithrombotic effect. This significantly reduces the risk of organ ischemia. More rapid normalization of platelet counts also reduces the risk of hemorrhagic complications. Its use also results in improved prognosis in low-response patients, including those with secondary TTP, for whom mortality from the disease remains high. In addition, polypeptides of the present invention, such as ALX 0081, are valuable in preventing relapse after recovery from an acute episode.

[0262] Table A-1: ​​Examples of polypeptides including ISVD and CDR for vWF Table A-1-1 Table A-1-2 Table A-1-3 Table A-1-4

[0263] Table A-2-1 Table A-2-2

Claims

1. A pharmaceutical composition comprising a polypeptide containing two anti-human von Willebrand factor (vWF) immunoglobulin monovariate domains (ISVDs), wherein the pharmaceutical composition is intended for use in reducing and / or preventing the risk of organ damage due to vWF-related disease in a person in need, and for restoring LDH levels, troponin I or T levels, and creatinine levels, and the step of administering the polypeptide to a human being at least 5 to 40 mg / day, preferably 10 mg / day; and At least one ISVD for vWF is represented by sequence number 19 (12A02H1), or The polypeptide is at least 90% identical to SEQ ID NO:

1. The aforementioned pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the polypeptide is ALX 0081 (SEQ ID NO: 1).

3. The pharmaceutical composition according to claim 1 or 2, wherein the aforementioned dose is administered once or twice a day.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the step of administering the polypeptide of the present invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or longer than 10 days, for example 20 days, preferably longer than 30 days, for example 2 months, 3 months, 4 months, 5 months, 6 months, or longer.

5. The pharmaceutical composition according to any one of claims 1 to 4, comprising repeatedly administering the polypeptide to the human until the platelet count in the human is at least 150,000 / μl.

6. The pharmaceutical composition according to claim 5, comprising repeatedly administering the polypeptide to the human until the platelet count in at least two consecutive measurements reaches at least 150,000 / μl.

7. The pharmaceutical composition according to claim 6, wherein the step of administering the polypeptide of the present invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or longer than 10 days, for example 20 days, preferably longer than 30 days, or longer, after the platelet count has reached at least 150,000 / μl in at least two continuous measurements.

8. The pharmaceutical composition according to claim 6 or 7, wherein the two consecutive measurements are separated by at least 24 hours, more preferably 48 hours, for example, at least 3 days, or longer, for example, 4, 5, 6 or 7 days, preferably 1 week.

9. The pharmaceutical composition according to any one of claims 1 to 8, for use in reducing and / or preventing organ damage due to vWF-related disease in a person in need thereof, further comprising the step of measuring the level of an organ damage marker, preferably the organ damage marker being selected from the group consisting of LDH, troponin T, troponin I and creatinine.

10. The pharmaceutical composition according to claim 9, comprising at least the step of administering to the human being a dose of 5 to 40 mg / day, preferably 10 mg / day, of the polypeptide until the level of the organ damage marker reaches a normal level.

11. The pharmaceutical composition according to claim 9, comprising at least the step of administering to the human being a dose of 5 to 40 mg / day, preferably 10 mg / day, of the polypeptide until the levels of the organ damage markers, such as LDH levels, troponin T levels, troponin I levels, and / or creatinine levels, return to normal levels.

12. The pharmaceutical composition according to claim 9, comprising at least the step of administering to the human being a dose of 5 to 40 mg / day, preferably 10 mg / day, of the polypeptide until the levels of the organ damage markers, such as LDH levels, troponin T levels, troponin I levels, and / or creatinine levels, are improved to normal levels.

13. A pharmaceutical composition comprising a polypeptide containing at least one immunoglobulin monovariate domain (ISVD) for von Willebrand factor (vWF), wherein the pharmaceutical composition is for use in reducing and / or preventing the risk of organ damage due to vWF-related disease in a person in need, and for restoring LDH levels, troponin I or T levels, and creatinine levels; wherein the administration of the polypeptide reduces and / or prevents the risk of organ damage by 10%, 20%, 30%, preferably at least 40%, or at least 50%, 60%, or at least 70% compared to the risk of organ damage in a person not administered the pharmaceutical composition; and wherein the pharmaceutical composition comprises a polypeptide containing at least one ISVD for vWF represented by SEQ ID NO: 19 (12A02H1), or wherein the polypeptide is at least 90% identical to SEQ ID NO:

1.

14. The pharmaceutical composition according to claim 13, wherein the step of administering the polypeptide is repeated for at least 1, 2, 3, 4, 5, 6, or 7 days, or longer, for example, 1 week, 2 weeks, 3 weeks, or longer, for example, 1 month or 2 months.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the vWF-related disease is selected from acute coronary syndrome (ACS), transient ischemic attack, unstable or stable angina pectoris, stroke, myocardial infarction, or thrombotic thrombocytopenic purpura (TTP), and is preferably TTP.

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